Endocrine Journal 2015
Original
Advance Publication
doi: 10.1507/endocrj. EJ15-0030
Role of the ubiquitin-proteasome system and autophagy in regulation of insulin sensitivity in serum-starved 3T3-L1 adipocytes Yemin Zhang1), Mao Ye2), Leyuan Jack Chen3), Mingxin Li1), Zhao Tang1) and Changhua Wang1) 1)
Department of Pathology & Pathophysiology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China Department of Endocrinology, The Central Hospital of Enshi Autonomous Prefecture, Enshi 445000, China 3) School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA 2)
Abstract. The ubiquitin-proteasome system (UPS) and autophagy are two conserved intracellular proteolytic pathways, responsible for degradation of most cellular proteins in living cells. Currently, both the UPS and autophagy have been suggested to be associated with pathogenesis of insulin resistance and diabetes. However, underlying mechanism remains largely unknown. The purpose of the present study is to investigate the impact of the UPS and autophagy on insulin sensitivity in serum-starved 3T3-L1 adipocytes. Our results show that serum depletion resulted in activation of the UPS and autophagy, accompanied with increased insulin sensitivity. Inhibition of the UPS with bortezomib (BZM), a highly selective, reversible 26S proteasome inhibitor induced compensatory activation of autophagy but did not affect significantly insulin action. Genetic and pharmacological inhibition of autophagy dramatically mitigated serum starvation-elevated insulin sensitivity. In addition, autophagy inhibition compromised UPS function and led to endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Inability of the UPS by BMZ exacerbated autophagy inhibition-induced ER stress and UPR. These results suggest that protein quality control maintained by the UPS and autophagy is required for preserving insulin sensitivity. Importantly, adaptive activation of autophagy plays a critical role in serum starvationinduced insulin sensitization in 3T3-L1 adipocytes. Key words: Autophagy, Ubiquitin-proteasome system, Insulin sensitivity, Adipocytes
Insulin resistance is a major hallmark of type 2 diabetes mellitus (T2DM). Recent studies suggest that insulin resistance combined with pancreas betacell dysfunction result in impaired glucose tolerance, hyperglycemia, and T2DM [1, 2]. However, current understanding of cellular mechanisms of insulin resistance remains limited. Several factors like oxidative stress, inflammation, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, hypoxia, and lipotoxicity have been proposed as triggers of insulin resistance [1]. Proteostasis, an important factor in keeping normal function of cells, is maintained by various competing and integrated cellular pathways controlling the biogenesis, folding, trafficking, modification, assem-
bly/disassembly, localization, and degradation of proteins presenting within and outside the cell. The ubiquitin-proteasome system (UPS) and macroautophagy (hereafter referred to as autophagy) are two conserved intracellular proteolytic pathways, responsible for degradation of most cellular proteins in living cells. Generally, the UPS degrades most of misfolded or damaged proteins while autophagy breaks down most aggregated proteins and unnecessary or dysfunctional cellular components [3, 4]. Previous studies have evidenced that dysfunction of the UPS or autophagy is implicated in pathogenesis of insulin resistance and diabetes. For example, insulin resistance can be induced by UPS-dependent degradation of important molecules
Submitted Jan. 19, 2015; Accepted Apr. 20, 2015 as EJ15-0030 Released online in J-STAGE as advance publication May 10, 2015
Abbreviations: shRNA, short hairpin RNA; Akt, protein kinase B (PKB); Glut4, glucose transporter type 4; 2-DG, 2-deoxy-Dglucose; UPS, ubiquitin-proteasome system; LC3, microtubuleassociated protein 1A/1B-light chain 3; BZM, bortezomib; BFA, bafilomycin A1; ER, endoplasmic reticulum; UPR, unfolded protein response; GFP, green fluorescent protein; RFP, red fluorescent protein
Correspondence to: Changhua Wang, M.D. & Ph.D., Department of Pathology & Pathophysiology, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Wuhan 430071, China. E-mail:
[email protected] ©The Japan Endocrine Society
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Zhang et al.
in the insulin signaling pathway such as insulin receptor substrate (IRS)-1/2 and protein kinase B (PKB or Akt) [5]. UPS-mediated down-regulation of IRS-2 is also found to contribute to a defective insulin secretion [6]. Similarly, accumulating evidence has established a direct link between autophagy and obesity, insulin resistance, and diabetes. ER stress-induced autophagy can result in pancreatic beta cell death [7] and insulin resistance [8]. Thus, overactivation of the UPS or autophagy was once considered as a deleterious factor for maintenance of glucose homeostasis. However, some recent studies reveal the necessary role of the UPS and autophagy in regulation of insulin action. Insufficient UPS function can induce hepatic steatosis, suppress hepatic insulin signaling, and increase hepatic glucose production [9]. Autophagy deficiency has also been found to promote ER stress and cause hepatic insulin resistance [10]. Moreover, Exerciseinduced autophagy has shown some beneficial metabolic effects [11]. All these evidences strongly suggest that both the UPS and autophagy are very important regulators of metabolic homeostasis. Adipose tissue is main site for energy storage in the body as well as an important endocrine organ, involving in the regulation of the synthesis and secretion of adipokines, and the uptake, storage, and synthesis of lipids. Adipose dysfunction underlies obesity, insulin resistance, and T2DM [12], whereas adipose function per se is also regulated by insulin [13]. Adipocyte insulin resistance will decrease insulin-mediated glucose uptake and promote lipolysis leading to ectopic lipid accumulation in other tissues such as liver and muscle, which will ultimately reduce insulin sensitivity in insulin-target tissues and cause systemic insulin resistance [13-15]. Although little is known about impact of the UPS on adipocytes function, current evidence demonstrates that autophagy activity is tightly associated with adipocyte development and function such as adipogenesis, lipolysis, and adipokines secretion [16-20]. Importantly, accumulating evidence has found a complex relationship between autophagy and adipocyte insulin resistance. Singh et al. reported that the specific inhibition of autophagy in adipose decreases body weight and enhances insulin sensitivity in high-fat diet (HFD)-fed mice [19]. In contrast, autophagy was found to be suppressed in the adipose tissue of insulin resistance mice and in hypertrophic 3T3-L1 adipocytes [21]. These conflicting results support a possibility that autophagy
has an important role in regulating adipocyte insulin action or systemic insulin sensitivity. In this present paper, we reveal the different impacts of the UPS and autophagy on insulin sensitivity in serum-starved 3T3-L1 adipocytes. We found that serum starvation activated the UPS and autophagy, along with an increase in insulin sensitivity. We further demonstrated that serum starvation-induced insulin sensitivity could be suppressed by autophagy inhibition but not by UPS inhibition. We also depicted the interaction between the UPS and autophagy in 3T3-L1 adipocytes and elucidated an ER stress mechanism of autophagy inhibition-reduced insulin action. These findings will deepen our understanding of adipocyte insulin signaling.
