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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.
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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.
The aim of the present study was to apply quantitative proteomics and biochemistry to brown bear skeletal muscle to test the hypothesis that changes in enzyme abundance and activity correspond to the expected decrease in metabolic rate and to determine if they could help to identify muscle fuel preferences (i.e. oxidation of lipid versus carbohydrate substrates). The results show that changes in enzyme activities do indeed appear to be relevant targets to gain insights into the biochemical mechanisms involved in metabolism suppression during hibernation. The metabolic reprogramming in muscles of hibernating brown bears does involve glycolysis although lipids remain the preferred fuels but with their rate of oxidation being reduced due to metabolic rate depression. Such regulations favour energy savings and the maintenance of muscle proteins in bears hibernating at a core body temperature that remains close to that of the summer-active period.
From label-free quantitative proteomics data (XIC), statistical analysis highlighted significant seasonal effects in the abundance of 146 muscle proteins, 67 of them being decreased and 79 increased in hibernating versus active bears (Fig. 1a, see also Additional file 1: Table S1).
Importantly, the overlap of quantitative data between the two proteomics approaches was highly consistent, as all of the eight commonly identified proteins exhibited very similar seasonal effects (Additional file 2: Table S2). From a merged list of differentially-expressed proteins coming from the two proteomics approaches, functional annotation analysis revealed that differences between hibernating and active bears involved mostly proteins known to play roles in muscle metabolism in a broad sense and in structural remodelling (Fig. 1c). Based on these data, we decided to focus our attention and extend the analysis for a more in-depth examination of muscle fuel metabolism.
Regarding muscle lipid metabolism, the expression levels of only two proteins remained essentially unchanged in winter versus summer bears, namely fatty acid translocase (CD36) and mitochondrial carnitine/acylcarnitine carrier protein (SLC25A20). Fatty acid binding protein 3 (FABP3) was detected in two different protein spots on 2D-DIGE gels, one exhibiting significantly lower (1.3 times) and the other higher (1.2 times) intensity in hibernating versus active bears. All other proteins involved in fatty acid beta-oxidation were found at significantly lower levels during hibernation, including long chain fatty acid-CoA ligase 1 (ACSL1; 2.2 times), carnitine O-palmitoyltransferases 1 (CPT1B; 3.4 times) and 2 (CPT2; 1.2 times), short/branched chain specific acyl-CoA dehydrogenase (ACADSB; 1.5 times), enoyl-CoA delta isomerases 1 (ECI1; 1.5 times) and 2 (ECI2; not detectable in winter), trifunctional enzyme subunit alpha (HADHA; 1.3 times) and beta (HADHB; 1.4 times), 3-hydroxyacyl-CoA dehydrogenase type-2 (HSD17B10; 1.6 times), enoyl-CoA hydratase (ECHS1; 1.5 times), hydroxyacyl-CoA dehydrogenase (HADH; 1.4 times), 3-ketoacyl-CoA thiolase (ACAA2; 1.5 times), and acetyl-CoA acetyltransferase (ACAT1; 1.6 times).
Similarly, all the proteins of the tricarboxylic acid cycle were downregulated in hibernating bear muscle, including citrate synthase (CS; 1.5 times), aconitate hydratase (ACO2; 1.5 times), isocitrate dehydrogenase (IDH2; 1.4 times) and isocitrate dehydrogenase subunits alpha, (IDH3A; 1.8 times), beta (IDH3B; 1.7 times) and gamma (IDHG; 2 times), 2-oxoglutarate dehydrogenase (OGDH; 1.4 times), succinyl-CoA ligase subunits alpha and beta (SUCA and SUCB1; 1.5 times), fumarate hydratase (FH; 1.5 times), and malate dehydrogenase (MDH2; 1.4 times).
