Irs2 and Irs4 synergize in non-LepRb neurons to control energy balance and glucose homeostasis

Abstract

Insulin receptor substrates (Irs1, 2, 3 and Irs4) mediate the actions of insulin/IGF1 signaling. They have similar structure, but distinctly regulate development, growth, and metabolic homeostasis. Irs2 contributes to central metabolic sensing, partially by acting in leptin receptor (LepRb)-expressing neurons. Although Irs4 is largely restricted to the hypothalamus, its contribution to metabolic regulation is unclear because Irs4-null mice barely distinguishable from controls. We postulated that Irs2 and Irs4 synergize and complement each other in the brain. To examine this possibility, we investigated the metabolism of whole body Irs4(-/y) mice that lacked Irs2 in the CNS (bIrs2(-/-)·Irs4(-/y)) or only in LepRb-neurons (Lepr (∆Irs2) ·Irs4 (-/y) ). bIrs2(-/-)·Irs4(-/y) mice developed severe obesity and decreased energy expenditure, along with hyperglycemia and insulin resistance. Unexpectedly, the body weight and fed blood glucose levels of Lepr (∆Irs2) ·Irs4 (-/y) mice were not different from Lepr (∆Irs2) mice, suggesting that the functions of Irs2 and Irs4 converge upon neurons that are distinct from those expressing LepRb.

Keywords: ARC, arcuate nucleus of the hypothalamus; CNS, central nervous system; ERK, extracellular signal-regulated kinase; Energy balance; Insulin receptor substrate 2; Insulin receptor substrate 4; Irs2, insulin receptor substrate 2; Irs4, insulin receptor substrate 4; LepRb, leptin receptor; Leptin; Nutrient homeostasis; Obesity; PI3K, phosphatidylinositol 3-kinase; POMC, proopiomelanocortin; Socs3, suppressor of cytokine signaling-3; Stat3, signal transducer and activator of transcription 3.

Figure 1

bIrs2^−/−·Irs4^−/y-mice are obese. (A) Average body weights of male bIrs2^−/−·Irs4^−/y-mice (open squares), Irs4^−/y-mice (closed squares), bIrs2^−/− mice (open circles) and control Irs2^L/L mice (closed circles) on regular chow diet was determined in each age group and compared by generalized linear regression (SPSS, v19). The number of mice in each group is indicated in parentheses (mean±SD; *, Bonferroni p<0.001). (B) Representative image of 3-months-old male mice. (C) Body length and (D) percent body fat was determined by DEXA using 12-week-old mice (mean±SEM; n=8–10/genotype, *p<0.05 vs. controls).

Figure 2

Representative H&E staining of (A) white adipose tissue (WAT) and (C) liver sections of 6-month-old bIrs2^−/−·Irs4^−/y-mice, Irs4^−/y-mice, bIrs2^−/− or Irs2^L/L mice. Liver sections of 9-month-old ob/ob mice are also shown. (B) Morphometric analysis of adipocyte cell size in epididymal adipose tissue (n=5 animals per genotype; *p<0.05 vs. control).

Figure 3

Energy expenditure in bIrs2^−/−·Irs4^−/y-mice. (A) Serum leptin levels of 9-month-old male bIrs2^−/−·Irs4^−/y-mice, Irs4^−/y-mice, bIrs2^−/− mice and Irs2^L/L mice (n=8–10, mean±SEM; *, p<0.05 for indicated comparison). (B) Food intake over 24 h in 3-month-old male bIrs2^−/−·Irs4^−/y-mice, Irs4^−/y mice, bIrs2^−/−-mice or Irs2^L/L-mice fed regular chow diet (mean±SEM; * p<0.05 for indicated comparison). Three month-old male mice of the indicated genotype were monitored for 72 h in the CLAMS (n=10/genotype) to assess (C) oxygen consumption (O₂, l/kg/h), and (D) heat production (kcal/h/kg). Dark phase is presented (mean±SD; * p<0.001 for indicated comparison). Heat production was analyzed by generalized linear regression (SPSS, v19), controlling for the effect of body weight (Bonferroni, p<0.001) and (E) RER (respiratory exchange ratio) during the light and dark phases. (F) Representative H&E staining of brown adipose tissue (BAT) of 3-month-old male bIrs2^−/−·Irs4^−/y-mice, Irs4^−/y-mice, bIrs2^−/−-mice and Irs2^L/L-mice.

Figure 4

Levels of mRNA (relative to actin) by RT-PCR of Npy (A), Agrp (B) and Pomc (C) from hypothalamus of 3-month-old chow-fed male mice of the indicated genotypes. (D) Ratio between Agrp and Pomc mRNAs (from B, C) for the indicated genotypes. Data are presented as mean±SEM; * p<0.05 for indicated comparisons. (n=4–5).

Figure 5

Glucose homeostasis in bIrs2^−/−·Irs4^−/y mice. (A) Fasted and (B) fed blood glucose for 3 month-old male mice. (C) Glucose tolerance test of 3 month-old male mice (n=10/genotype). (D) Insulin tolerance test of 4 month-old male mice (n=9–10/genotype). (E) HOMO2-IR index of insulin resistance (IR) and (F) fasting serum insulin levels (n=10/genotype) for 9 month-old male mice of the indicated genotypes. Data are expressed as mean±SEM. * p<0.05 for indicated comparisons.

Figure 6

(A) Representative H&E staining of pancreatic sections of bIrs2^−/−·Irs4^−/y mice, Irs4^−/y mice, bIrs2^−/− mice and Irs2^L/L mice at indicated ages. (B) Quantification of total pancreatic area occupied by β cells in bIrs2^−/−·Irs4^−/y mice, Irs4^−/y mice, bIrs2^−/− mice and Irs2^L/L mice. Data are presented as mean±SEM; * p<0.05. (n=4–5) compared to other genotypes.

Figure 7

Lepr^∆Irs2-mice and Lepr^∆Irs2·Irs4^−/y-mice display similar body weight and normal blood glucose levels. (A) Average body weights of 3 month-old male bIrs2^−/−·Irs4^−/y-mice, Irs4^−/y mice, bIrs2^−/− mice, Irs2^L/L mice and Lepr^∆Irs2-mice and Lepr^∆Irs2·Irs4^−/y-mice. (B) Fed blood glucose levels of 3 month-old male Irs2^L/L mice, Lepr^∆Irs2-mice and Lepr^∆Irs2·Irs4^−/y-mice. Mean±SD; n=6–8/genotype, *p<0.001 for indicated comparisons.