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note
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Male mice were used. Blood was collected from the orbital sinus of anesthetized mice, and serum was prepared. Serum glucose and triglyceride concentrations were determined with a Vitros analyzer, and a radioimmunoassay (Linco, St. Charles, MO) was used to measure insulin concentrations.
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0344148843
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note
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Glucose tolerance was performed after an overnight fast by oral administration of glucose [1 g per kilogram of body weight (1 g/kg)]. Insulin tolerance tests were performed after an overnight fast by intraperitoneal injection of 0.75 U/kg of human insulin (1 U ∼ 40 μg) (Lilly). Blood was withdrawn from the tail, and glucose concentrations were determined with a One Touch Basic glucometer (Lifescan Canada Ltd., Burnaby, Canada).
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19
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After an overnight fast, mice were anesthetized, the abdominal cavity was exposed, and human insulin (5 U) (Lilly) or saline was injected as a bolus into the inferior vena cava [E. Araki et al., Nature 372, 186 (1994); P. L. Rothenberg et al., J. Biol. Chem. 266, 8302 (1991)]. One minute after injection, a small piece of liver was excised and immediately frozen in liquid nitrogen. This was followed at 2 and 3 min for quadriceps muscle and abdominal fat, respectively, and the same procedure was again repeated at 5, 6, and 7 min. Immunoprecipitation of the insulin receptor β subunit was done as follows: the tissue (liver, muscle, or fat) was homogenized on ice in 50 mM tris (pH 7.5); 150 mM NaCl; 1 mM pyrophosphate; 100 μM pervanadate (a potent PTP inhibitor) [G. Huyer et al., J. Biol. Chem. 272, 843 (1997)]; and a protease inhibitor cocktail tablet. Complete (Boehringer Mannheim). A membrane fraction was prepared by centrifugation at 100,000g for 1 hour, and the protein concentration was determined. Membrane protein (200 μg from liver or muscle. 100 μg from fat) was solubilized in immunoprecipitation buffer (RIPA) [150 mM NaCl, 10 mM phosphate buffer (pH 7.5), 1% NP-40, 1% Na deoxycholate, and 0.1% SDS], and immunoprecipitation of the insulin receptor β sub- unit was done overnight at 4°C with the use of antibody to the insulin receptor (1 μg) (C-19, Santa Cruz Biotechnology, Santa Cruz, CA) followed by a 60-min incubation with 50 μl of a 50% slurry of protein G-Sepharose (Pharmacia Biotech). The sample was washed three times in 1 ml of RIPA buffer, and samples were resolved by SDS-polyacrylamide gel electrophoresis (8% gel). The gel was then transferred onto a polyvinyl difluoride membrane, and the phosphotyrosine was detected with antibody to phosphotyrosine (4G10) coupled to horseradish peroxidase (Upstate Biotech, Waltham, MA) and developed with enhanced chemiluminescence (NEN). The same blot was washed in 62.5 mM tris (pH 6.7), 2% w/v SDS, and 100 mM β-mercaptoethanol for 30 min at 55°C rinsed; reprobed with the antibody to the insulin receptor β subunit (C-19, Santa Cruz Biotechnology), followed by a second antibody to rabbit immunoglobulin G coupled to horseradish peroxidase (Amersham); and detected by enhanced chemiluminescence (NEN). The phosphotyrosine and β-subunit signals from the x-ray films of the exposed blots were quantified by densitometry (Molecular Dynamics), and amounts of phosphotyrosine were normalized to the amount of β subunit present in each sample.
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Araki, E.1
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20
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0025837881
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After an overnight fast, mice were anesthetized, the abdominal cavity was exposed, and human insulin (5 U) (Lilly) or saline was injected as a bolus into the inferior vena cava [E. Araki et al., Nature 372, 186 (1994); P. L. Rothenberg et al., J. Biol. Chem. 266, 8302 (1991)]. One minute after injection, a small piece of liver was excised and immediately frozen in liquid nitrogen. This was followed at 2 and 3 min for quadriceps muscle and abdominal fat, respectively, and the same procedure was again repeated at 5, 6, and 7 min. Immunoprecipitation of the insulin receptor β subunit was done as follows: the tissue (liver, muscle, or fat) was homogenized on ice in 50 mM tris (pH 7.5); 150 mM NaCl; 1 mM pyrophosphate; 100 μM pervanadate (a potent PTP inhibitor) [G. Huyer et al., J. Biol. Chem. 272, 843 (1997)]; and a protease inhibitor cocktail tablet. Complete (Boehringer Mannheim). A membrane fraction was prepared by centrifugation at 100,000g for 1 hour, and the protein concentration was determined. Membrane protein (200 μg from liver or muscle. 100 μg from fat) was solubilized in immunoprecipitation buffer (RIPA) [150 mM NaCl, 10 mM phosphate buffer (pH 7.5), 1% NP-40, 1% Na deoxycholate, and 0.1% SDS], and immunoprecipitation of the insulin receptor β sub- unit was done overnight at 4°C with the use of antibody to the insulin receptor (1 μg) (C-19, Santa Cruz Biotechnology, Santa Cruz, CA) followed by a 60-min incubation with 50 μl of a 50% slurry of protein G-Sepharose (Pharmacia Biotech). The sample was washed three times in 1 ml of RIPA buffer, and samples were resolved by SDS-polyacrylamide gel electrophoresis (8% gel). The gel was then transferred onto a polyvinyl difluoride membrane, and the phosphotyrosine was detected with antibody to phosphotyrosine (4G10) coupled to horseradish peroxidase (Upstate Biotech, Waltham, MA) and developed with enhanced chemiluminescence (NEN). The same blot was washed in 62.5 mM tris (pH 6.7), 2% w/v SDS, and 100 mM β-mercaptoethanol for 30 min at 55°C rinsed; reprobed with the antibody to the insulin receptor β subunit (C-19, Santa Cruz Biotechnology), followed by a second antibody to rabbit immunoglobulin G coupled to horseradish peroxidase (Amersham); and detected by enhanced chemiluminescence (NEN). The phosphotyrosine and β-subunit signals from the x-ray films of the exposed blots were quantified by densitometry (Molecular Dynamics), and amounts of phosphotyrosine were normalized to the amount of β subunit present in each sample.