Mitochondrial membrane respiratory chain protein complexes I to V are each composed of multiple different subunits that were similarly downregulated in hibernating bear muscles, including several subunits of NADH dehydrogenase (NDUFA9, NDUFA10, NDUFB9, NDUFB10, NDUFS1, NDUFS2, NDUFS3, NDUFS8, NDUFV1, NDUFV2; 1.4-2.1 times; as well as MT-ND5 not detectable in winter), succinate dehydrogenase (SDHA and SDHB; 1.3-1.4 times), cytochrome c reductase (UQCRC2, CYC1 and UQCRFS1; 1.3-1.5 times), cytochrome c oxidase (COX2 and COX5A; 1.4-2.7 times), and ATP synthase (ATP5A1, ATP5B, ATP5C1, ATP5F1, ATP5H and ATP5O; 1.2-2.7 times). Six other NADH dehydrogenase subunits, one other subunit of cytochrome c reductase, and two other ATP synthase subunits were detected and also showed a reduced trend in muscle of hibernating bears, although differences were not statistically significant. Levels of other proteins closely related to the mitochondrial respiratory chain and/or membrane potential were also reduced in winter bears, including phosphate carrier protein (SLC25A3; 1.9 times), ADP/ATP translocases 1 and 3 (SLC25A4 or ANT1 and SLC25A6 or ANT3; 1.4-1.7 times), NAD(P) transhydrogenase (NNT; 2 times), and mitochondrial carrier homolog 2 (MTCH2; 1.6 times). Finally, the abundance of mitochondrial creatine kinase was decreased during hibernation (CKMT2; 1.5 times), as well as that of voltage-dependent anion-selective channel proteins 1 and 2 (VDAC1 and VDAC2; 1.5 times), whereas voltage-dependent anion-selective channel protein 3 (VDAC3) remained unchanged.
Examining the different pathways of pyruvate metabolism, we observed decreased levels of pyruvate dehydrogenase E1 component subunits alpha and beta (PDHA1 and PDHB; 1.4 times) in winter versus summer bears, whereas increased levels were recorded for pyruvate dehydrogenase kinase isozyme 4 (PDK4; not detectable during summer) and malic enzyme (ME1; 2 times). The levels of lactate dehydrogenase A and B chains (LDHA and LDHB) and of alanine aminotransferase 1 (ALAT1) were not significantly different between seasons. In addition, we observed upregulation of cytosolic malate dehydrogenase (MDH1; 1.3 times) and aspartate aminotransferase (GOT1; 1.3 times) and downregulation of mitochondrial 2-oxoglutarate/malate carrier protein (SLC25A11; 1.6 times), calcium-binding mitochondrial carrier protein Aralar2 (SLC25A13; 1.4 times), and mitochondrial aspartate aminotransferase (GOT1; 1.3 times) in hibernating bears.
Plasma metabolite measurements assayed via enzymatic kits and those derived from nuclear magnetic resonance (NMR) analysis were in good agreement for glucose and lactate levels (Table 1). Thus, glycaemia tended to be higher in winter compared to summer, but the difference did not reach significance. Oppositely, lactate levels were significantly reduced in hibernating bear plasma (1.4-2 times). Free fatty acid levels were increased during hibernation (2.7 times) (Table 1). Concerning glycerol levels, the significant decrease (1.3 times) in winter, measured using NMR-based analysis, was also found using the enzymatic kit when considering exactly the same samples (see Table 1 and the red dots in Fig. 4C). Because the values were markedly variable among individuals, especially in summer, we extended the measurement of glycerol levels to 25 bears and still observed no significant seasonal effect (Table 1 and Fig. 4C). We also observed significantly higher circulating levels of triacylglycerols (1.3 times), 3-hydroxybutyrate (1.7 times), betaine (1.4 times), and creatinine (1.5 times) in hibernating bears. No significant change was recorded for plasma levels of pyruvate, glycine/sarcosine, and myo-inositol.
Concerning plasma amino acids (Table 1), although the 1.2-fold increase in glutamine levels measured using NMR-based analysis was not significant, specific measurements using liquid chromatography-based assay indicated a significant 1.3-fold increase in glutamine levels during the hibernation period. On the contrary, the levels of plasma alanine were significantly decreased (1.4 times) in hibernating bears.
Lipidomics analysis of polyunsaturated fatty acids (PUFA) highlighted significantly reduced levels of eicosapentaenoic acid (20:5n-3; EPA; 3.8 times) and increased levels of docosapentaenoic acid (22:5 n-3; DPA; 2.6 times) and docosahexaenoic acid (22:6 n-3; DHA; 4.7 times) in the serum of hibernating versus active bears (Fig. 5C).
Hibernation is a physiological fasting state where physical inactivity, metabolic rate depression, and a decrease in core body temperature allow for effective fuel and energy savings sufficient to sustain survival over the winter months. In such a metabolic context, energy supply appears to come primarily from lipid substrate oxidation, whereas glucose oxidation is reduced. However, contrasting data exist about the utilization of lipid versus carbohydrate fuels in the muscle of hibernators (see above in the Introduction section). Our data show that expression and activity levels of lipid oxidizing enzymes were lower in hibernating brown bears, whereas the expression levels of muscle glycolysis enzymes were higher during hibernation (see Fig. 2 and Additional file 1: Table S1 and Additional file 2: Table S2). 350c69d7ab