Knockout of STAT3 in skeletal muscle does not prevent high-fat diet-induced insulin resistance

Abstract

Objective: Increased signal transducer and activator of transcription 3 (STAT3) signaling has been implicated in the development of skeletal muscle insulin resistance, though its contribution, in vivo, remains to be fully defined. Therefore, the aim of this study was to determine whether knockout of skeletal muscle STAT3 would prevent high-fat diet (HFD)-induced insulin resistance.

Methods: We used Cre-LoxP methodology to generate mice with muscle-specific knockout (KO) of STAT3 (mKO). Beginning at 10 weeks of age, mKO mice and their wildtype/floxed (WT) littermates either continued consuming a low fat, control diet (CON; 10% of calories from fat) or were switched to a HFD (60% of calories from fat) for 20 days. We measured body composition, energy expenditure, oral glucose tolerance and in vivo insulin action using hyperinsulinemic-euglycemic clamps. We also measured insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake technique.

Results: STAT3 protein expression was reduced ∼75-100% in muscle from mKO vs. WT mice. Fat mass and body fat percentage did not differ between WT and mKO mice on CON and were increased equally by HFD. There were also no genotype differences in energy expenditure or whole-body fat oxidation. As determined, in vivo (hyperinsulinemic-euglycemic clamps) and ex vivo (2DOG uptake), skeletal muscle insulin sensitivity did not differ between CON-fed mice, and was impaired similarly by HFD.

Conclusions: These results demonstrate that STAT3 activation does not underlie the development of HFD-induced skeletal muscle insulin resistance.

Keywords: 2DOG, 2-deoxyglucose; AT, adipose tissue; Adgre1, adhesion G protein-coupled receptor E1; CON, normal chow, control diet; Clamp; Cre-LoxP; EDL, extensor digitorum longus; GA, gastrocnemius; GIR, glucose infusion rate; Glucose homeostasis; HFD, high-fat diet; HGP, hepatic glucose production; HYP-EUG, hyperinsulinemic-euglycemic; IL, interleukin; IS-GDR, insulin-stimulated glucose disposal rate; In vivo; KO, knockout; MCK, muscle creatine kinase; Obesity; STAT3; STAT3, signal transducer and activator of transcription 3; T2D, type 2 diabetes; WT, wild-type; mKO, muscle-specific knockout of STAT3.

Figure 1

mKO mice have decreased STAT3 protein expression in skeletal muscle: (A) PCR on genomic DNA from skeletal muscle (gastrocnemius [GA], soleus [SOL] and extensor digitorum longus [EDL]), adipose tissue (AT) and liver of WT and mKO (mKO) mice. Primers target a region within exon 20 of the STAT3 gene (Lane #1), which is present in all tissues (black arrow), or a region that spans exons 20–22 (Lane #2) and thus only prime when the floxed region is deleted (green arrow, note: band only in skeletal muscle from mKO mice). (B) Total STAT3 protein abundance measured in whole-cell lysates (WCL) of SOL, EDL and liver from WT and mKO mice. (C) Total STAT3 protein abundance in myofibers isolated from the tibialis anterior. (D) Activation of STAT3, as measured by pSTAT3, in WCL from gastrocnemius from mice before (−) and 10 min after (+) intravenous injection with IL-6 (55 μg/kg). (E) Total STAT3 protein abundance in nuclear (nuc) and cytosolic (cyto) fractions of GA muscle.

Figure 2

HFD feeding increases body fat and decreases respiratory exchange ratio (RER) in WT and mKO mice. WT and mKO mice were fed a CON or HFD for 20 d. (A) Body mass, lean mass, fat mass, and (B) percent body fat, as measured by MRI. n = 14–23/group. *, p < 0.05 vs. CON. (C–F) Food intake, energy expenditure, and spontaneous activity measurements were made using the CLAMS system over 3 consecutive days and averages for the light and dark cycles on days 2 and 3 are presented. (C) Cumulative food intake. (D) Total (x-total) activity was measured as all beam breaks on the horizontal axis. (E) $\dot{\text{V}}$O₂ and (F) RER were measured by indirect calorimetry. n = 6/group. *, p < 0.05 vs. CON. Data reported as mean ± SEM.

Figure 3

HFD-induced impairments in glucose tolerance or in vivo insulin action are similar in mKO and WT mice. WT and mKO mice were fed a CON or HFD for 20 days. (A) Blood glucose concentrations and (B) area under the curve (AUC) during an oral glucose tolerance test (OGTT; 5 g/kg). n = 6–15/group. *, p < 0.05 vs. CON. (C) Glucose infusion rate (GIR), (D) hepatic glucose production (HGP; basal and insulin-stimulated [clamp]), (E) percent suppression of HGP, and (F) insulin-stimulated glucose disposal rate (IS-GDR) during a HYP-EUG clamp. n = 7–10/group. *, p < 0.05 vs. CON. Data reported as mean ± SEM.

Figure 4

STAT3 knockout in muscle does not protect against HFD-induced impairments in skeletal muscle insulin sensitivity. WT and mKO mice were fed a CON or HFD for 20 days. Basal and insulin (60 μU/mL) 2-deoxyglucose (2DOG) uptake in paired (A) soleus and (B) EDL muscles. (C) Insulin-stimulated 2DOGU (calculated as insulin 2DOG uptake – basal 2DOG uptake) in isolated soleus and EDL muscles. n = 6–15/group. *, p < 0.05 vs. CON. Data reported as mean ± SEM.