Posts Tagged ‘Lipids’

Protein profiling in cancer and metabolic diseases

Larry H. Bernstein, MD, FCAP, Curator



Deep Protein Profiling Key

Company has encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment.

Proteome Sciences plc has strongly encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment. These highlight the growing need for more detailed, personal assessment of protein profiles to improve the management of cancer treatment.

In the first study two groups from University College London and Cancer Research UK demonstrated that genetic mutations in cancer can lead to changes in the proteins on the cell surface1. These are new sequences which are seen as foreign by the body’s immune system and, with appropriate immunotherapy, the level of response in lung cancer was greatly enhanced.

However many of the patients with these types of mutations unfortunately still did not respond which highlighted the need for deeper analysis of the protein expression in tumours in order to better appreciate the mechanisms that contribute to treatment failure.

The second study, led by Professor Nigel Bundred of Manchester University, reported that use of two drugs that act on the same breast cancer target, an over-expressing protein called Her-2, were able to eradicate detectable tumours in around 10% of those treated in just 11 days, with 87% of those treated having a proteomic change indicating cells had stopped growing and/or cell death had increased2.

Whilst these results appear very promising it is worth noting that the over-expressing Her-2 target is only present in about 20% of breast tumours meaning this combination therapy was successful in clearing tumours in just 2% of the total breast cancer population.

Dr. Ian Pike, Chief Operating Officer of Proteome Sciences commented, “Both these recent studies should rightly be recognised as important steps forward towards better cancer treatment. However, in order to overcome the limitations of current drug therapy programs, a much deeper and more comprehensive analysis of the complex protein networks that regulate tumour growth and survival is required and will be essential to achieve a major advance in the battle to treat cancer.

“Our SysQuant® workflows provide that solution. As an example, in pancreatic cancer3 we have successfully mapped the complex network of regulatory processes and demonstrate the ability to devise personalised treatment combinations on an individual basis for each patient. A retrospective study with SysQuant® to predict response to the targeted drug Sorafenib in liver cancer is in process and we are planning further prospective trials to guide personalised treatment selection in liver cancer.

“We are already delivering systems-wide biology solutions through SysQuant® and TMTcalibrator™ programs to our clients that are generating novel biological data and results using more sensitive profiling that are helping them to better understand their drug development programs and to provide new biomarkers for tracking patient response in clinical trials.

“We are strongly positioned to deliver more comprehensive analysis of proteins and cellular pathways across other areas of disease and in particular to extend the use of SysQuant® with other leading cancer research groups in liver and other cancers.”

Proteome Sciences has also expanded its offering in personalised medicine through the use of its TMTcalibrator™ technology to uniquely identify protein biomarkers that reveal active cancer and other disease processes in body fluid samples. The importance of these ‘mechanistic’ biomarkers is that they are essential to monitor that drugs are being effective and that they can be used as early biomarkers of disease recurrence.

Using SysQuant® and TMTcalibrator™, Proteome Sciences can deliver more comprehensive analysis and provide unparalleled levels of sensitivity and breadth of coverage of the proteome, enabling faster, more efficient drug development and more accurate disease diagnosis.


Discovering ‘Outlier’ Enzymes

Researchers at TSRI and Salk Institute have discovered ‘Outlier’ enzymes that could offer new targets to treat type 2 diabetes and inflammatory disorders.

A team led by scientists at The Scripps Research Institute (TSRI) and the Salk Institute for Biological Studies have discovered two enzymes that appear to play a role in metabolism and inflammation—and might someday be targeted with drugs to treat type 2 diabetes and inflammatory disorders. The discovery is unusual because the enzymes do not bear a resemblance—in their structures or amino-acid sequences—to any known class of enzymes.

The team of scientists nevertheless identified them as “outlier” members of the serine/threonine hydrolase class, using newer techniques that detect biochemical activity. “A huge fraction of the human ‘proteome’ remains uncharacterized, and this paper shows how chemical approaches can be used to uncover proteins of a given functionality that have eluded classification based on sequence or predicted structure,” said co-senior author Benjamin F. Cravatt, chair of TSRI’s Department of Chemical Physiology.

“In this study, we found two genes that control levels of lipids with anti-diabetic and anti-inflammatory activity, suggesting exciting targets for diabetes and inflammatory diseases,” said co-senior author Alan Saghatelian, who holds the Dr. Frederik Paulsen Chair at the Salk Institute. The study, which appeared as a Nature Chemical Biology Advance Online Publication on March 28, 2016, began as an effort in the Cravatt laboratory to discover and characterize new serine/threonine hydrolases using fluorophosphonate (FP) probes—molecules that selectively bind and, in effect, label the active sites of these enzymes.

Pulling FP-binding proteins out of the entire proteome of test cells and identifying them using mass spectrometry techniques, the team matched nearly all to known hydrolases. The major outlier was a protein called androgen-induced gene 1 protein (AIG1). The only other one was a distant cousin in terms of sequence, a protein called ADTRP. “Neither of these proteins had been characterized as an enzyme; in fact, there had been little functional characterization of them at all,” said William H. Parsons, a research associate in the Cravatt laboratory who was co-first author of the study.

Experiments on AIG1 and ADTRP revealed that they do their enzymatic work in a unique way. “It looks like they have an active site that is novel—it had never been described in the literature,” said Parsons. Initial tests with panels of different enzyme inhibitors showed that AIG1 and ADTRP are moderately inhibited by inhibitors of lipases—enzymes that break down fats and other lipids. But on what specific lipids do these newly discovered outlier enzymes normally work?

At the Salk Institute, the Saghatelian laboratory was investigating a class of lipids it had discovered in 2014. Known as fatty acid esters of hydroxy fatty acids (FAHFAs), these molecules showed strong therapeutic potential. Saghatelian and his colleagues had found that boosting the levels of one key FAHFA lipid normalizes glucose levels in diabetic mice and also reduces inflammation.

“[Ben Cravatt’s] lab was screening panels of lipids to find the ones that their new enzymes work on,” said Saghatelian, who is a former research associate in the Cravatt laboratory. “We suggested they throw FAHFAs in there—and these turned out to be very good substrates.” The Cravatt laboratory soon developed powerful inhibitors of the newly discovered enzymes, and the two labs began working together, using the inhibitors and genetic techniques to explore the enzymes’ functions in vitro and in cultured cells.

Co-first author Matthew J. Kolar, an MD-PhD student, performed most of the experiments in the Saghatelian lab. The team concluded that AIG1 and ADTRP, at least in the cell types tested, appear to work mainly to break down FAHFAs and not any other major class of lipid. In principle, inhibitors of AIG1 and ADTRP could be developed into FAHFA-boosting therapies.

“Our prediction,” said Saghatelian, “is that if FAHFAs do what we think they’re doing, then using an enzyme inhibitor to block their degradation would make FAHFA levels go up and should thus reduce inflammation as well as improve glucose levels and insulin sensitivity.” The two labs are now collaborating on further studies of the new enzymes—and the potential benefits of inhibiting them—in mouse models of diabetes, inflammation and autoimmune disease.

“One of the neat things this study shows,” said Cravatt, “is that even for enzyme classes as well studied as the hydrolases, there may still be hidden members that, presumably by convergent evolution, arrived at that basic enzyme mechanism despite sharing no sequence or structural homology.”

Other co-authors of the study, “AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs,” were Siddhesh S. Kamat, Armand B. Cognetta III, Jonathan J. Hulce and Enrique Saez, of TSRI; and co-senior author Barbara B. Kahn of Beth Israel Deaconess Medical Center and Harvard Medical School


New Weapon Against Breast Cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says.

Harvard Stem Cell Institute (HSCI) researchers at Dana-Farber Cancer Institute (DFCI) and collaborators at Brigham and Women’s Hospital (BWH) have identified a molecular marker in normal breast tissue that can predict a woman’s risk for developing breast cancer, the leading cause of death in women with cancer worldwide.

The work, led by HSCI principal faculty member Kornelia Polyak and Rulla Tamimi of BWH, was published in an early online release and in the April 1 issue of Cancer Research.

The study builds on Polyak’s earlier research finding that women already identified as having a high risk of developing cancer — namely those with a mutation called BRCA1 or BRCA2 — or women who did not give birth before their 30s had a higher number of mammary gland progenitor cells.

In the latest study, Polyak, Tamimi, and their colleagues examined biopsies, some taken as many as four decades ago, from 302 participants in the Nurses’ Health Study and the Nurses’ Health Study II who had been diagnosed with benign breast disease. The researchers compared tissue from the 69 women who later developed cancer to the tissue from the 233 women who did not. They found that women were five times as likely to develop cancer if they had a higher percentage of Ki67, a molecular marker that identifies proliferating cells, in the cells that line the mammary ducts and milk-producing lobules. These cells, called the mammary epithelium, undergo drastic changes throughout a woman’s life, and the majority of breast cancers originate in these tissues.

Doctors already test breast tumors for Ki67 levels, which can inform decisions about treatment, but this is the first time scientists have been able to link Ki67 to precancerous tissue and use it as a predictive tool.

“Instead of only telling women that they don’t have cancer, we could test the biopsies and tell women if they were at high risk or low risk for developing breast cancer in the future,” said Polyak, a breast cancer researcher at Dana-Farber and co-senior author of the paper.

“Currently, we are not able to do a very good job at distinguishing women at high and low risk of breast cancer,” added co-senior author Tamimi, an associate professor at the Harvard T.H. Chan School of Public Health and Harvard Medical School. “By identifying women at high risk of breast cancer, we can better develop individualized screening and also target risk reducing strategies.”

To date, mammograms are the best tool for the early detection, but there are risks associated with screening. False positive and negative results and over-diagnosis could cause psychological distress, delay treatment, or lead to overtreatment, according to the National Cancer Institute (NCI).

Mammography machines also use low doses of radiation. While a single mammogram is unlikely to cause harm, repeated screening can potentially cause cancer, though the NCI writes that the benefits “nearly always outweigh the risks.”

“If we can minimize unnecessary radiation for women at low risk, that would be good,” said Tamimi.

Screening for Ki67 levels would “be easy to apply in the current setting,” said Polyak, though the researchers first want to reproduce the results in an independent cohort of women.


AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs

William H ParsonsMatthew J Kolar, …., Barbara B KahnAlan Saghatelian & Benjamin F Cravatt

Nature Chemical Biology 28 March 2016          

Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions.

Figure 1: Discovery and characterization of AIG1 and ADTRP as FP-reactive proteins in the human proteome.

(a) Competitive ABPP-SILAC analysis to identify FP-alkyne-inhibited proteins, in which protein enrichment and inhibition were measured in proteomic lysates from SKOV3 cells treated with FP-alkyne (20 μM, 1 h) or DMSO using the FP-biotin…


  1. Willems, L.I., Overkleeft, H.S. & van Kasteren, S.I. Current developments in activity-based protein profiling. Bioconjug. Chem. 25, 11811191 (2014).
  2. Niphakis, M.J. & Cravatt, B.F. Enzyme inhibitor discovery by activity-based protein profiling.Annu. Rev. Biochem. 83, 341377 (2014).
  3. Berger, A.B., Vitorino, P.M. & Bogyo, M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am. J. Pharmacogenomics 4,371381 (2004).
  4. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. USA 96, 1469414699 (1999).
  5. Simon, G.M. & Cravatt, B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J. Biol. Chem. 285, 1105111055 (2010).
  6. Bachovchin, D.A. et al. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening. Proc. Natl. Acad. Sci. USA 107, 2094120946 (2010).
  7. Jessani, N. et al. A streamlined platform for high-content functional proteomics of primary human specimens. Nat. Methods 2, 691697 (2005).
  8. Higa, H.H., Diaz, S. & Varki, A. Biochemical and genetic evidence for distinct membrane-bound and cytosolic sialic acid O-acetyl-esterases: serine-active-site enzymes. Biochem. Biophys. Res. Commun. 144, 10991108 (1987).

Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesteras-1 inhibitors

Proc Natl Acad Sci U S A. 2011 Apr 26; 108(17): 6811–6816.    doi:  10.1073/pnas.1015248108
National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC50 values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

Protein phosphorylation is a pervasive and dynamic posttranslational protein modification in eukaryotic cells. In mammals, more than 500 protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues on proteins (1). A much more limited number of phosphatases are responsible for reversing these phosphorylation events (2). For instance, protein phosphatase 2A (PP2A) and PP1 are thought to be responsible together for > 90% of the total serine/threonine phosphatase activity in mammalian cells (3). Specificity is imparted on PP2A activity by multiple mechanisms, including dynamic interactions between the catalytic subunit (C) and different protein-binding partners (B subunits), as well as a variety of posttranslational chemical modifications (2, 4). Within the latter category is an unusual methylesterification event found at the C terminus of the catalytic subunit of PP2A that is introduced and removed by a specific methyltransferase (leucine carbxoylmethyltransferase-1 or LCMT1) (5, 6) and methylesterase (protein phosphatase methylesterase-1 or PME-1) (7), respectively (Fig. 1A). PP2A carboxymethylation (hereafter referred to as “methylation”) has been proposed to regulate PP2A activity, at least in part, by modulating the binding interaction of the C subunit with various regulatory B subunits (810). A predicted outcome of these shifts in subunit association is the targeting of PP2A to different protein substrates in cells. PME-1 has also been hypothesized to stabilize inactive forms of nuclear PP2A (11), and recent structural studies have shed light on the physical interactions between PME-1 and the PP2A holoenzyme (12).

