mTORC1 activation regulates beta-cell mass and proliferation by modulation of cyclin D2 synthesis and stability

Abstract

Growth factors, insulin signaling, and nutrients are important regulators of beta-cell mass and function. The events linking these signals to the regulation of beta-cell mass are not completely understood. The mTOR pathway integrates signals from growth factors and nutrients. Here, we evaluated the role of the mTOR/raptor (mTORC1) signaling in proliferative conditions induced by controlled activation of Akt signaling. These experiments show that the mTORC1 is a major regulator of beta-cell cycle progression by modulation of cyclin D2, D3, and Cdk4 activity. The regulation of cell cycle progression by mTORC1 signaling resulted from modulation of the synthesis and stability of cyclin D2, a critical regulator of beta-cell cycle, proliferation, and mass. These studies provide novel insights into the regulation of cell cycle by the mTORC1, provide a mechanism for the antiproliferative effects of rapamycin, and imply that the use of rapamycin could negatively impact the success of islet transplantation and the adaptation of beta-cells to insulin resistance.

FIGURE 1.

Overexpression of Akt in β-cells using a doxycycline-inducible system. A, immunoblotting for Akt and pGSK3α/β (Ser^21/9) in islets from ST (RIP-rtTA) and DT (RIP-rtTA/tetOcaAkt) mice treated with doxycycline for 6 and 24 h. B, intraperitoneal glucose tolerance test after overnight fasting in ST and DT mice before initiation of dox treatment. C, intraperitoneal glucose tolerance tests performed in ST and DT mice after 20 weeks of doxycycline treatment. D, 6-h fasting glucose measurements obtained from ST or DT mice. E, insulin levels in 6 h fasted ST and DT mice. F, β-cell mass measurements in ST and DT mice after 20 weeks on doxycycline or vehicle treatment. G, proliferation rate assessed by Ki67 in insulin-stained sections from ST and DT mice that received doxycycline treatment for 20 weeks. The data are presented as the means ± S.E. (n = 5). ^*, p < 0.05.

FIGURE 2.

Assessment of mTOR signaling in islets from ST and DT mice. A, experimental design used to assess rapamycin effect on ST and DT mice. B, immunostaining for insulin (green) and pS6 ribosomal protein (red) in islets from ST and DT mice at the end of the experimental protocol. C, immunoblotting for total Akt, p70S6K (phospho-p70 S6K Thr 389), phospho-S6 ribosomal protein (Ser^235/236) (pS6) and tubulin in islet lysates from ST and DT mice at the end of the experimental protocol described in A. Islets from all groups were harvested immediately after isolation and subjected to immunoblotting. Scale bar, 50 μm.

FIGURE 3.

Assessment of carbohydrate metabolism β-cell mass, proliferation, and apoptosis. A, intraperitoneal glucose tolerance test after overnight fasting in ST and DT mice before initiation of dox treatment. B, after 3 weeks of doxycycline treatment. C, after 2 weeks of Rap treatment (5 mg/kg) daily. D, β-cell mass in ST and DT mice treated with vehicle or Rap (5 mg/kg) daily for 2 weeks as indicated in Fig. 2A. E, frequency of β-cell proliferation assessed by Ki67 staining in insulin-stained sections from ST and DT mice treated with vehicle or Rap (5 mg/kg) daily for 2 weeks as indicated in Fig. 2A. Co-staining for insulin and Ki67 defined proliferating cells. F, apoptosis measured by cleaved-caspase 3 staining in insulin-stained sections from ST and DT mice at the end of the experimental protocol described for Fig. 2A. Apoptotic cells were determined as cells that were co-stained for insulin and cleaved-caspase 3. The data are presented as the means ± S.E. (n = 5). ^*, p < 0.05; #, p < 0.05 DT + Rap versus DT.

FIGURE 4.

Assessment of Akt activity. A, immunoblotting for pAKT (Thr³⁰⁸) and pAKT (Ser⁴⁷³) in islets lysates from ST and DT mice treated with vehicle or 50 nm Rap. For these experiments, islets from dox-treated ST and DT mice were cultured in medium containing vehicle or dox for 40 h. Rapamycin was added to the culture for the last 16 h before harvesting. B, in vitro Akt kinase activity in islets from ST and DT mice subjected to the same experimental conditions described in A (n = 4). C, in vitro Akt kinase activity in MIN6 cells overexpressing constitutive active Akt (n = 4). The cells were cultured in the presence of vehicle or rapamycin (50 nm) for 16 h before harvesting. Quantification data from in vitro Akt kinase activity in islets and MIN6 cells is presented as fold change with respect to that of vehicle-treated control. D, immunoblotting for pGSK α/β (Ser 21/9) and tubulin as loading control in islets from ST and DT mice treated as described for A. The data are presented as the means ± S.E. (n = 3). ^*, p < 0.05.