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Rothenberg, P.L.1
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21
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0031022433
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After an overnight fast, mice were anesthetized, the abdominal cavity was exposed, and human insulin (5 U) (Lilly) or saline was injected as a bolus into the inferior vena cava [E. Araki et al., Nature 372, 186 (1994); P. L. Rothenberg et al., J. Biol. Chem. 266, 8302 (1991)]. One minute after injection, a small piece of liver was excised and immediately frozen in liquid nitrogen. This was followed at 2 and 3 min for quadriceps muscle and abdominal fat, respectively, and the same procedure was again repeated at 5, 6, and 7 min. Immunoprecipitation of the insulin receptor β subunit was done as follows: the tissue (liver, muscle, or fat) was homogenized on ice in 50 mM tris (pH 7.5); 150 mM NaCl; 1 mM pyrophosphate; 100 μM pervanadate (a potent PTP inhibitor) [G. Huyer et al., J. Biol. Chem. 272, 843 (1997)]; and a protease inhibitor cocktail tablet. Complete (Boehringer Mannheim). A membrane fraction was prepared by centrifugation at 100,000g for 1 hour, and the protein concentration was determined. Membrane protein (200 μg from liver or muscle. 100 μg from fat) was solubilized in immunoprecipitation buffer (RIPA) [150 mM NaCl, 10 mM phosphate buffer (pH 7.5), 1% NP-40, 1% Na deoxycholate, and 0.1% SDS], and immunoprecipitation of the insulin receptor β sub- unit was done overnight at 4°C with the use of antibody to the insulin receptor (1 μg) (C-19, Santa Cruz Biotechnology, Santa Cruz, CA) followed by a 60-min incubation with 50 μl of a 50% slurry of protein G-Sepharose (Pharmacia Biotech). The sample was washed three times in 1 ml of RIPA buffer, and samples were resolved by SDS-polyacrylamide gel electrophoresis (8% gel). The gel was then transferred onto a polyvinyl difluoride membrane, and the phosphotyrosine was detected with antibody to phosphotyrosine (4G10) coupled to horseradish peroxidase (Upstate Biotech, Waltham, MA) and developed with enhanced chemiluminescence (NEN). The same blot was washed in 62.5 mM tris (pH 6.7), 2% w/v SDS, and 100 mM β-mercaptoethanol for 30 min at 55°C rinsed; reprobed with the antibody to the insulin receptor β subunit (C-19, Santa Cruz Biotechnology), followed by a second antibody to rabbit immunoglobulin G coupled to horseradish peroxidase (Amersham); and detected by enhanced chemiluminescence (NEN). The phosphotyrosine and β-subunit signals from the x-ray films of the exposed blots were quantified by densitometry (Molecular Dynamics), and amounts of phosphotyrosine were normalized to the amount of β subunit present in each sample.
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Huyer, G.1
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0344580065
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note
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2-terminus).
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24
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0344580062
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note
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-/- mice and their wild-type and heterozygous littermates at 7 to 8 weeks of age were fed a high-fat high-carbohydrate diet ad libidum (Diet F3282, Bioserve. NJ) and monitored for the following 10 weeks. Body weight was measured weekly, and food consumption was closely monitored. After 10 weeks, concentrations of glucose, insulin, and triglycerides in the serum of fasted animals were measured, and glucose and insulin tolerance tests were done.
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note
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We thank D. Moller, J. Mudgett, E. Asante-Appiah, and J. Evans for discussions. M.E. is a fellowship recipient and M.LT. is a Chercheur-Boursier Senior from the Fonds de la Recherche en Sante du Quebec. Supported in part by a grant from the Medical Research Council (MRC)/Pharmaceutical Manufacturers Association of Canada (M.L.T.) and the MRC (J.H.-H.).
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