There were several keys to the success of our probe development effort. First, screening for inhibitors of PME-1 benefited from the fluopol-ABPP technology, which circumvented the limited throughput of previously described substrate assays for this enzyme. Second, we were fortunate that the NIH compound library contained several members of the ABL class of small molecules. These chiral compounds, which represent an academic contribution to the NIH library, occupy an unusual portion of structural space that is poorly accessed by commercial compound collections. Although at the time of their original synthesis (23) it may not have been possible to predict whether these ABLs would show specific biological activity, their incorporation into the NIH library provided a forum for screening against many proteins and cellular targets, culminating in their identification as PME-1 inhibitors. We then used advanced chemoproteomic assays to confirm the remarkable selectivity displayed by ABLs for PME-1 across (and beyond) the serine hydrolase superfamily. That the mechanism for PME-1 inhibition involves acylation of the enzyme’s conserved serine nucleophile (Fig. 3) suggests that exploration of a more structurally diverse set of ABLs might uncover inhibitors for other serine hydrolases. In this way, the chemical information gained from a single high-throughput screen may be leveraged to initiate probe development programs for additional enzyme targets.

Projecting forward, this research provides an example of how public small-molecule screening centers can serve as a portal for spawning academic collaborations between chemical biology and synthetic chemistry labs. By continuing to develop versatile high-throughput screens and combining them with a small-molecule library of expanding structural diversity conferred by advanced synthetic methodologies, academic biologists and chemists are well-positioned to collaboratively deliver pharmacological probes for a wide range of proteins and pathways in cell biology.


New weapon against breast cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says

April 6, 2016 | Popular


New Group of Aging-Related Proteins Discovered

Scientists have discovered a group of six proteins that may help to divulge secrets of how we age, potentially unlocking new insights into diabetes, Alzheimer’s, cancer, and other aging-related diseases.

The proteins appear to play several roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading investigators to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, M.D., dean and professor of the University of Southern California Leonard Davis School of Gerontology, identified the proteins and observed their origin from mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Dr. Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria. Dr. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body. Of particular interest is SHLP 2, according to Dr. Cohen.  The protein appears to have insulin-sensitizing, antidiabetic effects as well as neuroprotective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

Proteins That May Protect Against Age Related Illnesses Discovered


The cell proliferation antigen Ki-67 organises heterochromatin

 Michal Sobecki, 

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.


A protein called Ki-67 is only produced in actively dividing cells, where it is located in the nucleus – the structure that contains most of the cell’s DNA. Researchers often use Ki-67 as a marker to identify which cells are actively dividing in tissue samples from cancer patients, and previous studies indicated that Ki-67 is needed for cells to divide. However, the exact role of this protein was not clear. Before cells can divide they need to make large amounts of new proteins using molecular machines called ribosomes and it has been suggested that Ki-67 helps to produce ribosomes.

Now, Sobecki et al. used genetic techniques to study the role of Ki-67 in mice. The experiments show that Ki-67 is not required for cells to divide in the laboratory or to make ribosomes. Instead, Ki-67 alters the way that DNA is packaged in the nucleus. Loss of Ki-67 from mice cells resulted in DNA becoming less compact, which in turn altered the activity of genes in those cells.


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Lipids link to breast cancer

Larry H. Bernstein, MD, FCAP, Curator



Lipids Found Critical to Breast Cancer Cell Proliferation


Scientists in Spain report finding that breast cancer cells need to take up lipids from the extracellular environment so that they can continue to proliferate. The main protein involved in this process is LIPG, an enzyme found in the cell membrane and without which tumor cell growth is arrested. Analyses of more than 500 clinical samples from patients with various kinds of breast tumors reveal that 85% have high levels of LIPG expression.

The research (“FoxA and LIPG Endothelial Lipase Control the Uptake of Extracellular Lipids for Breast Cancer Growth”) is published in Nature Communications.

In Spain, breast cancer is the most common tumor in women and the fourth most common type in both sexes (data from the Spanish Society of Medical Oncology, 2012), registering more than 25,000 new diagnoses each year. According to figures from the World Health Organization, every year 1.38 million new cases of breast cancer are diagnosed and 458,000 people die from this disease (International Agency for Research on Cancer Globocan, 2008).

It was already known that cancer cells require extracellular glucose to grow and that they reprogram their internal machinery to produce greater amounts of lipids. The relevance of this study is that it reveals for the first time that tumor cells must import extracellular lipids to grow.

“This new knowledge related to metabolism could be the Achilles heel of breast cancer,” explains ICREA researcher and Institute for Research in Biomedicine–Barcelona group leader Roger Gomis, Ph.D., co-leader of the study together with Joan J. Guinovart, Ph.D., director of IRB Barcelona and professor at the University of Barcelona. Using animal models and cancer cell cultures, the scientists have demonstrated that blocking of LIPG activity arrests tumor growth.

“What is promising about this new therapeutic target is that LIPG function does not appear to be indispensable for life, so its inhibition may have fewer side effects than other treatments,” explains the first author of the study, Felipe Slebe, a Ph.D. Fellow at IRB Barcelona.

According to Dr. Guinovart, “because LIPG is a membrane protein, it is potentially easier to design a pharmacological agent to block its activity.”

“If a drug were found to block its activity, it could be used to develop more efficient chemotherapy treatments that are less toxic than those currently available,” adds Dr. Gomis.

The scientists are now looking into international collaborations for developing LIPG inhibitors.

FoxA and LIPG endothelial lipase control the uptake of extracellular lipids for breast cancer growth

Felipe SlebeFederico RojoMaria Vinaixa,…Joan AlbanellJoan J. Guinovart & Roger R. Gomis

Nature Communications7,Article number:11199

The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids.

FoxA1 and FoxA2 in BCa growth

The importance of FoxA1 in BCa cells differentiation and its contribution to controlling the expression of metabolic genes in several other tissues makes this transcription factor a highly attractive target to explain the metabolic alterations reported in BCa. For these reason, we decided to ascertain the metabolic processes controlled by FoxA1 in BCa. We first confirmed the association between high FoxA1 expression (mRNA and protein) and luminal subtype (Fig. 1a). To this end, we used two cohorts of primary breast tumours with annotated clinical features and follow-up. The MSKCC/EMC BCa data set is based on gene expression profiles from an original series of 560 cases10, whereas the Spanish BCa data set (n=439) is a tissue microarray of formalin-fixed paraffin-embedded stage I–III breast tumour specimens11 (details provided in Methods Section). High FoxA1 gene expression significantly correlated with high expression of well-established luminal markers, such as GATA3 and ESR1, in primary tumours (Supplementary Fig. 1a). Next we explored FoxA1 expression beyond the luminal subtype. Lower FoxA1 expression was observed in non-luminal tumours (Fig. 1a,b); however, a subset also expressed higher FoxA1 levels (Supplementary Fig. 1b and Supplementary Table 1). Given that FoxA2, in conjunction with FoxA1, is also involved in the regulation of several metabolic pathways, we determined the expression of this factor in BCa samples. Unfortunately, no FoxA2 probes in the Affymetrix platform used in the MSKCC/EMC data set provided a reliable interpretation. To overcome this limitation, we used tissue arrays of early BCa samples (Spanish BCa set). Histological examination of FoxA2-stained tissue microarray slides from the Spanish BCa set revealed the expression of this factor in six non-luminal samples, which were scored as FoxA1 (examples in Fig. 1b and summarized inSupplementary Table 1). Collectively, the number of FoxA+ BCa samples detected by immunohistochemistry accounted for 81.3% of all samples in the Spanish BCa set (Supplementary Table 1), which represent a significant proportion of BCa and point to the participation of FoxA in this disease, beyond to its involvement in differentiation and control of hormonal responses.

Figure 1: FoxA1 and FoxA2 in BCa growth.

(a, top) FoxA1 mRNA expression in the MSKCC/EMC set. BCa samples were stratified in Luminal A, Luminal B, Her2, triple negative and unknown subgroups. The unknown group represents specimens that were not classified in any group. (bottom) FoxA1 protein levels by IHC staining in Luminal, Her2 and triple negative samples in the Spanish BCa set (cohort of 439 BCa patients). Data is average±s.d. (b) FoxA1 and FoxA2 IHC staining in FFPE human specimens representative of the different BCa subtypes. Six independent cases are depicted. FoxA1 and FoxA2 are expressed mainly in the nuclei of tumour cells. Scale bar, 50μm. (c) FoxA1 and FoxA2 mRNA expression analysis by qRT-PCR and protein expression by western blot in human BCa cell lines compared with HMECs. T-test was used. Data are average±s.e.m.; n= 3. Of note, MDA435 are of melanoma origin. (d) FoxA1 and FoxA2 expression in MCF7, MDA231 and their derivatives cells by qRT-PCR and western blot. FoxA1 and FoxA2 depletion was achieved with a doxycycline-inducible short hairpin vector. FoxA-depleted cells were rescued by expression of FoxA2 in MCF7 cells or FoxA1 in MDA231 cells. Cell populations were cultured in the presence or absence of doxycycline for 6 days. P value is the result of T-test. Data are average±s.e.m.;n=3. *P≤0.05, ***P≤0.001 (e, left) Schematic representation of MDA231 and MCF7 cells grown without doxycycline and inoculated in Balb/c nude mice treated with or without doxycycline to induce the expression of the indicated FoxA short hairpins. All tumour cell lines have GFP constitutive expression, and tRFP concomitantly with the short hairpin were expressed in doxycycline treated tumours. (right) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points. P value is the result of T-test. Data are average±s.e.m.; n= 5–8 tumours. *P≤0.05,**P≤0.01, ***P≤0.001. FFPE, formalin-fixed paraffin-embedded.

Next, we extended our analysis to BCa cell lines for further mechanistic studies. We compared FoxA1 and FoxA2 mRNA expression in four estrogen receptor positive (ER+) (MCF7, T47D, BT474 and ZR75) and four estrogen receptor negative (ER−) (SKBR3, MDA468, BT20 and MDA231) BCa cell lines, a cell line of melanoma origin (MDA435), and human mammary epithelial cells (HMECs). Of note, two of the BCa lines tested were HER2+ (BT474 and SKBR3) (Fig. 1c). All ER+ BCa cells (MCF7, T47D, BT474 and ZR75), the ER−/HER2+ SKBR3 and both triple negative-like MDA468 and BT20 cell lines expressed FoxA1. Interestingly, MDA231 triple negative-like cells expressed high levels of FoxA2 but not FoxA1, and the non-tumour HMECs did not express these factors (Fig. 1c). No BCa cells co-expressed these two proteins (Fig. 1c). Our results suggest that the expression of FoxA transcription factors is a common feature of breast tumours, as well as of BCa cell lines. This notion implies that FoxA factors play a major role in BCa growth, independently of luminal fate specification.

To examine the molecular basis of the contribution of FoxA1 and FoxA2 to BCa growth, we engineered constitutive GFP-luciferase-expressing MCF7 and MDA231 cells with a doxycycline-inducible short-hairpin RNA (sh-RNA) vector targeting either FoxA1 or FoxA2. Doxycycline addition to the cell culture media decreased FoxA expression in both cell lines compared with control cells (ShControl (Dox+) and Sh FoxA1 or Sh FoxA2 (Dox−))(Fig. 1d), with the concomitant expression of tRFP (Supplementary Fig. 1c). Of note, there was no gain of expression of FoxA2 in FoxA1-depleted cells or vice versa (Fig. 1d). Interestingly, cancer cell proliferation was impaired in vitroupon depletion of either FoxA1 or FoxA2 in MCF7 and MDA231 cells, respectively (Supplementary Fig. 1d,e). Similarly, when Balb/c nude mice implanted with xenograft tumours from the above described cellular populations were treated with doxycycline and the short hairpins were induced, striking differences in tumour growth were observed. FoxA1-depleted MCF7 and FoxA2-depleted MDA231 tumour growth was blunted (Fig. 1e and additional controls in Supplementary Fig. 1f. Experimental details in the Supplementary Methods Section). Collectively, these observations confirm that FoxA1 or FoxA2 expression is required for BCa growth.

Previous studies indicate that FoxA1 and FoxA2 transcriptionally regulate common genes in the liver and pancreas that are central to development and metabolism. We therefore hypothesized that crossed expression of FoxA factors could rescue tumour growth by restoring the expression of essential metabolic genes. To this end, we engineered doxycycline-driven shFoxA1 MCF7 cells to express exogenous FoxA2 and doxycycline-driven shFoxA2 MDA231 cells to express exogenous FoxA1 (Fig. 1d). Interestingly, when these BCa modified cells were implanted in Balb/c nude mice and FoxA depletion was induced with doxycycline, the sustained expression of another FoxA factor (FoxA2 in MCF7 and FoxA1 in MDA231 cells) was sufficient for tumours to continuously grow (Fig. 1e and additional controls in Supplementary Fig. 1f). Quantitative real-time PCR (qRT-PCR) analysis confirmed FoxA expression in the distinct tumour populations ex-vivo (Supplementary Fig. 1g). These results showed that retention of minimal levels of FoxA1 or FoxA2 expression is necessary for BCa cell growth.

FoxA1- and FoxA2-regulated transcripts for BCa growth

Figure 2: A genomic approach to identify FoxA1- and FoxA2-regulated transcripts in MCF7 and MDA231 cells.

(a) FACS profiling of MCF7 and MDA231 cells derived from tumours isolated from mice on the basis of the expression of GFP+ and RFP− (control group) or GFP+ and tRFP+ (knockdown and rescue groups). (b) Representation of the transcripts up- and downregulated by FoxA in MCF7 and MDA231 cells isolated from tumours. Up- and downregulated transcripts present a Bayesian false discovery rate below 5% and fold change >2.5. (c) LIPG, Bcl2 and Cdh11OB mRNA levels of the indicated genetically modified MCF7 and MDA231 tumour xenografts analysed by qRT-PCR. P value is the result of T-test. Data are average±s.e.m.; n= 5–8 tumours. *P≤0.05, ***P≤0.001. (d) LIPG protein expression in constitutive shFoxA1 MCF7 or shFoxA2 MDA231 cells. (e) Promoter reporter assay in HEK 293 cells. Cells were transfected with LIPG promoter reporter and FoxA1 or FoxA2 expressing vectors when indicated. P value is the result of T-test. Data are average±s.e.m.; n=3. ****P≤0.0001.

