Blood samples (60 l) were taken every 10 min from = 80 to 120 min and processed to determine glucose-specific activity. strains. Daily treatment of obese mice with rimonabant for 7 days resulted in significant and comparable reductions in body weight, serum leptin, free fatty acid, cholesterol, and triglyceride levels in the two strains. Rimonabant treatment improved glucose homeostasis and insulin sensitivity to the same extent in Adipo+/+ and Adipo?/? mice, whereas it reversed the HFD-induced hepatic steatosis, fibrosis, and hepatocellular damage only in the former. The adiponectin-dependent, antisteatotic effect of rimonabant was mediated by reduced uptake and increased -oxidation of fatty acids in the liver. We conclude that reversal of the HFD-induced hepatic steatosis and fibrosis by chronic CB1 blockade, but not the parallel reduction in adiposity and improved glycemic control, is usually mediated by adiponectin. = 4C5 mice/group). Representative images, offered in the figures at 10 magnification, were taken from the animal with the median value for each group. Glucose tolerance and insulin sensitivity assessments. Mice fasted overnight were injected with glucose (1.5 g/kg ip), followed by tail blood collection at 0, 15, 30, 45, 60, 90, and 120 min for determining blood glucose levels. On the following day, mice were fasted for 6 h before receiving insulin (0.75 U/kg ip; Eli Lilly), and blood glucose levels were decided at the same intervals as above. Hyperinsulinemic euglycemic clamp. Experiments were performed as explained previously (6) with modifications. Briefly, 5 days before the experiment, the left Aftin-4 common carotid artery and the right jugular vein of HFD-induced obese or slim control Adipo?/? and Adipo+/+ mice were catheterized under isofluorane anesthesia. Following a 14-h period of fasting, clamps were performed on unrestrained, conscious mice treated with rimonabant (10 mgkg?1day?1 ip) or vehicle for 7 days prior to the experiment. The clamp protocol consisted of a 120-min tracer equilibration period (from = ?120 to 0 min), followed by a 120-min clamp period (from = 0 to 120 min). A 5-Ci bolus of [3-3H]glucose (Perkin Elmer) was given at = ?120 min, followed by a 0.05 Ci/min infusion for 2 h at a pump rate of 0.1 l/min (CMA Microdialysis). The insulin clamp was begun at = 0 min with a priming bolus (64 mU/kg) of human insulin (Humulin R; Eli Lilly), followed by an infusion (3.6 mUkg?1min?1) delivered at a pump rate of 0.1 l/min from 0 to 120 min. The [3-3H]glucose infusion was increased to 0.1 Ci/min for the remainder of the experiment. Specific activity for individual time points did not vary by >15% from the average specific activity during the last 40 min of the clamp. Euglycemia (120C150 mg/dl) was maintained during clamps by measuring blood glucose every 10 min starting at = 0 min and infusing 40% dextrose as necessary. Blood samples (60 l) were taken every 10 min from = 80 to 120 min and processed to determine glucose-specific activity. Mice received saline-washed erythrocytes from donors throughout the experimental period (4 l/min) to prevent a fall of hematocrit by >5%. To estimate insulin-stimulated glucose fluxes in tissues, 2-deoxy-d-[1-14C]glucose (Perkin Elmer) was bolus administered Aftin-4 (10 Ci) at = 85 min, i.e., 45 min before the end of the experiment. At the end of the clamp, animals were anesthetized with intravenous injection of pentobarbital sodium. Within 5 min, gastrocnemius muscle from hindlimbs and liver and epididymal and subcutaneous fat were removed and frozen until analysis. To determine [3-3H]glucose flux, plasma samples were deproteinized using barium hydroxide and zinc sulfate. The glucose production and disappearance rates were determined using Steele’s non-steady-state equations (61). Clamp hepatic endogenous glucose production rate was determined by subtracting the glucose infusion rate (GIR) from total glucose turnover (Rd). The glucose uptake by tissues and glycogen synthesis rates were calculated as described previously (81). Cell culture. Human hepatoma HepG2 cells, purchased from the American Type Culture Collection, were plated in six-well plates at a density of 5 105 cells/ml and grown in Eagle’s Modified Essential Medium (EMEM) with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G sodium, and 100 g/ml streptomycin sulfate in a humidified atmosphere of 5% CO2 at 37C. Medium was changed every 