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Update on mitochondrial function, respiration, and associated disorders

Posted in Anaerobic Glycolysis, Ca2+ triggered activation, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Curation, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Experimental validation, Explanatory, Inferential analysis, Metabolomics, Na-K-ATPase, Nephrology, Neurohumoral Transmission, Oxidative phosphorylation, Phosphorylation, Proteomics, Translational Science, tagged 5HT1F, alpha ketoglutarate, ATP synthase, brown adipose tissue (BAT), C. Elegans, ca-uniporter (MCU), Ca2+-regulation, Cancer - General, CLL, coupled respiration, Glut4 transporter, inner membrane, K-depolarization, mitochondria, mTOR, NADPH, neurons, OCR, Oxidative phosphorylation, pyruvate -rate limiting, ROS, Sestrin2, TOR, UCP1 on July 8, 2014| Leave a Comment »

Larry H. Benstein, MD, FCAP, Gurator and writer

http://pharmaceuticalintelligence.com/7/8/2014/Update on mitochondrial function, respiration, and associated disorders

This is a condensed account of very recent published work on respiration and disturbed mitochondrail function. We know that their is an equilibrium between respiration and autophagy in eukaryotic cells. The Krebs Cycle produces 32 ATPs in oxidative phosphorylation, which is far more efficient than glycolysis. There is also a different contribution of mitochondrial metabolism, in the balance, between tissues that are synthetic and those that are catabolic. This is a subject long understood, essential for cellular energetics, and not adequately explored.

Gain-of-Function Mutant p53 Promotes Cell Growth and Cancer Cell Metabolism via Inhibition of AMPK Activation.

Zhou G¹, Wang J², Zhao M², Xie TX², Tanaka N², et al.
Mol Cell. 2014 Jun 19;54(6):960-974. doi: 10.1016/j.molcel.2014.04.024.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined.

We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells.

Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth.

Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation.

Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function. PMID:24857548

Investigating and Targeting Chronic Lymphocytic Leukemia Metabolism with the HIV Protease Inhibitor Ritonavir and Metformin.

Adekola KU, Aydemir SD, Ma S, Zhou Z, Rosen ST, Shanmugam M.
Leuk Lymphoma. 2014 May 14:1-23.

Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity.

Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal.

Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation.

CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL.

KEYWORDS: Basic Biology; Chemotherapeutic approaches; Lymphoid Leukemia; Signal transduction PMID: 24828872

Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

Lee ZW¹, Teo XY, Tay EY, Tan CH, Hagen T, Moore PK, Deng LW.
Br J Pharmacol. 2014 May 15. doi: 10.1111/bph.12773

Many disparate studies have reported the ambiguous role of hydrogen sulfide (H₂ S) in cell survival. The present study investigated the effect of H₂ S on viability of cancer and non-cancer cells.

Cancer and non-cancer cells were exposed to H₂ S (using sodium hydrosulfide, NaHS and GYY4137) and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis process by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi) was determined by ratiometric pHi measurement using BCECF staining.

Continuous, but not single, exposure to H₂ S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H₂ S-releasing donor, GYY4137, significantly increased glycolysis leading to overproduction of lactate. H₂ S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells.

Low and continuous exposure to H₂ S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

KEYWORDS: cancer cell death; cancer glucose metabolism; hydrogen sulfide; pH homeostasis PMID: 24827113

Agonism of the 5-Hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery from Acute Kidney Injury

Garrett SM, Whitaker RM, Beeson CC, and Schnellmann RG

Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina (S.M.G., R.M.W., C.C.B., R.G.S.); and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina (R.G.S.)
Address correspondence to: Dr. Rick G. Schnellmann, Department of Drug Discovery and Biomedical Sciences, MUSC, Charleston, SC 29425.
E-mail: schnell@musc.edu

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB.

The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT_1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT_1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT_1F receptor agonists,

LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and
LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1–100 nM)

increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone–uncoupled oxygen consumption in RPTC, and

validation studies confirmed both agonists increased mitochondrial proteins in vitro.
[e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)]

Small interfering RNA knockdown of the 5-HT_1F receptor

blocked agonist-induced MB.

Furthermore, LY344864 increased

peroxisome proliferator–activated receptor (PPAR) coactivator 1-α, Cox1, and
NDUFB8 transcript levels and
mitochondrial DNA (mtDNA) copy number

in murine renal cortex, heart, and liver.

Finally, LY344864 accelerated recovery of renal function, as indicated by

decreased blood urea nitrogen and kidney injury molecule 1 and
increased mtDNA copy number

following ischemia/reperfusion-induced acute kidney injury (AKI).

In summary, these studies reveal that

the 5-HT_1F receptor is linked to MB, 5-HT_1F receptor agonism promotes MB in vitro and in vivo, and

5-HT_1F receptor agonism promotes recovery from AKI injury.

