Metabolic reprogramming involving glycolysis in the hibernating brown bear skeletal muscle

Abstract

Background: In mammals, the hibernating state is characterized by biochemical adjustments, which include metabolic rate depression and a shift in the primary fuel oxidized from carbohydrates to lipids. A number of studies of hibernating species report an upregulation of the levels and/or activity of lipid oxidizing enzymes in muscles during torpor, with a concomitant downregulation for glycolytic enzymes. However, other studies provide contrasting data about the regulation of fuel utilization in skeletal muscles during hibernation. Bears hibernate with only moderate hypothermia but with a drop in metabolic rate down to ~ 25% of basal metabolism. To gain insights into how fuel metabolism is regulated in hibernating bear skeletal muscles, we examined the vastus lateralis proteome and other changes elicited in brown bears during hibernation.

Results: We show that bear muscle metabolic reorganization is in line with a suppression of ATP turnover. Regulation of muscle enzyme expression and activity, as well as of circulating metabolite profiles, highlighted a preference for lipid substrates during hibernation, although the data suggested that muscular lipid oxidation levels decreased due to metabolic rate depression. Our data also supported maintenance of muscle glycolysis that could be fuelled from liver gluconeogenesis and mobilization of muscle glycogen stores. During hibernation, our data also suggest that carbohydrate metabolism in bear muscle, as well as protein sparing, could be controlled, in part, by actions of n-3 polyunsaturated fatty acids like docosahexaenoic acid.

Conclusions: Our work shows that molecular mechanisms in hibernating bear skeletal muscle, which appear consistent with a hypometabolic state, likely contribute to energy and protein savings. Maintenance of glycolysis could help to sustain muscle functionality for situations such as an unexpected exit from hibernation that would require a rapid increase in ATP production for muscle contraction. The molecular data we report here for skeletal muscles of bears hibernating at near normal body temperature represent a signature of muscle preservation despite atrophying conditions.

Keywords: Brown bears; Enzymology; Glycolysis; Hibernation; Lipid oxidation; Metabolism shift; Omics; Skeletal muscle.

Fig. 1

Overview of bear muscle proteomic response to hibernation. Changes in the proteome of brown bear vastus lateralis muscle between active (summer) and hibernating (winter) periods (N = 7 per season) are shown as heatmaps of differentially expressed proteins that were produced by hierarchical clustering from the MS1 quantitative-based (panel a) and 2D-DIGE-based (panel b) analyses. Signal values between animals from the two seasons were successfully discriminated (green, black and red boxes represent downregulated, intermediate and upregulated proteins, respectively). Functional annotation analysis from differential proteins revealed enriched Gene Ontology terms, which allowed determination of broad functions significantly affected by hibernation (panel c; filled circles represent the broad functions depicted by proteins that are discussed in this paper). Detailed protein abundances and fold changes are given in Additional file 1: Table S1 and Additional file 2: Table S2. AA: amino acid metabolism; Dev.: development & differentiation; ECM: extracellular matrix; Hyp.: response to hypoxia; Ox. Stress: response to oxidative stress

Fig. 2

Regulation of metabolism-related factors in hibernating bear muscles. The relative abundance of muscle (vastus lateralis) proteins in winter (hibernating) versus summer (active) brown bears (N = 7 per season) is shown using the following colour code: significantly (Welch student and paired Student tests; P < 0.05) up- and down-regulated proteins are shown in red and green boxes, respectively; black boxes show proteins that did not change, and white boxes show proteins that were not detected (in black letters) or those for which slightly less reproducible up- (in red) or down- (in green) regulation was recorded. FABP3 (in orange) was identified in two distinct protein spots (2D-DIGE strategy) that exhibited opposite responses, suggesting the possible occurrence of post-translational modifications. Detailed protein abundances and fold changes are given in Additional file 1: Table S1 and Additional file 2: Table S2. WAT: white adipose tissue

Fig. 3

Repression of lipid metabolism and mitochondrial oxidative phosphorylation in winter bear muscles. Protein or mRNA expression levels of fatty acid translocase (CD36), mitochondrial hydroxyacyl-coenzyme A dehydrogenase (HADH), citrate synthase (CS) and subunits of the five oxidative phosphorylation (OXPHOS) complexes (C-I to C-V) were measured using RT-qPCR and/or Western-blot analysis in vastus lateralis muscle samples from brown bears in summer (white bars) and winter (black bars) (panels A and B; N = 8-12 per season). Corresponding blots are shown in Additional file 4: Figure S2. Maximum enzymatic activities (panels A and C) for HADH, CS and cytochrome c oxidase (COX) were measured at 33 °C and 37 °C (N = 7 per season). Data are expressed as means ± sem. Statistical significance is shown for paired student t-tests (* P < 0.05; ** P < 0.01) or post-hoc Tukey tests that followed type III ANOVA (values that do not share the same superscript letter are significantly different; P < 0.05)

Fig. 4

Changes related to carbohydrate metabolism in winter bears. Maximum enzymatic activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH) were measured in vastus lateralis muscle samples from brown bears in summer (white bars) and winter (black bars) at 33 °C and 37 °C (panel A; N = 7 per season). Gene expression levels of monocarboxylate transporter 1 (MCT1 or SLC16A1), monocarboxylate transporter 4 (MCT4 or SLC16A3), and aquaporin 7 (AQP7) were measured using RT-qPCR in bear vastus lateralis muscles (panel B and C; N = 8 per season). Data are expressed as means ± sem. Statistical significance is shown for paired student t-tests (** P < 0.01) and post-hoc Tukey tests that followed type III ANOVA (values that do not share the same superscript letter are significantly different; P < 0.05). Circulating levels of glycerol were assessed enzymatically in bear plasma using a commercial kit (panel C; N = 25 per season) and presented as individual values (circles, those in red correspond to plasma samples where glycerol was also measured using NMR-based analysis with values being shown in Table 1) along with the mean ± sem (in blue) and median (in green) values

Fig. 5

Muscle glycogen content is increased and serum fatty acid profiles are changed in winter bear muscles. As illustrated on representative electron micrographs of brown bear vastus lateralis muscle (panel A), intramyofibrillar (green) and especially intermyofibrillar (yellow) glycogen granules accumulate in skeletal muscle of hibernating bears (lower panel), whereas their presence was largely undetectable in active summer bears (upper panel) at this magnification level. Glycogen content was measured in bear muscles (panel B; N = 7 per group). Circulating levels of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were assessed in bear serum (panel C; N = 4 serum mixes per season). Data are expressed as means ± sem. Statistical significance is shown for paired student t-tests (* P < 0.05; ** P < 0.02) and post-hoc Tukey tests that followed one-way ANOVA (values that do not share the same superscript letter are significantly different; P < 0.05)