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

Journal: Cerebral Cortex (New York, NY)

Article Title: Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories

doi: 10.1093/cercor/bhy142

Figure Lengend Snippet: 2 populations of vGlut1+ and vGlut2+ DH neurons project to RSC. (a) Schematic of Cre-dependent AAV-DIO-hM4D(Gi)-mCherry infusion in DH of vGlut1-Cre and vGlut2-Cre mice (left). Labeling of vGlut1+ (middle) and vGlut2+ (right) DH neurons 6 weeks after virus expression. The mCherry labeling patterns were similar to the one of endogenous transporters (Supplementary Fig. 2). (b) Immunofluorescent staining for mCherry RSC (top) in vGlut1-Cre mice and vGlut2-Cre mice revealing terminal fields in RSC layers 1 and 3. Immunostaining in DH is shown below. (c) Retrograde labeling of DH neurons projecting to RSC using Fluoro-Gold (top and bottom left) or CTB (top and bottom right). Fluoro-Gold injected into RSC (top left) was detected predominantly in the subiculum (SUB, bottom left). Sparse signals were also found in dorsomedial CA1. Similarly, injection of CTB (blue) in RSC labels SUB neurons of vGlut1-Cre mice expressing Cre-dependent retrograde AAV-flex-dtTomato (red) (bottom left).

Article Snippet: The viral vector carrying a construct coding for the Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral, 2.25 mm ventral to bregma).

Techniques: Labeling, Expressing, Staining, Immunostaining, Injection

Journal: Cerebral Cortex (New York, NY)

Article Title: Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories

doi: 10.1093/cercor/bhy142

Figure Lengend Snippet: Chemogenetic silencing of DH→ RSC terminals impairs memory encoding of CFC. (a) Schematic of virus infusions and cannula implantations. The Cre-independent viral vector AAV-hM4D(Gi)-mCherry was injected into the DH 6 weeks before behavioral experiments. During the same surgery, cannulae were implanted into the RSC bilaterally. (b) Immunostaining for mCherry showing expression of hM4D(Gi) in DH and its projection throughout ventral RSC (RSCv) but not in ventral hippocampus (VH). (c) Effect of pretraining infusion of CNO on activity (cm/s) during context and shock exposure at training (left) and freezing during context test (right). Pretraining infusions of Veh and CNO did not affect locomotor activity (Veh: 14.7 ± 2.33; CNO: 16.6 ± 1.48) or activity burst (Veh: 71.5 ± 7.52; CNO: 70.1 ± 5.69) to the footshock (activity before shock: t = 0.71, P = 0.49; activity during shock: t = 0.15, P = 0.89; Veh n = 6, CNO n = 7). However, CNO significantly impaired freezing at test when compared to vehicle (Veh)-injected controls (t = 5.843, P 0.001; Veh: 62.3 ± 3.03; CNO: 25.3 ± 5.30).

Article Snippet: The viral vector carrying a construct coding for the Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral, 2.25 mm ventral to bregma).

Techniques: Plasmid Preparation, Injection, Immunostaining, Expressing, Activity Assay

Journal: Cerebral Cortex (New York, NY)

Article Title: Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories

doi: 10.1093/cercor/bhy142

Figure Lengend Snippet: Chemogenetic silencing of vGlut1+ DH→ RSC terminals impair encoding and retrieval of contextual fear conditioning. (a) Experimental design similar to Figure ​Figure11 except for infusion of a Cre-dependent AAV8-DIO-hM4D(Gi)-mCherry. Freezing during the context test was significantly reduced in vGlut1-Cre mice injected with CNO when compared to Veh (Veh: 61.5 ± 4.68; CNO: 37.13 ± 6.99; t = 2.87, P < 0.05 (n = 8/group)), but not in vGlut2-Cre mice (Veh: 49.48 ± 5.32; CNO: 45.66 ± 6.41; t = 0.45, P = 0.65; Veh n = 12, CNO n = 14). (b) Within-subject design was used to determine the effect of pretraining CNO on recent and remote memory. Significant treatment effects were found for each genotype (vGlut1-Cre: F1,16 = 43.78, P < 0.001; n = 9/group; vGlut2-Cre: F1,16 = 10.91, P < 0.01; Veh n = 12, CNO n = 14). However, vGlut1-Cre mice receiving CNO before training showed reduced freezing both at recent (Veh: 54.3 ± 6.36; CNO: 7.56 ± 2.29; P < 0.05) and remote memory tests (Veh: 55.8 ± 6.09; CNO: 22.7 ± 5.79; P < 0.01), whereas similarly treated vGlut2-Cre mice showed freezing deficits only at the remote (Veh: 61.3 ± 6.35; CNO: 30.9 ± 5.96; P < 0.01), but not recent test (Veh: 58.2 ± 3.86; CNO: 57.7 ± 5.06). (c) Infusion of CNO before the recent memory test impaired freezing to the conditioning context in vGlut1-Cre mice (Veh: 64.6 ± 3.08; CNO: 37.2 ± 6.98; t = 3.14, P < 0.01; n = 9/group) without affecting freezing in vGlut2-Cre mice (Veh: 47.5 ± 7.73; CNO: 53.9 ± 8.46; t = 0.56, P = 0.58; n = 7/group). Infusion of CNO before the remote memory test was ineffective in either mouse line (vGlut1-Cre, Veh: 36.2 ± 5.04; CNO: 38.2 ± 6.32; t = 0.61, P = 0.54; n = 15/group; vGlut2-Cre: Veh: 49.9 ± 8.62; CNO: 54.8 ± 8.78; t13 = 0.39, P = 0.7; n = 7/group).

Article Snippet: The viral vector carrying a construct coding for the Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral, 2.25 mm ventral to bregma).

Techniques: Injection