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Archive for the ‘Fatty acids’ Category


 Cholesterol Lowering Novel PCSK9 drugs: Praluent [Sanofi and Regeneron] vs Repatha [Amgen] – which drug cuts CV risks enough to make it cost-effective?

Reporter: Aviva Lev-Ari, PhD, RN

 

UPDATED on 1/15/2019

In the patent fight over PCSK9 inhibitors, the Supreme Court refused to hear Amgen’s appeal of a 2017 court decision allowing Sanofi and Regeneron to continue selling alirocumab (Praluent). Amgen still has a new patent trial starting in Delaware federal court next month, FiercePharma reports.

Amgen’s Repatha hits wall at SCOTUS but presses ahead—new price breaks included

Amgen has been trying since 2015 to protect its PCSK9 cholesterol drug Repatha by keeping Sanofi and Regeneron’s rival Praluent off the market, even going as far as to ask the U.S. Supreme Court to review an ongoing patent fight.

But that attempt fell short this week as SCOTUS refused to hear the company’s appeal of a 2017 court decision allowing Sanofi and Regeneron to continue selling its head-to-head rival.

Amgen isn’t giving up the fight, though. The company is prepping for a new patent trial starting in Delaware federal court next month. And it’s responding to long-standing criticism of the high cost of PCSK9 drugs, which hit the market in 2015 at list prices of about $14,000 a year.

Amgen had already brought the price of the biweekly version of Repatha down to $5,850 per year before discounts and rebates, and late Monday it said it would lower cost of the monthly injectable dose to that same level.

SOURCE

UPDATED on 11/13/2018

ODYSSEY OUTCOMES: Alirocumab Cost-effective at $6000 a Year

Marlene Busko

November 11, 2018

CHICAGO — Treatment with the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor alirocumab (Praluent, Sanofi/Regeneron) is cost-effective at $6319 a year when the willingness-to-pay threshold is the generally accepted $100,000 per quality-adjusted life-year (QALY), new research reports.

Deepak L. Bhatt, MD, MPH, Brigham and Women’s Hospital Heart and Vascular Center, Harvard Medical School, Boston, Massachusetts, presented these cost-effectiveness findings for alirocumab, based on data from the ODYSSEY OUTCOMES trial, here at the American Heart Association (AHA) 2018 Scientific Sessions

As previously reported, results from ODYSSEY OUTCOMES were presented at American College of Cardiology (ACC) 2018 Annual Scientific Session in March and the study was published November 7 in the New England Journal of Medicine.

Strengths of the current cost analysis include that it used actual trial data as opposed to modeling estimates, Bhatt pointed out to theheart.org | Medscape Cardiology.

SOURCE

https://www.medscape.com/viewarticle/904744?nlid=126063_3866&src=WNL_mdplsfeat_181113_mscpedit_card&uac=93761AJ&spon=2&impID=1799507&faf=1

 

Did Amgen’s Repatha cut CV risks enough to make it cost-effective? Analysts say no

Sanofi, Regeneron’s Praluent pulls off PCSK9 coup with 29% cut to death risks in most vulnerable patients
SEE our curations on PCSK9 drugs:

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LIVE 9/21 8AM to 2:40PM Targeting Cardio-Metabolic Diseases: A focus on Liver Fibrosis and NASH Targets at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

#BostonDOT16

@BostonDOT

 

Nonalcoholic Steatohepatitis (NASH)

 

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

 

Wednesday, September 21

7:30 am Registration Open and Morning Coffee

8:00 Chairperson’s Opening Remarks

Rebecca Taub, M.D., Ph.D., CEO, Madrigal Pharmaceuticals

  • Epidemic of NASH,
  • approaches to treating NASH – Fibrosis
  • NASH is a metabolic Disease of the Liver
  • Treating the HCV will treat the Fibrosis

8:10 FEATURED PRESENTATION: The Epidemic of Fatty Liver Disease: Silent, Serious and Still Growing?

Lee Kaplan, M.D., Ph.D., Director, Obesity, Metabolism and Nutrition Institute, Massachusetts General Hospital, Harvard Medical School

  • Silent, Serious and Growing
  • Obesity the Disease = BMI>30: Medical Complicastions for BMI >%) – On ANti-Obisity and Bariatric SUrgery, Type 2 Diabetis .. NAFLD .. NASH .. Cirrhosis .. HCC
  • Parkinson’s Disease
  • HIV/AIDS
  1. Medical Complications of Obisity =197 :
  2. NAFLD – Nonalcoholic Fatty Liver Disease >>> Liver transplantation replacing HCV
  3. Associated with obesity and type 2 diabetes
  4. NAFLD is UP 90% wiht Severe Obesity
  5. Viral hepatitis and Hemochromatosis
  6. NAFLD: Steatosis, Inflamamtion, Hepatocellular Necrosis, Fibrosis, Cirrhosis
  7. NASH: insulin resistence .. metabolic syndrom .. interaction
  8. Alternative Model: Metabolis Syndrom.. Steatosis .. NASH … FIbrosis
  9. Genetics of Liver DIsease
  10. PNPLA3 Associated with NAFLD – Not Weight Gain
  11. Other genes: A Partial List:
  12. Diagnosis of NASH: Liver biopsy macrovescicular fatty change: InflammationMollery bodies
  13. 75% Patients with Cirrhousis have obisity
  14. Alcoholoc hepatisis >> Progression to Cirrhousis
  15. Macrovesicular Steatosis
  16. NASH – inflammation
  17. Sinusoidal Pericellular Fibrosis –
  18. LAB Features of NAFLD
  • Transaminase elevation
  • Akaline phosphate
  1. Biomarkers – NASH – associated cirrhousis with lower rate 30% of elevation
  2. Fibrosure
  • Clinical Features of NASH: none presentation, Bright, Echo Fibroscan FibroscanScreen for HCC, Varices if Gray zone: Biopsy
  • Treatment of NASH
  • Treat liver disease: Treat steatosis then Inflamamtion and fibrosis
  1. NAFLD Treatment Strategy: Stepwise Approach
  • Treat the steatosis Piodlitazone
  • PPARalpha, delta,
  • Treat Inflammation: ANtioxidant
  • CCR2/CCR% inhibitors
  • Metabolic SUrgery
  • Weigh-independent for bariatric
  • Bariatic: improvrment of steatosis,effect on inflammationless clear
  • dramatic on weigh loss
  • NO clear is surgery improved cirhousis
  • If NASH developed >>>> progression s the rule
  • No great treatment of NASH

Medication-assciated NASH: Glucocorti

 

8:40 Non-Alcoholic Steatohepatitis and Cardiovascular Disease: Modulation by Novel PPAR Agonists

Bart Staels, Ph.D., Professor, INSERM, University of Lille, Pasteur Institute

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors which regulate lipid and glucose metabolism as well as inflammation. In this presentation, we will review recent findings on the pathophysiological role of PPARs in the different stages of non-alcoholic fatty liver disease (NAFLD), from steatosis development to steatohepatitis and fibrosis, as well as the preclinical and clinical evidences for potential therapeutical use of PPAR agonists in the treatment of NAFLD. PPARs play a role in modulating hepatic triglyceride accumulation, a hallmark of the development of NAFLD. Moreover, PPARs may also influence the evolution of reversible steatosis towards irreversible, more advanced lesions. Large controlled trials of long duration to assess the long-term clinical benefits of PPAR agonists in humans are ongoing.

Non-Alcoholic Steatohepatisis and CVD – Meta inflammatory disease

  • NAFL — abnormal Lipid accumulation
  • NASH >> Balooning, FIbrosis inflamation
  • Resolution of NASH is associated with reduction of Fibrosis (Golden – 505 trial)

CVD is linked to NAFLD: Lipids elevated and therosclerosis

  • TG – elevated, APO B elevated, VLDL – elevated HDL decrease
  • PPAR Alpha
  • Gamma
  • PPAR Beta/Delta agonist: GENFIT – Elafibranor
  • SPPARM

Trans-activation: Lipid and Glucose homeostasis: Trans-repression – anti-inflammatory properties

  • Hapatic mitochondrial activity deseases upon progression from NAFL to NASH: Obese NAFL and NASH
  1. Upregulated hepatic respiratory in obese humans with or without NAFL
  2. Impaired
  3. Hepatic PPARalpha Expression Decreases upon Progression of Nash and Fibrosis
  4. hepatic PPARalpha expression – target genes increase in patients with improved NASH histology after 1 year
  5. Metabolic Regualtion by thehepatic JNK Signaling Pathway
  6. Target gene transcription – miR-21 expression increases in human
  7. PPAR Delta: Elafibbranor: – effect on plasma lipids: A Dual PPAR alpha/Delts (GFT505): 80mg vs placebo and 120mg vs placebo, improves plasma apolipolipids and glucose HbA1C – insulin sensitivity
  8. efficacy in NASH acting on: Steatosis, fibrosis and cirrhosis
  9. inflammatory markers: RESOLVE-IT Phase 3 Study Desing: NASH ressolution without adverse on FIbrosis and Cirrhosis

GOLDEN505 Trial: Improves plasma lipid levels: Triglycerides

Inclusion Criteria:

ALT, AST, GGT, ALP

Improve atherogenic dyslipidemia

  • APOC3 – associated with CVD

9:10 PANEL DISCUSSION: Liver Fibrosis and NASH Targets

Moderator: H. James Harwood, Ph.D., Delphi BioMedical Consultants, LLC

Panelists:

Lee Kaplan, M.D., Ph.D., Director, Obesity, Metabolism and Nutrition Institute, Massachusetts General Hospital, Harvard Medical School

Bart Staels, Ph.D., Professor, INSERM, University of Lille, Pasteur Institute

Rebecca Taub, M.D., Ph.D., CEO, Madrigal Pharmaceuticals

Weilin Xie, Ph.D., Senior Principal Scientist, Biotherapeutics, Celgene Corp.

  • FDA’s view on surrogate endpoints
  • Biomarkers of NASH
  • Regulatory challenges
  1. Liver biopsy: gold standard, invasive direct measure of endpoints pros/cons
  2. non-invasive functional tests – plasma bioamrkers
  3. non-invasisve liver imaging techniques: MRI to assess hepatic fat content MRE to assess hepatic fibrosis, Fibroscan,
  4. Endpoints acceptable by FDA: Current vs Future
  • Pre clinical Translational animal models

Discussion by Panel members

Progression from NAFLD to NASH: Oxidative stress and toxic lipids

NASH and Steatosis are different populations

Alcoholoc Steatosis vs Non-Alcoholic Steatosis

  • Obesity cause of Fatty liver
  • NASH in Diabetes
  • NASH progresses
  • Steatosis is associated with NASH
  • Different types of NASH: HTN, Dislipedemia,
  • GENETICS underlining factors, more genes are discovered
  • Limitations of Animal Studies for inference on Humans – careful in over generalizing results
  • Metabolic SYndrom -not all progresses to NASH
  • Nonalcoholic Steatohepatitis (NASH) depend on Steatosis

 

9:40 Coffee Break in the Exhibit Hall with Poster Viewing

10:25 Targeting Fibroblast Activation Protein (FAP) and FGF21 to Treat Fatty Liver Disease

Diana Ronai Dunshee, Ph.D., Department of Molecular Biology, Senior Scientific Researcher, Genentech, Inc.

FGF21 is a hormone with anti-obesity and hepatoprotective properties. However, the beneficial effects of FGF21 are limited by a relatively short half-life in circulation. We discovered that fibroblast activation protein (FAP), an endopeptidase overexpressed in liver with cirrhosis, cleaves and inactivates FGF21. Pharmacological inhibition of FAP increases endogenous levels of active FGF21, thus making FAP a promising target for the treatment of non-alcoholic-steatohepatitis (NASH).

  • Medical complications of obisity: NASH and DM-2
  • energy consumption
  • white adipose tissue – energy storage
  • brown adipose tissue matochondia’s energy
  • FGF21 – Human activation of protein cleavage: A Homone beneficial on metabolic health circulation, weigh loss
  • it suppreses hepatic Steatohepatitis
  • One singleinjection in mice — leads to energy expenditure induced weigh loss and metabolic improvement in Obese Humans
  • Negative FGF21 is Rapidly Eliminated from the body – renal degradation and Inactivation of FGF21 Endopeptidase Cleavage Site – Fibroblast Activation Protein Matched FAP Endopeptidease Specificity
  • Closest relative of DPP4 upregulted during tissue injury in NASH
  • FAP is SUfficient to Cleave FGF21
  • Recombinant FGF21 with Recombinant FAP in Serum or Plasma
  • FAP Protease – Serum Immunodepleted Ablates FGF21 Cleavage Activity: Peptide IgG vs anti-FAP
  • FAP Cleavage Inactivates Human FGF21 dependent on KLB-FGFR1c placed on the site
  • hFGF21 in Not Cleaved in FAP KO Mice
  • Fc-hFGF21 is more stable in FAP KO mice
  • FAR cleaves Endogenously Produced FGF21 In Vivo in monkeys and in dogs
  • The FAP Cleavage Consensus GLY-Pro is COnserved in most mammalian FGF21
  • FAP Does not Cleave the C-Terminal Residues of Mouse FGF21
  • Human: FAP, DPPIV
  • Mouse: FAP, DPP4
  • FAP INhibition
  • FAP is Overexpressed in Liver with Steatohepatitis: Early NASH vs Late NASH
  • Proposal: FAP Inhibition for FGF21 Stabilization in NASH
  1. Fatty hepatocytes – e.g. NASH
  2. Activated stellate cells, e.g. NASH

 

10:55 Thyroid Hormone Receptor Beta (THR-ß) Agonist for NASH: Correcting a Primary Deficiency in NASH Livers

Rebecca Taub, M.D., Ph.D., CEO, Madrigal Pharmaceuticals

NASH patients typically have metabolic syndrome including diabetes, dyslipidemia, obesity, and primarily die of cardiovascular disease. Hypothyroidism at the level of the thyroid gland and liver-specific hypothyroidism are common in NASH. Based on clinical and preclinical data, Thyroid receptor beta agonists decrease insulin resistance, reduce LDL-C, triglycerides fatty liver, inflammation and fibrosis in NASH. The target will also provide CV benefit to patients with NASH. MGL-3196 is a highly THR-ß selective liver-directed once daily oral medication that has shown excellent safety and lipid-lowering efficacy in humans; unlike prior thyroid receptor agonist(s), no cartilage findings in chronic toxicology or ALT increases in human studies. MGL-3196 is being advanced in Phase II studies in patients with genetic dyslipidemia or NASH.

Madrigal Portfolio of drugs:

  • MGL-3196: First-in-Class THR-Beta Agonist – discovered first at ROCHE – THR-beta selective targeted to the Liver – regulated by THR-Alpha  – in Phase II – no side effects on bone
  • Large & underserved Markets in NASH
  • Phase 2 HeFH Patients
  1. Hypothyroidism common in NASH patients
  2. Liver-specific Hypothyroidism present in human NASH degradation of thyroid hormone increases deiodised 9DIO) 3 produced by Stelllate cells in NASH liver
  3. Treating NASHrather than fibrosis is key in addressing the disease – approvable endpoint
  4. THR – Thyroid hormone reduces Cholesterol
  5. Thyroid hormone T3 thyroxine – treatment amy cause osteoporosis
  6. MGL 0 3196: Liver size, Live Triglycerides, Improve Insulin tolerance, decrease ALT
  7. Reduction of key NASH, Fibrosis Pathway Genes at Human Comparable Drug levels
  8. THR-beta: Decreased Liver Fibrosis, Apoptosis in mice:

HUMAN DATA

  • Single ascending dose study
  • Multiple – ascending studies: LDL and TG decrease
  • decrease Non-HDL CHolesterol
  • Decrease Apolipoprotein B
  • Pleiotropic Pioglitazone Effect in NASH at 6 month treatment and biopsy of liver – dramatic effect in NASH – ten years ago study
  • PPAR gamma agonist – NEGATIVE SIDE EFFECTS: weight gain, CHF, Bone osteoporosis
  • Anti-inflammatory: well tolerated

No Single NASH Therapeutics – Conbination agents

MGL – 3196 Phase 2 – Study: Proposed Phase 2 Proof of COncepts NASH Protocol

  • Unmet needs in FH, a severeGenetic Dyslipedemia
  • Weight loss in 6 weeksreduction in cholesterol and TG
  • Likelihood of Success
  • second study after 9 months
  • is different on NASH Patients in 12 weeks using MRI on Liver
  • prevalence
  • HeFH, PCSK9 inhibitors plus standard care
  • Unique and Complementary Lipid Lowering Profile
  1. Lowers Lp(a) and severely atherogenic practice
  2. Proposed Phase 2 HeFH Patients

 

11:25 Enjoy Lunch on Your Own

 

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What drug interfered with the performance of Sharapova?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Meldonium — The Drug That Brought Down Sharapova

Gayle Nicholas Scott, PharmD

When tennis player Maria Sharapova recently revealed that she had tested positive for the banned drug meldonium, the reaction of most healthcare providers was, “What is it?”

Meldonium is manufactured and sold as Mildronate by the pharmaceutical company Grindeks in the Baltic nation of Latvia. Meldonium is not available in the United States or elsewhere in the European Union (it was grandfathered in Latvia) other than via purchase on the Internet.

The World Anti-Doping Agency classifies meldonium as a “metabolic modulator” and moved the drug from its watch list to its list of banned substances in January 2016.

Other “metabolic modulators” are insulin and trimetazidine, an anti-ischemic metabolic agent that increases myocardial glucose utilization through inhibition of fatty acid metabolism.[1,2] Trimetazidine is approved in the European Union for the treatment of angina, but it is not approved in the United States.

The chemical name for meldonium is trimethylhydrazinium propionate. Meldonium works by decreasing the availability of levocarnitine (L-carnitine). L-carnitine is found naturally in milk and meats, and also can be synthesized by the body from lysine and methionine with the help of gamma-butyrobetaine hydroxylase. L-carnitine helps move long-chain fatty acids into the mitochondria for oxidation and energy production in the muscles.

Ironically, L-carnitine, which meldonium inhibits, is taken as a dietary supplement alone and as an ingredient in energy drinks to increase athletic performance. (L-carnitine is available in the United States as the prescription drug Carnitor®, which is indicated for carnitine deficiency owing to inborn errors of metabolism and for end-stage renal disease requiring dialysis.) After two decades of research, no consistent evidence has emerged indicating that carnitine supplements can improve exercise or physical performance. Carnitine supplements do not appear to increase the body’s use of oxygen or improve metabolic status when exercising, and may not increase the amount of carnitine in muscle.[3,4]Carnitine is not on the list of banned substances.[1]

As a modulator of L-carnitine metabolism, meldonium inhibits gamma-butyrobetaine hydroxylase and L-carnitine transmembrane transport of long-chain fatty acids, thus decreasing L-carnitine levels in tissue and plasma. Reducing the amount of bioavailable L-carnitine shifts the source of metabolic energy production from fatty acid oxidation to glucose metabolism. Aerobic glucose oxidation consumes less oxygen than fatty acid oxidation and increases the effectiveness of adenosine triphosphate (ATP) generation. Additionally, meldonium appears to increase glucose uptake. In ischemic conditions (hypoxia), meldonium appears to restore the balance between cellular oxygen supply and demand, and prevents ATP transport impairment.[3,5]

All published clinical efficacy studies on meldonium, except one,[6] are in Russian. Abstracts of randomized controlled trials have reported the efficacy of meldonium in reducing angina, arrhythmias, and anxiety and other early sequelae of myocardial infarction[7-10]; as an “adaptogen” in patients with cardiovascular disease[11,12]; and in treating angina and reducing myocardial ischemia after percutaneous coronary intervention,[6,13,14] heart failure,[15] and diabetic peripheral neuropathy.[16] Doses, when included in the abstracts, ranged from 750 to 1000 mg per day. Only one abstract mentioned adverse effects, stating that none occurred.[7]

A pharmacokinetic study of meldonium showed that the drug has a dose-dependent half-life and volume of distribution with accumulation on multiple-dose administration. In eight healthy volunteers who received meldonium for 13 days, almost all reported insomnia, half reported burping, and one quarter reported “dreaminess.” No serious adverse effects were reported.[17]

A study in healthy, nonvegetarian volunteers receiving 1000 mg meldonium per day for 4 weeks showed that plasma concentrations of L-carnitine decreased by 18%. Urine samples showed an increase in L-carnitine excretion. Adverse effects were not mentioned.[18] Meldonium is excreted in the urine largely unchanged, making urine testing a valid monitor presence of meldonium.[19]

No long-term studies on the safety and efficacy of meldonium have been published. No studies on the effect of meldonium on athletic performance in humans have been published. One study on the reliability of urine testing in professional sports[19]mentions an article and an abstract, but neither of those appears in PubMed. The abstract purports to be a review of “recent studies on mildronate especially in fields associated with physical work capabilities and sport” but cites only the study mentioned in the urine testing review.[20] Most articles about meldonium cited on PubMed are by Latvian authors.

Animal research suggests the potential usefulness of meldonium in Alzheimer disease,[21-23] Parkinson disease,[24,25] and diabetes.[26-29] Meldonium increased sexual activity in boars[30] but not in male rats.[31] Research in rodents found that meldonium can cause carnitine deficiency in offspring, so the drug should not be taken in pregnancy.[32]

Because meldonium is excreted renally, serum levels may be higher in patients with reduced kidney function, and the drug may accumulate with repeated dosing.[19] L-carnitine appears to antagonize the effects of meldonium[33]; otherwise, drug interactions are not known.

To recap, meldonium is an interesting drug developed by Latvian researchers. Published research suggests that it may be an effective treatment for cardiovascular diseases, such as angina. Little information about its adverse effects has been published, however, and the long-term safety of meldonium is not known. And although reliable research on meldonium’s use for athletic performance is not available, the World Anti-Doping Agency has declared it a banned substance.

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Nuts and health in aging

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Nut consumption and age-related disease

Giuseppe GrossoRamon Estruch

MATURITAS · OCT 2015     http://dx.doi.org/10.1016/j.maturitas.2015.10.014

Current knowledge on the effects of nut consumption on human health has rapidly increased in recent years and it now appears that nuts may play a role in the prevention of chronic age-related diseases. Frequent nut consumption has been associated with better metabolic status, decreased body weight as well as lower body weight gain over time and thus reduce the risk of obesity. The effect of nuts on glucose metabolism, blood lipids, and blood pressure are still controversial. However, significant decreased cardiovascular risk has been reported in a number of observational and clinical intervention studies. Thus, findings from cohort studies show that increased nut consumption is associated with a reduced risk of cardiovascular disease and mortality (especially that due to cardiovascular-related causes). Similarly, nut consumption has been also associated with reduced risk of certain cancers, such as colorectal, endometrial, and pancreatic neoplasms. Evidence regarding nut consumption and neurological or psychiatric disorders is scarce, but a number of studies suggest significant protective effects against depression, mild cognitive disorders and Alzheimer’s disease. The underlying mechanisms appear to include antioxidant and anti-inflammatory actions, particularly related to their mono- and polyunsaturated fatty acids (MUFA and PUFA, as well as vitamin and polyphenol content. MUFA have been demonstrated to improve pancreatic beta-cell function and regulation of postprandial glycemia and insulin sensitivity. PUFA may act on the central nervous system protecting neuronal and cell-signaling function and maintenance. The fiber and mineral content of nuts may also confer health benefits. Nuts therefore show promise as useful adjuvants to prevent, delay or ameliorate a number of chronic conditions in older people. Their association with decreased mortality suggests a potential in reducing disease burden, including cardiovascular disease, cancer, and cognitive impairments.

 

Global life expectancy has increased from 65 years in 1990 to about 71 years in 2013 [1]. As life expectancy has increased, the number of healthy years lost due to disability has also risen in most countries, consistent with greater morbidity [2]. Reduction of mortality rates in developed countries has been associated with a shift towards more chronic non-communicable diseases [1]. Cardiovascular diseases (CVDs) and related risk factors, such as hypertension, diabetes mellitus, hypercholesterolemia, and obesity are the top causes of death globally, accounting for nearly one-third of all deaths worldwide [3]. Equally, the estimated incidence, mortality, and disability- adjusted life-years (DALYs) for cancer rose to 14.9 million incident cancer cases, 8.2 million deaths, and 196.3 million DALYs, with the highest impact of prostate and breast cancer in men and women, respectively [4]. Depression is a leading cause of disability worldwide (in terms of total years lost due to disability), especially in high-income countries, increasing from 15th to 11th rank (37% increase) and accounting for 18% of total DALYs (almost 100 million DALYs) [5]. Overall, the global rise in chronic non-communicable diseases is congruent with a similar rise in the elderly population. The proportion of people over the age of 60 is growing faster than any other age group and is estimated to double from about 11% to 22% within the next 50 years [6]. Public health efforts are needed to face this epidemiological and demographic transition, both improving the healthcare systems, as well as assuring a better health in older people. Accordingly, a preventive approach is crucial to dealing with an ageing population to reduce the burden of chronic disease.

In this context, lifestyle behaviors have demonstrated the highest impact for older adults in preventing and controlling the morbidity and mortality due to non- communicable diseases [7]. Unhealthy behaviors, such as unbalanced dietary patterns, lack of physical activity and smoking, play a central role in increasing both cardiovascular and cancer risk [7]. Equally, social isolation and depression in later life may boost health decline and significantly contribute to mortality risk [8]. The role of diet in prevention of disability and death is a well-established factor, which has an even more important role in geriatric populations. Research has focused on the effect of both single foods and whole dietary patterns on a number of health outcomes, including mortality, cardiovascular disease (CVD), cancer and mental health disorders (such as cognitive decline and depression) [9-13]. Plantbased dietary patterns demonstrate the most convincing evidence in preventing chronic non-communicable diseases [14-17]. Among the main components (including fruit and vegetables, legumes and cereals), only lately has attention focused on foods such as nuts. Knowledge on the effect of nut consumption on human health has increased rapidly in recent years. The aim of this narrative review is to examine recent evidence regarding the role of nut consumption in preventing chronic disease in older people.

Tree nuts are dry fruits with an edible seed and a hard shell. The most popular tree nuts are almonds (Prunus amigdalis), hazelnuts (Corylus avellana), walnuts (Juglans regia), pistachios (Pistachia vera), cashews (Anacardium occidentale), pecans (Carya illinoiensis), pine nuts (Pinus pinea), macadamias (Macadamia integrifolia), Brazil nuts (Bertholletia excelsa), and chestnuts (Castanea sativa). When considering the “nut” group, researchers also include peanuts (Arachis hypogea), which technically are groundnuts. Nuts are nutrient dense foods, rich in proteins, fats (mainly unsaturated fatty acids), fiber, vitamins, minerals, as well as a number of phytochemicals, such as phytosterols and polyphenols [18]. Proteins account for about 10-25% of energy, including individual aminoacids, such as L-arginine, which is involved in the production of nitric oxide (NO), an endogenous vasodilatator [19].

The fatty acids composition of nuts involves saturated fats for 415% and unsaturated fatty acids for 30-60% of the content. Unsaturated fatty acids are different depending on the nut type, including monounsaturated fatty acids (MUFA, such as oleic acid in most of nuts, whereas polyunsaturated fatty acids (PUFA, such as alpha-linolenic acid) in pine nuts and walnuts [20]. Also fiber content is similar among most nut types (about 10%), although pine nuts and cashews hold the least content. Vitamins contained in nuts are group B vitamins, such as B6 (involved in many aspects of macronutrient metabolism) and folate (necessary for normal cellular function, DNA synthesis and metabolism, and homocysteine detoxification), as well as tocopherols, involved in anti-oxidant mechanisms [21]. Among minerals contained in vegetables, nuts have an optimal content in calcium, magnesium, and potassium, with an extremely low amount of sodium, which is implicated on a number of pathological conditions, such as bone demineralization, hypertension and insulin resistance[22]. Nuts are also rich in phytosterols, non-nutritive components of certain plant-foods that exert both structural (at cellular membrane phospholipids level) and hormonal (estrogen-like) activities [23]. Finally, nuts have been demonstrated to be a rich source of polyphenols, which account for a key role in their antioxidant and anti-inflammatory effects.

 

Metabolic disorders are mainly characterized by obesity, hypertension, dyslipidemia, and hyperglycemia/ hyperinsulinemia/type-2 diabetes, all of which act synergistically to increase morbidity and mortality of aging population.

Obesity Increasing high carbohydrate and fat food intake in the last decades has contributed significantly to the rise in metabolic disorders. Nuts are energy-dense foods that have been thought to be positively associated with increased body mass index (BMI). As calorie-dense foods, nuts may contain 160–200 calories per ounce. The recommendation from the American Heart  Association to consume 5 servings per week (with an average recommended serving size of 28 g) corresponds to a net increase of 800–1000 calories per week, which may cause weight gain. However, an inverse relation between the frequency of nut consumption and BMI has been observed in large cohort studies [24]. Pooling the baseline observations of BMI by category of nut consumption in 5 cohort studies found a significant decreasing trend in BMI values with increasing nut intake [24]. While the evidence regarding nut consumption and obesity is limited, findings so far are encouraging [25, 26]. When the association between nut consumption and body weight has been evaluated longitudinally over time, nut intake was associated with a slightly lower risk of weight gain and obesity [25]. In the Nurses’ Health Study II (NHS II), women who eat nuts ≥2 times per week had slightly less weight gain (5.04 kg) than did women who rarely ate nuts (5.55 kg) and marginally significant 23% lower risk of obesity after 9-year follow-up [25]. Further evaluation of the NHS II data and the Physicians’ Health Study (PHS) comprising a total of 120,877 US women and men and followed up to 20 years revealed that 4-y weight change was inversely associated with a 1-serving increment in the intake of nuts (20.26 kg) [27]. In the “Seguimiento Universidad de Navarra” (SUN) cohort study, a significant decreased weight change has been observed over a period of 6 years [26]. After adjustment for potential confounding factors the analysis was no longer significant, but overall no weight gain associated with >2 servings per week of nuts has been observed. Finally, when considering the role of the whole diet on body weight, a meta-analysis of 31 clinical trials led to the conclusion of a null effect of nut intake on body weight, BMI, and waist circumference [28].

Glucose metabolism and type-2 diabetes The association between nut consumption and risk of type-2 diabetes in prospective cohort studies is controversial [29-32]. A pooled analysis relied on the examination of five large cohorts, including the NHS, the Shanghai Women’s Health Study, the Iowa Women’s Health Study, and the PHS, and two European studies conducted in Spain (the PREDIMED trial) and Finland including a total of more than 230,000 participants and 13,000 cases, respectively. Consumption of 4 servings per week was associated with 13% reduced risk of type-2 diabetes without effect modification by age [29]. In contrast, other pooled analyses showed non-significant reduction of risk for increased intakes of nuts, underlying that the inverse association between the consumption of nuts and diabetes was attenuated after adjustment for confounding factors, including BMI [30]. However, results from experimental studies showed promising results. Thus, nut consumption has been demonstrated to exert beneficial metabolic effects due to their action on post-prandial glycemia an insulin sensitivity. A number of RCTs have demonstrated positive effects of nut consumption on post-prandial glycemia in healthy individuals [33-38]. Moreover, a meta-analysis of RCTs on the effects of nut intake on glycemic control in diabetic individuals including 12 trials and a total of 450 participants showed that diets with an emphasis on nuts (median dose = 56 g/d) significantly lowered HbA1c (Mean Difference [MD] : -0.07%; 95% confidence interval [CI]: -0.10, -0.03%; P = 0.0003) and fasting glucose (MD : -0.15 mmol/L; 95% CI: -0.27, -0.02 mmol/L; P = 0.03) compared with control diets [39]. No significant treatment effects were observed for fasting insulin and homeostatic model assessment (HOMA-IR), despite the direction of effect favoring diet regimens including nuts.

Blood lipids and hypertension Hypertension and dyslipidemia are major risk factors for CVD. Diet alone has a predominant role in blood pressure and plasma lipid homeostasis. One systematic review [40] and 3 pooled quantitative analyses of RCTs [41-43] evaluated the effects of nut consumption on lipid profiles. A general agreement was relevant on certain markers, as daily consumption of nuts (mean = 67 g/d) induced a pooled reduction of total cholesterol concentration (10.9 mg/dL [5.1% change]), low-density lipoprotein cholesterol concentration (LDL-C) (10.2 mg/dL [7.4% change]), ratio of LDL-C to high-density lipoprotein cholesterol concentration (HDL-C) (0.22 [8.3% change]), and ratio of total cholesterol concentration to HDL-C (0.24 [5.6% change]) (P <0.001 for all) [42]. All meta-analyses showed no significant effects of nut (including walnut) consumption on HDL cholesterol or triglyceride concentrations in healthy individuals [41], although reduced plasma triglyceride levels were found in individuals with hypertriglyceridemia [42]. Interestingly, the effects of nut consumption were dose related, and different types of nuts had similar effects on blood lipid concentrations.

There is only limited evidence from observational studies to suggest that nuts have a protective role on blood pressure. A pooled analysis of prospective cohort studies on nut consumption and hypertension reported a decreased risk associated with increased intake of nuts [32]. Specifically, only a limited number of cohort studies have been conducted exploring the association between nut consumption and hypertension (n = 3), but overall reporting an 8% reduced risk of hypertension for individuals consuming >2 servings per week (Risk Ratio [RR] = 0.92, 95% CI: 0.87-0.97) compared with never/rare consumers, whereas consumption of nuts at one serving per week had similar risk estimates (RR = 0.97, 95% CI: 0.83, 1.13) [32]. These findings are consistent with results obtained in a pooled analysis of 21 experimental studies reporting the effect of consuming single or mixed nuts (in doses ranging from 30 to 100 g/d) on systolic (SBP) and diastolic blood pressure (DBP) [44]. A pooled analysis found a significant reduction in SBP in participants without type2 diabetes [MD: -1.29 mmHg; 95% CI: -2.35, -0.22; P = 0.02] and DBP (MD: -1.19; 95% CI: -2.35, -0.03; P = 0.04), whereas subgroup analyses of different nut types showed that pistachios, but not other nuts, significantly reduced SBP (MD: -1.82; 95% CI: -2.97, -0.67; P = 0.002) and SBP (MD: -0.80; 95% CI: -1.43, -0.17; P = 0.01) [44].

Nut consumption and CVD risk Clustering of metabolic risk factors occurs in most obese individuals, greatly increasing risk of CVD. The association between nut consumption and CVD incidence [29-31] and mortality [24] has been explored in several pooled analyses of prospective studies. The overall risk calculated for CVD on a total of 8,862 cases was reduced by 29% for individuals consuming 7 servings per week (RR = 0.71, 95% CI: 0.59, 0.85) [30]. A meta-analysis including 9 studies on coronary artery disease (CAD) including 179,885 individuals and 7,236 cases, reporting that 1-serving/day increment would reduce risk of CAD of about 20% (RR = 0.81, 95% CI: 0.72, 0.91) [31]. Similar risk estimates were calculated for ischemic heart disease (IHD), with a comprehensive reduced risk of about 25-30% associated with a daily intake of nuts [29, 30]. Findings from 4 prospective studies have been pooled to estimate the association between nut consumption and risk of stroke, and a non-significant/borderline reduced risk was found [29-31, 45]. CVD mortality was explored in a recent meta-analysis including a total of 354,933 participants, 44,636 cumulative incident deaths, and 3,746,534 cumulative person-years [24]. One serving of nuts per week and per day resulted in decreased risk of CVD mortality (RR = 0.93, 95% CI: 0.88, 0.99 and RR =0.61, 95% CI: 0.42, 0.91, respectively], primarily driven by decreased coronary artery disease (CAD) deaths rather than stroke deaths [24]. Overall, all pooled analyses demonstrated a significant association between nut consumption and cardiovascular health. However, it has been argued that nut consumption was consistently associated with healthier background characteristics reflecting overall healthier lifestyle choices that eventually lead to decreased CVD mortality risk.

