Sixteen healthy male captive Arctic foxes [body weight (BW) = 5.74 ± 0.10 kg] and 7 healthy male captive silver foxes (BW = 7.08 ± 0.43 kg) that were maintained in the research farm of Jilin Agricultural University were included in this study. The Arctic foxes were randomly assigned to either a 15% crude fat diet group (n = 8, LFA group) or a 40% crude fat diet group (n = 8, HFA group). Silver foxes were fed a 15% crude fat diet (n = 7, LFS group). The dietary components and composition are shown in Supplementary Table S1. Each animal was individually housed, and following a one-week adaptation period, then they were fed each diet for 11 weeks, respectively. The body weight was recorded weekly. All animal procedures were approved and authorized by the Animal Ethics Committee of Jilin Agricultural University (No.20210314002) and used the ARRIVE guidelines 2.0 (21).

At the end of the study, blood samples were collected from the hindlimb vein, and centrifuged at 3,500 g for 10 min at 4°C to obtain serum. Animals were then intravenously injected with 5 mL of succinylcholine (0.4 mg/mL), and subcutaneously adipose tissue and liver tissue were collected. Tissue samples were cut into small pieces (approximately 0.5 × 0.5 cm) and immediately frozen in liquid nitrogen for further analysis. Liver samples were also cut into small pieces (1.0 × 1.0 cm) and fixed in 10% neutral buffered formalin for histological assessment.

The intraperitoneal glucose tolerance test (IPGTT) was performed according to previous established methods (22). Briefly, 3 animals from each group at 9 weeks were selected to conduct the IPGTT. Following a 14 h overnight fast, animals were received intraperitoneal administration glucose (1 g/kg). Whole blood samples were collected from the hindlimb vein before administration (0 min) and at time points (15, 30, 60, 90, 120, 150, and 180 min) after administration, and blood glucose levels were measured using an Accu-Chek glucometer (Roche Diagnostics Shanghai Ltd., China).

Serum samples was used to determine the concentrations of glucose, aspartate transaminase (AST), alanine transaminase (ALT), TAG, total cholesterol (CHO), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) using the commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) with an automatic biochemical analyzer (Vitalab Selectra E, Vitalab, Dieren, Netherlands). The concentrations of growth hormone (GH), IGF-1, insulin and VLDL in serum were measured using the commercial ELISA kits (MLbio, Shanghai, China).

The liver samples fixed in 10% neutral buffered formalin were embedded in paraffin, cut to 5 μm sections, and stained using H&E (23), and then were visualized using an Olympus AX80 microscope (Olympus Optical, Tokyo, Japan) equipped with Nikon D2X high-resolution camera (Nikon, Tokyo, Japan). The lipid droplet areas were quantified by Image J 1.53a software, and the lipid droplet areas were divided by the area of image to calculate the percentage of lipid droplet areas (24). The concentrations of glucose, TAG, adenosine 3′, 5′-phosphate (cAMP), uridine, glycogen, insulin-like growth factor 1 receptor (IGF1R) and insulin receptor substrate 1 (IRS1) in the liver were measured by the commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Total RNA from liver samples was extracted using the RNeasy Mini Kit (QIAGEN, CA, United States). RNA purity, concentration and integrity were determined using a NanoPhotometer^® (IMPLEN, CA, United States), an Agilent 2,100 bioanalyzer in combination with the RNA Nano 6,000 assay kit (Agilent Technologies, CA, United States). A total of 1–3 μg of RNA from each sample with an integrity number greater than 7.0 was used to construct the RNA-Seq library using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina^®. The cDNA concentration of constructed library was measured using the Qubit^® RNA Assay Kit in Qubit^® 3.0. Each library was then sequenced on a Novaseq 6000 platform, producing 150 bp paired-end reads.

Trimmomatic (25) was used to remove low quality and adapter sequences, and HISAT2 was employed to align the remaining clean reads to the reference genomes of the Arctic fox and silver fox, respectively (8, 26, 27). Gene expression level was calculated using the raw count data. Differentially expressed genes (DEGs) were determined using the DeSeq2 package (28) based on the threshold |log₂ FC| > 0.5 and Benjamini-Hochberg adjusted p value <0.05. The weighted gene coexpression network analysis (WGCNA) was conducted to explore the correlation between DEGs and serum biochemical indices using the WGCNA package (29). The clusterProfiler package was utilized to conduct Gene Ontology (GO) term and Kyoto Encyclopedia of Gene and Genomics (KEGG) pathway enrichment analyses (30).

