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Objective: We investigated the regulation and involvement of microRNAs (miRNAs) in fat cell development and obesity., Research Design and Methods: Using miRNA microarrays, we profiled the expression of >370 miRNAs during adipogenesis of preadipocyte 3T3-L1 cells and adipocytes from leptin deficient ob/ob and diet-induced obese mice. Changes in key miRNAs were validated by RT-PCR. We further assessed the contribution of the chronic inflammatory environment in obese adipose tissue to the dysregulated miRNA expression by tumor necrosis factor (TNF)-alpha treatment of adipocytes. We functionally characterized two adipocyte-enriched miRNAs, miR-103 and miR-143, by a gain-of-function approach., Results: Similar miRNAs were differentially regulated during in vitro and in vivo adipogenesis. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice and vice versa. These changes are likely associated with the chronic inflammatory environment, since they were mimicked by TNF-alpha treatment of differentiated adipocytes. Ectopic expression of miR-103 or miR-143 in preadipocytes accelerated adipogenesis, as measured both by the upregulation of many adipogenesis markers and by an increase in triglyceride accumulation at an early stage of adipogenesis., Conclusions: Our results provide the first experimental evidence for miR-103 function in adipose biology. The remarkable inverse regulatory pattern for many miRNAs during adipogenesis and obesity has important implications for understanding adipose tissue dysfunction in obese mice and humans and the link between chronic inflammation and obesity with insulin resistance.

Despite extensive studies implicating tumor necrosis factor (TNF)-alpha as a contributing cause of insulin resistance, the mechanism(s) by which TNF-alpha alters energy metabolism in vivo and the tissue specificity of TNF-alpha action are unclear. Here, we investigated the effects of TNF-alpha infusion on gene expression and energy metabolism in adult rats. A 1-day TNF-alpha treatment decreased overall insulin sensitivity and caused a 70% increase (P = 0.005) in plasma levels of free fatty acids (FFAs) and a 46% decrease (P = 0.01) in ACRP30. A 4-day TNF-alpha infusion caused insulin resistance and significant elevation of plasma levels of FFAs and triglycerides and reduction of ACRP30. Plasma glucose concentration was not altered following TNF-alpha infusion for up to 4 days. As revealed by oligonucleotide microarrays, TNF-alpha evoked major and rapid changes in adipocyte gene expression, favoring FFA release and cytokine production, and fewer changes in liver gene expression, but favoring FFA and cholesterol synthesis and VLDL production. There was only a moderate repressive effect on skeletal muscle gene expression. We demonstrate that TNF-alpha antagonizes the actions of insulin, at least in part, through regulation of adipocyte gene expression including reduction in ACRP30 mRNA and induction of lipolysis resulting in increased plasma FFAs. TNF-alpha later alters systemic energy homeostasis that closely resembles the insulin resistance phenotype. Our data suggest that blockade of TNF-alpha action in adipose tissue may prevent TNF-alpha-induced insulin resistance in vivo.

Tumor necrosis factor-alpha (TNF-alpha) is a contributing cause of the insulin resistance seen in obesity and obesity-linked type 2 diabetes, but the mechanism(s) by which TNF-alpha induces insulin resistance is not understood. By using 3T3-L1 adipocytes and oligonucleotide microarrays, we identified 142 known genes reproducibly upregulated by at least threefold after 4 h and/or 24 h of TNF-alpha treatment, and 78 known genes downregulated by at least twofold after 24 h of TNF-alpha incubation. TNF-alpha-induced genes include transcription factors implicated in preadipocyte gene expression or NF-kappaB activation, cytokines and cytokine-induced proteins, growth factors, enzymes, and signaling molecules. Importantly, a number of adipocyte-abundant genes, including GLUT4, hormone sensitive lipase, long-chain fatty acyl-CoA synthase, adipocyte complement-related protein of 30 kDa, and transcription factors CCAAT/enhancer binding protein-alpha, receptor retinoid X receptor-alpha, and peroxisome profilerator-activated receptor gamma were significantly downregulated by TNF-alpha treatment. Correspondingly, 24-h exposure of 3T3-L1 adipocytes to TNF-alpha resulted in reduced protein levels of GLUT4 and several insulin signaling proteins, including the insulin receptor, insulin receptor substrate 1 (IRS-1), and protein kinase B (AKT). Nuclear factor-kappaB (NF-kappaB) was activated within 15 min of TNF-alpha addition. 3T3-L1 adipocytes expressing IkappaBalpha-DN, a nondegradable NF-kappaB inhibitor, exhibited normal morphology, global gene expression, and insulin responses. However, absence of NF-kappaB activation abolished suppression of >98% of the genes normally suppressed by TNF-alpha and induction of 60-70% of the genes normally induced by TNF-alpha. Moreover, extensive cell death occurred in IkappaBalpha-DN-expressing adipocytes after 2 h of TNF-alpha treatment. Thus the changes in adipocyte gene expression induced by TNF-alpha could lead to insulin resistance. Further, NF-kappaB is an obligatory mediator of most of these TNF-alpha responses.

Two species of glucose transporter (GT) are present in the liver: the erythroid/brain GT and the newly characterized liver GT. No information is available regarding the functional role of these two species or whether their expression is regulated concordantly in states in which hepatic glucose uptake or output are markedly altered. In this study, we analyzed the effect of fasting and refeeding and streptozocin-induced diabetes and subsequent insulin treatment on the expression of the erythroid/brain and liver GT polypeptides and their mRNAs in rat liver. The erythroid/brain GT mRNA in livers of control rats corresponded to 1-3% of the amount of liver GT mRNA. After a 4-day fast, its level increased approximately 3-fold and represented 8-10% of the liver GT mRNA, whereas the corresponding protein increased 4-fold. In livers from diabetic rats, levels of the erythroid/brain GT mRNA increased up to 2.4-fold and gradually returned to normal with chronic insulin treatment. Levels of the corresponding protein were minimally altered. Levels of immunoreactive liver GTs were not significantly changed by 2 days of fasting, 7 or 14 days of diabetes, or subsequent insulin treatment for 3, 5, or 7 days but increased up to 75% with refeeding for 3-48 h. Liver GT mRNA levels minimally decreased in diabetic or insulin-treated rats, decreased 45% after a 2-day fast, and increased up to 5-fold on refeeding for 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of cDNA encoding the human erythrocyte facilitated-diffusion glucose transporter (GT) has elucidated its structure and has permitted a careful study of its tissue distribution and of its involvement in processes such as insulin-stimulated glucose uptake by adipose cells or transformation-induced increase in glucose metabolism. An important outcome of these studies was the discovery that additional isoforms of this transporter were expressed in a tissue-specific manner; these comprise a family of structurally and functionally related molecules. Their tissue distribution, differences in kinetic properties, and differential regulation by ambient glucose and insulin levels suggest that they play specific roles in the control of glucose homeostasis. Herein, we will discuss the structure of three members of the GT family: erythroid/brain GT, liver GT, and adipose cell/muscle GT. In the light of their tissue-specific expression, kinetic parameters, and susceptibility to insulin action, we discuss their possible specific functions.