Dataset Information

Changes in the phosphoproteome of brown adipose tissue during hibernation in the ground squirrel, Ictidomys tridecemlineatus.

ABSTRACT: Mammalian hibernation is characterized by metabolic rate depression and a strong decrease in core body temperature that together create energy savings such that most species do not have to eat over the winter months. Brown adipose tissue (BAT), a thermogenic tissue that uses uncoupled mitochondrial respiration to generate heat instead of ATP, plays a major role in rewarming from deep torpor. In the present study we developed a label-free liquid chromatography mass spectrometry (LC-MS) strategy to investigate both differential protein expression and protein phosphorylation in BAT extracts from euthermic vs. hibernating ground squirrels (Ictidomys tridecemlineatus). In particular, we incorporated the filter-assisted sample preparation protocol, which provides a more in-depth analysis compared with gel-based and other LC-MS proteomics approaches. Surprisingly, mitochondrial membrane and matrix protein expression in BAT was largely constant between active euthermic squirrels and their hibernating counterparts. Validation by immunoblotting confirmed that the protein levels of mitochondrial respiratory chain complexes were largely unchanged in hibernating vs. euthermic animals. On the other hand, phosphoproteomics revealed that pyruvate dehydrogenase (PDH) phosphorylation increased during squirrel hibernation, confirmed by immunoblotting with phospho-specific antibodies. PDH phosphorylation leads to its inactivation, which suggests that BAT carbohydrate oxidation is inhibited during hibernation. Phosphorylation of hormone-sensitive lipase (HSL) was also found to increase during hibernation, suggesting that HSL would be active in BAT to produce the fatty acids that are likely the primary fuel for thermogenesis upon arousal. Increased perilipin phosphorylation along with that of a number of other proteins was also revealed, emphasizing the importance of protein phosphorylation as a regulatory mechanism during mammalian hibernation.

SUBMITTER: Herinckx G

PROVIDER: S-EPMC5625268 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Changes in the phosphoproteome of brown adipose tissue during hibernation in the ground squirrel, <i>Ictidomys tridecemlineatus</i>.

Herinckx Gaëtan G Hussain Nusrat N Opperdoes Fred R FR Storey Kenneth B KB Rider Mark H MH Vertommen Didier D

Physiological genomics 20170710 9

Mammalian hibernation is characterized by metabolic rate depression and a strong decrease in core body temperature that together create energy savings such that most species do not have to eat over the winter months. Brown adipose tissue (BAT), a thermogenic tissue that uses uncoupled mitochondrial respiration to generate heat instead of ATP, plays a major role in rewarming from deep torpor. In the present study we developed a label-free liquid chromatography mass spectrometry (LC-MS) strategy t ...[more]

PMID: 28698229

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Project description:BackgroundThirteen-lined ground squirrels (Ictidomys tridecemlineatus) experience dramatic changes in physiological and molecular parameters during winter hibernation. Notably, these animals experience reduced blood circulation during torpor, which can put numerous stresses on their hearts. The present study evaluates the role played by the epidermal growth factor receptor (EGFR) in signal transduction during hibernation at low body temperature to evaluate signaling mechanisms. By investigating the regulation of intracellular mitogen activated protein kinase (MAPK) pathway responses, anti-apoptosis signals, downstream transcription factors, and heat shock proteins in cardiac muscle we aim to determine the correlation between upstream tyrosine phosphorylation events and downstream outcomes.MethodsProtein abundance of phosphorylated EGFR, MAPKs and downstream effector proteins were quantified using immunoblotting and Luminex® multiplex assays.ResultsMonitoring five time points over the torpor/arousal cycle, EGFR phosphorylation on T654, Y1068, Y1086 was found to increase significantly compared with euthermic control values particularly during the arousal process from torpor, whereas phosphorylation at Y1045 was reduced during torpor. Phosphorylation of intracellular MAPK targets (p-ERK 1/2, p-JNK, p-p38) also increased strongly during the early arousal stage with p-p38 levels also rising during prolonged torpor. However, of downstream MAPK effector kinases that were measured, only p-Elk-1 levels changed showing a decrease during interbout arousal (IA). Apoptosis markers revealed a strong reduction of the pro-apoptotic p-BAD protein during entrance into torpor that remained suppressed through torpor and IA. However, active caspase-9 protein rose strongly during IA. Levels of p-AKT were suppressed during the transition phases into and out of torpor. Of four heat shock proteins assessed, only HSP27 protein levels changed significantly (a 40% decrease) during torpor.ConclusionWe show evidence of EGFR phosphorylation correlating to activation of MAPK signaling and downstream p-ELK1 suppression during hibernation. We also demonstrate a reduction in p-BAD mediated pro-apoptotic signaling during hibernation with active caspase-9 protein levels increasing only during IA. I. tridecemlineatus has natural mechanisms of tissue protection during hibernation that is largely due to cellular regulation through phosphorylation-mediated signaling cascade. We identify a possible link between EGFR and MAPK signaling via p-ERK, p-p38, and p-JNK in the cardiac muscle of these hibernating mammals that correlates with an apparent reduction in caspase-9 apoptotic signaling. This reveals a piece of the mechanism behind how these mammals are resilient to cardiac stresses during hibernation that would otherwise be damaging.

Project description:Background:Inflammation is generally suppressed during hibernation, but select tissues (e.g. lung) have been shown to activate both antioxidant and pro-inflammatory pathways, particularly during arousal from torpor when breathing rates increase and oxidative metabolism fueling the rewarming process produces more reactive oxygen species. Brown and white adipose tissues are now understood to be major hubs for the regulation of immune and inflammatory responses, yet how these potentially damaging processes are regulated by fat tissues during hibernation has hardly been studied. The advanced glycation end-product receptor (RAGE) can induce pro-inflammatory responses when bound by AGEs (which are glycated and oxidized proteins, lipids, or nucleic acids) or damage associated molecular pattern molecules (DAMPs, which are released from dying cells). Methods:Since gene expression and protein synthesis are largely suppressed during torpor, increases in AGE-RAGE pathway proteins relative to a euthermic control could suggest some role for these pro-inflammatory mediators during hibernation. This study determined how the pro-inflammatory AGE-RAGE signaling pathway is regulated at six major time points of the torpor-arousal cycle in brown and white adipose from a model hibernator, Ictidomys tridecemlineatus. Immunoblotting, RT-qPCR, and a competitive ELISA were used to assess the relative gene expression and protein levels of key regulators of the AGE-RAGE pathway during a hibernation bout. Results:The results of this study revealed that RAGE is upregulated as animals arouse from torpor in both types of fat, but AGE and DAMP levels either remain unchanged or decrease. Downstream of the AGE-RAGE cascade, nfat5 was more highly expressed during arousal in brown adipose. Discussion:An increase in RAGE protein levels and elevated mRNA levels of the downstream transcription factor nfat5 during arousal suggest the pro-inflammatory response is upregulated in adipose tissue of the hibernating ground squirrel. It is unlikely that this cascade is activated by AGEs or DAMPs. This research sheds light on how a fat-but-fit organism with highly regulated metabolism may control the pro-inflammatory AGE-RAGE pathway, a signaling cascade that is often dysregulated in other obese organisms.

Dataset Information

Changes in the phosphoproteome of brown adipose tissue during hibernation in the ground squirrel, Ictidomys tridecemlineatus.

Publications

Changes in the phosphoproteome of brown adipose tissue during hibernation in the ground squirrel, <i>Ictidomys tridecemlineatus</i>.

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