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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

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.
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Figure 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.

Mentions: We confirmed the integration of the transgene in three independent founder lines (R3, R20, and R35; accession no. CDB0441T). Immunoblotting of total cellular extracts from pancreatic islets using the anti-Rheb antibody demonstrated that the transgene product was detected at a slightly higher level than endogenous Rheb on SDS-PAGE (Fig. 1A). Similar results were observed when FLAG-tagged Rheb was expressed transiently in HEK293 cells (data not shown), suggesting that the addition of a FLAG sequence to Rheb lowers the mobility of the polypeptide on SDS-PAGE. Immunoprecipitation with the anti-FLAG antibody followed by immunoblotting with the same antibody failed to detect the expression of FLAG-Rheb in the hypothalamus, muscle, or liver, indicating that the expression of FLAG-Rheb is specific to the pancreatic β-cells (Fig. 1F).


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)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: We confirmed the integration of the transgene in three independent founder lines (R3, R20, and R35; accession no. CDB0441T). Immunoblotting of total cellular extracts from pancreatic islets using the anti-Rheb antibody demonstrated that the transgene product was detected at a slightly higher level than endogenous Rheb on SDS-PAGE (Fig. 1A). Similar results were observed when FLAG-tagged Rheb was expressed transiently in HEK293 cells (data not shown), suggesting that the addition of a FLAG sequence to Rheb lowers the mobility of the polypeptide on SDS-PAGE. Immunoprecipitation with the anti-FLAG antibody followed by immunoblotting with the same antibody failed to detect the expression of FLAG-Rheb in the hypothalamus, muscle, or liver, indicating that the expression of FLAG-Rheb is specific to the pancreatic β-cells (Fig. 1F).

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