Cyclin D2 is essential for the compensatory beta-cell hyperplastic response to insulin resistance in rodents

Abstract

Objective: A major determinant of the progression from insulin resistance to the development of overt type 2 diabetes is a failure to mount an appropriate compensatory beta-cell hyperplastic response to maintain normoglycemia. We undertook the present study to directly explore the significance of the cell cycle protein cyclin D2 in the expansion of beta-cell mass in two different models of insulin resistance.

Research design and methods: We created compound knockouts by crossing mice deficient in cyclin D2 (D2KO) with either the insulin receptor substrate 1 knockout (IRS1KO) mice or the insulin receptor liver-specific knockout mice (LIRKO), neither of which develops overt diabetes on its own because of robust compensatory beta-cell hyperplasia. We phenotyped the double knockouts and used RT-qPCR and immunohistochemistry to examine beta-cell mass.

Results: Both compound knockouts, D2KO/LIRKO and D2KO/IRS1KO, exhibited insulin resistance and hyperinsulinemia and an absence of compensatory beta-cell hyperplasia. However, the diabetic D2KO/LIRKO group rapidly succumbed early compared with a relatively normal lifespan in the glucose-intolerant D2KO/IRS1KO mice.

Conclusions: This study provides direct genetic evidence that cyclin D2 is essential for the expansion of beta-cell mass in response to a spectrum of insulin resistance and points to the cell-cycle protein as a potential therapeutic target that can be harnessed for preventing and curing type 2 diabetes.

FIG. 1.

D2KO/LIRKO mice exhibit severe hyperglycemia and early diabetes. A and B: Plasma insulin (A) and glucagon (B) levels were measured at random fed states in control (white), LIRKO (light gray), D2KO/LIRKO (black), and D2KO (dark gray) mice (n = 4–10). *P < 0.05 as indicated by bars. C: Glucose tolerance tests were performed in 7-week-old mice, and blood glucose was measured at 0, 15, 30, 60, and 120 min after intraperitoneal (IP) injection of glucose (2 g/kg body wt). *P < 0.05 vs. controls; n = 4–8.

FIG. 2.

Absence of cyclin D2 limits islet hyperplasia in LIRKO mice. A: Real-time RT-qPCR was performed on RNA extracted from islets of control, LIRKO, D2KO/LIRKO, and D2KO mice (n = 4–9). Results are normalized to TATA-binding protein (TBP) and expressed relative to controls. *P < 0.05 vs. groups as indicated. B: Triple immunostaining for insulin (blue), somatostatin (green), and glucagon (red) on pancreas sections from control, LIRKO, D2KO/LIRKO, and D2KO mice. A representative islet for each group at magnification 40× is presented (n = 4). C: Relative islet size was assessed from triple immunostaining (presented in B). Quantification with Image J software of islet area is presented (means ± SEM from n ≥10 islets counted per mouse; n = 4–6 in each group). D: β-Cell mass was assessed as described in research design and methods (n = 4–6). E: Relative β-cell size was assessed by coimmunostaining for β-catenin, insulin, and DAPI in pancreas sections from control, LIRKO, D2KO/LIRKO, and D2KO mice (n = 4–6). Quantification with Image J software of relative β-cell area is presented (means ± SEM from n ≥100 cells counted per mouse; n = 4–6 in each group). F: β-Cell proliferation was assessed by coimmunostaining for Ki-67, insulin, and DAPI in pancreas sections from control, LIRKO, D2KO/LIRKO, and D2KO mice. Respective replication index is presented. *P < 0.05 vs. groups as indicated; n = 4–6. In all cases, at least two to three pancreas sections were used for each animal. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Increased expression of cyclin D2 in LIRKO islets. A: Real-time RT-qPCR was performed on RNA extracted from islets of LIRKO (light gray) or control (white) mice (n = 4). Results are normalized to TATA-binding protein (TBP) and expressed relative to controls. *P < 0.05 for LIRKO vs. controls. B: Coimmunostaining of cyclin D2 (green) with insulin (red) and DAPI (blue) in pancreas sections from control and LIRKO mice. One to two representative islets for each group at magnification 40x are presented (n = 3). In all cases at least two to three pancreas sections were used for each animal. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Regulation of PDX-1 and GLUT2 expression in D2KO/LIRKO islets. A and C: Real time RT-qPCR was performed on RNA extracted from control, LIRKO, D2KO/LIRKO, and D2KO islets. Results are normalized to TATA-binding protein (TBP) and expressed relative to controls. *P < 0.05 in comparison with controls as indicated; n = 4. B: Coimmunostaining of PDX-1 (red) and FoxO1 (green) with DAPI (blue) in pancreas sections from control, LIRKO, D2KO/LIRKO, and D2KO mice. A representative islet for each group at magnification 40× is presented (n = 4). D: Coimmunostaining of GLUT2 (green) and insulin (red) with DAPI (blue) in pancreas sections from control, LIRKO, D2KO/LIRKO, and D2KO mice. A representative islet for each group at magnification 40× is presented (n = 4). In all cases, at least two to three pancreas sections were used for each animal. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

D2KO/IRS1KO animals are insulin resistant, hypoinsulinemic, and glucose intolerant. A: Insulin resistance was quantified by insulin tolerance tests. Insulin (0.75 mU/g body wt) was injected intraperitoneally, and blood was collected from tail veins of control (white), D2KO (gray), IRS1KO (striped), and D2KO/IRS1KO (hatched) mice. Data are expressed as area under the curve of glucose excursion. B: plasma insulin levels were measured before and 30 min after intraperitoneal injection of glucose in control, D2KO, IRS1KO, or D2KO/IRS1KO mice. C: Glucose tolerance test. Blood glucose was measured at 0, 15, 30, 60, and 120 min after intraperitoneal injection of glucose. *P < 0.05 and ***P < 0.005 in comparison with controls (A–C) or as indicated by bars. n = 7–8 for each group.

FIG. 6.

D2KO/IRS1KO mice fail to exhibit compensatory β-cell replication and islet hyperplasia in response to insulin resistance. A: Coimmunostaining for insulin (green) and glucagon (red) in pancreas sections from control, D2KO, IRS1KO, and D2KO/IRS1KO mice as described in research design and methods. A representative islet for each group at magnification 20× is presented; n = 3. B: Quantification of β-cell mass in control (white), D2KO (gray), IRS1KO (striped), and D2KO/IRS1KO (hatched) mice. *P = 0.03 compared with controls (n = 3–5). C: Quantification of relative β-cell diameter. A minimum of 100 cells were measured per genotype. *P < 0.05, **P < 0.01, and ***P < 0.005 in comparison with control. D: Quantification of the proliferation index of replicating β-cells for each group in control (white), D2KO (gray), IRS1KO (striped), and D2KO/IRS1KO (hatched) mice. *P < 0.05 compared with controls; n = 3–5. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Alterations in expression of D-type cyclins in D2KO/IRS1KO islets. Coimmunostaining for insulin (green) and cyclin (red) D1, D2, or D3 in pancreas sections from control, D2KO, IRS1KO, and D2KO mice. A representative islet for each group at magnification 20× is presented; n = 3. (A high-quality digital representation of this figure is available in the online issue.)