Tissue-specific response of carbohydrate-responsive element binding protein (ChREBP) to mammalian hibernation in 13-lined ground squirrels
Introduction
Hibernation is a state of greatly suppressed metabolic and physiological activity that is used by many small mammals to survive long and cold winters [3], [28]. Hibernators undergo torpor-arousal cycles over the hibernation period that begin with a lowering of metabolism and core body temperature (Tb). Hibernating animals must also drastically reduce their breathing, organ perfusion, and heart rates to achieve low metabolic rate. During deep torpor (which can last 5–15 days) metabolism can decrease by as much as 95–99% and Tb can decrease to as low as 0–5 °C before the animal spontaneously warms up during the arousal period [27], [28]. The arousal period occurs approximately every 3 weeks and persists 6–24 h before animals re-enter torpor, depending on the species [29], [36]. Hibernating animals save ∼90% of the energy they would need to maintain a core body temperature of 37 °C by adopting a heterothermic lifestyle during the hibernation months [13], [28].
The physiological changes that hibernators make are awe-inspiring, but the changes that accompany hibernation at the molecular level are vastly more complex. Hibernators like the 13-lined ground squirrel (Ictidomys tridecemlineatus) prepare for hibernation during the late summer by fattening up as they store high levels of triglycerides in their white adipose tissue. Even though metabolic rate is greatly reduced during torpor, hibernating animals still require a lot of metabolic fuel for both long term survival and to power periodic shivering and non-shivering thermogenesis. During hibernation, lipid catabolism becomes the primary fuel source since there is an absence of food intake and the only sources of glucose are liver and muscle glycogen reserves and glucose produced via gluconeogenesis [10], [34], [35]. Previous studies have shown an increase in the activity of lipogenic enzymes like fatty acid synthase (FAS), diacylglycerol acyltransferase (DGAT), and acyl-coA synthetase during the pre-hibernation phase and a decrease in the activity of glycolytic enzymes like phosphofructokinase (PFK), pyruvate kinase (PK), pyruvate dehydrogenase (PDH), and glycogen phosphorylase during torpor [4], [5], [34]. Further providing evidence for decreased carbohydrate catabolism during hibernation, glucose concentrations are found at reduced levels and radiolabeled glucose incorporation into CO2 is significantly suppressed during torpor [6], [31]. In fact, most hibernators rely almost entirely on the oxidation of stored lipid reserves as the primary fuel source during hibernation, with most organs switching to mitochondrial fatty acid oxidation as the main means of generating ATP. The exception to this is brain and a few other tissues that mainly catabolize glucose (e.g. erythrocytes and retina). Even brain, which does not take up and oxidize triglycerides directly, can oxidize ketone bodies which are produced from lipid catabolism in the liver and transported to the brain for use as a fuel to supplement glucose. The presence of fatty acids and their metabolites can activate many different pathways, influencing gene expression. For example, peroxisome proliferator–activated receptor (PPAR)α and PPARγ, hepatocyte nuclear factor 4α (HNF4α), sterol regulatory element binding protein 1c (SREBP1c), nuclear factor kappa B (NFκB) and ChREBP are a few of the transcription factors known to be associated with dietary fats [7], [17].
