Electronic supplementary material (ESM) MATERIALS AND METHODS. Study subjects.

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1 Electronic supplementary material (ESM) MATERIALS AND METHODS Study subjects. Twelve obese patients with type 2 diabetes carefully matched to ten healthy, lean and ten obese, non-diabetic volunteers participated in the study (Table 1). Patients with type 2 diabetes were treated either by diet alone (n=1) or diet in combination with metformin (n=7), insulin (n=1), metformin and insulin (n=2), or rosiglitazone and sulfonylurea (n=1). Oral antidiabetics were withdrawn one week prior to the study together with antihypertensive and lipid lowering drugs. Long-acting insulin was withdrawn one day before and rapid acting insulin the night before the study. The patients with type 2 diabetes were GAD65 antibody negative and without signs of diabetic retinopathy, nephropathy, neuropathy or macrovascular complications. All women were postmenopausal. The lean and obese controls had normal glucose tolerance (by 120 min OGTT with 75 g glucose) and no family history of diabetes, and none were treated with drugs known to affect glucose metabolism. All participants were sedentary, and had normal results on screening blood tests of hepatic and renal function. Informed consent was obtained from all subjects before participation. The study was approved by the Local Ethics Committee and was performed in accordance with the Helsinki Declaration. Study design. The participants were instructed to refrain from strenuous physical activity for a period of 48-h before the experiment. After an overnight fast the lean and obese controls underwent a euglycaemic-hyperinsulinaemic clamp, whereas the patients with type 2 diabetes were clamped twice. The diabetic patients were randomized to start with either a euglycaemic or an isoglycaemic-hyperinsulinaemic clamp, which were separated by 4-6 weeks. They were instructed to eat the same meal on the evening before each study day. Three of twelve patients with type 2 diabetes could not participate in the second clamp, leaving paired data of nine patients. A 2-h basal tracer equilibration period was followed by infusion of insulin at a rate of 40 mu m -2 min -1 for 4 hours. A primed-constant 3-3 H-glucose infusion was used throughout the 6-h study, and 3-3 H-glucose was

2 added to the glucose infusates to maintain plasma specific activity constant at baseline levels during the 4-h clamp period as described [1-3]. In patients with type 2 diabetes, plasma glucose was allowed to decline to 5.5 mmol/l during the euglycaemic clamp before glucose infusion was initiated, whereas during the isoglycaemic clamp, glucose infusion was started simultaneously with insulin infusion in order to clamp plasma glucose at their prevailing levels of fasting hyperglycaemia. Total glucose disposal rates (GDR) was calculated using Steele s non-steady-state-equations adapted for labelled glucose infusates [4]. Distribution volume of glucose was taken as 200 ml/kg body weight and pool fraction as The studies were combined with indirect calorimetry using a TrueOne 2400 Metabolic Measurement System for continuous gas exchange measurements (ParvoMedics, Sandy, UT, USA) with the ventilated hood technique to determine the respiratory exchange ratio (RER) and rates of glucose and lipid oxidation during the 30-min basal and insulin-stimulated steady-state periods as described [5,6]. Protein oxidation rates were estimated from urinary urea nitrogen excretion and corrected for changes in pool size as described [7]. Rates of nonoxidative glucose metabolism (NOGM) were calculated as the difference between GDR and glucose oxidation. Fat mass and fat free mass (FFM) were determined by impedance (Tanita TBF-300 GS, Tanita Corporation, Illinois, USA). Plasma glucose and lactate and serum insulin, C-peptide, cholesterols, triglycerides and NEFA were measured as described previously [8]. Muscle biopsies, homogenate and lysate preparation. Muscle biopsies were obtained from the vastus lateralis muscle before and after the 4-h insulin infusion period using a modified Bergström needle with suction under local anaesthesia (lidocaine). Muscle samples were immediately blotted free of blood, fat and connective tissue and frozen in liquid nitrogen within s. Homogenates and lysates were prepared from 70 mg (w.w.) muscle that was freeze-dried, and dissected free of visible fat, blood and connective [9]. Total homogenate and lysate protein content was analysed by the bicinchoninic acid method (Pierce Chem. Comp. IL, USA). Antibodies The following primary antibodies were used for Western blotting in the present study: Anti-Akt2 (#2967), anti-akt2 (#3063), anti-pakt Ser473 (#9271), anti-pakt Thr308 (#9275), anti-pgsk-3 Ser21/Ser9 (#9331), and anti-pgsk-3 Ser21(#9316) (Cell Signaling Technology, Beverly, MA, USA). The insulin