2C3 days until cells reached 80C90% confluence. Prior to adiponectin treatment, cells were starved in serum-free EMEM for 24 h. Fatty acid uptake. Palmitate uptake was initiated in HepG2 cells preincubated for 24 h with vehicle or adiponectin. Medium was replaced with an incubation medium containing serum-free EMEM supplemented with palmitic acid (final concentration: 500 M) bound to fatty acid-free bovine serum albumin (BSA) at a 6:1 molar ratio and a trace amount (0.2 Ci) of radiolabeled [14C]palmitate (Perkin Elmer) at a specific activity of 2,220 MBq/mmol. Palmitate uptake was terminated after 1 h by removing the medium and washing twice with ice-cold PBS containing 0.5 mM.Steele R, Wall JS, De Bodo RC, Altszuler N. Measurement of size and turnover rate of body glucose pool by the isotope dilution method. strains. Daily treatment of obese mice with rimonabant for 7 days resulted in significant and comparable reductions in body weight, serum leptin, free fatty acid, cholesterol, and triglyceride levels in the two strains. Rimonabant treatment improved glucose homeostasis and insulin sensitivity to the same extent in Adipo+/+ and Adipo?/? mice, whereas it reversed the HFD-induced hepatic steatosis, fibrosis, and hepatocellular damage only in the former. The adiponectin-dependent, antisteatotic effect of rimonabant was mediated by reduced uptake and increased -oxidation of fatty acids in the liver. We conclude that reversal of the HFD-induced hepatic steatosis and fibrosis by chronic CB1 blockade, but not the parallel reduction in adiposity and improved glycemic control, is mediated by adiponectin. = 4C5 mice/group). Representative images, presented in the figures at 10 magnification, were taken from the animal with the median value for each group. Glucose tolerance and insulin sensitivity tests. Mice fasted overnight were injected with glucose (1.5 g/kg ip), followed by tail blood collection at 0, 15, 30, 45, 60, 90, and 120 min for determining blood glucose levels. On the following day, mice were fasted for 6 h before receiving insulin (0.75 U/kg ip; Eli Lilly), and blood glucose levels were determined at the same intervals as above. Hyperinsulinemic euglycemic clamp. Experiments were performed as described previously (6) with modifications. Briefly, 5 days before the experiment, the left common carotid artery and the right jugular vein of HFD-induced obese or lean control Adipo?/? and Adipo+/+ mice were catheterized under isofluorane anesthesia. Following a 14-h period of fasting, clamps were performed on unrestrained, conscious mice treated with rimonabant (10 mgkg?1day?1 ip) or vehicle for 7 days prior to the experiment. The clamp protocol consisted of a 120-min tracer equilibration period (from = ?120 to 0 min), followed by a 120-min clamp period (from = 0 to 120 min). A 5-Ci bolus of [3-3H]glucose (Perkin Elmer) was given at = ?120 min, followed by a 0.05 Ci/min infusion for 2 h at a pump rate of 0.1 l/min (CMA Microdialysis). The insulin clamp was begun at = 0 min with a priming bolus (64 mU/kg) of human insulin (Humulin R; Eli Lilly), followed by an infusion (3.6 mUkg?1min?1) delivered at a pump rate of 0.1 l/min from 0 to 120 min. The [3-3H]glucose infusion was increased to 0.1 Ci/min for the remainder of the experiment. Specific activity for individual time points did not vary by >15% from the average specific activity during the last 40 min of the clamp. Euglycemia (120C150 mg/dl) was taken care of during clamps by measuring blood glucose every 10 min starting at = 0 min and infusing 40% dextrose as necessary. Blood samples (60 l) were taken every 10 min from = 80 to 120 min and processed to determine glucose-specific activity. Mice received saline-washed erythrocytes from donors throughout the experimental period (4 l/min) to prevent a fall of hematocrit by >5%. To estimate insulin-stimulated glucose fluxes in cells, 2-deoxy-d-[1-14C]glucose (Perkin Elmer) was bolus given (10 Ci) at = 85 min, i.e., 45 min before the end of the experiment. At the end of the clamp, animals were anesthetized with intravenous injection of pentobarbital sodium. Within 5 min, gastrocnemius muscle mass from hindlimbs and liver and epididymal and subcutaneous extra fat were removed and freezing until analysis. To determine [3-3H]glucose flux, plasma samples were deproteinized using barium hydroxide and zinc sulfate. The glucose production and disappearance rates were