Carbohydrate response element-binding protein (ChREBP) is an essential transcription factor that coordinates glucose uptake, glycolysis, and the synthesis of fats from carbohydrates [21], [39]. In the presence of glucose, but independent of insulin levels, ChREBP moves from the cytoplasm to the nucleus to bind to carbohydrate-responsive elements (ChoREs) within the promoters of genes involved in carbohydrate metabolism. ChREBP controls the expression of many glycolytic and lipogenic genes including L-type pyruvate kinase (Pkl), fatty acid synthase (Fasn) and acetyl coA carboxylase (Acc) [23], [30], [37]. As such, this protein is believed to have imperative roles in fatty acid synthesis during the preparatory phase of hibernation, in the coordinated expression of glycolytic and gluconeogenic enzymes, and in the maintenance of fatty acid stores throughout torpor [16]. This transcription factor has been well studied in model organisms for obesity, insulin resistance, and type 2 diabetes in an effort to better understand energy dysregulation [12], [23], [30]. However, ChREBP has not been studied in hibernating mammals, which are able to gain up to 50% of their body weight in fat stores and develop insulin insensitivity to endure hibernation, and naturally reverse these molecular changes upon emergence from torpor [24]. Studying ChREBP expression and activity in the late torpid 13-lined ground squirrel is important to better understand the molecular mechanisms that regulate energy metabolism under cellular stress, which could have applications in treatment development for patients with insulin resistance-related disorders such as obesity, diabetes, and heart disease [35]. ChREBP is regulated at two levels: nuclear entry and DNA binding and by a reversible phosphorylation mechanism. Under low glucose conditions, ChREBP translocation to the nucleus is inhibited by ChREBP phosphorylation on Ser196 by cAMP-dependent protein kinase (PKA) [2], [16], [39]. ChREBP DNA binding activity is inactivated by phosphorylation at Ser 626 and Thr 666 by PKA and at Ser 568 by AMP-activated protein kinase (AMPK) [2], [18], [19]. This study examines the organ-specific responses of ChREBP by assessing ChREBP total protein level, phosphorylation status, nuclear entry and DNA-binding activities during euthermia and late torpor in liver, kidney, heart and muscle tissues. Furthermore, we examined the tissue-specific responses of downstream genes encoding enzymes known to be under ChREBP control and involved in triglyceride synthesis and glycolysis, namely fatty acid synthase (Fasn) and liver type pyruvate kinase (Pkl).
Section snippets
Animal experiments and tissue collection
Thirteen-lined ground squirrels, Ictidomys tridecemlineatus (130–180 g), were captured by a licensed trapper (TLS Research, Michigan) and transported to the Animal Hibernation Facility at the National Institute of Neurological Disorders and Stroke (NIH, Bethesda, MD). All animal experiments were approved by the NIH Institutional Animal Care and Use Committee and conducted by the laboratory of Dr. J.M. Hallenbeck. Briefly, animals were kept on a fall day/night light cycle at 21 °C in shoebox
Response of ChREBP to late torpor
Total ChREBP expression levels were assessed in four tissues (liver, kidney, muscle, and heart) of ground squirrels via Western blotting. A goat anti-ChREBP antibody cross reacted with a single protein band at ∼95 kDa (Fig. 1B) that corresponded with the known size of the ChREBP protein in other mammals. ChREBP protein levels changed significantly (p < 0.05) in liver, muscle and heart during hibernation, but remained constant in kidney (Fig. 1A). Compared to EC values, ChREBP protein levels
Discussion
The activity of ChREBP has been shown to be dysregulated in diseases such as breast cancer, obesity, diabetes, fatty liver disease, etc. where it is thought to intensify processes such as aerobic glycolysis and lipid accumulation [1], [16], [37]. To better understand how to treat metabolic dysregulation, it is essential to study ChREBP in hibernating ground squirrels because they make extremely regulated molecular changes in their energy metabolism pathways to ensure cell survival throughout
Conclusion
The results of the current study show coordination between ChREBP total protein expression and serine phosphorylation levels, subcellular localization, and DNA-binding ability in a range of organs during hibernation. ChREBP behavior also correlated well with the transcriptional status of two downstream genes under ChREBP control, in all tissues studied. In particular, there seems to be a role for ChREBP in mediating homeostasis of lipid reserves in muscle and heart during late torpor, since
Acknowledgements
Thanks to Manimegala Mathialagan for aiding in the data collection for the creation of this manuscript. Thanks to J.M. Storey for editorial review of this manuscript. This work was supported by a Discovery grant (#6793) from the Natural Sciences and Engineering Research Council of Canada and a grant from the Heart and Stroke Foundation of Canada (#G-14-0005874). KBS holds the Canada Research Chair in Molecular Physiology and SML holds a Queen Elizabeth II Graduate Scholarship in Science and
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