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. 2011 Mar;60(3):827-37.
doi: 10.2337/db10-1194. Epub 2011 Jan 24.

Rictor/mTORC2 is essential for maintaining a balance between beta-cell proliferation and cell size

Yanyun Gu  1 , Jill Lindner, Anil Kumar, Weiping Yuan, Mark A Magnuson
Affiliations

Rictor/mTORC2 is essential for maintaining a balance between beta-cell proliferation and cell size

Yanyun Gu et al. Diabetes. 2011 Mar.

Abstract

Objective: We examined the role of Rictor/mammalian target of rapamycin complex 2 (mTORC2), a key component of the phosphotidylinositol-3-kinase (PI3K)/mTORC2/AKT signaling pathway, in regulating both β-cell mass and function.

Research design and methods: Mice with β-cell-specific deletions of Rictor or Pten were studied to determine the effects of deleting either or both genes on β-cell mass and glucose homeostasis.

Results: Rictor null mice exhibited mild hyperglycemia and glucose intolerance caused by a reduction in β-cell mass, β-cell proliferation, pancreatic insulin content, and glucose-stimulated insulin secretion. Islets from these mice exhibited decreased AKT-S473 phosphorylation and increased abundance of FoxO1 and p27 proteins. Conversely, Pten null (βPtenKO) mice exhibited an increase in β-cell mass caused by increased cellular proliferation and size. Although β-cell mass was normal in mice lacking both Rictor and Pten (βDKO), their β-cells were larger than those in the βPtenKO mice. Even though the β-cell proliferation rate in the βDKO mice was lower than in the βPtenKO mice, there was a 12-fold increase the phosphorylation of AKT-T308.

Conclusions: PI3K/AKT signaling through mTORC2/pAKT-S473 plays a key role in maintaining normal β-cell mass. The phosphorylation of AKT-S473, by negatively regulating that of AKT-T308, is essential for maintaining a balance between β-cell proliferation and cell size in response to proliferative stimuli.