identified using Steele’s non-steady-state equations (61). Clamp hepatic endogenous glucose production rate was determined by subtracting the glucose infusion rate (GIR) from total glucose turnover (Rd). The glucose uptake by cells and glycogen synthesis rates were calculated as explained previously (81). Cell tradition. Human being hepatoma HepG2 cells, purchased from your American Type Tradition Collection, were plated in six-well plates at a denseness of 5 105 OI4 cells/ml and cultivated in Eagle’s.Int J Clin Pract 65: 713C715, 2011 [PubMed] [Google Scholar] 74. fatty acids in the liver. We conclude that reversal of the HFD-induced hepatic steatosis and fibrosis by chronic CB1 blockade, but not the parallel reduction in adiposity and Aftin-4 improved glycemic control, is definitely mediated by adiponectin. = 4C5 mice/group). Representative images, offered in the numbers at 10 magnification, were taken from the animal with the median value for each group. Glucose tolerance and insulin level of sensitivity checks. Mice fasted over night were injected with glucose (1.5 g/kg ip), followed by tail blood collection at 0, 15, 30, 45, 60, 90, and 120 min for determining blood glucose levels. On the following day, mice were fasted for 6 h before receiving insulin (0.75 U/kg ip; Eli Lilly), and blood glucose levels were identified at the same intervals as above. Hyperinsulinemic euglycemic clamp. Experiments were performed as explained previously (6) with modifications. Briefly, 5 days before the experiment, the remaining common carotid artery and the right jugular vein of HFD-induced obese or slim control Adipo?/? and Adipo+/+ mice were catheterized under isofluorane Aftin-4 anesthesia. Following a 14-h period of fasting, clamps were performed on unrestrained, conscious mice treated with rimonabant (10 mgkg?1day?1 ip) or vehicle for 7 days prior to the experiment. The clamp protocol consisted of a 120-min tracer equilibration period (from = ?120 to 0 min), followed by a 120-min clamp period (from = 0 to 120 min). A 5-Ci bolus of [3-3H]glucose (Perkin Elmer) was given at = ?120 min, followed by a 0.05 Ci/min infusion for 2 h at a pump rate of 0.1 l/min (CMA Microdialysis). The insulin clamp was begun at = 0 min having a priming bolus (64 mU/kg) of human being insulin (Humulin R; Eli Lilly), followed by an infusion (3.6 mUkg?1min?1) delivered at a pump rate of 0.1 l/min from 0 to 120 min. The [3-3H]glucose infusion was increased to 0.1 Ci/min for the remainder of the experiment. Specific activity for individual time points did not vary by >15% from the average specific activity during the last 40 min of the clamp. Euglycemia (120C150 mg/dl) was taken care of during clamps by measuring blood glucose every 10 min starting at = 0 min and infusing 40% dextrose as necessary. Blood samples (60 l) were taken every 10 min from = 80 to 120 min and processed to determine glucose-specific activity. Mice received saline-washed erythrocytes from donors throughout the experimental period (4 l/min) to prevent a fall of hematocrit by >5%. To estimate insulin-stimulated glucose fluxes in cells, 2-deoxy-d-[1-14C]glucose (Perkin Elmer) was bolus given (10 Ci) at = 85 min, i.e., 45 min before the end of the experiment. At the end of the clamp, animals were anesthetized with intravenous injection of pentobarbital sodium. Within 5 min, gastrocnemius muscle mass from hindlimbs and liver and epididymal and subcutaneous extra fat were removed and freezing until analysis. To determine [3-3H]glucose flux, plasma samples were deproteinized using barium hydroxide and zinc sulfate. The glucose production and disappearance rates were identified using Steele’s non-steady-state equations (61). Clamp hepatic endogenous glucose production rate was determined by subtracting the glucose infusion rate (GIR) from total glucose turnover.Gary-Bobo M, Elachouri G, Gallas JF, Janiak P, Marini P, Ravinet-Trillou C, Chabbert M, Cruccioli N, Pfersdorff C, Roque C, Arnone M, Croci T, Soubrie P, Oury-Donat F, Maffrand JP, Scatton B, Lacheretz F, Le Fur G, Herbert JM, Bensaid M. Rimonabant reduces obesity-associated hepatic steatosis and features of metabolic syndrome in obese Zucker fa/fa rats. by reduced uptake and improved -oxidation of fatty acids in the liver. We conclude that reversal of the HFD-induced hepatic steatosis and fibrosis by chronic CB1 blockade, but not the parallel reduction in adiposity and improved