FIGURE 5.

Cdk4 activity and assessment of cell cycle components. A, in vitro Cdk4 kinase activity in islets from control and transgenic mice overexpressing a constitutively active Akt under the rat insulin promoter (caAkt). The islets were cultured for 40 h after isolation, and Rap (50 nm) was included in the culture medium for the last 16 h of the culture. B, in vitro Cdk4 kinase activity in stable MIN6 cells overexpressing constitutively active Akt. MIN6-GFP and MIN6-caAkt were cultured in regular medium for 16 h in the presence of vehicle or rapamycin (50 nm). C, in vitro Cdk4 kinase activity in islets from ST and DT mice. The islets were obtained from ST or DT mice treated with vehicle or dox in the drinking water for 3 weeks. After isolation, the islets were cultured in medium with vehicle (ST) or dox (DT) for 40 h. Rapamycin (50 nm) or vehicle was added to the medium for the last 16 h of culture before harvesting. D, immunoblotting for cyclin D1, cyclin D2, cyclin D3, Cdk4, and p27 in islets from ST and DT mice treated and nontreated with rapamycin. These experiments were performed using the experimental protocol described for C. E, immunostaining for insulin (green) and cyclin D2 (red) in islets from ST and DT mice, treated, and nontreated with rapamycin using the experimental protocol described for Fig. 2A. The data are presented as the means ± S.E. (n = 3). ^*, p < 0.05.

FIGURE 6.

Measurement of mRNA levels for G₁ components in islets. TaqMan reverse transcription-PCR for cyclin D1, cyclin D2, cyclin D3, Cdk4 p21, and p27 in islets from ST and DT mice treated and nontreated with rapamycin. For these experiments, the islets were obtained from ST or DT mice treated with vehicle or dox in the drinking water for 3 weeks. After isolation, the islets were cultured in medium with vehicle (ST) or dox (DT) for 40 h. Rapamycin (50 nm) or vehicle was added to the medium for the last 16 h of culture before harvesting. The data are presented as the means ± S.E. (n = 3). ^*, p < 0.05.

FIGURE 7.

Effect of rapamycin on cyclin D2 synthesis and stability. A, pulse-chase experiment in islets from ST and DT mice. Protein lysates were precipitated with monoclonal antibody to Cyclin D2. Islets were treated with Rap (50 nm) as indicated under “Experimental Procedures.” Immunoblotting for cyclin D2 was used as loading control. The blot is representative of two independent experiments. B, pulse-label experiment in control (MIN6-GFP) and MIN6 cells expressing a constitutively active Akt (MIN6-caAkt). MIN6 cells were preincubated with 50 nm rapamycin for 90 min before the pulse, and the treatment was continued during the pulse for 30 min. Immunoblotting for cyclin D2 was used as loading control. C, quantitation and statistical analysis of the pulse-label experiment performed in MIN6 cells (B). D, islets from ST and DT mice were treated with 12.5μg/ml cycloheximide (CHX) for 30 min, and the levels of cyclin D2 were determined by immunoblotting. Protein bands for cyclin D2 were quantified and normalized to the levels in islets treated with vehicle control. E, islets from DT mice were treated with Me₂SO or 50 nm rapamycin for 2, 4, 6, and 8 h, and the levels of cyclin D2 were determined by immunoblotting. Protein bands for cyclin D2 were quantified, normalized to levels in DT mice treated with Me₂SO, and represented as the average of three separate experiments. F, islets from DT mice were treated with cycloheximide (lanes C) in the presence or absence of rapamycin (lanes R) for the indicated times. The levels of cyclin D2 were determined by immunoblotting. Protein bands for cyclin D2 were quantified, normalized to levels in DT mice before treatment, and represented as the average of three separate experiments. The data are presented as the means ± S.E. (n = 3). ^*, p < 0.05.