Activation of Lipoprotein Lipase by Glucose-dependent Insulinotropic Polypeptide in Adipocytes

Glucose-dependent insulinotropic polypeptide (GIP) has been mainly studied because of its glucose-dependent insulinotropic action and its ability to regulate β-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 epidydimal 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. Glucose-dependent insulinotropic polypeptide (GIP) has been mainly studied because of its glucose-dependent insulinotropic action and its ability to regulate β-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 epidydimal 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. Glucose-dependent insulinotropic polypeptide (GIP) 2The abbreviations used are: GIP, glucose-dependent insulinotropic polypeptide; LPL, lipoprotein lipase; PKB, protein kinase B; AMPK, AMP-activated protein kinase; GLP-1, glucagon-like peptide-1; FA, fatty acid; VDF, Vancouver diabetic fatty; PI3K, phosphatidylinositol 3-kinase; CA, constitutively active; DN, dominant negative; TG, triglyceride(s); DMEM, Dulbecco's modified Eagle's medium; OGTT, oral glucose tolerance test; ANOVA, analysis of variance; siRNA, small interfering RNA. is a pleiotropic hormone that is released from gut endocrine cells in response to nutrient ingestion (1Brown J.C. Buchan A.M.J. McIntosh C.H.S. Pederson R.A. Schultz S.G. Makhlouf G.M. Rauner B.B. Handbook of Physiology. American Physiology Society, Bethesda, MD1989: 403-430Google Scholar, 2Pederson R.A. Walsh J. 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Rev. 2005; 21: 91-117Crossref PubMed Scopus (244) Google Scholar). Both incretins also exert powerful positive effects on pancreatic β-cell growth, development, and survival (5Ehses J.A. Casilla V.R. Doty T. Pospisilik J.A. Winter K.D. Demuth H.-U. Pederson R.A. McIntosh C.H.S. Endocrinology. 2003; 144: 4433-4445Crossref PubMed Scopus (149) Google Scholar, 6Drucker D.J. Endocrinology. 2003; 144: 5145-5148Crossref PubMed Scopus (243) Google Scholar). A number of studies have demonstrated that GIP plays an important role in the regulation of fat metabolism (7Morgan L.M. Biochem. Soc. Trans. 1996; 24: 585-591Crossref PubMed Scopus (50) Google Scholar, 8McIntosh C.H.S. Bremsak I. Lynn F.C. Gill R. Hinke S.A. Gelling R. McKnight G. Jaspers S. Pederson R.A. Endocrinology. 1999; 140: 398-404Crossref PubMed Scopus (51) Google Scholar, 9Yip R.G.C. Wolfe M.M. Life Sci. 2000; 66: 91-103Crossref PubMed Google Scholar). GIP is released in response to administration of triglycerides (TG) (1Brown J.C. Buchan A.M.J. McIntosh C.H.S. Pederson R.A. Schultz S.G. Makhlouf G.M. Rauner B.B. Handbook of Physiology. American Physiology Society, Bethesda, MD1989: 403-430Google Scholar, 2Pederson R.A. Walsh J. Dockray G. Gut Peptides: Biochemistry and Physiology. Raven Press, Ltd., New York1993: 217-259Google Scholar), with long chain fatty acids (FAs) being responsible for stimulating secretion (1Brown J.C. Buchan A.M.J. McIntosh C.H.S. Pederson R.A. Schultz S.G. Makhlouf G.M. Rauner B.B. Handbook of Physiology. American Physiology Society, Bethesda, MD1989: 403-430Google Scholar). In dogs, GIP has been shown to promote clearance of chylomicron-associated TG from blood (10Wasada T. McCorkle K. Harris V. Kawai K. Howard B. Unger R.H. J. Clin. Invest. 1981; 68: 1106-1107Crossref PubMed Scopus (130) Google Scholar), and in rats, it has been shown to promote infusion of GIP-lowered plasma TG responses to intraduodenal fat (11Ohneda A. Kobayashi T. Nihei J. Regul. Pept. 1984; 8: 123-130Crossref PubMed Scopus (9) Google Scholar). GIP enhanced FA synthesis from acetate in adipose tissue explants (12Oben J. Morgan L. Fletcher J. Marks V. J. Endocrinol. 1991; 130: 267-272Crossref PubMed Scopus (177) Google Scholar) as well as potentiating insulin-stimulated FA incorporation into adipose tissue (13Beck B. Max J.P. Regul. Pept. 1983; 7: 3-8Crossref PubMed Scopus (71) Google Scholar) and stimulating lipoprotein lipase (LPL) activity in cultured preadipocytes (14Eckel R.H. Fujimoto W.Y. Brunzell J.D. Diabetes. 