glycemic control, is definitely mediated by adiponectin. = 4C5 mice/group). Representative images, offered in the numbers at 10 magnification, were taken from the animal with the median value for each group. Glucose tolerance and insulin level of sensitivity checks. Mice fasted over night were injected with glucose (1.5 g/kg ip), followed by tail blood collection at 0, 15, 30, 45, 60, 90, and 120 min for determining blood glucose levels. On the following day, mice were fasted for 6 h before receiving insulin (0.75 U/kg ip; Eli Lilly), and blood glucose levels were identified at the same intervals as above. Hyperinsulinemic euglycemic clamp. Experiments were performed as explained previously (6) with modifications. Briefly, 5 days before the experiment, the remaining common carotid artery and the right jugular vein of HFD-induced obese Aftin-4 or slim control Adipo?/? and Adipo+/+ mice were catheterized under isofluorane anesthesia. Following a 14-h period of fasting, clamps were performed on unrestrained, conscious mice treated with rimonabant (10 mgkg?1day?1 ip) or vehicle for 7 days prior to the experiment. The clamp protocol consisted of a 120-min tracer equilibration period (from = ?120 to 0 min), followed by a 120-min clamp period (from = 0 to 120 min). A 5-Ci bolus of [3-3H]glucose (Perkin Elmer) was given at = ?120 min, followed by a 0.05 Ci/min infusion for 2 h at a pump rate of 0.1 l/min (CMA Microdialysis). The insulin clamp was begun at = 0 min having a priming bolus (64 mU/kg) of human being insulin (Humulin R; Eli Lilly), followed by an infusion (3.6 mUkg?1min?1) delivered at a pump rate of 0.1 l/min from 0 to 120 min. The [3-3H]glucose infusion was increased to 0.1 Ci/min for the remainder of the experiment. Specific activity for individual time points did not vary by >15% from the average specific activity during the last 40 min of the clamp. Euglycemia (120C150 mg/dl) was taken care of during clamps by measuring blood glucose every 10 min starting at = 0 min and infusing 40% dextrose as necessary. Blood samples (60 l) were taken every 10 min from = 80 to 120 min and processed to determine glucose-specific activity. Mice received saline-washed erythrocytes from donors throughout the experimental period (4 l/min) to prevent a fall of hematocrit by >5%. To estimate insulin-stimulated glucose fluxes in cells, 2-deoxy-d-[1-14C]glucose (Perkin Elmer) was bolus given (10 Ci) at = 85 min, i.e., 45 min before the end of the experiment. At the end of the clamp, animals were anesthetized with intravenous injection of pentobarbital sodium. Within 5 min, gastrocnemius muscle mass from hindlimbs and liver and epididymal and subcutaneous extra fat were removed and freezing until analysis. To determine [3-3H]glucose flux, plasma samples were deproteinized using barium hydroxide and zinc sulfate. The glucose production and disappearance rates were identified using Steele’s non-steady-state equations (61). Clamp hepatic endogenous glucose production rate was determined by subtracting the glucose infusion rate (GIR) from total glucose turnover (Rd). The glucose uptake by cells and glycogen synthesis rates were calculated as explained previously (81). Cell tradition. Human being hepatoma HepG2 cells, purchased from your American Type Tradition Collection, were plated in six-well plates at a denseness of 5 105 cells/ml and cultivated in Eagle’s Modified Essential Medium (EMEM) with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G sodium, and 100 g/ml streptomycin sulfate inside a humidified atmosphere of 5% CO2 at 37C. Medium was changed every 2C3 days until cells reached 80C90% confluence. Prior to adiponectin treatment, cells were starved in serum-free EMEM for 24 h. Fatty acid uptake. Palmitate uptake was initiated in HepG2 cells preincubated for 24 h with vehicle or adiponectin. Medium was replaced with an incubation medium made up of serum-free EMEM supplemented with palmitic acid (final concentration: 500 M).It is also possible that an adiponectin-dependent component in the insulin-sensitizing effect of CB1 blockade emerges only following more prolonged treatment, such as that used in the earlier study. HFD-induced obesity and its hormonal/metabolic consequences were indistinguishable in the two strains. Daily treatment of obese mice with rimonabant for 7 days resulted in significant and comparable reductions in body weight, serum leptin, free fatty acid, cholesterol, and triglyceride levels in the two strains. Rimonabant treatment improved glucose homeostasis and insulin sensitivity to the same extent in Adipo+/+ and Adipo?