Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.

Background Significant evidence indicates that the failing heart is “energy-starved”. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. Methods and Results Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of β-hydroxybutyrate dehydrogenase 1 (BDH1), a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation studies in an isolated heart preparation using ex vivo NMR combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. Conclusions These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Peroxisome proliferator-activated receptor-␥ co-activator 1␣ (PGC1␣) is a promiscuous co-activator that plays a key role in regulating mitochondrial biogenesis and fuel homeostasis. Emergent evidence links decreased skeletal muscle PGC1␣ activity and coincident impairments in mitochondrial performance to the development of insulin resistance in humans. Here we used rodent models to demonstrate that muscle mitochondrial efficiency is compromised by diet-induced obesity and is subsequently rescued by exercise training. Chronic high fat feeding caused accelerated rates of incomplete fatty acid oxidation and accumulation of ␤-oxidative intermediates. The capacity of muscle mitochondria to fully oxidize a heavy influx of fatty acid depended on factors such as fiber type and exercise training and was positively correlated with expression levels of PGC1␣. Likewise, an efficient lipid-induced substrate switch in cultured myocytes depended on adenovirus-mediated increases in PGC1␣ expression. Our results supported a novel paradigm in which a high lipid supply, occurring under conditions of low PGC1␣, provokes a disconnect between mitochondrial ␤-oxidation and tricarboxylic acid cycle activity. Conversely, the metabolic remodeling that occurred in response to PGC1␣ overexpression favored a shift from incomplete to complete ␤-oxidation. We proposed that PGC1␣ enables muscle mitochondria to better cope with a high lipid load, possibly reflecting a fundamental metabolic benefit of exercise training.Obesity and type 2 diabetes are two closely associated diseases that continue to affect Westernized societies at epidemic rates (1). Results from both epidemiological and animal studies suggest that the risk of developing these diseases is increased by consumption of a high fat diet (2), which has been attributed in part to increased intramuscular triacylglycerol storage and adverse changes in skeletal muscle energy metabolism (3). Moreover, mounting evidence from animal and cellbased models indicates that lipid oversupply to skeletal muscle results in functional perturbations that eventually manifest as impaired insulin action (2, 4, 5). Together, these and other reports have established a compelling connection between muscle lipid dysregulation and impaired metabolic control at both the cellular and systemic levels. Despite intense investigation, a clear understanding of the biochemical and molecular mechanisms that mediate lipid-induced muscle dysfunction has remained elusive.Similar to high fat feeding, exercise training increases skeletal muscle supply and storage of lipid substrates (6). However, in contrast to the adverse events provoked by a high fat diet, exercise is known to promote muscle as well as whole body metabolic fitness (7). These observations suggest that physical activity somehow enhances the capacity of the muscle to tolerate and/or adapt to a high lipid load. Indeed, contractile activity leads to dramatic adjustments in oxidative fuel metabolism that are largely mediated by an increase in muscle mitochondria...

Lipid infusion or ingestion of a high-fat diet results in insulin resistance, but the mechanism underlying this phenomenon remains unclear. Here we show that, in rats fed a high-fat diet, whole-animal, muscle and liver insulin resistance is ameliorated following hepatic overexpression of malonyl-coenzyme A (CoA) decarboxylase (MCD), an enzyme that affects lipid partitioning. MCD overexpression decreased circulating free fatty acid (FFA) and liver triglyceride content. In skeletal muscle, levels of triglyceride and long-chain acyl-CoA (LC-CoA)-two candidate mediators of insulin resistance-were either increased or unchanged. Metabolic profiling of 36 acylcarnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, beta-OH-butyrate, in muscle of MCD-overexpressing animals. The best explanation for our findings is that hepatic expression of MCD lowered circulating FFA levels, which led to lowering of muscle beta-OH-butyrate levels and improvement of insulin sensitivity.

Summary The concept of “metabolic inflexibility” was first introduced to describe the failure of insulin resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine dimunition was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete ␤-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.Disturbances in mitochondrial genesis, morphology, and function are increasingly recognized as components of insulin resistance and the metabolic syndrome (1-3). Still unclear is whether poor mitochondrial performance is a predisposing factor or a consequence of the disease process. The latter view is supported by recent animal studies linking diet-induced insulin resistance to a dysregulated mitochondrial phenotype in skeletal muscle, marked by excessive ␤-oxidation, impaired substrate switching during the fasted to fed transition, and coincident reduction of organic acid intermediates of the tricarboxylic acid cycle (4, 5). In these studies, both diet-induced and genetic forms of insulin resistance were specifically linked to high rates of incomplete fat oxidation and intramuscular accumulation of fatty acylcarnitines, byproducts of lipid catabolism that are produced under conditions of metabolic stress (5, 6). Most compelling, we showed that genetically engineered inhibition of fat oxidation lowered intramuscular acylcarnitine levels and preserved glucose tolerance in mice fed a high fat diet (5, 7). In aggregate, the findings established a stron...

OBJECTIVEIt has been proposed that skeletal muscle insulin resistance arises from the accumulation of intramyocellular lipid metabolites that impede insulin signaling, including diacylglycerol and ceramide. We determined the role of de novo ceramide synthesis in mediating muscle insulin resistance.RESEARCH DESIGN AND METHODSMice were subjected to 12 weeks of diet-induced obesity (DIO), and then treated for 4 weeks with myriocin, an inhibitor of serine palmitoyl transferase-1 (SPT1), the rate-limiting enzyme of de novo ceramide synthesis.RESULTSAfter 12 weeks of DIO, C57BL/6 mice demonstrated a doubling in gastrocnemius ceramide content, which was completely reversed (141.5 ± 15.8 vs. 94.6 ± 10.2 nmol/g dry wt) via treatment with myriocin, whereas hepatic ceramide content was unaffected by DIO. Interestingly, myriocin treatment did not alter the DIO-associated increase in gastrocnemius diacyglycerol content, and the only correlation observed between lipid metabolite accumulation and glucose intolerance occurred with ceramide (R = 0.61). DIO mice treated with myriocin showed a complete reversal of glucose intolerance and insulin resistance which was associated with enhanced insulin-stimulated Akt and glycogen synthase kinase 3β phosphorylation. Furthermore, myriocin treatment also decreased intramyocellular ceramide content and prevented insulin resistance development in db/db mice. Finally, myriocin-treated DIO mice displayed enhanced oxygen consumption rates (3,041 ± 124 vs. 2,407 ± 124 ml/kg/h) versus their control counterparts.CONCLUSIONSOur results demonstrate that the intramyocellular accumulation of ceramide correlates strongly with the development of insulin resistance, and suggests that inhibition of SPT1 is a potentially promising target for the treatment of insulin resistance.

Summary Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a novel regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a novel SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.