Hypoxia reduces the response of human adipocytes towards TNFα resulting in reduced NF-κB signaling and MCP-1 secretion.

OBJECTIVE: Obesity is associated with adipose tissue hypoxia, and is thought to be linked to the chronic low-grade inflammation of adipose tissue, although the precise mechanism has remained unclear. In this study, we investigated the effect of a prominent hypoxia on human primary adipocyte secretion and tumor necrosis factor alpha (TNFα)-induced nuclear factor-κB (NF-κB) signaling. RESULTS: Using cytokine array and ELISA analysis, we compared the secretion patterns of normoxic and hypoxic (1% O(2)) adipocytes and observed various alterations in adipokine release. We could reproduce known alterations like an induction of interleukin (IL)-6, vascular endothelial growth factor, leptin and a reduction in adiponectin release under hypoxia. Interestingly, we observed a significant reduction in the secretion of macrophage chemotactic protein (MCP)-1 and other NF-κB-related genes, such as growth-regulated oncogene-α, eotaxin and soluble TNF-Receptor1 (TNF-R1) under hypoxia. TNFα stimulation of hypoxic adipocytes resulted in a significantly reduced phosphorylation of NF-κB and its inhibitor IκBα compared with normoxic cells. Furthermore, chronic treatment of hypoxic adipocytes with TNFα resulted in an expected higher secretion of the chemokines MCP-1 and IL-8, but under hypoxia, the secretion level was substantially lower than that under normoxia. This reduction in protein release was accompanied by a reduced mRNA expression of MCP-1, whereas IL-8 mRNA expression was not altered. Additionally, we observed a significantly reduced expression of the TNF-receptor TNF-R1, possibly being one cause for the reduced responsiveness of hypoxic adipocytes towards TNFα stimulation. CONCLUSION: In conclusion, human primary adipocytes show a basal and TNFα-induced reduction of MCP-1 release under hypoxia. This effect may be due to a reduced expression of TNF-R1 and therefore attenuated TNFα-induced NF-κB signaling. These observations demonstrate a reduced responsiveness of hypoxic adipocytes towards inflammatory stimuli like TNFα, which may represent an adaptation process to maintain adipose tissue function under hypoxia and inflammatory conditions.

Pigment epithelium-derived factor (PEDF) is one of the most abundant proteins secreted by human adipocytes and induces insulin resistance and inflammatory signaling in muscle and fat cells.

OBJECTIVE: Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic and anti-angiogenic properties. More recently it became evident that PEDF is upregulated in patients with type 2 diabetes and also contributes to insulin resistance in mice. During characterization of the secretome of in vitro differentiated human adipocytes by two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-MS, we found that PEDF is one of the most abundant proteins released by adipocytes. The aim of this study was to investigate the regulation and autocrine function of PEDF in human adipocytes and to determine its paracrine effects on human skeletal muscle cells (hSkMC) and human smooth muscle cells (hSMC). METHODS AND RESULTS: Human primary adipocytes secrete 130 ng ml(-1) PEDF over 24 h from 1 million cells, which is extremely high as compared with adiponectin, interleukin-6 (IL-6) or IL-8. This release of PEDF is significantly higher than from other primary cells, such as adipose-tissue located macrophages (50-times), hSkMC and hSMC (5-times). PEDF protein expression significantly increases during adipogenesis, which is paralleled by increased PEDF secretion. Furthermore, tumor necrosis factor-α and hypoxia significantly downregulate PEDF protein levels. PEDF secretion was significantly reduced by troglitazone and hypoxia and significantly increased by insulin. Treatment of adipocytes and hSkMC with PEDF induced insulin resistance in adipocytes, skeletal and smooth muscle cells at the level of insulin-stimulated Akt phosphorylation, which was dose dependent and more prominent in adipocytes. Furthermore, inflammatory nuclear factor-κB (NF-κB) signaling was induced by PEDF. In hSMC, PEDF induced proliferation (1.7-fold) and acutely activated proliferative and inflammatory signaling pathways (NF-κB, p38 mitogen-activated protein kinase and mammalian target of rapamycin). CONCLUSION: PEDF is one of the most abundant adipokines and its secretion is inversely regulated by insulin and hypoxia. PEDF induces insulin resistance in adipocytes and hSkMC and leads to inflammatory signaling in hSMC. Because of these diverse actions, PEDF is a key adipokine, which could have an important role in diabetes and obesity-related disorders.

Cannabinoid type 1 receptors in human skeletal muscle cells participate in the negative crosstalk between fat and muscle.

AIMS/HYPOTHESIS: Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle. METHODS: The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyte-conditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated. RESULTS: We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulin-stimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt(Ser473) phosphorylation, but had no effect on Akt(Thr308) and glycogen synthase kinase 3alpha/beta phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed. CONCLUSIONS/INTERPRETATION: Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance.