Increased Reliance on Muscle-based Thermogenesis upon Acute Minimization of Brown Adipose Tissue Function

Abstract

Skeletal muscle has been suggested as a site of nonshivering thermogenesis (NST) besides brown adipose tissue (BAT). Studies in birds, which do not contain BAT, have demonstrated the importance of skeletal muscle-based NST. However, muscle-based NST in mammals remains poorly characterized. We recently reported that sarco/endoplasmic reticulum Ca(2+) cycling and that its regulation by SLN can be the basis for muscle NST. Because of the dominant role of BAT-mediated thermogenesis in rodents, the role of muscle-based NST is less obvious. In this study, we investigated whether muscle will become an important site of NST when BAT function is conditionally minimized in mice. We surgically removed interscapular BAT (iBAT, which constitutes ∼70% of total BAT) and exposed the mice to prolonged cold (4 °C) for 9 days. The iBAT-ablated mice were able to maintain optimal body temperature (∼35-37 °C) during the entire period of cold exposure. After 4 days in the cold, both sham controls and iBAT-ablated mice stopped shivering and resumed routine physical activity, indicating that they are cold-adapted. The iBAT-ablated mice showed higher oxygen consumption and decreased body weight and fat mass, suggesting an increased energy cost of cold adaptation. The skeletal muscles in these mice underwent extensive remodeling of both the sarcoplasmic reticulum and mitochondria, including alteration in the expression of key components of Ca(2+) handling and mitochondrial metabolism. These changes, along with increased sarcolipin expression, provide evidence for the recruitment of NST in skeletal muscle. These studies collectively suggest that skeletal muscle becomes the major site of NST when BAT activity is minimized.

Keywords: brown adipose tissue; calcium transport; cold adaptation; core body temperature; mitochondria; mitochondrial dynamics; mitochondrial metabolism; sarcoplasmic reticulum (SR); skeletal muscle.

FIGURE 1.

Body temperature maintenance, physical activity, and oxygen consumption in iBAT-ablated mice. A, iBAT-ablated mice (−BAT, n = 8) were able to maintain body temperature similar to sham controls (+BAT, n = 8) when housed at 29.0 ± 1.0 °C (green line) and at 22.0 ± 1.0 °C (blue line). Upon exposure to 4.0 °C (brown line) the −iBAT group maintained a slightly lower body temperature 0.8 ± 0.3 °C (inset) compared with the sham group during the first 4 h (black box). B, physical activity was measured at various housing temperatures. At 29.0 ± 1.0 °C (first inset) and 22.0 ± 1.0 °C (second inset), there was no significant difference in activity between the −iBAT and +iBAT groups. At 4 °C, the −iBAT group exhibited higher local activity with increased grooming behavior (third inset) for 3 days. Gradually, at 4 °C, they resumed long-range activity, and by day 8, physical activity of both the groups did not show any statistical difference (fourth inset). Unpaired Student's t test was applied. ***, p < 0.00001; ns, p > 0.05 (nonsignificant). C, VO₂ of the −iBAT (iBAT ablated) group was similar to that of the +iBAT (sham) group at housing temperatures of 29.0 ± 1.0 °C (first inset) and 22.0 ± 1.0 °C (second inset). Upon acute cold (4 °C) challenge, the −iBAT group up-regulated VO₂ significantly compared with the +iBAT group on the first day (third inset). By day 8 of cold adaptation, VO₂ of the −iBAT group was still higher than that of controls (fourth inset). Unpaired Student's t test was applied. *, p < 0.05; ****, p < 0.00001; ns, p < 0.05 (nonsignificant).

FIGURE 2.