LIPG expression in BCa

Next, we showed that LIPG expression in primary tumours was specific to BCa tumour cells and not to other stroma cellular entities (Fig. 3a). Subsequently, we tested LIPG expression in normal breast epithelia and interrogated 20 samples from mammoplasty reductions. Normal breast epithelial cells showed a lower expression of LIPG than cells from tumour specimens (Fig. 3b). Similar results were obtained for LIPG protein levels in a panel from BCa lines compared with HMEC cells. Of the cellular populations tested, the eight BCa cell lines expressing FoxA1 or FoxA2 had very high levels of LIPG protein compared with the melanoma MDA435 cell line and the human epithelial cell (Fig. 3c). Consistent with this observation, 83.8% of BCa samples in the Spanish tumour cohort were LIPG+ (Fig. 3d and Supplementary Table 3), and LIPG expression correlated with FoxA expression (Spearman correlation; r=0.477, P=0.000001; Fig. 3e). Further analysis showed that LIPG expression levels in primary tumours do not have the capacity to stratify patients for differential risk of overall or disease-free survival (Supplementary Fig. 2a) and are not dependent on estrogen signalling (Supplementary Fig. 2b), thus reinforcing the notion that LIPG is essential for BCa growth.

Figure 3: LIPG contributes to BCa growth.

a) Representative LIPG IHC staining on primary BCa tissues (cohort of 439 BCa patients). LIPG is expressed in the cytoplasm of tumour cells. Faint staining is also detected in the extracellular area. Scale bar, 50μm. (b) Representative LIPG IHC staining in normal breast tissue from mammoplasty reductions. Weak LIPG expression occurs in epithelial cells from ducts and lobuli. Scale bar, 50μm. (c) LIPG protein expression in human cancer cell lines compared with HMECs. Actin was used as loading control.*Unspecific band. Of note, MDA435 are of melanoma origin. (d) LIPG protein levels by IHC staining in Luminal, Her2, and triple negative samples in the Spanish BCa set (cohort of 439 BCa patients). Data is average±s.d. (e) Spearman correlation (P=0.000001) between FoxA and LIPG IHC staining intensities in Spanish BCa set (cohort of 439 BCa patients). (f) Left panel, in vitro proliferation curves of MCF7 and MDA231 cells transduced with a control or a LIPG short hairpin. Data are average±s.e.m.; n=3. (right) LIPG protein expression in shLIPG MCF7 and shLIPG MDA231 cells. The blot shown is representative of three independent experiments. P value is the result of T-test.**P≤0.01, ***P≤0.001. (g) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points.P value is the result of T-test. Data are average±s.e.m.; n= 6–8 tumours. *P≤0.05.

LIPG is a phospholipase located in the cytosol and cellular membrane and has been shown to hydrolyse extracellular phospholipids from high-density lipoprotein that are afterwards incorporated into intracellular lipid species thus providing lipid precursors of cell metabolism17, 18. Thus we questioned whether LIPG regulates essential lipid intake in BCa and whether it is necessary for proliferation. To validate this hypothesis, we genetically downregulated the expression of this protein in MCF7 and MDA231 cells by means of sh-RNA (Fig. 3f and Supplementary Fig. 2c). LIPG depletion blunted BCa cell capacity to proliferate in vitro (Fig. 3f), as previously observed in FoxA-depleted cells (Supplementary Fig. 1d,e), and caused a reduction in invasion and self-renewal properties (Supplementary Fig. 3a–d). Similarly, LIPG-depleted cells were unable to grow tumours in vivo (Fig. 3g).

LIPG induces BCa cells lipid metabolic reprograming

Figure 4: LIPG regulates the uptake of lipids in BCa cells inducing a lipid metabolic reprograming.

LIPG regulates the uptake of lipids in BCa cells inducing a lipid metabolic reprograming.

(a) Schematic representation of LIPG action. (b) Heat map representation of the downregulated (blue) lipids identified by MS/MS in the cell homogenates of MCF7 or MDA231 LIPG-depleted cells compared with shControl cells. Depicted lipids have a fold change >1.5 and P value<0.05 using the Welch’s t-testn=5. (c) Downregulated lipid species (previously identified in b) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG (blue box). P values are <0.05 and calculated using Welch’s t-test, n=5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. (d) Heat map representation of the upregulated (red) lipids identified by MS/MS in the media of MCF7 or MDA231 LIPG-depleted cells compared with the corresponding shControl cells. Characterized lipids have a fold change >1.5 and P value<0.05 using the Welch’s t-test n=5. (e) Upregulated lipid species in the media (previously identified in d) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG cells (blue box). P values are <0.05 and calculated using Welch’s t-test, n=5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. (f) Heat map representation of the MS/MS downregulated (blue) lipids in the cell media of MCF7/MDA231 LIPG-depleted or shControl cells (as described in d) compared with fresh medium (without cell incubation). Depicted lipid species have a log2 fold change>1.5 and P value<0.05 using the Welch’s t-test n=5. (g) MDA231 and MCF7 cell growth for 48h in complete medium: medium containing 10% FBS 10%); lipoprotein-free medium: medium containing 10% free lipoprotein FBS; and LPC (18:0): medium containing 10% free lipoprotein FBS and 20μM of LPC (18:0). P value is the result of T-test. Data are average±s.e.m.; n=3. **P≤0.01, ***P≤0.001, ****P≤0.0001. (h) Above, schematic representation of the experimental protocol used. (bottom) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice treated with high-fat diet (HFD) are determined at the indicated time points. P value is the result of T-test. Data are average±s.e.m.; n= 6–8 tumours. *P≤0.05, **P≤0.01. Inside graph, plasma cholesterol levels of animals treated with standard diet (SD) or HFD. P value is the result of T-test. Data are average±s.e.m.; n= 4 animals per group. **P≤0.01, ***P≤0.001.

LIPG location has been shown to be functional on the outer face of the cellular membrane (Fig. 4a)18, thus we postulated the possibility that BCa cells are dependent on LIPG function to access extracellular lipids to support their growth needs. To test this notion, we profiled the media of control and LIPG-depleted MCF7 and MDA231 cells following the same liquid chromatography-mass spectrometry-based untargeted lipidomic approach as for cell homogenates. LIPG depletion prevented the absorption of particular lipids from the media (Supplementary Fig. 4a). The structural identification of the lipids by MS/MS confirms the absence of degradation of glycerophospholipids belonging to the LPC class in both MCF7 and MDA231 cells, which is depicted by higher levels in the media of these species in LIPG-depleted when compared with control cells (Fig. 4d,e). Interestingly when we analysed the LPCs species in the media of control and LIPG-depleted cells and compared with fresh media (without cells), all LPC species from control cell media were decreased. This reduction was weaker in the media of Sh LIPG cells, indicating that LIPG-depleted cells have a defect in processing and importing of pre-existing lipid species from the medium (Fig. 4f).

Finally, we evaluated which of the commonly identified potential substrates of LIPG sustains BCa cell proliferation. Initially, we confirmed that the growth of MCF7 and MDA231 cells is impaired when grown in vitro in lipoprotein-depleted media (Fig. 4g). Next we tested the capacity of LPC (18:0) to rescue BCa cell growth in the absence of lipoproteins and confirmed that this lysophosphatidylcholine was able to restore the cells’ capacity to proliferate (Fig. 4g). In accordance, this process was dependent on LIPG expression (Fig. 4g). Similarly, LIPG-depleted cells were not able to grow in vivo in animals fed with high-fat diet (Fig. 4h) indicating that LIPG is indispensable to process the extracellular lipids and mediate their uptake by the cells, irrespectively of the concentration of lipid substrates in circulation, a phenotype also observed in FoxA-depleted cells (Fig. 4h).

LIPG activity supports BCa growth

Figure 5: LIPG activity is essential for BCa growth.

LIPG activity is essential for BCa growth.

(a, top) Homology 3D structural model of LIPG (backbone coloured according to the QMEANlocal parameter values; red residues with low error). The heavy atoms of the three catalytic residues are shown explicitly and the residue mutated in this study is shown in green (Asp 193). (b) FoxA1, FoxA2 and LIPG protein expression in MCF7, MDA231 and their derivative cells determine by western blot. FoxA1 and FoxA2 depletion was achieved with a doxycycline-inducible short hairpin vector. FoxA-depleted cells were rescued by expression of a WT or Inactive LIPG. Cell populations were cultured in the presence or absence of doxycycline for 6 days. *blots represent different exposition times. (c) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points. Pvalue is the result of T-test. Data are average±s.e.m.; n=5–8 tumours. *P≤0.05, **P≤0.01. (d) MDA231 and MCF7 cell growth for 48h treated with DMSO (control), FAS inhibitor (C75) and/or lipase inhibitor (Orlistat). For MDA231 cells C75 was used at a final concentration of 10μgml−1 and for MCF7 cells 8μgml−1. Orlistat was used at a final concentration of 30 or 10μgml−1 in MCF7 or MD231 respectively. Pvalue is the result of T-test. Data are average±s.e.m.; n=3.*P≤0.05, **P≤0.01, ***P≤0.001. (e) Forty-eight hours cell growth of MDA231 or MCF7 cells overexpressing exogenous WT or Inactive LIPG. Cells were treated with DMSO (control) and FAS inhibitor (C75) at a final concentration of 20μgml−1. P value is the result of T-test. Data are average±s.e.m.; n=3.***P≤0.001, ****P≤0.0001 (f) Schematic representation showing how FoxA controls LIPG and lipid metabolism to support tumour growth.

As previous reports showed that de novo lipid metabolism is necessary for BCa growth3, 22, we next questioned whether this lipid synthesis was sufficient or, instead, whether exogenous sources are also required to support BCa cell growth and proliferation, as suggested by our experimental data. To this end, we inhibited the activity of fatty acid synthase (FAS) in BCa cells by means of the chemical inhibitor C75 (ref. 23). FAS activity is crucial for de novo lipid synthesis in cancer cells3,22. To test the complementarity of both de novo and/or exogenous lipid supplies, we used a C75 concentration causing a 50% reduction in BCa cell growth in vitro 48h post incubation (Fig. 5d andSupplementary Fig. 5d). Similarly, we tested the contribution of LIPG inhibition by means of treatment with a lipase inhibitor, Orlistat21. A specific dose causing a 50% reduction in the growth of each BCa cell line was further used (Fig. 5d and Supplementary Fig. 5d). Interestingly, concomitant treatment with FAS and LIPG inhibitors caused an additive effect, blunting BCa cell growth (Fig. 5d). Next, we evaluated whether LIPG activity was sufficient to rescue the chemical inhibition of FAS. To this end, we overexpressed WT and inactive LIPG and grew MCF7 and MDA231 cells in the presence or absence of a high dose of C75 (20mgml−1), which blocks cell growth (Supplementary Fig. 5d). Complete blockade of FAS was not rescued by LIPG (Fig. 5e). Collectively, our results suggest that both exogenous lipid precursors provided by means of LIPG activity and de novo lipid synthesis mediated by FAS are necessary for BCa cell growth.


Here we reveal that FoxA factors provide a central metabolic growth function by specifically regulating LIPG expression, thereby allowing the acquisition of indispensable extracellular lipids for BCa tumour proliferation. FoxA family of transcription factors are expressed in the vast majority of BCa and FoxA1 is expressed across various BCa subtypes. Moreover we show that, in some cases, its absence is associated with the expression of FoxA2. Interestingly, in addition of FoxA1 contribution to luminal commitment24, 25, 26, 27 the factor may drive BCa growth by specifically regulating LIPG levels.

The catalytic activity of LIPG generates extracellular lipid precursors that are imported to fulfill the intracellular production of lipid species (Fig. 5f). LIPG downregulation blocks BCa cell growth, thereby indicating that the import of extracellular lipid precursors is important for the proliferation of these cells. This is a striking observation given that it is generally believed that de novo fatty acid synthesis is the main driver of tumour growth22. Indeed, our experimental data with LIPG-depleted BCa cells revealed a massive decrease of most intracellular glycerolipid intermediates in the synthesis of TG (PC, PE, PG and DG) and their derivatives (LPC and LPE). Accordingly, certain lipid species (LPC) in the media were not decreased in LIPG-depleted cells as much as in control cells, thus indicating that extracellular lipids are the substrates for intracellular lipid production. In particular, we demonstrate the relevance of extracellular LPC (18:0) for BCa cell proliferation in a lipoprotein-depleted medium, a process dependent on LIPG. In this context, a high-fat diet was shown to rescue the absence of a critical intracellular lipase, Monoacylglycerol lipase, for cancer pathogenesis given cancer cells ability to uptake lipids from the extracellular compartment was functional19. Herein, we showed that this rescue mechanism is not functional in BCa cells in the absence of FoxA2 or LIPG. In support of this notion, it is worth noting that extracellular LIPG activity releases fatty acids from high-density lipoprotein phospholipids and these acids are further employed for intracellular lipid production in the human hepatic cell line HepG2 (refs 28, 29).

In conclusion, BCa cells are dependent on a mechanism to supply precursors derived from extracellular sources for intracellular lipid production, and LIPG fulfills this function. Therefore, LIPG stands out as an important component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. Our results also suggest thatde novo lipid synthesis is necessary but not sufficient to support lipid production for BCa tumour growth. Accordingly, recent clinical studies demonstrate the association between lipids and lipoproteins in circulation and risk of BCa in women with extensive mammographic density. This observation implies that interventions aimed to reduce them may have effect on BCa risk30. All together, these observations make LIPG activity an Achilles heel of luminal and, more importantly, of triple negative/basal-like breast tumours, for which limited therapeutic options are currently available.