Nut consumption and cancer risk Cancer is one of the leading causes of death in the elderly population. After the evaluation of the impact on cancer burden of food and nutrients, it has been concluded that up to one third of malignancies may be prevented by healthy lifestyle choices. Fruit and vegetable intake has been the focus of major attention, but studies on nut consumption and cancer are scarce. A recent metaanalysis pooled together findings of observational studies on cancer incidence, including a total of 16 cohort and 20 casecontrol studies comprising 30,708 cases, compared the highest category of nut consumption with the lowest category and found a lower risk of any cancer of 25% (RR = 0.85, 95% CI: 0.86, 0.95) [46]. When the analysis was conducted by cancer site, highest consumption of nuts was associated with decreased risk of colorectal (RR = 0.76, 95% CI: 0.61, 0.96), endometrial (RR = 0.58, 95% CI: 0.43, 0.79), and pancreatic cancer (RR = 0.71, 95% CI: 0.51, 0.99), with only one cohort study was conducted on the last [46]. The potential protective effects of nut consumption on cancer outcomes was supported also by pooled analysis of 3 cohort studies [comprising the PREDIMED, the NHS, the HPS, and the Health Professionals Follow-Up Study (HPFS) cohorts] showing a decreased risk of cancer death for individuals consuming 3-5 servings of nuts per week compared with never eaters (RR = 0.86, 95% CI: 0.75, 0.98) [24]. The analysis was recently updated by including results from the Netherlands Cohort Study reaching a total of 14,340 deaths out of 247,030 men and women observed, confirming previous results with no evidence of between-study heterogeneity (RR = 0.85, 95% CI: 0.77, 0.93) [47]. However, a dose- response relation showed the non-linearity of the association, suggesting that only moderate daily consumption up to 5 g reduced risk of cancer mortality, and extra increased intakes were associated with no further decreased risk.

Nut consumption and affective/cognitive disorders Age-related cognitive decline is one of the most detrimental health problems in older people. Cognitive decline is a paraphysiological process of aging, but timing and severity of onset has been demonstrated to be affected by modifiable lifestyle factors, including diet. In fact, the nature of the age- related conditions leading to a mild cognitive impairment (MCI) differs by inflammation-related chronic neurodegenerative diseases, such as dementia, Alzheimer’s disease, Parkinson’s disease and depression. Evidence restricted to nut consumption alone is scarce, but a number of studies have been conducted on dietary patterns including nuts as a major component. A pooled analysis synthesizing findings of studies examining the association between adherence to a traditional Mediterranean diet and risk of depression (n = 9), cognitive decline (n = 8), and Parkinson’s disease (n = 1) showed a reduction of risk of depression (RR = 0.68, 95% CI: 0.54, 0.86) and cognitive impairment (RR = 0.60, 95% CI: 0.43, 0.83) in individuals with increased dietary adherence [10].

The study that first found a decreased risk of Alzheimer’s disease in individuals highly adherent to the Mediterranean diet was conducted in over 2,000 individuals in the Washington/Hamilton Heights-Inwood Columbia Aging Project (WHICAP), a cohort of non-demented elders aged 65 and older living in a multi-ethnic community of Northern Manhattan in the US (Hazard Ratio [HR] = 0.91, 95% CI: 0.83, 0.98) [48]. These results have been replicated in further studies on the Mediterranean diet, however nut consumption was not documented [49, 50]. A number of observational studies also demonstrated a significant association between this dietary pattern and a range of other cognitive outcomes, including slower global cognitive decline [51]. However, evidence from experimental studies is limited to the PREDIMED trial, providing interesting insights on the association between the Mediterranean diet supplemented with mixed nuts and both depression and cognitive outcomes. Regarding depression, the nutritional intervention with a Mediterranean diet supplemented with nuts showed a lower risk of about 40% in participants with type-2 diabetes (RR = 0.59, 95% CI: 0.36, 0.98) compared with the control diet [52]. However the effect was not significant in the whole cohort overall [52]. Regarding cognitive outcomes after a mean follow-up of 4.1 years, findings from the same trial showed significant improvements in memory and global cognition tests for individuals allocated to the Mediterranean diet supplemented with nuts [adjusted differences: -0.09 (95% CI: -0.05, 0.23), P = 0.04 and -0.05 (95% CI: -0.27, 0.18), P = 0.04, respectively], compared to control group, showing that Mediterranean diet plus mixed nuts is associated with improved cognitive function [53].

 

Potential mechanisms of protection of nut consumption Despite the exact mechanisms by which nuts may ameliorate human health being largely unknown, new evidence has allowed us to start to better understand the protection of some high-fat, vegetable, energy-dense foods such as nuts. Non- communicable disease burden related with nutritional habits is mainly secondary to exaggerated intakes of refined sugars and saturated fats, such as processed and fast- foods. Nuts provide a number of nutrient and non-nutrient compounds and it is only recently that scientists have tried to examine their effects on metabolic pathways.

Metabolic and cardiovascular protection With special regard to body weight and their potential effects in decreasing the risk of obesity (or weight gain, in general), nuts may induce satiation (reduction in the total amount of food eaten in a single meal) and satiety (reduction in the frequency of meals) due to their content in fibers and proteins, which are associated with increased release of glucagon-like protein 1 (GLP-1) and cholecystokinin (CCK), gastrointestinal hormones with satiety effects [54, 55]. The content in fiber of nuts may also increase thermogenesis and resting energy expenditure, and reduce post- prandial changes of glucose, thus ameliorating inflammation and insulin resistance. Moreover, the specific content profile of MUFA and PUFA provides readily oxidized fats than saturated or trans fatty acids, leading to reduced fat accumulation [56, 57]. The beneficial effects of nuts toward glucose metabolism may be provided by their MUFA content that improves the efficiency of pancreatic beta-cell function by enhancing the secretion of GLP1, which in turn helps the regulation of postprandial glycemia and insulin sensitivity [58]. MUFA and PUFA are also able to reduce serum concentrations of the vasoconstrictor thromboxane 2, which might influence blood pressure regulation. Together with polyphenols and anti-oxidant vitamins, nuts may also ameliorate inflammatory status at the vascular level, reducing circulating levels of soluble cellular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, which are released from the activated endothelium and circulating monocytes [59]. Moreover, nuts may improve vascular reactivity due to their content in L-arginine, which is a potent precursor of the endogenous vasodilator nitric oxide. Nuts content in microelements is characterized by a mixture that may exert a direct effect in modulating blood pressure, including low content of sodium and richness in magnesium, potassium and calcium, which may interact to beneficially influence blood pressure
Despite the exact mechanisms by which nuts may ameliorate human health being largely unknown, new evidence has allowed us to start to better understand the protection of some high-fat, vegetable, energy-dense foods such as nuts. Non- communicable disease burden related with nutritional habits is mainly secondary to exaggerated intakes of refined sugars and saturated fats, such as processed and fast- foods. Nuts provide a number of nutrient and non-nutrient compounds and it is only recently that scientists have tried to examine their effects on metabolic pathways.

Cancer protection The potential mechanisms of action of nuts that may intervene in the prevention of cancer have not been totally elucidated. Numerous hypotheses have been proposed on the basis of basic research exploring the antioxidant and anti-inflammatory compounds characterizing nuts [61]. Vitamin E can regulate cell differentiation and proliferation, whereas polyphenols (particularly flavonoids such as quercetin and stilbenes such as resveratrol) have been shown to inhibit chemically-induced carcinogenesis [62]. Polyphenols may regulate the inflammatory response and immunological activity by acting on the formation of the prostaglandins and pro-inflammatory cytokines, which may be an important mechanism involved in a number of cancers, including colorectal, gastric, cervical and pancreatic neoplasms [62]. Among other compounds contained in nuts, dietary fiber may exert protective effects toward certain cancers (including, but not limited to colorectal cancer) by the aforementioned metabolic effects as well as increasing the volume of feces and anaerobic fermentation, and reducing the length of intestinal transit. As a result, the intestinal mucosa is exposed to carcinogens for a reduced time and the carcinogens in the colon are diluted [62]. Finally, there is no specific pathway demonstrating the protective effect of PUFA intake against cancer, but their interference with cytokines and prostaglandin metabolism may inhibit a state of chronic inflammation that may increase cancer risk [63].

Cognitive aging and neuro-protection There is no universal mechanism of action for nuts with regard to age-related conditions. A number of systemic biological conditions, such as oxidative stress, inflammation, and reduced cerebral blood flow have been considered as key factors in the pathogenesis of both normal cognitive ageing and chronic neurodegenerative disease [64]. Nuts, alone or as part of healthy dietary patterns, may exert beneficial effects due to their richness in antioxidants, including vitamins, polyphenols and unsaturated fatty acids, that may be protective against the development of cognitive decline and depression [65, 66]. Both animal studies and experimental clinical trials demonstrated vascular benefits of nuts, including the aforementioned lowering of inflammatory markers and improved endothelial function, which all appear to contribute to improved cognitive function [67]. The antioxidant action may affect the physiology of the ageing brain directly, by protecting neuronal and cell-signaling function and maintenance. Moreover, certain compounds contained in nuts may directly interact with the physiology and functioning of the brain. For instance, walnuts are largely composed of PUFA, especially ALA, which have been suggested to induce structural change in brain areas associated with affective experience [66]. Moreover, PUFA have been associated with improved symptoms in depressed patients, suggesting an active role in the underlying pathophysiological mechanisms [68]. Thus, the mechanisms of action of nut consumption on age-related cognitive and depressive disorders are complex, involving direct effects on brain physiology at the neuronal and cellular level and indirect effects by influencing inflammation.

 

Summary From an epidemiological point of view, nut eaters have been associated with overall healthier lifestyle habits, such as increased physical activity, lower prevalence of smoking, and increased consumption of fruits and vegetables [24]. These variables represent strong confounding factors in determining the effects of nuts alone on human health and final conclusions cannot be drawn. Nevertheless, results from clinical trials are encouraging. Nuts show promise as useful adjuvants to prevent, delay or ameliorate a number of chronic conditions in older people.

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Seaweed in Diet

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

7 Ways to Eat More Seaweed

http://www.integrativenutrition.com/blog/2015/11/7-ways-to-eat-more-seaweed-and-why-you-should

 

http://amznstatic01.integrativenutrition.com/styles/panopoly_image_original/shutterstock_255225709.jpg?itok=0EzFp2t6

 

Seaweed, is making waves again thanks to the discovery of a seaweed that tastes like bacon and is better for you than kale. This could be good news given the World Health Organization’s recent declaration that eating processed meats such as bacon and sausage can increase the risk of colorectal cancer.

But why is seaweed so good for you? And can it actually taste good?

Nutrition-wise, it’s a powerhouse of vitamins and minerals. Seaweeds contain dietary fiber, essential amino acids, vitamins, A, B, C, and E, Omega-3 fats, and minerals such as iodine, calcium, iron, zinc, and magnesium. All of these nutrients combine to reduce inflammation, lift your energy, maintain strong bones and teeth, support thyroid health and hormonal balance, and even reduce your risk of cancer.

It’s no wonder seaweed has been a staple in Asian cuisine for centuries!

While some may have concerns in recent years that seaweed is becoming a potentially harmful food due to the increasing pollution of our oceans, there has not been any evidence to support this despite ongoing testing. That being said, it’s always best to seek out quality products. We recommend finding brands that grow seaweed in sustainable ways and in pure or tested waters.

Let’s get cookin’!

While seaweed hasn’t quite risen to popularity as a superfood like kale or other leafy greens, it’s so versatile and easy to prepare we have a feeling it’ll win you over.

Here are some delicious ways to incorporate more seaweed into your diet:

1. Cook your beans with kombu.
Try adding a strip of kombu when cooking your dried beans to add a rich array of vitamins and minerals to your dish while enhancing the flavor and making the beans more digestible!

2. Snack on nori.
Nori is lightweight and easy to pack yet also nutritious and surprisingly filling.  It’s the perfect thing to throw into your bag for a quick snack at work or on the go, and is a healthy alternative for those with salty cravings. Even your kids will enjoy its crispy texture. Want another great way to use nori? Try these delicious seaweed breakfast wraps!

3. Enhance your smoothies with spirulina.
Powdered seaweed such as spirulina is a great source of natural protein, making it a morning and pre- or post-workout favorite among Health Coaches and smoothie-lovers alike.  Start with a teaspoon added to your preferred smoothie combo (it goes especially well with avocado, banana, or pineapple) and adjust the amount as you get used to it.

4. Add a dash of seaweed flakes to every meal.
Health food stores or Asian markets will have a variety of packaged salts and seasoning that include seaweed. Or you can make your own by combining fine-chopped or ground nori, kombu, dulse, sea salt, black pepper, and sesame seeds. Keep this right along with your most frequently used seasonings and sprinkle it over daily meals.

 5. Mix in kelp or kombu to stocks, soups, and stews.
Add seaweed to any savory liquid-based foods! If you make your own vegetable stock from veggie scraps toss in a strip of seaweed and strain it out along with the other ingredients. If adding directly to soups or stews remove the solid strip before serving but don’t worry about any small pieces left in, they’re edible and will enhance the overall meal.

6. Stir it into your salad dressing.
Sprinkle some powdered seaweed into any salad dressing you’re using, allow it to sit for a minute to mix and absorb, shake well, then toss into your salad.

7. Toss together a seaweed salad.
Wakame and arame are best for a salad made predominantly of seaweed.  Combine with vinegar, sesame oil, garlic, and scallions. You can also incorporate other veggies like cucumbers, carrots, or radishes. Experiment to find your favorite combination and share it as a unique side dish at your next potluck.

 

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Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception


Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception

Curator: Larry H. Bernstein, MD, FCAP

Pasteur Effect – Warburg Effect – What its history can teach us today. 

José Eduardo de Salles Roselino

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur’s time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took a very long time to create, a rather selective anaerobic condition. This selective culture media was characterized by the higher carbon dioxide levels produced by fast growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric (heat generation) results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits. In much resumed form, these observations indicate the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million years that included volcanic activity, tectonic movements of the earth crust, and glaciation, and more recently the use of carbon fuels and the extensive deforestation of our land masses have had a large role in determining the biological speciation over time, in sea and on land. O2 is generated by plants utilizing energy from the sun and conversion of CO2. Remove the plants and we tip the balance. A large source of CO2 is from beneath the earth’s surface.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to increase in V (volume), all this occurring in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In biochemistry, reversible usually indicates a reaction that easily goes either from A to B or B to A. For instance, when it was required to search for an anti-ischemic effect of Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate system of increased biliary system pressure in a reversible manner to exclude a direct effect of this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res 1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken into consideration since, after several mutations (not affecting previous activity and function), a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an example, consider a Prion C from lamb getting similar to bovine Prion C while preserving  its normal role in the lamb when its ability to change Human Prion C is considered (Stanley Prusiner).

This observation is good enough, to confirm one of the most important contributions of Erwin Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its followers described its requirement for aerobic catabolism two major lines of research started the search for the understanding of the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched for a mechanism that could explain how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. One of the major leaders of this research line was Britton Chance. He took into account that relatively earlier in the series of Krebs cycle reactions, two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not important whether the released carbons were or were not exactly those originated from glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary that could act as an energy reservoir. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. A key intermediate involved in the transfer was identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of Peter Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for the energetic transfer mechanism required for ATP synthesis.
This diverted the attention from high energy (~P) phosphate bond to the transfer of electrons. During most of the time the harsh period of the two confronting points of view, Paul Boyer and followers attempted to act as a conciliatory third party, without getting good results, according to personal accounts (in L. A. or Latin America) heard from those few of our scientists who were able to follow the major scientific events held in USA, and who could present to us later. Paul  Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility).

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute, over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow, this outcome represents such a blow to Chance’s previous work that somehow it seems to have cast a shadow over very important findings obtained during his earlier career that should not be affected by one or another form of energy transfer mechanism.  For instance, Britton Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that brings us back to Pasteur.

This metabolic alternative result seems to have been neglected in the recent years of obesity epidemics, which led to a search for a single molecular mechanism required for the understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does not mean that here the role of central nervous system is neglected. In short, in respiring mitochondria the rate of electron transport linked to the rate of ATP production is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low respiratory rate.   [When skeletal muscle is stressed by high exertion, lactic acid produced is released into the circulation and is metabolized aerobically by the heart at the end of the activity].

This respiratory control of metabolism will lead to preservation of body carbon reserves and in case of high caloric intake in a diet, also shows increase in fat reserves essential for our biological ancestors survival (Today for our obesity epidemics). No matter how important this observation is, it is only one focal point of metabolic control. We cannot reduce the problem of obesity to the existence of metabolic control. There are numerous other factors but on the other hand, we cannot neglect or remove this vital process in order to correct obesity. However, we cannot explain obesity ignoring this metabolic control. This topic is so neglected in modern times that we cannot follow major research lines of the past that were interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic vision of biology could replace all previous knowledge by a new one based upon ATGC readings. For instance, in order to display bad consequences derived from the ignorance of these old scientific facts, we can take into account, for instance, how ion movements across membranes affects membrane protein conformation and therefore contradicts the wrong central dogma of molecular biology. This change in protein conformation (with unchanged amino acid sequence) and/or the lack of change in protein conformation is linked to the factors that affect vital processes as the heart beats. This modern ignorance could also explain some major pitfalls seen in new drugs clinical trials and in a small scale on bad medical practices.

The work of Britton Chance and of Peter Mitchell have deep and sound scientific roots that were made with excellent scientific techniques, supported by excellent scientific reasoning and that were produced in a large series of very important intermediary scientific results. Their sole difference was to aim at very different scientific explanations as their goals (They have different Teleology in their minds made by their previous experiences). When, with the use of mutants obtained in microorganisms P Mitchell´s goal was found to survive and B Chance to succumb to the experimental evidence, all those excellent findings of B Chance and followers were directed to the dustbin of scientific history as an example of lack of scientific consideration.  [On the one hand, the Mitchell model used a unicellular organism; on the other, Chance’s work was with eukaryotic cells, quite relevant to the discussion.]

We can resume the challenge faced by these two great scientists in the following form: The first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up with the name of Fritz Lipmann. Its central feature was the recognition that adenosine triphosphate, ATP, serves as a universal energy  “currency” much as money serves as economic currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason that we cannot consider that gene expression is equivalent to protein function (an acceptable error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to presume that to find out how the flow of metabolism (carbon flow) led to ATP production must be considered a major focal point of research of the two contenders. Consequently, what could be a minor fall of one of the contenders, in case we take into account all that was found during their entire life of research, the real failure in B Chance’s final goal was amplified far beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a very sound root. Metabolism may produce two forms of energy currency (I personally don´t like this expression*) and I use it here because it was used by both groups in order to express their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas): The second kind of energy currency is the current of ions passing from one side of a membrane to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley, Huxley &  Huxley, Huxley & Simmons

*ATP is produced under the guidance of cell needs and not by its yield. When glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is required to match cellular needs. On the other hand, when it may yield (thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar to an investor choice its least money yield form for its investment (1940s to 1972) as a solid support. B. Chance had the enzymologists involved in clarifying how ATP could be produced directly from NADH + H+ oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate intermediary. Both competitors had their work supported by different but, sound scientific roots and have produced very important scientific results while trying to present their hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance followers was to create a conflict between what would be expected by a restrictive role of proteins through its specificity ionic interactions and the general ability of ionic asymmetries that could be associated with mitochondrial ATP production. Chemical catalyzed protein activities do not have perfect specificity but an outstanding degree of selective interaction was presented by the lock and key model of enzyme interaction. A large group of outstanding “mitochondriologists” were able to show ATP synthesis associated with Na+, K+, Ca2+… asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to display the existence of antiporters that exchange X for hydrogen as the final common source of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow discontinued in the form of scientific research, when the final E. Coli mutant studies presented the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you will always be better off in case you have a mutant”

(Principles of Medical Genetics T D Gelehrter & F.S. Collins chapter 7, 1990).

However, let’s take the example of a mechanical wristwatch. It clearly indicates when the watch is working in an acceptable way, that its normal functioning condition is not the result of one of its isolated components – or something that can be shown by a reductionist molecular view.  Usually it will be considered that it is working in an acceptable way, in case it is found that its accuracy falls inside a normal functional range, for instance, one or two standard deviations bellow or above the mean value for normal function, what depends upon the rigor wisely adopted. While, only when it has a faulty component (a genetic inborn error) we can indicate a single isolated piece as the cause of its failure (a reductionist molecular view).

We need to teach in medicine, first the major reasons why the watch works fine (not saying it is “automatic”). The functions may cross the reversible to irreversible regulatory limit change, faster than what we can imagine. Latter, when these ideas about normal are held very clear in the mind set of medical doctors (not medical technicians) we may address the inborn errors and what we may have learn from it. A modern medical technician may cause admiration when he uses an “innocent” virus to correct for a faulty gene (a rather impressive technological advance). However, in case the virus, later shows signals that indicate that it was not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be found. Even those oscillatory changes in Ca2+ that were shown to affect gene expression (C. De Duve) were laid to rest since, they clearly indicate a source of biological information that despite the fact that it does not change nucleotides order in the DNA, it shows an opposing flux of biological information against the dogma (DNA to RNA to proteins). Another, line has shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used for Ca2+ uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L. Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in Ca2+ load. As some of Krebs cycle dehydrogenases were activated by Ca2+, this finding was used to propose a new control factor in addition to the one of ADP (B. Chance). All this was discontinued with the wrong use of calculus (today we could indicate bioinformatics in a similar role) in biochemistry that has established less importance to a mitochondrial role after comparative kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic medical courses that must urgently reverse the faulty trend that tries to impose a view that goes from the detail towards generalization instead of the correct form that goes from the general finding well understood towards its molecular details. The view that led to curious subjects as bioinformatics in medical courses as training in sequence finding activities can only be explained by its commercial value. The usual form of scientific thinking respects the limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as a form to surpass lack of mathematical equation that defines relationship of variables and the determination of its functional domains. It also uses old scientific roots, as its sound support never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of DNA was found as a technical advance to find amino acid sequence in proteins it was just a technical advance. This technical advance by no means could be considered a scientific result presented as an indication that DNA sequences alone have replaced the need to study protein chemistry, its responses to microenvironmental changes in order to understand its multiple conformations, changes in activities and function. As E. Schrodinger correctly describes the chemical structure responsible for the coded form stored of genetic information must have minimal interaction with its microenvironment in order to endure hundreds and hundreds years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent that no longer could be considered to be an inert bath for catalytic activity of the enzymes when the transfer of energy include a role for hydrogen transport. The great increase in understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough to confirm once more, one of the most important contribution of E. Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work conformational energy as a property derived from our atomic structure can be neutral, positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it possible that this knowledge may advance to little just a short of a complete understanding -of the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have occurred or have not occurred and yet does not present a proof about how it would have being occurred. It will be always be an indication of possible against highly unlike and never a scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings that indicates wrong directions in the future as was his case, or directed toward our past.

The Skeleton of Physical Time – Quantum Energies in Relative Space of S-labs

By Radoslav S. Bozov  Independent Researcher

WSEAS, Biology and BioSystems of Biomedicine

Space does not equate to distance, displacement of an object by classically defined forces – electromagnetic, gravity or inertia. In perceiving quantum open systems, a quanta, a package of energy, displaces properties of wave interference and statistical outcomes of sums of paths of particles detected by a design of S-labs.

The notion of S-labs, space labs, deals with inherent problems of operational module, R(i+1), where an imagination number ‘struggles’ to work under roots of a negative sign, a reflection of an observable set of sums reaching out of the limits of the human being organ, an eye or other foundational signal processing system.

While heavenly bodies, planets, star systems, and other exotic forms of light reflecting and/or emitting objects, observable via naked eye have been deduced to operate under numerical systems that calculate a periodic displacement of one relative to another, atomic clocks of nanospace open our eyes to ever expanding energy spaces, where matrices of interactive variables point to the problem of infinity of variations in scalar spaces, however, defining properties of minute universes as a mirror image of an astronomical system. The first and furthermost problem is essentially the same as those mathematical methodologies deduced by Isaac Newton and Albert Einstein for processing a surface. I will introduce you to a surface interference method by describing undetermined objective space in terms of determined subjective time.

Therefore, the moment will be an outcome of statistical sums of a numerical system extending from near zero to near one. Three strings hold down a dual system entangled via interference of two waves, where a single wave is a product of three particles (today named accordingly to either weak or strong interactions) momentum.

The above described system emerges from duality into trinity the objective space value of physical realities. The triangle of physical observables – charge, gravity and electromagnetism, is an outcome of interference of particles, strings and waves, where particles are not particles, or are strings strings, or  are waves waves of an infinite character in an open system which we attempt to define to predict outcomes of tomorrow’s parameters, either dependent or independent as well as both subjective to time simulations.

We now know that aging of a biological organism cannot be defined within singularity. Thereafter, clocks are subjective to apparatuses measuring oscillation of defined parameters which enable us to calculate both amplitude and a period, which we know to be dependent on phase transitions.

The problem of phase was solved by the applicability of carbon relative systems. A piece of diamond does not get wet, yet it holds water’s light entangled property. Water is the dark force of light. To formulate such statement, we have been searching truth by examining cooling objects where the Maxwell demon is translated into information, a data complex system.

Modern perspectives in computing quantum based matrices, 0+1 =1 and/or 0+0=1, and/or 1+1 =0, will be reduced by applying a conceptual frame of Aladdin’s flying anti-gravity carpet, unwrapping both past and future by sending a photon to both, placing present always near zero. Thus, each parallel quantum computation of a natural system approaching the limit of a vibration of a string defining 0 does not equal 0, and 1 does not equal 1. In any case, if our method 1+1 = 1, yet, 1 is not 1 at time i+1. This will set the fundamentals of an operational module, called labris operator or in simplicity S-labs. Note, that 1 as a result is an event predictable to future, while interacting parameters of addition 1+1 may be both, 1 as an observable past, and 1 as an imaginary system, or 1+1 displaced interactive parameters of past observable events. This is the foundation of Future Quantum Relative Systems Interference (QRSI), taking analytical technologies of future as a result of data matrices compressing principle relative to carbon as a reference matter rational to water based properties.

Goedel’s concept of loops exist therefore only upon discrete relative space uniting to parallel absolute continuity of time ‘lags’. ( Goedel, Escher and Bach: An Eternal Golden Braid. A Metaphorical Fugue on Minds and Machines in the Spirit of Lewis Carroll. D Hofstadter.  Chapter XX: Strange Loops, Or Tangled Hierarchies. A grand windup of many of the ideas about hierarchical systems and self-reference. It is concerned with the snarls which arise when systems turn back on themselves-for example, science probing science, government investigating governmental wrongdoing, art violating the rules of art, and finally, humans thinking about their own brains and minds. Does Gödel’s Theorem have anything to say about this last “snarl”? Are free will and the sensation of consciousness connected to Gödel’s Theorem? The Chapter ends by tying Gödel, Escher, and Bach together once again.)  The fight struggle in-between time creates dark spaces within which strings manage to obey light properties – entangled bozons of information carrying future outcomes of a systems processing consciousness. Therefore, Albert Einstein was correct in his quantum time realities by rejecting a resolving cube of sugar within a cup of tea (Henri Bergson 19th century philosopher. Bergson’s concept of multiplicity attempts to unify in a consistent way two contradictory features: heterogeneity and continuity. Many philosophers today think that this concept of multiplicity, despite its difficulty, is revolutionary.) However, the unity of time and space could not be achieved by deducing time to charge, gravity and electromagnetic properties of energy and mass.

Charge is further deduced to interference of particles/strings/waves, contrary to the Hawking idea of irreducibility of chemical energy carrying ‘units’, and gravity is accounted for by intrinsic properties of   anti-gravity carbon systems processing light, an electromagnetic force, that I have deduced towards ever expanding discrete energy space-energies rational to compressing mass/time. The role of loops seems to operate to control formalities where boundaries of space fluctuate as a result of what we called above – dark time-spaces.

Indeed, the concept of horizon is a constant due to ever expanding observables. Thus, it fails to acquire a rational approach towards space-time issues.

Richard Feynman has touched on issues of touching of space, sums of paths of particle traveling through time. In a way he has resolved an important paradigm, storing information and possibly studying it by opening a black box. Schroedinger’s cat is alive again, but incapable of climbing a tree when chased by a dog. Every time a cat climbs a garden tree, a fruit falls on hedgehogs carried away parallel to living wormholes whose purpose of generating information lies upon carbon units resolving light.

In order to deal with such a paradigm, we will introduce i+1 under square root in relativity, therefore taking negative one ( -1 = sqrt (i+1), an operational module R dealing with Wheelers foam squeezed by light, releasing water – dark spaces. Thousand words down!

What is a number? Is that a name or some kind of language or both? Is the issue of number theory possibly accountable to the value of the concept of entropic timing? Light penetrating a pyramid holding bean seeds on a piece of paper and a piece of slice of bread, a triple set, where a church mouse has taken a drop of tear, but a blood drop. What an amazing physics! The magic of biology lies above egoism, above pride, and below Saints.

We will set up the twelve parameters seen through 3+1 in classic realities:

–              discrete absolute energies/forces – no contradiction for now between Newtonian and Albert Einstein mechanics

–              mass absolute continuity – conservational law of physics in accordance to weak and strong forces

–              quantum relative spaces – issuing a paradox of Albert Einstein’s space-time resolved by the uncertainty principle

–              parallel continuity of multiple time/universes – resolving uncertainty of united space and energy through evolving statistical concepts of scalar relative space expansion and vector quantum energies by compressing relative continuity of matter in it, ever compressing flat surfaces – finding the inverse link between deterministic mechanics of displacement and imaginary space, where spheres fit within surface of triangles as time unwraps past by pulling strings from future.

To us, common human beings, with an extra curiosity overloaded by real dreams, value happens to play in the intricate foundation of life – the garden of love, its carbon management in mind, collecting pieces of squeezed cooling time.

The infinite interference of each operational module to another composing ever emerging time constrains unified by the Solar system, objective to humanity, perhaps answers that a drop of blood and a drop of tear is united by a droplet of a substance separating negative entropy to time courses of a physical realities as defined by an open algorithm where chasing power subdue to space becomes an issue of time.

Jose Eduardo de Salles Roselino

Some small errors: For intance an increase i P leads to a decrease in V ( not an increase in V)..

 

Radoslav S. Bozov  Independent Researcher

If we were to use a preventative measures of medical science, instruments of medical science must predict future outcomes based on observable parameters of history….. There are several key issues arising: 1. Despite pinning a difference on genomic scale , say pieces of information, we do not know how to have changed that – that is shift methylome occupying genome surfaces , in a precise manner.. 2. Living systems operational quo DO NOT work as by vector gravity physics of ‘building blocks. That is projecting a delusional concept of a masonry trick, who has not worked by corner stones and ever shifting momenta … Assuming genomic assembling worked, that is dealing with inferences through data mining and annotation, we are not in a position to read future in real time, and we will never be, because of the rtPCR technology self restriction into data -time processing .. We know of existing post translational modalities… 3. We don’t know what we don’t know, and that foundational to future medicine – that is dealing with biological clocks, behavior, and various daily life inputs ranging from radiation to water systems, food quality, drugs…

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Sirtuins

Writer and Curator: Larry H. Bernstein, MD, FCAP 

7.8  Sirtuins

7.8.1 Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia

7.8.2 Substrates and Regulation Mechanisms for the Human Mitochondrial Sirtuins- Sirt3 and Sirt5

7.8.3 The mTORC1 Pathway Stimulates Glutamine Metabolism and Cell Proliferation by Repressing SIRT4

7.8.4  Rab1A and small GTPases Activate mTORC1

7.8.5 PI3K.Akt signaling in osteosarcoma

7.8.6 The mTORC1-S6K1 Pathway Regulates Glutamine Metabolism through the eIF4B-Dependent Control of c-Myc Translation

7.8.7 Localization of mouse mitochondrial SIRT proteins

7.8.8 SIRT4 Has Tumor-Suppressive Activity and Regulates the Cellular Metabolic Response to DNA Damage by Inhibiting Mitochondrial Glutamine Metabolism

7.8.9 Mitochondrial sirtuins and metabolic homeostasis

7.8.10 Mitochondrial sirtuins

7.8.11 Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling

 

7.8.1 Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia

Gertz M1Steegborn C.
Biochim Biophys Acta. 2010 Aug; 1804(8):1658-65
http://dx.doi.org:/10.1016/j.bbapap.2009.09.011

Sirtuins are a family of protein deacetylases that catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent removal of acetyl groups from modified lysine side chains in various proteins. Sirtuins act as metabolic sensors and influence metabolic adaptation but also many other processes such as stress response mechanisms, gene expression, and organismal aging. Mammals have seven Sirtuin isoforms, three of them – Sirt3, Sirt4, and Sirt5 – located to mitochondria, our centers of energy metabolism and apoptosis initiation. In this review, we shortly introduce the mammalian Sirtuin family, with a focus on the mitochondrial isoforms. We then discuss in detail the current knowledge on the mitochondrial isoform Sirt5. Its physiological role in metabolic regulation has recently been confirmed, whereas an additional function in apoptosis regulation remains speculative. We will discuss the biochemical properties of Sirt5 and how they might contribute to its physiological function. Furthermore, we discuss the potential use of Sirt5 as a drug target, structural features of Sirt5 and of an Sirt5/inhibitor complex as well as their differences to other Sirtuins and the current status of modulating Sirt5 activity with pharmacological compounds.

removal of acetyl groups from modified lysine side chain

removal of acetyl groups from modified lysine side chain

http://ars.els-cdn.com/content/image/1-s2.0-S1570963909002593-gr1.sml
removal of acetyl groups from modified lysine side chain

sirtuin structure

sirtuin structure

http://ars.els-cdn.com/content/image/1-s2.0-S1570963909002593-gr2.sml
sirtuin structure

7.8.2 Substrates and Regulation Mechanisms for the Human Mitochondrial Sirtuins- Sirt3 and Sirt5

Schlicker C1Gertz MPapatheodorou PKachholz BBecker CFSteegborn C
J Mol Biol. 2008 Oct 10; 382(3):790-801
http://dx.doi.org/10.1016/j.jmb.2008.07.048

The enzymes of the Sirtuin family of nicotinamide-adenine-dinucleotide-dependent protein deacetylases are emerging key players in nuclear and cytosolic signaling, but also in mitochondrial regulation and aging. Mammalian mitochondria contain three Sirtuins, Sirt3, Sirt4, and Sirt5. Only one substrate is known for Sirt3 as well as for Sirt4, and up to now, no target for Sirt5 has been reported. Here, we describe the identification of novel substrates for the human mitochondrial Sirtuin isoforms Sirt3 and Sirt5. We show that Sirt3 can deacetylate and thereby activate a central metabolic regulator in the mitochondrial matrix, glutamate dehydrogenase. Furthermore, Sirt3 deacetylates and activates isocitrate dehydrogenase 2, an enzyme that promotes regeneration of antioxidants and catalyzes a key regulation point of the citric acid cycle. Sirt3 thus can regulate flux and anapleurosis of this central metabolic cycle. We further find that the N- and C-terminal regions of Sirt3 regulate its activity against glutamate dehydrogenase and a peptide substrate, indicating roles for these regions in substrate recognition and Sirtuin regulation. Sirt5, in contrast to Sirt3, deacetylates none of the mitochondrial matrix proteins tested. Instead, it can deacetylate cytochrome c, a protein of the mitochondrial intermembrane space with a central function in oxidative metabolism, as well as apoptosis initiation. Using a mitochondrial import assay, we find that Sirt5 can indeed be translocated into the mitochondrial intermembrane space, but also into the matrix, indicating that localization might contribute to Sirt5 regulation and substrate selection.

