OBJECTIVE: We examined the role of butyric acid, a short-chain fatty acid formed by fermentation in the large intestine, in the regulation of insulin sensitivity in mice fed a high-fat diet. RESEARCH DESIGN AND METHODS: In dietary-obese C57BL/6J mice, sodium butyrate was administrated through diet supplementation at 5% wt/wt in the high-fat diet. Insulin sensitivity was examined with insulin tolerance testing and homeostasis model assessment for insulin resistance. Energy metabolism was monitored in a metabolic chamber. Mitochondrial function was investigated in brown adipocytes and skeletal muscle in the mice. RESULTS: On the high-fat diet, supplementation of butyrate prevented development of insulin resistance and obesity in C57BL/6 mice. Fasting blood glucose, fasting insulin, and insulin tolerance were all preserved in the treated mice. Body fat content was maintained at 10% without a reduction in food intake. Adaptive thermogenesis and fatty acid oxidation were enhanced. An increase in mitochondrial function and biogenesis was observed in skeletal muscle and brown fat. The type I fiber was enriched in skeletal muscle. Peroxisome proliferator–activated receptor-γ coactivator-1α expression was elevated at mRNA and protein levels. AMP kinase and p38 activities were elevated. In the obese mice, supplementation of butyrate led to an increase in insulin sensitivity and a reduction in adiposity. CONCLUSIONS: Dietary supplementation of butyrate can prevent and treat diet-induced insulin resistance in mouse. The mechanism of butyrate action is related to promotion of energy expenditure and induction of mitochondria function.

Chronic inflammation and reduced adiponectin are widely observed in the white adipose tissue in obesity. However, the cause of the changes remains to be identified. In this study, we provide experimental evidence that hypoxia occurs in adipose tissue in obese mice and that adipose hypoxia may contribute to the endocrine alterations. The adipose hypoxia was demonstrated by a reduction in the interstitial partial oxygen pressure (Po₂), an increase in the hypoxia probe signal, and an elevation in expression of the hypoxia response genes in ob/ ob mice. The adipose hypoxia was confirmed in dietary obese mice by expression of hypoxia response genes. In the adipose tissue, hypoxia was associated with an increased expression of inflammatory genes and decreased expression of adiponectin. In dietary obese mice, reduction in body weight by calorie restriction was associated with an improvement of oxygenation and a reduction in inflammation. In cell culture, inflammatory cytokines were induced by hypoxia in primary adipocytes and primary macrophages of lean mice. The transcription factor NF-κB and the TNF-α gene promoter were activated by hypoxia in 3T3-L1 adipocytes and NIH3T3 fibroblasts. In addition, adiponectin expression was reduced by hypoxia, and the reduction was observed in the gene promoter in adipocytes. These data suggest a potential role of hypoxia in the induction of chronic inflammation and inhibition of adiponectin in the adipose tissue in obesity.

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor κB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser³⁰⁷ (equivalent to Ser³¹² in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-α or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser³¹² in response to tumor necrosis factor-α was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J₂ as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser³¹². Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser³¹² (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFκB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IκBα in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFκB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFκB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFκB activation and COX-2 expression. These results demonstrate that NFκB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFκB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.