Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: IRS2 is required for glucose-induced β-cell proliferation in vivo and ex vivo. A–D: Mice with generalized deletion of IRS2 or littermate controls were infused intravenously with saline or glucose for 4 days. B and C: β-Cell proliferation was increased by hyperglycemia in WT and HT controls (n = 10–16) but not in IRS2-KO mice (n = 1, 4, and 7). D: β-Cell mass was reduced in diabetic IRS2-KO mice (n = 4–9, except for KO BG 116–138 for which n = 1). E and F: Glucose induced proliferation in WT- but not KO-dispersed mouse islet cells cultured for 72 h (n = 6–9). G and H: Acute knockdown of IRS2 by adenovirus-delivered shRNA targeting IRS2 (MOI 25) in mouse islet cell cultures diminished glucose-induced β-cell proliferation (n = 4). Arrows point to BrdU-positive β-cell nuclei. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. BG, blood glucose; ND, not determined; ns, not signficant.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: In Vivo, Ex Vivo, Saline, Cell Culture, Knockdown, shRNA

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: Glucose-induced proliferation in mouse islets does not require the insulin receptor (IR). A–L: IR blockers S961 (A–F; 100 nmol/L added 120 min before glucose or insulin [100 nmol/L] stimulation) or HNMPA (G–L; 10 μmol/L added 60 min before glucose stimulation) prevented activation of insulin signaling 2 min after glucose treatment (whole islets, n = 4; A and B and G and H) but did not prevent the glucose-induced increase in PCNA abundance after 72 h of glucose treatment (whole islets, n = 3–4; C and D and I and J) or the glucose-induced increase in the percent of β-cells incorporating BrdU (dispersed islets, n = 4–8; E and F and K and L). Adenovirus expressing an shRNA targeting the mouse IR (MOI 50) in dispersed islets reduced IR expression 72 h after glucose exposure (n = 5; M and N) but did not prevent the glucose-induced increase in PCNA abundance (n = 5; O and P) or β-cell BrdU incorporation (n = 4; Q and R). Arrows point to BrdU-positive β-cell nuclei. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. 15 mmol/L glucose control condition (marked by dotted line); #P < 0.05 vs. 5 mmol/L control. gluc, glucose; ns, not significant; p, phosphorylated; tot, total; veh, vehicle.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: Activation Assay, Expressing, shRNA, BrdU Incorporation Assay, Control

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: IRS2 and MTOR but not the insulin receptor are required for glucose induction of cyclin D2; cyclin D2 is required for glucose-induced β-cell proliferation. A–C: IRS2-KO islets contained less IRS2 (n = 1–4; A) and cyclin D2 (n = 4–6; B–C). D and E: IRS2-KO islets failed to induce cyclin D2 protein when cultured in high glucose (n = 5–14). F–K: Blocking insulin receptor action using S961 (whole islets, n = 4; F–G), HNMPA (whole islets, n = 3; H–I), or adenovirus with shRNA targeting the insulin receptor (MOI 50) (dispersed islets, n = 5; J–K) did not prevent glucose induction of cyclin D2 protein. L and M: Treating whole islets with insulin (100 nmol/L) did not induce cyclin D2 protein expression (n = 4). N and O: Blocking MTOR but not PI3K or ERK in whole islets prevented glucose induction of cyclin D2 protein (n = 5–8). P–T: Reducing cyclin D2 expression by adenovirus with shRNA targeting cyclin D2 in dispersed islets (MOI 5, n = 3) prevented glucose induction of cyclin D2 (P and Q), PCNA (P and R), and proliferation (S and T). Panels D–T are at 72 h. Arrows point to BrdU-positive β-cell nuclei. Dotted line in O marks the high glucose control. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. cyc, cyclin; gluc, glucose; ins, insulin; IR, insulin receptor; PD, MEK inhibitor PD98059; RAP, rapamycin; veh, vehicle; WM, wortmannin.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: Cell Culture, Blocking Assay, shRNA, Expressing, Control

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: Restoring cyclin D2 expression rescues the proliferation defect due to loss of IRS2 and rapamycin (RAPA) treatment. A: In dispersed IRS2-WT islets, cyclin D2 overexpression (MOI 5) in 5 mmol/L glucose increased β-cell proliferation to levels achieved in 15 mmol/L glucose, and cyclin D2 overexpression in 15 mmol/L glucose further increased proliferation (n = 3–4). In dispersed IRS2-KO islets, 15 mmol/L glucose did not significantly increase β-cell proliferation compared with 5 mmol/L, but overexpression of cyclin D2 markedly increased proliferation in 15 mmol/L glucose. B and C: Reduced dispersed β-cell proliferation caused by adenovirus knockdown of IRS2 (MOI 50, n = 3–4; B) or RAPA treatment (n = 4–5; C) was rescued by overexpression of cyclin D2 (MOI 5). BrdU incorporation results were confirmed by a second proliferation measure, immunoblot for PCNA: loss of PCNA by IRS2 knockdown (dispersed islets, MOI 50, n = 8; D–F) or RAPA treatment (whole islets, n = 3; G–I) was rescued by overexpression of cyclin D2 (MOI 5). Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. cyc, cyclin; gluc, glucose; ns, not significant; p, phosphorylated; tot, total.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: Expressing, Over Expression, Knockdown, BrdU Incorporation Assay, Western Blot