3 receptor (IR) monoclonal CT3 antibody was raised against the COOH-terminal of the IRβ-subunit and was a gift from Dr. Ken Siddle (Cambridge University, Cambridge, UK). GS was detected as a single band at ~90 kda using a polyclonal GS antibody (kindly provided by Prof. Oluf Pedersen, Steno Diabetes Center, Denmark) [1,3,10]. For detection of GS site 3a+3b (Ser640 and Ser644) phosphorylation antibodies were raised against the peptide RYPRPA(Sx)VPP(Sx)PSLSR (residues of human GS), where Sx denote a phosphorylated or non-phosphorylated serine residue, as described [1]. The antibody toward site 2+2a (Ser7 and Ser10) was raised against the peptide PLSRSL(Sx)MS(Sx)LPGLED (residues 1-16 of rat GS) and the antibody toward site 1b (Ser710) was raised against the peptide CSGSKRNSpVDTATS ( residues of human GS as described [1,3]. The specificity of these antibodies has previously been investigated [1,3]. IRS-1 associated PI3-kinase activity IRS-1 associated PI3-kinase activity was measured on immunoprecipitates of IRS-1 from 300 µg of muscle lysate using an IRS-1 antibody raised against the COOHterminus of IRS-1 provided by Dr. K. Siddle (Cambridge University, UK). The kinase reaction ran for 15 min at 30 C with L-α-Phosphatidylinositol as substrate (Sigma-Aldrich, Broendby, Denmark) and [ - 33 P]- ATP as tracer (Perkin Elmer, Skovlunde, Denmark). After the reaction was terminated the organic phases were separated with methanol-chloroform and subjected to thin layer chromatography using TLC silica gel (Merck, Nottingham, UK). The TLC plates were analyzed for activity using a Storm 850 PhosphoImager (Molecular Dynamics). Isoform-specific Akt activity Akt2 activity was measured in immune-precipitates of Akt2 from 300 µg lysate protein using an anti-akt2 antibody (#AF23151, R&D Systems, Minneapolis, MN) and Akt1 activity was measured in 255 µg of the supernatant from the initial Akt2 immuno-precipitate. After an overnight IP at 4 C the IP was washed once in the IP-buffer (50 mm NaCl, 1% Triton X-100, 50 mm NaF, 5 mm Napyrophosphate, 20 mm Tris-base (ph 7.5), 500 μm PMSF, 2 mm DTT, 4 μg/ml Leupeptin, 50 μg/ml Soybean Trypsin inhibitor, 6 mm Benzamidine and 250 mm Sucrose), once in 480 mm Hepes (ph 7.0) and 240 mm NaCl, and twice in 240 mm Hepes (ph 7.0) and 120 mm NaCl. The kinase reaction ran for 30 min. at 30 C in a total of 30 µl containing 50 mm Tris-HCl (ph 7.4), 1 mm DTT, 5 mm MgCl 2, 90 µm substrate

4 peptid (#12-340, Millipore, Denmark), 200 µm ATP and 2.2 µci [ - 33 P]-ATP as tracer (Perkin Elmer, Skovlunde, Denmark). The reaction was stopped by addition of 10 µl 1% phosphoric acid and 20 µl was spotted onto P81 filter paper, which was washed and analyzed in a Storm 850 PhosphoImager (Molecular Dynamics, Struers Kebo lab, Denmark) and by using liquid scintillation (Tri-Carb 2000, Perkin Elmer, Denmark). Tyrosine phosphorylation of the insulin receptor The IR was immunoprecipitated from 300 µg lysate protein with 3 µg specific antibody (#sc-711, Santa Cruz Biotech., CA, USA) as described above for Akt immuneprecipitation. All of the immunoprecipitates were loaded onto a gel and blotted for tyrosine phosphorylation with the 4G10 antibody (#05-321, Millipore, Denmark). References 1. Højlund K, Staehr P, Hansen BF, Green KA, Hardie DG, Richter EA, Beck-Nielsen H, Wojtaszewski JF (2003) Increased phosphorylation of skeletal muscle glycogen synthase at NH2- terminal sites during physiological hyperinsulinemia in type 2 diabetes. Diabetes 52: Højlund K, Frystyk J, Levin K, Flyvbjerg A, Wojtaszewski JF, Beck-Nielsen H (2006) Reduced plasma adiponectin concentrations may contribute to impaired insulin activation of glycogen synthase in skeletal muscle of patients with type 2 diabetes. Diabetologia 49: Højlund K, Birk JB, Klein DK, Levin K, Rose AJ, Hansen BF, Nielsen JN, Beck-Nielsen H, Wojtaszewski JF (2009) Dysregulation of glycogen synthase COOH- and NH2-terminal phosphorylation by insulin in obesity and type 2 diabetes mellitus. J Clin Endocrinol Metab 94: Hother-Nielsen O, Henriksen JE, Holst JJ, Beck-Nielsen H (1996) Effects of insulin on glucose turnover rates in vivo: isotope dilution versus constant specific activity technique. Metabolism 45: Bassett DR Jr, Howley ET, Thompson DL, King GA, Strath SJ, McLaughlin JE, Parr BB (2001)Validity of inspiratory and expiratory methods of measuring gas exchange with a computerized system. J Appl Physiol 91:

5 6. Frayn KN (1983) Calculation of substrate oxidation rates in vivo from gaseous exchange. J Appl Physiol 55: Tappy L, Owen OE, Boden G (1988) Effect of hyperinsulinemia on urea pool size and substrate oxidation rates. Diabetes 37: Jørgensen GM, Vind B, Nybo M, Rasmussen LM, Højlund K (2009) Acute hyperinsulinemia decreases plasma osteoprotegerin with diminished effect in type 2 diabetes and obesity. Eur J Endocrinol 161: Birk JB, Wojtaszewski JF (2006) Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle. J Physiol 577: Glintborg D, Højlund K, Andersen NR, Hansen BF, Beck-Nielsen H, Wojtaszewski JF (2008) Impaired insulin activation and dephosphorylation of glycogen synthase in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment. J Clin Endocrinol Metab 93: