Growth Hormone Inhibition of Glucose Uptake in Adipocytes Occurs without Affecting GLUT4 Translocation through an Insulin Receptor Substrate-2-Phosphatidylinositol 3-Kinase-dependent Pathway

Abstract

Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4. Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4. Insulin is the major anabolic hormone whose action plays pivotal roles in tissue development, growth, and the maintenance of glucose homeostasis. Insulin regulates glucose metabolism at several levels, reducing hepatic glucose production and increasing the rate of glucose uptake into skeletal muscle and adipose tissue. Insulin-responsive glucose transporter isoform GLUT4 5The abbreviations used are: GLUT, glucose transporter; GH, growth hormone; PI, phosphatidylinositol; IRS, insulin receptor substrate; PIP3, phosphoinositide 3,4,5-triphosphate; PDK, phosphoinositide-dependent kinase; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum; NC, nonrelevant control; GFP, green fluorescent protein; PI, phosphatidylinositol; siRNA, small interfering RNA. is expressed in skeletal muscle and adipose tissue and is known to be responsible for the glucose uptake into these tissues (1Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (930) Google Scholar, 2Ducluzeau P.H. Fletcher L.M. Vidal H. Laville M. Tavare J.M. Diabetes Metab. 2002; 28: 85-92PubMed Google Scholar, 3Watson R.T. Kanzaki M. Pessin J.E. Endocr. Rev. 2004; 25: 177-204Crossref PubMed Scopus (355) Google Scholar). Thus, glucose uptake through GLUT4 plays a central role in the regulation of postprandial glucose clearance from the plasma. Dysfunction in the ability of insulin to stimulate glucose uptake results in states of insulin resistance that plays a major role in the development of type 2 diabetes mellitus. Insulin binding to the extracellular domain of the insulin receptor on the plasma membrane of target cells activates its intrinsic cytoplasmic tyrosine kinase activity (4Gronborg M. Wulff B.S. Rasmussen J.S. Kjeldsen T. Gammeltoft S. J. Biol. Chem. 1993; 268: 23435-23440Abstract Full Text PDF PubMed Google Scholar, 5Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4605) Google Scholar). The activated insulin receptor tyrosine kinase phosphorylates a variety of substrates, including insulin receptor substrates (IRSs) and Shc (6Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar, 7White M.F. Diabetologia. 1997; 40: 2-17Crossref PubMed Scopus (462) Google Scholar). Tyrosine phosphorylation of these substrates leads to their binding to several Src homology 2 domain-containing signaling molecules, including p85 PI 3-kinase regulatory subunit and Grb2 (6Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar, 8Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. 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Activated PI 3-kinase phosphorylates phosphatidylinositol and generates phosphoinositide 3,4,5-triphosphate (PIP3). PIP3 production recruits Akt kinase to the plasma membrane, where Akt is phosphorylated by phosphoinositide-dependent kinases, resulting in activation of the Akt kinase (17Galetic I. Andjelkovic M. Meier R. Brodbeck D. Park J. Hemmings B.A. Pharmacol. Ther. 1999; 82: 409-425Crossref PubMed Scopus (96) Google Scholar). Activated Akt phosphorylates various Akt substrates, including AS160, GTPase-activating protein for Rab10 (18Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). Recently, it was reported that phosphorylation and inhibition of AS160 and subsequent activation of small GTP-binding protein Rab10 are sufficient for GLUT4 translocation to plasma membrane (19Sano H. Eguez L. Teruel M.N. Fukuda M. Chuang T.D. Chavez J.A. Lienhard G.E. McGraw T.E. Cell Metab. 2007; 5: 293-303Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Growth hormone (GH) is well known to possess bioactivities regulating both growth and metabolism. In various tissues, GH is known to mediate anti-insulin effects on glucose and lipid metabolism. Insulin resistance is often observed in acromegalic patients, and GH administration to GH-deficient patients has been observed to increase the incidence of diabetes mellitus (20Beck P. Schalch D.S. Parker M.L. Kipnis D.M. Daughaday W.H. J. Lab. Clin. Med. 1965; 66: 366-379PubMed Google Scholar, 21Cerasi E. Luft R. Lancet. 