Upregulation of the Mammalian Target of Rapamycin Complex 1 Pathway by Ras Homolog Enriched in Brain in Pancreatic β-Cells Leads to Increased β-Cell Mass and Prevention of Hyperglycemia
Article Figures & Tables
Figures
- FIG. 1.
Activation of the mTORC1 pathway by overexpression of FLAG-Rheb in pancreatic β-cells. Islets isolated from mice of each genotype were incubated either in 50% RPMI medium without FCS (A, B, and D) or in RPMI medium (C), or they were stimulated with 100 nmol/l IGF-1 (B) or 15% FCS (D) as indicated. The same amounts of cellular extracts were analyzed by immunoblotting with the antibodies to Rheb, phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein, phospho-4EBP1 (Thr37/46 or Thr70), phospho-p70 S6 kinase (Thr389), p70 S6 kinase, phospho-PKB (Ser473), PKB, phospho-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, or IRS2. A representative experiment is shown. The bottom panel in A shows the same blot as the top panel, except that it was exposed longer to detect the endogenous Rheb (end-Rheb). E: Relative quantification of phospho-S6 (Ser235/236), phospho-4EBP1 (T37/46 or T70), phospho-PKB (Ser473), phospho-p44/42 MAP kinase (Thr202/Tyr204), and IRS2. The immunoblots were scanned, and the optical density for R3 with IGF1 or FCS stimulation (A, B, and D) or the optical density for R3 (C) was set to 100%. F: Immunoprecipitation with the anti-FLAG antibody was performed using lysates of the hypothalamus, muscle, or liver isolated from mice of each genotype and analyzed by immunoblotting with the same antibody. WT, wild type.
- FIG. 2.
Metabolic effects of overexpression of Rheb in pancreatic β-cells. A: Growth curves of R3 or R20 lines of transgenic mice (●) and their wild-type littermates (○). B: Blood glucose concentrations were measured in R3 and R20 transgenic mice and their wild-type littermates in the fed state at 8 and 50 weeks. C: Plasma insulin concentrations were measured in R3 and R20 transgenic mice and their wild-type littermates in the fed state at 8 weeks. D and E: Oral glucose tolerance tests were performed in R3 or R20 transgenic mice (●) and their wild-type littermates (○) at 8 weeks. Glucose concentrations (D) and plasma insulin concentrations (E) are shown. F: Oral glucose tolerance tests were performed in R20 transgenic mice (●) and wild-type littermates (○) at 40 weeks. G: Intraperitoneal insulin tolerance tests were performed in R20 transgenic mice (●) and wild-type littermates (○). H: Intraperitoneal glucose tolerance tests were performed in R20 transgenic mice (●) and wild-type littermates (○). Glucose concentrations and plasma insulin concentrations are shown. Data are the means ± SE of values from six (A), six to eight (B), eight (C), five (D and E), six (F), nine (G), and six (H) animals from each genotype. *P < 0.05; **P < 0.01. WT, wild type.
- FIG. 3.
Increased β-cell mass and cell growth in transgenic mice. A: Hematoxylin and eosin staining of representative pancreatic sections from 8-week-old R3 transgenic mice and their wild-type littermates. B and C: Immunostaining with the anti-FLAG antibody (B) and anti–phospho-S6 (Ser235/236) antibody (C, upper panels) of representative pancreatic sections from 8-week-old R3 transgenic mice and wild-type littermates. Ratio of the number of dark staining cells to the total number of nuclei in islets from transgenic mice and wild-type littermates is shown (C, lower panels). D: Hematoxylin and eosin (H-E) staining and immunostaining with the anti-FLAG antibody of representative pancreatic sections from 90-week-old R3 transgenic mice and their wild-type littermates. E: Immunostaining with the anti-insulin (red) and the anti-glucagon (green) antibodies of representative pancreatic sections from 8-week-old R3 transgenic mice and their wild-type littermates. F: Quantification of β- and α-cell area as a percentage of total pancreatic area in transgenic mice and their wild-type littermates. G: β-Cell mass was calculated by the β-cell area and pancreas weight. H: The relative size of β-cells in the transgenic mice and their wild-type littermates was calculated. I: Immunostaining with anti–Ki-67 antibody of representative pancreatic sections from 9-week-old R3 transgenic mice and their wild-type littermates. Data are the means ± SE of values from four (C), five (F), four (G), and five (H) animals from each genotype. * P < 0.05; ** P < 0.01. WT, wild type. (A high-quality digital representation of this figure is available in the online issue.).
- FIG. 4.
Transgenic mice were resistant to streptozotocin-induced hyperglycemia. A: Doses of 40 mg/kg or 50 mg/kg streptozotocin were administered to R3 line transgenic mice (●) or wild-type littermates (○) for 5 consecutive days (arrows in the figure). Blood glucose concentrations in the fed state were monitored. Data are the means ± SE of values from four (40 mg/kg) or five (50 mg/kg) animals from each genotype. B: A dose of 50 mg/kg streptozotocin was administered to R20 transgenic mice (●) or wild-type littermates (○), and blood glucose concentrations were monitored with mice in the fed state. Data are the means ± SE of values from four animals from each genotype. C and D: Plasma insulin concentration (C) and glucose-to-insulin ratio (D) after administration with or without 50 mg/kg streptozotocin as shown in A. E: Oral glucose tolerance tests were performed in R20 transgenic mice (●) and wild-type littermates (○) 4 weeks after streptozotocin administration as shown in B. Glucose and insulin concentrations are shown. F: Immunostaining with the anti-insulin (red) and anti-glucagon (green) antibodies of representative pancreatic sections from R3 transgenic mice (R3) and their wild-type littermates (WT) 4 weeks after 50 mg/kg streptozotocin administration as shown in A. G: Quantification of β-cell area as a percentage of total pancreatic area in transgenic mice and their wild-type littermates 4 weeks after administration with or without 50 mg/kg streptozotocin as shown in A. H: Immunostaining with anti-insulin (red) antibody and TUNEL assay (green) of representative pancreatic sections from the transgenic mice and their wild-type littermates 1 week after 50 mg/kg streptozotocin administration. Values represent the means ± SE. * P < 0.05; ** P < 0.01. (A high-quality digital representation of this figure is available in the online issue.).
- FIG. 5.
A: Transgenic mice were resistant to obesity-induced hyperglycemia. Oral glucose tolerance tests were performed in Ay/Rheb mice (●) and Aylittermates (○). Blood glucose concentrations and plasma insulin concentrations are shown. B: Elimination of the effect of Rheb expression in β-cells after administration with rapamycin. Oral glucose tolerance tests were performed in rapamycin-treated transgenic mice (●) and their rapamycin-treated wild-type littermates (○). Blood glucose and plasma insulin concentrations are shown. Data are the means ± SE of values from five (A and B) animals from each genotype. C: Islets were prepared from rapamycin- or vehicle-treated mice of each genotype and incubated for 1 h in RPMI, and the same amounts of cellular extracts were analyzed by immunoblotting with the antibodies to phospho-S6 ribosomal protein (Ser235/236), phospho-4EBP1 (Thr37/46), or S6 ribosomal protein. * P < 0.05; ** P < 0.01. WT, wild type.
Tables
- TABLE 1
Metabolic characteristics of the transgenic mice and their wild-type littermates
Plasma free fatty acids (mg/dl) Plasma triglycerides (mg/dl) Plasma leptin (ng/ml) Wild type 1.46 ± 0.13 154.6 ± 9.4 2.09 ± 0.30 Transgenic 1.44 ± 0.77 166.8 ± 16.6 1.57 ± 0.21 -
Data are means ± SE of values from 7–10 animals from each genotype at 8 weeks for plasma triglycerides and leptin or at 10 weeks for plasma free fatty acids.
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- TABLE 2
Numbers of total and Ki-67–positive nuclei in islets
Counted islets per animal Counted nuclei per animal Nuclei per islet Proportion of Ki-67–positive nuclei per islet (%) Wild type 68 6,297 ± 664.5 93 ± 7.6 1.10 ± 0.08 Transgenic 60 5,291 ± 567.7 89 ± 9.7 1.16 ± 0.13 -
Data are means ± SE of values from four animals from each genotype. Pancreatic sections were immunostained with the anti–Ki-67 antibody followed by counterstaining with hematoxylin as shown inFig. 3I. Numbers of all nuclei and of Ki-67–positive nuclei in the islets of wild-type and transgenic mice were counted.
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- TABLE 3
Numbers of insulin- and TUNEL-positive nuclei in islets
Counted islets per animal Counted nuclei per animal Nuclei per islet Proportion of TUNEL-positive nuclei per islet (%) Wild type 69 2,882 ± 567.8 43 ± 4.3 0.55 ± 0.15 Transgenic 65 3,233 ± 495.5 46 ± 4.0 0.42 ± 0.11 -
Data are means ± SE of values from five animals from each genotype. Pancreatic sections were double-stained with the anti-insulin antibody and TUNEL assay 1 week after 50 mg/kg streptozotocin administration as shown inFig. 4H. Numbers of all nuclei and of TUNEL-positive nuclei in the insulin-positive cells of wild-type and transgenic mice were counted.
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