Mitochondria are central organelles in cellular energy metabolism, but also in processes such as apoptosis, cellular senescence, and lifespan regulation.1 and 2 Failures in mitochondrial function and regulation contribute to aging-related diseases, such as atherosclerosis3 and Parkinson’s disease,4 likely by increasing cellular levels of reactive oxygen species and the damage they cause.1 Emerging players in metabolic regulation and cellular signaling are members of the Sirtuin family of homologs of “silent information regulator 2” (Sir2), a yeast protein deacetylase.5 and 6 Sir2 was found to be involved in aging processes and lifespan determination in yeast,7 and 8 and its homologs were subsequently identified as lifespan regulators in various higher organisms.89 and 10 Sirtuins form class III of the protein deacetylase superfamily and hydrolyze one nicotinamide adenine dinucleotide (NAD +) as cosubstrate for each lysine residue they deacetylate.11 and 12 The coupling of deacetylation to NAD + was proposed to link changes in cellular energy levels to deacetylation activity,13 and 14 which would indicate Sirtuins as metabolic sensors. Other known regulation mechanisms for Sirtuin activity are the modulation of the expression levels of their genes6 and the autoinhibitory effect of an N-terminal region on the yeast Sirtuin “homologous to SIR2 protein 2” (Hst2).15

The seven mammalian Sirtuin proteins (Sirt1–Sirt7) have various substrate proteins that mediate functions in genetic, cellular, and mitochondrial regulation.5 and 6 The best-studied mammalian Sir2 homolog, Sirt1, was shown to regulate, among others, transcription factor p53, nuclear factor-kappa B, and peroxisome proliferator-activated receptor gamma coactivator-1-alpha.6 Three human Sirtuin proteins are known to be located in the mitochondria, Sirt3, Sirt4, and Sirt5,161718 and 19 although Sirt3 was reported to change its localization to nuclear when coexpressed with Sirt5.20 The recent identification of the first substrates for mitochondrial Sirtuins—acetyl coenzyme A synthetase 221 and 22 and glutamate dehydrogenase (GDH)16—as targets of Sirtuins 3 and 4, respectively, revealed that these Sirtuins control a regulatory network that has implications for energy metabolism and the mechanisms of caloric restriction (CR) and lifespan determination.23 Sirt3 regulates adaptive thermogenesis and decreases mitochondrial membrane potential and reactive oxygen species production, while increasing cellular respiration.24 Furthermore, Sirt3 is down-regulated in several genetically obese mice,24 and variability in the human SIRT3 gene has been linked to survivorship in the elderly. 25 In contrast to the deacetylases Sirt3 and Sirt5, Sirt4 appears to be an ADP ribosyltransferase. 16 Through this activity, Sirt4 inhibits GDH and thereby down-regulates insulin secretion in response to amino acids. 16 For Sirt5, however, there is no report yet on its physiological function or any physiological substrate. It is dominantly expressed in lymphoblasts and heart muscle cells,17 and 26 and its gene contains multiple repetitive elements that might make it a hotspot for chromosomal breaks. 26 Interestingly, the Sirt5 gene has been located to a chromosomal region known for abnormalities associated with malignant diseases. 26

A proteomics study found 277 acetylation sites in 133 mitochondrial proteins;27 many of them should be substrates for the mitochondrial Sirtuins mediating their various functions, but up to now, only one physiological substrate could be identified for Sirt3,21 and 22 and none could be identified for Sirt5. Our understanding of substrate selection by Sirtuins is incomplete, and knowledge of specific Sirtuin targets would be essential for a better understanding of Sirtuin-mediated processes and Sirtuin-targeted therapy. A first study on several Sirtuins showed varying preferences among acetylated peptides.28 Structural and thermodynamic analysis of peptides bound to the Sirtuin Sir2Tm from Thermatoga maritima indicated that positions − 1 and + 2 relative to the acetylation site play a significant role in substrate binding. 29 However, these studies were conducted with nonphysiological Sirtuin/substrate pairs, and other studies indicated little sequence specificity; instead, the yeast Sirtuin Hst2 was described to display contextual and conformational specificity: Hst2 deacetylated acetyl lysine only in the context of a protein, and it preferentially deacetylated within flexible protein regions. 30 Finally, statistical analysis of a proteomics study on acetylated proteins identified preferences at various positions such as + 1, − 2, and − 3, and deacetylation sites appeared to occur preferentially in helical regions. 27 Thus, our present knowledge of Sirtuin substrates and of factors determining Sirtuin specificity is incomplete and insufficient for sequence-based identification of physiological substrates.

Here, we describe the identification of novel targets for the mitochondrial deacetylases Sirt3 and Sirt5. We show that Sirt3 can deacetylate and thereby activate the enzymes GDH and isocitrate dehydrogenase (ICDH) 2—two key metabolic regulators in the mitochondrial matrix. We find that the N- and C-terminal regions of Sirt3 influence its activity against GDH and a peptide substrate, indicating roles in regulation and substrate recognition for these regions. Furthermore, we find that Sirt5 can deacetylate cytochrome c, a protein of the mitochondrial intermembrane space (IMS) with a central function in oxidative metabolism and apoptosis.

The upstream sequence contributes to the target specificity of Sirt3 and Sirt5

Sirtuins have been reported to have little sequence specificity,30 but other studies indicated a sequence preference dominated by positions − 1 and + 2.29 We tested the importance of the amino acid pattern preceding the acetylation site for recognition by the mitochondrial Sirtuins Sirt3 and Sirt5 through a fluorescence assay. First, the fluorogenic and commercially available modified p53-derived tetrapeptide QPK-acetylK, originally developed for Sirt2 assays but also efficiently used by Sirt3, was tested. Even 60 μg of Sirt5 did not lead to any deacetylation signal, whereas 0.35 μg of Sirt3 efficiently deacetylated the peptide (Fig. 1a). We then tested Sirt3 and Sirt5 on a second modified p53-derived tetrapeptide, RHK-acetylK. Sirt3 (0.5 μg) showed a slightly increased activity against this substrate as compared to QPK-acetylK (Fig. 1b); more importantly, 0.5 μg of Sirt5 showed significant activity against this peptide. These results show that the mitochondrial Sirtuins Sirt3 and, especially, Sirt5 indeed recognize the local target sequence, and target positions further upstream of − 1 seem to be involved in substrate recognition. For identification of novel substrates for the mitochondrial Sirtuins and further characterization of their target recognition mechanisms, we then turned to testing full-length proteins, as the downstream sequence and the larger protein context of the deacetylation site might also contribute to substrate selection.

Sirtuin substrate specificity

Sirtuin substrate specificity

Fig. 1. Testing the substrate specificity of Sirt3 and Sirt5 with peptides. (a) Sirt3, but not Sirt5, deacetylates the fluorogenic peptide QPK-acetylK. (b) Sirt3 efficiently deacetylates the fluorogenic peptide RHK-acetylK, and Sirt5 also significantly deacetylates this substrate.
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Sirt3 deacetylates and activates GDH

In order to identify novel physiological substrates of the mitochondrial Sirtuins, we used proteins isolated in their partly acetylated form from natural sources (i.e., from mammalian mitochondria). These proteins, carrying physiological acetylations, were tested as Sirt3 and Sirt5 substrates in vitro in an ELISA system using an antibody specific for acetylated lysine. In a recent proteomics study, 27 GDH, a central regulator of mitochondrial metabolism, was identified to be acetylated in a feeding-dependent manner. With our ELISA, we found that Sirt3 and Sirt5 can both deacetylate pure GDH isolated from mitochondria, but with very different efficiencies ( Fig. 2a). Sirt3 significantly deacetylated GDH, but even large amounts of Sirt5 decreased the acetylation level of this substrate only slightly. We next tested the effect of GDH deacetylation on its activity. Deacetylation of GDH through incubation with Sirt3 and NAD + before its examination in a GDH activity assay increased its activity by 10%, and a stronger stimulation of GDH activity was seen when larger amounts of Sirt3 were used for deacetylation ( Fig. 2b). GDH is colocalized with Sirt3 in the mitochondrial matrix 1618 and 19 and, thus, likely could be a physiological substrate of this Sirtuin. Indeed, GDH from a Sirt3 knockout mouse was recently shown to be hyperacetylated compared to protein from wild-type mice. 31 Thus, Sirt3 deacetylates GDH in vivo, and our results show that this direct deacetylation of GDH by Sirt3 leads to GDH activation.

sirtuin structure

sirtuin structure

Fig. 2. Sirt3 can deacetylate and thereby activate GDH. (a) Deacetylation of GDH tested in ELISA. Sirt3 efficiently deacetylates GDH, whereas Sirt5 has only a small effect on the acetylation state. (b) GDH activity is increased after deacetylation of the enzyme by Sirt3. The increase in GDH activity depends on the amount of Sirt3 activity used for deacetylation.
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Sirt3 can deacetylate and thereby activate ICDH2

In the proteomics study by Kim et al., the mitochondrial citric acid cycle enzymes fumarase and ICDH2 (a key regulator of this metabolic cycle) were found to be acetylated in a feeding-dependent manner. 27 In our ELISA system, we found that Sirt3 efficiently deacetylated the ICDH2 substrate isolated from mitochondria ( Fig. 3a). Western blot analysis (data not shown) and mass spectrometry confirmed that, indeed, the ICDH2 fraction of the partially purified protein was deacetylated by Sirt3. In contrast, even large amounts of Sirt5 did not significantly decrease the acetylation level of this substrate ( Fig. 3a). As expected, deacetylation of ICDH2 by Sirt3 was dependent on NAD +. Fumarase, in contrast, could not be deacetylated as efficiently as ICDH2 through treatment with either Sirt3 or Sirt5 ( Fig. 3b). The low absolute values over background for the ELISA with fumarase, however, might indicate low acetylation levels of the natively purified protein, and a stronger effect might be attainable when testing fumarase with a higher acetylation level.

Fig. 3. Sirt3 deacetylates ICDH2, but not fumarase. (a) Deacetylation of ICDH2 by Sirt3 and Sirt5 tested in ELISA. Sirt3, but not Sirt5, deacetylates ICDH2 in a NAD +-dependent manner. (b) Fumarase acetylation determined through ELISA cannot be significantly decreased by incubation with recombinant Sirt3 or Sirt5. (c) ICDH2 activity measured in a spectrophotometric assay based on the formation of NADPH. ICDH2 activity (continuous line) is increased after deacetylation of the enzyme by Sirt3 (dashed line). (d) The stimulatory effect of deacetylation on ICDH2 activity depends on the amount of deacetylase activity added during pretreatment. (e) ICDH2 with and without Sirt3 treatment analyzed by mass spectrometry after proteolytic digest. The decrease in the signal at 962.3 Da and the increase in signal at 903.5 Da indicate deacetylation at either K211 or K212.

In order to analyze the potential physiological function of ICDH2 deacetylation, we tested the effect of Sirt3-mediated ICDH2 deacetylation on its activity. Incubation of ICDH2 with Sirt3 and NAD + prior to its analysis in an ICDH activity assay increased its activity (Fig. 3c). The stimulation of ICDH2 activity was further increased when larger amounts of Sirt3 were used for deacetylation (Fig. 3d), and no significant increase in ICDH2 activity was observed when the Sirtuin inhibitor dihydrocoumarin was present during incubation with Sirt3 (data not shown). Sirt3 and ICDH2 are colocalized in the mitochondrial matrix,1619 and 32 and we therefore assume that ICDH2 is likely a physiological substrate for Sirt3, which activates ICDH2 by deacetylation.
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Sirt3 can deacetylate KK motifs in substrate proteins

In order to identify the site of ICDH2 deacetylation upon treatment with Sirt3, we analyzed ICDH2 by mass spectrometry. For analyzing pure ICDH2, we excised its band from an SDS gel before mass spectrometry analysis. In the proteomics study by Kim et al., two acetylation sites were reported for ICDH2: K75 and K241 (numbering of the partial sequence of the unprocessed precursor; SwissProt entry P33198). 27 After digest of ICDH2, we could not detect peptides comprising K75 and, therefore, could not determine its acetylation status, and we only observed the deacetylated form of K241. We identified an additional acetylation site, however, by detecting signals at m/z = 903.5 and m/z = 962.3 for the peptide QYAIQKK (residues 206–212) carrying one and two acetyl groups, respectively ( Fig. 3e; calculated m/z = 903.5 and 962.5). Sirt3 treatment decreased the signal for the double-acetylated form and increased the signal for the single-acetylated form as compared to internal peptides [e.g., m/z = 890.5 (calculated m/z = 890.5) andm/z = 1041.4 (calculated m/z = 1041.5)]. These data indicate that Sirt3 deacetylates either position K211 or K212 of this KK motif located at a surface-exposed end of a helix that flanks the active site of ICDH2. 33Deacetylation of a KK motif by Sirt3 is consistent with the efficient use of the tested peptide substrates (see above) that both carry KK motifs.

Fig. 4. Increased activity of N- and C-terminally truncated Sirt3. (a) Specific activity against a peptide substrate of the longest Sirt3 form after proteolytic processing that covers residues 102–399. N-terminal truncation increases the specific activity dramatically, and an additional C-terminal truncation activates the catalytic core further. (b) Homology model of Sirt3 based on the crystal structure of Sirt2. The part comprising the catalytic core is shown in red. The NAD + and peptide ligands were manually placed into their binding sides based on the crystal structure of their complex with a bacterial Sir2 homolog from T. maritima. Parts removed in N- and C-terminal truncation constructs are shown in cyan and blue, respectively. (c) Level of acetylation of GDH tested in ELISA. The shortest Sirt3 form Sirt3(114–380) deacetylates more efficiently than Sirt3(114–399) and Sirt3(102–399), which show activities comparable to each other.

Sirt5 can deacetylate cytochrome c

Sirt5 can deacetylate cytochrome c

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Sirt5 can deacetylate cytochrome c

The Sirt5 protein that we used for our study comprises residues 34–302, corresponding to the fully active catalytic core determined for Sirt3 (see above). This protein is indeed active against a peptide substrate, but it showed no significant activity against the acetylated mitochondrial matrix proteins tested so far: GDH, ICDH2, and fumarase. We thus picked cytochrome c, a central protein in energy metabolism and apoptosis localized in the mitochondrial IMS, from the list of acetylated mitochondrial proteins 27 for testing as deacetylation substrate. Sirt5 showed deacetylation activity against pure cytochrome c in our ELISA system, whereas Sirt3 had almost no activity against this substrate ( Fig. 5a). Even the more active shortened form of Sirt3(114–380) showed no considerable activity against this substrate.

Fig. 5.  Sirt5 can deacetylate cytochrome c. (a) Deacetylation of cytochrome c tested in ELISA. Sirt5 uses cytochrome c as substrate for deacetylation, whereas Sirt3 treatment leaves the acetylation level of cytochrome c unchanged. (b) Model of the action of the mammalian Sirtuins Sirt3, Sirt4, and Sirt5 in mitochondria. CAC: citric acid cycle. (c) Digest of Sirt5 synthesized in vitro with PK. The protein is fully degraded at proteinase concentrations of 25 μg/ml and above. (d) Import of Sirt5 into isolated yeast mitochondria. Sirt5 reaches an inner mitochondrial compartment in the presence and in the absence of the mitochondrial membrane potential (ΔΨ), whereas Sirt3, as a control for a matrix-targeted protein, is not imported into uncoupled mitochondria. (e) Intramitochondrial localization of Sirt5. Part of the imported Sirt5 is sensitive to PK after swelling (SW) and thus localized in the IMS, but another part of the protein remains protease-resistant and therefore appears to be localized to the matrix. Atp3, a protein localized at the matrix site of the mitochondrial inner membrane, and an IMS-located domain of translocase of inner membrane 23 detected by Western blot analysis served as controls for matrix transport and swelling, respectively. aTim23: anti-Tim23. (f) Scheme of the domain organizations of Sirt3 and Sirt5. Numbers in brackets are residue numbers for boundaries of protein parts. NLS: nuclear localization sequence; MLS: mitochondrial localization sequence; R1, regulatory region 1; R2: regulatory region 2.
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Cytochrome c might be a physiological substrate of Sirt5 if this Sirtuin is localized to the mitochondrial IMS (Fig. 5b). A recent study on overexpressed tagged mouse Sirt5 in COS7 cells 20 indeed indicated that Sirt5, at least from mouse, is localized in the IMS. In order to test whether human Sirt5 can be localized to the IMS, we performed import experiments with human Sirt3 and Sirt5 using isolated yeast mitochondria as a model system. 3 Sirt3 and Sirt5 proteins were incubated with mitochondria, followed by PK treatment for degradation of nonimported protein ( Fig. 5d). In a parallel reaction, mitochondria were uncoupled prior to the import reaction by addition of valinomycin (− ΔΨ). Sirt3, a protein known to be located in the mitochondrial matrix, 19 was only efficiently imported in the presence of a membrane potential. Dependence on the mitochondrial potential is a hallmark of matrix import, 38 and the results thus show that Sirt3 is imported into the correct compartment in our experimental system. Sirt5, in contrast, reaches an inner-mitochondrial compartment both in the presence and in the absence of the membrane potential, suggesting that Sirt5 may accumulate in the IMS.

In order to further test the localization of Sirt5, we removed the outer mitochondrial membrane after the import reaction by osmotic swelling, followed by PK digest of then accessible proteins (Fig. 5e). Rupture of the outer membrane was confirmed by monitoring the accessibility of an IMS-exposed domain of endogenous translocase of inner membrane 23 (detected by Western blot analysis). Part of the imported Sirt5 was degraded by PK, indicating its localization in the IMS.

Sirtuins are involved in central physiological regulation mechanisms, many of them with relevance to metabolic regulation and aging processes.5 and 6 Therefore, the seven mammalian Sirtuin isoforms are emerging targets for the treatment of metabolic disorders and aging-related diseases.39 For most Sirtuin effects, however, the specific signaling mechanisms and molecular targets are not yet known. We have identified novel potential targets for Sirtuins in mitochondria, the major metabolic centers in cells. We found that Sirt3 can deacetylate and thereby activate ICDH2, a key regulation point for flux throughout the citric acid cycle. Interestingly, the ICDH isoform regulated by Sirt3 forms NADPH instead of the NADH used for ATP synthesis. This activity is assumed to be important for the NADPH-dependent regeneration of antioxidants,40 and its stimulation by Sirt3 should thus help to slow oxidative damage and cellular aging processes. Furthermore, Sirt3 deacetylates GDH in vitro (this study) and in vivo31 and we find that this modification also stimulates GDH activity that promotes glucose and ATP synthesis by enabling amino acids to be used as fuels for citric acid cycle and gluconeogenesis. 41 Consistently, Sirt3 was reported to increase respiration, 24 which is needed for ATP synthesis but also for conversion of amino acids into glucose and urea. 41 The enzyme previously identified to be activated by Sirt3, acetyl coenzyme A synthetase 2, 21 and 22 also fuels the citric acid cycle independently of glycolysis by activating free acetate (Fig. 5b). Interestingly, a shift away from liver glycolysis is one of the metabolic changes observed under CR, a feeding regimen with 20–40% fewer calories than consumed ad libitum that is found to extend the lifespan of a variety of organisms. 6 CR was previously reported to increase GDH activity in the liver, 42where Sirt3 is highly expressed, 17 and Sirt3 activity is known to be increased by CR. 6 and 24 It thus appears that Sirt3 mediates some of the effects of CR and lifespan regulation, consistent with its implication in survivorship in the elderly 25 and 43 and the prominent role of Sirtuins in CR found for various organisms,6 and 44 and it also appears that GDH activation likely contributes to the Sirt3-dependent effects.

Little is known about additional factors regulating the activity and specificity of Sirtuin enzymes. Their requirement for NAD + indicates that the NAD +/NADH ratio should regulate Sirtuins,13 and 14 but even changes to ratios observed under extreme conditions such as CR appear to influence Sirtuin activity only slightly.35 Furthermore, NAD + levels would influence all Sirtuins similarly, but a more specific tuning of individual Sirtuin activities appears necessary in order to orchestrate the many effects mediated by Sirtuins (see, e.g., discussion above).6 and 45 A deeper insight into the regulation of Sirtuin enzymes would also be required for the development of more specific Sirtuin inhibitors—a prerequisite for Sirtuin-targeted therapy.39 The regulatory parts flanking the catalytic cores might be interesting target sites (Fig. 5f). N-terminal extensions between ∼ 30 and 120 residues are present in all human Sirtuins but show little conservation, indicating that they might respond to various regulators. Our results indicate that the corresponding N-terminal region in Sirt3 also blocks productive binding for small peptides (Fig. 4a), but enables access for entire protein substrates (Fig. 4c). The C-terminal truncated part in our experiments (Sirt3 residues 380–399) is formed by α14 (secondary structure numbering for Sirt236) whose end corresponds to the N-terminus of Hst2 α13 that partly occupies the NAD +binding site.15 In Sirt3, however, the C-terminal truncation alone lowers activity only slightly, and we assume that it has no regulatory function on its own but might instead assist the N-terminal autoinhibitory region. This module of the N-terminus and the C-terminus (Figs. 4b and 5f) appears to contribute to the substrate specificity of the enzyme, and ligands binding to it might enable or block rearrangements opening up the active site and thereby regulate the enzyme’s activity. Alternatively, the flanking parts might be removed by proteolytic processing or alternative splicing, thereby changing Sirtuin activity and specificity.

7.8.3 The mTORC1 Pathway Stimulates Glutamine Metabolism and Cell Proliferation by Repressing SIRT4

Csibi A1Fendt SMLi CPoulogiannis GChoo AYChapski DJ, et al.
Cell. 2013 May 9; 153(4):840-54.
http://dx.doi.org:/10.1016/j.cell.2013.04.023

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mTORC1 activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP response element binding-2 (CREB2). Thus, a relationship between mTORC1, SIRT4 and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mTORC1 activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP response element binding-2 (CREB2). Thus, a relationship between mTORC1, SIRT4 and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Nutrient availability plays a pivotal role in the decision of a cell to commit to cell proliferation. In conditions of sufficient nutrient sources and growth factors (GFs), the cell generates enough energy and acquires or synthesizes essential building blocks at a sufficient rate to meet the demands of proliferation. Conversely, when nutrients are scarce, the cell responds by halting the biosynthetic machinery and by stimulating catabolic processes such as fatty acid oxidation and autophagy to provide energy maintenance (Vander Heiden et al., 2009). Essential to the decision process between anabolism and catabolism is the highly conserved, atypical Serine/Threonine kinase mammalian Target of Rapamycin Complex 1 (mTORC1), whose activity is deregulated in many cancers (Menon and Manning, 2008). This complex, which consists of mTOR, Raptor, and mLST8, is activated by amino acids (aa), GFs (insulin/IGF-1) and cellular energy to drive nutrient uptake and subsequently proliferation (Yecies and Manning, 2011). The molecular details of these nutrient-sensing processes are not yet fully elucidated, but it has been shown that aa activate the Rag GTPases to regulate mTORC1 localization to the lysosomes (Kim et al., 2008Sancak et al., 2008); and GFs signal through the PI3K-Akt or the extracellular signal-regulated kinase (ERK)-ribosomal protein S6 kinase (RSK) pathways to activate mTORC1 by releasing the Ras homolog enriched in brain (RHEB) GTPase from repression by the tumor suppressors, tuberous sclerosis 1 (TSC1)– TSC2 (Inoki et al., 2002Manning et al., 2002Roux et al., 2004). Finally, low energy conditions inhibit mTORC1 by activating AMPK and by repressing the assembly of the TTT-RUVBL1/2 complex. (Inoki et al., 2003Gwinn et al., 2008Kim et al., 2013).

Glutamine, the most abundant amino acid in the body plays an important role in cellular proliferation. It is catabolized to α-ketoglutarate (αKG), an intermediate of the tricarboxylic acid (TCA) cycle through two deamination reactions in a process termed glutamine anaplerosis (DeBerardinis et al., 2007). The first reaction requires glutaminase (GLS) to generate glutamate, and the second occurs by the action of either glutamate dehydrogenase (GDH) or transaminases. Incorporation of αKG into the TCA cycle is the major anaplerotic step critical for the production of biomass building blocks including nucleotides, lipids and aa (Wise and Thompson, 2010). Recent studies have demonstrated that glutamine is also an important signaling molecule. Accordingly, it positively regulates the mTORC1 pathway by facilitating the uptake of leucine (Nicklin et al., 2009) and by promoting mTORC1 assembly and lysosomal localization (Duran et al., 2012;Kim et al., 2013).

Commonly occurring oncogenic signals directly stimulate nutrient metabolism, resulting in nutrient addiction. Oncogenic levels of Myc have been linked to increased glutamine uptake and metabolism through a coordinated transcriptional program (Wise et al., 2008Gao et al., 2009). Hence, it is not surprising that cancer cells are addicted to glutamine (Wise and Thompson, 2010). Thus, considering the prevalence of mTORC1 activation in cancer and the requirement of nutrients for cell proliferation, understanding how mTORC1 activation regulates nutrient levels and metabolism is critical. Activation of the mTORC1 pathway promotes the utilization of glucose, another nutrient absolutely required for cell growth. However, no study has yet investigated if and how the mTORC1 pathway regulates glutamine uptake and metabolism. Here, we discover a novel role of the mTORC1 pathway in the stimulation of glutamine anaplerosis by promoting the activity of GDH. Mechanistically, mTORC1 represses the transcription of SIRT4, an inhibitor of GDH. SIRT4 is a mitochondrial-localized member of the sirtuin family of NAD-dependent enzymes known to play key roles in metabolism, stress response and longevity (Haigis and Guarente, 2006). We demonstrate that the mTORC1 pathway negatively controls SIRT4 by promoting the proteasome-mediated degradation of cAMP-responsive element-binding (CREB) 2. We reveal that SIRT4 levels are decreased in a variety of cancers, and when expressed, SIRT4 delays tumor development in a Tsc2−/− mouse embryonic fibroblasts (MEFs) xenograft model. Thus, our findings provide new insights into how mTORC1 regulates glutamine anaplerosis, contributing therefore to the metabolic reprogramming of cancer cells, an essential hallmark to support their excessive needs for proliferation.

The mTORC1 pathway regulates glutamine metabolism via GDH

The activation of the mTORC1 pathway has recently been linked to glutamine addiction of cancer cells (Choo et al., 2010), yet it remains to be resolved if mTORC1 serves as a regulator of glutamine anaplerosis. To investigate this possibility, we first determined the effect of mTORC1 activity on glutamine uptake. We measured glutamine uptake rates in Tsc2 wild-type (WT) and Tsc2−/− MEFs. We found that Tsc2−/− MEFs consumed significantly more glutamine (Figure 1A), showing that mTORC1 activation stimulates the uptake of this nutrient. In addition, re-expression of Tsc2 in Tsc2−/− cells reduced glutamine uptake (Figure S1A). Similarly, mTORC1 inhibition with rapamycin resulted in decreased glutamine uptake in MEFs (Figure 1A). The decreased on glutamine uptake was significantly reduced after 6h of rapamycin treatment when compared to control (data not shown). To further confirm the role of mTORC1 on glutamine uptake, we used human embryonic kidney (HEK) 293T cells stably expressing either WT-RHEB or a constitutively active mutant (S16H) of RHEB. Increased mTORC1 signaling, as evidenced by sustained phosphorylation of S6K1 and its target rpS6, was observed in RHEB-expressing cells (Figure S1B). The activation of the mTORC1 pathway nicely correlated with an increase in glutamine consumption, therefore confirming that changes in mTORC1 signaling are reflected in cellular glutamine uptake (Figure S1B). To determine whether the modulation of glutamine uptake by the mTORC1 pathway occurs in cancer cells, we examined glutamine uptake rates in conditions of mTORC1 inhibition in human epithelial tumor cell lines, including the colon carcinoma DLD1, and the prostate cancer DU145. Rapamycin treatment resulted in decreased proliferation (data not shown) and yielded a decreased glutamine uptake in both cell lines (Figure 1B & data not shown). Glutamine is the major nitrogen donor for the majority of ammonia production in cells (Figure 1C) (Shanware et al., 2011). Consistent with decreased glutamine uptake, we found that ammonia levels were also diminished after rapamycin treatment (Figure S1C).

Figure 1  The mTORC1 pathway regulates glutamine metabolism via glutamate dehydrogenase

We next examined the fate of glutamine in conditions of mTORC1 inhibition, using gas chromatography/mass spectrometry (GC/MS) analysis to monitor the incorporation of uniformly labeled [U-13C5]-Glutamine into TCA cycle intermediates. Direct glutamine contribution to I̧KG (m+5), succinate (m+4), malate (m+4) and citrate (m+4) was decreased in rapamycin treated cells (Figure S1D) indicating that rapamycin impaired glutamine oxidation and subsequent carbon contribution into the TCA cycle.

To test whether glutamine uptake or glutamine conversion is limiting, we measured the intracellular levels of glutamine and glutamate in DLD1 cells. Increased levels of glutamine and/or glutamate will show that the catalyzing enzyme activity is limiting and not glutamine transport itself (Fendt et al., 2010). Rapamycin treatment resulted in increased intracellular levels of both glutamine and glutamate, showing that glutamate to αKG conversion is the critical limiting reaction (Figures 1D & 1E). To further confirm the implication of the glutamate catalyzing reaction we also measured αKG levels. If glutamate conversion is indeed critical we expect no alteration in αKG levels. This is expected because αKG is downstream of the potentially limiting glutamate conversion step, and it has been shown that product metabolite concentrations of limiting metabolic enzymes stay unaltered, while the substrate metabolite concentrations change to keep metabolic homeostasis (Fendt et al., 2010). We found that αKG levels were unaltered after rapamycin treatment, corroborating that the limiting enzymatic step is glutamate conversion (Figure 1F). To further confirm the limitation in glutamate-to-αKG conversion, we measured flux through this reaction. Strikingly, this flux was significantly reduced during rapamycin treatment (Figure 1G). Additionally, the inhibition of mTORC1 resulted in increased glutamate secretion (Figure 1H), thus confirming that the glutamate-to-αKG conversion step is a major bottleneck in the glutamine pathway during rapamycin treatment.

Glutamate conversion can be conducted by GDH (Figure 1C), suggesting that the mTORC1 pathway potentially regulates this enzyme. In agreement, rapamycin treatment resulted in decreased GDH activity in DLD1 cells (Figure 1I). To exclude that transaminases play a role in the mTORC1-induced regulation of glutamine metabolism, we used amin ooxyacetate (AOA) at a concentration shown to effectively inhibit the two predominant transaminases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Figure 1C) (Wise et al., 2008), or rapamycin in the presence of α-15N-labeled glutamine. Subsequently, we measured 15N-labeling patterns and metabolite levels of alanine, an amino acid that is predominately produced by a transaminase-catalyzed reaction (Possemato et al., 2011). We found that AOA dramatically decreased 15N contribution and metabolite levels of alanine, while rapamycin only mildly affected the 15N contribution to this amino acid and showed no effect on alanine levels compared to the control condition (Figures 1J & S1E). In conclusion, these data demonstrate that GDH, not transaminases, plays a major role in the regulation of glutamine metabolism downstream of mTORC1.

mTORC1 controls GDH activity by repressing SIRT4

As our results show that mTORC1 regulates glutamate dehydrogenase, we sought to identify the molecular mechanism. SIRT4 is a negative regulator of GDH activity through ADP-ribosylation (Haigis et al., 2006), thus suggesting that mTORC1 potentially controls this step of glutamine metabolism via SIRT4. To test this possibility, we first assessed the ADP-ribosylation status of GDH by introducing biotin-labeled NAD followed by immunoprecipitation using avidin-coated beads. Rapamycin treatment led to an increase in the mono-ADP-ribosylation status of GDH, similar to that observed in cells stably expressing SIRT4 (Figure 2A). Importantly, we found that the knockdown of SIRT4 abrogated the rapamycin-induced decrease in the activity of GDH (Figures 2B & S2A). Strikingly, SIRT4 protein levels were increased upon mTORC1 inhibition in MEFs (Figures 2C). This regulation was confirmed in both DLD1 and DU145 cells (Figures 2D). Remarkably, rapamycin potently increased SIRT4 levels after 6h of treatment (Figure S2B), correlating with reduced glutamine consumption at the same time point (data not shown). In contrast, SIRT4 levels were not influenced by the treatment of MEFs with U0216, an inhibitor of MEK1/2 in the MAPK pathway (Figure S2C). All other mTOR catalytic inhibitors tested in Tsc2−/− MEFs also resulted in increased SIRT4 protein levels (Figure S2D). To evaluate a potential regulation of SIRT4 by mTORC2, we performed RNA interference (RNAi) experiments of either raptor or the mTORC2 component, rictor, in Tsc2−/− MEFs. The knockdown of raptor, but not rictor, was sufficient to increase SIRT4 protein levels, confirming the role of the mTORC1 pathway in the regulation of SIRT4 (Figure 2E). To investigate whether mTORC1 regulation of SIRT4 occurs in tumor samples, a TSC-xenograft model was used. We injected a TSC2−/− rat leiomyoma cell line; ELT3 cells, expressing either an empty vector (V3) or TSC2 (T3), in the flank of nude mice. SIRT4 levels were dramatically increased in TSC2-expressing tumors compared to empty vector samples (Figure S2E). In addition, we assessed the levels of SIRT4 in both ELT3 xenograft tumors and in mouse Tsc2+/− liver tumors after rapamycin treatment. As expected, these tumor samples exhibited robust elevation of SIRT4 after rapamycin treatment (Figures 2F & S2F). Thus, these data demonstrate that the mTORC1 pathway represses SIRT4 in several tumor systems.

Figure 2  mTORC1 controls glutamate dehydrogenase activity by repressing SIRT4

CREB2 regulates the transcription of SIRT4 in an mTORC1-dependent fashion

We next asked whether the mTORC1-dependent regulation of SIRT4 occurred at the mRNA level. Quantitative RT-PCR results show that rapamycin treatment significantly increased the expression of SIRT4mRNA in Tsc2−/− MEFs (Figure 3A). SIRT4 mRNA levels were dramatically reduced in Tsc2−/− MEFs compared to their WT counterpart (Figure 3B). Similar results were obtained from transcriptional profiling analysis of the SIRT4 gene from a previously published dataset (GSE21755) (Figure 3C) (Duvel et al. 2010). Altogether, our data demonstrate that mTORC1 negatively regulates the transcription of SIRT4. To determine whether CREB2 is involved in the mTORC1-dependent regulation of SIRT4, we performed RNAi experiments. The silencing of CREB2 abolished the rapamycin-induced expression of SIRT4 (Figures 3E & S3A). The knockdown of CREB1 did not affect the upregulation of SIRT4 upon mTORC1 inhibition, thus demonstrating the specificity of CREB2 to induce SIRT4 (Figure S3B), and the knockdown of CREB2 significantly abrogated the rapamycin-induced increase in the activity of the SIRT4 promoter.

Figure 3  SIRT4 is regulated at the mRNA level in an mTORC1-dependent fashion

mTORC1 regulates the stability of CREB2

We next investigated whether the mTORC1 pathway regulates CREB2. Although we did not observe major changes in Creb2 mRNA in normal growth conditions (Figure S4A), mTORC1 inhibition resulted in accumulation of CREB2 protein levels by 2h of rapamycin treatment (Figure 4A). U0126 failed to cause the accumulation of CREB2 (Figure S4B). In contrast, CREB1 protein levels were not affected after 24h rapamycin treatment (Figure S4C). As observed for SIRT4, mTOR catalytic inhibitors, and the specific knockdown of mTOR, resulted in upregulation of CREB2 protein levels (Figures S4D & S4E). CREB2 is upregulated in diverse cell types as a response to a variety of stresses, including hypoxia, DNA damage, and withdrawal of GFs, glucose, and aa (Cherasse et al., 2007Rouschop et al., 2010Yamaguchi et al., 2008;Whitney et al., 2009). Interestingly, mTORC1 is negatively regulated by all of these environmental inputs (Zoncu et al., 2011). Since mTORC1 signaling in Tsc2−/− MEFs is insensitive to serum deprivation, we assessed the role of aa withdrawal and re-stimulation on CREB2 levels. As shown in Fig. 4B, CREB2 accumulated upon aa deprivation, and was decreased following aa re-addition. This phenomenon required the action of the proteasome as MG132 efficiently blocked CREB2 degradation following aa re-addition. Importantly, we found that mTORC1 inhibition abrogated the aa-induced decrease of CREB2 (Figure 4B).

Figure 4  mTORC1 regulates the stability of CREB2

mTORC1 activation promotes the binding of CREB2 to βTrCP and modulates CREB2 ubiquitination

Next, we attempted to identify the E3 ubiquitin ligase that might be responsible for CREB2 turnover. Consistent with a recent study, we found CREB2 to bind the E3 ligase, βTrCP (Frank et al., 2010). However, other related E3 ligases including Fbxw2, Fbxw7a, and Fbxw9 did not bind to CREB2 (data not shown). The interaction of CREB2 with Flag-βTrCP1 was enhanced in the presence of insulin, and was abolished by rapamycin pretreatment (Figure 4D). Importantly, insulin treatment promoted the ubiquitination of CREB2 in an mTORC1-dependent fashion (Figure 4E). Altogether, our results support the notion that the mTORC1 pathway regulates the targeting of CREB2 for proteasome-mediated degradation. βTrCP binds substrates via phosphorylated residues in conserved degradation motifs (degrons), typically including the consensus sequence DpSGX(n)pS or similar variants. We found an evolutionary conserved putative βTrCP binding site (DSGXXXS) in CREB2 (Figure 4F). Interestingly, we noted a downward mobility shift in CREB2 protein with mTORC1 inhibition, consistent with a possible decrease in the phosphorylation of CREB2. (Figure 4A). Frank et al. (2010) showed that phosphorylation of the first serine in the degron motif corresponding to Ser218 is required for the CREB2/βTrCP interaction, and this modification acts as a priming site for a gradient of phosphorylation events on five proline-directed residues codons (T212, S223, S230, S234, and S247) that is required for CREB2 degradation during the cell cycle progression (Frank et al., 2010). Consistent with these observations, we found that the mutation of the five residues to alanine (5A mutant) resulted in strong stabilization of CREB2, comparable to the serine-to-alanine mutation on the priming Ser218 phosphorylation site (Figure S4G).

