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 (). 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.

Cytoprotection by a naturally occurring variant of ATP5G1 in Arctic ground squirrels

“…Mitochondrial metabolic dysfunction is central to ischemia and reperfusion injury. Physiologic, transcriptomic, and proteomic studies have highlighted the importance of ketone and fatty acid metabolism in hibernating states (37, 38) as well as pointed to a role for specific post-translational protein modifications in the differential regulation of metabolic pathways in hibernation (10, 39, 40). We expanded this body of knowledge, by identifying altered mitochondrial dynamics and enhanced spare respiratory capacity in cells of hibernating species as potentially adaptive cellular mechanisms in hibernating animals.…”

Hepatic lipid droplet breakdown through lipolysis during hibernation in Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

“…Besides, another brown bear paper in subcutaneous adipose tissue showed that increased expression of negative regulators of lipolysis could promote weight gain in summer [27]. As a thermogenic organ for maintaining body temperature, brown adipose tissue (BAT) has been found in the hibernators, such as thirteen-lined ground squirrel ( Ictidomys tridecemlineatus ) [32] and been implicated in therapy for obesity and type 2 diabetes [33]. However, it hasn’t been studied in Chinese Soft-Shelled Turtle ( Pelodiscus sinensis ).…”

Nature's fat-burning machine: brown adipose tissue in a hibernating mammal

“…6). Moreover, a recent study using label-free liquid chromatography mass spectrometry investigated changes in the phosphoproteome of BAT during hibernation in the thirteen-lined ground squirrel (Herinckx et al, 2017). Protein levels of mitochondrial membrane and matrix proteins and respiratory chain complexes in BAT are largely constant between active normothermic squirrels and their hibernating counterparts (Herinckx et al, 2017).…”

“…Cy c, cytochrome c; FADH, reduced flavin adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; TCA, tricarboxylic acid cycle; UCP1, uncoupling protein 1. et al, 2017). Furthermore, protein phosphorylation may have an underappreciated role as a regulatory mechanism in BAT given that it is likely involved in causing metabolic changes during hibernation (Herinckx et al, 2017). Overall, these -omic studies have identified essential components for the recruitment and control of BAT activation, which may provide novel targets for effective treatment of human obesity.…”