1964; 2: 769-771Abstract PubMed Scopus (31) Google Scholar, 22Yuen K. Cook D. Ong K. Chatelain P. Fryklund L. Gluckman P. Ranke M.B. Rosenfeld R. Dunger D. Clin. Endocrinol. 2002; 57: 333-341Crossref PubMed Scopus (23) Google Scholar). Recently, we have reported that the GH transgenic rat, in which human GH was highly expressed, showed an insulin-resistant phenotype in both muscle and adipose tissues (23Cho Y. Ariga M. Uchijima Y. Kimura K. Rho J.Y. Furuhata Y. Hakuno F. Yamanouchi K. Nishihara M. Takahashi S. Endocrinology. 2006; 147: 5374-5384Crossref PubMed Scopus (31) Google Scholar). Similarly, chronic GH treatment in cultured 3T3-L1 adipocytes was also found to impair insulin-induced glucose uptake (24Takano A. Haruta T. Iwata M. Usui I. Uno T. Kawahara J. Ueno E. Sasaoka T. Kobayashi M. Diabetes. 2001; 50: 1891-1900Crossref PubMed Scopus (82) Google Scholar). Together, these studies indicate that chronic GH treatment can result in insulin-resistant glucose uptake in vitro as well as in vivo. Thus, this study was undertaken to evaluate the molecular mechanism of GH-induced impairment of insulin-dependent glucose uptake in 3T3-L1 adipocytes. Here we showed that chronic GH pretreatment inhibits insulin-induced glucose uptake without affecting GLUT4 translocation, through the reduction of IRS-2-associated PI 3-kinase activity. Materials-Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and Hanks’ buffered salt solution were purchased from Nissui (Tokyo, Japan). Calf serum and fetal bovine serum (FBS) were obtained from JRH Bioscience (Tokyo, Japan). Penicillin and streptomycin were obtained from Ban’yu Pharmaceutical Co. (Tokyo, Japan). Recombinant human GH was kindly donated from Dainippon Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan), and CR Pharmaceuticals Co., Ltd. (Kobe, Japan). Bovine insulin was obtained from Sigma. Anti-IRS-1 antibody and anti-IRS-2 antibody were raised in rabbits as described previously (25Ariga M. Nedachi T. Akahori M. Sakamoto H. Ito Y. Hakuno F. Takahashi S. Biochem. J. 2000; 348: 409-416Crossref PubMed Scopus (60) Google Scholar). Anti-JAK2 antibody, anti-GLUT1 antibody, anti-GLUT4 antibody, and anti-IRβ antibody were obtained from Santa Cruz Biotechnology. Anti-PI 3-kinase p85 antibody and anti-c-Myc antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-phospho-Akt (Ser-473) antibody and anti-Akt antibody were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Anti-phospho-Akt (Thr-308) antibody, anti-β-actin antibody, and anti-phosphotyrosine antibody were obtained from Sigma. Texas Red dye-conjugated anti-mouse IgG was obtained from Jackson ImmunoResearch (West Grove, PA). Protein A-Sepharose and 2-deoxy-d-[2,6-3H]glucose (1 mCi/ml) were purchased from Amersham Biosciences. Wheat germ agglutinin-agarose was obtained from Seikagaku Co. (Tokyo, Japan). Random control, IRS-1-, and IRS-2-specific siRNAs were purchased from RNAi Corp. (Tokyo, Japan). The sequence of the IRS-1 siRNA used was CUC GAG AGC UGU UUC AAC AUC. The sequence of the IRS-2 siRNA used was GCC CGA ACC UCA AUA ACA ACA. The control siRNA sequence was GUA CCG CAC GUC AUU CGU AUC. Other chemicals were of reagent grade available commercially. Cell Culture of 3T3-L1 Adipocytes-Murine 3T3-L1 preadipocytes were purchased from the American Type Tissue Culture repository. 3T3-L1 preadipocytes were maintained at 37 °C in a humidified 5% CO2 controlled atmosphere in DMEM supplemented with Japan). 3T3-L1 preadipocytes were to into adipocytes as described previously J.S. D. Pessin J.E. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). were with DMEM FBS, and 4 the medium was to DMEM and insulin for an 2 The medium was to DMEM were used for at of cells an of 3T3-L1 of 3T3-L1 adipocytes was described previously F. S. R.T. Pessin J.E. Takahashi S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). 