Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant

https://doi.org/10.1016/j.ejcb.2011.06.004Get rights and content

Abstract

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and local inflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environment with the release of pro-inflammatory factors that stimulate macrophage migration and cause muscle insulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxia response by elevating protein expression of HIF-1α. This was associated with elevated mRNA expression and peptide release of cytokines TNFα, IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced. Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raised basal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Akt and AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H also caused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes. These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heat inactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulated cell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc traffic through its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversed the inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwell co-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H prevented macrophage migration towards it and partly reversed the effect of CM-H on insulin response in muscle cells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory response observed in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migration towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.

Introduction

Obesity is associated with chronic inflammation in adipose tissue (Sell and Eckel, 2010). However, how the inflammation is triggered is not well-defined. Recently adipose tissue has been characterized with low oxygen tension (hypoxia) in genetic and diet-induced obesity in mice and human obesity (Hosogai et al., 2007, Ye et al., 2007). In vivo, adipose tissue hypoxia is associated with elevated expression of a hypoxia gene profile, including elevated expression of the hypoxia inducible factor (HIF)1-α transcription factor and the GLUT1 glucose transporter, elevated expression of pro-inflammatory genes such as TNFα and IL-6, infiltration by macrophages, and reduction in adiponectin gene expression (Chen et al., 2006, Lolmede et al., 2003, Wang et al., 2007a). Indeed, cultured primary adipocytes and 3T3-L1 adipocytes increase expression of hypoxia-inducible genes when cultured in low oxygen conditions for several hours (Ye et al., 2007). Interestingly, cultured adipocytes also increase their pro-inflammatory gene expression when exposed to hypoxic conditions, suggesting that adipocytes contribute to the inflammatory response of adipose tissue in vivo. Yet, how hypoxia contributes to adipose tissue inflammation during obesity requires clarification.

Dysfunction of adipose tissue contributes to the pathophysiology of obesity-related metabolic diseases such as insulin resistance and type 2 diabetes, by promoting insulin resistance in liver and skeletal muscle (Guilherme et al., 2008). Skeletal muscle plays an important role in glucose homeostasis, since it is the major site for insulin-stimulated glucose disposal after a meal. Insulin acts on its surface receptors to elicit a cascade of intracellular signals through IRS1, PI3K, Akt and AS160/TBC1D4 to stimulate GLUT4 exocytosis to the muscle membrane and glucose removal from the circulation (Thong et al., 2005). Therefore, insulin resistance of glucose uptake in skeletal muscle can have a deleterious effect on whole-body glucose homeostasis. At the molecular level, insulin resistance in skeletal muscle is associated with blunted responsiveness of the insulin signaling pathway (Boura-Halfon and Zick, 2009, Kellerer et al., 1998) that is often manifest by elevated serine phosphorylation of IRS1 via activation of the stress kinase c-Jun NH2-terminal kinase (JNK) and/or the negative feed-back of p70S6-kinase (S6K) (Herschkovitz et al., 2007, Hotamisligil, 2006).

Given that adipose tissue is an endocrine organ it is postulated that, in obese animals, release of pro-inflammatory cytokines to the circulation with reduced production of insulin-sensitizing adipokines such as adiponectin and elevated release of free fatty acids, conveys insulin resistance to skeletal muscle (Plomgaard et al., 2005, Schenk et al., 2008, Tsuchiya et al., 2010). Macrophages are the main producers of cytokines in obese adipose tissue (Weisberg et al., 2003, Xu et al., 2003). Prolonged exposure of conditioned medium from adipocytes and macrophages renders muscle cells insulin resistant (Samokhvalov et al., 2009, Sell et al., 2008), but the effect of hypoxia on this communication has not been explored. Importantly, hypoxia in adipose tissue of genetically obese ob/ob mice is observed as early as 6 weeks of age (Yin et al., 2009), suggesting it could play a role in the pathophysiology of obesity.

Here we explore the hypothesis that hypoxic adipocytes release inflammatory factors that can attract macrophages and confer insulin resistance to skeletal muscle. To test the fundamentals of this hypothesis, we explored the effect of conditioned medium from adipocytes grown under hypoxic conditions, on macrophage migration and insulin-stimulated signaling and GLUT4 exocytosis in C2C12 myotubes.

Section snippets

Materials

The protease inhibitor cocktail, o-phenylenediamine dihydrochloride, anti-c-myc (epitope) polyclonal IgG, anti-α-Actinin-1, anti-β-actin, dexamethasone (Dex), 3-isobutyl-1-methylxanthine (IBMX), porcine insulin and all other chemicals unless otherwise noted were from Sigma Chemical (St. Louis, MO). Human insulin (Humulin R) was from Eli Lilly Canada (Toronto, ON, Canada). Dulbecco's modified Eagle's medium (DMEM), Horse serum (HS), penicillin/streptomycin and trypsin-EDTA were from Invitrogen

Hypoxia increases HIF-1α and GLUT1 expression in adipocytes

Increased HIF-1α and GLUT1 expression is a prototypical response of cells to a hypoxic environment (Bashan et al., 1993, Trayhurn and Wood, 2004, Wood et al., 2007, Yin et al., 2009). 3T3-L1 adipocytes were incubated in a low oxygen atmosphere (1% O2) for 4 h while 3T3-L1 control adipocytes were incubated in parallel under normal oxygen conditions (normoxia, 21% O2). Expression of the hypoxia-responsive protein, HIF-1α and GLUT1 were examined by immunoblotting of cell lysates. HIF-1α and GLUT1

Discussion

In obesity, the expanded adipose tissue develops localized hypoxia that is associated with adipose tissue inflammation (Trayhurn and Wood, 2004). Elevated adipose tissue production of pro-inflammatory cytokines and elevated numbers of macrophages and other cell types in this tissue contribute to localized insulin resistance of adipocytes that impairs the anti-lipolytic actions of the hormone. These cytokines also enter the circulation along with elevated free fatty acids and, coupled to a

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grants #30611120532 and #30570912), by the Tianjin Municipal Science and Technology Commission (grant # 09ZCZDSF04500) to W. Niu, and by a grant to A. Klip from the Canadian Diabetes Association. M. Constantine Samaan was supported by a grant from the Canadian Pediatric Endocrine Group (CPEG).

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