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Upregulation of the mammalian target of rapamycin complex 1 pathway by Ras homolog enriched in brain in pancreatic beta-cells leads to increased beta-cell mass and prevention of hyperglycemia.

Hamada S, Hara K, Hamada T, Yasuda H, Moriyama H, Nakayama R, Nagata M, Yokono K - Diabetes (2009)

Bottom Line: We examined the activation of the mTORC1 pathway and its effects on beta-cell mass, on glucose metabolism, and on protection against hyperglycemia.Immunoblots of islet extracts revealed that the phosphorylation levels of ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1, downstream effectors for mTORC1, were upregulated in transgenic beta-cells.Immunostaining of the pancreatic sections with anti-phospho-S6 antibody confirmed upregulation of the mTORC1 pathway in beta-cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal and Geriatric Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT

Objective: Components of insulin/IGF-1 receptor-mediated signaling pathways in pancreatic beta-cells have been implicated in the development of diabetes, in part through the regulation of beta-cell mass in vivo. Studies in vitro have shown that the protein Ras homolog enriched in brain (Rheb) plays a key role as a positive upstream regulator of the mammalian target of rapamycin complex 1 (mTORC1) pathway in integrating inputs from nutrients and growth factors for cell growth. Our objective was to investigate the role of the mTORC1 pathway in the regulation of beta-cell mass in vivo.

Research design and methods: We generated transgenic mice that overexpress Rheb in beta-cells. We examined the activation of the mTORC1 pathway and its effects on beta-cell mass, on glucose metabolism, and on protection against hyperglycemia.

Results: Immunoblots of islet extracts revealed that the phosphorylation levels of ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1, downstream effectors for mTORC1, were upregulated in transgenic beta-cells. Immunostaining of the pancreatic sections with anti-phospho-S6 antibody confirmed upregulation of the mTORC1 pathway in beta-cells in vivo. The mice showed improved glucose tolerance with higher insulin secretion. This arose from increased beta-cell mass accompanied by increased cell size. The mice also exhibited resistance to hyperglycemia induced by streptozotocin and obesity.

Conclusions: Activation of the mTORC1 pathway by Rheb led to increased beta-cell mass in this mouse model without producing obvious unfavorable effects, giving a potential approach for the treatment of beta-cell failure and diabetes.

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Related in: MedlinePlus

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.).
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Figure 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.).

Mentions: Hematoxylin and eosin staining of the sections revealed that the pancreatic islet architecture was grossly unchanged in mice from ages 8 to 90 weeks (Fig. 3A and D). Immunostaining of the sections with the anti-FLAG antibody confirmed the expression of FLAG-Rheb in the islets of transgenic mice from ages 8 to 90 weeks (Fig. 3B and D). Immunostaining with the anti–phospho-S6 antibody appeared lower in the islet areas than in the exocrine acinar areas. However, the dark-staining cells in the islets appeared more abundant in transgenic mice than in the wild-type littermates (Fig. 3C). The ratio of the number of the dark staining cells to the number of nuclei in the islets was significantly higher in transgenic mice than in wild-type littermates (19.48 ± 3.23 vs. 6.76 ± 1.14%).


Upregulation of the mammalian target of rapamycin complex 1 pathway by Ras homolog enriched in brain in pancreatic beta-cells leads to increased beta-cell mass and prevention of hyperglycemia.

Hamada S, Hara K, Hamada T, Yasuda H, Moriyama H, Nakayama R, Nagata M, Yokono K - Diabetes (2009)

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.).
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Related In: Results  -  Collection

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Figure 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.).
Mentions: Hematoxylin and eosin staining of the sections revealed that the pancreatic islet architecture was grossly unchanged in mice from ages 8 to 90 weeks (Fig. 3A and D). Immunostaining of the sections with the anti-FLAG antibody confirmed the expression of FLAG-Rheb in the islets of transgenic mice from ages 8 to 90 weeks (Fig. 3B and D). Immunostaining with the anti–phospho-S6 antibody appeared lower in the islet areas than in the exocrine acinar areas. However, the dark-staining cells in the islets appeared more abundant in transgenic mice than in the wild-type littermates (Fig. 3C). The ratio of the number of the dark staining cells to the number of nuclei in the islets was significantly higher in transgenic mice than in wild-type littermates (19.48 ± 3.23 vs. 6.76 ± 1.14%).

Bottom Line: We examined the activation of the mTORC1 pathway and its effects on beta-cell mass, on glucose metabolism, and on protection against hyperglycemia.Immunoblots of islet extracts revealed that the phosphorylation levels of ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1, downstream effectors for mTORC1, were upregulated in transgenic beta-cells.Immunostaining of the pancreatic sections with anti-phospho-S6 antibody confirmed upregulation of the mTORC1 pathway in beta-cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal and Geriatric Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT

Objective: Components of insulin/IGF-1 receptor-mediated signaling pathways in pancreatic beta-cells have been implicated in the development of diabetes, in part through the regulation of beta-cell mass in vivo. Studies in vitro have shown that the protein Ras homolog enriched in brain (Rheb) plays a key role as a positive upstream regulator of the mammalian target of rapamycin complex 1 (mTORC1) pathway in integrating inputs from nutrients and growth factors for cell growth. Our objective was to investigate the role of the mTORC1 pathway in the regulation of beta-cell mass in vivo.

Research design and methods: We generated transgenic mice that overexpress Rheb in beta-cells. We examined the activation of the mTORC1 pathway and its effects on beta-cell mass, on glucose metabolism, and on protection against hyperglycemia.

Results: Immunoblots of islet extracts revealed that the phosphorylation levels of ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1, downstream effectors for mTORC1, were upregulated in transgenic beta-cells. Immunostaining of the pancreatic sections with anti-phospho-S6 antibody confirmed upregulation of the mTORC1 pathway in beta-cells in vivo. The mice showed improved glucose tolerance with higher insulin secretion. This arose from increased beta-cell mass accompanied by increased cell size. The mice also exhibited resistance to hyperglycemia induced by streptozotocin and obesity.

Conclusions: Activation of the mTORC1 pathway by Rheb led to increased beta-cell mass in this mouse model without producing obvious unfavorable effects, giving a potential approach for the treatment of beta-cell failure and diabetes.

Show MeSH
Related in: MedlinePlus