/? mice, whereas it reversed the HFD-induced hepatic steatosis, fibrosis, and hepatocellular damage only in the former. The adiponectin-dependent, antisteatotic effect of rimonabant was mediated by reduced uptake and increased -oxidation of fatty acids in the liver. We conclude that reversal of the HFD-induced hepatic steatosis and fibrosis by chronic CB1 blockade, but not the parallel reduction in adiposity and improved glycemic control, is usually mediated by adiponectin. = 4C5 mice/group). Representative images, offered in the figures at 10 magnification, were taken from the animal with the median value for each group. Glucose tolerance and insulin sensitivity assessments. Mice fasted overnight were injected with glucose (1.5 g/kg ip), followed by tail blood collection at 0, 15, 30, 45, 60, 90, and 120 min for determining blood glucose levels. On the following day, mice were fasted for 6 h before receiving insulin (0.75 U/kg ip; Eli Lilly), and blood glucose levels were decided at the same intervals as above. Hyperinsulinemic euglycemic clamp. Experiments were performed as explained previously (6) with modifications. Briefly, 5 days before the experiment, the left common carotid artery and the right jugular vein of HFD-induced obese or slim control Adipo?/? and Adipo+/+ mice were catheterized under isofluorane anesthesia. Following a 14-h period of fasting, clamps were performed on unrestrained, conscious mice treated with rimonabant (10 mgkg?1day?1 ip) or vehicle for 7 days prior to the experiment. The clamp protocol consisted of a 120-min tracer equilibration period (from = ?120 to 0 min), followed by a 120-min clamp period (from = 0 to 120 min). A 5-Ci bolus of [3-3H]glucose (Perkin Elmer) was given at = ?120 min, followed by a 0.05 Ci/min infusion for 2 h at a pump rate of 0.1 l/min (CMA Microdialysis). The insulin clamp was begun at = 0 min with a priming bolus (64 mU/kg) of human insulin (Humulin R; Eli Lilly), followed by an infusion (3.6 mUkg?1min?1) delivered at a pump rate of 0.1 l/min from 0 to 120 min. The [3-3H]glucose infusion was increased to 0.1 Ci/min for the remainder of the experiment. Specific activity for individual time points did not vary by >15% from the average specific activity during the last 40 min of the clamp. Euglycemia (120C150 mg/dl) was maintained during clamps by measuring blood glucose every 10 min starting at = 0 min and infusing 40% dextrose as necessary. Blood samples (60 l) were taken every 10 min from = 80 to 120 min and processed to determine glucose-specific activity. Mice received saline-washed erythrocytes from donors throughout the experimental period (4 l/min) to prevent a fall of hematocrit by >5%. To estimate insulin-stimulated glucose fluxes in tissues, 2-deoxy-d-[1-14C]glucose (Perkin Elmer) was bolus administered (10 Ci) at = 85 min, i.e., 45 min before the end of the experiment. At the end of the clamp, animals were anesthetized with intravenous injection of pentobarbital sodium. Within 5 min, gastrocnemius muscle mass from hindlimbs and liver and epididymal and subcutaneous excess fat were removed and frozen until analysis. To determine [3-3H]glucose flux, plasma samples were deproteinized using barium hydroxide and zinc sulfate. The glucose production and disappearance rates were decided using Steele’s non-steady-state equations (61). Clamp hepatic endogenous glucose production rate was determined by subtracting the glucose infusion rate (GIR) from total glucose turnover (Rd). The glucose uptake by tissues and glycogen synthesis rates were calculated as explained previously (81). Cell culture. Human hepatoma HepG2 cells, purchased from your American Type Culture Collection, were plated in six-well plates at a density of 5 105 cells/ml and produced in Eagle’s Modified Essential Medium (EMEM) with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G sodium, and 100 g/ml streptomycin sulfate in a humidified atmosphere of 5% CO2 at 37C. Medium was transformed every 2C3 times until cells reached 80C90% confluence. Ahead of adiponectin treatment, cells had been starved in serum-free EMEM for 24 h. Fatty acidity uptake. Palmitate uptake was initiated in HepG2 cells preincubated for 24 h with automobile or adiponectin. Moderate was changed with an.