The effect of iBAT-ablation on food intake, body weight, fat mass, and serum metabolite levels. A, both the −iBAT and +iBAT groups consumed similar amounts of food when housed at 29.0 ± 1.0 °C and 22.0 ± 1.0 °C. On the first day at 4 °C, the − iBAT group consumed significantly more food than the +iBAT group, but there was no difference in food take by day 8. B, both the −iBAT and +iBAT groups weighed similarly at 29.0 ± 1.0 °C and 22.0 ± 1.0 °C. However, the −iBAT group lost a significant amount of weight compared with the +iBAT group after 9 days of cold adaptation. C, both the −iBAT and +iBAT groups had similar amounts of intraperitoneal fat mass at 29.0 ± 1.0 °C and 22.0 ± 1.0 °C, whereas, after 9 days of cold adaptation, the −iBAT group lost significant white fat. Two-way analysis of variance employing Tukey's multiple comparison tests were performed for statistical analysis. D, serum metabolites after cold adaptation. Glucose was higher and serum triglyceride was lower in iBAT-ablated mice. Serum ketone was found to be higher but did not reach statistical significance. Total cholesterol in serum was not altered. All data presented here were averaged from at least five mice. *, p < 0.05; ***, p < 0.0001; ns, p > 0.05 (nonsignificant).

FIGURE 3.

Cold adaptation modifies the expression of SERCA isoforms and increases the SLN level in skeletal muscle. A, cold induces a switch in the expression of SERCA isoforms in gastrocnemius muscle. SERCA 2a is up-regulated, but SERCA 1a is down-regulated. Expression of both calsequestrin isoforms (skeletal, CASQ 1, and cardiac, CASQ 2) involved in Ca²⁺ buffering was not altered. B, the levels of SERCA 1a and SERCA 2a were quantified using ImageJ software and normalized to the GAPDH level. GAPDH was used as a loading control. **, p < 0.001; ns, not significant. C, SLN, but not PLB, is induced during cold adaptation in the red portion of gastrocnemius. SLN expression was further up-regulated in cold-adapted iBAT-ablated mice. PLB remained undetectable in skeletal muscle even after cold exposure and iBAT removal. Samples were boiled at 95 °C for 5 min before separation on gels for detecting PLB. D, SLN level normalized to GAPDH level. All Western blotting experiments were repeated at least three times, and data were included for statistical analysis. E, cold adaptation increased SERCA activity (ATP hydrolysis) and showed a trend toward further enhancement upon iBAT ablation. Unpaired Student's t test was applied. *, p < 0.05; **, p < 0.05; ***, p < 0.01; ****, p < 0.001; ns, p > 0.05 (nonsignificant).

FIGURE 4.

Cold adaptation increases RyR1 expression and phosphorylation in skeletal muscle. A, expression of total CaMKIIα and Thr(P)²⁸⁶-CaMKIIα was measured in skeletal muscles from the cold-adapted mice. B, the level of Thr(P)²⁸⁶-CaMKIIα was measured and normalized using values from total CaMKIIα. C, conservation of sequences around potential CaMKII phosphorylation sites from RyR 1 and 2 isoforms from various vertebrate species. The percentage of occurrence of residues is shown as probability in the y axis. Acidic residues are shown in red, and basic residues are shown in blue. PKA and CaMKII phosphorylation sites on RyR 2 (Ser²⁸⁰⁸ and Ser²⁰¹⁴) are indicated with downward arrows. These residues correspond to Ser²⁸⁴⁴ and Thr²⁸⁴⁸, respectively, on RyR 1. D, cold adaptation leads to increased expression and phosphorylation (at Thr²⁸⁴⁸) of RyR 1. iBAT-ablated mice showed higher p-RyR 1-Thr²⁸⁴⁸ levels, indicating a higher SER Ca²⁺ leak. The Ponceau S (Pon S)-stained membrane is shown as a loading control. E, the density of total RyR1 and p-RyR 1-Thr²⁸⁴⁸ was quantified and plotted. All Western blotting experiments were repeated at least twice, and data were included for statistical analysis. Unpaired Student's t test was applied. ns, p > 0.05; *, p < 0.05; **, p < 0.001; ***, p < 0.0001.