In normal cells, the glucose carbon flow is directed into a de novo lipogenic pathway that is regulated, in part, via phosphoinositide-3 kinase (PI-3K)-dependent activation of ATP citrate lyase (ACL), a key rate-limiting, enzyme in de novo lipogenesis. ACL is a cytosolic enzyme that catalyzes the generation of acetyl CoA from citrate. Inhibition of ACL results in a loss of B-cell growth and cell viability [10] .
The plasma membrane and its constituent phosphoinositides form the basis of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway, which is crucial for cell proliferation and survival. Phosphatase and tensin-homolog deleted on chromosome 10 (PTEN) is a tumor-suppressor protein that regulates phosphatidylinositol 3-kinase (PI3-K) signaling by binding to the plasma membrane and hydrolyzing the 3′ phosphate from phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) to form phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Several loss-of-function mutations in PTEN that impair lipid phosphatase activity and membrane binding are oncogenic, leading to the development of a variety of cancers. Of these three residues, R335 was observed to interact with the membrane to the greatest extent across all of the simulations. R335L, in common with several other germline mutations, has been associated with the inherited cancer [11] .
ACLY is up-regulated or activated in several types of cancers, and its inhibition is known to induce proliferation arrest in cancer cells both in vitro and in vivo. The last studies were showed that BCR-mediated signaling is regulated in part by the amount of membrane cholesterol. It was observed that statins (Lovostatin), the pharmacological inhibitors of cholesterol synthesis, induce apoptosis of CLL cells in vitro and in vivo. Also the ectopic expression of CD5 in a B-cell line stimulates the transcription of genes involved in the synthesis of cholesterol [12] .

[10] Zaidi N, Swinnen JV, Smans K. ATP-citrate lyase: a key player in cancer metabolism Cancer Res; 2012 (11): 3709-14.

[11] Craig N, Mark S.P. Sansom. Defining the Membrane-Associated State of the PTEN Tumor Suppressor Protein. Biophys J 2013; 5; 104(3: 613–21.

[12] Tomowiak C, Kennel A, Gary-Gouy, Hadife N. High Membrane Cholesterol in CLL B-Cells and Differential Expression of Cholesterol Synthesis Genes in IG GENE Unmutated vs Mutated Cells. British Journal of Medicine & Medical Research 2012; 2(3): 313-26.


Cancer’s Vanguard

Exosomes are emerging as key players in metastasis.

By Catherine Offord | April 1, 2016

PREPARING THE TURF: Before tumor cells arrive at their metastatic destination, part of the site is readied for them. One recent study of liver metastasis in mice found that resident macrophages called Kupffer cells take up exosomes from the original tumor (1). Additionally, macrophages from the bone marrow show up upon the release of fibronectin by other liver cells called stellate cells (2). A current proposal for additional steps in metastatic niche development includes the recruitment of epithelial cells and fibroblasts, which contribute to angiogenesis, and, finally, the arrival of tumor cells themselves (3).© IKUMI KAYAMA/STUDIO KAYAMA

In 2005, David Lyden noticed something unexpected. He and his colleagues at Weill Cornell Medical College had been researching metastasis—the spread of cancer from one part of the body to another. The team had shown that bone marrow–derived cells (BMDCs) were recruited to future metastatic sites before the arrival of tumor cells, confirming that metastasis occurred after a habitable microenvironment, or “premetastatic niche,” had been prepared.1

But carefully studying images of this microenvironment in the lung tissue of mice, Lyden saw something else. Amongst the BMDCs, the micrographs showed tiny specks, far too small to be cells, gathering at the future site of metastasis. “I said, ‘What are these viruses doing here?’” recalls Lyden. “I had no idea about exosomes, microvesicles, and microparticles.”

Those specks, Lyden would come to realize, were in fact primary tumor–derived exosomes. These membrane-enclosed vesicles packed full of molecules are now attracting growing attention as important mediators of intercellular communication, particularly when it comes to cancer’s insidious capacity to spread from one organ to another.

Preparing the ground

Tumors require a community of support cells, including fibroblasts, BMDCs, and endothelial cells, to provide functional and structural assistance and to modulate immune system behavior. Bringing together the first members of this community before the arrival of tumor cells is all part of cancer’s survival strategy, says Joshua Hood, a cancer researcher at the University of Louisville.

“It wouldn’t be efficient for tumor cells to strike out on their own, and just say, ‘Oh, here we are!’” he says. “They would run the risk of being destroyed.” Preparing a “nest” in advance makes the process much safer. “Then the tumor can just efficiently come along and set up shop without ever having to fight much of a battle with the immune system.”

But although Lyden’s group had shown that this preparation was taking place, it remained unclear how such a process might be regulated. For the next few years, many cancer researchers believed that tumor cells must communicate with the premetastatic niche primarily through tumor-secreted signaling molecules such as cytokines.

Meanwhile, research into extracellular vesicles, previously considered biological garbage bags, was revealing new modes of intercellular communication. In 2007, a group of scientists in Sweden discovered that exosomes, tiny vesicles measuring just 30 nanometers to 100 nanometers across, transport mRNA and microRNAs intercellularly, with the potential to effect changes in protein synthesis in recipient cells.2 A new means for tumors to regulate distant cellular environments came into focus, and research on exosomes exploded. In 2011, Hood and his colleagues showed that exosomes facilitate melanoma metastasis through the lymphatic system.3 The following year, Lyden’s group demonstrated that tumor-derived exosomes can direct BMDCs to one of melanoma’s most common sites of metastasis, the lung.4 Exosomes, it seemed, had been underestimated.

Tiny terraformers

Armed with the knowledge that exosomes are involved in multiple stages of melanoma metastasis, Lyden’s lab went searching for the vesicles’ potential role in the metastasis of other cancers. Turning to pancreatic ductal adenocarcinoma (PDAC)—one of the most lethal cancers in humans—postdoctoral researcher Bruno Costa-Silva led a series of exhaustive in vitro and in vivo experiments in mouse models to detail the process of premetastatic niche formation in the liver, PDAC’s most common destination. The team’s results, published last May, reveal an intricate series of sequential steps—mediated by PDAC-derived exosomes (Nature Cell Biol, 17:816-26, 2015).

Using fluorescence labeling, Lyden’s group observed that PDAC-derived exosomes are taken up by Kupffer cells, specialized macrophages lining the outer walls of blood vessels in the liver. There, the exosomes trigger the cells’ secretion of transforming growth factor β (a type of cytokine involved in cell proliferation), plus the production of fibronectin by neighboring hepatic stellate cells, and the recruitment of BMDCs.

The researchers also showed that this cascade of events could be inhibited by depleting exosomal macrophage migratory inhibitory factor (MIF), an abundant protein in PDAC exosomes. “If you target the specific proteins of exosomes, you can reduce metastasis,” explains coauthor Héctor Peinado, leader of the microenvironment and metastasis group at the Spanish National Cancer Research Center.

For Hood, the findings add to a developing picture of exosomes’ vital role as “vanguard” in the progression of cancer. “It’s like the colonization of a new planet,” he says. “They’re terraforming the environment to make it hospitable.”



  • B. Costa-Silva et al., “Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver,”Nature Cell Biol, 17:816-26, 2015.
  • A. Hoshino et al., “Tumour exosome integrins determine organotropic metastasis,” Nature, 527:329-35, 2015.
  • L. Zhang et al., “Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth,”Nature, 527:100-04, 2015.

Internal mail

Although research was revealing the steps involved in forming premetastatic sites, it was less clear how these sites were being selected. “This has always been a great mystery in cancer,” says Ayuko Hoshino, a research associate in Lyden’s lab. “Why do certain cancers metastasize to certain organs?”

One theory, proposed in 1928 by pathologist James Ewing, suggested that anatomical and mechanical factors explained organ specificity in metastasis. The premetastatic niche, then, might form wherever exosomes are likely to land. But this couldn’t be the whole story, says Hoshino. “For instance, there’s eye melanoma. Thinking about that site, you could imagine it metastasizing to the brain. But actually, it almost only metastasizes to the liver.”

Because exosomes arrive at metastatic sites before tumor cells, the team reasoned, perhaps the exosomes themselves were organotropic (i.e., attracted to particular organs or tissues). Sure enough, Lyden says, when Hoshino and Costa-Silva began injecting tumor-derived exosomes into mice, “their preliminary findings were that wherever they injected the exosomes, the pancreatic cancer ones were ending up in the liver and the breast metastasis exosomes would end up in the lung.”

Using mass spectrometry, the researchers analyzed the protein content of exosomes from lung-tropic, liver-tropic, and brain-tropic tumors. They found that the composition of exosomes’ integrins—membrane proteins involved in cell adhesion—was destination-specific (Nature, 527:329-35, 2015). Exosomes bearing integrin α6β4, for example, were directed to the lung, where they could prepare a premetastatic niche potent enough even for normally bone-tropic tumor cells to colonize. Integrin αvβ5, meanwhile, directed metastasis to the liver.

The researchers also showed that exosomal integrins didn’t necessarily correspond to the parent-cell proteins, making exosomes potentially better indicators of where a cancer will spread than the tumor cells themselves. “We can show that an integrin that’s high in the tumor cell might be completely absent in the tumor exosome or vice versa,” says Lyden, adding that, taken together, the results point to a role for exosomes in “dictating the future sites of metastasis.”

“It’s a beautiful story,” says Dihua Yu, a molecular and cellular oncologist at the University of Texas MD Anderson Cancer Center. “This is a very novel finding that gives really good indicators for potential strategies to intervene in metastasis.”

Metastatic crosstalk

In the same month that Lyden’s group published its work on organotropism, Yu’s own lab published a different exosome study—one that told another side of the story.

Yu and her colleagues had found that when tumor cells in mice metastasized to the brain, they downregulated expression of a tumor suppressor gene called PTEN, and became primed for growth at the metastatic site. When the tumor cells were taken out of the microenvironment and put in culture, however, they restored normal PTEN expression.

The researchers demonstrated that a microRNA from astrocytes—star-shape glial cells in the brain—reversibly downregulated the levels of PTEN transcripts in the tumor cells, but they couldn’t figure out how the microRNA was getting into the tumor. Blocking “obvious signaling pathways,” such as gap junctions, failed to have an effect, Yu says.

Scrutinizing astrocyte-conditioned media using electron microscopy, the researchers identified spherical vesicles between 30 nanometers and 100 nanometers in diameter—the defining size of exosomes. Exposing mouse tumor cells to these vesicles increased cell microRNA content and reduced PTENexpression (Nature, 527:100-04, 2015). The study revealed yet another role for exosomes in the communication between tumors and their microenvironment.

The findings were a surprise, says Yu, not least because they showed a different perspective from the bulk of recent research. “We’re talking about astrocytes in the brain secreting exosomes to give welcome help to the cancer cells,” she says.

“I find it an extremely interesting paper because it shows that the astrocytes can change the whole phenotype of the tumor in the brain,” says Lyden. He adds that the results underline the importance of studying the mutational status of tumors at various sites. “All this work in exosomes, it adds to the complexity,” he says. “We can’t just target tumor cells at the primary site. We’ll have to understand all the details of metastasis if we’re really going to tackle it.”

What’s next?

The discovery of multiple roles for exosomes in metastasis has generated excitement about the potential for their use in diagnostics and treatment. As protective containers of tumor-derived genetic material, exosomes could provide information about the status of cancer progression. And as mediators of premetastatic niche formation, they make obvious targets for inhibition. (See “Banking on Blood Tests,”here.)

Exosomes might even be useful as vehicles to deliver drugs because they’re patient-matched and “naturally designed to function in a biocompatible way with living systems,” says Hood. “You could take them out of people, and at some point down the road try to have patients be their own nanofactory, using their own particles for treatment purposes.”

Pancreatic cancer exosomes initiate pre-metastatic nihe formation in the liver

Bruno Costa-SilvaNicole M. AielloAllyson J. Ocean, et al.   Nature Cell Biology 2015; 17,816–826

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Ayuko HoshinoBruno Costa-SilvaTang-Long ShenGoncalo RodriguesAyako HashimotoMilica Tesic Mark, et al. Nature Nov 2015; 527,329–335

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  1. Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 8, 98101 (1989)
  2. Hart, I. R. & Fidler, I. J. Role of organ selectivity in the determination of metastatic patterns of B16 melanoma. Cancer Res. 40, 22812287 (1980)
  3. Müller, A. et al. Involvement of chemokine receptors in breast cancer metastasis. Nature410, 5056 (2001)
  4. Weilbaecher, K. N., Guise, T. A. & McCauley, L. K. Cancer to bone: a fatal attraction. Nature Rev. Cancer 11, 411425 (2011)
  5. Zhou, W. et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 25, 501515 (2014)
  6. Chang, Q. et al. The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis.Neoplasia 15, 848862 (2013)
  7. Lu, X. & Kang, Y. Organotropism of breast cancer metastasis. J. Mammary Gland Biol. Neoplasia 12, 153162 (2007)


Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth

Lin ZhangSiyuan ZhangJun YaoFrank J. LoweryQingling ZhangWen-Chien Huang, et al.  Nature  Nov 2015; 527,100–104

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells’ adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites1. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs2. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth3, 4. Yet, it is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. Here we show that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. Furthermore, this adaptive PTEN loss in brain metastatic tumour cells leads to an increased secretion of the chemokine CCL2, which recruits IBA1-expressing myeloid cells that reciprocally enhance the outgrowth of brain metastatic tumour cells via enhanced proliferation and reduced apoptosis. Our findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth. Our findings signify the dynamic and reciprocal cross-talk between tumour cells and the metastatic niche; importantly, they provide new opportunities for effective anti-metastasis therapies, especially of consequence for brain metastasis patients.

  1. Quail, D. F. & Joyce, J.A. Microenvironmental regulation of tumor progression and metastasis. Nature Med. 19, 14231437 (2013)
  2. Park, E. S. et al. Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis. Proc. Natl Acad. Sci. USA 108, 1745617461 (2011)
  3. Joyce, J. A. & Pollard, J. W. Microenvironmental regulation of metastasis. Nature Rev. Cancer 9, 239252 (2009)
  4. Vanharanta, S. & Massagué, J. Origins of metastatic traits. Cancer Cell 24, 410421 (2013)
  5. Gray, J. Cancer: genomics of metastasis. Nature 464, 989990 (2010)
  6. Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 9921009 (2011)

Banking on Blood Tests

How close are liquid biopsies to replacing current diagnostics?