SIRT4 represses bioenergetics and cell proliferation

We observed that glutamine utilization is repressed by rapamycin treatment (Figure 1) and SIRT4 is induced by mTORC1 inhibition (Figure 2). Thus, we tested whether SIRT4 itself directly regulates cellular glutamine uptake. The stable expression of SIRT4 resulted in the repression of glutamine uptake in Tsc2−/− MEFs and DLD1 cells (Figures 5A & 5B). Glucose uptake was not affected by SIRT4 expression (data not shown). Because glutamine can be an important nutrient for energy production, we examined ATP levels in SIRT4 expressing cells. Consistent with reduced glutamine consumption, the expression of SIRT4 in Tsc2−/− cells resulted in decreased ATP/ADP ratio compared to control cells (Figure 5C). Cells produce ATP via glycolysis and oxidative phosphorylation (OXPHOS). To test the contribution of mitochondrial metabolism versus glycolysis to ATP, we measured the ATP/ADP ratio after the treatment with oligomycin, an inhibitor of ATP synthesis from OXPHOS. Importantly, the difference of the ATP/ADP ratio between control and SIRT4 expressing cells was abrogated by oligomycin (Figure 5C), further demonstrating that SIRT4 may repress the ability of cells to generate energy from mitochondrial glutamine catabolism. Mitochondrial glutamine catabolism is essential for energy production and viability in the absence of glucose (Yang et al., 2009Choo et al., 2010). Thus, we examined the effect of SIRT4 on the survival of Tsc2−/− MEFs during glucose deprivation. Control cells remained viable following 48h of glucose deprivation. Conversely, SIRT4 expressing cells showed a dramatic increase in cell death under glucose-free conditions, which was rescued by the addition of the cell permeable dimethyl-I̧KG (DM-I̧KG) (Figure 5D). Conversely, the expression of SIRT4 did not affect the viability of glucose-deprived Tsc2 WT MEFs (Figure S5A). Glucose deprivation also induced death of the human DU145 cancer cell line stably expressing SIRT4 (data not shown).

Figure 5  SIRT4 represses bioenergetics and proliferation

Glutamine is an essential metabolite for proliferating cells, and many cancer cells exhibit a high rate of glutamine consumption (DeBerardinis et al., 2007). Thus, decreased glutamine uptake in DLD1 and DU145 cancer cells expressing SIRT4 might result in decreased proliferation. Indeed, these cells grew significantly slower than did control cells. Remarkably, DM-I̧KG completely abrogated the decreased proliferation of SIRT4 expressing cells (Figure 5E & 5F), suggesting that repressed glutamine metabolism drove the reduced proliferation of cells expressing SIRT4. The expression of SIRT4 also slowed the proliferation of Tsc2−/− MEFs but did not affect Tsc2 WT MEFs (Figures S5B & S5C). Finally, to rule out that the effect on proliferation was due to aberrant localization and to off-target effects of the overexpressed protein, we examined the localization of HA-SIRT4. We found that SIRT4 is co-localized with the MitoTracker, a mitochondrial-selective marker (Figure S5D). Taken together, these data demonstrate that SIRT4 is a critical negative regulator of mitochondrial glutamine metabolism and cell proliferation.

SIRT4 represses TSC-tumor development

Recent studies have demonstrated a major role of glutamine metabolism in driving oncogenic transformation of many cell lines (Gao et al., 2009Wang et al., 2011). Since SIRT4 expression represses glutamine uptake and cell proliferation (Figure 5), we hypothesized that it could affect tumorigenesis. To test this idea, we assessed the role of SIRT4 in cell transformation by using an anchorage-independent growth assay. SIRT4 expression reduced the ability of Tsc2−/−p53−/− MEFs to grow in soft agar. However, the expression of SIRT4 in Tsc2+/+p53−/− did not impair their colony formation properties (Figure 6A). Tumor incidence in mice injected with Tsc2+/+p53−/− MEFs was not affected by SIRT4 (data not shown). Conversely, in the Tsc2−/−p53−/− cohort, SIRT4 reduced tumor incidence by 20 days at median (Figure 6B). SIRT4 expression inTsc2−/−p53−/− MEFs resulted in reduction of Ki-67 positivity by 60% (Figure 6E), consistent with the finding that SIRT4 inhibits the proliferation of these cells in vitro (Figure S5B). Finally, we performed a comprehensive meta-analysis of SIRT4 expression in human tumors and found significantly lower expression levels of SIRT4, relative to normal tissue, in bladder, breast, colon, gastric, ovarian and thyroid carcinomas (Figure 6F). Interestingly, loss of SIRT4 expression showed a strong association with shorter time to metastasis in patients with breast cancer (Figures 6G & 6H). Altogether, these data strongly suggest that SIRT4 delays tumorigenesis regulated by the mTORC1 pathway.

Figure 6
SIRT4 suppresses TSC-tumor development

The pharmacologic inhibition of glutamine anaplerosis synergizes with glycolytic inhibition to induce the specific death of mTORC1 hyperactive cells

The activation of mTORC1 leads to glucose and glutamine addiction as a result of increased uptake and metabolism of these nutrients (Choo et al., 2010Duvel et al., 2010 & Figure 1). These observations suggest that targeting this addiction offers an interesting therapeutic approach for mTORC1-driven tumors. The alkylating agent, mechlorethamine (Mechlo), incites cell toxicity in part by the inhibition of the GAPDH step of glycolysis via poly-ADP ribose polymerase (PARP)-dependent cellular consumption of cytoplasmic NAD+. The ultimate consequence is glycolytic inhibition, thus mimicking glucose deprivation (Zong et al., 2004). Treatment of Tsc2−/− MEFs with Mechlo decreased both NAD levels and lactate production (Figure 7A and data not shown). The decrease in NAD+ levels was rescued by addition of DPQ (Figure 7A), a PARP inhibitor (Zong et al., 2004). We next tested the ability of glutamine inhibition to determine the sensitivity of Tsc2−/− MEFs to Mechlo. As shown in Figure 7B, the treatment with EGCG, a GDH inhibitor (Figure 1G), potently synergized with Mechlo to kill Tsc2−/− MEFs with the greatest effect observed at 30μM (Figure 7B). As a result, this combination dramatically increased the cleavage of PARP, an apoptotic marker (Figure 7E). Similarly, glutamine deprivation sensitized Tsc2−/− MEFs to Mechlo (data not shown). The RNAi-mediated knockdown of GDH also synergized with Mechlo to induce death of Tsc2−/− MEFs (Figure 7D). Importantly, at these concentrations the combination did not induce death of a Tsc2-rescued cell line (Figure 7C).

Figure 7 The combination of glutamine metabolism inhibitors with glycolytic inhibition is an effective therapy to kill Tsc2−/− and PTEN−/− cells

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Because the metabolic properties of cells with activated mTORC1 by Tsc2– deficiency can be efficiently targeted, we also examined other cell types in which mTORC1 is hyperactive by the loss of PTEN. We found that the combination of Mechlo and EGCG was also effective to induce specific toxicity of PTEN−/− MEFs, while PTEN+/+ MEFs were not affected (Figures S7A & S7B). In addition, the PTEN-deficient human prostate adenocarcinoma cell line, LNCaP, was also sensitive to treatment with Mechlo and EGCG (Figure 7F). This effect was specifically due to lack of TCA cycle replenishment as pyruvate supplementation completely reversed the synergistic effect (Figure 7F). The combination of Mechlo with the GLS1 inhibitor, BPTES (Figure 1G), also resulted in decreased viability of Tsc2−/− cells but not of Tsc2-reexpressing cells (Figures S7C & S7D). Again, death in Tsc2−/− cells was rescued with pyruvate or OAA (Figure S7E). To further investigate if the potent cell death in Tsc2−/− was restricted to Mechlo, we used 2-DG, a glycolytic inhibitor. The combination of 2-DG with either EGCG or BPTES resulted in enhanced cell death of Tsc2−/− MEFs compared to single agent treatments (Figure S7F). This effect was also specific to Tsc2−/− cells, since this combination was less toxic in Tsc2-reexpressing MEFs (Figure S7G). Taken together, our results demonstrate that the combination treatments aimed at inhibiting glycolysis and glutaminolysis potently synergize to kill cells with hyperactive mTORC1 signaling.

Here, we define a novel mTORC1-regulated pathway that controls glutamine-dependent anaplerosis and energy metabolism (Figure 7G). We discovered that the mTORC1 pathway regulates glutamine metabolism by promoting the activity of GDH (Figures 1​-3).3). We show that this regulation occurs by repressing the expression of SIRT4, an inhibitor of GDH (Figures 2 & 3). Molecularly, this is the result of mTORC1-dependent proteasome-mediated degradation of the SIRT4 transcriptional regulator, CREB2 (Figure 4). Interestingly, the modulation of CREB2 levels correlates with increased sensitivity to glutamine deprivation (Ye et al., 2010Qing et al., 2012), fitting with our model of glutamine addiction as a result of mTORC1 activation (Choo et al., 2010). Our data suggest that mTORC1 promotes the binding of the E3 ligase, βTrCP, to CREB2 (Figure 4D), promoting CREB2 degradation by the proteasome (Figure 4E). A previous study has demonstrated that five residues in CREB2 located next to the βTrCP degron are required for its stability (Frank et al., 2010). Accordingly, the mutation of these residues to alanine resulted in stabilization of CREB2 and SIRT4 following insulin and aa-dependent mTORC1 activation (Figure 4G). Future work is aimed at determining if mTORC1 and/or downstream kinases are directly responsible for the multisite phosphorylation of CREB2.

The identification of CREB2 as an mTORC1-regulated transcription factor increases the repertoire of transcriptional regulators modulated by this pathway including HIF1α (glycolysis), Myc (glycolysis) and SREBP1 (lipid biosynthesis) (Duvel et al., 2010Yecies and Manning, 2011). The oncogene Myc has also been linked to the regulation of glutamine metabolism by increasing the expression of the surface transporters ASCT2 and SN2, and the enzyme GLS. Thus, enhanced activity of Myc correlates with increased glutamine uptake and glutamate production (Wise et al., 2008Gao et al., 2009). Our findings describe a new level of control to this metabolic node as shown by the modulation of the glutamate-to-αKG flux (Figure 2). This regulation is particularly relevant as some cancer cells produce more than 50% of their ATP by oxidizing glutamine-derived αKG in the mitochondria (Reitzer et al JBC, 1979). Therefore, these studies support the notion that Myc and CREB2/SIRT4 cooperate to regulate the metabolism of glutamine to αKG.

7.8.4  Rab1A and small GTPases Activate mTORC1

7.8.4.1 Rab1A Is an mTORC1 Activator and a Colorectal Oncogene

Thomas JD1Zhang YJ2Wei YH3Cho JH3Morris LE3Wang HY4Zheng XF5.
Cancer Cell. 2014 Nov 10; 26(5):754-69.
http://dx.doi.org:/10.1016/j.ccell.2014.09.008.

Highlights

  • Rab1A mediates amino acid signaling to activate mTORC1 independently of Rag
  • Rab1A regulates mTORC1-Rheb interaction on the Golgi apparatus
  • Rab1A is an oncogene that is frequently overexpressed in human cancer
  • Hyperactive amino acid signaling is a common driver for cancer

Amino acid (AA) is a potent mitogen that controls growth and metabolism. Here we describe the identification of Rab1 as a conserved regulator of AA signaling to mTORC1. AA stimulates Rab1A GTP binding and interaction with mTORC1 and Rheb-mTORC1 interaction in the Golgi. Rab1A overexpression promotes mTORC1 signaling and oncogenic growth in an AA- and mTORC1-dependent manner. Conversely, Rab1A knockdown selectively attenuates oncogenic growth of Rab1-overexpressing cancer cells. Moreover, Rab1A is overexpressed in colorectal cancer (CRC), which is correlated with elevated mTORC1 signaling, tumor invasion, progression, and poor prognosis. Our results demonstrate that Rab1 is an mTORC1 activator and an oncogene and that hyperactive AA signaling through Rab1A overexpression drives oncogenesis and renders cancer cells prone to mTORC1-targeted therapy.

7.8.4.2 Regulation of TOR by small GTPases

Raúl V Durán1 and Michael N Halla,1
EMBO Rep. 2012 Feb; 13(2): 121–128.
http://dx.doi.org/10.1038%2Fembor.2011.257

TOR is a conserved serine/threonine kinase that responds to nutrients, growth factors, the bioenergetic status of the cell and cellular stress to control growth, metabolism and ageing. A diverse group of small GTPases including Rheb, Rag, Rac1, RalA and Ryh1 play a variety of roles in the regulation of TOR. For example, while Rheb binds to and activates TOR directly, Rag and Rac1 regulate its localization and RalA activates it indirectly through the production of phosphatidic acid. Here, we review recent findings on the regulation of TOR by small GTPases.

The growth-controlling TOR signalling pathway is structurally and functionally conserved from unicellular eukaryotes to humans. TOR, an atypical serine/threonine kinase, was originally discovered inSaccharomyces cerevisiae as the target of rapamycin (Heitman et al, 1991). It was later described in many other organisms including the protozoan Trypanosoma brucei, the yeast Schizosaccharomyces pombe, photosynthetic organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii, and in metazoans such as Caenorhabditis elegansDrosophila melanogaster and mammals. TOR integrates various stimuli to control growth, metabolism and ageing (Avruch et al, 2009Kim & Guan, 2011Soulard et al, 2009;Wullschleger et al, 2006Zoncu et al, 2011a). In mammals, mTOR is activated by nutrients, growth factors and cellular energy, and is inhibited by stress. Thus, the molecular regulation of TOR is complex and diverse. Among the increasing number of TOR regulators, small GTPases are currently garnering much attention. Small GTPases (20–25 kDa) are either in an inactive GDP-bound form or an active GTP-bound form (Bos et al, 2007). GDP–GTP exchange is regulated by GEFs, which mediate the replacement of GDP by GTP, and by GAPs, which stimulate the intrinsic GTPase activity of a cognate GTPase to convert GTP into GDP (Fig 1). Upon activation, small GTPases interact with effector proteins, thereby stimulating downstream signalling pathways. Small GTPases constitute a superfamily that comprises several subfamilies, such as the Rho, Ras, Rab, Ran and Arf families. Rheb, Rag, RalA, Rac1 and Ryh1, all members of the small GTPase superfamily, play a role in the concerted regulation of TOR by different stimuli. This review summarizes recent advances in the understanding of TOR regulation by these small GTPases.

Regulation of small GTPases by GEFs and GAPs

Regulation of small GTPases by GEFs and GAPs

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Figure 1 Regulation of small GTPases by GEFs and GAPs. A guanine nucleotide exchange factor (GEF) replaces GDP with GTP to activate the signalling function of the GTPase. Conversely, a GTPase-activating protein (GAP) stimulates hydrolysis of GTP into GDP

The TOR complexes

TOR is found in two functionally and structurally distinct multiprotein complexes, named TORC1 and TORC2 (Avruch et al, 2009Kim & Guan, 2011Soulard et al, 2009Wullschleger et al, 2006Zoncu et al, 2011a). TORC1 regulates several cellular processes including protein synthesis, ribosome biogenesis, nutrient uptake and autophagy. TORC2, in turn, regulates actin cytoskeleton organization, cell survival, lipid synthesis and probably other processes. TORC1 and TORC2 are rapamycin-sensitive and rapamycin-insensitive, respectively, although in some organisms, for example A. thaliana and T. brucei, this rule does not apply (Barquilla et al, 2008Mahfouz et al, 2006). Nevertheless, long-term treatment with rapamycin can also indirectly inhibit TORC2 in mammalian cell lines (Sarbassov et al, 2006). Furthermore, there is accumulating evidence that not all TORC1 readouts are rapamycin-sensitive (Choo & Blenis, 2009Dowling et al, 2010Peterson et al, 2011).

Upstream of TOR

Four main inputs regulate mTORC1: nutrients, growth factors, the bioenergetic status of the cell and oxygen availability. It is well established that growth factors activate mTORC1 through the PI3K–AKT pathway. Once activated, AKT phosphorylates and inhibits the heterodimeric complex TSC1–TSC2, a GAP for Rheb and thus an inhibitor of mTORC1 (Avruch et al, 2009). The TSC1–TSC2 heterodimer is a ‘reception centre’ for various stimuli that are then transduced to mTORC1, including growth factor signals transduced through the AKT and ERK pathways, hypoxia through HIF1 and REDD1, and energy status through AMPK (Wullschleger et al, 2006). In addition to the small GTPases Rheb and Rag (see below), PA also binds to and activates mTORC1 (Fang et al, 2001). Pharmacological or genetic inhibition of PA production, through the inhibition of PLD, impairs activation of mTORC1 by nutrients and growth factors (Fang et al, 2001). Moreover, elevated PLD activity leads to rapamycin resistance in human breast cancer cells (Chen et al, 2003), further supporting a role for PA as an mTORC1 regulator. As discussed below, the small GTPase RalA participates in the mechanism by which PA activates mTORC1 (Maehama et al, 2008Xu et al, 2011).

In the case of nutrients, amino acids in particular, several elements mediate the activation of TORC1. As discussed below, the Rag GTPases are necessary to activate TORC1 in response to amino acids (Binda et al, 2009Kim et al, 2008Sancak et al, 2008). In mammals, it has also been proposed that amino acids stimulate an increase in intracellular calcium concentration, which in turn activates mTORC1 through the class III PI3K Vps34 (Gulati et al, 2008).

Downstream of TOR

TORC1 regulates growth-related processes such as transcription, ribosome biogenesis, protein synthesis, nutrient transport and autophagy (Wullschleger et al, 2006). In mammals, the best-characterized substrates of mTORC1 are S6K and 4E-BP1, through which mTORC1 stimulates protein synthesis. mTORC1 activates S6K, which is a positive regulator of protein synthesis, and inhibits 4E-BP1, which is a negative regulator of protein synthesis. Upon phosphorylation by mTORC1, 4E-BP1 releases eIF4E. Once released from 4E-BP1, eIF4E interacts with the eIF4G subunit of the eIF4F complex, allowing initiation of translation. In mammals, 4E-BP1 participates mainly in the regulation of cell proliferation and metabolism (Dowling et al, 2010). In S. cerevisiae, the main substrate of TORC1 is the S6K orthologue Sch9 (Urban et al, 2007). Sch9 is required for the activation of ribosome biogenesis and translation initiation stimulated by TORC1. Furthermore, it participates in TORC1-dependent inhibition of G0 phase entry.

Regulation of TOR by Rheb

The small GTPase Rheb was first identified in 1994 in a screen for genes induced in neurons in response to synaptic activity (Yamagata et al, 1994), and was first described to interact with the Raf1 kinase (Yee & Worley, 1997). A later report showed that loss of Rhb1, the Rheb orthologue in S. pombe, causes a starvation-like growth arrest (Mach et al, 2000). In 2003, several independent groups working with mammalian cells in vitro and Drosophila in vivo demonstrated that Rheb is the target of the TSC1–TSC2 GAP and a TORC1 activator (Avruch et al, 2009).

Interestingly, the Rheb–mTOR interaction both in vivo and in vitro does not depend on GTP loading of Rheb. This is unusual for GTPases as GTP loading usually regulates effector binding. However, GTP loading of Rheb is crucial for the activation of mTOR kinase activity (Sancak et al, 2007). Conversely, mTOR becomes inactive after association with a nucleotide-deficient Rheb (Long et al, 2005a; Fig 2). Similar results were obtained in S. pombe, making use of mutations that hyperactivate Rheb by increasing its overall GTP : GDP binding ratio (Urano et al, 2005). In contrast to the situation in mammals, interaction of Rheb with SpTOR2 in fission yeast is detected only with a hyperactive Rheb mutant. This suggests that, in S. pombe, Rheb binds to SpTOR2 in a GTP-dependent manner.

Rheb activates TORC1

Rheb activates TORC1

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271343/bin/embor2011257f2.gif

Figure 2 Rheb activates TORC1 both directly and indirectly. GTP-bound Rheb interacts directly with TORC1 to activate TORC1 kinase. GTP-bound Rheb also activates RalA, which activates PLD to increase production of PA. PA in turn interacts with TORC1

In addition to the direct interaction between mTOR and Rheb, activation of PA production by Rheb is an additional mechanism by which Rheb might regulate mTORC1. Rheb binds to and activates PLD in a GTP-dependent manner (Sun et al, 2008). PLD produces PA, which binds directly to and upregulates mTORC1. This finding reveals cross-talk between the TSC–Rheb and the PA pathways in the regulation of mTORC1 signalling. A recent study by Yoon and colleagues further demonstrated the role of PLD in mTORC1 regulation (Yoon et al, 2011). They showed that amino acids activate PLD through translocation of PLD to the lysosomal compartment. This translocation is positively regulated by human Vps34 and is necessary for the activation of mTORC1 by amino acids. These authors propose the existence of a Vps34–PLD1 pathway that activates mTORC1 in parallel to the Rag pathway (Yoon et al, 2011).

Although Rheb is required for the activation of mTORC1 by amino acids, Rheb itself does not participate in amino acid sensing, and GTP-loading of Rheb is not affected by amino acid depletion (Long et al, 2005b). Furthermore, amino acid depletion inhibits mTORC1 even in TSC2−/− fibroblasts (Roccio et al, 2006). Nevertheless, interaction of mTORC1 with Rheb depends on amino acid availability (Long et al, 2005b). As discussed below, the current model proposes that amino acids mediate translocation of mTORC1 to the lysosomal surface where mTORC1 interacts with and is activated by GTP-loaded Rheb (Sancak et al, 2008).

Regulation of TOR by Rag

Rag GTPases have unique features among the Ras GTPase subfamily members: they form heterodimers and lack a membrane-targeting sequence (Nakashima et al, 1999Sekiguchi et al, 2001). Gtr1 in S. cerevisiaewas the first member of this GTPase subfamily to be identified (Bun-Ya et al, 1992). The mammalian RagA and RagB GTPases were later described as Gtr1 orthologues (Hirose et al, 1998). Gtr2 in yeast (Nakashima et al, 1999) and its mammalian orthologues RagC and RagD (Sekiguchi et al, 2001) were subsequently discovered due to their ability to form heterodimers with Gtr1 in yeast and RagA and RagB in mammals, respectively. The crystal structure of the Gtr1–Gtr2 complex has been determined recently (Gong et al, 2011). Gtr1 and Gtr2 have similar structures, organized in two domains: an amino-terminal GTPase domain (designated as the G domain) and a carboxy-terminal domain. The Gtr1–Gtr2 heterodimer presents a pseudo-twofold symmetry resembling a horseshoe. The crystal structure reveals that Gtr1–Gtr2 dimerization results from extensive contacts between the C-terminal domains of both proteins, while the G domains do not contact each other (Gong et al, 2011).

Rag proteins mediate the activation of TORC1 in response to amino acids.

Rag proteins mediate the activation of TORC1 in response to amino acids.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271343/bin/embor2011257f3.gif

Figure 3 Rag proteins mediate the activation of TORC1 in response to amino acids. The RagA/B–RagC/D heterodimer is anchored to the MP1–p14–p18 complex on the surface of the lysosome.

Overexpressed Rheb is mislocalized throughout the cell, and therefore interaction of mTORC1 with Rheb does not require amino-acid-induced translocation of mTORC1 to the lysosome. The model is further supported by observations in Drosophila showing that expression of a constitutively active mutant of RagA significantly increases the size of individual cells, whereas expression of a dominant negative mutant of RagA reduces cell size (Kim et al, 2008). Moreover, Rag plays a role in TORC1-mediated inhibition of autophagy both in Drosophila (Kim et al, 2008) and in human cells (Narita et al, 2011).

mTOR and small GTPases are therapeutic targets in the treatment of cancer (Berndt et al, 2011Dazert & Hall, 2011). Aberrant activation of GTPases, including Ras, Rho, Rab or Ran GTPases, promotes cell transformation and cancer (Agola et al, 2011Ly et al, 2010Pylayeva-Gupta et al, 2011), in some cases by acting in the mTOR pathway. Targeting GTPases by using farnesyltransferase inhibitors or geranylgeranyltransferase inhibitors affects signal transduction pathways, cell cycle progression, proliferation and cell survival. Both types of inhibitor are currently under investigation for cancer therapy, although only a small subset of patients responds to these inhibitors (Berndt et al, 2011). A better understanding of the relationship between GTPases and mTOR is essential for the design of combined therapies.

From a mechanistic point of view, research on TOR in different systems is continually adding new insight on the role of TOR in cell biology. However, what is lacking is an integration of the various proposed regulators of TOR, in particular small GTPases (see Sidebar A).

Sidebar A | In need of answers

  1. How are amino acids sensed by the cell?
  2. What is the mechanism by which amino acids regulate the GTP-loading of Rag proteins? What are the GEF and GAP for the Rag proteins?
  3. Is there a GEF that regulates the GTP-loading of Rheb?
  4. What is the molecular mechanism by which Rheb activates TORC1?
  5. How is the dual effect of Rac1 being both upstream and downstream from TOR regulated?
  6. How are the diverse GTPases that impinge on TOR integrated?

7.8.5 PI3K.Akt signaling in osteosarcoma

Zhang J1Yu XH2Yan YG1Wang C1Wang WJ3.
Clin Chim Acta. 2015 Apr 15; 444:182-192.
http://dx.doi.org:/10.1016/j.cca.2014.12.041

Highlights

  • Activation of the PI3K/Akt signaling regulates various cellular functions.
  • The PI3K/Akt signaling may play a key role in the progression of osteosarcoma.
  • Targeting the PI3K/Akt signaling has therapeutic potential for osteosarcoma.

Osteosarcoma (OS) is the most common nonhematologic bone malignancy in children and adolescents. Despite the advances of adjuvant chemotherapy and significant improvement of survival, the prognosis remains generally poor. As such, the search for more effective anti-OS agents is urgent. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is thought to be one of the most important oncogenic pathways in human cancer. An increasing body of evidence has shown that this pathway is frequently hyperactivated in OS and contributes to disease initiation and development, including tumorigenesis, proliferation, invasion, cell cycle progression, inhibition of apoptosis, angiogenesis, metastasis and chemoresistance. Inhibition of this pathway through small molecule compounds represents an attractive potential therapeutic approach for OS. The aim of this review is to summarize the roles of the PI3K/Akt pathway in the development and progression of OS, and to highlight the therapeutic potential of targeting this signaling pathway. Knowledge obtained from the application of these compounds will help in further understanding the pathogenesis of OS and designing subsequent treatment strategies.

PK.Akt signaling

PK.Akt signaling

http://ars.els-cdn.com/content/image/1-s2.0-S0009898115001059-gr1.sml

PI3K/Akt signaling

PI3K.Akt signaling pathway

PI3K.Akt signaling pathway

http://ars.els-cdn.com/content/image/1-s2.0-S0009898115001059-gr2.sml

PI3K/Akt signaling pathway

PK.Akt therapeutic target

PK.Akt therapeutic target

http://ars.els-cdn.com/content/image/1-s2.0-S0009898115001059-gr3.sml

PK/Akt therapeutic target

7.8.6 The mTORC1-S6K1 Pathway Regulates Glutamine Metabolism through the eIF4B-Dependent Control of c-Myc Translation

Csibi A1Lee G1Yoon SO1Tong H2,…, Fendt SM4Roberts TM2Blenis J5.
Curr Biol. 2014 Oct 6; 24(19):2274-80.
http://dx.doi.org:/10.1016/j.cub.2014.08.007

Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation. Glutamine is catabolyzed to α-ketoglutarate (αKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. Here we demonstrate that mTORC1 positively regulates GLS and glutamine flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5′ untranslated region (5’UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN.

Highlights

  • The mTORC1 pathway positively regulates GLS and glutamine flux
  • mTORC1 controls the translation efficiency of Myc mRNA
  • S6K1 regulates Myc translation through eIF4B phosphorylation
  • Inhibition of GLS decreases the growth of pancreatic cancer cells

Figure 1. The mTORC1 Pathway Regulates GLS1 (A–C and E) GLS protein levels in whole cell lysates from Tsc2 WT and Tsc22/2 MEFs treated with rapamycin (Rapa) for 8 hr (A); HEK293T cells stably expressing Rheb WT, the mutant S16H Rheb, or EV and treated with rapamycin for 24 hr (B); Tsc22/2 MEFs treated with rapamycin at the indicated time points (C); and Tsc2 WT and Tsc22/2 MEFs treated with the indicated compounds for 8 hr (E). The concentrations of the compounds were as follows: rapamycin, 20 ng/ml; LY294002 (LY), 20 mM; and BEZ235, 10 mM. (D) Time course of glutamine consumption in Tsc22/2 MEFs incubated with or without 20ng/ml rapamycin for 24 hr. Each time data point is an average of triplicate experiments. (F) Intracellular glutamine levels in Tsc22/2 MEFs treated with rapamycin for 24 hr. (G) Glutamineflux inTsc22/2 MEFs expressing an EV or re-expressingTSC2 treated with theindicated compounds for 24hr.The concentrations of the compounds were as follows: rapamycin 20 ng/ml; LY294002, 20 mM; BEZ235, 10 mM; BPTES, 10 mM; and 6-diazo-5-oxo-l-norleucine, 1mM. The mean is shown. Error bars represent the SEM from at least three biological replicates. Numbers below the immunoblot image represent quantification normalized to the loading control. See also Figure S1.

Figure2. The mTORC1 Pathway Regulates GLS1 via Myc GLS and Myc protein levels in whole cell lysates from BxPC3 cells transfected with a nontargeting control (NTC) siRNA or four independent siRNAs against Myc for 72 hr (A), Tsc2 WT and Tsc22/2 MEFs treated with rapamycin (20 ng/ml) for 8 hr (B), and Tsc22/2 MEFs stably expressing Myc or EV and treated with rapamycin (20 ng/ml) for 24 hr (C).

Figure 3. The mTORC1 Substrate S6K1 Controls GLS through Myc mRNA Translation (A) Normalized luciferase light units of Tsc22/2 MEFs stably expressing a Myc-responsive firefly luciferase construct (Myc-Luc) or vector control (pCignal Lenti-TRE Reporter). Myc transcriptional activity was measured after treatment with rapamycin (20 ng/ml) or PF4708671 (10 mM) for 8 hr. (B) GLS and Myc protein levels in whole cell lysates from HEK293T cells expressing HA-S6K1-CA (F5A-R3A-T389E) or EV treated with rapamycin (20 ng/ml) for 24 hr. HA, hemagglutinin. (CandD) Intracellular glutamine levels of Tsc22/2 MEFs stably expressing S6K-CA(F5A/R5A/T389E, mutating either the three arginines or all residues within the RSPRR motif to alanines shows the same effect; [10]) or empty vector and treated with rapamycin (20 ng/ml) or DMSO for 48 hr (C) or transfected with NTC siRNA or siRNA against both S6K1/2 (D). 24 hr posttransfection, cells transfected with NTC siRNA were treated with PF4708671 (10 mM) or DMSO for 48 hr. (E) Glutamine consumption of Tsc22/2 MEFs transfected with NTC siRNA or siRNA against both S6K1/2. 72 hr posttransfection, media were collected, and levels of glutamine in the media were determined. (F) Normalized luciferase light units of Tsc2WTMEFs transfected with thepDL-N reporter construct containing the 50 UTR of Myc under the control of Renilla luciferase. Firefly luciferase was used as an internal control. 48hr posttransfection, cells were treated with rapamycin (20ng/ml) or PF4708671 (10mM) for 8h. (G) Relative levels of Myc, Gls, and Actin mRNA in each polysomal gradient fraction. mRNA levels were measured by quantitative PCR and normalized to the 5S rRNA level. HEK293T cells were treated with rapamycin (20 ng/ml) for 24 hr, and polysomes were fractionated on sucrose density gradients. The values are averaged from two independent experiments performed in duplicate, and the error bars denote SEM (n = 4). (Hand I) GLS and Myc protein levels in whole cell lysates from Tsc22/2 MEFs transfected with NTC siRNA or two independent siRNAs against eIF4B for 72hr (H) and Tsc22/2 MEFs stably expressing eIF4B WT, mutant S422D, or EV) and treated with rapamycin for 24 hr (I). The mean is shown. Error bars represent the SEM from at least three biological replicates. The asterisk denotes a nonspecific band. The numbers below the immunoblot image represent quantification normalized to the loading control. See also Figures S2 and S3.

Figure 4. Inhibition of GLS Reduces the Growth of Pancreatic Cancer Cells (A) GLS and Myc protein levels in whole cell lysates from BxPC3, MIAPaCa-2, or AsPC-1 cells treated with rapamycin (20 ng/ml) or BEZ235 (1 mM) for 24 hr. (B) Glutamine consumption of BxPC3 or AsPC-1 cells 48 hr after plating. (Cand D) Soft agar assays with BxPC3 or AsPC-1 cells treated with BPTES (10 mM), the combination of BPTES (10 mM) + OAA (2 mM) (C) and BxPC3 or AsPC-1 cells treated with BPTES, and the combination of BPTES (10 mM) + NAC (10 mM) (D). NS, not significant. The mean is shown. Error bars represent the SEM from at least three biological replicates.

7.8.7 Localization of mouse mitochondrial SIRT proteins

Nakamura Y1Ogura MTanaka DInagaki N.
Biochem Biophys Res Commun. 2008 Feb 1; 366(1):174-9
http://www.ncbi.nlm.nih.gov/pubmed/18054327#

Yeast silent information regulator 2 (SIR2) is involved in extension of yeast longevity by calorie restriction, and SIRT3, SIRT4, and SIRT5 are mammalian homologs of SIR2 localized in mitochondria. We have investigated the localization of these three SIRT proteins of mouse. SIRT3, SIRT4, and SIRT5 proteins were localized in different compartments of the mitochondria. When SIRT3 and SIRT5 were co-expressed in the cell, localization of SIRT3 protein changed from mitochondria to nucleus. These results suggest that the SIRT3, SIRT4, and SIRT5 proteins exert distinct functions in mitochondria. In addition, the SIRT3 protein might function in nucleus

Fig. 1. Localization of SIRT3, SIRT4, and SIRT5 in mitochondria. (A) Confocal microscopy. SIRT3-myc (upper panels), SIRT4-myc (middle panels), and SIRT5-FLAG (lower panels) were expressed in COS7 cells and immunostained with anti-myc antibody or anti-FLAG antibody. Mitochondria and nuclei were stained by MitoTracker Red and DAPI, respectively, and fluorescent images were obtained using a confocal microscope. (B) Fractionation of post-nuclear supernatant. SIRT3-myc, SIRT4-myc, and SIRT5-FLAG proteins each was expressed in COS7 cells, and the obtained PNS was fractionated into mitochondria-enriched precipitate (P1), microsome-enriched precipitate (P2), and supernatant (S) fractions. The three fractions were separated by SDS–PAGE and then analyzed by Western blotting using anti-myc antibody for SIRT3-myc and SIRT4-myc or anti-FLAG antibody for SIRT5-FLAG. Hsp60, calnexin, and GAPDH were used as endogenous markers for mitochondria, microsome, and cytosol, respectively. (C) Alkaline treatment of mitochondria. Mitochondria prepared from the COS7 cells expressing each of the SIRT3-myc, SIRT4-myc, and SIRT5-FLAG proteins were treated with Na2CO3. The reaction mixture was centrifuged to separate the precipitate and supernatant fractions, containing membrane-integrated proteins and soluble proteins, respectively. The two fractions were analyzed by Western blotting. Cytochrome c (cytc) and hsp60 were used as endogenous protein markers for mitochondrial soluble protein. (D) Submitochondrial fractionation. The mitochondria from COS7 cells expressing one of three SIRT proteins were treated with either H2O (hypotonic) or TX-100, and then treated with trypsin. The reaction mixtures were analyzed by Western blotting. Cytochrome c and hsp60 were used as endogenous markers for mitochondrial intermembrane space protein and matrix protein, respectively.

