Metabolic rescue in pluripotent cells from patients with mtDNA disease.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Energy metabolism in the acquisition and maintenance of stemness.

Energy metabolism is traditionally considered a reactive homeostatic system addressing stage-specific cellular energy needs. There is however growing appreciation of metabolic pathways in the active control of vital cell functions. Case in point, the stem cell lifecycle--from maintenance and acquisition of stemness to lineage commitment and specification--is increasingly recognized as a metabolism-dependent process. Indeed, metabolic reprogramming is an early contributor to the orchestrated departure from or reacquisition of stemness. Recent advances in metabolomics have helped decipher the identity and dynamics of metabolic fluxes implicated in fueling cell fate choices by regulating the epigenetic and transcriptional identity of a cell. Metabolic cues, internal and/or external to the stem cell niche, facilitate progenitor pool restitution, long-term tissue renewal or ensure adoption of cytoprotective behavior. Convergence of energy metabolism with stem cell fate regulation opens a new avenue in understanding primordial developmental biology principles with future applications in regenerative medicine practice.

1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells.

Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength.

Myocardial fatty acid metabolism in health and disease.

There is a constant high demand for energy to sustain the continuous contractile activity of the heart, which is met primarily by the beta-oxidation of long-chain fatty acids. The control of fatty acid beta-oxidation is complex and is aimed at ensuring that the supply and oxidation of the fatty acids is sufficient to meet the energy demands of the heart. The metabolism of fatty acids via beta-oxidation is not regulated in isolation; rather, it occurs in response to alterations in contractile work, the presence of competing substrates (i.e., glucose, lactate, ketones, amino acids), changes in hormonal milieu, and limitations in oxygen supply. Alterations in fatty acid metabolism can contribute to cardiac pathology. For instance, the excessive uptake and beta-oxidation of fatty acids in obesity and diabetes can compromise cardiac function. Furthermore, alterations in fatty acid beta-oxidation both during and after ischemia and in the failing heart can also contribute to cardiac pathology. This paper reviews the regulation of myocardial fatty acid beta-oxidation and how alterations in fatty acid beta-oxidation can contribute to heart disease. The implications of inhibiting fatty acid beta-oxidation as a potential novel therapeutic approach for the treatment of various forms of heart disease are also discussed.

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Metabolic determinants of embryonic development and stem cell fate.

Decoding stem cell metabolism has implicated a tight linkage between energy metabolism and cell fate regulation, a dynamic interplay vital in the execution of developmental and differentiation programs. The inherent plasticity in energy metabolism enables prioritisation of metabolic pathways in support of stage-specific demands. Beyond traditional support of energetic needs, intermediate metabolism may also dictate cell fate choices through regulation of cellular signalling and epigenetic regulation of gene expression. The notion of a 'metabolism-centric' control of stem cell differentiation has been informed by developmental embryogenesis based upon an on-demand paradigm paramount in defining diverse developmental behaviours, from a post-fertilisation nascent zygote to complex organogenesis leading to adequate tissue formation and maturation. Monitored through natural or bioengineered stem cell surrogates, nutrient-responsive metabolites are identified as mediators of cross-talk between metabolic flux, cell signalling and epigenetic regulation charting, collectively, whether a cell will self-renew to maintain progenitor pools, lineage specify to ensure tissue (re)generation or remain quiescent to curb stress damage. Thus, bioenergetics are increasingly recognised as integral in governing stemness and associated organogenic decisions, paving the way for metabolism-defined targets in control of embryology, stem cell biology and tissue regeneration.

Stem cell systems informatics for advanced clinical biodiagnostics: tracing molecular signatures from bench to bedside.

