Inflammation stimulates hypoxia-inducible factor-1 alpha regulatory activity in 3T3-L1 adipocytes with conditioned medium from lipopolysaccharide-activated RAW 264.7 macrophages
Obesity is a multifactorial, chronic, inflammatory disease that involves different processes, such as adipose tissue hypoxia. The aim of the current study was to characterize the effects of conditioned medium (CM) from lipopolysaccharide (LPS)-activated macrophages on the regulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha)-related genes in murine adipocytes. For the in vitro analyses, 3T3-L1 murine adipocytes (9 days postdifferentiation) were incubated either in CM (25% medium of RAW 264.7 murine macrophages with 24hr 500ng/ml LPS), LPS at 500ng/ml, or hypoxia (Hx; 1% O-2, 94% N-2, 5% CO2) for 24hr. For the in vivo experiments, mice were fed a high-fat diet. Both epididymal white adipose tissue (eWAT) and adipocytes in CM showed upregulation of Glut1, Mcp1, Il10, Tnf, and Il1b. The secretion of IL-6, TNF-alpha, and MCP-1 was also increased in CM-treated adipocytes. Moreover, increased levels of HIF-1 alpha subunit and nuclear factor kappa B p65 were found after CM treatment, linking Hx, and inflammation. HIF-1 alpha directly bound vascular endothelial growth factor A (Vegfa) and uncoupling protein 2 (Ucp2) genes, up- and downregulating its expression, respectively. Furthermore, the oxygen consumption rate was 30% lower in CM. The siRNA knockdown of mammalian target of rapamycin (Mtor) reversed the induction of HIF-1 alpha found in CM. The macrophage infiltration simulated through CM seems to be a similar environment to an abnormally enlarged eWAT. We have evidenced that HIF-1 alpha plays a regulatory role in the expression of Vegfa and Ucp2 in CM. Finally, the inhibition of the mTOR pathway prevented the HIF-1 alpha activation induced by CM. The involvement of HIF-1 alpha under proinflammatory conditions provides insight into the origins of Hx in obesity.