Fig. 2. Localization of SIRT3 when co-expressed with SIRT5. (A) Confocal microscopic analysis of COS7 cells expressing two of the three mitochondrial SIRT proteins. SIRT3-myc and SIRT5-FLAG (upper panels), SIRT3-myc and SIRT4-FLAG (middle panels), and SIRT4-myc and SIRT5-FLAG (lower panels) were co-expressed in COS7 cells, and immunostained using antibodies against myc tag and FLAG tag. Nuclei were stained by DAPI. (B) Subcellular fractionation of PNS. PNS of COS7 cells co-expressing SIRT3-myc and SIRT5-FLAG was fractionated into mitochondria-enriched precipitate (P1), microsome-enriched precipitate (P2), and supernatant (S) fractions, and these fractions along with whole cell lysate were analyzed by Western blotting. (C) Subcellular fractionation using digitonin. COS7 cells expressing either SIRT3-myc (left) or SIRT5-FLAG (middle) or both (right) were solubilized by digitonin, and the obtained lysate was centrifuged and fractionated into nuclear-enriched insoluble (INS), and soluble (SOL) fractions. Hsp60 and laminA/C were used as endogenous markers for mitochondria protein and nucleus protein, respectively.

Because the segment containing amino acid residues 66– 88 potentially forms a basic amphiphilic a-helical structure, it could serve as a MTS. To examine the role of this segment, SIRT3 mutant SIRT3mt, in which the four amino acid residues 72–75 were replaced by four alanine residues, was constructed (Fig. 3A). When SIRT3mt alone was expressed in COS7 cells, SIRT3mt protein was not detected in mitochondria but was widely distributed in the cell in confocal microscopic analysis (Fig. 3B, upper panels). In addition, when SIRT3mt and SIRT5 were co-expressed, the distribution of SIRT3mt protein was not changed compared to that expressed alone (Fig. 3B, lower panels). In fractionation of PNS, SIRT3mt protein was fractionated into S fraction both when SIRT3mt was expressed alone and when SIRT3mt and SIRT5 were co-expressed. SIRT5 protein was localized in mitochondria when SIRT3mt and SIRT5 were co-expressed (Fig. 3C). These results indicate that the MTS is necessary not only for targeting SIRT3 to mitochondria in the absence of SIRT5 but also for targeting SIRT3 to nucleus in the presence of SIRT5.

Fig. 3. Effect of disruption of putative mitochondrial targeting signal of SIRT3. (A) Alanine replacement of putative MTS of SIRT3. Four residues of the putative MTS of SIRT3 (amino acid residues 72–75) were replaced with four alanine residues. In the SIRT3mt sequence, amino acid residues identical with wild-type SIRT3 protein are indicated with dots. (B) Confocal microscopy. Immunofluorescent images of COS7 cells expressing SIRT3mt-myc alone (upper panels) or both SIRT3mt-myc and SIRT5-FLAG (lower panels) are shown. Mitochondria and nuclei were stained by MitoTracker Red and DAPI, respectively. (C) Subcellular fractionation of PNS. PNSs of COS7 cells expressing SIRT3mt-myc alone (an upper panel) or co-expressing SIRT3mt-myc and SIRT5-FLAG (middle and lower panels) were centrifuged and fractionated into mitochondria-enriched precipitate (P1), microsome-enriched precipitate (P2), and supernatant (S) fractions. The fractions were analyzed by Western blotting.

Fig. 4. Effect of disruption of putative nuclear localization signal of SIRT3. (A) Comparison of the amino acid sequences of putative NLS of SIRT3, SIRT3nu, and SV40 large T antigen. Three basic amino acid residues of the putative NLS of SIRT3 (amino acid residues 214–216) were replaced with three alanine residues. In the SIRT3nu sequence, amino acid residues identical with wild-type SIRT3 protein are indicated with dots. The classical NLS of SV40 large T antigen also is shown (SV40). (B) Confocal microscopy. Immunofluorescent images of COS7 cells expressing SIRT3nu-myc alone (upper panels) or both SIRT3nu-myc and SIRT5-FLAG (lower panels) are shown. Mitochondria and nuclei were stained by MitoTracker Red and DAPI, respectively. (C) Subcellular fractionation of PNS. PNSs of the COS7 cells expressing SIRT3nu-myc alone (an upper panel) or co-expressing SIRT3numyc and SIRT5-FLAG (middle and lower panels) were fractionated into mitochondria-enriched precipitate (P1), microsome-enriched precipitate (P2), and supernatant (S) fractions. The fractions were analyzed by Western blotting.

The sequence containing amino acid sequence 213-219 of the SIRT3 closely resembles the putative protein classical NLS of the SV40 T antigen (Fig. 4A). To examine whether this sequence functions as a NLS, the mutant SIRT3 protein SIRT3nu, in which the three basic amino acid residues (214–216) in the putative NLS of SIRT3 were replaced by three alanine residues (Fig. 4A), was constructed. When SIRT3nu alone was expressed in COS7 cells, it was localized in mitochondria (Fig. 4B, upper panels). In the cells co-expressing SIRT3nu and SIRT5, a shift of SIRT3nu protein to the nucleus was not observed, and SIRT3nu protein and a part of SIRT5 protein were scattered widely in the cell in confocal microscopic analysis (Fig. 4B, lower panels). In fractionation of PNS, all of the SIRT3nu protein and nearly half of the SIRT5 protein were shifted from P1 fraction to S fraction by co-expression (Figs. 1B and 4C). These results suggest that the segment containing amino acid residues 213–219 of SIRT3 plays an important role in the localization shift of SIRT3 protein to nucleus when co-expressed with SIRT5. Furthermore, SIRT5 may well hamper SIRT3nu localization in mitochondria through interaction with SIRT3nu. However, further study is required to elucidate the mechanism of the localization shift of SIRT3 protein. Interestingly, recent study has reported that human prohibitin 2 (PHB2), known as a repressor of estrogen receptor (ER) activity, is localized in the mitochondrial inner membrane, and translocates to the nucleus in the presence of ER and estradiol [18]. Although the mechanism of regulation of the expression level of SIRT5 remains unknown, SIRT3 might play a role in communication between nucleus and mitochondria in a SIRT5-dependent manner. The function of mitochondrial SIRT proteins is still not well known. In the present study, we determined the exact localization of mouse SIRT3, SIRT4, and SIRT5 proteins in mitochondria. In addition, we demonstrated that SIRT3 can be present in nucleus in the presence of SIRT5. It has been reported that SIRT3 deacetylates proteins that are not localized in mitochondria in vitro such as histone-4 peptide and tubulin [14]. Thus, if SIRT3 is present in nucleus in vivo, SIRT3 protein might well deacetylate nuclear proteins. These results provide useful information for the investigation of the function of these proteins.

References

[1] J.C. Tanny, G.J. Dowd, J. Huang, H. Hilz, D. Moazed, An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing, Cell 99 (1999) 735–745.
[2] S. Imai, C.M. Armstrong, M. Kaeberlein, L. Guarente, Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase, Nature 403 (2000) 795–800.
[3] M. Gotta, S. Strahl-Bolsinger, H. Renauld, T. Laroche, B.K. Kennedy, M. Grunstein, S.M. Gasser, Localization of Sir2p: the nucleolus as a compartment for silent information regulators, EMBO J. 16 (1997) 3243–3255.
[4] I. Muller, M. Zimmermann, D. Becker, M. Flomer, Calendar life span versus budding life span of Saccharomyces cerevisiae, Mech. Aging Dev. 12 (1980) 47–52.
[5] S.J. Lin, M. Kaeberlein, A.A. Andalis, L.A. Sturtz, P.A. Defossez, V.C. Culotta, G.R. Fink, L. Guarente, Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration, Nature 418 (2002) 344–348.
[6] S.J. Lin, P.A. Defossez, L. Guarente, Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae, Science 289 (2000) 2126–2128.

7.8.8 SIRT4 Has Tumor-Suppressive Activity and Regulates the Cellular Metabolic Response to DNA Damage by Inhibiting Mitochondrial Glutamine Metabolism

Jeong SM1Xiao CFinley LWLahusen TSouza ALPierce KLi YH, et al.
Cancer Cell. 2013 Apr 15; 23(4):450-63.
http://www.ncbi.nlm.nih.gov/pubmed/23562301#
http://dx.doi.org:/10.1016/j.ccr.2013.02.024

DNA damage elicits a cellular signaling response that initiates cell cycle arrest and DNA repair. Here we find that DNA damage triggers a critical block in glutamine metabolism, which is required for proper DNA damage responses. This block requires the mitochondrial SIRT4, which is induced by numerous genotoxic agents and represses the metabolism of glutamine into TCA cycle. SIRT4 loss leads to both increased glutamine-dependent proliferation and stress-induced genomic instability, resulting in tumorigenic phenotypes. Moreover, SIRT4 knockout mice spontaneously develop lung tumors. Our data uncover SIRT4 as an important component of the DNA damage response pathway that orchestrates a metabolic block in glutamine metabolism, cell cycle arrest and tumor suppression.

DNA damage initiates a tightly coordinated signaling response to maintain genomic integrity by promoting cell cycle arrest and DNA repair. Upon DNA damage, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related protein (ATR) are activated and induce phosphorylation of Chk1, Chk2 and γ-H2AX to trigger cell cycle arrest and to initiate assembly of DNA damage repair machinery (Abraham, 2001Ciccia and Elledge, 2010Su, 2006). Cell cycle arrest is a critical outcome of the DNA damage response (DDR) and defects in the DDR often lead to increased incorporation of mutations into newly synthesized DNA, the accumulation of chromosomal instability and tumor development (Abbas and Dutta, 2009Deng, 2006Negrini et al., 2010).

The cellular metabolic response to DNA damage is not well elucidated. Recently, it has been shown that DNA damage causes cells to upregulate the pentose phosphate pathway (PPP) to generate nucleotide precursors needed for DNA repair (Cosentino et al., 2011). Intriguingly, a related metabolic switch to increase anabolic glucose metabolism has been observed for tumor cells and is an important component of rapid generation of biomass for cell growth and proliferation (Jones and Thompson, 2009Koppenol et al., 2011). Hence, cells exposed to genotoxic stress face a metabolic challenge; they must be able to upregulate nucleotide biosynthesis to facilitate DNA repair, while at the same time limiting proliferation and inducing cell cycle arrest to limit the accumulation of damaged DNA. The molecular events that regulate this specific metabolic program in response to DNA damage are still unclear.

Sirtuins are a highly conserved family of NAD+-dependent deacetylases, deacylases, and ADP-ribosyltransferases that play various roles in metabolism, stress response and longevity (Finkel et al., 2009;Haigis and Guarente, 2006). In this study, we studied the role of SIRT4, a mitochondria-localized sirtuin, in cellular metabolic response to DNA damage and tumorigenesis.

DNA damage represses glutamine metabolism

To investigate how cells might balance needs for continued nucleotide synthesis, while also preparing for cell cycle arrest, we assessed the metabolic response to DNA damage by monitoring changes in the cellular consumption of two important fuels, glucose and glutamine, after DNA-damage. Strikingly, treatment of primary mouse embryonic fibroblasts (MEFs) with camptothecin (CPT), a topoisomerase 1 inhibitor that causes double-stranded DNA breaks (DSBs), resulted in a pronounced reduction in glutamine consumption (Figure 1A). Glutamine metabolism in mammalian cells is complex and contributes to a number of metabolic pathways. Glutamine is the primary nitrogen donor for protein and nucleotide synthesis, which are essential for cell proliferation (Wise and Thompson, 2010). Additionally, glutamine provides mitochondrial anaplerosis. Glutamine can be metabolized via glutaminase (GLS) to glutamate and NH4+, and further converted to the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate via glutamate dehydrogenase (GDH) or aminotransferases. This metabolism of glutamine provides an important entry point of carbon to fuel the TCA cycle (Jones and Thompson, 2009), and accounts for the majority of ammonia production in cells (Yang et al., 2009). CPT-induced reduction of glutamine consumption was accompanied by a reduction in ammonia secretion from cells (Figure 1B). Notably, under these conditions, we observed no obvious decrease in glucose uptake and lactate production (Figures 1C and 1D), consistent with previous studies showing that intact glucose utilization through the PPP is important for a normal DNA damage response (Cosentino et al., 2011). Preservation of glucose uptake also suggests that repression of glutamine consumption may be a specific metabolic response to genotoxic stress and not reflective of a non-specific metabolic crisis.

Figure 1 Glutamine metabolism is repressed by genotoxic stress

To examine the metabolic response to other forms of genotoxic stress, we monitored the metabolic response to ultra-violet (UV) exposure in primary MEFs. Similar to CPT treatment, UV exposure reduced glutamine uptake, without significant changes in glucose consumption (Figures 1E and 1F). Similarly two human cell lines, HepG2 and HEK293T, also demonstrated marked reductions in glutamine uptake in response to DNA damaging agents without comparable changes in glucose uptake (Figures 1G and 1HFigures S1A and S1B). Taken together, these results suggest that a variety of primary and tumor cell lines (from mouse or human) respond to genotoxic stress by down-regulating glutamine metabolism.

To examine in more detail the changes in cellular glutamine metabolism after genotoxic stress, we performed a global metabolomic analysis with transformed MEFs before and after DNA damage. As previously reported, we observed that PPP intermediates were increased in response to DNA damage (Figures 1I and 1J). Remarkably, we observed a decrease in measured TCA cycle intermediates after UV exposure (Figures 1I and 1K). Moreover, we found that HepG2 cells showed a similar metabolomic shift in response to DNA damage (Figure S1D). We did not observe a clear, coordinated repression of nucleotides or glutamine-derived amino acids after exposure to DNA damage (Figure S1C).

To determine whether reduction in TCA cycle metabolites was the consequence of reduced glutamine metabolism, we performed a time-course tracer study to monitor the incorporation of [U-13C5]glutamine into TCA cycle intermediates at 0, 2 and 4 hr after UV treatment. We observed that after UV exposure, cells reduced contribution of glutamine to TCA cycle intermediates in a time-dependent manner (Figure 1L). Moreover, the vast majority of the labeled fumarate and malate contained four carbon atoms derived from [U-13 C5]glutamine (Figure S1F, M+3 versus M+4), indicating that most glutamine was used in the non-reductive direction towards succinate, fumarate and malate production. We were able to observe little contribution of glutamine flux into nucleotides or glutathione in control or UV-treated cells at these time points (data not shown), suggesting that the mitochondrial metabolism of glutamine accounts for the majority of glutamine consumption in these cells. Taken together, the metabolic flux analysis demonstrates that DNA damage results in a reduction of mitochondrial glutamine anaplerosis, thus limiting the critical refueling of carbons into the TCA cycle.

To assess the functional relevance of decreased glutamine metabolism after DNA damage, we deprived cells of glucose, thereby shifting cellular dependence to glutamine to maintain viability (Choo et al., 2011Dang, 2010). If DNA damage represses glutamine usage, we reasoned that cells would be more sensitive to glucose deprivation. Indeed, following 72 hr of glucose deprivation, cell death in primary MEFs was significantly elevated at 10 hr after UV exposure (Figure S1E). However, cells cultured with glucose remained viable in these conditions. Thus, these data demonstrate that genotoxic stress limits glutamine entry into the central mitochondrial metabolism of the TCA cycle.

SIRT4 is induced in response to genotoxic stress

Because sirtuins regulate both cellular metabolism and stress responses (Finkel et al., 2009Schwer and Verdin, 2008), we examined whether sirtuins were involved in the metabolic adaptation to DNA damage. We first examined the expression of sirtuins in the response to DNA damage. Specifically, we probed SIRT1, which is involved in stress responses (Haigis and Guarente, 2006), as well as mitochondrial sirtuins (SIRT3–5), which have been shown to regulate amino acid metabolism (Haigis et al., 2006Hallows et al., 2011Nakagawa et al., 2009). Remarkably, SIRT4 mRNA levels were induced by nearly 15-fold at 15 hr after CPT treatment and 5-fold after etoposide (ETS), a topoisomerase 2 inhibitor, in HEK293T cells (Figure 2A). Interestingly, the induction of SIRT4 was significantly higher than the induction of SIRT1 and mitochondrial SIRT3 (~2-fold), sirtuins known to be induced by DNA damage and regulate cellular responses to DNA damage (Sundaresan et al., 2008Vaziri et al., 2001Wang et al., 2006). Moreover, overall mitochondrial mass was increased by only 10% in comparison with control cells (Figure S2A), indicating that the induction of SIRT4 is not an indirect consequence of mitochondrial biogenesis. These data hint that SIRT4 may have an important, previously undetermined role in the DDR.

Figure 2 SIRT4 is induced by DNA damage stimuli

To test the induction of SIRT4 in the general genotoxic stress response, we treated cells with other types of DNA damage, including UV and gamma-irradiation (IR). SIRT4 mRNA levels were also increased by these genotoxic agents (Figures S2B and S2C) and low doses of CPT and UV treatment also induced SIRT4expression (Figures S2D and S2E). We observed similar results with MEFs (Figures 2B and 2DFigure S2F) and HepG2 cells (Figure S2G). DNA damaging agents elevated SIRT4 in p53-inactive HEK293T cells (Figures 2A and 2C) and in p53-null PC3 human prostate cancer cells (Figure S2H), suggesting that SIRT4can be induced in a p53-independent manner.

To examine whether the induction of SIRT4 occurred as a result of cell cycle arrest, we measured SIRT4levels after the treatment of nocodazole, which inhibits microtubule polymerization to block mitosis. While treatment with nocodazole completely inhibited cell proliferation (data not shown), SIRT4 expression was not elevated (Figure S2I). In addition, we analyzed SIRT4 expression in distinct stages of the cell cycle in HepG2 cells synchronized with thymidine block (Figure S2J, Left). SIRT4 mRNA levels were measured at different times after release and were not elevated during G1 or G2/M phases (Figure S2J, Right), suggesting thatSIRT4 is not induced as a general consequence of cell cycle arrest. Next, we re-examined the localization of SIRT4 after DNA damage. SIRT4 localizes to the mitochondria of human and mouse cells under basal, unstressed conditions (Ahuja et al., 2007Haigis et al., 2006). Following CPT treatment, SIRT4 colocalized with MitoTracker, a mitochondrial-selective marker, indicating that SIRT4 retains its mitochondrial localization after exposure to DNA damage (Figure S2K). Taken together, our findings demonstrate that SIRT4 is induced by multiple forms of DNA damage in numerous cell types, perhaps to coordinate the mitochondrial response to genotoxic stress.

SIRT4 represses glutamine anaplerosis

We observed that glutamine anaplerosis is repressed by genotoxic stress (Figure 1) and SIRT4 is induced by DNA damage (Figure 2). Additionally, previous studies reported that SIRT4 represses glutamine anaplerosis (Haigis et al., 2006). We next tested whether SIRT4 directly regulates cellular glutamine metabolism and contribution of glutamine to the TCA cycle. Like DNA damage, SIRT4 overexpression (SIRT4-OE) in HepG2, HeLa or HEK293T cells resulted in the repression of glutamine consumption (Figure 3AFigures S3A–C). Conversely, SIRT4 knockout (KO) MEFs consumed more glutamine than did wild-type (WT) cells (Figure 3B).

Figure 3 SIRT4 represses mitochondrial glutamine metabolism in response to DNA damage

Mitochondrial glutamine catabolism refuels the TCA cycle and is essential for viability in the absence of glucose (Choo et al., 2011Yang et al., 2009). Thus, we examined the effect of SIRT4 on cell survival during glucose deprivation. Overexpression of SIRT4 in HEK293T or HeLa cells increased cell death in glucose-free media compared to control cells (Figure 3CFigure S3D). Importantly, this cell death was completely rescued by the addition of pyruvate or cell permeable dimethyl α-ketoglutarate (DM-KG), demonstrating that SIRT4 overexpression reduced the ability of cells to utilize glutamine for mitochondrial energy production. Moreover, cell death was equally maximized in the absence of glucose and presence of the mitochondrial ATPase inhibitor oligomycin (Figure 3C). These findings are in line with the model that SIRT4 induction with DNA damage limits glutamine metabolism and utilization by the TCA cycle

We next utilized a metabolomic approach to interrogate glutamine usage in the absence of SIRT4. SIRT4 KO MEFs demonstrated elevated levels of TCA cycle intermediates (Figure 3J, WT versus KO), whereas intermediates of glycolysis were comparable with WT cells (data not shown). Nucleotides and other metabolites downstream of glutamine metabolism were not coordinately regulated by SIRT4 loss (Figure S3E and data not shown). Next, we analyzed glutamine flux in WT and SIRT4 KO MEFs in medium containing [U-13C5]glutamine for 2 or 4 hours and measured isotopic enrichment of TCA cycle intermediates. Loss of SIRT4 promoted a higher rate of incorporation of 13C-labeled metabolites derived from [U-13C5]glutamine in all TCA cycle intermediates measured (Figure 3D). These data provide direct evidence that SIRT4 loss drives increased entry of glutamine-derived carbon into the TCA cycle.

Next, we examined the mechanisms involved in this repression of glutamine anaplerosis. GLS is the first required enzyme for mitochondrial glutamine metabolism (Curthoys and Watford, 1995) and its inhibition limits glutamine flux into the TCA cycle (Wang et al., 2010; Le et al., 2012; Yuneva et al., 2012). Treatment with bis-2-(5-phenylacetoamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) (Robinson et al., 2007), an inhibitor of GLS1, repressed glutamine uptake and completely rescued the increased glutamine consumption of SIRT4 KO cells (Figure 3E). Moreover, SIRT4 overexpression no longer inhibited glutamine uptake when GLS1 was reduced by using short hairpin RNAs (shRNAs) (Figures 3F and 3G), demonstrating that SIRT4 regulates mitochondrial glutamine metabolism. SIRT4 is a negative regulator of GDH activity (Haigis et al., 2006) and SIRT4 KO MEFs exhibited increased GDH activity in comparison with WT MEFs (Figure S3F). To test whether SIRT4 regulates mitochondrial glutamine metabolism via inhibiting GDH activity, we measured glutamine uptake in WT and SIRT4 KO cells in the presence of EGCG, a GDH inhibitor (Choo et al., 2011Li et al., 2006). The treatment of EGCG partially rescued the increased glutamine uptake of KO cells (Figure S3G), suggesting that GDH contributes to the role of SIRT4 in glutamine metabolism.

SIRT4 represses mitochondrial glutamine metabolism after DNA damage

SIRT4 regulates cell cycle progression and genomic fidelity in response to DNA damage

Figure 4 SIRT4 is involved in cellular DNA damage responses

SIRT4 represses tumor proliferation

Figure 5 SIRT4 has tumor suppressive function

(A and B) Growth curves of WT and SIRT4 KO MEFs (n = 3) cultured in standard media (A) or media supplemented with BPTES (10 μM) (B). Data are means ±SD.

(C and D) Growth curves of Vector and SIRT4-OE HeLa cells (n = 3) cultured in standard media (C) or media supplemented with BPTES (10 μM) (D). Data are means ±SD.

(E) Focus formation assays with transformed WT and SIRT4 KO MEFs (left). Cells were cultured with normal medium or medium without glucose or glutamine for 10 days and stained with crystal violet. The number of colonies was counted (right) (n =3 samples of each condition). n.d., not determined.

(F) Focus formation assays with transformed KO MEFs reconstituted with SIRT4 or a catalytic mutant of SIRT4 (n = 3). Cells were cultured for 8 days and stained with crystal violet.

(G) Contact inhibited cell growth of transformed WT and SIRT4 KO MEFs cultured in the presence of DMSO or BPTES (10 μM) for 14 days (left). The number of colonies was counted (right). Data are means ±SEM. n.s., not significant. *p < 0.05, **p < 0.005. See also Figure S5.

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SIRT4 represses tumor formation in vivo

To investigate SIRT4 function in human cancers, we examined changes in SIRT4 expression. SIRT4 mRNA level was reduced in several human cancers, such as small cell lung carcinoma (Garber et al., 2001), gastric cancer (Wang et al., 2012), bladder carcinoma (Blaveri et al., 2005), breast cancer (TCGA) and leukemia (Choi et al., 2007) (Figure 6A). Of note, lower SIRT4 expression associated with shorter time to death in lung tumor patients (Shedden et al., 2008) (Figure 6B). Overall the expression data is consistent with the model that SIRT4 may play a tumor suppressive role in human cancers.

Figure 6 SIRT4 is a mitochondrial tumor suppressor

SIRT4 regulates glutamine metabolism in lung tissue

To test further the biological relevance of this pathway in lung, we examined whether SIRT4 is induced in vivo after exposure to DNA damaging IR treatment. Remarkably, Sirt4 was significantly induced in lung tissue after IR exposure (Figure 7A). We next examined whether IR repressed glutamine metabolism in vivo, as observed in cell culture by examining GDH activity in lung tissue from WT and SIRT4 KO mice with or without IR exposure. GDH activity was elevated in lung tissue extracts from SIRT4 KO mice compared with WT lung tissue (Figure 7B). Importantly, GDH activity was significantly decreased in lung tissue from WT mice after IR exposure, whereas not in lung tissue from KO mice (Figure 7C). Thus, these findings recapitulate our cellular studies and are in line with the model that SIRT4 induction with DNA damage limits mitochondrial glutamine metabolism and utilization.

SIRT4 inhibits mitochondria glutamine metabolism in vivo

SIRT4 inhibits mitochondria glutamine metabolism in vivo

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Figure 7 SIRT4 inhibits mitochondria glutamine metabolism in vivo

To assess whether the functions of SIRT4 can be reproduced in these lung tumors, cells derived from SIRT4 KO lung tumors were reconstituted with wild type SIRT4 (Figure S7A). As expected, SIRT4 reconstitution reduced glutamine uptake, but not glucose uptake (Figures 7D and 7E) and repressed proliferation (Figure S7B) of lung tumor cells.

Here, we report that SIRT4 has an important role in cellular metabolic response to DNA damage by regulating mitochondrial glutamine metabolism with important implication for the DDR and tumorigenesis. First, we discovered that DNA damage represses cellular glutamine metabolism (Figure 1). Next, we found that SIRT4 is induced by genotoxic stress (Figure 2) and is required for the repression of mitochondrial glutamine metabolism (Figure 3). This metabolic response contributes to the control of cell cycle progression and the maintenance of genomic integrity in response to DNA damage (Figure 4). Loss of SIRT4 increased glutamine-dependent tumor cell proliferation and tumorigenesis (Figure 5). In mice, SIRT4 loss resulted in spontaneous tumor development (Figure 6). We demonstrate that SIRT4 is induced in normal lung tissue in response to DNA damage where it represses GDH activity. Finally, the glutamine metabolism-genomic fidelity axis is recapitulated in lung tumor cells derived from SIRT4 KO mice via SIRT4 reconstitution (Figure 7). Our studies therefore uncover SIRT4 as a important regulator of cellular metabolic response to DNA damage that coordinates repression of glutamine metabolism, genomic stability and tumor suppression.

The DDR is a highly orchestrated and well-studied signaling response that detects and repairs DNA damage. Upon sensing DNA damage, the ATM/ATR protein kinases are activated to phosphorylate target proteins, leading to cell cycle arrest, DNA repair, transcriptional regulation and initiation of apoptosis (Ciccia and Elledge, 2010Su, 2006). Dysregulation of this pathway is frequently observed in many tumors. Emerging evidence has suggested that cell metabolism also plays key roles downstream of the DDR-induced pathways.

 

7.8.9 Mitochondrial sirtuins and metabolic homeostasis

Pirinen E1Lo Sasso GAuwerx J.
Best Pract Res Clin Endocrinol Metab. 2012 Dec; 26(6):759-70. http://dx.doi.org:/10.1016/j.beem.2012.05.001

The maintenance of metabolic homeostasis requires the well-orchestrated network of several pathways of glucose, lipid and amino acid metabolism. Mitochondria integrate these pathways and serve not only as the prime site of cellular energy harvesting but also as the producer of many key metabolic intermediates. The sirtuins are a family of NAD+-dependent enzymes, which have a crucial role in the cellular adaptation to metabolic stress. The mitochondrial sirtuins SIRT3, SIRT4 and SIRT5 together with the nuclear SIRT1 regulate several aspects of mitochondrial physiology by controlling posttranslational modifications of mitochondrial protein and transcription of mitochondrial genes. Here we discuss current knowledge how mitochondrial sirtuins and SIRT1 govern mitochondrial processes involved in different metabolic pathways.

Mitochondria are organelles composed of a matrix enclosed by a double (inner and outer) membrane (1). Major cellular functions, such as nutrient oxidation, nitrogen metabolism, and especially ATP production, take place in the mitochondria. ATP production occurs in a process referred to as oxidative phosphorylation (OXPHOS), which involves electron transport through a chain of protein complexes (I-IV), located in the inner mitochondrial membrane. These complexes carry electrons from electron donors (e.g. NADH) to electron acceptors (e.g. oxygen), generating a chemiosmotic gradient between the mitochondrial intermembrane space and matrix. The energy stored in this gradient is then used by ATP synthase to produce ATP (1). One well-known side effect of the OXPHOS process is the production of reactive oxygen species (ROS) that can generate oxidative damage in biological macromolecules (1). However, to neutralize the harmful effects of ROS, cells have several antioxidant enzymes, including superoxide dismutase, catalase, and peroxidases (1). The sirtuin silent information regulator 2 (Sir2), the founding member of the sirtuin protein family, was identified in 1984 (2). Sir2 was subsequently characterized as important in yeast replicative aging (3) and shown to posses NAD+-dependent histone deacetylase activity (4), suggesting it could play a role as an energy sensor. A family of conserved Sir2-related proteins was subsequently identified. Given their involvement in basic cellular processes and their potential contribution to the pathogenesis of several diseases (5), the sirtuins became a widely studied protein family.

In mammals the sirtuin family consists of seven proteins (SIRT1-SIRT7), which show different functions, structure, and localization. SIRT1 is mostly localized in the nucleus but, under specific physiological conditions, it shuttles to the cytosol (6). Similar to SIRT1, also SIRT6 (7) and SIRT7 (8) are localized in the nucleus. On the contrary, SIRT2 is mainly present in the cytosol and shuttles into the nucleus during G2/M cell cycle transition (9). Finally, SIRT3, SIRT4, and SIRT5, are mitochondrial proteins (10).

The main enzymatic activity catalyzed by the sirtuins is NAD+-dependent deacetylation, as known for the progenitor Sir2 (4,11). Along with histones also many transcription factors and enzymes were identified as targets for deacetylation by the sirtuins. Remarkably, mammalian sirtuins show additional interesting enzymatic activities. SIRT4 has an important ADP-ribosyltransferase activity (12), while SIRT6 can both deacetylate and ADP-ribosylate proteins (13,14). Moreover, SIRT5 was recently shown to demalonylate and desuccinylate proteins (15,16), in particular the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) (16). The (patho-)physiological context in which the seven mammalian sirtuins exert their functions, as well as their biochemical characteristics, are extensively discussed in the literature (17,18) and will not be addressed in this review; here we will focus on the emerging roles of the mitochondrial sirtuins, and their involvement in metabolism. Moreover, SIRT1 will be discussed as an important enzyme that indirectly affects mitochondrial physiology.

Sirtuins are regulated at different levels. Their subcellular localization, but also transcriptional regulation, post-translational modifications, and substrate availability, all impact on sirtuin activity. Moreover, nutrients and other molecules could affect directly or indirectly sirtuin activity. As sirtuins are NAD+-dependent enzymes, the availability of NAD+ is perhaps one of the most important mechanisms to regulate their activity. Changes in NAD+ levels occur as the result of modification in both its synthesis or consumption (19). Increase in NAD+ amounts during metabolic stress, as prolonged fasting or caloric restriction (CR) (2022), is well documented and tightly connected with sirtuin activation (4,19). Furthermore, the depletion and or inhibition of poly-ADP-ribose polymerase (PARP) 1 (23) or cADP-ribose synthase 38 (24), two NAD+consuming enzymes, increase SIRT1 action.

Analysis of the SIRT1 promoter region identified several transcription factors involved in up- or down-regulation of SIRT1 expression. FOXO1 (25), peroxisome proliferator-activated receptors (PPAR) α/β (26,27), and cAMP response element-binding (28) induce SIRT1 transcription, while PPARγ (29), hypermethylated in cancer 1 (30), PARP2 (31), and carbohydrate response element-binding protein (28) repress SIRT1 transcription. Of note, SIRT1 is also under the negative control of miRNAs, like miR34a (32) and miR199a (33). Furthermore, the SIRT1 protein contains several phosphorylation sites that are targeted by several kinases (34,35), which may tag the SIRT1 protein so that it only exerts activity towards specific targets (36,37). The beneficial effects driven by the SIRT1 activation – discussed below- led the development of small molecules modulators of SIRT1. Of note, resveratrol, a natural plant polyphenol, was shown to increase SIRT1 activity (38), most likely indirectly (22,39,40), inducing lifespan in a range of species ranging from yeast (38) to high-fat diet fed mice (41). The beneficial effect of SIRT1 activation by resveratrol on lifespan, may involve enhanced mitochondrial function and metabolic control documented both in mice (42) and humans (43). Subsequently, several powerful synthetic SIRT1 agonists have been identified (e.g. SRT1720 (44)), which, analogously to resveratrol, improve mitochondrial function and metabolic diseases (45). The precise mechanism of action of these compounds is still under debate; in fact, it may well be that part of their action is mediated by AMP-activated protein kinase (AMPK) activation (21,22,46), as resveratrol was shown to inhibit ATP synthesis by directly inhibiting ATP synthase in the mitochondrial respiratory chain (47), leading to an energy stress with subsequent activation of AMPK. However, at least in β-cells, resveratrol-mediated SIRT1 activation and AMPK activation seem to regulate glucose response in the opposite direction, pointing to the existence of alternative molecular targets (48).

Another hypothesis to explain the pleitropic effects of resveratrol suggests it inhibits cAMP-degrading phosphodiesterase 4 (PDE4), resulting in the cAMP-dependent activation of exchange proteins activated by cyclic AMP (Epac1) (40). The consequent Epac1-mediated increase of intracellular Ca2+ levels may then activate of CamKKβ-AMPK pathway (40), which ultimately will result in an increase in NAD+ levels and SIRT1 activation (21). Interestingly, also PDE4 inhibitors reproduce some of the metabolic benefits of resveratrol representing yet another putative way to activate SIRT1.

The regulation of the activity of the mitochondrial sirtuins is at present poorly understood. SIRT3 expression is induced in white adipose (WAT) and brown adipose tissues upon CR (49), while it is down-regulated in the liver of high-fat fed mice (50). SIRT3 activity changes also in the muscle after fasting (51) and chronic contraction (52). All these processes are associated with increase (20,53) or decrease (50) in NAD+ levels. From a transcriptional point of view, SIRT3 gene expression in brown adipocytes seems under the control of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) -estrogen-related receptor α (ERRα) axis, and this effect is crucial for full brown adipocyte differentiation (54,55). SIRT4 expression is reported to be reduced during CR (12), while the impact of resveratrol on SIRT4 is still under debate (56). Finally, upon ethanol exposure, SIRT5 gene expression was shown to be decreased together with the NAD+levels (57), probably explaining the protein hyperacetylation caused by alcohol exposure (58).

Metabolic homeostasis

The maintenance of metabolic homeostasis is critical for the survival of all species to sustain body structure and function. Metabolic homeostasis is achieved through complicated interactions between metabolic pathways that govern glucose, lipid and amino acid metabolism. Mitochondria are organelles, which integrate these metabolic pathways by serving a physical site for the production and recycling of metabolic intermediates.

Glucose metabolism

Overview

Glucose homeostasis is regulated through various complex processes including hepatic glucose output, glucose uptake, glucose utilization and storage. The main hormones regulating glucose homeostasis are insulin and glucagon, and the balance between these hormones determines glucose homeostasis. Insulin promotes glucose uptake in peripheral tissues (muscle and WAT), glycolysis and storage of glucose as glycogen in the fed state, while glucagon stimulates hepatic glucose production during fasting. Sirtuins influence many aspects of glucose homeostasis in several tissues such as muscle, WAT, liver and pancreas.

Gluconeogenesis

The body’s ability to synthesise glucose is vital in order to provide an uninterrupted supply of glucose to the brain and survive during starvation. Gluconeogenesis is a cytosolic process, in which glucose is formed from non-carbohydrate sources, such as amino acids, lactate, the glycerol portion of fats and tricarboxylic acid (59) cycle intermediates, during energy demand. This process, which occurs mainly in liver and kidney, shares some enzymes with glycolysis but it employs phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase to control the flow of metabolites towards glucose production. These three enzymes are stimulated by glucagon, epinephrine and glucocorticoids, whereas their activity is suppressed by insulin.

