Rom distal to proximal areas of the quadriceps. The biopsy was

Rom distal to proximal areas of the quadriceps. The biopsy was snap-frozen in liquid nitrogen and stored at 280uC until further analysis. Insulin was infused at 40 mU. min21.m22 body surface area followed by a variable infusion of 20 glucose to maintain plasma glucose concentration at 5.2 mmol/l. A second muscle biopsy was taken through a separate incision after one hour of the clamp. The clamp was maintained for a further hour to assess insulin sensitivity (M-value, see below).Analytical MethodsPlasma. Plasma was separated from whole blood by centrifugation (300 g) immediately after collection. Plasma glucose was Microcystin-LR supplier measured with a YSI Stat2300 (Yellow Spring Instruments, Yellow Spring, OH) immediately after collection of each sample; the remainder of the plasma sample was frozen until further analysis. Plasma insulin was measured by the Clinical Biochemistry Department at Ninewells Hospital, Dundee, using a Siemens Immulite 2000 Immunoassay system. Preparation of Protein Extracts for Western Blotting or Immunoprecipitation. Protein extracts were obtained asMaterials and Methods Ethics StatementThe subjects were informed of the experimental protocol both verbally and in writing before giving their informed consent. The experimental protocol was approved by the Tayside Ethics Committee and was carried out according to the Helsinki Declaration.Participant characteristicsTwenty two healthy men, 2968y (SEM) participated in the study. None were taking regular medication. Height (metres) and weight (kilograms) were measured to determine the body mass index (BMI). The cohort was recruited to include four groups of individuals: seven of normal weight (BMI, 20?4 kg/m2), five overweight (BMI 25?9) and ten obese (BMI 30 kg/m2). The subjects were not habitually active. They were instructed to adhere to their usual diet and to refrain from any strenuous physical activity for 2 days before the study.Study protocolAll subjects had a screening visit to assess family history of diabetes and to confirm normal glucose tolerance with a standard (75 g of powdered glucose) 2-hour oral glucose tolerance test. Those individuals with a first-degree relative known to have diabetes or with diabetes according to the World Health Organisation diagnostic criteria (fasting glucose 7.0 mmol/l or 2 h glucose 11.1 mmol/l) were excluded from the study. Body composition (fat mass, fat free mass and bone mineral content) was determined by dual energy photon X-ray absorptiometry (DEXA; HOLOGIC Discovery W, Bedford, MA, version 12.1). Eligible subjects attended the Clinical Investigation Unit, Ninewells Hospital at 08:00 having not eaten for 12 hours, for a study protocol 18204824 involving skeletal muscle biopsies and a hyperinsulinaemic, euglycaemic clamp [15]. A cannula was introduced into a forearm vein for infusion of insulin (Actrapid, NovoNordisk Copenhagen, Denmark) and 20 dextrose while on the contradetailed purchase 80-49-9 previously[11]. In brief, muscle biopsies were thawed in ice and homogenized (Dounce, 10?5 strokes) in 0.5 ml ice-cold lysis buffer (25 mM Tris-HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 1 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1 (v/v) Triton X-100, 10 mM sodium pyrophosphate, 0.27 M sucrose, Complete Protease inhibitor cocktail tablets (1 tablet/ 10 ml), and 0.1 (v/v) 2-mercaptoethanol). Protein lysates were obtained from the supernatant fraction after 10 min centrifugation at 13,000 r.p.m., and then pre-cleared for 1 h at 4uC with Protein G-Sepharose in PBS 50 (v/v).Rom distal to proximal areas of the quadriceps. The biopsy was snap-frozen in liquid nitrogen and stored at 280uC until further analysis. Insulin was infused at 40 mU. min21.m22 body surface area followed by a variable infusion of 20 glucose to maintain plasma glucose concentration at 5.2 mmol/l. A second muscle biopsy was taken through a separate incision after one hour of the clamp. The clamp was maintained for a further hour to assess insulin sensitivity (M-value, see below).Analytical MethodsPlasma. Plasma was separated from whole blood by centrifugation (300 g) immediately after collection. Plasma glucose was measured with a YSI Stat2300 (Yellow Spring Instruments, Yellow Spring, OH) immediately after collection of each sample; the remainder of the plasma sample was frozen until further analysis. Plasma insulin was measured by the Clinical Biochemistry Department at Ninewells Hospital, Dundee, using a Siemens Immulite 2000 Immunoassay system. Preparation of Protein Extracts for Western Blotting or Immunoprecipitation. Protein extracts were obtained asMaterials and Methods Ethics StatementThe subjects were informed of the experimental protocol both verbally and in writing before giving their informed consent. The experimental protocol was approved by the Tayside Ethics Committee and was carried out according to the Helsinki Declaration.Participant characteristicsTwenty two healthy men, 2968y (SEM) participated in the study. None were taking regular medication. Height (metres) and weight (kilograms) were measured to determine the body mass index (BMI). The cohort was recruited to include four groups of individuals: seven of normal weight (BMI, 20?4 kg/m2), five overweight (BMI 25?9) and ten obese (BMI 30 kg/m2). The subjects were not habitually active. They were instructed to adhere to their usual diet and to refrain from any strenuous physical activity for 2 days before the study.Study protocolAll subjects had a screening visit to assess family history of diabetes and to confirm normal glucose tolerance with a standard (75 g of powdered glucose) 2-hour oral glucose tolerance test. Those individuals with a first-degree relative known to have diabetes or with diabetes according to the World Health Organisation diagnostic criteria (fasting glucose 7.0 mmol/l or 2 h glucose 11.1 mmol/l) were excluded from the study. Body composition (fat mass, fat free mass and bone mineral content) was determined by dual energy photon X-ray absorptiometry (DEXA; HOLOGIC Discovery W, Bedford, MA, version 12.1). Eligible subjects attended the Clinical Investigation Unit, Ninewells Hospital at 08:00 having not eaten for 12 hours, for a study protocol 18204824 involving skeletal muscle biopsies and a hyperinsulinaemic, euglycaemic clamp [15]. A cannula was introduced into a forearm vein for infusion of insulin (Actrapid, NovoNordisk Copenhagen, Denmark) and 20 dextrose while on the contradetailed previously[11]. In brief, muscle biopsies were thawed in ice and homogenized (Dounce, 10?5 strokes) in 0.5 ml ice-cold lysis buffer (25 mM Tris-HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 1 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1 (v/v) Triton X-100, 10 mM sodium pyrophosphate, 0.27 M sucrose, Complete Protease inhibitor cocktail tablets (1 tablet/ 10 ml), and 0.1 (v/v) 2-mercaptoethanol). Protein lysates were obtained from the supernatant fraction after 10 min centrifugation at 13,000 r.p.m., and then pre-cleared for 1 h at 4uC with Protein G-Sepharose in PBS 50 (v/v).