1979; 28: 1141-1142Crossref PubMed Scopus (0) Google Scholar) and mature adipocytes (15Knapper J.M.E. Puddicombe S.M. Morgan L.M. Fletcher J.M. J. Nutr. 1995; 125: 183-188PubMed Google Scholar). These studies pointed to a significant role for GIP in the regulation of adipogenesis, and its physiological importance was emphasized by the demonstration by Miyawaki et al. (16Miyawaki K. Yamada Y. Ban N. Ihara Y. Tsukiyama K. Zhou H. Fujimoto S. Oku A. Tsuda K. Toyokuni S. Hiau H. Mizunoya W. Fushiki T. Holst J.J. Makino M. Tashita A. Kobara Y. Tsubamoto Y. Jinnouchi T. Jomori T. Seino Y. Nat. Med. 2002; 8: 738-742Crossref PubMed Scopus (725) Google Scholar) that GIP receptor knock-out mice exhibited reduced adipose tissue accretion on a high fat diet. The GIP receptor is a member of the class B seven-transmembrane G protein-coupled family to which the receptors for glucagon, GLP-1, and secretin belong (17Wheeler M.B. Gelling R.W. McIntosh C.H.S. Georgiou J. Brown J.C. Pederson R.A. Endocrinology. 1995; 136: 4629-4639Crossref PubMed Google Scholar, 18Usdin T.B. Mezey E. Button D.C. Brownstein M.J. Bonner T.I. Endocrinology. 1993; 133: 2861-2870Crossref PubMed Scopus (307) Google Scholar). The majority of studies on the mode of action of GIP have been performed in islets, dissociated β-cells, or β-cell lines, and these have shown that receptor activation results in the stimulation of adenylyl cyclase (17Wheeler M.B. Gelling R.W. McIntosh C.H.S. Georgiou J. Brown J.C. Pederson R.A. Endocrinology. 1995; 136: 4629-4639Crossref PubMed Google Scholar) and phospholipase A2 (19Ehses J.A. Lee S.S. Pederson R.A. McIntosh C.H.S. J. Biol. Chem. 2001; 276: 23667-23673Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Downstream signaling involves a number of enzyme modules, including protein kinase A/cAMP-response element-binding protein, Rap1/Raf-A/Mek/Erk1/2 (20Ehses J.A. Pelech S.L. Pederson R.A. McIntosh C.H.S. J. Biol. Chem. 2002; 277: 37088-37097Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/Foxo1 (21Kim S.J. Winter K. Nian C. Tsuneoka M. Koda Y. McIntosh C.H.S. J. Biol. Chem. 2005; 280: 22297-22307Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 22Trümper A. Trümper K. Trusheim H. Arnold R. Göke B. Hörsch D. Mol. Endocrinol. 2001; 15: 1559-1570Crossref PubMed Scopus (240) Google Scholar). The mechanism by which GIP acts on adipocytes is largely unknown. GIP receptor expression has been demonstrated in rat adipocytes (23Yip R.G.C. Boylan M.O. Kieffer T.J. Wolfe M.M. Endocrinology. 1998; 139: 4004-4007Crossref PubMed Scopus (143) Google Scholar) and differentiated 3T3-L1 cells (8McIntosh C.H.S. Bremsak I. Lynn F.C. Gill R. Hinke S.A. Gelling R. McKnight G. Jaspers S. Pederson R.A. Endocrinology. 1999; 140: 398-404Crossref PubMed Scopus (51) Google Scholar), and, in the absence of insulin, activation results in the stimulation of adenylyl cyclase and lipolysis (8McIntosh C.H.S. Bremsak I. Lynn F.C. Gill R. Hinke S.A. Gelling R. McKnight G. Jaspers S. Pederson R.A. Endocrinology. 1999; 140: 398-404Crossref PubMed Scopus (51) Google Scholar). However, since this lipolytic action is inhibited by insulin (8McIntosh C.H.S. Bremsak I. Lynn F.C. Gill R. Hinke S.A. Gelling R. McKnight G. Jaspers S. Pederson R.A. Endocrinology. 1999; 140: 398-404Crossref PubMed Scopus (51) Google Scholar), it was considered likely that the lipogenic effects of GIP are mediated through alternative pathways. AMP-activated protein kinase (AMPK) is a serine/threonine kinase that acts as an intracellular energy sensor (24Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 25Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (651) Google Scholar) or “fuel gauge” (26Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar). AMPK exists as a heterotrimeric protein complex consisting of a catalytic subunit (α) and two regulatory subunits (β and γ) (24Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 25Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (651) Google Scholar). Two α isoforms exist, and they are both found in 3T3-L1 adipocytes (27Salt I.P. Connell J.M.C. Gould G.W. Diabetes. 