Development of innovative high throughput technologies has enabled a variety of molecular landscapes to be interrogated with an unprecedented degree of detail. Emergence of next generation nucleotide sequencing methods, advanced proteomic techniques, and metabolic profiling approaches continue to produce a wealth of biological data that captures molecular frameworks underlying phenotype. The advent of these novel technologies has significant translational applications, as investigators can now explore molecular underpinnings of developmental states with a high degree of resolution. Application of these leading-edge techniques to patient samples has been successfully used to unmask nuanced molecular details of disease vs healthy tissue, which may provide novel targets for palliative intervention. To enhance such approaches, concomitant development of algorithms to reprogram differentiated cells in order to recapitulate pluripotent capacity offers a distinct advantage to advancing diagnostic methodology. Bioinformatic deconvolution of several "-omic" layers extracted from reprogrammed patient cells, could, in principle, provide a means by which the evolution of individual pathology can be developmentally monitored. Significant logistic challenges face current implementation of this novel paradigm of patient treatment and care, however, several of these limitations have been successfully addressed through continuous development of cutting edge in silico archiving and processing methods. Comprehensive elucidation of genomic, transcriptomic, proteomic, and metabolomic networks that define normal and pathological states, in combination with reprogrammed patient cells are thus poised to become high value resources in modern diagnosis and prognosis of patient disease.

Metabolic Remodeling during Early Cardiac Lineage Specification of Pluripotent Stem Cells.

Growing evidence indicates that metabolites and energy metabolism play an active rather than consequential role in regulating cellular fate. Cardiac development requires dramatic metabolic remodeling from relying primarily on glycolysis in pluripotent stem cells (PSCs) to oxidizing a wide array of energy substrates to match the high bioenergetic demands of continuous contraction in the developed heart. However, a detailed analysis of how remodeling of energy metabolism contributes to human cardiac development is lacking. Using dynamic multiple reaction monitoring metabolomics of central carbon metabolism, we evaluated temporal changes in energy metabolism during human PSC 3D cardiac lineage specification. Significant metabolic remodeling occurs during the complete differentiation, yet temporal analysis revealed that most changes occur during transitions from pluripotency to mesoderm (day 1) and mesoderm to early cardiac (day 5), with limited maturation of cardiac metabolism beyond day 5. Real-time metabolic analysis demonstrated that while hPSC cardiomyocytes (hPSC-CM) showed elevated rates of oxidative metabolism compared to PSCs, they still retained high glycolytic rates, confirming an immature metabolic phenotype. These observations support the opportunity to metabolically optimize the differentiation process to support lineage specification and maturation of hPSC-CMs.

Extracellular Flux Analysis of Mitochondrial Function in Pluripotent Stem Cells.

Mitochondrial function and energy metabolism are increasingly recognized not only as regulators of pluripotent stem cell function and fate, but also as critical targets in disease pathogenesis and aging. Therefore across the downstream applications of pluripotent stem cells, including development and disease modeling, drug screening, and cell-based therapies, it is crucial to be able to measure mitochondrial function and metabolism in a high-throughput, real-time and label-free manner. Here we describe the application of Seahorse extracellular flux analysis to measure mitochondrial function in pluripotent stem cells and their derivatives. Specifically, we highlight two assays, the Mitochondrial Stress Test, which quantifies overall mitochondrial function including basal, maximal and ATP-couple oxygen consumption rates, and the Electron Transport Chain Complex Specific assay, that quantifies function of individual complexes within the electron transport chain.

Uncoupling of Proliferative Capacity from Developmental Stage During Directed Cardiac Differentiation of Pluripotent Stem Cells.

Lineage-specific differentiation of human-induced pluripotent stem cells (hiPSCs) into cardiomyocytes (CMs) offers a patient-specific model to dissect development and disease pathogenesis in a dish. However, challenges exist with this model system, such as the relative immaturity of iPSC-derived CMs, which evoke the question of whether this model faithfully recapitulates in vivo cardiac development. As in vivo cardiac developmental stage is intimately linked with the proliferative capacity (or maturation is inversely correlated to proliferative capacity), we sought to understand how proliferation is regulated during hiPSC CM differentiation and how it compares with in vivo mouse cardiac development. Using standard Chemically Defined Media 3 differentiation, gene expression profiles demonstrate that hiPSC-derived cardiomyocytes (hiPSC-CMs) do not progress past the equivalent of embryonic day 14.5 of murine cardiac development. Throughout differentiation, overall DNA synthesis rapidly declines with <5% of hiPSC-CMs actively synthesizing DNA at the end of the differentiation period despite their immaturity. Bivariate cell cycle analysis demonstrated that hiPSC-CMs have a cell cycle profile distinct from their non-cardiac counterparts from the same differentiation, with significantly fewer cells within G1 and a marked accumulation of cells in G2/M than their non-cardiac counterparts throughout differentiation. Pulse-chase analysis demonstrated that non-cardiac cells progressed completely through the cell cycle within a 24-h period, whereas hiPSC-CMs had restricted progression with only a small proportion of cells undergoing cytokinesis with the remainder stalling in late S-phase or G2/M. This cell cycle arrest phenotype is associated with abbreviated expression of cell cycle promoting genes compared with expression throughout murine embryonic cardiac development. In summary, directed differentiation of hiPSCs into CMs uncouples the developmental stage from cell cycle regulation compared with in vivo mouse cardiac development, leading to a premature exit of hiPSC-CMs from the cell cycle despite their relative immaturity.

Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function.

Energy deprivation in the myocardium is associated with impaired heart function and increased morbidity. LKB1 is a kinase that is required for activation of AMP-activated protein kinase (AMPK) as well as 13 AMPK-related protein kinases. AMPK stimulates ATP production during ischemia and prevents post-ischemic dysfunction. We used the Cre-Lox system to generate mice where LKB1 was selectively knocked out in cardiomyocytes and muscle cells (LKB1-KO) to assess the role of LKB1 on cardiac function in these mice. Heart rates of LKB1-KO mice were reduced and ventricle diameter was increased. Ex vivo, cardiac function was impaired during aerobic perfusion of isolated working hearts, and recovery of function after ischemia was reduced. Although oxidative metabolism and mitochondrial function were normal, the AMP/ATP ratio was increased in LKB1-KO hearts. This was associated with a complete ablation of AMPKalpha2 activity, and a stimulation of signaling through the mammalian target of rapamycin. Our results establish a critical role for LKB1 for normal cardiac function under both aerobic conditions and during recovery after ischemia. Ablation of LKB1 leads to a decreased cardiac efficiency despite normal mitochondrial oxidative metabolism.

Energy Metabolism Regulates Stem Cell Pluripotency.

Pluripotent stem cells (PSCs) are characterized by their unique capacity for both unlimited self-renewal and their potential to differentiate to all cell lineages contained within the three primary germ layers. While once considered a distinct cellular state, it is becoming clear that pluripotency is in fact a continuum of cellular states, all capable of self-renewal and differentiation, yet with distinct metabolic, mitochondrial and epigenetic features dependent on gestational stage. In this review we focus on two of the most clearly defined states: "naïve" and "primed" PSCs. Like other rapidly dividing cells, PSCs have a high demand for anabolic precursors necessary to replicate their genome, cytoplasm and organelles, while concurrently consuming energy in the form of ATP. This requirement for both anabolic and catabolic processes sufficient to supply a highly adapted cell cycle in the context of reduced oxygen availability, distinguishes PSCs from their differentiated progeny. During early embryogenesis PSCs adapt their substrate preference to match the bioenergetic requirements of each specific developmental stage. This is reflected in different mitochondrial morphologies, membrane potentials, electron transport chain (ETC) compositions, and utilization of glycolysis. Additionally, metabolites produced in PSCs can directly influence epigenetic and transcriptional programs, which in turn can affect self-renewal characteristics. Thus, our understanding of the role of metabolism in PSC fate has expanded from anabolism and catabolism to include governance of the pluripotent epigenetic landscape. Understanding the roles of metabolism and the factors influencing metabolic pathways in naïve and primed pluripotent states provide a platform for understanding the drivers of cell fate during development. This review highlights the roles of the major metabolic pathways in the acquisition and maintenance of the different states of pluripotency.

High rates of residual fatty acid oxidation during mild ischemia decrease cardiac work and efficiency.