3T3-L1 adipocytes were from the tissue by in and with a of of or of siRNA the at and of adipocytes were with the of insulin in for at 37 2-deoxy-d-[2,6-3H]glucose was and cells were for at 37 The was by of were with and by the cells was by adipocytes were and in with a were at for The resulting obtained from this was in and at for This a at the plasma membrane The plasma membrane was in and at for The resulting were in and of Cell Protein and of adipocytes with or without GH were with insulin. were with and with in for at 4 The was by the with in The cells were and with were with and the was at 4 °C The were and with with with in and with in The as protein and as protein were J.F. Lee E. 28: PubMed Scopus Google Scholar, Morris N.J. F. Lienhard G.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. R. S. E. K. E. Diabetologia. 1997; 40: PubMed Scopus Google Scholar). and adipocytes were with or without siRNA with fluorescent protein cells were for 2 and with or without GH by insulin were without and with were Japan). GLUT4 translocation to the we the of cells to of Insulin adipocytes were for 2 in DMEM bovine serum were with or without GH by treatment with insulin. were by of the at for at 4 was as The were to protein protein of was used for with were by and with the PI 3-kinase activity was as described T. Akahori M. Ariga M. Sakamoto H. K. Hakuno F. Takahashi Endocrinology. 2000; PubMed Scopus Google Scholar). 3T3-L1 adipocytes were for 2 in DMEM bovine serum by pretreatment with or without GH for were with or without insulin for were by and of was with antibody or anti-IRS-2 were with and and and and in of and was by of in with and phosphatidylinositol at °C for was by lipid was and with into phosphatidylinositol was by as PI 3-kinase activity. and to 3T3-L1 active of and were described H. T. M. M. K. H. Y. Y. H. M. M. Y. T. Diabetes. 2000; PubMed Scopus Google Scholar). The to IRS-2 was as described S. M. Y. S. Y. K. I. S. A. 1996; PubMed Scopus Google Scholar). were in human 3T3-L1 adipocytes were with by cells with of of were used for of of data were by or of by The results are the S.E. was GH in 3T3-L1 has been reported that chronic GH pretreatment inhibits insulin-dependent glucose uptake in cultured adipocytes (24Takano A. Haruta T. Iwata M. Usui I. Uno T. Kawahara J. Ueno E. Sasaoka T. Kobayashi M. Diabetes. 2001; 50: 1891-1900Crossref PubMed Scopus (82) Google Scholar). these 3T3-L1 adipocytes were with various of GH for and to an stimulation with insulin. In the of GH insulin stimulation resulted in a increase in glucose uptake pretreatment with GH inhibited insulin-stimulated glucose uptake in a GH insulin-stimulated glucose uptake occurred in an insulin in the of GH pretreatment GH inhibited insulin-stimulated glucose uptake at insulin but at the of glucose uptake was data that GH pretreatment results in an insulin-dependent in but not the of GH pretreatment on insulin-stimulated glucose uptake GH pretreatment for to significant effect on glucose uptake. of GH the of insulin-stimulated glucose uptake was The GH reduction of insulin-stimulated glucose uptake to with GH and by glucose uptake was to insulin In the effect of GH was also in that of GH of GH resulted in a time-dependent of insulin-stimulated glucose uptake these data that GH pretreatment of 3T3-L1 adipocytes results in a and inhibition of insulin-stimulated glucose uptake. GH GLUT4 to glucose are expressed in 3T3-L1 adipocytes with the isoform to glucose uptake and GLUT4 to insulin-stimulated glucose uptake P.H. Fletcher L.M. Vidal H. Laville M. Tavare J.M. Diabetes Metab. 