Intracellular negative feedback mechanisms in blubber and muscle moderate acute stress responses in fasting seals

The physiological stress response is a critical animal adaptation for survival in the face of change. In mammals, extrinsic or intrinsic stressors activate the hypothalamic-pituitary–adrenal (HPA) axis, triggering secretion of corticotropin-releasing hormone by the hypothalamus, which induces adrenocorticotropic hormone (ACTH) release from the anterior pituitary, which, in turn, stimulates glucocorticoid (GC; e.g. cortisol) synthesis by the adrenal cortex. The resultant increase in circulating GCs enables adaptive changes necessary for the response to stress, including mobilization of energy stores and suppression of energetically expensive functions (e.g. reproduction, immunity; Sapolsky et al., 2000). Stress responses are costly, however, and may be detrimental if experienced repeatedly and/or during critical life history stages such as development or reproduction. Therefore, animals have evolved robust negative feedback mechanisms to limit the extent of the stress response, and their ability to tune stress responses to environmental or physiological conditions, or HPA flexibility, is a critical factor influencing fitness (Lattin and Kelly, 2020; Taff and Vitousek, 2016; Vitousek et al., 2019; Zimmer et al., 2020).

Negative feedback mechanisms operate at the levels of GC secretion, GC turnover and tissue sensitivity to GCs. When not bound to the carrier protein corticosteroid-binding globulin (CBG), free GCs bind to intracellular glucocorticoid receptors (GRs) in target cells, which translocate to the nucleus and alter transcription of genes that enable appropriate tissue responses to stress (e.g. lipolysis in adipose tissue, proteolysis in muscle; Sapolsky et al., 2000). GRs also function via non-genomic and epigenetic mechanisms (Oakley and Cidlowski, 2013). At basal levels, the physiological effects of GCs are mediated by the mineralocorticoid receptor (MR), while higher GC concentrations activate GRs. GCs feed back on the HPA axis to limit their own synthesis, a response that has been used to assess stress resilience in animals (Lattin and Kelly, 2020). Tissue exposure to GCs is limited by the cortisol-inactivating enzyme HSD11B2 (Chapman et al., 2013), and tissue sensitivity by the density and activity of GRs. These are regulated by intracellular negative feedback loops: ligand-bound GR downregulates its own expression and upregulates HSD11B2 and FKBP5, a nuclear receptor co-chaperone that inhibits GR activity (Oakley and Cidlowski, 2013). FKBP5 was recently proposed as a driver of HPA axis flexibility in wild songbirds (Zimmer et al., 2021, 2020). Specifically, low baseline levels and lower stress-induced increases in FKBP5 expression in the hypothalamus were correlated with higher HPA axis flexibility and enhanced stress-coping behaviors (Zimmer et al., 2021, 2020). However, the role of intracellular negative feedback in stress resilience across life history challenges has not been studied in wild mammals.

Capital breeding marine mammals are an ideal system for examining how animals modulate stress responses during energetically demanding life history stages. The northern elephant seal (Mirounga angustirostris) is a deep-diving pelagic pinniped that undergoes biannual, prolonged fasting periods during reproduction and molting on land. High rates of lipid catabolism, maintained in part by elevation of baseline GC levels across the fast, are used to meet the energetic demands of up to 3 months of fasting without substantial impact on protein stores; basal GC elevation may also facilitate milk synthesis during lactation and skin shedding during molting (Crocker et al., 2014). The endocrine response to administration of ACTH varies by life history stage and fasting state in northern elephant seals, potentially due to differences in baseline GC levels. The magnitude of the cortisol response to ACTH administration is higher during late compared with early fasting in molting adult females (Northey et al., 2023), and its duration is higher in adults than in juveniles (Ensminger et al., 2014; Khudyakov et al., 2015; McCormley et al., 2018; Northey et al., 2023); in adult males, ACTH elevates cortisol levels for >48 h during early but not late breeding (Ensminger et al., 2014). Whether these changes in HPA axis responsiveness and negative feedback across life history stages are mirrored by intracellular feedback mechanisms is currently unknown. We previously showed that FKBP5 is upregulated in response to ACTH administration in juvenile northern elephant seals (Deyarmin et al., 2019; Khudyakov et al., 2017), but have not examined its expression in adults during prolonged fasting.

We hypothesized that (1) fasting adult northern elephant seals induce robust intracellular negative feedback in metabolically active GC target tissues (blubber, skeletal muscle) in response to acute HPA axis activation by ACTH administration, and that (2) the magnitude of the negative feedback response changes across fasting. We collected blubber and muscle from early- and late-fasted molting adult female northern elephant seals before and 2 and 24 h after ACTH administration and measured the expression of NR3C1 (which encodes GR, the receptor type activated at stress-induced GC levels), FKBP5 and HSD11B2 (Fig. 1A). We predicted that (3) baseline expression of NR3C1 would decrease, and HSD11B2 and FKBP5 expression would increase over fasting. We also predicted that the magnitude of their fold-change in response to ACTH would be lower when energy reserves are high (early fasting), enabling more robust responses to GCs, and higher when energy reserves are limited (late fasting), necessitating constraint of stress responses.

Adult female northern elephant seals, Mirounga angustirostris (Gill 1866), were sampled at Año Nuevo State Reserve (San Mateo County, CA, USA). Independent cohorts of females (n=4 each) were sampled at the beginning and end of their fasting period associated with molting, as determined by the percentage of visibly molted pelage (<10% for early molt, >90% for late molt). All animal handling procedures were approved by Sonoma State University Institutional Animal Care and Use Committee and were conducted under National Marine Fisheries Service Permit No. 19108.

Chemical sedation, ACTH administration (0.2 units kg⁻¹ corticotropin gel administered via intramuscular injection; Wedgewood Pharmacy, Swedesboro, NJ, USA) and blood sampling from the extradural vein were conducted as previously described (Northey et al., 2023). Blubber and muscle samples were collected from the posterior flank of each animal using a 6.0 mm diameter biopsy punch (Miltex, Plainsboro, NJ, USA) within 30 min of initial sedation (‘0 h’ samples) and 2 and 24 h after ACTH administration. After sampling, biopsy sites were cleaned with sterile saline and sterile gauze was applied with pressure to promote coagulation. Only the inner half of blubber cores was used for gene expression analyses. Tissue samples were frozen on dry ice and stored at −80°C. Muscle samples were not obtained at 2 and 24 h for several females (figshare, Supplementary File 1: https://figshare.com/s/5c7ff8cb3ef9643b6509). Blood samples were processed and used for cortisol measurement using radioimmunoassay as described previously (Northey et al., 2023).

Samples from all animals and time points were randomized prior to RNA extraction and reverse transcription–real-time PCR (RT-qPCR). Blubber and muscle samples were processed separately. Total RNA was isolated from blubber and muscle tissue using RNeasy Lipid Mini Kit (Qiagen) or Trizol (Ambion) with RNeasy Mini Kit (Qiagen), respectively, as previously described (Khudyakov et al., 2022; Piotrowski et al., 2021). RNA samples were treated with DNase I (Qiagen) for 15 min. RNA concentrations were determined using Qubit 2.0 Fluorometer (Thermo Scientific). RNA integrity was assessed using RNA 6000 Pico Kit on a 2100 Bioanalyzer (Agilent; all RNA integrity numbers, RINs >7.0).

cDNA was synthesized from 500 ng of total RNA using SuperScript IV VILO Master Mix with ezDNase digest (Thermo Fisher). cDNA samples were diluted 1:10 and 2 μl were used in each 20 μl qPCR reaction using PowerUp SYBR Green Master Mix (Thermo Fisher), as previously described (Khudyakov et al., 2022). All primers were used at 400 nmol l⁻¹ final concentration.

Primers targeting coding sequences of NR3C1, FKBP5 and HSD11B2 genes (figshare, Supplementary File 1: https://figshare.com/s/5c7ff8cb3ef9643b6509) were designed using northern elephant seal blubber and muscle transcriptomes (Deyarmin et al., 2019; Khudyakov et al., 2015) with PrimerQuest™ Tool (Integrated DNA Technologies). Primer specificity was confirmed by melt curve analysis and gel electrophoresis. The absence of primer-dimers was assessed using a no-template control. The absence of genomic DNA was confirmed using no-reverse transcriptase controls. Primer efficiency values were calculated from standard curves of five serial 1:2 dilutions of pooled cDNAs (Svec et al., 2015). YWHAZ and NONO were used as reference genes for blubber and EF2 and CCNG1 were used as reference genes for muscle, after testing for expression stability as described previously (Piotrowski et al., 2021). Inter-assay and intra-assay coefficients of variation (CV) for all qPCR assays were <0.44% and <0.63%, respectively. Relative gene expression values (ΔCt) were obtained by subtracting the Ct of the gene of interest from the geometric mean of the Cts of two reference genes (Schmittgen and Livak, 2008). Fold-changes in gene expression were calculated using a modified Pfaffl method (Pfaffl, 2001), as follows: fold-change=RQ^GOI/GeoMean(RQ^REF), where RQ=E^{(Ct_time1–Ct_time2)}, GOI is the gene of interest, REF is the reference gene, and E is primer efficiency. Mean (±s.d.) relative expression (ΔCt) and fold-change levels are reported for biological replicates.

