Nikolas Dedousis

Inhibition or deletion of the lipopolysaccharide receptor Toll-like receptor-4 confers partial protection against lipid-induced insulin resistance in rodent skeletal muscle

Aims/hypothesisA role for increased activity of the innate immune system in the pathogenesis of insulin resistance is supported by a number of studies. The current study assessed the potential role of the lipopolysaccharide receptor known as Toll-like receptor-4 (TLR-4), a component of the innate immune system, in mediating lipid-induced insulin resistance in skeletal muscle.MethodsThe effects of TLR-4 inhibition/deletion on lipid-induced insulin resistance was determined in skeletal muscle of TLR-4 null mice in vivo and in rat L6 myotubes in vitro.ResultsIn mice, acute hyperlipidaemia induced skeletal muscle insulin resistance, but a deletion of TLR-4 conferred significant protection against these effects. In L6 myotubes, inhibition of TLR-4 activity substantially reduced the capacity of the saturated fatty acid palmitate to induce insulin resistance. Importantly, palmitate activated the nuclear factor κB (NFκB) pathway in L6 myotubes and mouse skeletal muscle, and these effects were blocked by inhibition of TLR-4 in L6 myotubes and absence of TLR-4 in skeletal muscle. Furthermore, inhibition of the NFκB pathway downstream of TLR-4 in L6 myotubes also protected against the induction of insulin resistance by palmitate.Conclusions/interpretationInhibition or absence of TLR-4 confers protection against the detrimental effects of lipids on skeletal muscle insulin action, and these effects are associated with a prevention of the activation of the NFκB pathway by lipids. Importantly, inhibition of the NFκB pathway in myotubes downstream of TLR-4 also protects against lipid-induced insulin resistance, suggesting a mechanism by which reduced TLR-4 activity confers beneficial effects on insulin action.

Mice Lacking NKT Cells but with a Complete Complement of CD8+ T-Cells Are Not Protected against the Metabolic Abnormalities of Diet-Induced Obesity

The contribution of natural killer T (NKT) cells to the pathogenesis of metabolic abnormalities of obesity is controversial. While the combined genetic deletion of NKT and CD8+ T-cells improves glucose tolerance and reduces inflammation, interpretation of these data have been complicated by the recent observation that the deletion of CD8+ T-cells alone reduces obesity-induced inflammation and metabolic dysregulation, leaving the issue of the metabolic effects of NKT cell depletion unresolved. To address this question, CD1d null mice (CD1d−/−), which lack NKT cells but have a full complement of CD8+ T-cells, and littermate wild type controls (WT) on a pure C57BL/6J background were exposed to a high fat diet, and glucose intolerance, insulin resistance, dyslipidemia, inflammation, and obesity were assessed. Food intake (15.5±4.3 vs 15.3±1.8 kcal/mouse/day), weight gain (21.8±1.8 vs 22.8±1.4 g) and fat mass (18.6±1.9 vs 19.5±2.1 g) were similar in CD1d−/− and WT, respectively. As would be expected from these data, metabolic rate (3.0±0.1 vs 2.9±0.2 ml O2/g/h) and activity (21.6±4.3 vs 18.5±2.6 beam breaks/min) were unchanged by NKT cell depletion. Furthermore, the degree of insulin resistance, glucose intolerance, liver steatosis, and adipose and liver inflammatory marker expression (TNFα, IL-6, IL-10, IFN-γ, MCP-1, MIP1α) induced by high fat feeding in CD1d−/− were not different from WT. We conclude that deletion of NKT cells, in the absence of alterations in the CD8+ T-cell population, is insufficient to protect against the development of the metabolic abnormalities of diet-induced obesity.

Impaired Activation of Phosphatidylinositol 3-Kinase by Leptin Is a Novel Mechanism of Hepatic Leptin Resistance in Diet-induced Obesity

Obesity is associated with the development of leptin resistance. However, the effects of leptin resistance on leptin-regulated metabolic processes and the biochemical defects that cause leptin resistance are poorly understood. We have addressed in rats the effect of dietinduced obesity (DIO), a situation of elevated tissue lipid levels, on the well described lipid-lowering effect of leptin in liver, an action that is proposed to be important for the prevention of tissue lipotoxicity and insulin resistance. In addition, we have addressed the role of phosphatidylinositol 3-kinase (PI 3-kinase) in mediating the acute effects of leptin on hepatic lipid levels in lean and DIO animals. A 90-min leptin (∼10 ng/ml) perfusion of isolated livers from lean animals decreased triglyceride levels by 42 ± 5% (p = 0.006). However, leptin concentrations ranging from ∼10 to ∼90 ng/ml had no effect on triglyceride levels in livers from DIO animals. The acute lipid-lowering effect of leptin on livers from lean animals was mediated by a PI 3-kinase-dependent mechanism, because wortmannin and LY294002, the PI 3-kinase inhibitors, blocked the effects of leptin on hepatic triglyceride levels and leptin increased liver PI 3-kinase activity by 183 ± 6% (p = 0.003) and insulin receptor substrate 1 tyrosine phosphorylation by 185 ± 30% (p = 0.02) in the absence of PI 3-kinase inhibitors. Contrary to the effects of leptin in lean livers, leptin did not activate PI 3-kinase in livers from DIO rats. These data present evidence for a role for 1) leptin resistance in contributing to the excessive accumulation of tissue lipid in obesity, 2) PI 3-kinase in mediating the acute lipid-lowering effects of leptin in liver, and 3) defective leptin activation of PI 3-kinase as a novel mechanism of leptin resistance.

