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.

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Progression through the cell division cycle is orchestrated by a complex network of interacting genes and proteins. Some of these proteins are known to fluctuate periodically during the cell cycle, but a systematic study of the fluctuations of a broad sample of cell-cycle proteins has not been made until now. Using time-lapse fluorescence microscopy, we profiled 16 strains of budding yeast, each containing GFP fused to a single gene involved in cell cycle regulation. The dynamics of protein abundance and localization were characterized by extracting the amplitude, period, and other indicators from a series of images. Oscillations of protein abundance could clearly be identified for Cdc15, Clb2, Cln1, Cln2, Mcm1, Net1, Sic1, and Whi5. The period of oscillation of the fluorescently tagged proteins is generally in good agreement with the inter-bud time. The very strong oscillations of Net1 and Mcm1 expression are remarkable since little is known about the temporal expression of these genes. By collecting data from large samples of single cells, we quantified some aspects of cell-to-cell variability due presumably to intrinsic and extrinsic noise affecting the cell cycle.

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Obesity is associated with chronic inflammation and the development of type 2 diabetes. Skeletal muscle from obese humans is resistant to insulin and characterized by abnormal substrate metabolism, i.e. increased glycolytic activity, reduced oxidative capacity, and elevated lipid accumulation. Toll‐like receptors (TLRs) are transmembrane receptors that are important in the induction of inflammatory responses. TLRs have been linked to lipid‐induced skeletal muscle insulin resistance and may contribute to reduced oxidative capacity and skeletal muscle lipid accumulation associated with obesity. In the current study, we demonstrate that the TLR4 signaling pathway is up regulated in skeletal muscle of obese humans and regulates substrate metabolism in a manner that reproduces the metabolic phenotype of skeletal muscle observed with obesity. TLR4 gene expression is increased in skeletal muscle of obese humans (p<0.001) and significantly related to BMI (r=0.92, p<0.001) and intramyocellular triglyceride synthesis (r=0.86, p<0.006). In addition, treatment of skeletal muscle cells with Lipopolysaccharide results in a 15% reduction in fatty acid oxidation (p<0.002), a 30% increase in glucose oxidation (p=0.04), and a 33% increase in triglyceride synthesis (p<0.002). These results provide a potential mechanism linking innate immunity with abnormal substrate metabolism associated with obesity.Supported by the American Diabetes Association (Junior Faculty Award, MWH) and the National Institutes of Health (DK078765‐01, MWH).