Data analyses were conducted using JMP v16.0 (SAS, Cary, NC, USA). Gene expression data were analyzed separately for blubber and muscle as Ct values for genes of interest were normalized to different reference genes. The effects of ACTH administration and molting stage on circulating cortisol and normalized gene expression levels (ΔCt) were evaluated using linear mixed-effect models (LMM) with time post-ACTH (0, 2, 24 h), molt stage (early, late), adiposity, and the interaction between time and molt (time×molt) as fixed effects and animal ID as a random effect. Parameters for full models are shown in Table 1. Parameters for parsimonious models (without non-significant effects) are presented in the text. Model residuals were visually assessed for normality and homoscedasticity; cortisol was log-transformed to meet model assumptions. Tukey's post hoc test was used to determine differences in markers between time points. Data from muscle sampled at 2 h post-ACTH were not included in post hoc tests because of insufficient sample size. Fold-changes in gene expression between baseline and 24 h post-ACTH were compared between molt stages using two-tailed t-tests.

The endocrine response of molting adult female northern elephant seals to an ACTH challenge was recently described (Northey et al., 2023). In the subset of animals used here, cortisol levels were higher during late compared with early molt (F_1,19=8.47, P=0.0090) and were significantly elevated by ACTH (F_2,19=65.28, P<0.0001; Fig. 1B). Cortisol increased by 10.40-fold 2 h after ACTH administration (P<0.0001), decreased by 1.51-fold between 2 and 24 h post-ACTH (P=0.027), and remained elevated by 6.91-fold relative to baseline at 24 h (P<0.0001). The previous study found that the magnitude of the cortisol response to ACTH was higher during late compared with early molt and was negatively associated with adiposity, suggesting greater HPA axis responsiveness in late-fasted animals (Northey et al., 2023). However, fold-changes in metabolites and immune markers in response to ACTH did not differ between early and late molt (Northey et al., 2023), suggesting that the effects of cortisol may be regulated by intracellular feedback mechanisms throughout fasting.

We therefore examined the effect of ACTH administration on the expression of three genes associated with intracellular negative feedback (NR3C1, FKBP5, HSD11B2) in blubber and skeletal muscle from the same females. ACTH treatment significantly decreased NR3C1 expression in blubber (F_2,21=6.48, P=0.0064) and muscle (F_2,14=6.95, P=0.0080; Fig. 2). NR3C1 expression was downregulated 1.36±0.15-fold (mean±s.d.) in blubber (P=0.013) and 1.44±0.20-fold in muscle (P=0.0080), 24 h after ACTH administration. NR3C1 expression was not significantly associated with molt stage or the interaction term in either tissue (P>0.05). However, baseline NR3C1 expression in blubber was higher in three of four early molt animals (Fig. 2).

FKBP5 expression was significantly affected by ACTH administration in blubber (F_2,21=42.77, P<0.0001) and muscle (F_2,11=81.96, P<0.0001; Fig. 2). Blubber FKBP5 expression was upregulated 2.62±1.88-fold within 2 h of ACTH administration (P=0.013) and by a further 4.28±1.67-fold between 2 and 24 h (P<0.0001); it was elevated 10.48±7.49-fold relative to baseline 24 h post-ACTH (P<0.0001). Muscle FKBP5 expression was upregulated 31.72±26.52-fold relative to baseline 24 h post-ACTH (P<0.0001). While blubber FKBP5 expression was not significantly associated with molt stage (P=0.48), baseline FKBP5 was higher during late molt in three of four animals (Fig. 2). Muscle FKBP5 expression was significantly higher during late compared with early molt (F_1,11=11.35, P=0.0063), and the interaction between time and molt was significant (F_2,11=4.59, P=0.036); the latter was primarily driven by higher baseline FKBP5 expression in muscle during late molt (P=0.053).

HSD11B2 expression was upregulated by 2.03±0.99-fold 24 h after ACTH administration in blubber (F_2,19=9.34, P=0.0015; post hoc test P=0.0017), but was unaffected by ACTH in muscle (P=0.74; Fig. 2). Expression of HSD11B2 was significantly higher in early compared with late molt in blubber (F_1,19=22.11, P=0.0002) and muscle (F_1,15=7.39, P=0.016). There was no effect of the interaction term on HSD11B2 expression in either tissue (P>0.05). Blubber HSD11B2 expression was the only variable that was positively correlated with adiposity (F_1,19=11.30, P=0.0033; data not shown).

