Volume 6, Issue 1, Pages 87-97 (July 2000) Loss of Insulin Signaling in Hepatocytes Leads to Severe Insulin Resistance and Progressive Hepatic Dysfunction  M.Dodson Michael, Rohit N. Kulkarni, Catherine Postic, Steven F. Previs, Gerald I. Shulman, Mark A. Magnuson, C.Ronald Kahn  Molecular Cell  Volume 6, Issue 1, Pages 87-97 (July 2000) DOI: 10.1016/S1097-2765(05)00015-8

Figure 1 Insulin Receptor Content and Insulin-Stimulated Signaling Are Selectively Abolished in LIRKO Liver (a) Protein extracts were prepared from tissues of 24-day-old wild-type (WT), IR(lox/lox), alb-Cre, and LIRKO mice and subjected to immunoprecipitation (WAT, 1.5 mg; other tissues, 5 mg) with an IR-specific antiserum followed by Western blot analysis with the same antiserum. Signal intensity was quantified using a Storm 840 Phosphorimager (Molecular Dynamics) with ImageQuant 4.0 software. (b) Twenty-four-day-old WT, IR(lox/lox), alb-Cre, and LIRKO mice were stimulated with either saline (−) or 5 U of regular insulin (+) via the inferior vena cava. Liver protein extracts were prepared and subjected to immunoprecipitation of IR (upper panels), IRS-1 (middle panels), and IRS-2 (lower panels) followed by Western blotting with anti-phosphotyrosine antibody (PY), IR antiserum, IRS-1 antiserum, IRS-2 antiserum, or an antiserum to the p85 regulatory subunit of PI 3′-kinase. (c) The experiment was performed as described in (b) with the exception that skeletal muscle was used to prepare the protein lysates. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)

Figure 2 LIRKO Mice Display Hyperglycemia and Hyperinsulinemia, Glucose Intolerance, and Insulin Resistance (a) Fed blood glucose and serum insulin concentrations were determined from 2-month-old male WT, IR(lox/lox), alb-Cre, and LIRKO mice at 2200–2300 hr (light cycle: 0700 on–1900 off). Each bar represents the mean ± SEM of at least 12 animals of each genotype. (b) Glucose tolerance tests were performed on 2-month-old male WT, IR(lox/lox), alb-Cre, and LIRKO mice that had been fasted for 16 hr. Animals were injected intraperitoneally with 2 g/kg body weight of glucose. Blood glucose was measured immediately before injection and 15, 30, 60, and 120 min after the injection. Results are expressed as mean blood glucose concentration ± SEM from at least eight animals per genotype. (c) Insulin tolerance tests were performed on random-fed, 2-month-old male WT, IR(lox/lox), alb-Cre, and LIRKO mice (performed at 1400 hr). Animals were injected intraperitoneally with 1 U/kg body weight of human regular insulin. Blood glucose was measured immediately before injection and 15, 30, and 60 min after the injection. Results are expressed as mean percent of basal blood glucose concentration ± SEM from at least eight animals per genotype. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)

Figure 3 Hyperinsulinemia in LIRKO Mice Is Likely Due to Both Increased Insulin Secretion and Decreased Insulin Clearance (a) Pancreatic insulin content was measured in acid-ethanol extracts of pancreas from 6-month-old female WT, IR(lox/lox), alb-Cre, and LIRKO mice by RIA (Linco). Values are expressed as mean ± SEM in pg/mg wet weight of pancreas from four animals per genotype. The lower panel shows a representative Northern blot analysis of total RNA isolated from 60–120 freshly isolated islets from control and LIRKO mice and hybridized with a radiolabeled rat insulin-1 cDNA as probe. (b) Immunostaining for non–β cell hormones was carried out in pancreatic sections from 6-month-old female WT, IR(lox/lox), alb-Cre control mice, and LIRKO mice using a cocktail of antibodies to glucagon, somatostatin, and pancreatic polypeptide. Representative sections from a control and a LIRKO pancreas are shown. (c) To estimate insulin clearance, a ratio of serum levels of C-peptide to insulin was calculated. Serum was obtained from 16-hour-fasted, 11-week-old male WT, IR(lox/lox), alb-Cre, and LIRKO mice. Insulin and C-peptide were determined by RIA (Linco). The decreased C-peptide:insulin ratio in the LIRKO mice reflects decreased insulin clearance. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)