Materials and Methods Antibodies and reagents Biochemical reagents and antibodies were obtained from the following sources: bafilomycin A1 (BFA), Sigma-Aldrich Corp. (St. Louis, MO, USA); CbzLeu-Leu-norleucinal (MG132), epoxomicin, lactacystin, Calbiochem (Billerica, MA, USA); bortezomib (BZM), LC Laboratories (Woburn, MA, USA); antibodies to AKT, GSK3β, Glut4, IRS-1, JNK, caveolin-1, Atg5, Atg7, and phospho-specific antibodies to AKT Thr308, IRS-1 Ser307, and GSK3β Ser9, Cell Signaling Technology (Beverly, MA, USA); antibodies to GFP and RFP, Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); monoclonal anti-ubiquitin antibody, Covance Research Products, Inc. (Berkeley, CA, USA); secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase, Promega (Madison, WI, USA). 2-deoxy-D-2-[3H] glucose was obtained from HTA Co. Ltd. (Beijing, China). Cell culture and treatment 3T3-L1 preadipocytes were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (p/s). To convert preadipocytes into mature adipocytes, 3T3-L1 preadipocytes were allowed to grow for 3 days after 100% confluence. Differentiation was induced by incubating cells in fresh DMEM containing 1 μmol/L dexamethasone, 0.5 mmol/L isobutylmethylxanthine (IBMX), 1.67 μmol/L insulin, and 10% FBS for 3 days, and then the cells were cultured in DMEM containing 0.41 μmol/L insulin and 10% FBS for another 3 days, followed by main-
Endocrine Journal Advance Publication
Autophagy sensitizes insulin action
taining in growth medium (DMEM containing 10% FBS and 1% p/s) for an additional 4 days, which at that time the mature 3T3-L1 adipocytes were obtained. All cells were maintained in a humidified incubator with 5% CO2 and 95% air at 37°C. For serum starvation treatment, cells were washed twice with warm phosphate-buffered saline (PBS) and then incubated in serum-free medium (DMEM containing 1% p/s) pending for experiments. DMEM medium used in this study contains 4500 mg/L glucose. Virus infection Lentiviruses carrying mouse Atg5 shRNA, Atg7 shRNA, and control shRNA were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Adenoviruses encoding GFPu, RFP, GFP-LC3, or β-galactosidase (β-gal), respectively, were kindly provided by Dr. Xuejun Wang (The University of South Dakota Sanford School of Medicine, USA). 3T3-L1 preadipocytes or fully differentiated 3T3-L1 adipocytes were incubated in serum-free medium containing lentiviruses or adenoviruses for 6 h, and then grown in growth medium for another 36 h. Forty-two hours late after virus infection, the cells were pending for experimental study. Adenovirus/β-gal and lentivirus/control shRNA served as negative controls, respectively. Optimal multiplicity of infection (MOI) used in experiments were as follows: adenovirus/GFPu, 50; adenovirus/RFP, 50; adenovirus/GFP-LC3, 50; adenovirus/βgal, 50; lentivirus/Atg5 shRNA: 120; lentivirus/Atg7 shRNA: 120; lentivirus/control shRNA: 120. Measurement of glucose transporter type 4 (Glut4) on plasma membrane The plasma membrane fraction from the cells was prepared using Plasma Membrane Protein Extraction Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Purity of membrane fraction was determined by expression of membrane marker caveolin and ER maker calnexin. Glut4 expression on plasma membrane was determined by western blot. Caveolin served as internal control. Glucose uptake assay Glucose uptake assay was performed as described previously [22]. In brief, 3T3-L1 adipocytes were incubated with serum-free medium for 6 h, washed twice with wash buffer (containing 20 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 5 mmol/L KCl,
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2.5 mmol/L MgSO4, 1 mmol/L CaCl2), and then incubated in 1 mL of wash buffer. After stimulation with 100 nmol/L insulin for 20 min, 0.5 μCi/mL of 2-deoxyD-2-[3H] glucose (HTA Co. Ltd., Bejing, China) and 10 μmol/L 2-deoxyglucose (2-DG) were added to each well. 2-DG uptake was allowed at 37°C for 10 min. Glucose uptake was stopped by adding 2 mL of icecold 50 mmol/L glucose in PBS. Cells were washed twice with 1 mL of wash buffer and then lysed in 0.5 mL of 0.1 mol/L NaOH. The nonspecific uptake (
