Insulin receptor substrates Irs1 and Irs2 coordinate skeletal muscle growth and metabolism via the Akt and AMPK pathways

Abstract

Coordination of skeletal muscle growth and metabolism with nutrient availability is critical for metabolic homeostasis. To establish the role of insulin-like signaling in this process, we used muscle creatine kinase (MCK)-Cre to disrupt expression of insulin receptor substrates Irs1 and Irs2 in mouse skeletal/cardiac muscle. In 2-week-old mice, skeletal muscle masses and insulin responses were slightly affected by Irs1, but not Irs2, deficiency. In contrast, the combined deficiency of Irs1 and Irs2 (MDKO mice) severely reduced skeletal muscle growth and Akt→mTOR signaling and caused death by 3 weeks of age. Autopsy of MDKO mice revealed dilated cardiomyopathy, reflecting the known requirement of insulin-like signaling for cardiac function (P. G. Laustsen et al., Mol. Cell. Biol. 27:1649-1664, 2007). Impaired growth and function of MDKO skeletal muscle were accompanied by increased Foxo-dependent atrogene expression and amino acid release. MDKO mice were resistant to injected insulin, and their isolated skeletal muscles showed decreased insulin-stimulated glucose uptake. Glucose utilization in MDKO mice and isolated skeletal muscles was shifted from oxidation to lactate production, accompanied by an elevated AMP/ATP ratio that increased AMP-activated protein kinase (AMPK)→acetyl coenzyme A carboxylase (ACC) phosphorylation and fatty acid oxidation. Thus, insulin-like signaling via Irs1/2 is essential to terminate skeletal muscle catabolic/fasting pathways in the presence of adequate nutrition.

FIG. 1.

Irs1 and Irs2 in skeletal and cardiac muscle. (A and B) Protein content of Irs1 (A) and Irs2 (B) analyzed by specific immunoblotting of gastrocnemius muscle lysates from 2-week-old mice of the indicated genotype (n = 6; *, P < 0.05). Error bars represent standard error of the mean (SEM). (C) Representative immunoblot of IRS1 and IRS2 proteins in EDL and soleus muscles of control (Irs1/2^L/L) and MDKO mice. (D) Survival curve of MDKO mice and Irs1/2^L/L mice. (E to H) Protein concentration and tyrosine phosphorylation of Irs1 and Irs2 in cardiac muscle extracts from 2-week-old control and MDKO mice (n = 2; *, P < 0.05). (I) Transverse sections of cardiac muscle from two Irs1/2^L/L and two MDKO mice at 2 weeks of age. RV, right ventricle; LV, left ventricle. (J). Relative sizes of the left and right ventricles (ventricle area/heart area) from Irs1/2^L/L and MDKO mice (n = 4; *, P < 0.05).

FIG. 2.

Skeletal muscle protein content and cross-sectional area. (A) Relative average weight (mg/g body weight [BW]) of gastrocnemius (Gas), tibialis anterior (TA), and quadriceps (Quad) muscle in 2-week-old male mice (n = 6; *, P < 0.05; **, P < 0.005). (B) Relative protein content (mg/g body weight) in gastrocnemius isolated from 2-week-old male mice. (C) Representative cross sections of tibialis anterior (TA) muscle of control and MDKO mice stained with dystrophin; cross-sectional area of TA muscle (n = 6; *, P < 0.05). (D, E) Representative cross sections of TA muscle of control and MDKO mice (D) stained with dystrophin and DAPI (4′,6-diamidino-2-phenylindole) to determine the distribution of the myofiber cross-sectional area (E) in 2-week-old male mice (n = 6; *, P < 0.05; **, P < 0.005).

FIG. 3.

The effect of skeletal muscle deficiency of Irs1 and Irs2 upon signaling. (A to F) MKO1, MKO2, or MDKO mice were treated with saline or insulin (Ins) for 5 min, followed by an immunoblot assay of Akt phosphorylation in gastrocnemius muscle with phosphospecific antibodies against Thr308 (A to C) or Ser473 (D to F). (G to K) EDL muscles isolated from control or MDKO mice were treated without or with IGF1 for 30 min; tissue extracts were analyzed by immunoblotting with phospho-specific and non-phospho-specific antibodies against Akt, Tsc2, S6k, S6, or 4EBP1. Error bars represent SEM (n = 4; *, P < 0.05; **, P < 0.01).