FIGURE 5.

iBAT ablation causes a significant up-regulation of oxidative metabolism in skeletal muscle. A, low magnification electron micrographs of cold-adapted TA, a fast-twitch skeletal muscle. Removal of iBAT leads to an increase in the abundance of intramyofibrilar mitochondria and lipid droplets (yellow arrowheads). B, high-magnification electron micrographs of cold-adapted skeletal muscles. The intramyofibrilar mitochondria in the skeletal muscles from iBAT-ablated mice are ultrastructurally much better organized with densely packed cristae. C, representative images showing SDH activity in cold-adapted TA-skeletal muscle samples. The −iBAT group shows denser staining, indicating higher SDH activity, suggesting higher oxidative metabolism. D, muscles from iBAT-ablated mice show higher oxygen consumption. Glut, glutamate; Mal, malate; wt, weight. E, representative Western blot showing the expression of the electron transport complex proteins peroxisome proliferator-activated receptor β, lipoprotein lipase, CD36, and citrate synthase (CS) in gastrocnemius muscle. 20 μg of muscle homogenate was analyzed. F, densitometric quantification of the proteins shown in E. The levels of complexes II, III, IV, and ATP synthase (V) were higher in muscles from sham mice compared with controls from 22 °C. The levels of complexes I, III, and V were higher in muscles from iBAT-ablated mice compared with sham controls. Values are normalized to the GAPDH level. All Western blotting experiments were repeated at least twice, and data were included for statistical analysis. Unpaired Student's t test was applied. *, p < 0.05; **, p < 0.001; ***, p < 0.0001; ns, p < 0.05 (nonsignificant).

FIGURE 6.

Increased expression of proteins associated with mitochondrial fusion in iBAT-ablated mice. A, Mfn 1, Mfn 2, and Opa 1 associated with mitochondrial fusion were up-regulated during cold adaptation and further increased upon removal of iBAT. B, densitometric quantification of mitochondrial fusion proteins. The values were normalized to GAPDH. C, the expression of mitochondrial fission proteins was not altered in skeletal muscle. D, cold adaptation and iBAT removal did not alter level of mitochondrial fission proteins. E, the mitochondrial proteins VDAC 1 (Ca²⁺ channel), ANT 1 (ATP transporter), and TFAM (regulator of mitochondrial DNA synthesis) were increased. F, quantification of VDAC 1, TFAM, and ANT 1. All Western blotting experiments were repeated at least three times, and data were included for statistical analysis. Values were normalized to the GAPDH level. Unpaired Student's t test was applied. *, p < 0.05; **, p < 0.001; ***, p < 0.0001; ns, p < 0.05 (nonsignificant).

FIGURE 7.

Schematic showing the structural remodeling of the SR and mitochondria during adaptation to cold in iBAT-ablated mice. iBAT ablation leads to significant recruitment of skeletal muscle-based NST during cold adaptation. NST in muscle relies on increased heat production through uncoupling of SERCA activity by SLN. Increased SLN/SERCA interaction promotes uncoupling of SERCA activity, leading to heat production but creating increased energy demand. To support the increased energy demand, there is remodeling of mitochondria and the SR structure, including increased mitochondrial size (elaborate cristae), up-regulation of oxidative phosphorylation proteins, and increased SR-mitochondrial contacts that will allow increased Ca²⁺ influx into mitochondria, leading to elevated ATP production. In addition, SLN uncoupling of SERCA results in increased local cytosolic Ca²⁺ concentration, thereby activating Ca²⁺-dependent signaling pathways, including CAMKII, known to hyperphosphorylate RyR. Phosphorylation of RyR1 leads to spontaneous Ca²⁺ release from the SER, leading to increased SERCA activity, ATP hydrolysis, and heat production. This model proposes how increased Ca²⁺ leaking from the SR and reuptake of Ca²⁺ by SERCA can provide the basis for NST in muscle.