2002; 28: 85-92PubMed Google Scholar, A. A. J. 2004; 28: PubMed Scopus Google Scholar). The expression of and GLUT4 were by chronic GH treatment Insulin stimulation glucose uptake by the translocation of the GLUT4 isoform from to the plasma membrane R.T. Pessin J.E. Res. 2001; PubMed Scopus Google Scholar). of a plasma the of the GLUT4 protein insulin stimulation and chronic GH pretreatment significant effect on the of insulin-stimulated GLUT4 in the plasma membrane and GH pretreatment the fusion of the GLUT4 protein with the plasma membrane, adipocytes were with to plasma membrane with of with an insulin-stimulated increase in the of GLUT4 and to the translocation insulin was of the plasma membrane fusion of GLUT4 in cells with GH and was a in the of GLUT4 in the Moreover, was of the GLUT4 protein by without to data from are in of the these we expressed the GLUT4 protein an 3T3-L1 adipocytes were with and the of the was various by fluorescent Insulin stimulation the of the of the and this was by chronic GH pretreatment. The inhibition of insulin-stimulated glucose uptake by GH pretreatment with GLUT4 translocation and plasma membrane fusion have from a reduction in activity that phosphorylates to the this insulin-induced glucose uptake was in the cells with various of that the GH-induced inhibition of glucose uptake was of glucose that glucose metabolism was not inhibited by GH pretreatment. these data indicate that chronic GH pretreatment not insulin-stimulated GLUT4 translocation or with the plasma membrane but that the reduction in glucose uptake in glucose transport activity. GH of PI to IRS-2 and Akt binding to the GH receptor activates the tyrosine kinase pathway that can also through tyrosine the effect of chronic GH pretreatment on insulin signaling 3T3-L1 adipocytes were with GH for by stimulation with and the activation of target in to insulin stimulation was that GH pretreatment tyrosine and insulin receptor tyrosine which is known to tyrosine kinase was not by GH pretreatment IRS-1 and IRS-2 tyrosine phosphorylation was by GH pretreatment GH stimulation both IRS-1 and IRS-2 tyrosine The of the p85 PI 3-kinase regulatory subunit with IRS-1 and IRS-2 was also by GH which of tyrosine phosphorylation PI 3-kinase activity was also by GH pretreatment Surprisingly, IRS-2-associated PI 3-kinase activity was by GH treatment the of PI 3-kinase with IRS-2 was activation of the PI 3-kinase generates the production of PIP3 that is for the phosphorylation of Akt on and resulting in the activation of Akt kinase activity. in Akt phosphorylation was also inhibited by chronic GH pretreatment and that inhibition with the suppression of IRS-2-associated PI 3-kinase activity. IRS-2 the GH of in that IRS-2 to Akt activation be the target of GH that is responsible for the inhibition of insulin-stimulated glucose uptake. observed GH pretreatment insulin-stimulated Akt phosphorylation in cells In contrast, overexpression of IRS-2 by the inhibition of insulin-stimulated Akt phosphorylation by GH pretreatment The of insulin-stimulated Akt phosphorylation by IRS-2 overexpression occurred with the of insulin-stimulated glucose uptake in the cells In parallel, expression of a constitutively active Akt mutant resulted in the increase in glucose uptake that was expression the cells from the GH inhibition of insulin-stimulated glucose uptake. regulation of Akt activation is the of GH-induced insulin reduction of IRS-2 the effect of GH pretreatment. this we IRS-1 or IRS-2 protein by 3T3-L1 adipocytes were with an or IRS-2-specific siRNA that resulted in a reduction in protein without significant effect on the protein IRS-1 knockdown significant effect on insulin-stimulated Akt GLUT4 translocation, or glucose uptake the in IRS-2 knockdown was a reduction in insulin-stimulated Akt phosphorylation GLUT4 translocation to plasma membrane in to insulin was not inhibited by IRS-2 knockdown insulin-induced glucose uptake was inhibited by the reduction in IRS-2 protein to that observed for chronic GH pretreatment In this we have observed that chronic GH pretreatment not impair insulin-stimulated GLUT4 translocation to plasma membrane, results in a reduction in insulin-stimulated glucose uptake. Recently, several studies have also observed an of GLUT4 translocation and glucose uptake. in skeletal muscle insulin-stimulated glucose uptake GLUT4 translocation G. J. R. D. R. Klip A. Endocrinology. 2001; PubMed Scopus Google Scholar). B.A. Robinson M.G. Diabetes. 2000; PubMed Scopus Google in 3T3-L1 glucose impaired GLUT4 intrinsic activity. J.A. L. Biochem. Biophys. 