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, 114 (7), 908-16

Dysregulation of Insulin Receptor Substrate 2 in Beta Cells and Brain Causes Obesity and Diabetes

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Dysregulation of Insulin Receptor Substrate 2 in Beta Cells and Brain Causes Obesity and Diabetes

Xueying Lin et al. J Clin Invest.

Abstract

The molecular link between obesity and beta cell failure that causes diabetes is difficult to establish. Here we show that a conditional knockout of insulin receptor substrate 2 (Irs2) in mouse pancreas beta cells and parts of the brain--including the hypothalamus--increased appetite, lean and fat body mass, linear growth, and insulin resistance that progressed to diabetes. Diabetes resolved when the mice were between 6 and 10 months of age: functional beta cells expressing Irs2 repopulated the pancreas, restoring sufficient beta cell function to compensate for insulin resistance in the obese mice. Thus, Irs2 signaling promotes regeneration of adult beta cells and central control of nutrient homeostasis, which can prevent obesity and diabetes in mice.

Figures

Figure 1
Figure 1
Creation of fIrs2 mice and cr2-mediated recombination/deletion of fIrs2. (A) A schematic diagram of the 4 steps in the generation of the fIrs2 containing embryonic stem cells (see Methods for details). Homol recomb, homologous recombination. (B) Recombination/deletion of fIrs2 was revealed in various mouse tissues at 4 weeks of age by PCR analysis of genomic DNA. L, liver; K, kidney; H, heart; S, spleen; B, brain, F, fat; M, muscle. The intact fIrs2 allele (fIrs2+) yields a 750-bp fragment, whereas the recombined/deleted allele (fIrs2_) yields a 250-bp fragment. (C) Real-time PCR analysis of Irs2 expression in isolated islets, normalized by cyclophilin expression. (D) PCR analysis of hypothalamic DNA to reveal recombination event. (E) Immunoprecipitation and Western blot analysis of hypothalamus with antibodies against Irs2. (F) Brain sections were immunostained with antibodies against Irs2 (red) and Cre (green), and the images were merged in Openlab Image software (version 3.14; Improvision). Similar results were obtained with multiple sections from 2 to 8 mice. Asterisks indicate third ventricle. Magnification, ×20.
Figure 2
Figure 2
Growth and nutrient homeostasis of the fIrs2:cr2 mice. (A) Male littermates fed regular or low-fat chow were weighed weekly from postnatal day 21 until 32 weeks of age; each point represents the average ± SE of at least 6 mice. Omitted error bars are smaller than the symbol. (B) Food intake was determined with 8-week-old male mice over 24 hours using a Comprehensive Lab Animal Monitoring System; average ± SE for 4 animals per genotype is reported. (C) Cumulative water intake was determined with 8-week-old male mice over 24 hours using a Comprehensive Lab Animal Monitoring System; average ± SE for 3 animals per genotype is reported. (D) Average lean and fat body mass ± SE was determined on three 8-week-old male of the indicated genotypes using a dual energy X-ray absorptiometry (DEXA) scanner (GE Medical Systems Lunar) according to the manufacturer’s instructions. (E) Average ± SE plasma insulin and leptin levels were determined on at least fifteen 8-week-old random fed male mice of each genotype using a rat insulin or mouse leptin ELISA Kit. *P < 0.05; **P < 0.01.
Figure 3
Figure 3
Glucose homeostasis in the fIrs2:cr2 mice. (A) Average ± SE random-fed blood glucose levels were determined using a Glucometer Elite in at least fifteen 8-week-old male mice of the indicated genotypes. (B and C) Glucose-disposal rates and hepatic glucose output before and during hyperinsulinemia (average ± SE) were determined on 6_10 fasted and conscious 10-week-old male mice of the indicated genotypes. (D) Pancreas insulin content was determined using rat insulin ELISA Kit following acid-ethanol extraction. Results are average ± SE for five 8-week-old mice per genotype. (E) After a 16-hour overnight fast, blood glucose (average ± SE) was determined on at least fifteen 8-week-old male mice of the indicated genotypes following intraperitoneal injection of 2 g D-glucose/kg body weight. (F) The integrated glucose tolerance (GTT, t = 0 Ø 120 min) for the 8-week old male mice fed the ordinary diet (data in E) was compared with that for 16-week-old male mice fed a low-fat diet (LFD, 5%); these ages were selected for comparison to match the body weights. The glucose tolerance curves were integrated using the trapezoid rule implemented in Sigmaplot 8.0 (Systat Software Inc.). *P < 0.05; **P < 0.01.
Figure 4
Figure 4
Pancreas β cell function in aged fIrs2:cr2 and fIrs2:cr2:Irs1+/_ mice.(A) Random-fed blood glucose levels were determined at the indicated ages. Averages ± SE were determined from at least 5 male mice per genotype. (B) Random insulin levels were determined in male mice of the indicated genotypes and ages. Averages ± SE were determined from at least 5 mice per genotype. (C) Cre-mediated recombination/deletion of fIrs2 alleles was determined by real-time PCR analysis in single islets isolated from male fIrs2:cr2 mice at the indicated ages; or determined in hypothalamus from 10-month-old fIrs2 or fIrs2:cr2 mice. (D) Real-time PCR analysis of cr2 expression in isolated fIrs2:cr2 islets at the indicated ages; mRNA levels were normalized against cyclophilin expression, and the averages ± SE were determined for 2 male mice at each age. (E) Body weight of male littermates of the indicated genotypes that were fed regular chow and weighed weekly from postnatal day 21 until 16 weeks of age; each point represents the average ± SE of at least 6 mice. (F) Three representative pancreatic sections obtained from 6-month-old mice of the indicated genotypes immunostained with antibodies against insulin (green) or glucagon (red).
Figure 5
Figure 5
Analysis of hypothalamic function in the fIrs2:cr2 mice. (A) Immunoblots of hypothalamus lysates. After an overnight fasting, anesthetized 8-week-old mice were injected intravenously with 50 μl of saline or saline containing 5 units of insulin. Fifteen minutes later, the hypothalamus was isolated and lysed. Lysates were immunoprecipitated with anti_Irs2 antibody, resolved by SDS-PAGE, and then immunoblotted with antibodies against Irs2, phosphotyrosine (pY20), or p85. Lysates were also blotted independently with antibodies against Akt or phospho-Akt [Akt(Pi)], or Stat3. Data are representative of at least three independent experiments. (B) The relative expression of various mRNAs_Npy (1421690_s_at), Agrp (1419127_at), Cart (1422825_at) or Pomc1 (1455858_x_at; 1433800_a_at), Stat3 (1426587_a_at; 1426587_a_at; 1460700_at), Socs3 (1455899_x_at; 1456212_x_at) or PcSK2 (1448312_at; 1444147_at) was determined in hypothalamic extracts using Affymetrix GeneChip Mouse Expression Array 430A. The fold change is shown with the 90% confidence interval for expression in 3 fIrs2:cr2 mice compared with the average control values from two WT, 2 cr2 and 2 fIrs2 mice. Where more than one probe occurs on the Genechip, the average is reported. Cntr, control. (C) Brain sections from WT or fIrs2:cr2 mice were immunostained with antibodies against αMSH (green) or Irs2 (red). Yellow appears in the digitally merged images where αMSH and Irs2 are coexpressed. Magnification, ×20; *3rd ventricle. (D) Body weight and blood glucose levels of fIrs2:cr2, [fIrs2:cr2]:rip13Irs2, and WT male mice at 8 weeks; average ± SE was determined from 5 animals of each genotype. No tg, no transgene.

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