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Figures

FIG. 1.
FIG. 1.
Verification of tissue-specific deletion of Rictor or Pten and effects of Rictor or Pten deficiency on mouse growth and glucose metabolism. A: PCR analysis of islet DNA. Upper: Conditional Rictor and Pten alleles in which the LoxP sites are indicated by pink triangles. Expression of Ins2Cre results in the Cre-mediated deletion of exon 3 or 5 in the Rictor or Pten genes, respectively. Lower: Photographs of PCR analysis showing the loxed, wild-type, and deleted alleles of both genes (lane 1, βRicKO, Rictorlox/lox; Ptenlox/WT; Ins2Cre+; lane 2, βPtenKO, Rictorlox/WT; Ptenlox/lox; Ins2Cre+; lane 3, βDKO, Rictorlox/lox; Ptenlox/lox; Ins2Cre+; and lane 4, Dlox, Rictorlox/lox; Ptenlox/lox). B: Western blot analysis of protein lysates isolated from control and mutant islets. Blots were probed with Rictor and Pten antibodies to verify the deletion of both genes. GAPDH was used as the loading control. C: Mouse body weight and fed blood glucose (D) were measured every 4 weeks between 4 and 16 weeks of age. †P < 0.05, βPtenKO vs. other groups; ‡P < 0.05, βPtenKO and βDKO vs. other groups. #P < 0.05, βRicKO vs. βPtenKO and Dlox; *P < 0.05, βRicKO vs. all other groups. Purple diamonds, βhet (n = 12–24); blue squares, βPtenKO (n = 7–20); green triangles, βRicKO (n = 10–19); red circles, βDKO (n = 13–37); and black squares, Dlox (n = 13–23). E: Intraperitoneal glucose tolerance test. Blood glucose concentrations were tested 0, 15, 30, 60, and 120 min after glucose injection. Blood glucose in βRicKO mice was significantly higher than Dlox at 15 and 30 min. βDKO mice had significantly improved blood glucose concentration compared with βRicKO at 30 and 60 min. #P < 0.05, βRicKO vs. βPtenKO and Dlox; **P < 0.01, βRicKO vs. all other groups, βDhet (n = 13), βPtenKO (n = 7), βRicKO (n = 15), βDKO (n = 9), and Dlox (n = 13). F: AUGC and IAUGC were calculated following the trapezoid rule, to evaluate total blood glucose excursion after a glucose bolus. IAUGC was equal to AUGC minus the area beneath the fasting concentrations. βRicKO mice had higher AUGC and IAUGC value than Dlox. AUCG and IAUGC in βDKO mice showed significant improvement compared with βRicKO. G: Curve of in vivo glucose-stimulated insulin secretion at 0, 15, and 30 min after glucose injection. Insulin secretion after peritoneal glucose injection in the βRicKO mice trended lower at 15 min compared with the Dlox. Insulin secretion between βRicKO and βDKO mice showed no significant difference. βPtenKO mice had the lowest insulin secretion at 15 min after glucose bolus. *P < 0.05, βPtenKO and Dlox vs. all other groups; **P < 0.05, βPtenKO vs. all other groups, βDhet (n = 13), βPtenKO (n = 7), βRicKO (n = 15), βDKO (n = 9), and Dlox (n = 13). H: Curve of insulin secretion per 100 islet equivalents in ex vivo perifusion test in all genotypes. Isolated islets were incubated with 5.6 mmol/L and 16.7 mmol/L glucose, and insulin secretion was measured every 3 min up to 42 min (n = 3). *P < 0.05, βRicKO and βPtenKO vs. Dlox; **P < 0.01, βRicKO and βPtenKO vs. Dlox. All data presented as mean ± SEM. EE, extended exposure. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Effects of Rictor or Pten deficiency on pancreatic islet characteristics and insulin content. Paraffin sections from 3-month-old mice were immunolabeled with insulin and BrdU, Ki67, or Glut2. A: β-Cell mass adjusted for body weight. βPtenKO mice showed higher β-cell mass when compared with other groups. βRicKO showed a significantly reduced β-cell mass compared with control group. *P < 0.05; **P < 0.01. B: Whole pancreatic insulin content was compared among all the groups. βRicKO showed the lowest insulin content among all genotypes. *P < 0.05. C: Quantification of the percentage of β-cells labeled for both Ki67 and insulin. βRicKO had lowest percentage of double-labeled cells. At least 1,000 cells from three embryonic pancreata were counted for each genotype to determine the number of insulin positive and insulin/Ki67 double-positive cells. *P < 0.05; **P < 0.01. D: Quantification of β-cell size. βDKO had largest cell size compared with other groups. In all cases, three to five mice per genotype were analyzed. In total, 500–1,000 cells were counted, and there were at least three animals per group. *P < 0.05 vs. all other groups or between indicated groups. **P < 0.01 vs. all other groups or between indicated groups. Data presented as mean ± SEM. E: Representative immunolabeled sections from embryonic day 17.5 pancreata for insulin (green), Ki67 (red), and Topo3 (blue) nuclear staining. Insulin/Ki67 immunolabeling in pancreatic islets was used to identify proliferating β-cells. Scale bar = 20 μm. F: Representative immunolabeled sections of pancreas from adult mice for Glut2 (green) and DAPI (blue) nuclear staining. Glut2 was used to label the β-cell membrane to measure cell size. Scale bar = 25 μm. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 3.
FIG. 3.
Western blot and densitometry analysis of islet proteins in different genotypes. A: Western blotting for determining total and phosphorylated AKT. The phosphorylation of AKT at T308 and S473, and total AKT protein were examined in islets of mutant and control groups. B: Densitometry analysis for AKT phosphorylation. C: Immunoblot of p27 and FoxO1 using GAPDH as a loading control. D: Densitometry analysis for p27 and FoxO1 protein adjusted with GAPDH. E: Immunoblot for phosphorylation of S6 (top) and densitometry analysis (bottom). Figures represent blots from multiple mice in each genotype. Results are presented as relative folds of change in mutant compared with control mice. Control is Dlox. *P < 0.05 vs. all other groups or the indicated group; **P < 0.01 vs. all other groups or indicated group; §P = 0.07. Four to five mice were studied per groups. Data are presented as mean ± SEM. ADU, arbitrary density unit.
FIG. 4.
FIG. 4.
Differential cellular distribution of FoxO1 in pancreatic β-cells with different genotypes. A–D: Immunolabeling of FoxO1 (red) in pancreatic islets. E–H: Double immunolabeling of FoxO1 (red) and insulin (green). A and E: FoxO1 immunolabeling in control islets showing its localization mainly in the nucleus. B and F: FoxO1 immunolabeling in βRicKO islets. βRicKO islets showing a more intense nuclear FoxO1 staining than the controls. C and G: FoxO1 immunolabeling in βPtenKO islets showing the lack of nuclear FoxO1 accumulation. D and H: FoxO1 immunolabeling in βDKO islets. Nuclear staining pattern in βDKO islets is similar to βRicKO islets. Scale bar = 20 μm. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
Deletion of Rictor or Pten changed gene expressions of pancreatic transcription factors and genes related to cell cycle and insulin secretion in pancreatic islets. A: Heat map representing comparison of gene expression profile determined by TaqMan RT-PCR and calculated by -ΔΔCT method with 18S RNA as the internal control. A total of 43 genes from the four categories indicated on the far left were analyzed. Gene expression abundance was shown in varying color levels of red and green, with red representing upregulation, green representing downregulation, and black representing the level in the control group. B: Comparisons of some cell cycle-related genes: p27, CDK4, and p21 expression profiles were significantly different among all genotypes. C: Comparison of genes regulating insulin secretion machinery; Vamp-2, Connexin-36, and Syntaxin-1 had differential gene expression patterns in all four genotypes. D: Comparisons of critical pancreatic transcription factors; Pdx1, MafB, and Neurog3 showed differential expression patterns in all four genotypes. Control is Dlox. *P < 0.05; **P < 0.01. Data are presented as mean ± SEM. Three mice were analyzed for each genotype.
FIG. 6.
FIG. 6.
Model for the differential roles of the two AKT phosphorylation sites in regulating pancreatic β-cell size and proliferation. A: Balance between β-cell size and cell number is maintained by the site-specific phosphorylation of AKT at T308 and S473 and dual negative feedback loops. B: PI3K/AKT signaling is activated by growth factors/insulin binding to their receptors. In the case of insulin, insulin receptor activation stimulates insulin receptor substrate, which then activates PI3K and phosphorylation of PI2P to PI3P. The intracellular accumulation of PI3P then stimulates PDK1 and mTORC2 to phosphorylate AKT at T308 and S473, respectively. AKT is phosphorylated at both T308 and S473. Phosphorylation of AKT-T308 (pAKT-T308) selectively targets mTORC1/S6, whereas phosphorylation of AKT-S473 (pAKT-S473) selectively targets FoxO1/p27 signaling. These AKT phosphorylations mutually regulate to maintain β-cell mass homeostasis. Solid arrows indicate the known signaling events; dashed lines indicate a newly proposed inhibitory feedback mechanism. CM, cell membrane; IR, insulin receptor; IRS, insulin receptor substrate; PH, pleckstrin homology domain.
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