The correlations between intramyocellular lipid (IMCL), decreased fatty acid oxidation (FAO), and insulin resistance have led to the hypothesis that impaired FAO causes accumulation of lipotoxic intermediates that inhibit muscle insulin signaling. Using a skeletal muscle-specific carnitine palmitoyltransferase-1 KO model, we show that prolonged and severe mitochondrial FAO inhibition results in increased carbohydrate utilization, along with reduced physical activity; increased circulating nonesterified fatty acids; and increased IMCLs, diacylglycerols, and ceramides. Perhaps more importantly, inhibition of mitochondrial FAO also initiates a local, adaptive response in muscle that invokes mitochondrial biogenesis, compensatory peroxisomal fat oxidation, and amino acid catabolism. Loss of its major fuel source (lipid) induces an energy deprivation response in muscle coordinated by signaling through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) to maintain energy supply for locomotion and survival. At the whole-body level, these adaptations result in resistance to obesity.carnitine palmitoyltransferase | muscle | fatty acid | lipid | carbohydrate C onsiderable evidence suggests that when oversupply of dietary fat exceeds the storage capacity of adipose tissue, ectopic lipids accumulate in skeletal muscle, leading to "metabolic stress" that induces insulin resistance. One prevailing theory is that impaired skeletal muscle fatty acid oxidation (FAO) (1-4) leads to cytosolic accumulation of lipotoxic intermediates that are directly linked to defects in insulin signaling (5-11). Recent findings counter this premise because they have shown that models of insulin resistance consistently exhibit enhanced (not reduced) FAO, as demonstrated by elevated incomplete β-oxidation and accumulation of excess lipid-derived acylcarnitines (12, 13). Supporting evidence was obtained using a whole-body genetic approach to elevate levels of the endogenous carnitine palmitoyltransferase-1 (Cpt1) inhibitor, malonyl-CoA (12). These studies have led to the contrasting theory that lipid overload and incomplete FAO within the mitochondria accelerate the progression of insulin resistance (14). Faced with a plethora of studies supporting both hypotheses, a crucial question remains: "Is inhibition of mitochondrial FAO in skeletal muscle sufficient to initiate development of insulin resistance?" Cpt1 is essential for long-chain acyl-CoA transport into the mitochondria, and lies at the nexus of both the lipotoxicity and the mitochondrial overload hypotheses. If decreased FAO is a root cause of lipotoxicity, then muscle-specific ablation of Cpt1b activity should lead to impaired FAO, intramyocellular lipid (IMCL) accumulation, and insulin resistance. In stark contrast, the mitochondrial overload hypothesis suggests that decreased Cpt1b activity would preserve insulin sensitivity by preventing unbalanced overfueling of β-oxidation. Characterization of the rare genetic disorders o...

Toll-like receptor 4 (TLR4), a protein integral to innate immunity, is elevated in skeletal muscle of obese and type 2 diabetic humans and has been implicated in the development of lipid-induced insulin resistance. The purpose of this study was to examine the role of TLR4 as a modulator of basal (non-insulin-stimulated) substrate metabolism in skeletal muscle with the hypothesis that its activation would result in reduced fatty acid oxidation and increased partitioning of fatty acids toward neutral lipid storage. Human skeletal muscle, rodent skeletal muscle, and skeletal muscle cell cultures were employed to study the functional consequences of TLR4 activation on glucose and fatty acid metabolism. Herein, we demonstrate that activation of TLR4 with low (metabolic endotoxemia) and high (septic conditions) doses of LPS results in increased glucose utilization and reduced fatty acid oxidation in skeletal muscle and that these changes in metabolism in vivo occur in concert with increased circulating triglycerides. Moreover, animals with a loss of TLR4 function possess increased oxidative capacity in skeletal muscle and present with lower fasting levels of triglycerides and nonesterified free fatty acids. Evidence is also presented to suggest that these changes in substrate metabolism under metabolic endotoxemic conditions are independent of skeletal muscle-derived proinflammatory cytokine production. This report illustrates that skeletal muscle is a target for circulating endotoxin and may provide critical insight into the link between a proinflammatory state and dysregulated metabolism as observed with obesity, type 2 diabetes, and metabolic syndrome.