The role of mitochondrial sirtuins in the control of gluconeogenesis is not well established. SIRT3 is suggested to induce fasting-dependent hepatic glucose production from amino acids by deacetylating and activating the mitochondrial conversion of glutamate into the TCA cycle intermediate α-ketoglutarate, via the enzyme glutamate dehydrogenase (GDH) (Fig. 1A) (60,61). As SIRT3−/− mice do not display changes in GDH activity (62), the mechanism requires further clarification. In contrast to SIRT3, SIRT4 inhibits GDH via ADP-ribosylation under basal dietary conditions (Fig. 1A-B) (12). Conversely, SIRT4 activity is suppressed during CR resulting in activation of GDH, which fuels the TCA cycle and possibly also gluconeogenesis (12). Therefore, mitochondrial sirtuins may function to support gluconeogenesis during energy limitation, but further research is required to understand the exact roles of mitochondrial sirtuins in gluconeogenesis.

Summary of mitochondrial sirtuins’ role in mitochondrial pathways

Summary of mitochondrial sirtuins’ role in mitochondrial pathways

Figure 1 Summary of mitochondrial sirtuins’ role in mitochondrial pathways

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Glucose utilization

 Lipid metabolism

Urea metabolism

The recent discoveries in the biology of mitochondria have shed light on the metabolic regulatory roles of the sirtuin family. To maintain proper metabolic homeostasis, sirtuins sense cellular NAD+ levels, which reflect the nutritional status of the cells, and translate this information to adapt the activity of mitochondrial processes via posttranslational modifications and transcriptional regulation. SIRT1 and SIRT3 function to stimulate proper energy production via FAO and SIRT3 also protects from oxidative stress and ammonia accumulation during nutrient deprivation. SIRT4 seems to play role in the regulation of gluconeogenesis, insulin secretion and fatty acid utilization during times of energy limitation, while SIRT5 detoxifies excess ammonia that can accumulate during fasting. However, we are only at the beginning of our understanding of the roles of the mitochondrial sirtuins, SIRT3, SIRT4 and SIRT5 in complex metabolic processes. In the coming years, further research should identify and verify novel sirtuin targets in vivo and in vitro. We need also to elucidate the regulation and tissue-specific functions of these mitochondrial sirtuins, as well as to understand the potential crosstalk and synchrony between the different sirtuins in different subcellular compartments. Ultimately, the understanding of mitochondrial sirtuin functions may open new possibilities, not only for treatment of cancer and metabolic diseases characterized by mitochondrial dysfunction, but also for disease prevention and health maintenance.

7.8.10 Mitochondrial sirtuins

Huang JY1Hirschey MDShimazu THo LVerdin E.
Biochim Biophys Acta. 2010 Aug; 1804(8):1645-51. http://dx.doi.org:/10.1016/j.bbapap.2009.12.021

Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2′-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD(+) onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation. Here, we review and discuss recent progress in the study of mitochondrial sirtuins and their targets.

mitochondrial sirtuins

mitochondrial sirtuins

http://www.sciencedirect.com/science/article/pii/S1570963909003902

mitochondrial sirtuins
Fig.1 .NAD+ -dependent deacetylation of sirtuins. The two step catalytic reaction mechanism. In this diagram ADPR = acetyl-ADP-ribose, NAM = nicotinamide, 1-O-AADPR = 1-O-acetyl ADP-ribose and βNAD = beta nicotinamide adenine dinucleotide.

Table 1 Shows subcellular localization, substrates and functions of different types of sirtuins.

Fig.2. Sirt3 regulated pathways in mitochondrial metabolism. Schematic diagram demonstrating the different roles of Sirt3 in the regulation of the main metabolic pathways of mitochondria.In this diagram LCAD = long-chain acyl-CoA dehydrogenase, ACeS2 = acetyl coenzyme synthetase 2, Mn SOD = manganese superoxide dismutase, CypD = cyclophilin D, ICDH2 = isocitrate dehydrogenase 2, OTC = ornithine transcarbomylase,TCA = tricaboxylic acid, ROS = reactive oxygen species, mPTP = membrane permeability transition pore, I–V = respiratory chain complex I–V

Fig. 3.(A) Schematic diagram showing different roles of Sirt4 in the regulation of various metabolic pathways. The diagram shows the Sirt4 regulated decrease in insulin level and the increase in availability of ATP inside mitochondria via upregulation of insulin degrading enzyme (IDE) and adenine translocator (ANT). The diagram also shows the Sirt4 regulated decrease in the efficiency of fatty acid oxidation and tricarboxylic acid cycle (TCA) via inhibition of glutamate dehydrogenase (GDH) and malonyl CoA decarboxylase (MCoAD). (B) Schematic diagram indicating the different roles of Sirt5 in regulation of various metabolic pathways. Sirt5 regulates urea production, fatty acid oxidation, tricarboxylic acid cycle (TCA), glycolysis, reactive oxygen species (ROS) metabolism, purine metabolism via regulating carbamoyl phosphate synthetase (CPS), hydroxyl-coenzyme A dehydrogenase (HADH), pyruvate dehydrogenase (PDH), pyruvate kinase (PK), succinate dehydrogenase(SDH) andurate oxidase (UO) respectively

Conclusion and future perspectives

Sirtuins are highly conserved NAD+-dependent protein deacetylases or ADP ribosyl transferases involved in many cellular processes including genome stability, cell survival, oxidative stress responses, metabolism, and aging. Mitochondrial sirtuins, Sirt3, Sirt4 and Sirt5 are important energy sensors and thus can be regarded as master regulators of mitochondrial metabolism. But it is still not known whether specific sirtuins can only function within particular metabolic pathways or two or more sirtuins could affect the same pathways. One of the mitochondrial sirtuins, Sirt3 is a major mitochondrial deacetylase that plays a pivotal role in the acetylation based regulation of numerous mitochondrial proteins. However, the question how mitochondrial proteins become acetylated is still unsolved and the identity of mitochondrial acetyltransferases is mysterious. Although the predominant function of the sirtuins is NAD+ dependent lysine deacetylation, but along with this major function another less characterized activity of these sirtuins includes ADP ribosylation which is mainly done by Sirt4. Moreover, in the case when the mitochondrial sirtuins exhibit both deacetylase and ADP ribosyl transferase activity, the conditions that determine the relative contribution of both of these activities in same or different metabolic pathways require further investigation. Sirt5 another mitochondrial sirtuin, was a puzzle until the recent finding as it possesses unique demalonylase and desuccinylase activities. However, most of the malonylated or succinylated proteins are important metabolic enzymes but as the significance of lysine malonylation and succinylation is still unknown thus it would be interesting to know how lysine malonylation and succinylation alter the functions of various metabolic enzymes. The mitochondrial sirtuins Sirt3, Sirt4 and Sirt5 serve as critical junctions and are required to exert many of the beneficial effect in mitochondrial metabolism. The emerging multidimensional role of mitochondrial sirtuins in regulation of mitochondrial metabolism and bioenergetics may have far-reaching consequences for many diseases associated with mitochondrial dysfunctions. However it is very important to fully elucidate the functions of mitochondrial sirtuins in different tissues to achieve the goal of therapeutic intervention in different metabolic diseases. Although several proteomic studies have provided detailed information that how mitochondrial sirtuin driven modification takes place on various targets in response to different environmental conditions, still the role of sirtuins in mitochondrial physiology and human diseases requires further exploration. Hopefully the progress in the field of sirtuin biology will soon provide insight into the therapeutic applications for targeting mitochondrial sirtuins by bioactive compounds to treat various human age-related diseases.

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7.8.11 Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling

Verdin E1Hirschey MDFinley LWHaigis MC.
Trends Biochem Sci. 2010 Dec; 35(12):669-75.
http://dx.doi.org:/10.1016/j.tibs.2010.07.003

Sirtuins are a highly conserved family of proteins whose activity can prolong the lifespan of model organisms such as yeast, worms and flies. Mammals contain seven sirtuins (SIRT1-7) that modulate distinct metabolic and stress response pathways. Three sirtuins, SIRT3, SIRT4 and SIRT5, are located in the mitochondria, dynamic organelles that function as the primary site of oxidative metabolism and play crucial roles in apoptosis and intracellular signaling. Recent findings have shed light on how the mitochondrial sirtuins function in the control of basic mitochondrial biology, including energy production, metabolism, apoptosis and intracellular signaling.

Mitochondria play critical roles in energy production, metabolism, apoptosis, and intracellular signaling [13]. These highly dynamic organelles have the ability to change their function, morphology and number in response to physiological conditions and stressors such as diet, exercise, temperature, and hormones [4]. Proper mitochondrial function is crucial for maintenance of metabolic homeostasis and activation of appropriate stress responses. Not surprisingly, changes in mitochondrial number and activity are implicated in aging and age-related diseases, including diabetes, neurodegenerative diseases, and cancer [1]. Despite the important link between mitochondrial dysfunction and human diseases, in most cases, the molecular causes for dysfunction have not been identified and remain poorly understood.

One of the principal bioenergetic functions of mitochondria is to generate ATP through the process of oxidative phosphorylation (OXPHOS), which occurs in the inner-mitochondrial membrane. Mitochondria are unique bi-membrane organelles that contain their own circular genome (mtDNA) encoding 13 protein subunits involved in electron transport. The remainder of the estimated 1000-1500 mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria from the cytoplasm [56]. These imported proteins can be found either in the matrix, associated with inner or outer mitochondrial membranes or in the inner membrane space (Figure 1). Dozens of nuclear-encoded protein subunits form complexes with the mtDNA-encoded subunits to form electron transport complexes I-IV and ATP synthase, again highlighting the need for precise coordination between these two genomes. The transcriptional coactivator PGC-1α, a master regulator of mitochondrial biogenesis and function, is responsive to a variety of metabolic stresses, ensuring that the number and capacity of mitochondria keeps pace with the energetic demands of tissues [7].

Network of mitochondrial sirtuins

Network of mitochondrial sirtuins

http://www.ncbi.nlm.nih.gov/pmc/articles/instance/2992946/bin/nihms239607f1.gif

Network of mitochondrial sirtuins. Mitochondria can metabolize fuels, such as fatty acids, amino acids, and pyruvate, derived from glucose. Electrons pass through electron transport complexes (I-IV; red) generating a proton gradient, which is used to drive ATP synthase (AS; red) to generate ATP. SIRT3 (gold) binds complexes I and II, regulating cellular energy levels in the cell [4355]. Moreover, SIRT3 binds and deacetylates acetyl-CoA synthetase 2 (AceCS2) [3940] and glutamate dehydrogenase (GDH) [3347], thereby activating their enzymatic activities. SIRT3 also binds and activates long-chain acyl-CoA dehydrogenase (LCAD) [46]. SIRT4 (light purple) binds and represses GDH activity via ADP-ribosylation [21]. In the rate-limiting step of the urea cycle, SIRT5 (light blue) deacetylates and activates carbamoyl phosphate synthetase 1 (CPS1) [4849].

As high-energy electrons derived from glucose, amino acids or fatty acids fuels are passed through a series of protein complexes (I-IV), their energy is used to pump protons from the mitochondrial matrix through the inner membrane into the inner-membrane space, generating a proton gradient known as the mitochondrial membrane potential (Dψm) (Figure 1). Ultimately, the electrons reduce oxygen to form water, and the protons flow down their gradient through ATP synthase, driving the formation of ATP from ADP. Protons can also flow through uncoupling proteins (UCPs), dissipating their potential energy as heat. Reactive oxygen species (ROS) are a normal side-product of the respiration process [18]. In addition, an increase in Dψm, whether caused by impaired OXPHOS or by an overabundance of nutrients relative to ADP, will result in aberrant electron migration in the electron transport chain and elevated ROS production [1]. ROS react with lipids, protein and DNA, generating oxidative damage. Consequently, cells have evolved robust mechanisms to guard against an increase in oxidative stress accompanying ROS production [9].

Mitochondria are the primary site of ROS production within the cell, and increased oxidative stress is proposed to be one of the causes of mammalian aging [1210]. Major mitochondrial age-related changes are observed in multiple tissues and include decreased Dψm, increased ROS production and an increase in oxidative damage to mtDNA, proteins, and lipids [1114]. As a result, mitochondrial bioenergetic changes that occur with aging have been extensively reviewed [1517].

Silent information regulator (SIR) 2 protein and its orthologs in other species, termed sirtuins, promote an increased lifespan in model organisms such as yeast, worms and flies. Mammals contain seven sirtuins (SIRT1–7) that are characterized by an evolutionary conserved sirtuin core domain [1819]. This domain contains the catalytic activity and invariant amino acid residues involved in binding NAD+, a metabolic co-substrate. All sirtuins exhibit two major enzymatic activities in vitro: NAD+-dependent protein deacetylase activity and ADP-ribosyltransferase activity. Except for SIRT4, well-defined acetylated substrates have been identified for the other sirtuins. So far, only ADP-ribosyltransferase activity has been described for SIRT4 [2021]. Thus, these enzymes couple their biochemical and biological functions to an organism’s energetic state via their dependency on NAD+. A decade of research, largely focused on SIRT1, has revealed that mammalian sirtuins regulate metabolism and cellular survival. In brief, SIRT1–7 target distinct acetylated protein substrates and are localized in distinct subcellular compartments. SIRT1, SIRT6 and SIRT7 are found in nucleus, SIRT2 is primarily cytosolic and SIRT3, 4 and 5 are found in the mitochondria. The mitochondrial-only localization of SIRT3 is controversial and other groups have reported non-mitochondrial localization of this sirtuin [2223]. The biology and biochemistry of the seven mammalian sirtuins have been extensively discussed in the literature [2426] and is not the topic of this review. Instead, we focus on the mitochondrial sirtuins, their substrates, and their impact on mitochondrial biology.

The mitochondrial sirtuins, SIRT3–5 [212729], participate in the regulation of ATP production, metabolism, apoptosis and cell signaling. Unlike SIRT1, a 100 kDa protein, the mitochondrial sirtuins are small, ranging from 30–40 kDa. Thus, their amino acid sequence consists mostly of an N-terminal mitochondrial targeting sequence and the sirtuin core domain, with small flanking regions. Whereas, SIRT3 and SIRT5 function as NAD+-dependent deacetylases on well defined substrates, SIRT4 has no identified acetylated substrate and only shows ADP-ribosyltransferase activity. It is likely, however, that SIRT4 possesses substrate-specific NAD+-dependent deacetylase activity, as has been demonstrated for SIRT6 [30,31]. The three-dimensional structures for the core domains of human SIRT3 and human SIRT5 have been solved and reveal remarkable structural conservation with other sirtuins, such as the ancestral yeast protein and human SIRT2 (Figure 2) [3234]. Given its sequence conservation with the other sirtuins [18], it is likely that SIRT4 adopts a similar three-dimensional conformation.

Figure 2 Structure and alignment of sirtuins

Role of mitochondrial sirtuins in metabolism and energy production

The NAD+ dependence of sirtuins provided the first clue that these enzymes function as metabolic sensors. For instance, sirtuin activity can increase when NAD+ levels are abundant, such as times of nutrient deprivation. In line with this model, mass spectrometry studies have revealed that metabolic proteins, such as tricarboxylic acid (TCA) cycle enzymes, fatty acid oxidation enzymes and subunits of oxidative phosphorylation complexes are acetylated in response to metabolic stress [3537].

Fatty acid oxidation

Consistent with the hypothesis that nutrient stress alters sirtuin activity, a recent report identified significant metabolic abnormalities in Sirt3-/- mice during fasting [38]. In this study, hepatic SIRT3 protein expression increased during fasting, suggesting that both its levels and enzymatic activity are elevated during nutrient deprivation. SIRT3 activates hepatic lipid catabolism via deacetylation of long-chain acyl-CoA dehydrogenase (LCAD), a central enzyme in the fatty acid oxidation pathway. Sirt3-/- mice have diminished fatty acid oxidation, develop fatty liver, have low ATP production, and show a defect in thermogenesis and hypoglycemia during a cold test [38].

Surprisingly, many of the phenotypes observed in Sirt3-/- mice were also observed in mice lacking acetyl-CoA synthetase 2 (AceCS2), a previously identified substrate of SIRT3 [3940]. For example, fasting ATP levels were reduced by 50% in skeletal muscle of AceCS2-/- mice, in comparison to wild type (WT) mice. As a result, fasted AceCS2-/- mice were hypothermic and had reduced capacity for exercise. By converting acetate into acetyl CoA, AceCS2 provides an alternate energy source during times of metabolic challenges, such as thermogenesis or fasting. Interestingly, Acadl-deficient mice (Acadl encodes LCAD) also show cold intolerance, reduced ATP, and hypoglycemia under fasting conditions [41]. These overlapping phenotypes between Sirt3-/-AceCS2-/- and Acadl-/- mice indicate that the regulation of LCAD and AceCS2 acetylation by SIRT3 represents an important adaptive signal during the fasting response (Figure 2).

Electron transport chain

Of all mitochondrial proteins, oxidative phosphorylation complexes are among the most heavily acetylated. One study reported that 511 lysine residues in complexes I-IV and ATP synthase are modified by acetylation [37], hinting that a mitochondrial sirtuin might deacetylate these residues. Indeed, SIRT3 interacts with and deacetylates complex I subunits (including NDUFA9) [42], succinate dehydrogenase (complex II) [43]. SIRT3 has also been shown to bind ATP synthase in a proteomic analysis [44]. SIRT3 also regulates mitochondrial translation, a process which can impact electron transport [45]. Mice lacking SIRT3 demonstrate reduced ATP levels in many tissues [42 46]; however, additional work is required to determine if reduced ATP levels in Sirt3-/- mice is a direct result of OX PHOS hyperacetylation or an indirect effect, via decreased fatty acid oxidation, or a combination of both effects.

Less is known about the roles of SIRT4 and SIRT5 in electron transport. SIRT4 binds adenine nucleotide translocator (ANT), which transports ATP into the cytosol and ADP into the mitochondrial matrix, thereby providing a substrate for ATP synthase [20]. SIRT5 physically interacts with cytochrome C. The biological significance of these interactions, however, remains unknown [21].

TCA cycle

Enzymes for the TCA cycle (also called the Kreb’s cycle) are located in the mitochondrial matrix; this compartmentalization provides a way for cells to utilize metabolites from carbohydrates, fats and proteins. Numerous TCA cycle enzymes are modified by acetylation, although the functional consequences of acetylation have been examined for only a few of these proteins. SIRT3 interacts with several TCA cycle enzymes, including succinate dehydrogenase (SDH, see above [43]) and isocitrate dehydrogenase 2 (ICDH2) [33]. ICDH2 catalyzes the irreversible oxidative decarboxylation of isocitrate to form alpha-ketoglutarate and CO2, while converting NAD+ to NADH. Although the biological significance of these interactions is not yet known, it seems possible that SIRT3 might regulate flux through the TCA cycle.

Role of mitochondrial sirtuins in signaling

During cellular stress or damage, mitochondria release a variety of signals to the cytosol and the nucleus to alert the cell of changes in mitochondrial function. In response, the nucleus generates transcriptional changes to activate a stress response or repair the damage. For example, mitochondrial biogenesis requires a sophisticated transcriptional program capable of responding to the energetic demands of the cell by coordinating expression of both nuclear and mitochondrial encoded genes [4]. Unlike anterograde transcriptional control of mitochondria from nuclear transcription regulators such as PGC-1α, the retrograde signaling pathway, from the mitochondria to the nucleus is poorly understood in mammals. Although there is no evidence directly linking sirtuins to a mammalian retrograde signaling pathway, changes in mitochondrial sirtuin activity could influence signals transmitted from the mitochondria. Interestingly, the nuclear sirtuin SIRT1 deacetylates and activates PGC-1α, a key factor in the transcriptional regulation of genes involved in fatty acid oxidation and oxidative phosphorylation (Figure 3) [5051]. Thus, mitochondrial and nuclear sirtuins might exist in a signaling communication loop to control metabolism.

mitochondria-at-nexus-of-cellular-signaling-nihms239607f3

mitochondria-at-nexus-of-cellular-signaling-nihms239607f3

http://www.ncbi.nlm.nih.gov/pmc/articles/instance/2992946/bin/nihms239607f3.gif

Mitochondria at nexus of cellular signaling. Mitochondria and mitochondrial sirtuins play a central role in intra- and extra-cellular signaling. Circulating fatty acids and acetate provide whole body energy homeostasis. The mitochondrial metabolites NAD+, NADH, ATP, Ca2+, ROS, ketone bodies, and acetyl-CoA participate in intracellular signaling.

Numerous signaling pathways are activated by changes in mitochondrial release of metabolites and molecules, such as Ca2+, ATP, NAD+, NADH, nitric oxide (NO), and ROS (Figure 3). Of these, Ca2+ is the best studied as a mitochondrial messenger. Mitochondria are important regulators of Ca2+ storage and homeostasis, and mitochondrial Ca2+ uptake is directly tied to the membrane potential of the organelle. Membrane potential serves as a gauge of mitochondrial function: disruption of OXPHOS, interruption in the supply or catabolism of nutrients or loss of structural integrity generally result in a fall in membrane potential, and, in turn, decreased mitochondrial Ca2+ uptake. Subsequent increases in cytosolic free Ca2+ will activate calcineurin and several Ca2+-dependent kinases [52] and affect a wide variety of transcription factors to produce appropriate cell-specific transcriptional responses [53]. Through regulation of nutrient oxidation and electron transport or yet to be identified target(s), mitochondrial sirtuins could influence mAlthough the effect of sirtuins on intracellular calcium signaling has not been studied directly, sirtuin effects on ATP production have been shown. ANT facilitates the exchange of mitochondrial ATP with cytosolic ADP. As a result the cytosolic ATP:ADP ratio reflects changes in mitochondrial energy production. A fall in ATP production activates AMP-activated protein kinase (AMPK), which directly stimulates mitochondrial energy production, inhibits protein synthesis through regulation of mammalian target of rapamycin (mTOR), and influences mitochondrial transcriptional programs [54]. SIRT3 regulates ATP levels in a variety of tissues, suggesting that its activity could have an important role in ATP-mediated retrograde signaling [46,55]. Indeed, recent studies have shown that SIRT3 regulates AMPK activation [5658]. Furthermore, SIRT4 interacts with ANT [20], raising the possibility that SIRT4 activity also influences the ATP:ADP ratio or membrane potential and modulates important mitochondrial signals.

NAD+ and NADH levels are intimately connected with mitochondrial energy production and regulate mitochondrial sirtuin activity. Unlike NAD+, however, NADH is not a sirtuin co-substrate. Indeed, changes in the NAD+:NADH ratio can change the redox state of the cell and alter the activity of enzymes such as poly-ADP-ribose polymerases and sirtuins, with subsequent effects on signaling cascades and gene expression [5961]. Changes in mitochondrial sirtuin activity could change the balance of these metabolites within the mitochondria. For example, fatty acid oxidation reduces NAD+ to NADH, which is oxidized back to NAD+ by OXPHOS. However, it is unclear whether changes in NAD+/NADH can be transmitted outside the organelle. The inner mitochondrial membrane is impermeable to NAD+ and NADH; however, the mitochondrial malate-aspartate shuttle could transfer reducing equivalents across the mitochondrial membranes.

Mitochondrial sirtuin control of apoptosis

Apoptosis is a cellular process of programmed cell death. Mitochondria play an important role in apoptosis by the activation of mitochondrial outer membrane permeabilization, which represents the irrevocable point of no return in committing a cell to death. Outer membrane permeabilization leads to the release of caspase-activating molecules, caspase-independent death effectors, and disruption of ATP production. Despite the central role for mitochondria in the control of apoptosis, surprisingly little is known about how mitochondrial sirtuins participate in apoptotic programs. SIRT3 plays a pro-apoptotic role in both BCL2-53- and JNK-regulated apoptosis [63]. Additionally, cells lacking SIRT3 show decreased stress-induced apoptosis, lending further support for a pro-apoptotic role for SIRT3 [62]. Furthermore, recent work points to a tumor suppressive role for SIRT3: SIRT3 levels are decreased in human breast cancers and Sirt3 null mice develop mammary tumors after 12 months [62]. The mechanism for the tumor suppressive function of SIRT3 is incompletely understood, but involves repression of ROS and protection against DNA damage [62]. In conflicting studies, SIRT3 has been shown to be anti-apoptotic. For example, in the cellular response to DNA damage when mitochondrial NAD+ levels fall below critical levels, SIRT3 and SIRT4 display anti-apoptotic activity, protecting cells from death [64]. SIRT3 has also been shown to be cardioprotective, in part by activation of ROS clearance genes [65]. In future studies, it will be important to elucidate the balance achieved by SIRT3 between stress resistance (anti-apoptosis) and tumor suppression (pro-apoptosis). Additionally, the role of SIRT4 and SIRT5 in regulating metabolism suggests that these mitochondrial sirtuins could also contribute to apoptosis in tumor suppressive or stress resistant manners.

Concluding remarks

An elegant coordination of metabolism by mitochondrial sirtuins is emerging where SIRT3, SIRT4 and SIRT5 serve at critical junctions in mitochondrial metabolism by acting as switches to facilitate energy production during nutrient adaptation and stress. Rather than satisfy, these studies lead to more questions. How important are changes in global mitochondrial acetylation to mitochondrial biology and is acetylation status a readout for sirtuin activity? What are other substrates for SIRT4 and SIRT5? What molecular factors dictate substrate specificity for mitochondrial sirtuins? Moreover, further studies will provide insight into the therapeutic applications for targeting mitochondrial sirtuins to treat human diseases. It is clear that many discoveries have yet to be made in this exciting area of biology.

Body of review in energetic metabolic pathways in malignant T cells

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-B is required for T cell proliferation and differentiation of effector cells.
The TCR-to-NF-B pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-B.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation.
Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.

Dr. Aurel,
Targu Jiu

  1. sjwilliamspa

    Wouldn’t then the preferred target be mTORC instead of Sirtuins if mTORC represses Sirtuin activity?

  2. The answer may not be so simple, perhaps a conundrum.

    In conflicting studies, SIRT3 has been shown to be anti-apoptotic. For example, in the cellular response to DNA damage when mitochondrial NAD+ levels fall below critical levels, SIRT3 and SIRT4 display anti-apoptotic activity, protecting cells from death [64].

    For anti-cancer activity, apoptosis is a desired effect. This reminds me of the problem 15 years ago with the drug that would be effective against sepsis, the best paper of the year in NEJM. It failed.

    We tend to not appeciate the intricacies of biological interactions and fail to see bypass reactions. Pleotropy comes up again and again. The seminal work from Britton Chances lab on the NAD+/NADH ratio have been overlooked.

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Pathway Specific Targeting in Anticancer Therapies

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

7.7 Pathway specific targeting in anticancer therapies

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

 

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

Thangavelua, CQ Pana, …, BC Lowa, and J. Sivaramana
Proc Nat Acad Sci 2012; 109(20):7705–7710
http://dx.doi.org:/10.1073/pnas.1116573109

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA’s active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

The Warburg effect in cancer biology describes the tendency of cancer cells to take up more glucose than most normal cells, despite the availability of oxygen (12). In addition to altered glucose metabolism, glutaminolysis (catabolism of glutamine to ATP and lactate) is another hallmark of cancer cells (23). In glutaminolysis, mitochondrial glutaminase catalyzes the conversion of glutamine to glutamate (4), which is further catabolized in the Krebs cycle for the production of ATP, nucleotides, certain amino acids, lipids, and glutathione (25).

Humans express two glutaminase isoforms: KGA (kidney-type) and LGA (liver-type) from two closely related genes (6). Although KGA is important for promoting growth, nothing is known about the precise mechanism of its activation or inhibition and how its functions are regulated under physiological or pathophysiological conditions. Inhibition of rat KGA activity by antisense mRNA results in decreased growth and tumorigenicity of Ehrlich ascites tumor cells (7), reduced level of glutathione, and induced apoptosis (8), whereas Myc, an oncogenic transcription factor, stimulates KGA expression and glutamine metabolism (5). Interestingly, direct suppression of miR23a and miR23b (9) or activation of TGF-β (10) enhances KGA expression. Similarly, Rho GTPase that controls cytoskeleton and cell division also up-regulates KGA expression in an NF-κB–dependent manner (11). In addition, KGA is a substrate for the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1, linking glutaminolysis to cell cycle progression (12). In comparison, function and regulation of LGA is not well studied, although it was recently shown to be linked to p53 pathway (1314). Although intense efforts are being made to develop a specific KGA inhibitor such as BPTES [bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] (15), its mechanism of inhibition and selectivity is not yet understood. Equally important is to understand how KGA function is regulated in normal and cancer cells so that a better treatment strategy can be considered.

The previous crystal structures of microbial (Mglu) and Escherichia coli glutaminases show a conserved catalytic domain of KGA (1617). However, detailed structural information and regulation are not available for human glutaminases especially the KGA, and this has hindered our strategies to develop inhibitors. Here we report the crystal structure of the catalytic domain of human apo KGA and its complexes with substrate (L-glutamine), product (L-glutamate), BPTES, and its derived inhibitors. Further, Raf-Mek-Erk module is identified as the regulator of KGA activity. Although BPTES is not recognized in the active site, its binding confers a drastic conformational change of a key loop (Glu312-Pro329), which is essential in stabilizing the catalytic pocket. Significantly, EGF activates KGA activity, which can be abolished by the kinase-dead, dominant negative mutants of Mek2 (Mek2-K101A) or its upstream activator Raf-1 (Raf-1-K375M), which are the kinase components of the growth-promoting Raf-Mek2-Erk signaling node. Furthermore, coexpression of phosphatase PP2A and treatment with Mek-specific inhibitor or alkaline phosphatase all abolished enhanced KGA activity inside the cells and in vitro, indicating that stimulation of KGA is phosphorylation dependent. Our results therefore provide mechanistic insights into KGA inhibition by BPTES and its regulation by EGF-mediated Raf-Mek-Erk module in cell growth and possibly cancer manifestation.

Structures of cKGA and Its Complexes with L-Glutamine and L-Glutamate.
The human KGA consists of 669 amino acids. We refer to Ile221-Leu533 as the catalytic domain of KGA (cKGA) (Fig. 1A). The crystal structures of the apo cKGA and in complex with L-glutamine or L-glutamate were determined (Table S1). The structure of cKGA has two domains with the active site located at the interface. Domain I comprises (Ile221-Pro281 and Cys424 -Leu533) of a five-stranded anti-parallel β-sheet (β2↓β1↑β5↓β4↑β3↓) surrounded by six α-helices and several loops. The domain II (Phe282-Thr423) mainly consists of seven α-helices. L-Glutamine/L-glutamate is bound in the active site cleft (Fig. 1B and Fig. S1B). Overall the active site is highly basic, and the bound ligand makes several hydrogen-bonding contacts to Gln285, Ser286, Asn335, Glu381, Asn388, Tyr414, Tyr466, and Val484 (Fig. 1C and Fig. S1C), and these residues are highly conserved among KGA homologs (Fig. S1D). Notably, the putative serine-lysine catalytic dyad (286-SCVK-289), corresponding to the SXXK motif of class D β-lactamase (17), is located in close proximity to the bound ligand. In the apo structure, two water molecules were located in the active site, one of them being displaced by glutamine in the substrate complex. The substrate side chain is within hydrogen-bonding distance (2.9 Å) to the active site Ser286. Other key residues involved in catalysis, such as Lys289, Tyr414, and Tyr466, are in the vicinity of the active site. Lys289 is within hydrogen-bonding distance to Ser286 (3.1 Å) and acts as a general base for the nucleophilic attack by accepting the proton from Ser286. Tyr466, which is close to Ser286 and in hydrogen-bonding contact (3.2 Å) with glutamine, is involved in proton transfer during catalysis. Moreover, the carbonyl oxygen of the glutamine is hydrogen-bonded with the main chain amino groups of Ser286 and Val484, forming the oxyanion hole. Thus, we propose that in addition to the putative catalytic dyad (Ser286 XX Lys289), Tyr466 could play an important role in the catalysis (Fig. 1Cand Fig. S2).

structure of the cKGA-L-glutamine complex

structure of the cKGA-L-glutamine complex

http://www.pnas.org/content/109/20/7705/F1.medium.gif

Fig. 1.  Schematic view and structure of the cKGA-L-glutamine complex. (A) Human KGA domains and signature motifs (refer to Fig. S1A for details). (B) Structure of the of cKGA and bound substrate (L-glutamine) is shown as a cyan stick. (C) Fourier 2Fo-Fc electron density map (contoured at 1 σ) for L-glutamine, that makes hydrogen bonds with active site residues are shown.

Allosteric Binding Pocket for BPTES. The chemical structure of BPTES has an internal symmetry, with two exactly equivalent parts including a thiadiazole, amide, and a phenyl group (Fig. S3A), and it equally interacts with each monomer. The thiadiazole group and the aliphatic linker are well buried in a hydrophobic cluster that consists of Leu321, Phe322, Leu323, and Tyr394 from both monomers, which forms the allosteric pocket (Fig. 2 B–E). The side chain of Phe322 is found at the bottom of the allosteric pocket. The phenyl-acetamido moiety of BPTES is partially exposed on the loop (Asn324-Glu325), where it interacts with Phe318, Asn324, and the aliphatic part of the Glu325 side chain. On the basis of our observations we synthesized a series of BPTES-derived inhibitors (compounds2–5) (Fig. S3 AF and SI Results) and solved their cocrystal structure of compounds 2–4. Similar to BPTES, compounds 24 all resides within the hydrophobic cluster of the allosteric pocket (Fig. S3 CF).

Fig. 2. Structure of cKGA: BPTES complex and the allosteric binding mode of BPTES.

Allosteric Binding of BPTES Triggers Major Conformational Change in the Key Loop Near the Active Site.  The overall structure of these inhibitor complexes superimposes well with apo cKGA. However, a major conformational change at the Glu312 to Pro329 loop was observed in the BPTES complex (Fig. 2F). The most conformational changes of the backbone atoms that moved away from the active site region are found at the center of the loop (Leu316-Lys320). The backbone of the residues Phe318 and Asn319 is moved ≈9 Å and ≈7 Å, respectively, compared with the apo structure, whereas the side chain of these residues moved ≈14 Å and ≈12 Å, respectively. This loop rearrangement in turn brings Phe318 closer to the phenyl group of the inhibitor and forms the inhibitor binding pocket, whereas in the apo structure the same loop region (Leu316-Lys320) was found to be adjacent to the active site and forms a closed conformation of the active site.

Binding of BPTES Stabilizes the Inactive Tetramers of cKGA.  To understand the role of oligomerization in KGA function, dimers and tetramers of cKGA were generated using the symmetry-related monomers (Fig. 2 A–E and Fig. S4 D and E). The dimer interface in the cKGA: BPTES complex is formed by residues from the helix Asp386-Lys398 of both monomers and involves hydrogen bonding, salt bridges, and hydrophobic interactions (Phe389, Ala390, Tyr393, and Tyr394), besides two sulfate ions located in the interface (Fig. 2E). The dimers are further stabilized by binding of BPTES, where it binds to loop residues (Glu312-Pro329) and Tyr394 from both monomers (Fig. 2 D and E). Similarly, residues from Lys311-Asn319 loop and Arg454, His461, Gln471, and Asn529-Leu533 are involved in the interface with neighboring monomers to form the tetramer in the BPTES complex.

BPTES Induces Allosteric Conformational Changes That Destabilize Catalytic Function of KGA

Fig. 3A shows that 293T cells overexpressing KGA produced higher level of glutamate compared with the vector control cells. Most significantly, all of these mutants, except Phe322Ala, greatly diminished the KGA activity.

Fig. 3. Mutations at allosteric loop and BPTES binding pocket abrogate KGA activity and BPTES sensitivity.

Raf-Mek-Erk Signaling Module Regulates KGA Activity. Because KGA supports cell growth and proliferation, we first validated that treatment of cells with BPTES indeed inhibits KGA activity and cell proliferation (Fig. S5 A–D and SI Results). Next, as cells respond to various physiological stimuli to regulate their metabolism, with many of the metabolic enzymes being the primary targets of modulation (18), we examined whether KGA activity can be regulated by physiological stimuli, in particular EGF, which is important for cell growth and proliferation. Cells overexpressing KGA were made quiescent and then stimulated with EGF for various time points. Fig. 4A shows that the basal KGA activity remained unchanged 30 min after EGF stimulation, but the activity was substantially enhanced after 1 h and then gradually returned to the basal level after 4 h. Because EGF activates the Raf-Mek-Erk signaling module (19), treatment of cells with Mek-specific inhibitor U0126 could block the enhanced KGA activity with parallel inhibition of Erk phosphorylation (Fig. 4A). Interestingly, such Mek-induced KGA activity is specific to EGF and lysophosphatidic acid (LPA) but not with other growth factors, such as PDGF, TGF-β, and basic FGF (bFGF), despite activation of Mek-Erk by bFGF (Fig. S6A).