By Jyoti Madhusoodanan | April 1, 2016

No matter where a tumor lurks in the body, its secrets circulate in the blood. Stray tumor cells begin metastatic migrations by slipping into the vasculature. Vesicles secreted by cancer cells and free-floating DNA are also released into the bloodstream. Because these bits of cellular debris are a grab-bag of biomarkers that could both signal a cancer’s presence and predict its progression and response to treatment, the use of blood-based tests, or liquid biopsies, to detect and evaluate them is now drawing significant commercial interest.

Last year, San Diego–based Pathway Genomics began advertising a screen “for the early detection of up to 10 different cancer types in high-risk populations.” But the screen had only been tested in already-diagnosed patients, not in at-risk individuals, and within weeks of making it commercially available, the company received an FDA notice to provide more information about their promotional claims before further marketing. “We . . . have not found any published evidence that this test or any similar test has been clinically validated as a screening tool for early detection of cancer in high risk individuals,” the agency wrote.

The Forces of Cancer

A tumor’s physical environment fuels its growth and causes treatment resistance.

By Lance L. Munn and Rakesh K. Jain | April 1, 2016

Ahelium balloon tugs gently at the end of its string. The tension in the string resists the buoyant force of the helium, and the elastic nature of the balloon’s rubber contains the helium gas as it tries   to expand. Cutting the string or poking the rubber with a pin reveals the precarious balance between the forces, upsets the equilibrium, and sets the system into motion.

Some biological tissues also exist in such a state of offsetting forces. The most familiar example is the balance between blood pressure and the elastic tension in the cardiovascular system that contains and conveys blood without bursting or collapsing. And in tumors, both solid and fluid forces are generated that make the cancerous tissue a lot like that helium balloon: cut a tumor with a scalpel and it rapidly swells and deforms as pent-up forces break free from structural elements that are severed.1

One force that is notably higher in tumors than in healthy tissues is fluid pressure, resulting from hyperpermeable, leaky blood vessels and a dearth of draining lymphatic vessels. Researchers have known since the 1950s that tumors exhibit elevated fluid pressure, but the implications for tumor progression and drug delivery were not realized until the late 1980s. That was when we (R.K.J. and colleagues) used a mathematical model to predict—and subsequently validate in animal and human tumors—that a precipitous drop in fluid pressure at the tumor–normal tissue interface causes interstitial fluid to ooze out of the tumor.2 This seeping fluid pushes drugs, growth factors, and cancer cells into the surrounding tissue and lymphatics, reducing drug delivery and facilitating local tumor invasion and distant metastasis.

Based on this insight, we suggested in 2001 that anti-angiogenic drugs could be used to lower a tumor’s fluid pressure and improve treatment outcome.3 This hypothesis changed the thinking about how existing anti-angiogenesis therapies actually work and spurred research into other physical forces acting in cancer.4 In the last 15 years, researchers have identified diverse sources of increased pressure in tumors, which may serve as possible targets for cancer therapy.5 For example, solid forces exerted by the extracellular matrix can be reduced by treatment with drugs approved by the US Food and Drug Administration (FDA) for controlling hypertension (angiotensin blockers) or diabetes (metformin). Retrospective clinical studies have found improved survival in cancer patients who were treated with these agents, which are now being tested in prospective trials for a variety of solid tumors.6,7

Tumors under pressure

In vitro experiments showing that cancer cells actively migrate in response to fluid flow have supported the hypothesis that fluid escaping from the boundary of a tumor may guide the invasive migration of cancer cells toward lymphatic or blood vessels, potentially encouraging metastasis. There remains controversy over how the fluid forces induce the migration; the cells may respond to chemical gradients created by the cells and distorted by the flowing fluid,8 or the fluid may activate cell mechanosensors.9 Because of the potential for new therapeutic interventions, the transduction of mechanical fluid forces into biochemical signals by cell mechanosensors is an active area of investigation. In a more direct manner, the fluid flow can physically carry cancer cells to lymph nodes.

Fluid forces may also promote tumor progression by recruiting blood vessels into the cancerous mass.10 Because tumor blood vessels are leaky, plasma can pass freely between vessels that have different pressures. When this happens at the periphery of a tumor, where angiogenic growth factors are prevalent, there can be synergistic induction of new vessel sprouts.


And fluid pressure is just one of the many forces in a tumor that can influence its development and progression. Tumors also develop increased solid pressure, as compared with normal tissue, stemming from the uncontrolled division of cancer cells and from the infiltration and proliferation of stromal and immune cells from the surrounding tissue and circulation. High-molecular-weight polysaccharides known as hydrogels found in the extracellular matrix (ECM) also add pressure on a tumor. The most well-studied of these hydrogels is hyaluronan; when the polysaccharide absorbs water, it swells, pressing on surrounding cells and structural elements of the tissue.

The ECM contains a highly interconnected network of collagen and other fibers and is normally very good at resisting and containing such tension. It also has support from infiltrating myofibroblasts, which detect areas where the ECM density or tension is not normal and initiate actomyosin-based contraction of collagen and elastin matrix structures to restore tensional homeostasis. But while this repair effort is typically effective in healthy tissues, uncooperative tumor cells interfere with these efforts, both by themselves generating pressure and by hyperactivating cancer-associated fibroblasts to produce more ECM and thus produce even more force.11

Because cell growth and ECM composition are not spatially uniform in cancer, tumors are subjected to multiple, dispersed sources of pressure associated with matrix “containers” of various sizes. This solid pressure from within the tumor deforms the surrounding normal tissue, potentially facilitating the metastatic escape of cancer cells. The physical forces also compress blood vessels and lymphatic vessels in the tumor and adjacent normal tissue,12 increasing the fluid pressure in the tumor13  and interrupting the delivery of nutrients, removal of waste, and entry of tumor-targeted drugs via the blood.4 Insufficient blood flow also results in poor oxygenation, which has been linked to immunosuppression, inflammation, invasion, and metastasis, as well as lowered efficacy of chemo-, radio-, and immunotherapies.4 These are all indirect consequences of solid stresses in and on tumors.

Such forces can also have direct effects on cancer cells, and may serve as independent triggers for tumor invasion. Mechanical forces are central to many of our sense systems, such as hearing, touch, and pain, and to tissue maintenance programs, such as bone regeneration and blood vessel remodeling. In these systems, mechanical forces are transduced by mechanosensors to activate downstream biochemical and genetic pathways. (See “Full Speed Ahead,” The Scientist, December 2009.) Cancer cells may similarly be able to sense and respond to dynamic forces in tumors. We have shown, for example, that metastatic cancer cells exposed to compressive stresses in a culture dish undergo a phenotypic transformation to become more invasive,14 and others have shown that compressive forces applied in vivo can also induce oncogenes in normal epithelium of the mouse colon.15

It is thus becoming quite clear that the physical environment can influence a tumor’s development and spread, and it may even be possible for physical forces to kick-start cancerous growth.


Full Speed Ahead

Physical forces acting in and around cells are fast—and making waves in the world of molecular biology.

By Jef Akst | December 1, 2009

When it comes to survival, few things are more important than being able to respond quickly to a change of circumstances. And when it comes to fast-acting indicators, it turns out that signals induced by physical forces acting in and around cells, appropriately dubbed biomechanical signals, are the champions of the cellular world.

“If you look at this mechanical signaling, it’s about 30 meters per second—that’s very fast,” says bioengineer Ning Wang of the University of Illinois at Urbana-Champaign. That’s faster than most family-owned speedboats, and second only to electrical (e.g., nerve) impulses in biological signaling. By comparison, small chemicals moving by diffusion average a mere 2 micrometers per second—a speed even the slowest row boater could easily top.

Indeed, when the two signal types were pitted against each other in a cellular race last year, the mechanical signals left chemical signals in their wake, activating proteins at distant sites in the cytoplasm in just a fraction of a second, at least 40 times faster than their growth factor opponent.1 Mechanical signals are so fast, Wang adds, they are “beyond our resolution,” meaning that current imaging techniques cannot capture the very first cellular changes that result from mechanical stress, which occur within nanoseconds.

For centuries, scientists have scrutinized the molecular inner workings of the body, with little or no regard to the physical environment in which these biological reactions take place. But the growing realization that physical forces have a pervasive presence in physiology (operating in a variety of bodily systems in thebone, blood, kidney, and ear, for instance), and act with astonishing speed, has caused many to consider the possibility that mechanical signaling may be just as important as chemical communication in the life of a cell.

“Biologists have traditionally ignored the role of mechanics in biology,” says biomechanical engineer Mohammad Mofrad of the University of California, Berkley, “[but] biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”

The wave within: Mechanical forces acting inside the cell

Once believed to be little more than sacks of chemically active goop, cells didn’t seem capable of transmitting physical forces into their depths, and researchers largely limited their search for molecules or structures that respond to physical forces, or mechanosensors, to the plasma membrane.

Mechanical signaling may be just as important as chemical communication in the life of a cell.

In the late 1990s, however, closer examination revealed that the cell’s interior is in fact a highly structured environment, composed of a network of filaments.2 Pull on one side of the cell, and these filaments will transmit the force all the way to other side, tugging on and bumping into a variety of cellular structures along the way—similar to how a boat’s wake sends a series of small waves lapping up on a distant and otherwise peaceful shoreline. Scientists are now realizing the potential of such intracellular jostling to induce molecular changes throughout the cell, and the search for mechanosensing molecules has escalated dramatically in scope, including, for example, several proteins of the nucleus.

It’s a search that will likely last a while, predicts cell biologist Donald Ingber, director of the Wyss Institute for Biologically Inspired Engineering at Harvard University. “To try to find out what’s the mechanosensor is kind of crazy at this point,” he says. As scientists are now learning, “the whole cell is the mechanosensor.”

A key player, most agree, is the cytoskeleton, which is comprised of a variety of microfilaments, including rigid actin filaments and active myosin motors—the two principle components of muscle. Activation of the so-called nonmuscle myosins causes the cytoskeleton to contract, much like an arm muscle does when it lifts a heavy object.

The first intimation that the cytoskeleton could go beyond its established inner-cell duties (molecule transport and cell movement and division) came in 1997, when Ingber did the logical (in hindsight, at least) experiment of pulling on the cells to see what happened inside.2 Using a tiny glass micropipette coated in ligands, Ingber and his team gently probed the surface proteins known as integrins, which secure the cell to the extracellular matrix. When they quickly pulled the micropipette away, they saw an immediate cellular makeover: cytoskeletal elements turned 90 degrees, the nucleus distorted, and the nucleolus—a small, dense structure within the nucleus that functions primarily in ribosome assembly—aligned itself with the direction of the applied force.

“That kind of blew people away,” Ingber recalls. “It revealed that cells have incredible levels of structure not only in the cytoplasm but in the nucleus as well.”

Wang (once a postdoc in Ingber’s lab at the Harvard School of Public Health) and other collaborators combined a similar technique with fluorescent imaging technology to visualize how these forces were channeled within the cell’s interior. Upping the resolution and further refining these techniques, Wang began mapping these intracellular forces as they made their way through the cell. In 2005, the maps confirmed the physical connection between the cell-surface integrins and the nucleus, and showed that these external forces follow a nonrandom path dictated by the tension of the cytoskeletal elements.3

“Biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”
–Mohammad Mofrad

The end point of these mechanical pathways is likely a mechanosensitive protein, which changes shape in response to the force, thereby exposing new binding areas or otherwise changing the protein’s function. In mitochondria, for example, mechanical forces may trigger the release of reactive oxygen species and activation of signaling molecules that contribute to inflammation and atherosclerosis.

Similarly, proteins on the nuclear membrane may pass mechanical signals into the nucleus by way of a specialized structure known as LINC (linker of nucleoskeleton and cytoskeleton), which physically links the actin cytoskeleton to proteins important in nuclear organization and gene function. To determine if mechanical forces directly affect gene expression, last year scientists began exploiting the increasingly popular fluorescence resonance energy transfer (FRET) technology,1 in which energy emitted by one fluorescent molecule can stimulate another, resulting in a visible energy transfer that can track enzymatic activities in live cells. By combining FRET technology with the techniques that apply physical forces to specific cell membrane proteins, scientists can visualize entire mechanochemical transduction pathways, Wang says.

“The big issue right now in the field of mechanotransduction is whether the genes in the nucleus can be directly activated by forces applied to the cell surface,” Wang explains. While the physical maps of the cytoskeleton tentatively sketch out a path that supports this possibility, confirmatory data is lacking. This combination of new technologies will be “tremendously” helpful in answering that question, he says, and “push the field” towards a more complete understanding of how mechanical forces can influence cellular life.

An early start: Mechanical forces in development

In the world of developmental biology, the cytoskeleton’s role in biomechanics really comes into its own. As the embryo develops, the cells themselves are the force generators, and by contracting at critical times, the cytoskeleton can initiate many key developmental steps, from invagination and gastrulation to proliferation and differentiation, and overall cellular organization.

The idea that physical forces play a role in development is not a new one. In the early 20th century, back when Albert Einstein was first developing the molecular basis of viscosity and scientists were realizing molecules are distinct particles, biologist and mathematician D’Arcy Thompson of the University of Dundee in Scotland suggested that mechanical strain is a key player in morphogenesis. Now, nearly a century later, biologists are finally beginning to agree.

Because Thompson “couldn’t measure [the forces] at that time, that kind of thinking got pushed to the wayside as genetic thinking took over biology,” says bioengineer Christopher Chen of the University of Pennsylvania. That is, until 2003, when Emmanuel Farge of the Curie Institute in France squeezedDrosophila embryos to mimic the compression experienced during early development and activated twist—a critical gene in the formation of the digestive tract.4 These results gave weight to Thompson’s idea that stress in the embryo stimulates development and growth, and inspired developmental scientists to begin considering mechanical effects, Chen says. “Now we’re at the stage where there’s a lot of interest and willingness to consider the fact that mechanical forces are not only shaping the embryo, but are linked to the differentiation programs that are going on.”

