Abstract
Glucose-dependent insulinotropic polypeptide (GIP) has been mainly studied because of its glucose-dependent insulinotropic action and its ability to regulate beta-cell proliferation and survival. Considerably less is known about the effects of GIP on fat metabolism, and the present study was directed at identifying the mechanisms underlying its stimulatory action on lipoprotein lipase (LPL). In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased LPL activity and triglyceride accumulation through a pathway involving increased phosphorylation of protein kinase B (PKB) and reductions in phosphorylated LKB1 and AMP-activated protein kinase (AMPK). Knockdown of AMPK using RNA interference and application of the AMPK inhibitor, Compound C, supported this conclusion. In contrast, the other major incretin hormone, glucagon-like peptide-1, exhibited no significant effects on LPL activity or PKB, LKB1, or AMPK phosphorylation. Cultured subcutaneous human adipocytes showed similar responses to GIP but with greater sensitivity. Chronic elevation of circulating GIP levels in the Vancouver diabetic fatty Zucker rat in vivo resulted in increased LPL activity and elevated triglyceride accumulation in epididymal fat tissue, combined with a modulation of PKB, LKB1, and AMPK phosphorylation similar to that observed in vitro. This appears to be the first demonstration of a GIP-stimulated signal transduction pathway involved in increasing fat storage in adipocytes.
Highlights
Glucose-dependent insulinotropic polypeptide (GIP)2 is a pleiotropic hormone that is released from gut endocrine cells in response to nutrient ingestion [1,2,3]
GIP, but Not glucagon-like peptide-1 (GLP-1), Strongly Increases lipoprotein lipase (LPL) Activity in 3T3-L1 Adipocytes—The effect of the incretins, GIP and GLP-1, on LPL activity was first studied in 3T3-L1 adipocytes
Concentration-dependent effects of GIP on LPL activity were observed with EC50 values of 15.3 Ϯ 0.1 nM (Fig. 1B)
Summary
Cell Culture and Differentiation of 3T3-L1 Adipocytes— 3T3-L1 cells (American Type Culture Collection; ATCC) were cultured in DMEM containing high glucose and supplemented with 5% newborn calf serum plus penicillin/streptomycin (standard medium) in 6-well culture plates. Institutional review board approval and informed consent for use of the adipose tissue were obtained from the patients by Zen-Bio Inc. Western Blot Analysis—For studies on the effect of GIP on PKB, LKB1, and AMPK phosphorylation, 3T3-L1 adipocytes or human adipocytes were incubated with GIP in the presence of 1 nM insulin, as indicated in the figure legends. Transfected cells were selected with G418 (Invitrogen), and cell clones were combined, differentiated into adipocytes, and analyzed by Western blotting to confirm AMPK protein expression levels. The specific interference of AMPK protein expression was confirmed by Western blot hybridization using antibody against phospho-AMPK, phospho-LKB1, and phospho-PKB. Determination of Intracellular TG Content—A TG assay kit (Zen-Bio Inc.) was used to measure intracellular TG content of human adipocytes and epididymal fat tissues, according to the manufacturer’s protocol. Data were analyzed using the nonlinear regression analysis program PRISM (GraphPad, San Diego, CA), and significance was tested using analysis of variance (ANOVA) with Newman-Keuls post hoc test (p Ͻ 0.05) as indicated in the figure legends
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