Simultaneous Glutamate and Perfusion fMRI Responses to Regional Brain Stimulation

Functional magnetic resonance imaging (fMRI) rests on the assumption that regional brain activity is closely coupled to regional cerebral blood flow (rCBF) in vivo. To test the degree of coupling, cortical brain activity was locally stimulated in rats by reversed microdialysis infusion of picrotoxinin, a γ-aminobutyric acid-A antagonist. Before and during the first 30 minutes of infusion, simultaneous fMRI (rCBF) and neurochemical (interstitial glutamate concentration) measures of brain activity were highly correlated (r = 0.83). After 30 minutes of picrotoxinin-induced stimulation, glutamate levels decreased but rCBF remained elevated, suggesting that additional factors modulate the relationship between neuronal neurotransmitters and hemodynamics at these later stages.

Leptin Augments the Acute Suppressive Effects of Insulin on Hepatic Very Low-Density Lipoprotein Production in Rats

It is well established that leptin increases the sensitivity of carbohydrate metabolism to the effects of insulin. Leptin and insulin also have potent effects on lipid metabolism. However, the effects of leptin on the regulation of liver lipid metabolism by insulin have not been investigated. The current study addressed the effects of leptin on insulin-regulated hepatic very low-density lipoprotein (VLDL) metabolism in vivo in rats. A 90-min hyperinsulinemic/euglycemic clamp (4 mU/kg x min(-1)) reduced plasma VLDL triglyceride (TG) by about 50% (P < 0.001 vs. saline control). Importantly, a leptin infusion (0.2 microg/kg x min(-1)) in combination with insulin reduced plasma VLDL-TG by about 80% (P < 0.001 vs. insulin alone). These effects did not require altered skeletal muscle lipoprotein lipase activity but did include differential effects of insulin and leptin on liver apolipoprotein (apo) B and TG metabolism. Thus, insulin decreased liver and plasma apoB100/B48 levels (approximately 50%, P < 0.01), increased liver TGs (approximately 20%, P < 0.05), and had no effect on fatty acid oxidation. Conversely, leptin decreased liver TGs (approximately 50%, P < 0.01) and increased fatty acid oxidation (approximately 50%, P < 0.01) but had no effects on liver or plasma apoB levels. Importantly, the TG-depleting and prooxidative effects of leptin were maintained in the presence of insulin. We conclude that leptin additively increases the suppressive effects of insulin on hepatic and systemic VLDL metabolism by stimulating depletion of liver TGs and increasing oxidative metabolism. The net effect of the combined actions of insulin and leptin is to decrease the production and TG content of VLDL particles.

DNA methylation alters transcriptional rates of differentially expressed genes and contributes to pathophysiology in mice fed a high fat diet

Objective Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity. Methods RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in vivo knockdown of an identified driver gene, Phlda1, were used to validate the results. Results A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype. Conclusions We conclude that a high fat diet alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity.

Adipose Tissue Derived Free Fatty Acids Initiate Myeloid Cell Accumulation in Mouse Liver in States of Lipid Oversupply

Accumulation of myeloid cells in the liver, notably dendritic cells (DCs) and monocytes/macrophages (MCs), is a major component of the metainflammation of obesity. However, the mechanism(s) stimulating hepatic DC/MC infiltration remain ill defined. Herein, we addressed the hypothesis that adipose tissue (AT) free fatty acids (FFAs) play a central role in the initiation of hepatic DC/MC accumulation, using a number of mouse models of altered FFA supply to the liver. In two models of acute FFA elevation (lipid infusion and fasting) hepatic DC/MC and triglycerides (TGs) but not AT DC/MC were increased without altering plasma cytokines (PCs; TNFα and monocyte chemoattractant protein 1) and with variable effects on oxidative stress (OxS) markers. However, fasting in mice with profoundly reduced AT lipolysis (AT-specific deletion of adipose TG lipase; AAKO) failed to elevate liver DC/MC, TG, or PC, but liver OxS increased. Livers of obese AAKO mice that are known to be resistant to steatosis were similarly protected from inflammation. In high-fat feeding studies of 1, 3, 6, or 20-wk duration, liver DC/MC accumulation dissociated from PC and OxS but tracked with liver TGs. Furthermore, decreasing OxS by ~80% in obese mice failed to decrease liver DC/MC. Therefore, FFA and more specifically AT-derived FFA stimulate hepatic DC/MC accumulation, thus recapitulating the pathology of the obese liver. In a number of cases the effects of FFA can be dissociated from OxS and PC but match well with liver TG, a marker of FFA oversupply.