The results show that KGA could interact equally well with the wild-type or mutant forms of Raf-1 and Mek2 (Fig. 4C). Importantly, endogenous Raf-1 or Erk1/2, including the phosphorylated Erk1/2 (Fig. 4 C and D), could be detected in the KGA complex. Taken together, these results indicate that the activity of KGA is directly regulated by Raf-Mek-Erk downstream of EGF receptor. To further show that Mek2-enhanced KGA activity requires both the kinase activity of Mek2 and the core residues for KGA catalysis, wild-type or triple mutant (Leu321Ala/Phe322Ala/Leu323Ala) of KGA was coexpressed with dominant negative Mek2-KA or the constitutive active Mek2-SD and their KGA activities measured. The result shows that the presence of Mek2-KA blocks KGA activity, whereas the triple mutant still remains inert even in the presence of the constitutively active Mek2 (Fig. 4E), and despite Mek2 binding to the KGA triple mutant (Fig. S7B). Consequently, expressing triple mutant did not support cell proliferation as well as the wild-type control (Fig. S7C).

Fig. 4. EGFR-Raf-Mek-Erk signaling stimulates KGA activity.

When cells expressing both KGA and Mek2-K101A were treated with subthreshold levels of BPTES, there was a synergistic reduction in cell proliferation (Fig. S6C and SI Results). Lastly, to determine whether regulation of KGA by Raf-Mek-Erk depends on its phosphorylation status, cells were transfected with KGA with or without the protein phosphatase PP2A and assayed for the KGA activity. PP2A is a ubiquitous and conserved serine/threonine phosphatase with broad substrate specificity. The results indicate that KGA activity was reduced down to the basal level in the presence of PP2A (Fig. 5A). Coimmunoprecipitation study also revealed that KGA interacts with PP2A (Fig. 5B), suggesting a negative feedback regulation by this protein phosphatase. Furthermore, treatment of immunoprecipitated and purified KGA with calf-intestine alkaline phosphatase (CIAP) almost completely abolished the KGA activity in vitro (Fig. S6D). Taken together, these results indicate that KGA activity is regulated by Raf-Mek2, and KGA activation by EGF could be part of the EGF-stimulated Raf-Mek-Erk signaling program in controlling cell growth and proliferation (Fig. 5C).

KGA activity is regulated by phosphorylation

KGA activity is regulated by phosphorylation

http://www.pnas.org/content/109/20/7705/F5.medium.gif

Fig. 5. KGA activity is regulated by phosphorylation. (C) Schematic model depicting the synergistic cross-talk between KGA-mediated glutaminolysis and EGF-activated Raf-Mek-Erk signaling. Exogenous glutamine can be transported across the membrane and converted to glutamate by glutaminase (KGA), thus feeding the metabolite to the ATP-producing tricarboxylic acid (TCA) cycle. This process can be stimulated by EGF receptor-mediated Raf-Mek-Erk signaling via their phosphorylation-dependent pathway, as evidenced by the inhibition of KGA activity by the kinase-dead and dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-specific inhibitor U0126. Consequently, inhibiting KGA with BPTES and blocking Raf-Mek pathway with Mek2-K101A provide a synergistic inhibition on cell proliferation.

Small-molecule inhibitors that target glutaminase activity in cancer cells are under development. Earlier efforts targeting glutaminase using glutamine analogs have been unsuccessful owing to their toxicities (2). BPTES has attracted much attention as a selective, nontoxic inhibitor of KGA (15), and preclinical testing of BPTES toward human cancers has just begun (20). BPTES selectively suppresses the growth of glioma cells (21) and inhibits the growth of lymphoma tumor growth in animal model studies (22). Wang et al. (11) reported a small molecule that targets glutaminase activity and oncogenic transformation. Despite extensive studies, nothing is known about the structural and molecular basis for KGA inhibitory mechanisms and how their function is regulated during normal and cancer cell metabolism. Such limited information impedes our effort in producing better generations of inhibitors for better treatment regimens.

Comparison of the complex structures with apo cKGA structure, which has well-defined electron density for the key loop, we provide the atomic view of an allosteric binding pocket for BPTES and elucidate the inhibitory mechanism of KGA by BPTES. The key residues of the loop (Glu312-Pro329) undergo major conformational changes upon binding of BPTES. In addition, structure-based mutagenesis studies suggest that this loop is essential for stabilizing the active site. Therefore, by binding in an allosteric pocket, BPTES inhibits the enzymatic activity of KGA through (i) triggering a major conformational change on the key residues that would normally be involved in stabilizing the active sites and regulating its enzymatic activity; and (ii) forming a stable inactive tetrameric KGA form. Our findings are further supported by two very recent reports on KGA isoform (GAC) (2324), although these studies lack full details owing to limitation of their electron density maps. BPTES is specific to KGA but not to LGA (15). Sequence comparison of KGA with LGA (Fig. S8A) reveals two unique residues on KGA, Phe318 and Phe322, which upon mutation to LGA counterparts, become resistant to BPTES. Thus, our study provides the molecular basis of BPTES specificity.

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

Islam SS, Mokhtari RB, Noman AS, …, van der Kwast T, Yeger H, Farhat WA.
Molec Carcinogenesis mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22300

shh sonic hedgehog signaling pathway nri2151-f1

shh sonic hedgehog signaling pathway nri2151-f1

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis

Sonic_hedgehog_pathway

Sonic_hedgehog_pathway

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Gaikwad SM, Thakur B, Sakpal A, Singh RK, Ray P.
Int J Biochem Cell Biol. 2015 Apr; 61:90-102
http://dx.doi.org:/10.1016/j.biocel.2015.02.001

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

Kuboki MIto ASimizu SUmezawa K.
Biochem Biophys Res Commun. 2015 Jan 16; 456(3):810-4
http://dx.doi.org:/10.1016/j.bbrc.2014.12.024

Activation of caspase 3 and caspase-dependent apoptosis  nrmicro2071-f1

Activation of caspase 3 and caspase-dependent apoptosis nrmicro2071-f1

Highlights

  • We have prepared RelB mutants that are resistant to caspase 3-induced scission.
  • Vinblastine induced caspase 3-dependent site-specific RelB cleavage in cancer cells.
  • Cancer cells expressing cleavage-resistant RelB showed less sensitivity to vinblastine.
  • Caspase 3-induced RelB cleavage may provide positive feedback mechanism in apoptosis.

DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.

apoptotic-caspases-control-microglia-activation-cdd2011107f3

apoptotic-caspases-control-microglia-activation-cdd2011107f3

 

 

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

He GDhar DNakagawa HFont-Burgada JOgata HJiang Y, et al.
Cell. 2013 Oct 10; 155(2):384-96
http://dx.doi.org/10.1016%2Fj.cell.2013.09.031

Il-6 signaling in cancer cells

Il-6 signaling in cancer cells

Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from pre-malignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.

Clonal evolution and selective pressure may cause some descendants of the initial progenitor to cross the bridge of no return and form a premalignant lesion. Cancer genome sequencing indicates that most cancers require at least five genetic changes to evolve (Wood et al., 2007). It has been difficult to isolate and propagate cancer progenitors prior to detection of tumor masses. Further, it is not clear whether cancer progenitors are the precursors for the  cancer stem cells (CSCs)isolated from cancers. An answer to these critical questions depends on identification and isolation of cancer progenitors, which may also enable definition of molecular markers and signaling pathways suitable for early detection and treatment.

Hepatocellular carcinoma (HCC), the end product of chronic liver diseases, requires several decades to evolve (El-Serag, 2011). It is the third most deadly and fifth most common cancer worldwide, and in the United States its incidence has doubled in the past two decades. Furthermore, 8% of the world’s population are chronically infected with hepatitis B or C viruses (HBV and HCV) and are at a high risk of new HCC development (El-Serag, 2011). Up to 5% of HCV patients will develop HCC in their lifetime, and the yearly HCC incidence in patients with cirrhosis is 3%–5%. These tumors may arise from premalignant lesions, ranging from dysplastic foci to dysplastic hepatocyte nodules that are often seen in damaged and cirrhotic livers and are more proliferative than the surrounding parenchyma (Hytiroglou et al., 2007). There is no effective treatment for HCC and, upon diagnosis, most patients with advanced disease have a remaining lifespan of 4–6 months. Premalignant lesions, called foci of altered hepatocytes (FAH), were described in chemically induced HCC models (Pitot, 1990), but it was questioned whether these lesions harbor tumor progenitors or result from compensatory proliferation (Sell and Leffert, 2008). The aim of this study was to determine whether HCC progenitor cells (HcPCs) exist and if so, to isolate these cells and identify some of the signaling networks that are involved in their maintenance and progression.

We now describe HcPC isolation from mice treated with the procarcinogen diethyl nitrosamine (DEN), which induces poorly differentiated HCC nodules within 8 to 9 months (Verna et al., 1996). The use of a chemical carcinogen is justified because the finding of up to 121 mutations per HCC genome suggests that carcinogens may be responsible for human HCC induction (Guichard et al., 2012). Furthermore, 20%–30% of HCC, especially in HBV-infected individuals, evolve in noncirrhotic livers (El-Serag, 2011). Nonetheless, we also isolated HcPCs fromTak1Δhep mice, which develop spontaneous HCC as a result of progressive liver damage, inflammation, and fibrosis caused by ablation of TAK1 (Inokuchi et al., 2010). Although the etiology of each model is distinct, both contain HcPCs that express marker genes and signaling pathways previously identified in human HCC stem cells (Marquardt and Thorgeirsson, 2010) long before visible tumors are detected. Furthermore, DEN-induced premalignant lesions and HcPCs exhibit autocrine IL-6 production that is critical for tumorigenic progression. Circulating IL-6 is a risk indicator in several human pathologies and is strongly correlated with adverse prognosis in HCC and cholangiocarcinoma (Porta et al., 2008Soresi et al., 2006). IL-6 produced by in-vitro-induced CSCs was suggested to be important for their maintenance (Iliopoulos et al., 2009). Little is known about the source of IL-6 in HCC.

DEN-Induced Collagenase-Resistant Aggregates of HCC Progenitors

A single intraperitoneal (i.p.) injection of DEN into 15-day-old BL/6 mice induces HCC nodules first detected 8 to 9 months later. However, hepatocytes prepared from macroscopically normal livers 3 months after DEN administration already contain cells that progress to HCC when transplanted into the permissive liver environment of MUP-uPA mice (He et al., 2010), which express urokinase plasminogen activator (uPA) from a mouse liver-specific major urinary protein (MUP) promoter and undergo chronic liver damage and compensatory proliferation (Rhim et al., 1994). HCC markers such as α fetoprotein (AFP), glypican 3 (Gpc3), and Ly6D, whose expression in mouse liver cancer was reported (Meyer et al., 2003), were upregulated in aggregates from DEN-treated livers, but not in nonaggregated hepatocytes or aggregates from control livers (Figure S1A). Using 70 μm and 40 μm sieves, we separated aggregated from nonaggregated hepatocytes (Figure 1A) and tested their tumorigenic potential by transplantation into MUP-uPA mice (Figure 1B). To facilitate transplantation, the aggregates were mechanically dispersed and suspended in Dulbecco’s modified Eagle’s medium (DMEM). Five months after intrasplenic (i.s.) injection of 104 viable cells, mice receiving cells from aggregates developed about 18 liver tumors per mouse, whereas mice receiving nonaggregated hepatocytes developed less than 1 tumor each (Figure 1B). The tumors exhibited typical trabecular HCC morphology and contained cells that abundantly express AFP (Figure S1B).

Only liver tumors were formed by the transplanted cells. Other organs, including the spleen into which the cells were injected, remained tumor free (Figure 1B), suggesting that HcPCs progress to cancer only in the proper microenvironment. Indeed, no tumors appeared after HcPC transplantation into normal BL/6 mice. But, if BL/6 mice were first treated with retrorsine (a chemical that permanently inhibits hepatocyte proliferation [Laconi et al., 1998]), intrasplenically transplanted with HcPC-containing aggregates, and challenged with CCl4 to induce liver injury and compensatory proliferation (Guo et al., 2002), HCCs readily appeared (Figure 1C). CCl4 omission prevented tumor development. Notably, MUP-uPA or CCl4-treated livers are fragile, rendering direct intrahepatic transplantation difficult. CCl4-induced liver damage, especially within a male liver, generates a microenvironment that drives HcPC proliferation and malignant progression. To examine this point, we transplanted GFP-labeled HcPC-containing aggregates into retrorsine-treated BL/6 mice and examined their ability to proliferate with or without subsequent CCl4 treatment. Indeed, the GFP+ cells formed clusters that grew in size only in CCl4-treated host livers (Figure S1E). Omission of CC14 prevented their expansion.

Because CD44 is expressed by HCC stem cells (Yang et al., 2008Zhu et al., 2010), we dispersed the aggregates and separated CD44+ from CD44 cells and transplanted both into MUP-uPA mice. Whereas as few as 103 CD44+ cells gave rise to HCCs in 100% of recipients, no tumors were detected after transplantation of CD44 cells (Figure 1E). Remarkably, 50% of recipients developed at least one HCC after receiving as few as 102 CD44+ cells.

HcPC-Containing Aggregates in Tak1Δhep Mice

We applied the same HcPC isolation protocol to Tak1Δhep mice, which develop HCC of different etiology from DEN-induced HCC. Importantly, Tak1Δhep mice develop HCC as a consequence of chronic liver injury and fibrosis without carcinogen or toxicant exposure (Inokuchi et al., 2010). Indeed, whole-tumor exome sequencing revealed that DEN-induced HCC contained about 24 mutations per 106 bases (Mb) sequenced, with B-RafV637E being the most recurrent, whereas 1.4 mutations per Mb were detected inTak1Δhep HCC’s exome (Table S1). By contrast, Tak1Δhep HCC exhibited gene copy number changes. HCC developed in 75% of MUP-uPA mice that received dispersed Tak1Δhep aggregates, but no tumors appeared in mice receiving nonaggregated Tak1Δhep or totalTak1f/f hepatocytes (Figure 2B). bile duct ligation (BDL) or feeding with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), treatments that cause cholestatic liver injuries and oval cell expansion (Dorrell et al., 2011), did increase the number of small hepatocytic cell aggregates (Figure S2A). Nonetheless, no tumors were observed 5 months after injection of such aggregates into MUP-uPA mice (Figure S2B). Thus, not all hepatocytic aggregates contain HcPCs, and HcPCs only appear under tumorigenic conditions.

The HcPC Transcriptome Is Similar to that of HCC and Oval Cells

To determine the relationship between DEN-induced HcPCs, normal hepatocytes, and fully transformed HCC cells, we analyzed the transcriptomes of aggregated and nonaggregated hepatocytes from male littermates 5 months after DEN administration, HCC epithelial cells from DEN-induced tumors, and normal hepatocytes from age- and gender-matched littermate controls. Clustering analysis distinguished the HCC samples from other samples and revealed that the aggregated hepatocyte samples did not cluster with each other but rather with nonaggregated hepatocytes derived from the same mouse (Figure S3A). 57% (583/1,020) of genes differentially expressed in aggregated relative to nonaggregated hepatocytes are also differentially expressed in HCC relative to normal hepatocytes (Figure 3B, top), a value that is highly significant (p < 7.13 × 10−243). More specifically, 85% (494/583) of these genes are overexpressed in both HCC and HcPC-containing aggregates (Figure 3B, bottom table). Thus, hepatocyte aggregates isolated 5 months after DEN injection contain cells that are related in their gene expression profile to HCC cells isolated from fully developed tumor nodules.

Figure 3 Aggregated Hepatocytes Exhibit an Altered Transcriptome Similar to that of HCC Cells

We examined which biological processes or cellular compartments were significantly overrepresented in the induced or repressed genes in both pairwise comparisons (Gene Ontology Analysis). As expected, processes and compartments that were enriched in aggregated hepatocytes relative to nonaggregated hepatocytes were almost identical to those that were enriched in HCC relative to normal hepatocytes (Figure 3C). Several human HCC markers, including AFP, Gpc3 and H19, were upregulated in aggregated hepatocytes (Figures 3D and 3E). Aggregated hepatocytes also expressed more Tetraspanin 8 (Tspan8), a cell-surface glycoprotein that complexes with integrins and is overexpressed in human carcinomas (Zöller, 2009). Another cell-surface molecule highly expressed in aggregated cells is Ly6D (Figures 3D and 3E). Immunofluorescence (IF) analysis revealed that Ly6D was undetectable in normal liver but was elevated in FAH and ubiquitously expressed in most HCC cells (Figure S3C). A fluorescent-labeled Ly6D antibody injected into HCC-bearing mice specifically stained tumor nodules (Figure S3D). Other cell-surface molecules that were upregulated in aggregated cells included syndecan 3 (Sdc3), integrin α 9 (Itga9), claudin 5 (Cldn5), and cadherin 5 (Cdh5) (Figure 3D). Aggregated hepatocytes also exhibited elevated expression of extracellular matrix proteins (TIF3 and Reln1) and a serine protease inhibitor (Spink3). Elevated expression of such proteins may explain aggregate formation. Aggregated hepatocytes also expressed progenitor cell markers, including the epithelial cell adhesion molecule (EpCAM) (Figure 3E) and Dlk1 (Figure 3D). We compared the HcPC and HCC (Figure 3A) to the transcriptome of DDC-induced oval cells (Shin et al., 2011). This analysis revealed a striking similarity between the HCC, HcPC, and the oval cell transcriptomes (Figure S3B). Despite these similarities, some genes that were upregulated in HcPC-containing aggregates and HCC were not upregulated in oval cells. Such genes may account for the tumorigenic properties of HcPC and HCC.

Figure 4  DEN-Induced HcPC Aggregates Express Pathways and Markers Characteristic of HCC and Hepatobiliary Stem Cells

We examined the aggregates for signaling pathways and transcription factors involved in hepatocarcinogenesis. Many aggregated cells were positive for phosphorylated c-Jun and STAT3 (Figure 4A), transcription factors involved in DEN-induced hepatocarcinogenesis (Eferl et al., 2003He et al., 2010). Sox9, a transcription factor that marks hepatobiliary progenitors (Dorrell et al., 2011), was also expressed by many of the aggregated cells, which were also positive for phosphorylated c-Met (Figure 4A), a receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF) and is essential for liver development (Bladt et al., 1995) and hepatocarcinogenesis (Wang et al., 2001). Few of the nonaggregated hepatocytes exhibited activation of these signaling pathways. Despite different etiology, HcPC-containing aggregates from Tak1Δhep mice exhibit upregulation of many of the same markers and pathways that are upregulated in DEN-induced HcPC-containing aggregates. Flow cytometry confirmed enrichment of CD44+ cells as well as CD44+/CD90+ and CD44+/EpCAM+ double-positive cells in the HcPC-containing aggregates from either DEN-treated or Tak1Δhep livers (Figure S4B).

HcPC-Containing Aggregates Originate from Premalignant Dysplastic Lesions

FAH are dysplastic lesions occurring in rodent livers exposed to hepatic carcinogens (Su et al., 1990). Similar lesions are present in premalignant human livers (Su et al., 1997). Yet, it is still debated whether FAH correspond to premalignant lesions or are a reaction to liver injury that does not lead to cancer (Sell and Leffert, 2008). In DEN-treated males, FAH were detected as early as 3 months after DEN administration (Figure 5A), concomitant with the time at which HcPC-containing aggregates were detected. In females, FAH development was delayed. FAH contained cells positive for the same progenitor cell markers and activated signaling pathways present in HcPC-containing aggregates, including AFP, CD44, and EpCAM (Figure 5C). FAH also contained cells positive for activated STAT3, c-Jun, and PCNA (Figure 5C).

HcPCs Exhibit Autocrine IL-6 Expression Necessary for HCC Progression

In situ hybridization (ISH) and immunohistochemistry (IHC) revealed that DEN-induced FAH contained IL-6-expressing cells (Figures 6A, 6B, and S5), and freshly isolated DEN-induced aggregates contained more IL-6 messenger RNA (mRNA) than nonaggregated hepatocytes (Figure 6C). We examined several factors that control IL-6 expression and found that LIN28A and B were significantly upregulated in HcPCs and HCC (Figures 6D and 6E). LIN28-expressing cells were also detected within FAH (Figure 6F). As reported (Iliopoulos et al., 2009), knockdown of LIN28B in cultured HcPC or HCC cell lines decreased IL-6 expression (Figure 6G). LIN28 exerts its effects through downregulation of the microRNA (miRNA) Let-7 (Iliopoulos et al., 2009).

Figure 6  Liver Premalignant Lesions and HcPCs Exhibit Elevated IL-6 and LIN28 Expression

Figure 7  HCC Growth Depends on Autocrine IL-6 Production

The isolation and characterization of cells that can give rise to HCC only after transplantation into an appropriate host liver undergoing chronic injury demonstrates that cancer arises from progenitor cells that are yet to become fully malignant. Importantly, unlike fully malignant HCC cells, the HcPCs we isolated cannot form s.c. tumors or even liver tumors when introduced into a nondamaged liver. Liver damage induced by uPA expression or CCl4 treatment provides HcPCs with the proper cytokine and growth factor milieu needed for their proliferation. Although HcPCs produce IL-6, they may also depend on other cytokines such as TNF, which is produced by macrophages that are recruited to the damaged liver. In addition, uPA expression and CCl4 treatment may enhance HcPC growth and progression through their fibrogenic effect on hepatic stellate cells. Although HCC and other cancers have been suspected to arise from premalignant/dysplastic lesions (Hruban et al., 2007Hytiroglou et al., 2007), a direct demonstration that such lesions progress into malignant tumors has been lacking. Based on expression of common markers—EpCAM, CD44, AFP, activated STAT3, and IL-6—that are not expressed in normal hepatocytes, we postulate that HcPCs originate from FAH or dysplastic foci, which are first observed in male mice within 3 months of DEN exposure.

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

Lin R1Tao RGao XLi TZhou XGuan KLXiong YLei QY.
Mol Cell. 2013 Aug 22; 51(4):506-18
http://dx.doi.org:/10.1016/j.molcel.2013.07.002

Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.

Fatty acid synthesis occurs at low rates in most nondividing cells of normal tissues that primarily uptake lipids from circulation. In contrast, increased lipogenesis, especially de novo lipid synthesis, is a key characteristic of cancer cells. Many studies have demonstrated that in cancer cells, fatty acids are preferred to be derived from de novo synthesis instead of extracellular lipid supply (Medes et al., 1953Menendez and Lupu, 2007;Ookhtens et al., 1984Sabine et al., 1967). Fatty acids are key building blocks for membrane biogenesis, and glucose serves as a major carbon source for de novo fatty acid synthesis (Kuhajda, 2000McAndrew, 1986;Swinnen et al., 2006). In rapidly proliferating cells, citrate generated by the tricarboxylic acid (TCA) cycle, either from glucose by glycolysis or glutamine by anaplerosis, is preferentially exported from mitochondria to cytosol and then cleaved by ATP citrate lyase (ACLY) (Icard et al., 2012) to produce cytosolic acetyl coenzyme A (acetyl-CoA), which is the building block for de novo lipid synthesis. As such, ACLY couples energy metabolism with fatty acids synthesis and plays a critical role in supporting cell growth. The function of ACLY in cell growth is supported by the observation that inhibition of ACLY by chemical inhibitors or RNAi dramatically suppresses tumor cell proliferation and induces differentiation in vitro and in vivo (Bauer et al., 2005Hatzivassiliou et al., 2005). In addition, ACLY activity may link metabolic status to histone acetylation by providing acetyl-CoA and, therefore, gene expression (Wellen et al., 2009).

While ACLY is transcriptionally regulated by sterol regulatory element-binding protein 1 (SREBP-1) (Kim et al., 2010), ACLY activity is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Berwick et al., 2002Migita et al., 2008Pierce et al., 1982). Akt can directly phosphorylate and activate ACLY (Bauer et al., 2005Berwick et al., 2002Migita et al., 2008Potapova et al., 2000). Covalent lysine acetylation has recently been found to play a broad and critical role in the regulation of multiple metabolic enzymes (Choudhary et al., 2009Zhao et al., 2010). In this study, we demonstrate that ACLY protein is acetylated on multiple lysine residues in response to high glucose. Acetylation of ACLY blocks its ubiquitinylation and degradation, thus leading to ACLY accumulation and increased fatty acid synthesis. Our observations reveal a crosstalk between protein acetylation and ubiquitylation in the regulation of fatty acid synthesis and cell growth.

Acetylation of ACLY at Lysines 540, 546, and 554

Recent mass spectrometry-based proteomic analyses have potentially identified a large number of acetylated proteins, including ACLY (Figure S1A available online; Choudhary et al., 2009Zhao et al., 2010). We detected the acetylation level of ectopically expressed ACLY followed by western blot using pan-specific anti-acetylated lysine antibody. ACLY was indeed acetylated, and its acetylation was increased by nearly 3-fold after treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases, and trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) class I and class II (Figure 1A). Experiments with endogenous ACLY also showed that TSA and NAM treatment enhanced ACLY acetylation (Figure 1B).

Figure 1  ACLY Is Acetylated at Lysines 540, 546, and 554

Ten putative acetylation sites were identified by mass spectrometry analyses (Table S1). We singly mutated each lysine to either a glutamine (Q) or an arginine (R) and found that no single mutation resulted in a significant reduction of ACLY acetylation (data not shown), indicating that ACLY may be acetylated at multiple lysine residues. Three lysine residues, K540, K546, and K554, received high scores in the acetylation proteomic screen and are evolutionarily conserved from C. elegans to mammals (Figure S1A). We generated triple Q and R mutants of K540, K546, and K554 (3KQ and 3KR) and found that both 3KQ and 3KR mutations resulted in a significant (~60%) decrease in ACLY acetylation (Figure 1C), indicating that 3K are the major acetylation sites of ACLY.  Further, we found that the acetylation of endogenous ACLY is clearly increased after treatment of cells with NAM and TSA (Figure 1D). These results demonstrate that ACLY is acetylated at K540, K546, and K554.

Glucose Promotes ACLY Acetylation to Stabilize ACLY

In mammalian cells, glucose is the main carbon source for de novo lipid synthesis. We found that ACLY levels increased with increasing glucose concentration, which also correlated with increased ACLY 3K acetylation (Figure 1E). Furthermore, to confirm whether the glucose level affects ACLY protein stability in vivo, we intraperitoneally injected glucose in BALB/c mice and found that high glucose resulted in a significant increase of ACLY protein levels (Figure 1F).

To determine whether ACLY acetylation affects its protein levels, we treated HeLa and Chang liver cells with NAM and TSA and found an increase in ACLY protein levels (Figure S1G, upper panel). ACLY mRNA levels were not significantly changed by the treatment of NAM and TSA (Figure S1G, lower panel), indicating that this upregulation of ACLY is mostly achieved at the posttranscriptional level. Indeed, ACLY protein was also accumulated in cells treated with the proteasome inhibitor MG132, indicating that ACLY stability could be regulated by the ubiquitin-proteasome pathway (Figure 1G). Blocking deacetylase activity stabilized ACLY (Figure S1H). The stabilization of ACLY induced by high glucose was associated with an increase of ACLY acetylation at K540, K546, and K554. Together, these data support a notion that high glucose induces both ACLY acetylation and protein stabilization and prompted us to ask whether acetylation directly regulates ACLY stability. We then generated ACLYWT, ACLY3KQ, and ACLY3KRstable cells after knocking down the endogenous ACLY. We found that the ACLY3KR or ACLY3KQmutant was more stable than the ACLYWT (Figures 1I and S1I). Collectively, our results suggest that glucose induces acetylation at K540, 546, and 554 to stabilize ACLY.

Acetylation Stabilizes ACLY by Inhibiting Ubiquitylation

To determine the mechanism underlying the acetylation and ACLY protein stability, we first examined ACLY ubiquitylation and found that it was actively ubiquitylated (Figure 2A). Previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). In order to identify the ubiquitylation sites, we tested the ubiquitylation levels of double mutants 540R–546R and 546–554R (Figure S2A). We found that the ubiquitylation of the 540R-546R and 546R-554R mutants is partially decreased, while mutation of K540, K546, and K554 (3KR), which changes all three putative acetylation lysine residues of ACLY to arginine residues, dramatically reduced the ACLY ubiquitylation level (Figures 2B and S2A), indicating that 3K lysines might also be the ubiquitylation target residues. Moreover, inhibition of deacetylases by NAM and TSA decreased ubiquitylation of WT but not 3KQ or 3KR mutant ACLY (Figure 2C). These results implicate an antagonizing role of the acetylation towards the ubiquitylation of ACLY at these three lysine residues.

Figure 2  Acetylation Protects ACLY from Proteasome Degradation by Inhibiting Ubiquitylation

We found that ACLY acetylation was only detected in the nonubiquitylated, but not the ubiquitylated (high-molecular-weight), ACLY species. This result indicates that ACLY acetylation and ubiquitylation are mutually exclusive and is consistent with the model that K540, K546, and K554 are the sites of both ubiquitylation and acetylation. Therefore, acetylation of these lysines would block ubiquitylation.

We also found that glucose upregulates ACLY acetylation at 3K and decreases its ubiquitylation (Figure S2B). High glucose (25 mM) effectively decreased ACLY ubiquitylation, while inhibition of deacetylases clearly diminished its ubiquitylation (Figure 2E). We conclude that acetylation and ubiquitylation occur mutually exclusively at K540, K546, and K554 and that high-glucose-induced acetylation at these three sites blocks ACLY ubiquitylation and degradation.

UBR4 Targets ACLY for Degradation

UBR4 was identified as a putative ACLY-interacting protein by affinity purification coupled with mass spectrometry analysis (data not shown). To address if UBR4 is a potential ACLY E3 ligase, we determined the interaction between ACLY and UBR4 and found that ACLY interacted with the E3 ligase domain of UBR4; this interaction was enhanced by MG132 treatment (Figure 3A). UBR4 knockdown in A549 cells resulted in an increase of endogenous ACLY protein level (Figure 3C). Moreover, UBR4 knockdown significantly stabilized ACLY (Figure 3D) and decreased ACLY ubiquitylation (Figure 3E). Taken together, these results indicate that UBR4 is an ACLY E3 ligase that responds to glucose regulation.

Figure 3  UBR4 Is the E3 Ligase of ACLY

PCAF Acetylates ACLY

PCAF knockdown significantly reduced acetylation of 3K, indicating that PCAF is a potential 3K acetyltransferase in vivo (Figure 4C, upper panel). Furthermore, PCAF knockdown decreased the steady-state level of endogenous ACLY, but not ACLY mRNA (Figure 4C, middle and lower panels). Moreover, we found that PCAF knockdown destabilized ACLY (Figure 4D). In addition, overexpression of PCAF decreases ACLY ubiquitylation (Figure 4E), while PCAF inhibition increases the interaction between UBR4 E3 ligase domain and wild-type ACLY, but not 3KR (Figure 4F). Together, our results indicate that PCAF increases ACLY protein level, possibly via acetylating ACLY at 3K.

Figure 4  PCAF Is the Acetylase of ACLY

SIRT2 Deacetylates ACLY

Figure 5  SIRT2 Decreases ACLY Acetylation and Increases Its Protein Levels In Vivo

Acetylation of ACLY Promotes Cell Proliferation and De Novo Lipid Synthesis

The protein levels of ACLY 3KQ and 3KR were accumulated to a level higher than the wild-type cells upon extended culture in low-glucose medium (Figure S6A, right panel), indicating a growth advantage conferred by ACLY stabilization resulting from the disruption of both acetylation and ubiquitylation at K540, K546, and K554. Cellular acetyl-CoA assay showed that cells expressing 3KQ or 3KR mutant ACLY produce more acetyl-CoA than cells expressing the wild-type ACLY under low glucose (Figures 6B and S6B), further supporting the conclusion that 3KQ or 3KR mutation stabilizes ACLY.

Figure 6  Acetylation of ACLY at 3K Promotes Lipogenesis and Tumor Cell Proliferation

ACLY is a key enzyme in de novo lipid synthesis. Silencing ACLY inhibited the proliferation of multiple cancer cell lines, and this inhibition can be partially rescued by adding extra fatty acids or cholesterol into the culture media (Zaidi et al., 2012). This prompted us to measure extracellular lipid incorporation in A549 cells after knockdown and ectopic expression of ACLY. We found that when cultured in low glucose (2.5 mM), cells expressing wild-type ACLY uptake significantly more phospholipids compared to cells expressing 3KQ or 3KR mutant ACLY (Figures 6C, 6D, and S6D). When cultured in the presence of high glucose (25 mM), however, cells expressing either the wild-type, 3KQ, or 3KR mutant ACLY all have reduced, but similar, uptake of extracellular phospholipids (Figures 6C, 6D, and S6D). The above results are consistent with a model that acetylation of ACLY induced by high glucose increases its stability and stimulates de novo lipid synthesis.

3K Acetylation of ACLY Is Increased in Lung Cancer

ACLY is reported to be upregulated in human lung cancer (Migita et al., 2008). Many small chemicals targeting ACLY have been designed for cancer treatment (Zu et al., 2012). The finding that 3KQ or 3KR mutant increased the ability of ACLY to support A549 lung cancer cell proliferation prompted us to examine 3K acetylation in human lung cancers. We collected a total of 54 pairs of primary human lung cancer samples with adjacent normal lung tissues and performed immunoblotting for ACLY protein levels. This analysis revealed that, when compared to the matched normal lung tissues, 29 pairs showed a significant increase of total ACLY protein using b-actin as a loading control (Figures 7A and S7A). The tumor sample analyses demonstrate that ACLY protein levels are elevated in lung cancers, and 3K acetylation positively correlates with the elevated ACLY protein. These data also indicate that ACLY with 3K acetylation may be potential biomarker for lung cancer diagnosis.

Figure 7
  Acetylation of ACLY at 3K Is Upregulated in Human Lung Carcinoma

Dysregulation of cellular metabolism is a hallmark of cancer (Hanahan and Weinberg, 2011Vander Heiden et al., 2009). Besides elevated glycolysis, increased lipogenesis, especially de novo lipid synthesis, also plays an important role in tumor growth. Because most carbon sources for fatty acid synthesis are from glucose in mammalian cells (Wellen et al., 2009), the channeling of carbon into de novo lipid synthesis as building blocks for tumor cell growth is primarily linked to acetyl-CoA production by ACLY. Moreover, the ACLY-catalyzed reaction consumes ATP. Therefore, as the key cellular energy and carbon source, one may expect a role for glucose in ACLY regulation. In the present study, we have uncovered a mechanism of ACLY regulation by glucose that increases ACLY protein level to meet the enhanced demand of lipogenesis in growing cells, such as tumor cells (Figure 7C). Glucose increases ACLY protein levels by stimulating its acetylation.

Upregulation of ACLY is common in many cancers (Kuhajda, 2000Milgraum et al., 1997Swinnen et al., 2004Yahagi et al., 2005). This is in part due to the transcriptional activation by SREBP-1 resulting from the activation of the PI3K/AKT pathway in cancers (Kim et al., 2010Nadler et al., 2001Wang and Dey, 2006). In this study, we report a mechanism of ACLY regulation at the posttranscriptional level. We propose that acetylation modulated by glucose status plays a crucial role in coordinating the intracellular level of ACLY, hence fatty acid synthesis, and glucose availability. When glucose is sufficient, lipogenesis is enhanced. This can be achieved, at least in part, by the glucose-induced stabilization of ACLY. High glucose increases ACLY acetylation, which inhibits its ubiquitylation and degradation, leading to the accumulation of ACLY and enhanced lipogenesis. In contrast, when glucose is limited, ACLY is not acetylated and thus can be ubiquitylated, leading to ACLY degradation and reduced lipogenesis. Moreover, our data indicate that acetylation and ubiquitylation in ACLY may compete with each other by targeting the same lysine residues at K540, K546, and K554. Consistently, previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). Similar models of different modifications on the same lysine residues have been reported in the regulation of other proteins (Grönroos et al., 2002Li et al., 20022012). We propose that acetylation and ubiquitylation have opposing effects in the regulation of ACLY by competitively modifying the same lysine residues. The acetylation-mimetic 3KQ and the acetylation-deficient 3KR mutants behaved indistinguishably in most biochemical and functional assays, mainly due to the fact that these mutations disrupt lysine ubiquitylation that primarily occurs on these three residues.