FIG. 4.

FoxO signaling and gene expression in skeletal muscle. (A and B) Skeletal muscle Foxo1 (A) or Foxo3 (B) phosphorylation in gastrocnemius muscle lysates from control, MKO1, MKO2, or MDKO mice. Muscles were isolated from 2-week-old male mice and analyzed by immunoblotting with phospho-specific or protein-specific antibodies. The mean quantified signals are compared; error bars represent SEM (n = 6; *, P < 0.05). (C) Relative mRNA concentrations of genes involved in skeletal muscle atrophy in 2-week-old Irs1/2^L/L or MDKO mice were determined by real-time PCR (n = 6; *, P < 0.05; **, P < 0.005).

FIG. 5.

Metabolic homeostasis in MDKO mice. (A) The rate of amino acid release from EDL or soleus muscles of 2-week-old Irs1/2^L/L or MDKO mice (n = 5 or 6; **, P < 0.05). (B) Protein content of EDL and soleus muscles of Irs1/2^L/L or MDKO mice (n = 5 or 6; **, P < 0.05). (C and D) Blood glucose and lactate concentrations in 2-week-old Irs1/2^L/L or MDKO mice (n = 6; *, P < 0.05; **, P < 0.005) sampled in the morning during random feeding. Error bars represent SEM. (E and F) Lactate and blood glucose concentrations in fasted 2-week-old Irs1/2^L/L or MDKO mice after a morning bolus glucose injection (n = 9 or 10; *, P < 0.05; **, P < 0.005). Error bars represent SEM. (G and H) Blood glucose and lactate concentrations in 2-week-old Irs1/2^L/L or MDKO mice fasted 4 h before injection with insulin (n = 6 or 7; *, P < 0.05; **, P < 0.005). Error bars represent SEM.

FIG. 6.

Activation of the AMPK pathway and altered metabolism in skeletal muscle from MDKO mice. (A) Rate of glucose oxidation in isolated EDL and soleus muscles from Irs1/2^L/L or MDKO mice (n = 5 or 6; *, P < 0.05; **, P < 0.01) (B) AMP/ATP ratio in Irs1/2^L/L or MDKO muscle (gastrocnemius) (n = 5 or 6; *, P < 0.05). (C) Expression and phosphorylation, analyzed by immunoblotting, of AMPK, ACC, Raptor, and Tsc2 in gastrocnemius muscle lysates from 2-week-old control, MKO1, MKO2, or MDKO mice (n = 6). (D) Rate of glucose uptake in isolated EDL and soleus muscles from Irs1/2^L/L or MDKO mice without or with insulin stimulation (n = 5 or 6; **, P < 0.01). Error bars represent SEM. (E to G) Protein contents of PDK4, GLUT1, and GLUT4 in gastrocnemius muscles from Irs1/2^L/L or MDKO mice (n = 5 or 6, *, P < 0.05) (H) Rate of lactate release from isolated EDL and soleus muscles from Irs1/2^L/L or MDKO mice (n = 5 or 6; *, P < 0.05; **, P < 0.01). (I) Gastrocnemius muscle glycogen content in Irs1/2^L/L or MDKO mice (n = 8; *, P < 0.05). (J) Rate of palmitate oxidation (Palm. OX) in EDL and soleus muscles from Irs1/2^L/L or MDKO mice (n = 5 or 6; *, P < 0.05; **, P < 0.01). Error bars represent SEM.

FIG. 7.

Model of Irs-dependent regulation of skeletal muscle growth and metabolism. Arrows indicate upregulation/activation, and blunt ends indicate downregulation/repression in normal tissue. Those pathways enhanced in MDKO muscle are presented as boldfaced green lines/type and those diminished are presented as thin and dashed red lines/red type. Pathways/intermediate steps not directly assayed are shown as solid gray lines/type. Proteins are capitalized, and the upregulation of transcription is shown in italics. ETC, mitochondrial electron transport chain; FAO, fatty acid oxidation; a.a., amino acids; Pdh, pyruvate dehydrogenase.