Non-shivering thermogenesis is differentially regulated during the hibernation season in Arctic ground squirrels

Abstract

Arctic ground squirrels are small mammals that experience physiological extremes during the hibernation season. Body temperature rises from 1°C to 40°C during interbout arousal and requires tight thermoregulation to maintain rheostasis. Tissues from wild-caught Arctic ground squirrels were sampled over 9 months to assess the expression of proteins key to thermogenic regulation. Animals were sacrificed while aroused, and the extensor digitorum longus, diaphragm, brown adipose tissue, and white adipose tissue were probed using Western blots to assess protein expression and blood was sampled for metabolite analysis. Significant seasonal expression patterns emerged showing differential regulation. Contrary to our prediction, white adipose tissue showed no expression of uncoupling protein 1, but utilization of uncoupling protein 1 peaked in brown adipose tissue during the winter months and began to taper after terminal arousal in the spring. The opposite was true for muscular non-shivering thermogenesis. Sarco/endoplasmic reticulum calcium ATPase 1a and 2a expressions were depressed during the late hibernation season and rebounded after terminal arousal in diaphragm tissues, but only SERCA2a was differentially expressed in the extensor digitorum longus. The uncoupler, sarcolipin, was only detected in diaphragm samples and had a decreased expression during hibernation. The differential timing of these non-shivering pathways indicated distinct functions in maintaining thermogenesis which may depend on burrow temperature, availability of endogenous resources, and other seasonal activity demands on these tissues. These results could be impacted by fiber type makeup of the muscles collected, the body weight of the animal, and the date of entrance or exit from hibernation.

Keywords: ground squirrel; hibernation; metabolism; non-shivering thermogenesis; sarcolipin.

FIGURE 1

Tissue collection scheme based on body temperature of an Arctic ground squirrel during hibernation. Internal body temperature during hibernation season is shown as a solid black line. Animals were sampled aroused or active from November 2016 to June 2017. Early hibernation was defined as undergoing at least 2–3 full torpor bouts shown in light blue ranging from 15 November to 8 December. Late hibernation was defined as continued regular torpor bouts after the turn of the new year, with IBAs shown in dark blue ranging from 22 January to 18 February. Post hibernation (days 3,8, and 15) defined as arousal from torpor without re-entrance into torpor shown in shades of green ranging from 28 February to 3 April. Summer active defined as a return to euthermic phenotype but still exposed to cold shown in yellow ranging from 3 June to 13 June.

FIGURE 2

Enrichment analysis of metabolites at two time points during hibernation. (A) Late hibernation had energy pathways linked to glyoxylate and citric acid cycles enriched when compared to early (p < 0.05). (B) Creatine, citric acid, and acetylcholine were three metabolites that were identified as key metabolites influencing the enrichment of pathways between early and late hibernation (p < 0.05). Values assessed with MetaboAnalyst enrichment analysis with KEGG 2019 (Kanehisa, 2000; 2019; Kanehisa et al., 2023) as the comparative library and significance threshold of Holms' p-value < 0.05.

FIGURE 3

Enrichment analysis of metabolites during hibernation vs. terminal arousal. (A) Early hibernation compared to 15 days post hibernation showing fatty acid pathways linked to biosynthesis (saturated and unsaturated), elongation, degradation, and linoleic acid metabolism to be enriched during early hibernation (p < 0.05). (B) Palmitic and linoleic acids are two metabolites that were significantly increased for multiple fatty acid utilization pathways during early hibernation (p < 0.05). Values assessed with MetaboAnalyst enrichment analysis with KEGG 2019 (Kanehisa, 2000; 2019; Kanehisa et al., 2023) as the comparative library and significance threshold of Holms' p-value <0.05.

FIGURE 4

UCP1 expression in brown adipose tissue. (A) Early, late, and 3 days post hibernation had an increased UCP1 expression when compared to 15 days post hibernation (p < 0.001, p < 0.01, p < 0.001) and early, late, and 3 days post hibernation had greater expression than 8 days post hibernation (p < 0.05, p < 0.05, and p < 0.01). Symbols indicate a significant difference based on a one-way ANOVA with Tukey's post hoc analysis, with significance set at p ≤ 0.05. Symbols indicate significance as follows: &, SA; @, early; #, late; $, 3 days; *, 8 days; +, 15 days. All values are represented as box and whiskers.

FIGURE 5

Expression of SERCA regulator SLN in AGS skeletal muscle. (A) Expression of SLN in AGS diaphragm shows a decrease in expression during early hibernation when compared to 8 (p < 0.05) and 15 days post hibernation (p < 0.01). Symbols indicate a significant difference based on a one-way ANOVA with Tukey's post hoc analysis, with significance set at p ≤ 0.05. Symbols are as follows: *, 8 days; and +, 15 days. All values are represented as box and whiskers.

FIGURE 6

Expression of SERCA1a and SERCA2a in AGS skeletal muscle. (A) Expression of SERCA1a in AGS diaphragm shows a decrease in expression during late hibernation when compared to SA (p < 0.05), 3 days post hibernation (p < 0.01), and 8 days post hibernation (p < 0.01) (B) SERCA1a was not detectably different at any time point in EDL. (C) SERCA2a shows an increased expression in the diaphragm during 8 days post hibernation when compared to summer active (p < 0.05), early (p < 0.001), late (p < 0.01), and 3 days post hibernation (p < 0.05). 15 days post hibernation was increased when compared to early hibernation (p < 0.05). (D) SERCA2a in EDL had a decrease in expression at late hibernation (p < 0.001), 3 days post hibernation (p < 0.001), 8 days post hibernation (p < 0.001), and 15 days post hibernation (p < 0.001) when compared to SA. 15 days post hibernation had decreased when compared to early hibernation (p < 0.05). Symbols indicate a significant difference based on a one-way ANOVA with Tukey's post hoc analysis, with significance set at p ≤ 0.05. Symbols are as follows: &, SA; @, early; #, late; $, 3 days; *, 8 days; +, 15 days. All values are represented as box and whiskers.