Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels

Abstract

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3.

Keywords: Ictidomys tridecemlineatus; autophagy; mitochondria; starvation.

Fig. 1.

Seasonal homeothermy and heterothermy in circannual hibernation. A: the different physiological states examined in this study are indicated on this representative body temperature (T_b) trace: 2 homeothermic groups, Sp (spring) and SA (summer active); 4 heterothermic, winter hibernating groups, IBA (interbout aroused), Ent (entrance into torpor), LT (late torpor), and Ar (early arousing), comprised the base states. Two fall transition groups, FT1 and FT2, were distinguished by whether the animal had previously been torpid (see methods for details). B: model depicting heterothermic states, including all 4 winter hibernation states and FT2 (the fall transition animals that had used torpor based on T_b records), constrained to a distinct baseline physiology (light blue shading).

Fig. 2.

Key parameters of animals used in this study. Individuals are represented by (●) and the mean of each group by a horizontal line; groups that differed are indicated by small letters. A: date, Aug. 1 = day 1. B: body mass at time of liver collection. C: number of males (black bar) and females (gray bar) in each group.

Fig. 3.

Random forests classification by liver protein spot intensities. A: classification of individual samples from all 8 groups using the relative intensities of 630 protein spots present on all gels (n = 6 per state). B: as in A, except using the 186 identified protein spots that were present on all gels (n = 6 per state). Nine of the top 10 classifiers from the full analysis (A) were identified and also appeared in the top 10 named protein classifiers (B). These proteins are (ID_spot number): HIBADH_1664, DMGDH_407, DBT_1007, SND1_767, PRDX3_1839, MPST_1552, GPT_1010, APOA1_1849, and IVD_1237.

Fig. 4.

Identification of key proteins that distinguish among ground squirrels in different stages of the seasonal and torpor-arousal cycles of hibernation. A: Venn diagram separates the classifiers identified in the 6-state RF analysis (Sp, SA, IBA, Ent, LT, and Ar: red) from those identified using just the winter states (IBA, Ent, LT and Ar: blue). Each RF was run 5 times: for the 4-state winter dataset these 5 protein spots were recovered 100% of the time. For the base state analyses, the 3 spots shared with the winter analysis were recovered in all 5 attempts, but HIBADH and DBT were additionally recovered 3 and 2 times, respectively. B–H: boxplots show the distribution of relative intensity values for animals from each state for each of these top classifying proteins, identified by their gene name and spot number. Proteins vary: B–D across season and hibernation state; E and F seasonally; G and H within winter hibernation. The horizontal lines, triangles, circles, rectangles, and whiskers represent the median, mean, outliers, 25–75th percentile, and 5–95th percentile, respectively.

Fig. 5.

Liver protein dynamics in 6 stages of hibernation. Heat map plots relative intensity (blue, gold, and white represent least, most, and missing values) for each protein spot (rows, ordered based on divisive clustering into 6 abundance patterns) for each individual (columns, ordered by physiological state). The mean spot intensity value ± 95% confidence interval for each of the 6 patterns is plotted to the right of the heat-map. The gene enrichments for the 3 most populated clusters (–3) are given in Table 2.

Fig. 6.

Liver proteome dynamics through the fall transition. Line plots (means ± SE) for 6 patterns of protein change occurring among SA, FT, and IBA in the proteins comprising cluster 1 (A) and cluster 3 (B) in Fig. 5, i.e., decreased and increased in winter heterothermy, respectively. Among these SA-FT-IBA patterns, A plots cluster 1 with 42 spots, 22 unique proteins, enriched for mitochondria, glucose catabolism, gluconeogenesis, and Aln, Ala, Asp, Arg, and Pro metabolism; cluster 2 with 44 spots, 23 unique proteins, enriched for mitochondria and glucose metabolism; and cluster 3 with just 10 spots and 9 unique proteins (Supplemental Table S2). In B, cluster 4 with 39 spots, 35 unique proteins, enriched for peroxisome and fatty acid metabolism; cluster 5 with 17 spots, 16 unique proteins, enriched for regulation of apoptosis, mitochondria, and ion homeostasis; and cluster 6 had only 4 spots, all unique proteins.

Fig. 7.

Posttranslational modification of liver proteins: protein acetylation is increased during winter hibernation. A: overall phosphorylation level (vs. SYPRO Ruby total protein stain intensity) in 2D gels is constant among Sp, Ent, and Ar (means ± SD, n = 3) ground squirrels. B: the same 2D gel stained for phospho- and total protein, using ProQ Diamond and SyproRuby, respectively, shows that spot 1144 with NSFL1C is a phosphoprotein. C: Western blot to detect acetyl-lysine reveals several bands with increased acetylation in winter heterotherms. Subsequent IP identified proteins in those bands as indicated on the left. D: relative abundance (vs. β-tubulin) of all acetylated bands detected by Western blot (mean ± SD, n = 3 from each state, small letters indicate distinct groups). E: overall acetylation labeling (vs. background) in 2D Western blots is most intense in winter hibernators, particularly LT (n = 2 SA, IBA, n = 3 LT; mean ± SD). F: the same 2D gel labeled by acetyl-lysine antibody for acetylated proteins (Ac-Protein, top) and for total protein with Cy2 (bottom) shows differential acetylation of 2 spots containing DMGDH (spot 413 is acetylated, 407 is not). G: Western blot of CPS1 shows decreased abundance in winter heterotherms, consistent with DiGE quantification. H: representative Western blot of SIRT3, quantified in I after normalization to β-tubulin (n = 3 per state, mean ± SD), shows decrease of SIRT3 during the winter fast [ANOVA P = 0.03; IBA, LT, and Ar ≠ SA (*), P < 0.04].