Cultured myocytes from severely obese women express perturbations in FA metabolism and insulin signaling reminiscent of those observed in vivo. The obesity phenotype can be recapitulated in muscle cells from lean subjects via exposure to excess lipid, but not by overexpressing the FAT/CD36 FA transporter.

OBJECTIVETo determine whether the obesity-related decrement in fatty acid oxidation (FAO) in primary human skeletal muscle cells (HSkMC) is linked with lower mitochondrial content and whether this deficit could be corrected via overexpression of peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α).RESEARCH DESIGN AND METHODSFAO was studied in HSkMC from lean (BMI 22.4 ± 0.9 kg/m2; N = 12) and extremely obese (45.3 ± 1.4 kg/m2; N = 9) subjects. Recombinant adenovirus was used to increase HSkMC PGC-1α expression (3.5- and 8.0-fold), followed by assessment of mitochondrial content (mtDNA and cytochrome C oxidase IV [COXIV]), complete (14CO2 production from labeled oleate), and incomplete (acid soluble metabolites [ASM]) FAO, and glycerolipid synthesis.RESULTSObesity was associated with a 30% decrease (P < 0.05) in complete FAO, which was accompanied by higher relative rates of incomplete FAO ([14C]ASM production/14CO2), increased partitioning of fatty acid toward storage, and lower (P < 0.05) mtDNA (−27%), COXIV (−35%), and mitochondrial transcription factor (mtTFA) (−43%) protein levels. PGC-1α overexpression increased (P < 0.05) FAO, mtDNA, COXIV, mtTFA, and fatty acid incorporation into triacylglycerol in both lean and obese groups. Perturbations in FAO, triacylglycerol synthesis, mtDNA, COXIV, and mtTFA in obese compared with lean HSkMC persisted despite PGC-1α overexpression. When adjusted for mtDNA and COXIV content, FAO was equivalent between lean and obese groups.CONCLUSIONReduced mitochondrial content is related to impaired FAO in HSkMC derived from obese individuals. Increasing PGC-1α protein levels did not correct the obesity-related absolute reduction in FAO or mtDNA content, implicating mechanisms other than PGC-1α abundance.

Recent reports suggest that short-term pharmacological Cpt1 inhibition improves skeletal muscle glucose tolerance and insulin sensitivity. While this appears promising for the treatment of diabetes these Cpt1 inhibitors are not specific to skeletal muscle and target multiple Cpt1 isoforms. To assess the effects of inhibiting the Cpt1b isoform we generated mice with a heart and skeletal muscle specific deletion of the Cpt1b, Cpt1bHM−/−. These mice seem to develop normally with similar bodyweights as control mice. However, by 15 weeks of age the Cpt1bHM−/− mice begin to die. The hearts of Cpt1bHM−/− mice were 4-times the size of controls. Cpt1bHM−/− mice were also subject to stress-induced seizures that accompanied an increased risk for premature mortality. Our data suggests that prolonged Cpt1b inhibition poses severe cardiac risk and emphasizes that attempts to improve insulin sensitivity by targeting Cpt1 with current inhibitors is not viable.

ObjectiveThe purpose of this investigation was to understand the metabolic adaptations to a short-term (5 days), isocaloric, high fat diet (HFD) in healthy, young males.MethodsTwo studies were undertaken with 12 subjects. Study 1 investigated the effect of the HFD on skeletal muscle substrate metabolism and insulin sensitivity. Study 2 assessed the metabolic and transcriptional response in skeletal muscle to the transition from a fasted-to-fed state using a high fat meal challenge prior to and following 5 days of HFD.ResultsStudy 1 showed no effect of a HFD on skeletal muscle metabolism or insulin sensitivity in fasting samples. Study 2 showed that a HFD elicits significant increases in fasting serum endotoxin, and disrupts the normal postprandial excursions of serum endotoxin, and metabolic and transcriptional responses in skeletal muscle. These effects following 5 days of HFD were accompanied by an altered fasting and postprandial response in the ratio of phosphorylated to total p38 protein. These changes all occurred in the absence of alterations in insulin sensitivity.ConclusionsOur findings provide evidence for early biological adaptations to high fat feeding that proceed and possibly lead to insulin resistance.

