The role of insulin receptor substrate 2 in hypothalamic and beta cell function

Abstract

Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in beta cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and beta cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced beta cell mass. Overt diabetes did not ensue, because beta cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced beta cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in beta cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis.

Figure 1

Generation of Irs2flox mice and characteristics of RIPCre and POMCCre mice. (A) Schema of targeting construct design, simplified restriction map of the Irs2 locus, the locus after homologous recombination and the deletion of neomycin cassette (Neo), and Southern blotting and PCR genotyping strategies used to identify these events. External probe A was used to identify homologous recombination (HR), probe B to detect the selection cassette, and probe C to detect the coding region of Irs2. HSV-tk, herpes simplex virus thymidine kinase. (B) Southern blot analysis with probe A demonstrating homologous recombination after targeting. (C and D) Southern blots using probe B after Cre-mediated recombination demonstrating deletion (Del) of the neomycin cassette and using probe C to demonstrate retention of Irs2 coding region confirming type 2 recombination. (E) PCR analysis with primers P1 and P2 of HR clone that has lost the neomycin cassette but retained the loxP site downstream of the Irs2 coding region. (F–H) We examined Cre expression in RIPCre mice by analyzing GFP expression in pancreatic sections costained with insulin and hypothalamic sections from RIPCreZEG mice. (I) We examined Cre expression in POMCCre mice by analyzing hypothalamic sections from POMCCreZEG mice. (J) Combined ISH for POMC and ICC for GFP was performed in POMCCreZEG mice to confirm Cre expression in POMC neurons. (K) Immunohistochemistry for IRS2 (red) was performed in hypothalamic sections from POMCCreZEG mice. Scale bars: 50 μm (H, I, and K) and 10 μm (J). 3V, third ventricle; ME, median eminence.

Figure 2

Analysis of deletion of Irs2 in islets and hypothalami from RIPCreIrs2KO, POMCCreIrs2KO, and NesCreIrs2KO mice. (A) We performed PCR analysis to detect recombination of the Irs2 locus in DNA from islets and hypothalami (hypo) of control and knockout mice. The presence of a 1.3-kb PCR product indicates recombination and deletion of the Irs2 gene. (B and C) Western blot analysis of Irs2 in islets from control and RIPCreIrs2KO mice (B) and whole brain and hypothalamic lysates from control and NesCreIrs2KO mice (C). (D) Immunofluorescence analysis for Irs2 expression in the hypothalami of RIPCreZEG and RIPCreIrs2KOZEG mice. Colocalization of GFP (green) and Irs2 (red) is seen in RIPCreZEG mice (indicated by white arrows) and no colocalization in RIPCreIrs2KOZEG mice. RIPCreGFP, GFP expression in RIPCre cells. (E) Colocalization of POMCCre expression and Irs2 in POMCCreZEG mice. (F) Colocalization of POMCCre expression and Irs2 in POMCCrelacZ mice and no colocalization of GFP and Irs2 in POMCCreIrs2KOlacZ mice. Confocal images of representative arcuate nucleus fields are shown in D–F. Scale bars: 10 μm.

Figure 3

Glucose homeostasis in RIPCreIrs2KO, NesCreIrs2KO, and POMCCreIrs2KO mice. (A) Fasting blood glucose levels of 12-week-old male mice of the indicated genotypes were measured after a 16-hour overnight fast. (B). Glucose tolerance tests were performed on 12-week-old male RIPCreIrs2KO (filled squares) and control mice (open squares). (C) Glucose tolerance tests were performed on 12-week-old male NesCreIrs2KO (filled squares) and control mice (open squares). (D) Fasting blood glucose levels of 6-month-old male mice of the indicated genotypes were measured after a 16-hour overnight fast. (E) Fasting blood insulin levels of 12-week-old male mice of the indicated genotypes were measured after a 16-hour overnight fast. Data in A–E represent the mean ± SEM for 8–10 animals of each genotype. (F) We calculated the percentage of the total pancreatic area occupied by β cells in 12-week-old male mice of the indicated genotypes using insulin-stained pancreatic sections. The right side shows data for 9-month-old RIPCreIrs2KO and control mice. Four pancreata were analyzed per genotype at each time point, and for each pancreas, 4 sections were analyzed. The data presented are mean ± SEM for 4 mice of each genotype. *P < 0.05, **P < 0.01, and ***P < 0.001. Cont, control.

