Muscle-specific loss of Bmal1 leads to disrupted tissue glucose metabolism and systemic glucose homeostasis

Abstract

Background: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 (-/-) mouse (iMSBmal1 (-/-)).

Results: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 (-/-) mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 (-/-) muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 (-/-) muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle.

Conclusions: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.

Keywords: Circadian rhythm; Glucose metabolism; Skeletal muscle.

Fig. 1

Body composition but not behavior was altered in the iMSBmal1^−/− mice. a Body weight and composition (lean and fat mass) at 3–5 weeks post-treatment (n = 17 iMSBmal1^+/+, n = 20 iMSBmal1^−/−). b Body weight and composition (lean and fat mass) at 10–12 weeks post-treatment (n = 12 iMSBmal1^+/+, n = 15 iMSBmal1^−/−). c Feeding (4 days) at 5 weeks post-treatment (n = 4 iMSBmal1^+/+, n = 6 iMSBmal1^−/−) and activity (3 days) at 5 and 10 weeks post-treatment (n = 5 iMSBmal1^+/+, n = 5 iMSBmal1^−/−)

Fig. 2

iMSBmal1^−/− mice display altered systemic glucose handling. a Fasted blood glucose (n = 8/group (2 females and 6 males in both groups)). b Fasted blood insulin n = 7/group (3 females and 4 males in both groups). c Glucose tolerance depicted as blood glucose versus time post-glucose injection and area under the curve (n = 8/group (2 females and 6 males) in both groups). d Non-fasted blood glucose measured every 4 h for 24 h [n = 4 per time point: CT18 iMSBmal1^+/+ n = 2 F/2 M; iMSBmal1^−/− n = 2 F/2 M; CT22 iMSBmal1^+/+ n = 4 M; iMSBmal1^−/− n = 2 F/2 M; CT26 iMSBmal1^+/+ n = 2 F/2 M; iMSBmal1^−/− n = 1 F/3 M; CT30 iMSBmal1^+/+ n = 1 F/3 M; iMSBmal1^−/− n = 1 F/3 M; CT34 iMSBmal1^+/+ n = 1 F/3 M; iMSBmal1^−/− n = 1 F/3 M]

Fig. 3

Glucose uptake was diminished in the iMSBmal1^−/− mice. a Insulin-stimulated glucose uptake in the EDL muscles using a maximum insulin dose and 20 min of exposure [n = 8 (2 F/6 M)/group]. b AICAR-stimulated glucose uptake in the EDL muscles after 40 min of exposure incubation media with or without AICAR n = 5 (5 F)/group

Fig. 4

Both mRNA and protein expression of the glucose transporter type 4 (Glut4/GLUT4) were decreased in the iMSBmal1 ^-/- mice. a mRNA expression of Glut4 in the GTN muscle measured using RT PCR. (n = 6). b Protein content of GLUT4 in the GTN muscle was measured as integrated intensity of the fluorescent bands (n = 7)

Fig. 5

Activity of rate-limiting glycolytic enzymes (HK2 and PFK1) was significantly decreased in the iMSBmal1^−/− mice. a mRNA expression of Hk2 obtained from microarray data (n = 6). b Enzymatic activity of the rate-limiting enzyme HK2 in the GTN muscle measured using a spectrophotometric assay (n = 7). c mRNA expression of the rate-limiting enzyme Pfkm in the GTN muscle (n = 7). d Enzymatic activity of PFKM in GTN muscles

Fig. 6

Genes involved in fat metabolism are upregulated in the iMSBmal1^−/− GTN. a Array data revealed that genes important in fat metabolism in the skeletal muscle are significantly upregulated in the iMSBmal1 ^−/− GTN. b Triglyceride levels trended toward a decrease in the iMSBmal1 ^−/− GTN

Fig. 7

Summary of changes linked to substrate oxidation in the skeletal muscle. Metabolomics data, enzymatic activity data, glycogen data, and triglyceride data are included. Green upregulated, red downregulated, green italicized trending up, red italicized trending down, and black no change