Deletion of Hypoxia-Inducible Factor-1α in Adipocytes Enhances Glucagon-Like Peptide-1 Secretion and Reduces Adipose Tissue Inflammation

<div><p>It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.</p></div

Involvement of GLP-1 in improvement of glucose tolerance in ahKO mice.

<p>(A) Serum GLP-1 and GIP levels under free-feeding condition. Values are means ±S.E.M. (n = 6). *<i>P</i><0.01. (B) Time-dependent changes of blood glucose concentrations in ipGTT. After a 24-h fast, 2 g/kg glucose was injected intraperitoneally. Values are means ±S.E.M. (n = 5). *<i>P</i><0.05 and **<i>P</i><0.01. (C) AUCs of glucose concentrations in ipGTT. Values are means ±S.E.M. (n = 5). *<i>P</i><0.01. (D) Time-dependent changes of blood glucose concentrations in OGTT with pre-injection of Ex9–39. Twenty five μg of Ex9–39 per kg body weight was injected. Fifteen minutes after Ex9–39 injection, 2 g/kg glucose was administered orally. Values are means ±S.E.M. (n = 5). (E) AUCs of glucose concentrations in OGTT with pre-injection of Ex9–39. Values are means ±S.E.M. (n = 5). *<i>P</i><0.01. (F) Serum concentrations of leptin, resistin, tPAI-1, and adiponectin are shown. Values are means ±S.E.M. (n = 6). *<i>P</i><0.01. (G) GLUTag cells were treated with 100 nM leptin, 10 ng/ml resistin, or 30 μg/ml adiponectin for 2 h and GLP-1 secreted into the incubation buffer was measured with ELISA. Ten uM Fsk/IBMX was used as positive control for GLP-1 secretion. Values are means ±S.E.M. (n = 5). *<i>P</i><0.01.</p

Inflammation of epididymal adipose tissues.

<p>(A) Immunostaining for F4/80. Isolated epididymal fat pads were immunostained with anti-F4/80 antibody (green). Adipocytes were counterstained with BODIPY (red) and nuclei, with Hoechst (blue). Bars indicate 200 μm. Arrowheads indicate CLS. (B) The number of CLS in a 1 mm² visual field was counted. Values are means ±S.E.M. (n = 6). *<i>P</i><0.01. (C) Real-time PCR analyses of inflammation-related genes in epididymal adipose tissues. Values are means ±S.E.M. (n = 6). *<i>P</i><0.05.</p

Expression of HIF-1α in epididymal adipose tissue and glucose and insulin tolerance tests.

<p>(A) Western blot images of HIF-1α. Epididymal fat pads isolated from ahKO and WT mice were solubilized in lysis buffer and subjected to western blotting. (B) Quantification of protein levels of HIF-1α. Values are means ±S.E.M. (n = 6). *<i>P</i><0.01. (C) Time-dependent changes of blood glucose concentrations in OGTT. After a 24-h fast, 2 g/kg glucose was injected orally and blood glucose concentrations were measured at the indicated time points. Values are means ±S.E.M. (n = 6∼8). *<i>P</i><0.05 and **<i>P</i><0.01. (D) AUCs of glucose concentration in OGTT. Values are means ±S.E.M. (n = 6∼8). *<i>P</i><0.01. (E) Time-dependent changes of blood glucose concentrations in ITT. After a 4-h fast, 1 U/kg insulin was injected intraperitoneally and blood glucose concentrations were measured. Values are means ±S.E.M. (n = 6∼7). *<i>P</i><0.05. (F) AUCs of glucose concentration in ITT. Values are means ±S.E.M. (n = 6∼7). *<i>P</i><0.01.</p

Characteristics of ahKO mice.

<p>Body weights, epididymal (Epi) fat weights, subcutaneous (Sc) fat weights, blood glucose concentrations, and adipocyte sizes of 25-week-old mice are indicated. Values are means ±S.E.M.</p><p>*<i>P</i><0.01 vs. WT-ND mice and †<i>P</i><0.01 vs. ahKO-ND mice. No category is significantly different between WT-ND mice and ahKO-ND mice and between WT-HFD mice and ahKO-HFD mice.</p