Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

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