Growth Hormone Induces Cellular Insulin Resistance by Uncoupling Phosphatidylinositol 3-Kinase and Its Downstream Signals in 3T3-L1 Adipocytes

A. Takano, T. Haruta, M. Iwata, I. Usui, T. Uno, J. Kawahara, E. Ueno, T. Sasaoka, M. Kobayashi
2001 Diabetes  
Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin
more » ... rylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1-associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110 CAAX ) or Akt activation stimulated by plateletderived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1-associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1-associated PI 3-kinase. Materials. Porcine insulin and human GH were kindly provided by Eli Lilly (Indianapolis, IN) and Novo Nordisk (Bagsvaerd, Denmark), respectively. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and human recombinant platelet-derived growth factor (PDGF)-BB were purchased from Life Technologies/Gibco (Burlington, ON, Canada). 2-Deoxy-D-[ 3 H]glucose was purchased from New England Nuclear (Boston, MA). [␥-32 P] ATP and protein G-Sepharose were purchased from Amersham Pharmacia Biotech. Anti-IR-␤ subunit (C-19), anti-JAK2, anti-STAT5b, and horseradishperoxidase-linked anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rat carboxyl-terminal IRS-1 and anti-human IGF-1 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phosphotyrosine (PY20, PY20H) and anti-p85 antibodies were purchased from Transduction Laboratories (Lexington, KY). Anti-Akt, anti-phospho-Akt (Ser473), and anti-phospho-Akt (Thr308) antibodies were purchased from New England Biolabs (Beverly, MA). Cell culture. 3T3-L1 fibroblasts were obtained from the American Type Culture Collection (Rockville, MD) and maintained at 37°C in DMEM/high glucose supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 g/ml) in a 10% CO 2 environment. Cells were differentiated 2 days postconfluence by the addition of the same media supplemented with 1 mol/l insulin, 1 mol/l dexamethasone, and 500 mol/l isobutyl-methylxanthine for 3 days, and then the medium containing 0.8 mol/l insulin for another 3 days. The medium was then changed every 3 days until the cells were used for the experimentation (i.e., 13-16 days after the induction of differentiation), when Ͼ95% of the cells had the morphorogical and biochemical properties of adipocytes. Infection of recombinant adenovirus. The recombinant adenoviruses Ad5-p110 CAAX (containing bovine p110␣ cDNA with the CAAX motif at the COOH terminus) and Ad5-CT (which has no insert) were gifts from Dr. Olefsky (University of California, San Diego, CA). They were amplified in human embryonic kidney 293 cells, and viral stock solutions with viral titers of Ͼ10 8 plaque-forming units/ml were prepared. 3T3-L1 adipocytes were infected with the adenoviruses by incubating the cells at the indicated multiplicity of infection (MOI) of viral stock solution in DMEM containing 2% heat-inactivated FBS for 16 h. The media were changed to the regular culture media, and the cells were used for experiments after 48 h of infection (48). 2-Deoxyglucose uptake. 3T3-L1 adipocytes were washed with PBS and incubated with Krebs-Ringer phosphate (KRP)-HEPES buffer (10 mmol/l HEPES, pH 7.4, 131.2 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l MgSO 4 , 2.5 mmol/l CaCl 2 , and 2.5 mmol/l NaH 2 PO 4 ) containing 1% bovine serum albumin (BSA) with the indicated concentration of insulin for 15 min at 37°C. The adipocytes were then incubated with 2-deoxy-D-[ 3 H]glucose for 4 min, and the reaction was terminated by the addition of 10 mol/l cytochalasin B (Sigma, St Louis, MO). Cells were washed three times with ice-cold PBS. Radioactivity taken up by the cells was measured by a liquid scintillation counter. Subcellular fractionation. 3T3-L1 adipocytes were rinsed twice with PBS and once with HES buffer (255 mmol/l sucrose, 20 mmol/l HEPES, pH 7.4, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride [PMSF], 1 mmol/l Na 3 VO 4 , 2 g/ml aprotinin, and 50 nmol/l okadaic acid) (49) and immediately homogenized by 20 strokes with a motor-driven Teflon/glass homogenizer in HES buffer at 4°C. The homogenates (two 10-cm dishes per condition) were subjected to subcellular fractionation as previously described to isolate PM, high-density microsomes (HDMs), low-density microsomes (LDMs), and cytosol (49,50), with some modification. Briefly, the homogenate was centrifuged at 19,000g for 20 min. The resulting supernatant was centrifuged at 41,000g for 20 min, yielding a pellet of HDMs. The supernatant from this spin was centrifuged at 250,000g for 90 min, yielding a pellet of LDMs. The remaining supernatant was concentrated by Centricon-30 (Amicon, Beverly, MA) and used as cytosol. The pellet obtained from the initial spin was resuspended in HES buffer, layered onto a 1.12 mol/l sucrose cushion, and centrifuged at 100,000g in swing rotor for 60 min. A white fluffy band at the interface was collected, resuspended in HES buffer, and centrifuged at 40,000g for 20 min, yielding a pellet of PM. All fractions were adjusted to a final protein
doi:10.2337/diabetes.50.8.1891 pmid:11473053 fatcat:vyienz4nwjh6fm7cphn7wpkowe