Toll-like Receptor 4 Negatively Mediates Islet Function And Insulin Resistance In Diet-Induced Obesity

Toll-like Receptor 4 Negatively Mediates Islet Function and Insulin Resistance in Diet-Induced ObesityIn conditions of nutrient excess such as obesity and diabetes,elevated free fatty acid (FFA) levels are implicated in the pathogenesis of both inflammation and insulin resistance in a variety of tissues, including islet beta cells.At the cellular level, utrient excess is linked to insulin resistance via activation of IKK? and, subsequently, NF-kB, a key transcriptional mediator of inflammation. In peripheral insulin-sensitive tissues such as muscle and liver, the pattern recognition receptor, Toll-like receptor-4 (TLR4) is implicated as a mediator of this effect. The current work was undertaken to determine whether obesity induced by high-fat(HF) feed- ing causes inflammation and insulin secretion dysfunction in islet in vivo, and whether TLR4 contributes to this effect.The bacterial endotoxin, lipopolysaccharide (LPS), is a potent activator of IKK? and NF-kB in most cell types. The majority of the biologi- cal activity of LPS is contained within a moiety ("lipid A") that is acylated with saturated fatty acids,and removal of these fatty acids results in complete loss of endotoxic activity.Recently, TLR4 was shown to be required not only for LPS- induced inflammatory responses,but for responses to nonbacterial ligands such as palmitate (C 18:0), a saturated fatty acid. These in vitro studies suggest that activation of TLR4 by certain FFA species can trigger cellular inflammatory responses. Whether TLR4 signaling contributes to the link between nutrient excess, inflamma- tion,and islet beta cell dysfunction in vivo is an important unanswered question.TLR4 is expressed on virtually all human cells and binds a wide spectrum of exogenous and endogenous ligands (including bacterial LPS) and is involved in innate immune responses to various infectious agents and stressors. In the presence of LPS, the TLR4 receptor complex recruits the adaptor protein, myeloid differentiation factor-88 (MyD88). MyD88 in turn activae IKK? and NF-kB, ultimately induces the expression of numerous inflammatory mediators including TNFa,IL-6. Recent evidence suggests that inflammatory processes induced by nutrient excess cause systemic insulin resistance via a mechanism involving TLR4.Based on evidence that TLR4 can be activated by saturated FFAs, we investigate whether TLR4 play a key role with islet beta cell function in diet-induced obesity.To explore the interaction between toll-like receptor 4 with islet beta cell function in high-fat diet-induced obesity mice. After high fat diet, weight,insulin sensitivity, Triglyceride(TG), total cholesterol (TC) and fasting serum insulin were measured; Fatty degeneration in the liver was observed; Glucose tolerance test and insulin releasing test were performed; the level of TLR4 mRNA in the islet of mice was determined with realtime PCR or immunohistochemical. High-fat-diet can increase the expressions of TLR4.Activation of TLR4 induces the expression of numerous inflammatory mediators which impair islet function.PartⅠA Model to Obese Mice of Insulin Resistance Induced by High-Fat-DietObesity is associated with insulin resistance and a state of abnormal inflammatory response.To establish insulin resistance obese mice model induced by high-fat–diet,40 Eight-week-old male C57BJ/L2 are randomly divided into two groups(n = 20): a standard rodent chow and high fat diet for24weeks ( HF24, and ND24). Before and after the experiment, the body weights of mice are weighed. Mice epididymal adipose weight is weighed at the end of the experiment.Insulin sensitivity is evaluated by insulin tolerance test and glucose tolerance test. Triglyceride (TG),total cholesterol (TC) are measured by Biochemical analyzer. Serum levels of insulin and leptin are determined by RIA assay. Pancreatic beta-cell function is judged by glucose tolerance test, insulin release test, insulin content of pancreas, effect ion of high glucose on secretion insulin of islets. Fatty degeneration in the liver is observed by