Induction of MB through 5-HT_1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Footnotes

Portions of this work have been presented previously: Garrett SM, Wills LP, and Schnellmann RG (2012) Serotonin (5-HT) 1F receptor agonism as a potential treatment for acceleration of recovery from acute kidney injury.American Society of Nephrology Annual Meeting; 2012 Nov 1–4; San Diego, CA.
dx.doi.org/10.1124/jpet.114.214700.

Ca2+ regulation of mitochondrial function in neurons.

Rueda CB¹, Llorente-Folch I¹, Amigo I¹, Contreras L¹, González-Sánchez P¹, Martínez-Valero P¹, Juaristi I¹, Pardo B¹, Del Arco A², Satrústegui J³

Biochim Biophys Acta. 2014 May 10. pii: S0005-2728(14)00126-1.
doi: 10.1016/j.bbabio.2014.04.010.

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca²⁺ signal or to Ca²⁺ itself.

[Na⁺]_i, [Ca²⁺]_i and [ATP]_i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca²⁺ clearly showed that

Ca²⁺-stimulation of coupled respiration is required to maintain [ATP]_i levels.

Ca²⁺ may regulate respiration by

activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or
after Ca²⁺ entry in mitochondria through the calcium uniporter (MCU).

Two Ca²⁺-regulated mitochondrial metabolite transporters are expressed in neurons,

the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, with a Kd for Ca²⁺ activation of 300nM, and
the ATP-Mg/Pi exchanger SCaMC-3/Slc25a23, with S_0.5 for Ca²⁺ of 300nM and 3.4μM, respectively.

The lack of SCaMC-3 results in a smaller Ca²⁺-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas

the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca²⁺-dependent responses to all workloads (veratridine, K⁺-depolarization, carbachol).

The lack of ARALAR caused a reduction of about 65-70% in the response to the high workload imposed by veratridine, and

completely suppressed the OCR responses to moderate (K⁺-depolarization) and small (carbachol) workloads,
effects reverted by pyruvate supply.

For K⁺-depolarization, this occurs in spite of the presence of large [Ca²⁺]_mit signals and increased reduction of mitochondrial NAD(P)H.

These results show that ARALAR-MAS is a major contributor of Ca²⁺-stimulated respiration in neurons

by providing increased pyruvate supply to mitochondria.

In its absence and under moderate workloads, matrix Ca²⁺ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions.

KEYWORDS: ATP-Mg/Pi transporter; Aspartate–glutamate transporter; Calcium; Calcium-regulated transport; Mitochondrion; Neuronal respiration PMID: 24820519

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Ro SH¹, Nam M², Jang I¹, Park HW¹, Park H¹, Semple IA¹, Kim M¹, et al.
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7849-54.
doi: 10.1073/pnas.1401787111.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans.

Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation.

Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation.

Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments.

Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

KEYWORDS: aging; homeostasis; mouse; β-adrenergic signaling PMID: 24825887 PMCID: PMC4040599

Mitochondrial EF4 links respiratory dysfunction and cytoplasmic translation in Caenorhabditis elegans.

Yang F¹, Gao Y¹, Li Z², Chen L³, Xia Z⁴, Xu T⁵, Qin Y⁶Biochim Biophys Acta. 2014 May 15. pii: S0005-2728(14)00499-X.
doi: 10.1016/j.bbabio.2014.05.353.

How animals coordinate cellular bioenergetics in response to stress conditions is an essential question related to aging, obesity and cancer. Elongation factor 4 (EF4/LEPA) is a highly conserved protein that promotes protein synthesis under stress conditions, whereas its function in metazoans remains unknown.

Here, we show that, in Caenorhabditis elegans, the mitochondria-localized CeEF4 (referred to as mtEF4) affects mitochondrial functions, especially at low temperature (15°C).

At worms’ optimum growing temperature (20°C), mtef4 deletion leads to self-brood size reduction, growth delay and mitochondrial dysfunction.

Transcriptomic analyses show that mtef4 deletion induces retrograde pathways, including mitochondrial biogenesis and cytoplasmic translation reorganization.

At low temperature (15°C), mtef4 deletion reduces mitochondrial translation and disrupts the assembly of respiratory chain supercomplexes containing complex IV.

These observations are indicative of the important roles of mtEF4 in mitochondrial functions and adaptation to stressful conditions.

KEYWORDS: C. elegans; EF4(LepA/GUF1); Mitochondrial dysfunction; Retrograde pathways; Translation PMID: 24837196

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Chin RM¹, Fu X², Pai MY³, Vergnes L⁴, Hwang H⁵, Deng G⁶, Diep S², et al.
Nature 2014 Jun 19;509(7505):397-401. doi: 10.1038/nature13264.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits.

Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans.

ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution.

Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan.

We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells.

We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream.

Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction.

Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

PMID: 24828042