2000; 49: 1649-1656Crossref PubMed Scopus (107) Google Scholar). In keeping with its energy sensor role, starvation activates AMPK in adipose tissue (28Sponarova J. Mustard K.J. Horakova O. Flachs P. Rossmeisl M. Brauner P. Bardova K. Thomason-Hughes M. Braunerova R. Janovska P. Hardie D.G. Kopecky J. FEBS Lett. 2005; 579: 6105-6110Crossref PubMed Scopus (41) Google Scholar, 29Daval M. Foufelle F. Ferré P. J. Physiol. (Lond.). 2006; 574: 55-62Crossref Scopus (311) Google Scholar), and AMPK exerts antilipolytic effects (28Sponarova J. Mustard K.J. Horakova O. Flachs P. Rossmeisl M. Brauner P. Bardova K. Thomason-Hughes M. Braunerova R. Janovska P. Hardie D.G. Kopecky J. FEBS Lett. 2005; 579: 6105-6110Crossref PubMed Scopus (41) Google Scholar, 29Daval M. Foufelle F. Ferré P. J. Physiol. (Lond.). 2006; 574: 55-62Crossref Scopus (311) Google Scholar, 30Daval M. Diot-Dupuy F. Bazin R. Hainault I. Viollet B. Vaulont S. Hajduch E. Ferré P. Foufelle F. J. Biol. Chem. 2005; 280: 25250-25257Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), as well as inhibiting adipocyte fatty acid synthesis, by phosphorylating acetyl-CoA-carboxylase-1 (29Daval M. Foufelle F. Ferré P. J. Physiol. (Lond.). 2006; 574: 55-62Crossref Scopus (311) Google Scholar) and inhibiting insulin-induced glucose uptake (30Daval M. Diot-Dupuy F. Bazin R. Hainault I. Viollet B. Vaulont S. Hajduch E. Ferré P. Foufelle F. J. Biol. Chem. 2005; 280: 25250-25257Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The overall effect of AMPK is to convert adipocytes into lipid oxidizing cells with suppressed lipolysis and lipogenesis (29Daval M. Foufelle F. Ferré P. J. Physiol. (Lond.). 2006; 574: 55-62Crossref Scopus (311) Google Scholar). LPL catalyzes the hydrolysis of TG associated with chylomicrons and very low density lipoproteins in the circulation, thus generating 2-monoacylglycerol and fatty acids, that undergo re-esterification in adipocytes (31Preiss-Landl K. Zimmermann R. Hämmerle G. Zechner R. Curr. Opin. Lipidol. 2002; 13: 471-881Crossref PubMed Scopus (197) Google Scholar, 32Goldberg I.J. J. 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Food deprivation results in down-regulation of adipose tissue LPL, whereas insulin increases overall activity, possibly by acting at several levels (31Preiss-Landl K. Zimmermann R. Hämmerle G. Zechner R. Curr. Opin. Lipidol. 2002; 13: 471-881Crossref PubMed Scopus (197) Google Scholar, 32Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar, 33Braun J.E. Severson D.L. Biochem. J. 1992; 287: 337-347Crossref PubMed Scopus (267) Google Scholar, 34Merkel M. Eckel R.H. Goldberg I.J. J. Lipid Res. 2002; 43: 1997-2006Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 35Berbö M. Wu G. Ruge T. Olivecrona T. J. Biol. Chem. 2002; 277: 11927-11932Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Since GIP increases adipocyte LPL activity (14Eckel R.H. Fujimoto W.Y. Brunzell J.D. Diabetes. 1979; 28: 1141-1142Crossref PubMed Scopus (0) Google Scholar, 15Knapper J.M.E. Puddicombe S.M. Morgan L.M. Fletcher J.M. J. Nutr. 1995; 125: 183-188PubMed Google Scholar), we postulated that it might act by suppressing AMPK levels, thus promoting fatty acid and 2-monoacylglycerol delivery to the cell and contributing to increased adipogenesis (6Drucker D.J. Endocrinology. 2003; 144: 5145-5148Crossref PubMed Scopus (243) Google Scholar, 7Morgan L.M. Biochem. Soc. Trans. 