HE staining and light microscope.Apoptosis is investigated by TUNEL. Weights, TC, TG, fasting serum insulin level, visceral fat content (epididymal fat / weight), of HF groups are significantly higher than the control groups. There is a significant decrease in insulin sensitivity. The acute response of the glucose-stimulated insulin secretion is inhabited. There are a varying degrees of hepatic steatosis observed in liver in the high-fat-diet obesity mice. Compared with ND24 groups, the degree of insulin resistance, the acute response of the glucose-stimulated insulin secretion defection, and fatty liver degeneration further accentuat. This provides an ideal animal model to investigate the molecular mechanism of incidence of obesity and islet dysfunction.PartⅡExpression of TLR4 in Islet of Obese Mice Induced by High-Fat-DietToll-like receptor4 (TLR4) play a critical role in the activation of innate immune responses in mammals. TLR4 can also play an additional role as well, as a"sensor"for endogenous lipids that may contribute to the pathogenesis of lipid-induced insulin resistance.To investigate the expression of TLR4 in islet of high-fat diet- induced obese mice,40 C57BJ/L2 mice were randomly divided into HF24 and ND24 groups (the same as the methods described in partⅠ).The expressions of TLR4 and insulin in islet were detected by immunohistochemical methods.The expressions of TLR4 in islet in HF24 were significantly higher than those in ND24. Obesity leads to elevated plasma free fatty acids (FFAs).Acute FFA exposure stimulates insulin secretion, but prolonged FFA exposure decreases glucose-stimulated insulin secretion(GSIS). To determine whether TLR4 signaling mediates deleterious effects of FFAs in beta cells, we in- cubated mouse beta cell lines MIN6 cells in the presence of palmitate and investi- gated the effects of prolonged exposure to palmitate on GSIS in MIN6 beta cells.MIN6 cells showed a decreased insulin response to FFAs such as palmitate .Real-time RT-PCR showed increased mRNA levels of cytokine genes including IL-6,TNFa,MCP-1 in MIN6 cells after palmiate exposure. TLR4mRNA levels were also increased in MIN6 cells after palmiate exposure.In conclusion, we have shown here that TLR4 is expressed on islet beta cells.High-fat-diet can increase the expressions of TLR4.Activation of TLR4 induces the expression of numerous inflammatory mediators which impair islet function. PartⅢToll-like Receptor 4 Negatively Mediates Islet Function and Insulin Resistance in Diet-Induced ObesityThe Toll-like receptor (TLR)4 has an important role in inflammation and immunity, and its expression has been reported in most tissues including islet beta cells.Because it is activated by lipopolysacch- aride and saturated fatty acids,which are inducers of insulin resistance, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals. At the cellular level, utrient excess is linked to insulin resistance via activation of IKK? and, subsequently, NF-kB, a key transcriptional mediator of inflammation. In peripheral insulin-sensitive tissues such as muscle and liver, the pattern recognition receptor, Toll-like receptor-4 (TLR4) is implicated as a mediator of this effect. The current work was undertaken to determine whether obesity induced by high-fat(HF) feeding causes inflammation and insulin resistance in islet in vivo, and whether TLR4 contributes to this effect. Recently, TLR4 was shown to berequired not only for LPS-induced inflammatory responses,but for responses to nonbacterial ligands such as palmitate (C 18:0), a saturated fatty acid. Whether TLR4 signaling contributes to the link between nutrient excess, inflammation,and islet beta cell dysfunction in vivo is an important unanswered question.Here, we show that NJ mice,which have been knock out in TLR4, are protected against the development of diet-induced obesity.In addition, these mice demonstrate decreased adiposity, improved insulin sensitivity. Moreover, in islet, control mice fed a high-fat diet show an increase in TNFa mRNA,IL-6mRNA and MCP-1mRNA. In isolated islets from NJ mice, protection from saturated fatty acid induced islet dysfunction is observed.