Muscle Arnt/Hif1β Is Dispensable in Myofiber Type Determination, Vascularization and Insulin Sensitivity

Abstract

Aryl Hydrocarbon Receptor Nuclear Translocator/ hypoxia-inducible factor 1 beta (ARNT/ HIF1β), a member of bHLH-PAS family of transcriptional factors, plays a critical role in metabolic homeostasis, insulin resistance and glucose intolerance. The contributions of ARNT in pancreas, liver and adipose tissue to energy balance through gene regulation have been described. Surprisingly, the impact of ARNT signaling in the skeletal muscles, one of the major organs involved in glucose disposal, has not been investigated, especially in type II diabetes. Here we report that ARNT is expressed in the skeletal muscles, particularly in the energy-efficient oxidative slow-twitch myofibers, which are characterized by increased oxidative capacity, mitochondrial content, vascular supply and insulin sensitivity. However, muscle-specific deletion of ARNT did not change myofiber type distribution, oxidative capacity, mitochondrial content, capillarity, or the expression of genes associated with these features. Consequently, the lack of ARNT in the skeletal muscle did not affect weight gain, lean/fat mass, insulin sensitivity and glucose tolerance in lean mice, nor did it impact insulin resistance and glucose intolerance in high fat diet-induced obesity. Therefore, skeletal muscle ARNT is dispensable for controlling muscle fiber type and metabolic regulation, as well as diet-induced weight control, insulin sensitivity and glucose tolerance.

Fig 1. ARNT expression in different tissues.

(A) ARNT protein expression in different organs [sub-cutaneous adipose tissue (Sc), perigonadic adipose tissue (Pg), brown adipose tissue (Bat), heart (He), liver (Li), brain (Br), kidney (Ki), pancreas (Pa), gastrocnemius muscle (Ga)] of 4 months old mice (N = 1). (B) Arnt gene expression in the soleus and the extensor digitorum longus (EDL) of 3 month old mice (N = 4–5). (C-D) ARNT expression in control and MKO muscle groups ranging from the most oxidative (soleus) to most glycolytic (EDL) (N = 3). (C) Representative images. (D) Densitometry for protein expression. (E) Arnt gene expression in EDL control and MKO muscles of 4 months old mice (N = 4–5). (*p<0.05,**p<0.01,***p<0.001, Unpaired Student’s t-test or One-way ANOVA with Tukey’s multiple comparison post-hoc test.)

Fig 2. Characterization of the MKO mice.

Following parameters were measured in wild type control and MKO mice. (A) Weight gain measured from 5 to 20 weeks of age (N = 10–12). (B-C) Body composition measured as fat mass (B) and lean mass (C) (N = 4). (D) H&E staining showing histomorphology of the tibialis anterior (TA) muscle cross-sections (N = 4). (Scale bar = 500 μm.) (***p<0.001, Unpaired Student’s t-test.)

Fig 3. Fiber type and oxidative capacity in NCD-fed mice.

Following parameters were measured in 4 months old control and MKO mice on NCD. (A) Representative images of medial (left) and outer (right) TA cross-sections stained for MyHC IIA (green), IIX (red), and IIB (purple) (N = 4–6). (B) Mitochondrial DNA content in EDL (N = 4–6). (C-D) Succinate dehydrogenase (SDH) staining activity in cross-sections of TA. (C) Representative cross-sectional images of the outer and medial TA (N = 4–6). (D) Percentage of positive fibers quantified in the outer and medial TA cross-sections for the SDH staining (N = 4–6). (E) Representative cross-sectional images of the outer and medial TA with NADH-TR staining (N = 4–6). (Scale bar = 200 μm.) The differences between groups were not statistically significant (Unpaired Student’s t-test).

Fig 4. Fiber type and oxidative capacity in HFD-fed mice.

Following parameters were measured in 5 months old control and MKO mice on HFD. (A) Representative images of medial (left) and outer (right) TA cross-sections stained for MyHC IIA (green), IIX (red), and IIB (purple) (N = 6–7). (B) Mitochondrial DNA content measured in EDL (N = 6–7). (C-D) SDH staining activity in TA. (C) Representative images of the outer and medial TA muscle (N = 6–7). (D) Percentage of positive myofibers measured in the outer and medial TA for the SDH staining (N = 6–7). (E) Representative images of the NADH-TR staining activity in the outer and medial TA muscle (N = 6–7). (Scale bar = 200 μm.) The differences between groups were not statistically significant (Unpaired Student’s t-test).

Fig 5. Insulin and Glucose Tolerance.

Following parameters were measured in control and MKO mice on HFD. (A) Weight gain (N = 6–12). (B) Fat mass in 4 month old mice (N = 6–12). (C) Lean mass in 4 month old mice (N = 6–12). (D) Insulin tolerance test (ITT) in 4 months old mice (N = 6–11). (E) Area Above the Curve (AAC) for ITT. (F) Glucose tolerance test (GTT) in 4 months old mice (N = 6–11). (G) Area Under the Curve (AUC) for GTT. (H) Ex vivo p-AKT ser473/panAKT stimulation by insulin measured in the gastrocnemius muscles of the 5 months old mice (N = 3–5). $ indicates the treatment effect; * indicate the genotype effect. $ p<0.05; $ $ $/*** p<0.001 (Unpaired Student’s t-test or Two-way ANOVA with a Bonferroni’s repeated measure test).