Abstract
Studies on the physiological roles of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) have largely focused on its insulinotropic action and ability to regulate beta-cell mass. In previous studies on the stimulatory effect of GIP on adipocyte lipoprotein lipase (LPL), a pathway was identified involving increased phosphorylation of protein kinase B (PKB) and reduced phosphorylation of LKB1 and AMP-activated protein kinase (AMPK). The slow time of onset of the responses suggested that GIP may have induced release of an intermediary molecule, and the current studies focused on the possible contribution of the adipokine resistin. In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased resistin secretion through a pathway involving p38 mitogen-activated protein kinase (p38 MAPK) and the stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK). The other major incretin hormone, glucagon-like peptide-1 (GLP-1), exhibited no significant effects. Chronic elevation of circulating GIP levels in the Vancouver Diabetic Fatty (VDF) Zucker rat resulted in increases in circulating resistin levels and activation of p38 MAPK or SAPK/JNK in epididymal fat tissue, suggesting the existence of identical pathways in vivo as well as in vitro. Administration of resistin to 3T3-L1 adipocytes mimicked the effects of GIP on the PKB/LKB1/AMPK/LPL pathway: increasing phosphorylation of PKB, reducing levels of phosphorylated LKB1 and AMPK, and increasing LPL activity. Knockdown of resistin using RNA interference attenuated the effect of GIP on the PKB/LKB1/AMPK/LPL pathway in 3T3-L1 adipocytes, supporting a role for resistin as a mediator.
Highlights
There is strong evidence that glucose-dependent insulinotropic polypeptide (GIP) plays an important regulatory role in fat metabolism [7, 8, 15]
In an earlier study we demonstrated that GIP increased lipoprotein lipase (LPL) activity and TG accumulation in differentiated 3T3-L1 cells and human subcutaneous adipocytes through a pathway involving increased phosphorylation of protein kinase B (PKB) and decreases in LKB1 and AMP-activated protein kinase (AMPK) phosphorylation [23]
GIP, but Not glucagon-like peptide-1 (GLP-1) Increases the Secretion of Resistin by 3T3-L1 Adipocytes—The effect of GIP and GLP-1 on the secretion of resistin in vitro was first examined in differentiated 3T3-L1 adipocytes
Summary
Cell Culture—3T3-L1 cells (American Type Culture Collection) were cultured in 25 mM glucose DMEM (Invitrogen) supplemented with 5% newborn calf serum, 100 unit/ml penicillin G-sodium, and 100 g/ml streptomycin sulfate. INS-1 (832/ 13) cells were cultured in 11 mM glucose RPMI 1640 (Sigma) supplemented with 2 mM glutamine, 50 M -mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 10% fetal bovine serum, 100 unit/ml penicillin G-sodium, and 100 g/ml streptomycin sulfate. Resistin ELISA (Enzyme-linked Immunosorbent Assay)—For studies on the effect of GIP or GLP-1 on the secretion of resistin, INS-1 -cells or 3T3-L1 adipocytes were treated with GIP or GLP-1 as indicated in the figure legends. 100 ng of cDNA were used in the real-time PCR to measure resistin expression, whereas 10 ng of cDNA were used for the -actin internal control. Western Blot Analysis—For studies on the effect of GIP or GLP-1 on intracellular resistin and protein kinase levels, INS-1 -cells or 3T3-L1 adipocytes were incubated with GIP or GLP-1, as indicated in the figure legends. Data were analyzed using the non-linear regression analysis program PRISM (GraphPad, San Diego, CA) and significance was tested using analysis of variance (ANOVA) with Newman-Keuls hoc test (p Ͻ 0.05) as indicated in the figure legends
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