Again, the cytoskeleton is a key player in this process. In fruit flies and frogs, for example, nonmuscle myosins contract the actin filaments to generate the compressive forces necessary for successful gastrulation—the first major shape-changing event of development. Myosins similarly influence proliferation in the development of the Drosophila egg chamber, with increased myosin activity resulting in increased cell division.

Cytoskeleton contractility also appears to direct stem cell differentiation. In 2006, Dennis Discher of the University of Pennsylvania demonstrated that the tension of the substrate on which cells are grown in culture is important for determining what type of tissue the cells will form.5 Cells grown on soft matrices that mimic brain tissue tended to grow into neural cells, while cells grown on stiffer matrices grew into muscle cell precursors, and hard matrices yielded bone. In this case, it seems that stiffer substrates increased the expression of nonmuscle myosin, generating greater tension in the actin cytoskeleton and affecting differentiation. (Altering or inhibiting myosin contraction can also affect differentiation.)

“To try to find out what’s the mechanosensor is kind of crazy at this point. As scientists are now learning, the whole cell is the mechanosensor.”
–Donald Ingber
Shaping a tumor

In addition to the influence of physical forces on cancer growth and invasion, forces can alter a tumor’s mechanical properties, and vice versa. Tumors are more rigid, or stiffer, than surrounding tissues, usually because they contain excess collagen in the ECM,5 and this can contain and amplify local forces produced by proliferating cancer cells. On the other hand, tumor rigidity can be further enhanced if the cells exert tension on ECM collagen fibers by pulling on them, or by stretching them, as occurs when tumors grow uncontrollably. Fluid forces can also influence the assembly of collagen fibers within and around tumors,8potentially increasing stiffness.

Importantly, tumor stiffness tends to be associated with poor prognoses, though the reasons for this are not fully understood. Cells are known to differentiate into different lineages depending on the local rigidity;16 for example, stem cells differentiate into bone on stiff substrates, but make adipose (fat) cells on softer substrates. Similar mechanisms are thought to affect tumor progression when the ECM changes rigidity, inducing cancer cells to become more invasive as well as more likely to metastasize. Indeed, longer collagen fibers in the matrix are associated with increased invasion and metastasis, as well as reduced survival, in mice.17

In addition, the abnormal ECM in tumors can affect cancer progression by activating normal stromal cells, such as macrophages and fibroblasts, that accelerate tumor growth and treatment resistance. These activated stromal cells further strengthen and stretch the ECM, causing a snowball effect.

The biochemical composition and organization of the ECM also influences tumor biology. Dysregulation of normal matrix signals can lead to tumor progression, characterized by excessive cell proliferation, immortality, enhanced migration, changes in metabolism, and evasion of the immune response. More research is needed to dissect the relationships between the ECM’s mechanical properties, forces, and cell signaling pathways.

Targeting the ECM

Because unchecked proliferation of cancer cells increases solid stress in the tumor, anticancer therapies should decrease the compressive forces in tumors and reopen collapsed blood and lymphatic vessels.11 This is exactly what happens when tumors are treated with certain doses of paclitaxel or docetaxel, two widely used cancer drugs. Shrinking tumors increases blood flow and allows more efficient fluid movement through the extravascular space, lowering the tumor interstitial fluid pressure in mouse models and in patients with breast cancer.5 However, cancer cells invariably develop resistance to treatment and begin to regrow, increasing solid stress again. As a result, other targets for reducing solid stresses are needed.

Because of its role in containing and concentrating the forces in a tumor, the collagen matrix within and around the tumor is another potential target for relieving tumor-related stresses. Indeed, solid stress in tumors can be reduced by drugs that selectively reprogram activated fibroblasts or modify the assembly of matrix components such as collagen and hyaluronan. In rodent studies, targeting these force-altering components in the tumor microenvironment has been shown to decrease solid stress, improve blood perfusion and drug delivery, and improve tumor response to chemotherapy and animal survival.6 We have found, for example, that injecting tumors with a collagen-digesting enzyme increases the diffusion of antibodies and viral particles and improves drug penetration in the tumor. Similarly, treatments that target transforming growth factor–beta (TGF-β), which controls the production of collagen by myofibroblasts, increase perfusion, improve the delivery of drugs of all sizes in mammary tumors, and improve treatment outcomes in mice.5

A class of drugs that is widely used to control blood pressure in hypertensive patients also blocks the TGF-β pathway. These drugs, known as angiotensin receptor 1 blockers, can reduce collagen production in and around the tumor by reducing the activity of TGF-β, as well as by blocking the function of connective tissue growth factor (CTGF), which is involved in stabilizing collagen and inducing resistance to chemotherapy.6Losartan and other angiotensin inhibitors reduce levels of collagen in various experimental models of fibrosis, and decrease renal and cardiac fibrosis in hypertensive patients. When given to mice with one of four different types of tumors characterized by high levels of cancer-associated fibroblasts (CAFs) and excess extracellular matrix—pancreatic ductal adenocarcinoma, breast cancer, sarcoma, and melanoma—losartan treatment caused a decrease in collagen content in a dose-dependent manner, enhanced penetration of nanoparticles into the tumor, and improved efficacy of diverse anticancer drugs. This is supported by a number of retrospective studies in patients with pancreatic, lung, and kidney cancers.6Researchers at Massachusetts General Hospital are now running a Phase 1/2 clinical trial to test losartan in pancreatic cancer patients.

THE TUMOR ENVIRONMENT: The extracellular matrix and stromal cells within a tumor’s microenvironment influence the physical forces a tumor experiences. Left: The immunofluorescent image shows stromal cells (red and green) surrounding tumor cells (red cluster with blue nuclei); the cells were isolated from a mouse model of lung adenocarcinoma. Right: In this immunofluorescent image of triple-negative breast cancer, tumor cells (blue) are in close contact with matrix collagen (purple). Immune cells are labeled in red and green.VASILENA GOCHEVA, JACKS LAB, KOCH INSTITUTE AT MIT; DONGMEI ZUO, LABORATORY DR. MORAG PARK

Another potential cancer treatment target is hyaluronan, which is abundant in 20 percent to 30 percent of human tumors, most notably breast, colon, and prostate cancers. In addition to its role as a pressure-creating gel, hyaluronan can sequester growth factors and inhibit interstitial fluid movement within the tumor. Hyaluronidase, an enzyme that digests hyaluronan, reduces mechanical stress in tumors grown in mice.1 And San Diego–based Halozyme Therapeutics’s PEGPH20, a formulation of hyaluronidase coated with polyethylene glycol to enhance bioavailability, can decompress blood vessels and improve treatment outcome in genetically engineered mouse models of pancreatic ductal adenocarcinoma. Based on these studies, Halozyme researchers are now testing PEGPH20 in a randomized clinical trial of pancreatic cancer patients. Another matrix-altering drug is the widely-prescribed antidiabetic drug metformin, which has been shown to decrease collagen and hyaluronan levels in pancreatic tumors in obese mice and patients.7 Metformin is currently being tested in more than 200 clinical trials worldwide as a treatment for different types of cancer.

Clearly, tumors should be studied not only in light of their biochemical processes and genetic underpinnings, but also for the specific physical forces and mechanical properties that may influence progression. Understanding the physical microenvironment of tumors, as well as its interplay with the biochemical environment, is necessary to improve cancer detection, prevention, and treatment.

  1. T. Stylianopoulos et al., “Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors,” PNAS, 109:15101-08, 2012.
  2. R.K. Jain, L.T. Baxter, “Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: Significance of elevated interstitial pressure,” Cancer Res, 48:7022-32, 1988.
  3. R.K. Jain, “Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy,”Science, 307:58-62, 2005.
  4. R.K. Jain, “Antiangiogenesis strategies revisited: From starving tumors to alleviating hypoxia,”Cancer Cell, 26:605-22, 2014.
  5. R.K. Jain et al., “The role of mechanical forces in tumor growth and therapy,” Annu Rev Biomed Eng, 16:321-46, 2014.
  6. V.P. Chauhan et al., “Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels,” Nat Commun, 4:2516, 2013.
  7. J. Incio et al., “Metformin reduces desmoplasia in pancreatic cancer by reprogramming stellate cells and tumor-associated macrophages,” PLOS ONE, 10:e0141392, 2015.
  8. M.A. Swartz, A.W. Lund, “Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity,” Nat Rev Cancer, 12:210-19, 2012.
  9. H. Qazi et al., “Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion,”Integr Biol, 5:1334-43, 2013.
  10. J.W. Song, L.L. Munn, “Fluid forces control endothelial sprouting,” PNAS, 108:15342-47, 2011.

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Introduction to Lipid Metabolism

Author, Curator: Larry H. Bernstein, MD, FCAP 

Introduction to Lipid Metabolism

This series of articles is concerned with lipid metabolism. These discussions lay
the groundwork to proceed to discussions that will take on a somewhat different
approach, but they are critical to developing a more complete point of view of life
processes.  I have indicated that there are protein-protein interactions or protein-membrane interactions and associated regulatory features, but the focus of the
discussion or points made were different, and will be returned to.  The role of
lipids in circulating plasma proteins as biomarkers for coronary vascular disease
can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and
disease has had much less attention, despite the enormous importance,
especially in the nervous system.

This portion of the discussions of metabolism will have several topics on lipid
metabolism.  The first is concerned with the basic types of lipids -which are defined structurally and have different carbon chain length, and have
two basic types of indispensible fatty acid derivations – along pro-inflammatory
and anti-inflammatory pathways:

  1. Alpha-linolenic acid (ALA) and LA, n-3 polyunsaturated fatty acids LCPUFAs (EPA, DHA, and AA), eicosanoids,
    delta-3-desaturase, prostaglandins, and leukotrienes.
  2. the role of the mitochondrial electron transport chain in hydrogen transfers
    and oxidative phosphorylation with respect to the oxidation of fatty acids
    and fatty acid synthesis.
  3. The membrane structures of the cell, including
  • the cytoskeleton, essential organelles, and the intercellular matrix, which
    is a critical consideration for
  • cell motility, membrane conductivity, flexibility, and  signaling.
  • The membrane structure involves aggregation of lipids with proteins,
  • and is associated with hydrophobicity.
  1. The pathophysiology of systemic circulating lipid disorders.
  2. The fifth is the pathophysiology of cell structures under oxidative
  3. Lipid disposal and storage diseases.

Read Full Post »

Compilation of References in Leaders in Pharmaceutical Intelligence about proteomics, metabolomics, signaling pathways, and cell regulation

Compilation of References in Leaders in Pharmaceutical Intelligence about
proteomics, metabolomics, signaling pathways, and cell regulation

Curator: Larry H. Bernstein, MD, FCAP



  1. The Human Proteome Map Completed
    Reporter and Curator: Larry H. Bernstein, MD, FCAP
  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Synthesizing Synthetic Biology: PLOS Collections
    Reporter: Aviva Lev-Ari



  1. Extracellular evaluation of intracellular flux in yeast cells
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator 
  2. Metabolomic analysis of two leukemia cell lines. I.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator 
  3. Metabolomic analysis of two leukemia cell lines. II.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator 
  4. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
    Reviewer and Curator, Larry H. Bernstein, MD, FCAP 
  5. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator


Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief
    Reviewer and Curator: Larry H. Bernstein, MD, FCAP
  2. Mitochondria: More than just the “powerhouse of the cell”
    Reviewer and Curator: Ritu Saxena
  3. Mitochondrial fission and fusion: potential therapeutic targets?
    Reviewer and Curator: Ritu saxena 
  4. Mitochondrial mutation analysis might be “1-step” away
    Reviewer and Curator: Ritu Saxena
  5. Selected References to Signaling and Metabolic Pathways in
    Curator: Larry H. Bernstein, MD, FCAP
  6. Metabolic drivers in aggressive brain tumors
    Prabodh Kandal, PhD 
  7. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
    Author and Curator: Aviva Lev-Ari, PhD, RD
  8. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
    Author and curator:Larry H Bernstein, MD, FCAP
  9. Therapeutic Targets for Diabetes and Related Metabolic Disorders
    Reporter, Aviva Lev-Ari, PhD, RD
  10. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator
  11. The multi-step transfer of phosphate bond and hydrogen exchange energy
    Curator:Larry H. Bernstein, MD, FCAP,
  12. Studies of Respiration Lead to Acetyl CoA
    Author and Curator: Larry H. Bernstein, MD, FCAP
  13. Lipid Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
  14. Carbohydrate Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
  15. Prologue to Cancer – e-book Volume One – Where are we in this journey?
    Author and Curator: Larry H. Bernstein, MD, FCAP
  16. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?
    Author and Curator: Larry H. Bernstein, MD, FCAP
  17. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K
    Author and Curator: Larry H. Bernstein, MD, FCAP
  18. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
    Author and Curator: Larry H. Bernstein, MD, FCAP
  19. Mitochondrial Metabolism and Cardiac Function
    Author and Curator: Larry H. Bernstein, MD, FCAP
  20. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia
    Curator: Larry H. Bernstein, MD, FCAP
  21. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
    Author and Curator: SJ. Williams
  22. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
    Author and Curator: Larry H. Bernstein, MD, FCAP
  23. Overview of Posttranslational Modification (PTM)
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  24. Malnutrition in India, high newborn death rate and stunting of children age under five years
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  25. Update on mitochondrial function, respiration, and associated disorders
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  26. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
    Larry H. Bernstein, MD, FCAP, Curator 
  27. Late Onset of Alzheimer’s Disease and One-carbon Metabolism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.
  28. Problems of vegetarianism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Signaling Pathways