It is unknown what effects high levels of fatty acids have on energy metabolism and cardiac efficiency during milder forms of ischemia. To address this issue, isolated working rat hearts perfused with Krebs-Henseleit solution (5 mM glucose, 100 muU/mL insulin, and 0.4 (Normal Fat) or 1.2 mM palmitate (High Fat)) were subjected to 30 min of aerobic perfusion followed by 30 min of mild ischemia (39% reduction in coronary flow). Both groups had similar aerobic function and rates of glycolysis, however the High Fat group had elevated rates of palmitate oxidation (150%), and decreased rates of glucose oxidation (51%). Mild ischemia decreased cardiac work (56% versus 40%) and efficiency (29% versus 11%) further in High Fat hearts. Palmitate oxidation contributed a greater percent of acetyl-CoA production during mild ischemia in the High Fat group (81% versus 54%). During mild ischemia glycolysis remained at aerobic levels in the Normal Fat group, but was accelerated in the High Fat group. Triglyceride, glycogen and adenine nucleotide content did not differ at the end of mild ischemia, however glycogen turnover was double in the High Fat group (248%). Addition of the pyruvate dehydrogenase inhibitor dichloroacetate to the High Fat group resulted in a doubling of the rate of glucose oxidation and improved cardiac efficiency during mild ischemia. We demonstrate that fatty acid oxidation dominates as the main source of residual oxidative metabolism during mild ischemia, which is accompanied by suppressed cardiac function and efficiency in the presence of high fat.

Suppression of 5'-AMP-activated protein kinase activity does not impair recovery of contractile function during reperfusion of ischemic hearts.

Activation of 5'-AMP-activated protein kinase (AMPK) may benefit the heart during ischemia-reperfusion by increasing energy production. While AMPK stimulates glycolysis, mitochondrial oxidative metabolism is the major source of ATP production during reperfusion of ischemic hearts. Stimulating AMPK increases mitochondrial fatty acid oxidation, but this is usually accompanied by a decrease in glucose oxidation, which can impair the functional recovery of ischemic hearts. To examine the relationship between AMPK and cardiac energy substrate metabolism, we subjected isolated working mouse hearts expressing a dominant negative (DN) alpha(2)-subunit of AMPK (AMPK-alpha(2) DN) to 20 min of global no-flow ischemia and 40 min of reperfusion with Krebs-Henseleit solution containing 5 mM [U-(14)C]glucose, 0.4 mM [9, 10-(3)H]palmitate, and 100 microU/ml insulin. AMPK-alpha(2) DN hearts had reduced AMPK activity at the end of reperfusion (82 +/- 9 vs. 141 +/- 7 pmol.mg(-1).min(-1)) with no changes in high-energy phosphates. Despite this, AMPK-alpha(2) DN hearts had improved recovery of function during reperfusion (14.9 +/- 0.8 vs. 9.4 +/- 1.4 beats.min(-1).mmHg.10(-3)). During reperfusion, fatty acid oxidation provided 44.0 +/- 2.8% of total acetyl-CoA in AMPK-alpha(2) DN hearts compared with 55.0 +/- 3.2% in control hearts. Since insulin can inhibit both AMPK activation and fatty acid oxidation, we also examined functional recovery in the absence of insulin. Functional recovery was similar in both groups despite a decrease in AMPK activity and a decreased reliance on fatty acid oxidation during reperfusion (66.4 +/- 9.4% vs. 85.3 +/- 4.3%). These data demonstrate that the suppression of cardiac AMPK activity does not produce an energetically compromised phenotype and does not impair, but may in fact improve, the recovery of function after ischemia.

Cardiac energy metabolism in obesity.

Obesity results in marked alterations in cardiac energy metabolism, with a prominent effect being an increase in fatty acid uptake and oxidation by the heart. Obesity also results in dramatic changes in the release of adipokines, such as leptin and adiponectin, both of which have emerged as important regulators of cardiac energy metabolism. The link among obesity, cardiovascular disease, lipid metabolism, and adipokine signaling is complex and not well understood. However, optimizing cardiac energy metabolism in obese subjects may be one approach to preventing and treating cardiac dysfunction that can develop in this population. This review discusses what is presently known about the effects of obesity and the impact adipokines have on cardiac energy metabolism and insulin signaling. The clinical implications of obesity and energy metabolism on cardiac disease are also discussed.

Fatty acids attenuate insulin regulation of 5'-AMP-activated protein kinase and insulin cardioprotection after ischemia.

