Oup, ten minutes prior to the finish of the in vivo studies, [U- 14C]-lactate (5- i bolus, 0.4 i/min; New England Nuclear) was administered to determine the contribution of gluconeogenesis for the hepatic glucose-6-phosphate pool. Consecutive samples were pooled together for the assessment on the plasma insulin and resistin levels. Steady state circumstances for each plasma glucose concentration and distinct activity had been accomplished by 40 minutes in these studies. At the finish on the in vivo research, mice were anesthetized (pentobarbital 60 mg/kg i.v.), the abdomen was immediately opened and adipose tissue and liver had been freeze-clamped in situ with aluminum tongs that have been cooled in liquid nitrogen. The time in between the injection of anesthesia as well as the freeze clamping of tissue samples was significantly less than 60 seconds. Tissue samples were stored at 0 for additional analysis. Analytical procedures. Liver triglycerides were measured as described. Plasma glucose was measured by the glucose oxidase technique on a Glucose Analyzer II (Beckman Instruments Inc., Fullerton, California, USA). Under steady-state circumstances for plasma glucose concentration, the glucose Rd equals the rate of glucose look (Ra). Ra was determined from the ratio with the infusion rate for [3H-3]-glucose (disintegrations per minute) plus the particular activity of plasma [3H-3]-glucose (disintegrations per minute per milligram glucose) under steady-state circumstances. The rate of GP was, thus, obtained from the distinction among Ra as well as the rate of glucose infusion. The hepatic [14C]-PEP and [3H]/[14C]-UDP-glucose pecific activities had been measured by HPLC, as well as the rates of PEP-gluconeogenesis (GNG) had been calculated. The percentage with the hepatic glucose-6-phos1. Kahn, B.B., and Flier, J.S. 2000. Obesity and insulin resistance. J. Clin. Filovirus site Invest. 106:47381. 2. Kopelman, P.G., and Hitman, G.A. 1998. Diabetes. Exploding kind II [review]. Lancet. 352(Suppl. 4): SIV5. three. Porte, D., Jr., et al. 1998. Obesity, diabetes as well as the central nervous technique. Diabetologia. 41:86381. 4. Flegal, K.M., Carroll, M.D., Ogden, C.L., and Johnson, C.L. 2002. Prevalence and trends in obesity among US adults, 1999-2000. JAMA. 288:1723727. five. Ogden, C.L., Flegal, K.M., Carroll, M.D., and Johnson, C.L. 2002. Prevalence and trends in overweight among US children and adolescents, 1999-2000. JAMA. 288:1728732.phate pool directly derived from plasma glucose (direct pathway) was Thyroid Hormone Receptor Accession calculated because the ratio of liver [3H]-UDP-glucose and plasma [3H-3]-glucose pecific activities. Gluconeogenesis was estimated from the particular activities of [14C]-labeled hepatic UDP-glucose (assumed to reflect the particular activity of hepatic glucose-6phosphate) and hepatic PEP soon after the infusion of [U-14C]-lactate and [3H-3]-glucose by application on the following formula: GNG = TGO [14C]-UDP-glucose SA/[14C]-PEP SA two, where SA is definitely the precise activity, and TGO is definitely the total glucose output. Western blot analyses. Liver tissues were homogenized in 20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 40 mM -glycerophosphate, ten mM sodium pyrophosphate, two mM orthovanadate, 0.5 NP-40, Total phosphatase inhibitor cocktail (Roche). Protein concentration was measured by use of a BCA protein quantification kit (Pierce, Rockford, Illinois, USA). Extracts were fractionated by use of either a four to 12 BisTris NuPAGE (Invitrogen) or Criterion XT (Bio-Rad Laboratories Inc.) gel and were blotted as described (39). Major antibodies against the phosphorylated.
