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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

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.
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Figure 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.

Mentions: Second, as a model of obesity-induced diabetes, we generated R3 transgenic mice with the lethal yellow agouti (Ay) mutation by breeding R3 transgenic mice with KK-Ay mice. For analysis we used F1 offspring littermates carrying the Ay mutation with or without a FLAG-Rheb transgene on a C57BL/6 and KK mouse strain hybrid background (designated Ay/Rheb and Ay, respectively). The rate of increase in body weight was not significantly different between Ay/Rheb and Ay mice (35.5 ± 0.94 vs. 37.5 ± 1.06 g at 10 weeks). Oral glucose tolerance testing revealed that the Ay/Rheb mice had improved glucose tolerance (Fig. 5A). Although blood glucose concentrations in the fasting state were not significantly different between Ay/Rheb and Ay mice, the Ay/Rheb mice had significantly lower blood glucose levels at 60 and 120 min after glucose loading. Thus, the glucose concentration in Ay mice remained at 424 ± 39.8 mg/dl at 60 min and 363.4 ± 47.9 mg/dl at 120 min, whereas that in Ay/Rheb mice was 210 ± 46.6 mg/dl at 60 min and returned to 116.2 ± 17.91 mg/dl at 120 min. The improved glucose tolerance in Ay/Rheb mice was attributable to higher insulin release, with a significant 2.2-fold level at 15 min and a 1.9-fold level at 30 min after glucose loading compared with Ay mice.


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)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: Second, as a model of obesity-induced diabetes, we generated R3 transgenic mice with the lethal yellow agouti (Ay) mutation by breeding R3 transgenic mice with KK-Ay mice. For analysis we used F1 offspring littermates carrying the Ay mutation with or without a FLAG-Rheb transgene on a C57BL/6 and KK mouse strain hybrid background (designated Ay/Rheb and Ay, respectively). The rate of increase in body weight was not significantly different between Ay/Rheb and Ay mice (35.5 ± 0.94 vs. 37.5 ± 1.06 g at 10 weeks). Oral glucose tolerance testing revealed that the Ay/Rheb mice had improved glucose tolerance (Fig. 5A). Although blood glucose concentrations in the fasting state were not significantly different between Ay/Rheb and Ay mice, the Ay/Rheb mice had significantly lower blood glucose levels at 60 and 120 min after glucose loading. Thus, the glucose concentration in Ay mice remained at 424 ± 39.8 mg/dl at 60 min and 363.4 ± 47.9 mg/dl at 120 min, whereas that in Ay/Rheb mice was 210 ± 46.6 mg/dl at 60 min and returned to 116.2 ± 17.91 mg/dl at 120 min. The improved glucose tolerance in Ay/Rheb mice was attributable to higher insulin release, with a significant 2.2-fold level at 15 min and a 1.9-fold level at 30 min after glucose loading compared with Ay mice.

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