1996; 24: 585-591Crossref PubMed Scopus (50) Google Scholar, 13Beck B. Max J.P. Regul. Pept. 1983; 7: 3-8Crossref PubMed Scopus (71) Google Scholar). Using differentiated 3T3-L1 cells and human subcutaneous adipocytes, we have demonstrated that GIP increases phosphorylation of PKB and decreases LKB1 and AMPK phosphorylation in the presence of insulin, resulting in activation of LPL and TG accumulation. Knockdown of AMPK using RNA interference and application of the AMPK inhibitor Compound C supported this conclusion. Chronic elevation of circulating GIP levels in the Vancouver diabetic fatty (VDF) Zucker rat in vivo resulted in activation of LPL in epidydimal fat tissue by a similar pathway. This appears to be the first description of a signaling pathway by which GIP stimulates FA storage in adipocytes. 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. Cells were induced to differentiate into the adipocyte phenotype as previously described (8McIntosh C.H.S. Bremsak I. Lynn F.C. Gill R. Hinke S.A. Gelling R. McKnight G. Jaspers S. Pederson R.A. Endocrinology. 1999; 140: 398-404Crossref PubMed Scopus (51) Google Scholar). In brief, 2 days after cells were confluent, medium was supplemented with dexamethasone (0.6 μm), 3-isobutyl-1-methylxanthine (0.1 mm), and insulin (16 μm) for 72 h, after which cells were cultured in DMEM high glucose medium plus 10% fetal calf serum. Differentiation was complete in 7 days. Differentiated cells were confirmed by Oil Red O staining, and fully differentiated cells (>85% adipose cells) from passages 3–8 were used in all experiments. Cell Culture and Differentiation of Human Adipocytes—Subcutaneous human preadipocytes were from Zen-Bio Inc. (Research Triangle Park, NC). They were obtained from healthy, nondiabetic women (n = 7; average body mass index, 25.17 kg/m2 (range 22.5–28.2); average age, 41 years (range 27–51)) and differentiated into adipocytes according to the supplier's protocol. 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. Where appropriate, the AMPK inhibitor, Compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine) (Calbiochem), was added at a final concentration of 40 μm. Total cellular extracts from each sample were separated on a 13% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Bio-Rad). Probing of the membranes was performed with phospho-PKB (serine 473), PKB, phospho-LKB1 (serine 428), phospho-AMPK (threonine 172), AMPK (Cell Signaling Technology, Beverly, MA), and β-tubulin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-conjugated IgG secondary antibodies. Generation of Stable Cell Lines—3T3-L1 preadipocytes were grown in DMEM (Invitrogen), supplemented with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (50 IU/ml, 50 μg/ml; Invitrogen) and transfected with constitutively active AMPK (CA-AMPK) and dominant negative AMPK (DN-AMPK) cDNAs, expressing the constitutively active and dominant negative forms of AMPK, respectively. For CA-AMPK, the cDNA encoded residues 1–312 of AMPK subunit α1, containing a mutation resulting in a change of threonine 172 to aspartic acid. A cDNA encoding subunit α1 containing a mutation altering an aspartic acid residue 157 to alanine was used. CA- and DN-AMPK constructs were kindly provided by Dr. David Carling (Imperial College School of Medicine, London, UK). Transfections were performed using Lipofectamine 2000™ transfection reagent (Invitrogen) for 4 h according to the manufacturer's instructions. Stably 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. Knockdown of AMPKα by RNA Interference—To reduce levels of endogenous AMPKα, 3T3-L1 adipocytes were transfected with a pool of three siRNAs for AMPKα (sc-45313; Santa Cruz Biotechnology) using Lipofectamine 2000™ transfection reagent and incubated for 72 h. The specific interference of AMPK protein expression was confirmed by Western blot hybridization using antibody against phospho-AMPK, phospho-LKB1, and phospho-PKB. LPL Enzyme Activity Assays—The LPL activity assay kit (Roar Biomedical Inc.) was used to measure enzyme activity, according to the manufacturer's