Together, our data suggest that molting female northern elephant seals exhibit robust intracellular negative feedback in blubber and muscle tissue in response to acute HPA axis activation, as predicted by our first hypothesis, and that this feedback is maintained throughout their fasting period. We propose three potential mechanisms by which GR target tissues may limit the duration and impacts of acute stress responses in fasting northern elephant seals. (1) Downregulation of NR3C1 expression may lead to a decrease in GR density, decreasing tissue sensitivity to cortisol despite its sustained elevation for >24 h after HPA axis activation. (2) Upregulation of FKBP5 expression and the presumed increased abundance of FKBP5 protein may reduce GR responsiveness to cortisol by preventing its access to target genes. (3) Upregulation of HSD11B2 expression and the consequent increase in enzyme abundance may limit tissue exposure to cortisol by converting it to the inactive metabolite cortisone. Together, these changes may serve to constrain lipid- and protein-mobilizing and immunosuppressive effects of acute GC elevation in animals that rely on fat stores to sustain prolonged fasting periods in crowded colony conditions (Peck et al., 2016). Downregulation of NR3C1 expression and GR inhibition by FKBP5 may also serve to reduce the inhibitory effects of GCs on mitochondrial respiration and connectivity in muscle (Torres-Velarde et al., 2021), especially during late molt, when animals are preparing to embark on a long-distance foraging trip.

Our studies in northern elephant seals suggest that the mechanisms used to regulate the duration and impact of acute stress responses vary over life history stages. In juveniles, negative feedback may primarily operate at the level of hormone secretion, limiting stress-induced cortisol elevation to <24 h (i.e. cortisol levels return to baseline within 24 h of ACTH administration), enabling robust but short stress responses during a life history stage characterized by rapid growth, but not extended fasting (Deyarmin et al., 2019; Khudyakov et al., 2017). In molting adults, negative feedback at the level of the HPA axis is reduced, potentially as a consequence of its already elevated activity during this life history stage. Instead, intracellular negative feedback mechanisms may enable adults to regulate the effects of basal and stress-induced GCs on a tissue- and life history stage-specific basis (Quax et al., 2013). For example, these mechanisms may facilitate lipid mobilization from blubber, while buffering skeletal muscle from the proteolytic effects of GCs during acute stress responses while fasting. Multiple feedback mechanisms operating on different time scales (i.e. receptor sequestration, hormone inactivation, receptor downregulation) may enable animals to precisely tune the duration of tissue responses to GCs.

Contrary to our second hypothesis, we found that the magnitude of the transcriptional response to ACTH (i.e. fold-change in gene expression) did not differ between early and late molt in adult female northern elephant seals (P>0.05), suggesting that animals maintain similar intracellular negative feedback capacity despite changes in baseline hormone and gene expression levels and an increase in HPA axis reactivity over fasting (Northey et al., 2023). The differences in baseline gene expression levels between early and late molt supported our third hypothesis, with the exception of HSD11B2, which was expressed more highly in early-fasted animals. This implies that while baseline tissue sensitivity and responsiveness to GCs may be higher during early fasting (as a consequence of higher GR and lower FKBP5 expression compared with that in late molt), tissues may be exposed to lower concentrations of cortisol at this time (as a result of higher HSD11B2 expression). The functional significance of this change in gene expression should be further examined by measuring tissue-specific cortisol and cortisone concentrations. Furthermore, as MR has been shown to mediate the effects of basal GC levels and to modulate GR responses to GCs in mice (Carceller-Zazo et al., 2023), future studies should also examine whether expression of NR3C2, which encodes MR, varies with life history stage and stress state in seals.

Larger sample sizes and cross-sectional sampling will undoubtedly be necessary to understand how the feedback mechanisms examined in this study vary across other life history stages in northern elephant seals, as well as whether individual variation in their magnitude influences fitness, as has been suggested in other species (Zimmer et al., 2019, 2021). Whether intracellular feedback mechanisms also operate within the HPA axis (i.e. in the hypothalamus) in northern elephant seals, as has been shown in birds (Zimmer et al., 2021), remains unknown, as brain samples cannot be obtained from free-ranging marine mammals. Nevertheless, our data suggests additional, tissue-specific mechanisms of endocrine flexibility by which vertebrates may modulate downstream impacts of acute stress responses on peripheral tissues during key life history stages. We also propose that NR3C1, FKBP5 and HSD11B2 expression may serve as proxy of recent HPA axis stimulation and GR activation in blubber and muscle, as GC measurements alone are increasingly recognized as unreliable indicators of stress and health in wildlife (Romero and Beattie, 2022).

The authors thank D. Costa, P. Robinson and Año Nuevo Reserve for facilitating animal sampling.