1993; 300: PubMed Scopus Google reported that an of tyrosine inhibited insulin-stimulated glucose transport without affecting translocation of GLUT4 in adipocytes. a which to GLUT4 translocation to the plasma membrane, without increase in glucose uptake M. P. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). with these data indicate that GLUT4 translocation and fusion with the plasma membrane are not sufficient to glucose uptake but that activation are Thus, by glucose uptake with GLUT4 translocation, we have glucose uptake into GLUT4 translocation and activation we have reported that adipose and muscle tissue from human GH transgenic insulin resistance and that this resulted from an impairment of GLUT4 activation in (23Cho Y. Ariga M. Uchijima Y. Kimura K. Rho J.Y. Furuhata Y. Hakuno F. Yamanouchi K. Nishihara M. Takahashi S. Endocrinology. 2006; 147: 5374-5384Crossref PubMed Scopus (31) Google Scholar). the we the effect of GH pretreatment in cultured 3T3-L1 adipocytes. The data that chronic GH pretreatment impaired insulin-induced activation of PI 3-kinase bound to but not to to inhibition of insulin-induced glucose uptake without affecting GLUT4 IRS-2 overexpression or expression GH-induced impairment of insulin-dependent glucose uptake. In addition, IRS-2 knockdown showed phenotype to chronic GH pretreatment. data that chronic GH pretreatment and this reduction was the glucose uptake was impaired in with this IRS-1 knockdown not insulin-induced glucose that it is that PI 3-kinase with IRS-1 or IRS-2 roles in glucose uptake. Similarly, studies for IRS-1 and IRS-2 in cells Huang Klip A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have reported that IRS-1 and IRS-2 showed in cells T. Hakuno F. T. Takahashi S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google that IRS-1 and IRS-2 are not and result in of PI of insulin activation that chronic GH treatment impaired IRS-2-associated PI 3-kinase activity the of PI 3-kinase p85 regulatory subunit bound to IRS-2 was are several that account for the suppression of IRS-2-associated PI 3-kinase activity a increase in the of PI 3-kinase which to but not to in a inhibited PI 3-kinase It is well known that several including that have phosphatidylinositol activity the PI 3-kinase activity. be for GH-induced IRS-2-associated chronic GH treatment of IRS-2 and not IRS-1 to the of IRS-2 to PI Recently, we reported that impaired GLUT4 translocation to the plasma membrane F. S. R.T. Pessin J.E. Takahashi S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). that to the insulin through inhibition of IRS-2-associated PI 3-kinase activity. Surprisingly, chronic GH pretreatment or IRS-2 knockdown impaired insulin-induced activation of Akt but not GLUT4 translocation to plasma membrane. It is well established that insulin results in a activation of Akt that is in for GLUT4 translocation A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S.E. Kahn J. Clin. 1999; PubMed Scopus Google Scholar, S. Kahn J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). it is that a of Akt activation is sufficient for GLUT4 translocation, Akt activity is for activation of glucose uptake. In addition, it is also that of Akt activation is to substrate for GLUT4 we reported that IRS-1 and IRS-2 are and the of Akt with these by chronic GH treatment result in substrate This account for the of activated Akt to Akt substrates for GLUT4 data that chronic GH pretreatment insulin-stimulated Akt activation and substrate phosphorylation in the plasma membrane with Akt activation in the in which GLUT4 is K. F. and of plasma membrane Akt substrates, whose phosphorylation is by chronic GH is that the signaling pathways of and Akt studies be to these data that chronic GH pretreatment of adipocytes GLUT4 glucose transport activity without affecting translocation to the plasma membrane. In addition, chronic GH pretreatment inhibits IRS-2-associated PI 3-kinase to an impairment of GLUT4 transport activity on the plasma membrane. of for and the with Japan), and we H. of of for the in this