  1. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
    Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN
  2. Epilogue: Envisioning New Insights in Cancer Translational Biology
    Series C: e-Books on Cancer & Oncology
    Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant
  3. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter  Writer and Curator: Larry H Bernstein, MD, FCAP and Curator and Content Editor: Aviva Lev-Ari, PhD, RN
  4. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
    Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
    Author and Curator: Larry H Bernstein, MD, FCAP and Article Curator: Aviva Lev-Ari, PhD, RN
  5. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
    Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN
  6. Identification of Biomarkers that are Related to the Actin Cytoskeleton
    Larry H Bernstein, MD, FCAP, Author and Curator
  7. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
    Author and Curator: Larry H Bernstein, MD, FCAP
  8. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology
    Demet Sag, PhD, Author and Curator
  9. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase
    Demet Sag, PhD, Author and Curator
  10. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad
    Author and Curator: Demet Sag, PhD, CRA, GCP
  11. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute
    Reporter: Aviva Lev-Ari, PhD, RN
  12. Naked Mole Rats Cancer-Free
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  13. Amyloidosis with Cardiomyopathy
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  14. Liver endoplasmic reticulum stress and hepatosteatosis
    Larry H Bernstein, MD, FACP
  15. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III
    Curator and Author: Larry H Bernstein, MD, FACP
  16. Nitric Oxide Function in Coagulation – Part II
    Curator and Author: Larry H. Bernstein, MD, FCAP
  17. Nitric Oxide, Platelets, Endothelium and Hemostasis
    Curator and Author: Larry H Bernstein, MD, FACP
  18. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium
    Curator and Author: Larry H Bernstein, MD, FACP
  19. Nitric Oxide and Immune Responses: Part 1
    Curator and Author:  Aviral Vatsa PhD, MBBS
  20. Nitric Oxide and Immune Responses: Part 2
    Curator and Author:  Aviral Vatsa PhD, MBBS
  21. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
    Curator and Author: Larry H Bernstein, MD, FACP
  22. New Insights on Nitric Oxide donors – Part IV
    Curator and Author: Larry H Bernstein, MD, FACP
  23. Crucial role of Nitric Oxide in Cancer
    Curator and Author: Ritu Saxena, Ph.D.
  24. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
    Curator and Author: Larry H Bernstein, MD, FACP
  25. Nitric Oxide and Immune Responses: Part 2
    Author and Curator: Aviral Vatsa, PhD, MBBS
  26. Mitochondrial Damage and Repair under Oxidative Stress
    Author and Curator: Larry H. Bernstein, MD, FCAP
  27. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
    Curator and Author: Larry H Bernstein, MD, FACP
  28. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
    Curator and Author: Ziv Raviv, PhD, RN 04/06/2013
  29. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
    Curator and Author: Larry H Bernstein, MD, FACP
  30. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III
    Curator and Author: Larry H Bernstein, MD, FACP
  31. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I
    Curator and Author: Larry H Bernstein, MD, FACP


Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  2. RNA and the transcription the genetic code
    Larry H. Bernstein, MD, FCAP, Writer and Curator
  3. A Primer on DNA and DNA Replication
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  4. Pathology Emergence in the 21st Century
    Author and Curator: Larry Bernstein, MD, FCAP
  5. RNA and the transcription the genetic code
    Writer and Curator, Larry H. Bernstein, MD, FCAP
  6. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP
    Author: Larry H Bernstein, MD, FCAP
  7. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies
    Author an Curator: Larry H Bernstein, MD, FCAP
  8. Silencing Cancers with Synthetic siRNAs
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  9. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points
    Reporter: Aviva Lev-Ari, PhD, RN
  10. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  11. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis
    Reporter: Aviva Lev-Ari, PhD, RN
  12. CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics
    Genomics Curator, Larry H Bernstein, MD, FCAP
  13. Big Data in Genomic Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
  14.  From Genomics of Microorganisms to Translational Medicine
    Author and Curator: Demet Sag, PhD
  15.  Summary of Genomics and Medicine: Role in Cardiovascular Diseases
    Author and Curator, Larry H Bernstein, MD, FCAP

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Lipid Metabolism

Lipid Metabolism

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


This is fourth of a series of articles, lipid metabolism, that began with signaling and signaling pathways. These discussion lay the groundwork to proceed in later discussions that will take on a somewhat different approach. These are critical to develop a more complete point of view of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.  The role of lipids in circulating plasma proteins as biomarkers for coronary vascular disease can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and disease has had much less attention, despite the enormous importance, especially in the nervous system.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism
  2. Protein synthesis and degradation
  3. Subcellular structure
  4. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.


Lipid Metabolism

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidationto produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.


metabolism of fats

metabolism of fats

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.





 Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.


Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

 Step  ATP produced
7  1
Step 4 (NAD+ to E.T.C.) 3
Step 6 (NAD+ to E.T.C.)  3
Step10 (NAD+ to E.T.C.)  3
Step 8 (FAD to E.T.C.) 2



 ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

 Step  ATP (used -) (produced +)
Step 1 (FAD to E.T.C.) +2
Step 4 (NAD+ to E.T.C.) +3
Total ATP  +5
 7 turns  7 x 5 = 35
initial step  -1
 NET  34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

 Step  ATP produced
One acetyl CoA per turn C.A.C. +12 ATP
8 Acetyl CoA = 8 turns C.A.C. 8 x 12 = 96 ATP
Fatty Acid Spiral 34 ATP


Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.


This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.


  • The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
  • the second was that the adenosine polyphosphate system plays a vital part in the cell,
    • not only in energy transfer, but
    • also in the regulation of the metabolic processes.



This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.


My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.


The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.


This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid  is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

  • fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

  • This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

  • we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme  A.

Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

Fyodor Lynen

Fyodor Lynen


SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1

1Department of Molecular Genetics, and
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (14). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).


Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1


Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.

SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.


Figure 1


When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).

When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).


Figure 2


major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2


Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Knockout and transgenic mice

Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (916). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.


Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs

Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1–/– mice is due to the loss of the SREBP-1a transcript (16).

To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.

Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.

In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.

Function of individual SREBP isoforms in vivo

To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.

Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).

We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.

The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).

The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.

Transcriptional regulation of SREBP genes

Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).

The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).

Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (2224). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.

LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.

SREBP-1c and the insulin/glucagon ratio

The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).

In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [3H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).

Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).

SREBPs in disease

Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).

Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.

The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).

Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).

In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because

humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,

it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.

SREBPs in liver: unanswered questions

The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.

Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.

Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.



Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.


  1. Brown, MS, Goldstein, JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997. 89:331-340.

View this article via: PubMed

  1. Horton, JD, Shimomura, I. Sterol regulatory element-binding proteins: activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol 1999. 10:143-150.

View this article via: PubMed

  1. Edwards, PA, Tabor, D, Kast, HR, Venkateswaran, A. Regulation of gene expression by SREBP and SCAP. Biochim Biophys Acta 2000. 1529:103-113.

View this article via: PubMed

  1. Sakakura, Y, et al. Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis. Biochem Biophys Res Commun 2001. 286:176-183.

View this article via: PubMed

  1. Goldstein, JL, Rawson, RB, Brown, MS. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Arch Biochem Biophys 2002. 397:139-148.

View this article via: PubMed

  1. Shimomura, I, Shimano, H, Horton, JD, Goldstein, JL, Brown, MS. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J Clin Invest 1997. 99:838-845.

View this article via: PubMed

  1. Moon, Y-A, Shah, NA, Mohapatra, S, Warrington, JA, Horton, JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem 2001. 276:45358-45366.

View this article via: PubMed

  1. Shimomura, I, Shimano, H, Korn, BS, Bashmakov, Y, Horton, JD. Nuclear sterol regulatory element binding proteins activate genes responsible for entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem 1998. 273:35299-35306.

View this article via: PubMed

  1. Shimano, H, et al. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 1996. 98:1575-1584.

View this article via: PubMed

  1. Shimano, H, et al. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 1997. 99:846-854.

View this article via: PubMed

  1. Horton, JD, et al. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 1998. 101:2331-2339.

View this article via: PubMed

  1. Korn, BS, et al. Blunted feedback suppression of SREBP processing by dietary cholesterol in transgenic mice expressing sterol-resistant SCAP(D443N). J Clin Invest 1998. 102:2050-2060.

View this article via: PubMed

  1. Shimano, H, et al. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene. J Clin Invest 1997. 100:2115-2124.

View this article via: PubMed

  1. Matsuda, M, et al. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev 2001. 15:1206-1216.

View this article via: PubMed

  1. Yang, J, et al. Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene. Proc Natl Acad Sci USA 2001. 98:13607-13612.

View this article via: PubMed

Liang, G, et al. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 2002. 277:9520-9528.


Structural Biochemistry/Lipids/Membrane Lipids

< Structural Biochemistry‎ | Lipids

Membrane proteins rely on their interaction with membrane lipids to uphold its structure and maintain its functions as a protein. For membrane proteins to purify and crystallize, it is essential for the membrane protein to be in the appropriate lipid environment. Lipids assist in crystallization and stabilize the protein and provide lattice contacts. Lipids can also help obtain membrane protein structures in a native conformation. Membrane protein structures contain bound lipid molecules. Biological membranes are important in life, providing permeable barriers for cells and their organelles. The interaction between membrane proteins and lipids facilitates basic processes such as respiration, photosynthesis, transport, signal transduction and motility. These basic processes require a diverse group of proteins, which are encoded by 20-30% of an organism’s annotated genes.

There exist a great number of membrane lipids. Specifically, eukaryotic cells have a very complex collection of lipids that rely on many of the cell’s resources for its synthesis. Interactions between proteins and lipids can be very specific. Specific types of lipids can make a structure stable, provide control in insertion and folding processes, and help to assemble multisubunit complexes or supercomplexes, and most importantly, can significantly affect a membrane protein’s functions. Protein and lipid interactions are not sufficiently tight, meaning that lipids are retained during membrane protein purification. Since cellular membranes are fluid arrangements of lipids, some lipids affect interesting changes to membrane due to their characteristics. Glycosphigolipids and cholesterol tend to form small islands within the membranes, called lipid rafts, due to their physical properties. Some proteins also tend to cluster in lipid raft, while others avoid being in lipid rafts. However, the existence of lipid rafts in cells seems to be transitory.

Recent progress in determining membrane protein structure has brought attention to the importance of maintaining a favorable lipid environment so proteins to crystallize and purify successfully. Lipids assist in crystallization by stabilizing the protein fold and the relationships between subunits or monomers. The lipid content in protein-lipid detergent complexes can be altered by adjusting solubilisation and purification protocols, also by adding native or non-native lipids.

There are three type of membrane lipids: 1. Phospholipids: major class of membrane lipids. 2. glycolipids. 3. Cholesterols. Membrane lipids were started with eukaryotes and bacteria.

Types of Membrane Lipids

Lipids are often used as membrane constituents. The three major classes that membrane lipids are divided into are phospholipids, glycolipids, and cholesterol. Lipids are found in eukaryotes and bacteria. Although the lipids in archaea have many features that are related to the membrane formation that is similar with lipids of other organisms, they are still distinct from one another. The membranes of archaea differ in composition in three major ways. Firstly, the nonpolar chains are joined to a glycerol backbone by ether instead of esters, allowing for more resistance to hydrolysis. Second, the alkyl chains are not linear, but branched and make them more resistant to oxidation. The ability of archaeal lipids to resist hydrolysis and oxidation help these types of organisms to withstand the extreme conditions of high temperature, low pH, or high salt concentration. Lastly, the stereochemistry of the central glycerol is inverted. Membrane lipids have an extensive repertoire, but they possess a critical common structural theme in which they are amphipathic molecules, meaning they contain both a hydrophilic and hydrophobic moiety.

Membrane lipids are all closed bodies or boundaries separating substituent parts of the cell. The thickness of membranes is usually between 60 and 100 angstroms. These bodies are constructed from non-covalent assemblies. Their polar heads align with each other and their non-polar hydrocarbon tails align as well. The resulting stability is credited to hydrophobic interaction which proves to be quite stable due to the length of their hydrocarbon tails.


Membrane Lipids

Lipid Vesicles

Lipid vesicles, also known as liposomes, are vesicles that are essentially aqueous vesicles that are surrounded by a circular phospholipid bilayer. Like the other phospholipid structures, they have the hydrocarbon/hydrophobic tails facing inward, away from the aqueous solution, and the hydrophilic heads facing towards the aqueous solution. These vesicles are structures that form enclosed compartments of ions and solutes, and can be utilized to study the permeability of certain membranes, or to transfer these ions or solutes to certain cells found elsewhere.

Liposomes as vesicles can serve various clinical uses. Injecting liposomes containing medicine or DNA (for gene therapy) into patients is a possible method of drug delivery. The liposomes fuse with other cells’ membranes and therefore combine their contents with that of the patient’s cell. This method of drug delivery is less toxic than direct exposure because the liposomes carry the drug directly to cells without any unnecessary intermediate steps.

Because of the hydrophobic interactions among several phospholipids and glycolipids, a certain structure called the lipid bilayer or bimolecular sheet is favored. As mentioned earlier, phospholipids and glycolipids have both hydrophilic and hydrophobic moieties; thus, when several phospholipids or glycolipids come together in an aqueous solution, the hydrophobic tails interact with each other to form a hydrophobic center, while the hydrophilic heads interact with each other forming a hydrophilic coating on each side of the bilayer.











Evidence Report/Technology Assessment   Number 89


Effects of Omega-3 Fatty Acids on Lipids and Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis


Prepared for:

Agency for Healthcare Research and Quality

U.S. Department of Health and Human Services

540 Gaither Road

Rockville, MD 20850

Contract No. 290-02-0003


Chapter 1. Introduction

This report is one of a group of evidence reports prepared by three Agency for Healthcare Research and Quality (AHRQ)-funded Evidence-Based Practice Centers (EPCs) on the role of omega-3 fatty acids (both from food sources and from dietary supplements) in the prevention or treatment of a variety of diseases. These reports were requested and funded by the Office of Dietary Supplements, National Institutes of Health. The three EPCs – the Southern California EPC (SCEPC, based at RAND), the Tufts-New England Medical Center (NEMC) EPC, and the University of Ottawa EPC – have each produced evidence reports. To ensure consistency of approach, the three EPCs collaborated on selected methodological elements, including literature search strategies, rating of evidence, and data table design.