protocol. Enzyme activity is presented as relative activity normalized to protein concentration. GIP Infusion—Obese VDF rats and their lean littermate controls (12 weeks old) were subjected to a 2-week continuous infusion of GIP (10 pmol/kg·min). The infusion was performed using an Alzet miniosmotic pump (Alzet Corp., Minneapolis, MN) implanted in the intraperitoneal region under pentobarbital (40 mg/kg) anesthesia. Rats were sacrificed at the end of the infusion, and epidydimal fat tissues were harvested for Western blotting. Experiments were conducted in accordance with guidelines of the University of British Columbia Animal Care Committee and Canadian Council on Animal Care. Oral Glucose Tolerance Tests (OGTTs) and Measurements of Blood Glucose and Plasma Insulin Levels—Blood glucose levels were measured using a SureStep Glucose analyzer (LifeScan Canada, Burnaby, Canada). Following an approximate 16-h overnight fast, OGTTs (2 g/kg) were performed, with blood glucose levels following the glucose challenge measured at the time points indicated in Fig. 8E. Plasma insulin levels were determined using a radioimmunoassay kit (Linco Research Inc., St. Charles, MO). Oil Red O Staining—After overnight serum starvation, human adipocytes were treated for 24 h with GIP (100 nm) or GLP-1 (100 nm) in the presence of insulin (1 nm). Cells were then fixed and stained for 2 h by complete immersion in a working solution of Oil Red O. The method of Ramirez-Zacarias et al. (36Ramirez-Zacarias J.L. Castro-Mufiozledo F. Kuri-Harcuch W. Histochemistry. 1992; 97: 493-497Crossref PubMed Scopus (836) Google Scholar) was used to determine the level of staining. Isopropyl alcohol was added to the stained culture dish and dye-extracted by gentle pipetting, and the absorbance at 490 nm was measured spectrophotometically. 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. Statistical Analysis—Data are expressed as means ± S.E. with the number of individual experiments presented in the figure legend. 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 < 0.05) as indicated in the figure legends. GIP, but Not GLP-1, Strongly Increases 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. Treatment with GIP (100 nm) in the presence of insulin (1 nm) for 24 h resulted in ∼2.6-fold increases in LPL activity, compared with basal. In contrast, treatment of 3T3-L1 adipocytes with GLP-1 (100 nm) under identical conditions resulted in only small increases in mean LPL activity that did not reach significance (Fig. 1A). Concentration-dependent effects of GIP on LPL activity were observed with EC50 values of 15.3 ± 0.1 nm (Fig. 1B). GIP Treatment of 3T3-L1 Adipocytes Results in Increased PKB Phosphorylation and Decreased Phosphorylation of AMPK and LKB1—The mechanisms involved in the activation of LPL by GIP treatment were next studied. Phosphorylation of AMPK at Thr172 by upstream kinase AMPK kinases is essential for its activation (24Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 25Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (651) Google Scholar), resulting in increases in activity of at least 50-fold. The major upstream kinase for activation of AMPK in most tissues, including adipose (25Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (651) Google Scholar), has recently been identified as LKB1 (37Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1340) Google Scholar, 38Carling D. Trends Mol. Med. 2006; 12: 144-147Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Treatment of 3T3-L1 adipocytes with GIP (100 nm) in the presence of insulin (1 nm) resulted in profound decreases in phosphorylation of AMPK at Thr172 (Fig. 2A) and LKB1 at Ser428 (Fig. 2C). GIP-induced responses were concentration-dependent, with EC50 values of 34.7 ± 0.2 nm for AMPK (Fig. 2B) and 25.5 ± 0.2 nm for LKB1 (Fig. 2D). In parallel experiments, GIP stimulated phosphorylation of PKB at Ser473 (Fig. 