Figure 4 Hepatic Glucose Production and Expression of Genes Involved in Gluconeogenesis Are Not Suppressed by Insulin in LIRKO Mice (a) Two-month-old male control (WT, IR(lox/lox), and alb-Cre; n = 9) and LIRKO (n = 4) mice were studied by the euglycemic, hyperinsulinemic clamp technique. Mice were infused with [U-13C6]-glucose for 90 min (basal) and then clamped using 2.5 mU human insulin/kg/min (insulin) with glucose maintained at ∼125 mg/dl. Hepatic glucose production during the clamp was determined by subtracting the glucose infusion rate from whole-body glucose appearance. (b) RNA was prepared from liver of 8-week-old, random-fed control (WT, IR(lox/lox), and alb-Cre), and LIRKO mice and was subjected to Northern blotting using probes specific for phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), liver pyruvate kinase (L-PK), and glucokinase (GK). Results are expressed as mean arbitrary phosphorimager units corrected for RNA loading by hybridization to a cyclophilin probe ± SEM from at least four animals per genotype. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)

Figure 5 Changes in Glucose Metabolism in Older LIRKO Mice May Be Related to Liver Dysfunction (a) Blood glucose concentrations were determined from 2-, 4-, and 6-month-old male wild-type (WT), IR(lox/lox), alb-Cre, and LIRKO mice that had been subjected to a 16 hr fast. Each bar represents the mean ± SEM of at least 12 animals of each genotype. LIRKO mice showed fasting hyperglycemia at 2 months of age, normoglycemia at 4 months of age, and hypoglycemia at 6 months of age. (b) Glucose tolerance tests were repeated as described in Figure 2 on 6-month-old male WT, IR(lox/lox), alb-Cre, and LIRKO mice. Results are expressed as mean blood glucose concentration ± SEM from at least eight animals per genotype. LIRKO mice demonstrated significant normalization of glucose tolerance compared to the GTT performed at 2 months of age (see Figure 2 for comparison). (c) Serum albumin levels were measured from 6-month-old male WT, IR(lox/lox), alb-Cre, and LIRKO mice using an albumin determination kit (Sigma). (d) Liver enzymes were measured in the serum of 6-month-old male IR(lox/lox) control mice (n = 6) and LIRKO mice (n = 6) to assess liver function. Alkaline phosphatase (ALP) and aspartate aminotransferase (AST) were significantly elevated in LIRKO mice. Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were not significantly different in LIRKO mice versus controls. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)

Figure 6 The Liver of Young LIRKO Mice Displays Focal Dysplasia and Impaired Glycogen Storage (a) Hematoxylin and eosin staining of liver sections from random-fed, two-month-old male control (upper panel) and LIRKO (lower panel) mice. The arrow denotes an area of dysplastic cells. Magnification, 10×. (b) Periodic acid-Schiff reagent (PAS) staining of liver sections from random-fed, two-month-old male control (upper panel) and LIRKO (lower panel) mice. Magnification, 4×. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)

Figure 7 The Liver of Aged LIRKO Mice Displays Focal Dysplasia, Hyperplastic Nodules, and Altered Ultrastructural Features (a) Gross appearance of 12-month-old control (upper panel) and LIRKO (lower panel) liver showing hyperplastic nodules and enlarged gallbladder. Magnification, 1.25×. (b) Hematoxylin and eosin staining of liver sections from random-fed, 12-month-old control (upper panel) and LIRKO (lower panel) mice. Note that the nodules appear to compress the adjacent tissue, suggesting that the cells of the nodule are hyperplastic. Magnification, 10×. (c) Electron microscopic analysis of liver sections from random-fed, 6-month-old control (upper panel) and LIRKO (lower panel) mice. Note that the LIRKO hepatocytes exhibit giant mitochondria, few electron-dense glycogen granules, and a moderate increase in lipid content. Magnification, 9690×. Molecular Cell 2000 6, 87-97DOI: (10.1016/S1097-2765(05)00015-8)