ACLY is increased in lung cancer tissues compared to adjacent tissues. Consistently, ACLY acetylation at 3K is also significantly increased in lung cancer tissues. These observations not only confirm ACLY acetylation in vivo, but also suggest that ACLY 3K acetylation may play a role in lung cancer development. Our study reveals a mechanism of ACLY regulation in response to glucose signals.

 

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

Nomura DK1Long JZNiessen SHoover HSNg SWCravatt BF.
Cell. 2010 Jan 8; 140(1):49-61
http://dx.doi.org/10.1016.2Fj.cell.2009.11.027

Highlights

  • Monoacylglycerol lipase (MAGL) is elevated in aggressive human cancer cells
  • Loss of MAGL lowers fatty acid levels in cancer cells and impairs pathogenicity
  • MAGL controls a signaling network enriched in protumorigenic lipids
  • A high-fat diet can restore the growth of tumors lacking MAGL in vivo
monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

http://www.cell.com/cms/attachment/1082768/7977146/fx1.jpg

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity—phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.

We show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in non-aggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity — phenotypes that are reversed by an MAGL inhibitor. Interestingly, impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of pro-tumorigenic signals.

The conversion of cells from a normal to cancerous state is accompanied by reprogramming of metabolic pathways (Deberardinis et al., 2008Jones and Thompson, 2009Kroemer and Pouyssegur, 2008), including those that regulate glycolysis (Christofk et al., 2008Gatenby and Gillies, 2004), glutamine-dependent anaplerosis (DeBerardinis et al., 2008DeBerardinis et al., 2007Wise et al., 2008), and the production of lipids (DeBerardinis et al., 2008Menendez and Lupu, 2007). Despite a growing appreciation that dysregulated metabolism is a defining feature of cancer, it remains unclear, in many instances, how such biochemical changes occur and whether they play crucial roles in disease progression and malignancy.

Among dysregulated metabolic pathways, heightened de novo lipid biosynthesis, or the development a “lipogenic” phenotype (Menendez and Lupu, 2007), has been posited to play a major role in cancer. For instance, elevated levels of fatty acid synthase (FAS), the enzyme responsible for fatty acid biosynthesis from acetate and malonyl CoA, are correlated with poor prognosis in breast cancer patients, and inhibition of FAS results in decreased cell proliferation, loss of cell viability, and decreased tumor growth in vivo (Kuhajda et al., 2000Menendez and Lupu, 2007Zhou et al., 2007). FAS may support cancer growth, at least in part, by providing metabolic substrates for energy production (via fatty acid oxidation) (Buzzai et al., 2005Buzzai et al., 2007Liu, 2006). Many other features of lipid biochemistry, however, are also critical for supporting the malignancy of cancer cells, including:

Prominent examples of lipid messengers that contribute to cancer include:

Here, we use functional proteomic methods to discover a lipolytic enzyme, monoacylglycerol lipase (MAGL), that is highly elevated in aggressive cancer cells from multiple tissues of origin. We show that MAGL, through hydrolysis of monoacylglycerols (MAGs), controls free fatty acid (FFA) levels in cancer cells. The resulting MAGL-FFA pathway feeds into a diverse lipid network enriched in pro-tumorigenic signaling molecules and promotes migration, survival, and in vivo tumor growth. Aggressive cancer cells thus pair lipogenesis with high lipolytic activity to generate an array of pro-tumorigenic signals that support their malignant behavior.

Activity-Based Proteomic Analysis of Hydrolytic Enzymes in Human Cancer Cells

To identify enzyme activities that contribute to cancer pathogenesis, we conducted a functional proteomic analysis of a panel of aggressive and non-aggressive human cancer cell lines from multiple tumors of origin, including melanoma [aggressive (C8161, MUM2B), non-aggressive (MUM2C)], ovarian [aggressive (SKOV3), non-aggressive (OVCAR3)], and breast [aggressive (231MFP), non-aggressive (MCF7)] cancer. Aggressive cancer lines were confirmed to display much greater in vitro migration and in vivo tumor-growth rates compared to their non-aggressive counterparts (Figure S1), as previously shown (Jessani et al., 2004;Jessani et al., 2002Seftor et al., 2002Welch et al., 1991). Proteomes from these cancer lines were screened by activity-based protein profiling (ABPP) using serine hydrolase-directed fluorophosphonate (FP) activity-based probes (Jessani et al., 2002Patricelli et al., 2001). Serine hydrolases are one of the largest and most diverse enzyme classes in the human proteome (representing ~ 1–1.5% of all human proteins) and play important roles in many biochemical processes of potential relevance to cancer, such as proteolysis (McMahon and Kwaan, 2008Puustinen et al., 2009), signal transduction (Puustinen et al., 2009), and lipid metabolism (Menendez and Lupu, 2007Zechner et al., 2005). The goal of this study was to identify hydrolytic enzyme activities that were consistently altered in aggressive versus non-aggressive cancer lines, working under the hypothesis that these conserved enzymatic changes would have a high probability of contributing to the pathogenic state of cancer cells.

Among the more than 50 serine hydrolases detected in this analysis (Tables S13), two enzymes, KIAA1363 and MAGL, were found to be consistently elevated in aggressive cancer cells relative to their non-aggressive counterparts, as judged by spectral counting (Jessani et al., 2005Liu et al., 2004). We confirmed elevations in KIAA1363 and MAGL in aggressive cancer cells by gel-based ABPP, where proteomes are treated with a rhodamine-tagged FP probe and resolved by 1D-SDS-PAGE and in-gel fluorescence scanning (Figure 1A). In both cases, two forms of each enzyme were detected (Figure 1A), due to differential glycoslyation for KIAA1363 (Jessani et al., 2002), and possibly alternative splicing for MAGL (Karlsson et al., 2001). We have previously shown that KIAA1363 plays a role in regulating ether lipid signaling pathways in aggressive cancer cells (Chiang et al., 2006). On the other hand, very little was known about the function of MAGL in cancer.

Figure 1  MAGL is elevated in aggressive cancer cells, where the enzyme regulates monoacylgycerol (MAG) and free fatty acid (FFA) levels

The heightened activity of MAGL in aggressive cancer cells was confirmed using the substrate C20:4 MAG (Figure 1B). Since several enzymes have been shown to display MAG hydrolytic activity (Blankman et al., 2007), we confirmed the contribution that MAGL makes to this process in cancer cells using the potent and selective MAGL inhibitor JZL184 (Long et al., 2009a).

MAGL Regulates Free Fatty Acid Levels in Aggressive Cancer Cells

MAGL is perhaps best recognized for its role in degrading the endogenous cannabinoid 2-arachidonoylglycerol (2-AG, C20:4 MAG), as well as other MAGs, in brain and peripheral tissues (Dinh et al., 2002Long et al., 2009aLong et al., 2009bNomura et al., 2008). Consistent with this established function, blockade of MAGL by JZL184 (1 μM, 4 hr) produced significant elevations in the levels of several MAGs, including 2-AG, in each of the aggressive cancer cell lines (Figure 1C and Figure S2). Interestingly, however, MAGL inhibition also caused significant reductions in the levels of FFAs in aggressive cancer cells (Figure 1D and Figure S2). This surprising finding contrasts with the function of MAGL in normal tissues, where the enzyme does not, in general, control the levels of FFAs (Long et al., 2009aLong et al., 2009b;Nomura et al., 2008).

Metabolic labeling studies using the non-natural C17:0-MAG confirmed that MAGs are converted to LPC and LPE by aggressive cancer cells, and that this metabolic transformation is significantly enhanced by treatment with JZL184 (Figure S1). Finally, JZL184 treatment did not affect the levels of MAGs and FFAs in non-aggressive cancer lines (Figure 1C, D), consistent with the negligible expression of MAGL in these cells (Figure 1A, B).

We next stably knocked down MAGL expression by RNA interference technology using two independent shRNA probes (shMAGL1, shMAGL2), both of which reduced MAGL activity by 70–80% in aggressive cancer lines (Figure 2A, D and Figure S2). Other serine hydrolase activities were unaffected by shMAGL probes (Figure 2A, D and Figures S2), confirming the specificity of these reagents. Both shMAGL probes caused significant elevations in MAGs and corresponding reductions in FFAs in aggressive melanoma (Figure 2B, C), ovarian (Figure 2E, F), and breast cancer cells (Figure S2).

Figure 2  Stable shRNA-mediated knockdown of MAGL lowers FFA levels in aggressive cancer cells.

Together, these data demonstrate that both acute (pharmacological) and stable (shRNA) blockade of MAGL cause elevations in MAGs and reductions in FFAs in aggressive cancer cells. These intriguing findings indicate that MAGL is the principal regulator of FFA levels in aggressive cancer cells. Finally, we confirmed that MAGL activity (Figure 3A, B) and FFA levels (Figure 3C) are also elevated in high-grade primary human ovarian tumors compared to benign or low-grade tumors. Thus, heightened expression of the MAGL-FFA pathway is a prominent feature of both aggressive human cancer cell lines and primary tumors.

Figure 3  High-grade primary human ovarian tumors possess elevated MAGL activity and FFAs compared to benign tumors.

Disruption of MAGL Expression and Activity Impairs Cancer Pathogenicity

shMAGL cancer lines were next examined for alterations in pathogenicity using a set of in vitro and in vivo assays. shMAGL-melanoma (C8161), ovarian (SKOV3), and breast (231MFP) cancer cells exhibited significantly reduced in vitro migration (Figure 4A, F and Figure S2), invasion (Figure 4B, G and Figure S2), and cell survival under serum-starvation conditions (Figure 4C, H and Figure S2). Acute pharmacological blockade of MAGL by JZL184 also decreased cancer cell migration (Figure S2), but not survival, possibly indicating that maximal impairments in cancer aggressiveness require sustained inhibition of MAGL.

Figure 4  shRNA-mediated knockdown and pharmacological inhibition of MAGL impair cancer aggressiveness.

MAGL Overexpression Increases FFAs and the Aggressiveness of Cancer Cells

Stable MAGL-overexpressing (MAGL-OE) and control [expressing an empty vector or a catalytically inactive version of MAGL, where the serine nucleophile was mutated to alanine (S122A)] variants of MUM2C and OVCAR3 cells were generated by retroviral infection and evaluated for their respective MAGL activities by ABPP and C20:4 MAG substrate assays. Both assays confirmed that MAGL-OE cells possess greater than 10-fold elevations in MAGL activity compared to control cells (Figure 5A and Figure S4). MAGL-OE cells also showed significant reductions in MAGs (Figure 5B andFigure S4) and elevated FFAs (Figure 5C and Figure S4). This altered metabolic profile was accompanied by increased migration (Figure 5D and Figure S4), invasion (Figure 5E and Figure S4), and survival (Figure S4) in MAGL-OE cells. None of these effects were observed in cancer cells expressing the S122A MAGL mutant, indicating that they require MAGL activity.  MAGL-OE MUM2C cells also showed enhanced tumor growth in vivo compared to control cells (Figure 5F). Notably, the increased tumor growth rate of MAGL-OE MUM2C cells nearly matched that of aggressive C8161 cells (Figure S4). These data indicate that the ectopic expression of MAGL in non-aggressive cancer cells is sufficient to elevate their FFA levels and promote pathogenicity both in vitro and in vivo.

Figure 5 Ectopic expression of MAGL elevates FFA levels and enhances the in vitro and in vivo pathogenicity of MUM2C melanoma cells.

Metabolic Rescue of Impaired Pathogenicity in MAGL-Disrupted Cancer Cells

MAGL could support the aggressiveness of cancer cells by either reducing the levels of its MAG substrates, elevating the levels of its FFA products, or both. Among MAGs, the principal signaling molecule is the endocannabinoid 2-AG, which activates the CB1 and CB2 receptors (Ahn et al., 2008Mackie and Stella, 2006). The endocannabinoid system has been implicated previously in cancer progression and, depending on the specific study, shown to promote (Sarnataro et al., 2006Zhao et al., 2005) or suppress (Endsley et al., 2007Wang et al., 2008) cancer pathogenesis. Neither a CB1 or CB2 antagonist rescued the migratory defects of shMAGL cancer cells (Figure S5). CB1 and CB2 antagonists also did not affect the levels of MAGs or FFAs in cancer cells (Figure S5).

We then determined whether increased FFA delivery could rectify the tumor growth defect observed for shMAGL cells in vivo. Immune-deficient mice were fed either a normal chow or high-fat diet throughout the duration of a xenograft tumor growth experiment. Notably, the impaired tumor growth rate of shMAGL-C8161 cells was completely rescued in mice fed a high-fat diet. In contrast, shControl-C8161 cells showed equivalent tumor growth rates on a normal versus high-fat diet. The recovery in tumor growth for shMAGL-C8161 cells in the high-fat diet group correlated with significantly increases levels of FFAs in excised tumors (Figure 6D). Collectively, these results indicate that MAGL supports the pathogenic properties of cancer cells by maintaining tonically elevated levels of FFAs.

Figure 6  Recovery of the pathogenic properties of shMAGL cancer cells by treatment with exogenous fatty acids.

MAGL Regulates a Fatty Acid Network Enriched in Pro-Tumorigenic Signals

Studies revealed that neither

  • the MAGL-FFA pathway might serve as a means to regenerate NAD+ (via continual fatty acyl glyceride/FFA recycling) to fuel glycolysis, or
  • increased lipolysis could be to generate FFA substrates for β-oxidation, which may serve as an important energy source for cancer cells (Buzzai et al., 2005), or
  • CPT1 blockade (reduced expression of CPT1 in aggressive cancer cells (data not shown) has been reported previously (Deberardinis et al., 2006))

providing evidence against a role for β-oxidation as a downstream mediator of the pathogenic effects of the MAGL-fatty acid pathway.

Considering that FFAs are fundamental building blocks for the production and remodeling of membrane structures and signaling molecules, perturbations in MAGL might be expected to affect several lipid-dependent biochemical networks important for malignancy. To test this hypothesis, we performed lipidomic analyses of cancer cell models with altered MAGL activity, including comparisons of:

  1. MAGL-OE versus control cancer cells (OVCAR3, MUM2C), and
  2. shMAGL versus shControl cancer cells (SKOV3, C8161).

Complementing these global profiles, we also conducted targeted measurements of specific bioactive lipids (e.g., prostaglandins) that are too low in abundance for detection by standard lipidomic methods. The resulting data sets were then mined to identify a common signature of lipid metabolites regulated by MAGL, which we defined as metabolites that were significantly increased or reduced in MAGL–OE cells and showed the opposite change in shMAGL cells relative to their respective control groups (Figure 7A, B and Table S4).

Figure 7  MAGL regulates a lipid network enriched in pro-tumorigenic signaling molecules.

Most of the lipids in the MAGL-fatty acid network, including several lysophospholipids (LPC, LPA, LPE), ether lipids (MAGE, alkyl LPE), phosphatidic acid (PA), and prostaglandin E2 (PGE2), displayed similar profiles to FFAs, being consistently elevated and reduced in MAGL-OE and shMAGL cells, respectively. Only MAGs were found to show the opposite profile (elevated and reduced in shMAGL and MAGL-OE cells, respectively). Interestingly, virtually this entire lipidomic signature was also observed in aggressive cancer cells when compared to their non-aggressive counterparts (e.g., C8161 versus MUM2C and SKOV3 versus OVCAR3, respectively; Table S4). These findings demonstrate that MAGL regulates a lipid network in aggressive cancer cells that consists of not only FFAs and MAGs, but also a host of secondary lipid metabolites. Increases (rather than decreases) in LPCs and LPEs were observed in JZL184-treated cells (Figure S1 and Table S4). These data indicate that acute and chronic blockade of MAGL generate distinct metabolomic effects in cancer cells, likely reflecting the differential outcomes of short- versus long-term depletion of FFAs.

Within the MAGL-fatty acid network are several pro-tumorigenic lipid messengers, including LPA and PGE2, that have been reported to promote the aggressiveness of cancer cells (Gupta et al., 2007Mills and Moolenaar, 2003). Metabolic labeling studies confirmed that aggressive cancer cells can convert both MAGs and FFAs (Figure S1) to LPA and PGE2 and, for MAGs, this conversion was blocked by JZL184 (Figure S1). Interestingly, treatment with either LPA or PGE2 (100 nM, 4 hr) rescued the impaired migration of shMAGL cancer cells at concentrations that did not affect the migration of shControl cells (Figure 7E).

Heightened lipogenesis is an established early hallmark of dysregulated metabolism and pathogenicity in cancer (Menendez and Lupu, 2007). Cancer lipogenesis appears to be driven principally by FAS, which is elevated in most transformed cells and important for survival and proliferation (De Schrijver et al., 2003;Kuhajda et al., 2000Vazquez-Martin et al., 2008). It is not yet clear how FAS supports cancer growth, but most of the proposed mechanisms invoke pro-tumorigenic functions for the enzyme s fatty acid products and their lipid derivatives (Menendez and Lupu, 2007). This creates a conundrum, since the fatty acid molecules produced by FAS are thought to be rapidly incorporated into neutral- and phospho-lipids, pointing to the need for complementary lipolytic pathways in cancer cells to release stored fatty acids for metabolic and signaling purposes (Prentki and Madiraju, 2008Przybytkowski et al., 2007). Consistent with this hypothesis, we found that acute treatment with the FAS inhibitor C75 (40 μM, 4 h) did not reduce FFA levels in cancer cells (data not shown). Furthermore, aggressive and non-aggressive cancer cells exhibited similar levels of FAS (data not shown), indicating that lipogenesis in the absence of paired lipolysis may be insufficient to confer high levels of malignancy.

Here we show that aggressive cancer cells do indeed acquire the ability to liberate FFAs from neutral lipid stores as a consequence of heightened expression of MAGL. MAGL and its FFA products were found to be elevated in aggressive human cancer cell lines from multiple tissues of origin, as well as in high-grade primary human ovarian tumors. These data suggest that the MAGL-FFA pathway may be a conserved feature of advanced forms of many types of cancer. Further evidence in support of this premise originates from gene expression profiling studies, which have identified increased levels of MAGL in primary human ductal breast tumors compared to less malignant medullary breast tumors (Gjerstorff et al., 2006). The key role that MAGL plays in regulating FFA levels in aggressive cancer cells contrasts with the function of this enzyme in normal tissues, where it mainly controls the levels of MAGs, but not FFAs (Long et al., 2009b). These data thus provide a striking example of the co-opting of an enzyme by cancer cells to serve a distinct metabolic purpose that supports their pathogenic behavior.

Taken together, our results indicate that MAGL serves as key metabolic hub in aggressive cancer cells, where the enzyme regulates a fatty acid network that feeds into a number of pro-tumorigenic signaling pathways.

 

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.8.1  Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling

Komai K1Niwa Y1Sasazawa Y1Simizu S2.
FEBS Lett. 2015 Mar 12; 589(6):738-43
http://dx.doi.org:/10.1016/j.febslet.2015.01.040

Highlights

  • Pirin decreases E-cadherin expression and induces EMT.
  • The induction of EMT by Pirin is achieved through a Bcl3 independent pathway.
  • Pirin may be a novel target for cancer therapy.

Epithelial to mesenchymal transition (EMT) is an important mechanism for the initial step of metastasis. Proteomic analysis indicates that Pirin is involved in metastasis. However, there are no reports demonstrating its direct contribution. Here we investigated the involvement of Pirin in EMT. In HeLa cells, Pirin suppressed E-cadherin expression and regulated the expression of other EMT markers. Furthermore, cells expressing Pirin exhibited a spindle-like morphology, which is reminiscent of EMT. A Pirin mutant defective for Bcl3 binding decreased E-cadherin expression similar to wild-type, suggesting that Pirin regulates E-cadherin independently of Bcl3-Slug signaling. These data provide direct evidence that Pirin contributes to cancer metastasis.

Pirin regulates the expression of E-cadherin and EMT markers

In melanoma, Pirin enhances NF-jB activity and increases Slug expression by binding Bcl3 [31], and it may also be involved in adenoid cystic tumor metastasis [23]. Since Slug suppresses E-cadherin transcription and is recognized as a major EMT inducer, we hypothesized that Pirin may regulate EMT through inducing Slug expression. To investigate whether Pirin regulates EMT, we measured E-cadherin expression following Pirin knockdown. As shown in Fig. 1A and B, E-cadherin expression was significantly increased following Pirin knockdown indicating that it may promote EMT. To confirm this, we established Pirin-expressing HeLa cells (Fig. 1C), which inhibited the expression of E-cadherin (Fig. 1D). Additionally, the expression of Occludin, an epithelial marker, was decreased, and several mesenchymal markers, including Fibronectin, N-cadherin, and Vimentin, were increased by Pirin expression (Fig. 1D). These data suggest that Pirin promotes EMT.

Pirin induces EMT-associated cell morphological changes

As mentioned above, cells undergo morphological changes during EMT. Therefore, we next analyzed whether Pirin expression affects cell morphology. Quantitative analysis of morphological changes was based on cell circularity, {4p(area)/(perimeter)2}100, which decreases during EMT-associated morphological changes [34–36]. Indeed, TGF-b or TNF-a exposure induced EMTassociated cell morphological changes in HeLa cells (data not shown). Employing this parameter of circularity, we compared the morphology of our established HeLa/Pirin-GFP cells with control HeLa/GFP cells. Although the control HeLa/GFP cells displayed a cobblestone-like morphology, HeLa/Pirin-GFP cells were elongated in shape (Fig. 2A). Indeed, compared with control cells, the circularity of HeLa/Pirin-GFP cells was significantly decreased (Fig. 2B). To confirm that these observations were dependent on Pirin expression, HeLa/Pirin-GFP cells were treated with an siRNA targeting Pirin. HeLa/Pirin-GFP cells recovered a cobblestone-like morphology (Fig. 2C) and circularity (Fig. 2D) when treated with Pirin siRNA indicating that Pirin expression induces EMT.

Pirin induces cell migration

During EMT cells acquire migratory capabilities. Therefore, we analyzed whether Pirin affects cell migration. HeLa cells were treated with an siRNA targeting Pirin and migration was assessed using a wound healing assay. Although Pirin knockdown had no effect on cell proliferation (data not shown), wound repair was inhibited in Pirin-depleted HeLa cells (Fig. 3A and B) suggesting that Pirin promoted cell migration. Furthermore, camptothecin treatment of HeLa/GFP cells caused decreased cell viability in a dose-dependent manner, whereas HeLa/Pirin-GFP cells were more resistantto drugtreatment (datanot shown).These results suggest that Pirin induces EMT-like phenotypes, such as cell migration and anticancer drug resistance.
Pirin regulates EMT independently of Bcl3-Slug signaling

To investigate whether Pirin controls E-cadherin expression at the transcriptional level, we measured E-cadherin promoter activity with a reporter assay. Indeed, the luciferase reporter analysis indicated that Pirin inhibited E-cadherin promoter activity (Fig. 4A and B). To determine if Bcl3 is involved in Pirin-induced EMT, we tested whether a Pirin mutant defective in Bcl3 binding could inhibit E-cadherin expression. We generated a mutation in the metal-binding cavity of Pirin(E103A) and confirmed that it disrupted Bcl3 binding. In vitro GST pull-down analysis using recombinant Pirin and Bcl3/ARD demonstrated that the Pirin mutant was defective for Bcl3 binding compared to wild-type (Fig. 5A). Interestingly, expression of both wild-type Pirin and the mutant defective in Bcl3 binding reduced E-cadherin gene and protein expression (Fig. 5B and C). Taken together these results indicate that Pirin decreases E-cadherin expression without binding Bcl3, and suggest that Pirin regulates EMT independently of Bcl3-Slug signaling.

Discussion

A characteristic feature of EMT is the disruption of epithelial cell–cell contact, which is achieved by reduced E-cadherin expression. Therefore, revealing the regulatory pathways controlling E-cadherin expression may elucidate the mechanisms of EMT. Several transcription factors regulate E-cadherin transcription. For instance,Snail,Slug,Twist,and Zebact as mastertranscriptional regulators that bind the consensus E-box sequence in the E-cadherin gene promoter and decrease the transcriptional activity [38]. Since Pirin regulates the transcription of Slug [31], we hypothesized that Pirin may also regulate EMT. In this study we demonstrated that Pirin decreases E-cadherin expression, and induces EMT and cancer malignant phenotypes. Since EMT is an initial step of metastasis, Pirin may contribute to cancer progression. We next examined whether the regulation of EMT by Pirin is attributed to Bcl3 binding and the induction of Slug. To this end, we generated a Pirin mutant (E103A) defective for Bcl3 binding (Fig. 5A). Single Fe2+ ion chelating is coordinated by His56, His58, His101, and Glu103 of Pirin, and the N-terminal domain containing these residues is highly conserved between mammals, plants, fungi, and prokaryotic organisms [15,27]. Therefore, it has been predicted that this N-terminal domain containing the metal-binding cavity is important for Pirin function [20,26,31]. Indeed, TPh A inserts into the metal-binding cavity and inhibits binding to Bcl3 suggesting that the interaction occurs with the metal-binding cavity of Pirin [31]. In contrast, Hai Pang suggests that a Pirin–Bcl3– (p50)2 complex forms between acidic regions of the N-terminal Pirin domain at residues 77–82, 97–103 and 124–128 with a basic patch of Bcl3 [27]. In this study, we mutated Glutamic acid 103, a residue common between Hai Pang’s model and Pirin’s metalbinding cavity. Pull-down analysis indicated that an E103A mutant is defectiveinfor Bcl3binding(Fig.5A). Thisis the firstexperimental demonstration showing that Glu103 of Pirin is important Bcl3 binding. However, expression of the E103A mutant suppressed Ecadherin gene expression similarly to wild-type Pirin (Fig. 5B and C). Although the Bcl3–(p50)2 complex participates in oncogene addiction in cervical cells [39,40], expression of Pirin in HeLa cells did not increase Slug expression (data not shown). Therefore, we concludethatPirindecreasesE-cadherinexpressionindependently of Bcl3-Slug signaling. To understand how Pirin suppresses E-cadherin gene expression, we analyzed E-cadherin promoter activity (Fig. 4). Since Pirin decreased the activity of the E-cadherin promoter (995+1), we constructed a series of promoter deletion mutants (795+1, 565+1, 365+1, 175+1) to identify a region important for Pirin-mediated regulation. Expression of Pirin decreased the transcriptional activity of all constructs (Supplementary Fig. S1A), suggesting that Pirin may suppress E-cadherin expression through element(s) in region 175+1. Yan-Nan Liu and colleagues proposed that this region contains four Sp1-binding sites and two E-boxes that regulate E-cadherin expression.

Fig. 1. Pirin regulates E-cadherin gene expression. (A, B) HeLa cells were transfected with siRNA targeting Pirin (siPirin#1 or #2) or control siRNA (siCTRL). Forty-eight hours after transfection, cDNA was used for PCR using primer sets specific against Pirin, E-cadherin and GAPDH (A). Forty-eight hours after transfection, HeLa cells were lysed and the lysates were analyzed by Western blot with the indicated antibodies (B). (C) Lysates from HeLa/Pirin-GFP and HeLa/GFP cells were analyzed by Western blot with the indicated antibodies. (D) cDNA from HeLa/GFP or HeLa/Pirin-GFP cells was used for PCR to determine the effect of Pirin on the expression of EMT marker genes.

Fig. 2. Pirin induces cell morphological changes associated with EMT. (A) Phase contrast and fluorescence microscopic images were taken of HeLa/GFP and HeLa/Pirin-GFP cells. (B) Cell circularity was defined as form factor, {4p(area)/(perimeter)2}100 [%], and calculated using Image J software. A random selection of 100 cells from each condition was measured. (C, D) Phase contrast and fluorescence microscopic images were taken of siRNA-treated HeLa/GFP and HeLa/Pirin-GFP cells. Each cell line was transfected with siPirin#2 or siCTRL. Cells were observed by microscopy 48 h after transfection (C) and circularity was measured (D). Data shown are means ± s.d. ⁄P <0.05, bars 100lm.

Fig. 3. Pirin knockdown suppresses cell migration. (A, B) HeLa cells were transfected with siPirin#2 or siCTRL. An artificial wound was created with a tip 24h after transfection and cells were cultured for an additional 12 h. For quantification, the cells were photographed after 12h of incubation (A) and the area covered by cells was measured using Image J and normalized to control cells (B).

Fig. 4. Pirin regulates E-cadherin promoter activity.(A). HeLacells were transfected with siPirin#2 or siGFP (control) and cultured for 24 h. The E-cadherin promoter construct (995+1) and phRL-TK vectorwere transfected and cellswere cultured for an additional 24 h. Luciferase activities were measured and normalized to Renilla luciferase activity. (B) HeLa cells were transfected with the promoter construct (995+1), phRL-TK vector, and a Pirin expression vector. After 24 h, luciferase activities were measured and normalized to Renilla luciferase activity. Data are the mean ± s.d. ⁄P < 0.05.

Fig. 5. Pirin decreases E-cadherin expression in a Bcl3-independent manner. (A) Purified His6-Pirin and His6-Pirin(E103A) were incubated with Glutathione-Sepharose beads conjugated to GST or GST-Bcl3/ARD. The samples were analyzed by Western blot. (B, C) HeLa cells were transfected with vectors encoding GFP, Pirin-GFP, or Pirin(E103A)GFP. Cells were lysed 48 h after transfection and lysates were analyzed by Western blot (B). RNA collected at 48h was used for RT-PCR with the specified primer sets for each gene (C).

7.7.8.2 1324 PIRIN DOWN-REGULATES THE EAF2/U19 SIGNALING AND RETARDS THE GROWTH INHIBITION INDUCED BY EAF2/U19 IN PROSTATE CANCER CELLS

Zhongjie Qiao, Dan Wang, Zhou Wang
The Journal of Urology Apr 2013; 189(4), Supplement: e541
http://dx.doi.org/10.1016/j.juro.2013.02.2678
EAF2/U19, as the tumor suppressor, has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth in vivo, and its expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification and characterization of the binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells.

7.7.8.3 Pirin Inhibits Cellular Senescence in Melanocytic Cells

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression

Cellular senescence is a physiological process through which normal somatic cells lose their ability to divide and enter an irreversible state of cell cycle arrest, although they remain viable and metabolically active.1,2The specific molecular circuitry underlying the onset of cellular senescence is dependent on the type of stimulus and on the cellular context. A central role is held by the activation of the tumor suppressor proteins p53 and retinoblastoma susceptibility protein (pRB),3–5 which act by interfering with the transcriptional program of the cell and ultimately arresting cell cycle progression.

In the last decade, senescence has been recognized as a major barrier against the development of tumors in mammals.6–8 One of the most prominent in vivo examples is represented by nevi, in which cells proliferate after oncogene activation and then become senescent. Melanoma is a highly aggressive form of neoplasm often observed to derive from nevi, and the transition implies suppression of the mechanisms that sustain the onset and maintenance of senescence.9 In fact, many of the melanoma-associated tumor suppressor genes identified to date are themselves involved in control of senescence, including BRAF (encoding serine/threonine-protein kinase B-raf), CKD4 (cyclin-dependent kinase 4), and CDKN2A (encoding cyclin-dependent kinase inhibitor 2A isoforms p16INK4a and p19ARF).3,10

Nevi frequently harbor oncogenic mutations of the tyrosine kinase BRAF gene, particularly V600E,11 andBRAFV600E is also found in approximately 70% of cutaneous melanomas.12 Expression of BRAFV600E in human melanocytes leads to oncogene-induced senescence,8 which can be considered as a mechanism that protects from malignant progression. In time, some cells may eventually escape senescence, probably through the acquisition of additional genetic abnormalities, thus favoring transformation to melanoma.13

Pirin (PIR) is a highly conserved nuclear protein belonging to the Cupin superfamily14 whose function is, to date, poorly characterized. It has been described as a putative transcriptional regulator on the basis of its physical association with the nuclear I/CCAAT box transcription factor NFI/CTF115 and with the B-cell lymphoma protein, BCL-3, a regulator of NF-κB/Rel activity. A recent report shows that PIR controls melanoma cell migration through the transcriptional regulation of snail homolog 2, SNAI2 (previously SLUG).16 Other reports described quercetinase enzymatic activity,17 and regulation of apoptosis18,19 and stress response, unveiling a high degree of cell-type and species specificity in PIR function.

There is evidence of variations in PIR expression levels in different types of malignancies, but a systematic analysis of PIR expression in human tumors has been lacking. We analyzed PIR expression pattern in a collection of normal and neoplastic human tissues and found that it is expressed in scattered melanocytes, virtually absent in more mature regions of nevi, and present at high levels in a subset of melanomas. Functional studies performed in normal and transformed melanocytic cells revealed that PIR ablation results in cellular senescence, and that PIR levels decrease in response to senescence stimuli. Our results suggest that PIR may be a relevant player in the negative control of cellular senescence in PIR-expressing melanomas.

PIR overexpression in melanoma

Figure 3  PIR overexpression in PIR melanoma cells has no effect on proliferation.
PIR Expression Is Down-Regulated by BRAF Activation and Camptothecin Treatment

BRAF mutations are frequent in nevi, and are directly linked to the induction of oncogene-induced senescence. Variations in PIR expression levels were therefore investigated in an experimental model of senescence induced by oncogenic BRAF. Human diploid fibroblasts (TIG3–hTERT) expressing a conditional form of constitutively activated BRAF fused to the ligand-binding domain of the estrogen receptor (ER) rapidly undergo oncogene-induced senescence on treatment with 4-hydroxytamoxifen (OHT).28,29 PIR protein and mRNA levels were measured in TIG3-BRAF-ER cells at different time points of treatment with 800 nmol/L OHT. PIR expression was significantly repressed both at the mRNA and at the protein level after BRAF activation (Figure 6A), and remained at low levels after 120 hours, suggesting that a significant reduction of PIR expression is associated with the establishment of oncogene-induced senescence in different cell types.

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

Brian A. Lewis
Biochim et Biophys Acta (BBA) – Gene Regulatory Mechanisms Nov 2013; 1829(11): 1202–1206
http://dx.doi.org/10.1016/j.bbagrm.2013.09.003

Highlights

  • This review article discusses recent advances in the links between O-GlcNAc and transcriptional regulation.
  • Discusses several systems to illustrate O-GlcNAc dynamics: Tet proteins, MLL complexes, circadian clock proteins and RNA pol II.
  • Suggests that promoters are nutrient sensors.

Post-translational modifications play important roles in transcriptional regulation. Among the less understood PTMs is O-GlcNAcylation. Nevertheless, O-GlcNAcylation in the nucleus is found on hundreds of transcription factors and coactivators and is often found in a mutually exclusive ying–yang relationship with phosphorylation. O-GlcNAcylation also links cellular metabolism directly to the proteome, serving as a conduit of metabolic information to the nucleus. This review serves as a brief introduction to O-GlcNAcylation, emphasizing its important thematic roles in transcriptional regulation, and highlights several recent and important additions to the literature that illustrate the connections between O-GlcNAc and transcription.

links between O-GlcNAc and transcriptional regulation.

links between O-GlcNAc and transcriptional regulation.

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr1.sml
links between O-GlcNAc and transcriptional regulation.

systems to illustrate O-GlcNAc dynamics

systems to illustrate O-GlcNAc dynamics

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr2.sml
systems to illustrate O-GlcNAc dynamics

7.7.10 O-GlcNAcylation in cellular functions and human diseases

Yang YR1Suh PG2.
Adv Biol Regul. 2014 Jan; 54:68-73
http://dx.doi.org:/10.1016/j.jbior.2013.09.007

O-GlcNAcylation is dynamic and a ubiquitous post-translational modification. O-GlcNAcylated proteins influence fundamental functions of proteins such as protein-protein interactions, altering protein stability, and changing protein activity. Thus, aberrant regulation of O-GlcNAcylation contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, metabolic disorders, and Alzheimer’s disease. Diverse cellular signaling systems are involved in pathogenesis of these diseases. O-GlcNAcylated proteins occur in many different tissues and cellular compartments and affect specific cell signaling. This review focuses on the O-GlcNAcylation in basic cellular functions and human diseases.

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr2.sml
O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

aberrant regulation of O-GlcNAcylation in disease

aberrant regulation of O-GlcNAcylation in disease

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr3.sml
aberrant regulation of O-GlcNAcylation in disease

 Comment:

Body of review in energetic metabolic pathways in malignant T cells

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-B is required for T cell proliferation and differentiation of effector cells.
The TCR-to-NF-B pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-B.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation.
Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.

Dr. Aurel,
Targu Jiu

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