2E) with an EC50 of 35.6 ± 0.2 nm (Fig. 2F). Decreased phosphorylation of both AMPK and LKB1 (Fig. 2, A and C) and increased phosphorylation of PKB (Fig. 2E) were evident by 6 h following GIP treatment and sustained for 24 h. Treatment of 3T3-L1 adipocytes with GLP-1 (100 nm) in the presence of 1 nm insulin resulted in no significant changes in phosphorylation of PKB, LKB1, or AMPK (Fig. 3). These results correlated well with the lack of effect of GLP-1 on LPL (Fig. 1).FIGURE 3GLP-1 had no significant effects on phosphorylation of AMPK, LKB1, or PKB in 3T3-L1 adipocytes. Protocols for treatment of 3T3-L1 adipocytes with GIP were as described in the legend to Fig. 2. Shown is the time course of phosphorylation of AMPK (A), LKB1 (C), and PKB (E) in the presence of GIP. Shown are concentration-response effects of GIP on AMPK (B), LKB1 (D), and PKB (F). Western blots were quantified using densitometric analysis and are representative of n = 3. Significance was tested using ANOVA with Newman-Keuls post hoc test. **, p < 0.05 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PI3K Is Involved in GIP Regulation of the PKB/LKB1/AMPK/LPL Signaling Pathway—Since phosphorylation of both Thr308 and Ser473 by PI3K is essential for PKB activation (39Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 5: 6541-6551Crossref Scopus (2517) Google Scholar), the relationship between PI3K and the PKB/LKB1/AMPK signaling modules was next studied using the selective pharmacological inhibitors of PI3K, LY294002 and wortmannin. Inhibition of PI3K greatly reduced basal levels of phospho-PKB and ablated PKB responses to GIP (Fig. 4A). On the other hand, inhibition of PI3K slightly increased basal levels of both phosphorylated LKB1 and AMPK, and GIP treatment had no significant effect on levels of either phosphorylated kinase (Fig. 4, B and C). Under the same conditions of PI3K inhibition, basal LPL activity was decreased, and responses to GIP were severely attenuated (Fig. 4D). These results suggest that PI3K is an upstream component of the GIP-stimulated PKB/AMPK/LPL signaling pathways. PKB and LKB1 Act Upstream of AMPK and LPL—To define further the relationship between PKB/LKB1 and AMPK signaling modules in the regulation of LPL activity, 3T3-L1 adipocytes stably expressing CA or DN forms of AMPK were generated. 3T3-L1 adipocytes stably expressing CA-AMPK demonstrated increased basal phospho-AMPK levels that were reduced by treatment with GIP or high concentrations of insulin (100 nm) (Fig. 5A). In contrast, levels of phospho-AMPK were greatly reduced in 3T3-L1 adipocytes expressing DN-AMPK (Fig. 5A). Surprisingly, the residual phospho-AMPK was ablated by GIP or insulin (100 nm). There were no significant changes in the phosphorylation levels of PKB and LKB1 with the expression of CA- or DN-AMPK, when compared with 3T3-L1 adipocytes transfected with empty vector (Fig. 5, B and C). In parallel experiments, 3T3-L1 adipocytes expressing CA-AMPK had decreased basal LPL that was still responsive to GIP stimulation. Expression of DN-AMPK increased basal LPL activity, and GIP treatment resulted in a further increase (Fig. 5D). Taken together, these results strongly suggest that PKB/LKB1 is an upstream signaling module of AMPK. However, since LPL activity of CA- and DN-AMPK cells was still responsive to GIP, alternative approaches were applied to establish that phospho-AMPK was involved in GIP-mediated LPL activation. First, pretreatment of cells with an AMPK inhibitor, Compound C, resulted in a marked decrease in phospho-AMPK levels (Fig. 6A) without changes in phosphorylation levels of PKB and LKB1 (Fig. 6, B and C). Phospho-AMPK levels were too low to assess effects of GIP. Under the same conditions, AMPK inhibitor increased LPL activity, but it was not further increased by GIP treatment (Fig. 6D). RNA interference-mediated knockdown of AMPKα resulted in similar results: a substantial decre