Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

Abstract

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast.

Fig. 1.

Seasonal core body temperature (T_b) transitions in ground squirrels. Graph of T_b over time shows summer homeothermy and winter heterothermy (hibernation). Summer active (SA) animals maintain T_b at 37 ± 1°C until late in the fall. Hibernation consists of multiday periods of torpor, during which T_b is near ambient, 4°C, punctuated by short interbout arousals to ∼37°C. Animals at the entrance (Ent) phase of winter hibernation were reentering torpor after a spontaneous interbout arousal.

Fig. 2.

Liver proteins fractionated by 2-dimensional (2-D) gel electrophoresis. In a representative pick gel stained with SYPRO Ruby, protein spots for which a unique identification (ID) was obtained are indicated with arrows and their gene ID is given. A: protein spots elevated in Ent. B: protein spots elevated in SA. Approximate molecular mass (in kDa) is shown at left, and approximate isoelectric point is shown at top. •, Reflective reference markers necessary for robotic picking. See Tables 1 and 2 for explanation of abbreviations.

Fig. 3.

Fold changes revealed by difference gel electrophoresis (DiGE) and Western blot results. A: DiGE quantification of ATP citrate lyase (ACLY) in 12 individual 13-lined ground squirrels (6 SA and 6 Ent). Cy2-normalized value for each of 3 spots (186, 193, and 197) is plotted. B: Western blot of ACLY showing the gel region from 75 to 150 kDa for 12 animals in A (top) and Western blot of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) showing the region from 37 to 50 kDa of the gel used for ACLY detection (bottom). Only a single band with chemifluorescent product was detected with either antibody, but the reverse image artifact bands observed with anti-HMGCS2 confirm equal protein loading for each sample. M, mol wt marker.C: quantification of HMGCS2 from the bottom Western blot in B. Average values of SA and Ent were normalized to SA. Error bars, SD. HMGCS2 is increased 2.01-fold in Ent compared with SA. ***P < 0.001 (Student's t-test).

Fig. 4.

Metabolic enzyme differences between SA and Ent livers centered on the tricarboxylic acid (TCA) cycle. Schematic depicts TCA cycle intermediates within the mitochondrion. Liver proteins, differing between SA and Ent animals (see Tables 1 and 2), that connect directly (solid arrow) or indirectly (dashed arrow) to the indicated metabolites are identified by their Entrez gene identifiers (in italics). If underlined, the enzyme is increased in Ent liver; if not, it is decreased, i.e., elevated in SA compared with Ent. FA, fatty acid; PEP, phosphoenolpyruvate; see Tables 1 and 2 for explanation of other abbreviations.