Metabolic reprogramming involving glycolysis in the hibernating brown bear skeletal muscle.

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.

Circadian Analysis of the Mouse Cerebellum Proteome.

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.

Litter size manipulation in laboratory mice: an example of how proteomic analysis can uncover new mechanisms underlying the cost of reproduction.

BACKGROUND: Life history theories predict that investment in current reproduction comes at a cost for future reproduction and survival. Oxidative stress is one of the best documented mechanisms underlying costs of reproduction to date. However, other, yet to be described molecular mechanisms that play a short term role during reproduction may explain the negative relationships underlying the cost of reproduction. To identify such new mechanisms, we used a global proteomic determination of liver protein profiles in laboratory adult female mice whose litter size had been either reduced or enlarged after birth. This litter size manipulation was expected to affect females by either raising or decreasing their current reproductive effort. At the same time, global parameters and levels of oxidative stress were also measured in all females. RESULTS: Based on plasma analyses, females with enlarged litters exhibited increased levels of oxidative stress at the date of weaning compared to females with reduced litters, while no significant difference was found between both the latter groups and control females. None of the liver proteins related to oxidative balance were significantly affected by the experimental treatment. In contrast, the liver protein profiles of females with enlarged and reduced litters suggested that calcium metabolism and cell growth regulation were negatively affected by changes in the number of pup reared. CONCLUSIONS: Plasma oxidative stress levels in reproductive mice revealed that the degree of investment in reproduction can actually incur a cost in terms of plasmatic oxidative stress, their initial investment in reproduction being close to maximum and remaining at a same level when the energy demand of lactation is reduced. Liver proteomic profiles in reproductive females show that hepatic oxidative stress is unlikely to be involved in the cost of reproduction. Reproductive females rather exhibited liver protein profiles similar to those previously described in laboratory ageing mice, thus suggesting that hepatic cell pro-ageing processes may be involved in the cost of reproduction. Overall, our data illustrate how a proteomic approach can unravel new mechanisms sustaining life-history trade-offs, and reproduction costs in particular.

Mass spectrometry-based sequencing and SRM-based quantitation of two novel vitellogenin isoforms in the leatherback sea turtle (Dermochelys coriacea).

No biomarker has yet been discovered to identify the reproductive status of the endangered leatherback sea turtle (Dermochelys coriacea). Although vitellogenin (VTG) could be used for this, its sequence is not known in D. coriacea and no quantitative assay has been carried out in this species to date. Using de novo sequencing-based proteomics, we unambiguously characterized sequences of two different VTG isoforms that we named Dc-VTG1 and Dc-VTG2. To our knowledge, this is the first clear evidence of different VTG isoforms and the structural characterization of derived yolk proteins in reptiles. This work illustrates how massive de novo sequencing can characterize novel sequences when working on "exotic" nonmodel species in which even nucleotide sequences are not available. We developed assays for absolute quantitation of these two isoforms using selected reaction monitoring (SRM) mass spectrometry, thus providing the first SRM assays developed specifically for a nonsequenced species. Plasma levels of Dc-VTG1 and Dc-VTG2 decreased as the nesting season proceeded, and were closely related to the increased levels of reproductive effort. The SRM assays developed here therefore provide an original and efficient approach for the reliable monitoring of reproduction cycles not only in D. coriacea, but potentially in other turtle species.

Daytime Restricted Feeding Affects Day-Night Variations in Mouse Cerebellar Proteome.