The cardioprotective effect of insulin during ischemia-reperfusion has been associated with stimulation of glucose uptake and glycolysis. Although fatty acids and 5'-AMP activated protein kinase (AMPK) are regulators of glucose metabolism, it is unknown what effect insulin has on postischemic function and AMPK activity in the presence of high levels of fatty acid. Isolated ejecting mouse hearts were perfused with Krebs-Henseleit solution containing 5 mmol x L(-1) glucose and 0, 0.2, or 1.2 mmol x L(-1) palmitate, with or without 100 microU/mL insulin. During aerobic perfusion in the absence of palmitate, insulin stimulated glycolysis by 73% and glucose oxidation by 54%, while inhibiting AMPK activity by 43%. In the presence of 0.2 or 1.2 mmol x L(-1) palmitate, insulin stimulated glycolysis by 111% and 105% and glucose oxidation by 72% and 274% but no longer inhibited AMPK activity. During reperfusion of hearts in the absence of palmitate, insulin increased recovery of cardiac power by 47%. This was associated with a 97% increase in glycolysis and a 160% increase in glucose oxidation. However, in the presence of 1.2 mmol x L(-1) palmitate, insulin now decreased recovery of cardiac power by 42%. During reperfusion, glucose oxidation was inhibited by high fat, but insulin-stimulated glycolysis remained high, resulting in increased proton production. In the absence of fatty acids, insulin blunted the ischemia-induced activation of AMPK, but this effect was lost in the presence of fatty acids. We demonstrate that the cardioprotective effect of insulin and its ability to inhibit AMPK activity are lost in the presence of high concentrations of fatty acids.

Role of malonyl-CoA in heart disease and the hypothalamic control of obesity.

Obesity is an important contributor to the risk of developing insulin resistance, diabetes, and heart disease. Alterations in tissue levels of malonyl-CoA have the potential to impact on the severity of a number of these disorders. This review will focus on the emerging role of malonyl-CoA as a key "metabolic effector" of both obesity and cardiac fatty acid oxidation. In addition to being a substrate for fatty acid biosynthesis, malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase (CPT) 1, a key enzyme involved in mitochondrial fatty acid uptake. A decrease in myocardial malonyl-CoA levels and an increase in CPT1 activity contribute to an increase in cardiac fatty acid oxidation. An increase in malonyl-CoA degradation due to increased malonyl-CoA decarboxylase (MCD) activity may be one mechanism responsible for this decrease in malonyl-CoA. Another mechanism involves the inhibition of acetyl-CoA carboxylase (ACC) synthesis of malonyl-CoA, due to AMP-activated protein kinase (AMPK) phosphorylation of ACC. Recent studies have demonstrated a role of malonyl-CoA in the hypothalamus as a regulator of food intake. Increases in hypothalamic malonyl-CoA and inhibition of CPT1 are associated with a decrease in food intake in mice and rats, while a decrease in hypothalamic malonyl-CoA increases food intake and weight gain. The exact mechanism(s) responsible for these effects of malonyl-CoA are not clear, but have been proposed to be due to an increase in the levels of long chain acyl CoA, which occurs as a result of malonyl-CoA inhibition of CPT1. Both hypothalamic and cardiac studies have demonstrated that control of malonyl-CoA levels has an important impact on obesity and heart disease. Targeting enzymes that control malonyl-CoA levels may be an important therapeutic approach to treating heart disease and obesity.

Glycolytic Stimulation Is Not a Requirement for M2 Macrophage Differentiation.

Enhanced glucose uptake and a switch to glycolysis are key traits of M1 macrophages, whereas enhanced fatty acid oxidation and oxidative phosphorylation are the main metabolic characteristics of M2 macrophages. Recent studies challenge this traditional view, indicating that glycolysis may also be critically important for M2 macrophage differentiation, based on experiments with 2-DG. Here we confirm the inhibitory effect of 2-DG on glycolysis, but also demonstrate that 2-DG impairs oxidative phosphorylation and significantly reduces 13C-labeled Krebs cycle metabolites and intracellular ATP levels. These metabolic derangements were associated with reduced JAK-STAT6 pathway activity and M2 differentiation marker expression. While glucose deprivation and glucose substitution with galactose effectively suppressed glycolytic activity, there was no effective suppression of oxidative phosphorylation, intracellular ATP levels, STAT6 phosphorylation, and M2 differentiation marker expression. These data indicate that glycolytic stimulation is not required for M2 macrophage differentiation as long as oxidative phosphorylation remains active.