Figure 4

Hypothalamic function in RIPCreIrs2KO, NesCreIrs2KO, and POMCCreIrs2KO mice. (A) Body weight was measured in 12-week-old male mice of the indicated genotypes. (B) Total body fat was determined by MRI in 16-week-old male mice of the indicated genotypes. (C) Cumulative 24-hour food intake was measured in 12-week-old male mice of the indicated genotypes. (D) Fasting blood leptin levels of 12-week-old male mice of the indicated genotypes were measured after a 16-hour overnight fast. (E–G) Cumulative food intake, percent reduction in food intake compared with baseline, and reduction in bodyweight were measured over a 3-day period of treatment with leptin (5 mg/kg) or vehicle in 6–8-week-old male mice of the indicated genotypes. (H) Food intake and fasting leptin levels were determined in 5-week-old male mice of the indicated genotypes. Data in A–H data represent the mean ± SEM for 8–10 animals of each genotype. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

Analysis of recombination and duct-marker expression in islets from RIPCreIrs2KOZEG mice. (A–D) Immunofluorescence staining for insulin (red) in islets from 4-week-old (A and B) and 9-month-old (C and D) RIPCreIrs2KOZEG mice stained for DNA (DAPI, blue). White arrows indicate β cells that do not express GFP and have not undergone recombination. Many cells in the 9-month-old islet do not express GFP. (E–G) Labeling of ducts with DBA in islets from 9-month-old RIPCreIrs2KOZEG mice stained blue for either DNA (E and F) or insulin (G). No duct marker is expressed in GFP-negative β cells. Scale bars: 100 μm.

Figure 6

Characterization of RIPCre neurons by ICC, ISH, and electrophysiological analysis. (A and B) Dual ISH for POMC and ICC for GFP was performed on hypothalamic sections from RIPCreZEG mice. Representative low- (A) and high-power (B) views are shown. (C and D) Dual ISH for NPY and ICC for GFP was performed on hypothalamic sections from RIPCreZEG mice. Representative low- (C) and high-power (D) views are shown. ARC, arcuate nucleus. (E) Fluorescence ICC for POMC was performed on hypothalamic sections from RIPCreZEG mice. Neurons expressing POMC are red and those expressing GFP are green. Arrows indicate noncolocalized neurons and GFP neurons that are abutted by POMC fibers. A representative confocal image is shown. Scale bars: 50 μm (A and C) and 10 μm (B, D, and E). (F–I) Current-clamp recordings were made from RIPCreZEG neurons in the absence and presence of the mixed melanocortin 3/4 receptor agonist MTII, as indicated above the traces. (F) Representative recording demonstrating MTII depolarization, resulting in a small increase in spike frequency (G). This depolarization is more clearly represented in neurons that have sub-spike thresholds, achieved by application of a constant hyperpolarizing current (H). MTII also depolarizes neurons in the presence of TTX (I), indicative of a direct action. The action potentials in panel H have been truncated to show clearly the depolarizing effect of MTII. Note that the MTII depolarization was irreversible over the time course of the recordings in cells represented in H and I.

Figure 7

Electrophysiological characteristics of RIPCre and POMC neurons in response to insulin and leptin. (A) Representative recordings from individual arcuate neurons are shown for POMCCreZEG (A–C, I, and J) and RIPCreZEG (D–H). Leptin (50 nM) and insulin (10 nM) were pressure-ejected (2–5 psi) for 60 seconds (as denoted by arrows). (A) Leptin depolarizes the POMCCreZEG neuron, and this is associated with significant attenuation of spike amplitude. The recording from the neuron shown in B was hyperpolarized to subthreshold potentials by injection of constant current (2–5 pA) through the recording electrode. Application of leptin depolarized and increased the firing rate of the neuron. (C) Diary plot of spike frequency for the neuron shown in B. In contrast, leptin had no effect on membrane potential (D) and spike frequency (E) in RIPCreZEG neurons. Insulin caused depolarization of the RIPCreZEG neuron (F) and increased spike frequency (G), with spike attenuation. As shown in H, the neuron was hyperpolarized to subthreshold potentials as above, and application of insulin clearly depolarizes this neuron with a large increase in spike frequency. In contrast, insulin caused hyperpolarization of POMCCreZEG neurons (I), which was accompanied by a reduction in firing rate (J).