The aim of these reports is to summarize the current evidence on the effects of omega-3 fatty acids on prevention and treatment of cardiovascular diseases, cancer, child and maternal health, eye health, gastrointestinal/renal diseases, asthma, immune- mediated diseases, tissue/organ transplantation, mental health, and neurological diseases and conditions. In addition to informing the research community and the public on the effects of omega-3 fatty acids on various health conditions, it is anticipated that the findings of the reports will also be used to help define the agenda for future research.

This report focuses on the effects of omega-3 fatty acids on immune- mediated diseases, bone metabolism, and gastrointestinal/renal diseases. Subsequent reports from the SCEPC will focus on cancer and neurological diseases and conditions.

This chapter provides a brief review of the current state of knowledge about the metabolism, physiological functions, and sources of omega-3 fatty acids.


The Recognition of Essential Fatty Acids

Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.

These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2

Fatty Acid Nomenclature

The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).

The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).

PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule. The trivial names, chemical names and abbreviations for the omega-3 fatty acids are detailed in Table 1.1.  Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).

Fatty Acid Metabolism

Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.

No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.

Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).

The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.

The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.

Physiological Functions of EPA and AA

As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.

Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3

The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.

EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.

DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).

Dietary Sources and Requirements

Both ALA and LA are present in a variety of foods. LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA is present in some commonly used oils, including canola and soybean oil, and in some leafy green vegetables. Thus, the major dietary sources of ALA and LA are PUFA-rich vegetable oils. The proportion of LA to ALA as well as the proportion of those PUFAs to others varies considerably by the type of oil. With the exception of flaxseed, canola, and soybean oil, the ratio of LA to ALA in vegetable oils is at least 10 to 1. The ratios of LA to ALA for flaxseed, canola, and soy are approximately 1: 3.5, 2:1, and 8:1, respectively; however, flaxseed oil is not typically consumed in the North American diet. It is estimated that on average in the U.S., LA accounts for 89% of the total PUFAs consumed, and ALA accounts for 9%. Another estimate suggests that Americans consume 10 times more omega-6 than omega-3 fatty acids.4 Table 1.2 shows the proportion of omega 3 fatty acids for a number of foods.

Syntheis and Degradation

Source of Acetyl CoA for Fatty Acid Synthesis

Source of Acetyl CoA for Fatty Acid Synthesis

step 1

step 1

condensation reaction with malonyl ACP

ACP (acyl carrier protein)

ACP (acyl carrier protein)

synthesis requires acetyl CoA from citrate shuttle

synthesis requires acetyl CoA from citrate shuttle

conversion to fatty acyl co A in cytoplasm

conversion to fatty acyl co A in cytoplasm

ACP (acyl carrier protein)

ACP (acyl carrier protein)

FA synthesis not exactly reverse of catabolism

FA synthesis not exactly reverse of catabolism


Fatty Acid Synthase

Fatty Acid Synthase

complete FA synthesis

complete FA synthesis



Elongation and Desaturation of Fatty Acids

Elongation and Desaturation of Fatty Acids

release of FAs from adiposites

release of FAs from adiposites

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

ketone bodies

ketone bodies

metabolism of ketone bodies

metabolism of ketone bodies



Arachidonate pathways

Arachidonate pathways

arachidonic acid derivatives

arachidonic acid derivatives

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

Model for the sterol-mediated proteolytic release of SREBPs from membrane

Model for the sterol-mediated proteolytic release of SREBPs from membrane

hormone regulation

hormone regulation

 insulin receptor and and insulin receptor signaling pathway (IRS)

insulin receptor and and insulin receptor signaling pathway (IRS)

 islet brain glucose signaling

islet brain glucose signaling









Fish source

Fish source

omega FAs

omega FAs


Excessive omega 6s

Excessive omega 6s

omega 6s

omega 6s

diet and cancer

diet and cancer

Patients at risk of FA deficiency

Patients at risk of FA deficiency

PPAR role

PPAR role

PPAR role

PPAR role

Omega 6_3 pathways

Omega 6_3 pathways

n3 vs n6 PUFAs

n3 vs n6 PUFAs

triene-teraene ratio

triene-teraene ratio

arachidonic acid, leukotrienes, PG and thromboxanes

arachidonic acid, leukotrienes, PG and thromboxanes

Cox 2 and cancer

Cox 2 and cancer

Lipidomics of atherosclerotic plaques

Lipidomics of atherosclerotic plaques
















Effect of TPN on EFAD

Effect of TPN on EFAD

benefits of omega 3s

benefits of omega 3s

food consumption

food consumption


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Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP


We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

Treatment Options for Left Ventricular Failure – Temporary Circulatory Support: Intra-aortic balloon pump (IABP) – Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical)
Author: Larry H Bernstein, MD, FCAP And Curator: Justin D Pearlman, MD, PhD, FACC

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN 

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:


This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)


“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.    …/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.


Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960, 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.


Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx.

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.


2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.


Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.


Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14. 


Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor


3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.

Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109. 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.




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Curated by: Dr. Venkat S. Karra, Ph.D.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease resulting in chronic activation of self-reactive lymphocytes and pro-inflammatory myeloid cells. SLE may also be caused by certain drugs called drug-induced lupus erythematosus.  People with SLE have abnormal deposits in the kidney cells. This leads to a condition called lupus nephritis. Patients with this condition may eventually develop kidney failure and need dialysis or a kidney transplant. The underlying cause of autoimmune diseases is not fully known and so far there is no cure for SLE.

SLE effects multiple end organs including the kidneys, brain, joints and skin and causes damage to many different parts of the body, including:

1. Blood clots in the legs (deep vein thrombosis) or lungs (pulmonary embolism)

2. Destruction of red blood cells (hemolytic anemia) or anemia of chronic disease

3. Fluid around the heart, pericarditis, endocarditis or inflammation of the heart (myocarditis)

4. Fluid around the lungs (pleural effusions) and damage to lung tissue

5. Pregnancy complications, including miscarriage

6. Stroke

7. Severely low blood platelets (thrombocytopenia)

8. Inflammation of the blood vessels

The molecular basis for the various manifestations of this autoimmune disease and the impact of the systemic autoimmune process on basic metabolic processes in the body are currently obscure.

However, recently a metabolomic study was executed first to understand the metabolic disturbances that underlie systemic lupus erythematosus (SLE). The study compared the sera of 20 SLE patients against that of healthy controls, using LC/MS and GC/MS platforms. Validation of key differences was performed using an independent cohort of 38 SLE patients and orthogonal assays.

The SLE metabolome exhibited profound lipid peroxidation, reflective of oxidative damage. Deficiencies were noted in the cellular anti-oxidant, glutathione, and all methyl group donors, including cysteine, methionine, and choline, as well as phosphocholines.

SLE sera showed evidence of profoundly dampened glycolysis, Krebs cycle, fatty acid β oxidation and amino acid metabolism, alluding to reduced energy biogenesis from all sources.

Whereas long-chain fatty acids, including the n3 and n6 essential fatty acids, were significantly reduced, medium chain fatty acids  and serum free fatty acids were elevated.

The best discriminators of SLE included elevated lipid peroxidation products, MDA, gamma-glutamyl peptides, GGT, leukotriene B4 and 5-HETE.

Comprehensive profiling of the SLE metabolome reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles and a pro-thrombotic state.

From this study it is evident that first supplementing the diet with essential fatty acids, vitamins and methyl group donors offers novel opportunities for disease modulation in this disabling systemic autoimmune ailment.

Second quickly identifying selected molecules/ therapies is another opportunity to resetting the SLE metabolome. One such opportunity is to use adrenocorticotropic hormone (ACTH) analogue.

With Prednisone, up to 90% of adults with minimal change disease (MCD) will respond to initial therapy and may require further immunosuppression. But with diseases such as idiopathic membranous nephropathy (iMN) and focal segmental glomerulosclerosis (FSGS), for which first-line therapies produce substantially lower response rates than for MCD and physicians are often compelled to use second-, third-, and even fourth-line therapies to achieve remission.

ACTH usage is not new, it was widely used way back in 1950s for the treatment of childhood nephrotic syndrome. Now there is a renewed interest in using ACTH as treatment for nephrotic syndrome as a second, third or even fourth line treatment, particularly in patients who are resistant to conventional therapies.

Subsequent clinical studies demonstrated that ACTH has prominent antiproteinuric and renoprotective effects that are not entirely explained by steroidogenic actions.

Adrenocorticotropic hormone (ACTH), also known as corticotropin, is a polypeptide tropic hormone produced and secreted by the anterior pituitry gland. It is an important component of the hypothalamic-pituitary-adrenal axis (HPA) and is often produced in response to biological stress. Its principal effects are increased production and release of corticosteriods. HPA is a complex set of direct influences and feedbackk interactions among the hypothalamus, the pituitary gland  and the adrenal glands.

A deficiency of ACTH is a cause of secondary adrenal insufficiency and an excess of it is a cause of Cushing’s syndrome.

Steroid hormones ( steriod that acts as a hormone) can be grouped into five groups by the receptors to which they bind: glycocorticoids, mineralcarticoids, androgens, estrogens, and progestrogens.

Steroid hormones help control metabolism, inflammation, immune functions, salt and water balance, development of sexual characteristics, and the ability to withstand illness and injury.

As a potent physiological agonist of melanocortin system that could directly target renal parenchymal cells, such as podocytes, ACTH might serve as a promising therapy for nephrotic glomerulopathies (a disease affecting the renal glomeruli – inflammatory or non-inflammatory).

Mineralocorticoids are hormones that were involved in the retention of sodium. The primary endogenous mineralocorticoid is aldosterone. Aldosterone acts on the kidneys to provide active reabsorption of sodium and an associated passive reabsorption of water, as well as the active secretion of potassium in the principal cells of the cortical collecting tubule and active secretion of protons via proton ATPases in the lumenal membrane of the intercalated cells of the collecting tubule. This in turn results in an increase of blood pressure and blood volume.

Aldosterone is produced in the cortex of the adrenal gland and its secretion is mediated principally by angiotensin II but also by adrenocorticotropic hormone (ACTH) and local potassium levels.

Aldosterone and cortisol (a glucosteroid) have similar affinity for the mineralocorticoid receptor; however, glucocorticoids circulate at roughly 100 times the level of mineralocorticoids. Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the HPA axis, and peripheral metabolism, for example by the enzyme 11beta-hydroxysteroid dehydrogenase type1 (11B-HSD1), which catalyses the reduction of inactive cortisone (11-DHC in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11B-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11B-HSD1 is implicated in the development of obesity.

Knock out of H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). Knock out of 11B-HSD1 alone or in combination with H6PDH led to a significant increase (36% and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11B-HSD1/H6PDH heterozygotes maintained a normal HPA axis.

Urinary steroid metabolite profile by GC/MS as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11B-HSD1/H6PDH deficiency

ACTH acts through the stimulation of cell surface ACTH receptors, which are located primarily on adrenocortical cells of the adrenal cortex. This results in the synthesis and secretion of gluco- and mineralo-corticosteriods and androgenic steroids.

An enzyme exists in mineralocorticoid target tissues to prevent overstimulation by glucocorticoids. This enzyme, 11-beta hydroxysteriod dehydrogenase type II (protein: HSD11B2), catalyzes the deactivation of glucocorticoids to 11-dehydro metabolites.

ACTH acts at several key steps to influence the steroidogenic pathway in the adrenal cortex:

ACTH stimulates lipoprotein uptake into cortical cells. This increases the bio-availability of cholestrol in the cells of the adrenal cortex.

ACTH increases the transport of cholesterol into the mitochondria and activates its hydrolysis.

ACTH Stimulates cholesterol side-chain cleavage enzyme, which makes the rate-limiting step in steroidogenesis. This results in the production of pregnenolone.

Receptor-binding studies have revealed that mineralcorticoids show a strong affinity for ACTH thereby establishing the potential for this hormone to activate mineralocorticoid receptors (MCRs). There are five MCRs and all of them show affinity for ACTH.

MCRs are expressed in kidney cells and that indicates that kidney is a target organ for the affects of ACTH.

Functions include:

1. Steroidogenic and adrenotropic activity

2. A multifaceted extra adrenal action that is mediated by the different MCRs present in the peripheral tissues and CNS

3. Has a lipostatic effect and stimulates lipolysis – (thus ACTH deficiency leads to obesity)

4. Its administration lowers levels of plasma lipids including Triglycerides, Total cholestrol, LDL-cholestrol and phospholipids

5. Its administration (complete ACTH molecule) rapidly increases the plasma insulin

Other activities include:

1. regulation of skin and hair pigmentation,

2. modulation of sebacious gland function and

3. anti-inflammatory and immunomodulatory functions

The total adrenocorticotropic hormone (ACTH) analogue is available as H.P. Acthar Gel (repository corticotropin injection) and is used for:

1. Monotherapy treatment of infantile spasms (IS) in infants and children under 2 years of age.

2. The treatment of exacerbations of multiple sclerosis in adults.

3. For inducing a diuresis or a remission of proteinuria in the nephrotic syndrome without uremia of the idiopathic type or that due to lupus erythematosus.

4. Also:: rheumatic disorders; collagen diseases; dermatologic diseases; allergic states; ophthalmic diseases and respiratory diseases.

FDA approved indications for the above prodcut are available at the following URL:

Disclaimer: This is for information purpose only, not a medical advise.

For a full list of warnings, precautions, and adverse events related to Acthar, please refer to the full Prescribing Information including the Medication Guide for the treatment of Infantile Spasms and discuss this information with your healthcare provider.


The renaissance of corticotropin therapy in proteinuric nephropathies

Metabolic Disturbances Associated with Systemic Lupus Erythematosus

H.P. Acthar Gel and Cosyntropin Review

Childhood nephrotic syndrome—current and future therapies

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