The cerebellum harbors a circadian clock that can be shifted by scheduled mealtime and participates in behavioral anticipation of food access. Large-scale two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry was used to identify day-night variations in the cerebellar proteome of mice fed either during daytime or nighttime. Experimental conditions led to modified expression of 89 cerebellar proteins contained in 63 protein spots. Five and 33 spots were changed respectively by time-of-day or feeding conditions. Strikingly, several proteins of the heat-shock protein family (i.e., Hsp90aa1, 90ab1, 90b1, and Hspa2, 4, 5, 8, 9) were down-regulated in the cerebellum of daytime food-restricted mice. This was also the case for brain fatty acid protein (Fabp7) and enzymes involved in oxidative phosphorylation (Ndufs1) or folate metabolism (Aldh1l1). In contrast, aldolase C (Aldoc or zebrin II) and pyruvate carboxylase (Pc), two enzymes involved in carbohydrate metabolism, and vesicle-fusing ATPase (Nsf) were up-regulated during daytime restricted feeding, possibly reflecting increased neuronal activity. Significant feeding × time-of-day interactions were found for changes in the intensity of 20 spots. Guanine nucleotide-binding protein G(o) subunit alpha (Gnao1) was more expressed in the cerebellum before food access. Neuronal calcium-sensor proteins [i.e., parvalbumin (Pvalb) and visinin-like protein 1 (Vsnl1)] were inversely regulated in daytime food-restricted mice, compared to control mice fed at night. Furthermore, expression of three enzymes modulating the circadian clockwork, namely heterogeneous nuclear ribonucleoprotein K (Hnrnpk), serine/threonine-protein phosphatases 1 (Ppp1cc and Ppp1cb subunits) and 5 (Ppp5), was differentially altered by daytime restricted feeding. Besides cerebellar proteins affected only by feeding conditions or daily cues, specific changes in in protein abundance before food access may be related to behavioral anticipation of food access and/or feeding-induced shift of the cerebellar clockwork.

In-Depth Proteome Analysis Highlights HepaRG Cells as a Versatile Cell System Surrogate for Primary Human Hepatocytes.

Of the hepatic cell lines developed for in vitro studies of hepatic functions as alternatives to primary human hepatocytes, many have lost major liver-like functions, but not HepaRG cells. The increasing use of the latter worldwide raises the need for establishing the reference functional status of early biobanked HepaRG cells. Using deep proteome and secretome analyses, the levels of master regulators of the hepatic phenotype and of the structural elements ensuring biliary polarity were found to be close to those in primary hepatocytes. HepaRG cells proved to be highly differentiated, with functional mitochondria, hepatokine secretion abilities, and an adequate response to insulin. Among differences between primary human hepatocytes and HepaRG cells, the factors that possibly support HepaRG transdifferentiation properties are discussed. The HepaRG cell system thus appears as a robust surrogate for primary hepatocytes, which is versatile enough to study not only xenobiotic detoxification, but also the control of hepatic energy metabolism, secretory function and disease-related mechanisms.

Differential proteomics reveals age-dependent liver oxidative costs of innate immune activation in mice.

UNLABELLED: Individual response to an immune challenge results from the optimization of a trade-off between benefits and costs of immune cell activation. Age-related immune disorders may have several mechanistic bases, from immune cell defects to chronic pro-inflammatory status and oxidative imbalance, but we are still lacking experimental data showing the relative importance of each of these mechanisms. Using a proteomic approach and subsequent biochemical validations of proteomics-derived hypotheses, we found age-dependent regulations in the liver of 3-months and 1-year old-mice in response to an acute innate immune activation. Old mice presented a chronic up-regulation of several proteins involved in pathways related to oxidative stress control. Interestingly, these pathways were weakly affected by the innate immune activation in old compared to young individuals. In addition, old mice suffered from lower glutathione-S-transferase activity and from higher oxidative damage at the end of the experiment, thus suggesting that they paid a higher immune-related cost than young individuals. On the whole, our data showed that a substantial fraction of the liver costs elicited by an activation of the innate immune response is effectively related to oxidative stress, and that ageing impairs the capacity of old individuals to control it. SIGNIFICANCE: Our paper tackles the open question of the cost of mounting an innate immune response. Evolutionary biologists are familiar since a long time with the concept of trade-offs among key traits of an organism, trade-offs that shape life history trajectories of species and individuals, ultimately in terms of reproduction and survival. On the other hand, medicine and molecular biologists study the intimate mechanisms of immune senescence and underline that oxidative imbalance is probably playing a key role in the progressive loss of immune function with age. This paper merges the two fields by exploring the nature of the cellular pathways that are mainly affected by age when the innate immunity is triggered. To this purpose, a proteomic approach was used to explore liver protein profiles and provide for the first time convincing data supporting the idea that oxidative stress constitutes a cost of innate immune response in old mice, possibly contributing to senescence. Proteomics-derived hypotheses were furthermore validated using biochemical assays. This paper therefore illustrates the added value of using proteomics to answer evolutionary biology questions, and opens a promising way to study the inter-specific variability in the rates of immune-senescence.