Aims Bioactives of Artemisia dracunculus L. (termed PMI 5011) have been shown to improve insulin action by increasing insulin signalling in skeletal muscle. However, it has not known if PMI 5011’s effects are retained during an inflammatory condition. We examined the attenuation of insulin action and whether PMI 5011 enhances insulin signalling in the inflammatory environment with elevated cytokines. Methods Muscle cell cultures derived from lean, overweight and diabetic obese subjects were used. Expression of pro-inflammatory genes and inflammatory response of human myotubes were evaluated by RT-PCR. Insulin signalling and activation of inflammatory pathways in human myotubes were evaluated by Multiplex protein assays. Results We found increased gene expression of MCP1 and TNFα, and basal activity of the NFkB pathway in myotubes derived from diabetic-obese subjects as compared to myotubes derived from normal-lean subjects. In line with this, basal Akt phosphorylation (Ser473) was significantly higher, while insulin-stimulated phosphorylation of Akt (Ser473) was lower in myotubes from normal-overweight and diabetic-obese subjects compared to normal-lean subjects. PMI 5011 treatment reduced basal phosphorylation of Akt and enhanced insulin-stimulated phosphorylation of Akt in the presence of cytokines in human myotubes. PMI 5011 treatment led to an inhibition of cytokine-induced activation of inflammatory signalling pathways such as Erk1/2 and IkBα-NFkB and moreover, NFkB target gene expression, possibly by preventing further propagation of the inflammatory response within muscle tissue. Conclusions PMI 5011 improved insulin sensitivity in diabetic-obese myotubes to the level of normal-lean myotubes despite the presence of pro-inflammatory cytokines.

Obesity‐associated, low‐grade, systemic inflammation is major contributor to obesity‐induced insulin resistance. Impaired insulin signalling in skeletal muscle is a key feature of type 2 diabetes. Bioactives of Artemisia dracunculus L. (termed PMI 5011) have been shown to improve insulin action by increasing insulin signalling in skeletal muscle. However, it has not known if PMI 5011’s effects are retained during an inflammatory condition. We examined the attenuation of insulin action and whether PMI 5011 enhances insulin signalling in the inflammatory environment with elevated cytokines. We found increased inflammation in skeletal muscle tissue of diet‐induced obese mice with upregulated Mcp1, Tnfα, Cxcl5, Cxcl10 and Tnfr1, Tnfr2 gene expressions. In line with this we detected increases in gene expression of MCP1 and TNFα, and basal activity of the NFkB pathway in myotubes derived from diabetic‐obese subjects as compared to myotubes derived from normal‐lean subjects. Multiplex protein analysis revealed that basal Akt phosphorylation (Ser473) was significantly higher, while insulin‐stimulated phosphorylation of Akt (Ser473) was lower in myotubes from normal‐overweight and diabetic‐obese subjects compared to normal‐lean subjects. PMI 5011 treatment reduced basal phosphorylation of Akt and enhanced insulin‐stimulated phosphorylation of Akt in the presence of cytokines in human myotubes. PMI 5011 treatment led to an inhibition of cytokine‐induced activation of inflammatory signalling pathways such as Erk1/2 and IkBα‐NFkB and moreover, NFkB target gene expression, possibly by preventing further propagation of the inflammatory response within muscle tissue. Thus PMI 5011 improved insulin sensitivity in diabetic‐obese myotubes to the level of normal‐lean myotubes despite the presence of pro‐inflammatory cytokines. Grant Funding Source: Supported by grants NCAMM (P50AT002776 NIH), ADA (1‐10‐BS‐129), NIH (RO1DK089641), COBRE (NIH 8P20GM103528), NORC (NIH 2P30‐DK072476)