A total of 20 mg adipose tissue from each animal in the LFA and LFS groups was dissolved in 400 μL water, vortexed for 60 s, and homogenized at 45 Hz for 4 min, and sonicated for 5 min in ice-water bath. The homogenization and sonication circle were repeated for 3 times. A 20 μL portion of the homogenate was mixed with 180 μL water, and then 480 μL extract solution (MTBE: MeOH = 5:1) containing internal standard (Supplementary Table S2) was added. After vortexing for 60 s, the samples were sonicated for 10 min in ice-water bath. The samples were then centrifuged at 3,000 rpm for 15 min at 4°C. A 250 μL supernatant was transferred to a fresh tube. The remaining sample was mixed with 250 μL of MTBE, followed with vortex, sonication and centrifugation, and another 250 μL supernatant was collected. The supernatants were combined and dried in a vacuum concentrator at 37°C and were reconstituted in 200 μL resuspension buffer (DCM: MeOH: H₂O = 60: 30: 4.5). The samples were vortexed for 30 s and sonicated for 10 min in ice-water bath. The mixture was then centrifuged at 12,000 rpm for 15 min at 4°C, and 35 μL of supernatant was transferred to a fresh glass vial for LC/MS analysis.

The ultra-high-performance liquid chromatography (UHPLC) separation was performed using a SCIEX ExionLC series UHPLC System. The mobile phase A consisted of 40% water, 60% acetonitrile, and 10 mmol/L ammonium formate. The mobile phase B consisted of 10% acetonitrile and 90% isopropanol, and 10 mmol/L ammonium formate. The column temperature was set to 45°C, while the auto-sampler temperature was set to 6°C. The injection volume was 2 μL. The Biobud-v2.1.4.1 Software (Biotree Biotech Co., Ltd., Shanghai, China) was used to quantify the metabolites. The absolute content of individuals lipids corresponding to the internal standard was calculated based on peak area and the actual concentration of the identical lipid class internal standard. Principal component analysis (PCA) was applied to reveal the differences of lipids. The value of variable importance in the projection (VIP) of the first principal component in orthogonal projections to latent structures discriminant analysis (OPLS-DA) was determined. Metabolites with VIP > 1 and a Benjamini-Hochberg adjusted p value <0.05 (student’s t-test) were considered as significantly changed metabolites (31).

The concentrations of LPCAT4 and FAS were measured by commercial kits from Jiancheng Bioengineering Institute (Nanjing, China) and BiotechPack (Beijing, China), respectively, according to the manufacturer’s instructions.

One-way ANOVA was utilized to determine the statistical significance of final body weights, serum biochemical indices and hormones, areas under curves of IPGTT, liver lipid droplets areas, metabolites and proteins among the LFA, HFA, and LFS groups. The unpaired t-test was performed to identify the statistical significance of adipose metabolites and proteins between the LFS and LFA groups. The statistical analysis was performed using Graphpad Prism (version 9.0.0, GraphPad Software Inc., San Diego, CA, United States). All p value < 0.05 were considered to indicate statistical significance.

We monitored body weight of Arctic and silver foxes fed a 15% crude fat diet and found that the body weight gain rate of Arctic foxes was significantly greater than that of silver foxes over the 11-week monitoring period. In addition, consumption of a 40% crude fat diet led to further increases in the body weight and the body weight gain rate of Arctic foxes (Figure 1B). Consequently, the thickness of subcutaneous adipose tissue in Arctic foxes was approximately 3 times greater than that in silver foxes (Supplementary Figure S1), indicating that the weight gain observed in Arctic foxes was due primarily to fat deposition. We measured serum biochemical parameters and found that the serum concentrations of CHO, VLDL and HDL-C in Arctic foxes were significantly greater than those in silver foxes fed a 15% crude fat diet (p < 0.05; Figure 1C). Notably, no differences were observed in these serum biochemical parameters between Arctic foxes fed a 15% crude fat diet and those fed a 40% crude fat diet, indicating that Arctic foxes can maintain normal blood lipid levels on a 40% crude fat diet. The serum glucose and insulin levels did not differ significantly among the three experimental groups (Figure 1D). In addition, we conducted an IPGTT and found that the glucose metabolism capacity did not differ significantly among the groups (Figure 1D), suggesting that liver function and glucose homeostasis remained largely unaffected.

Histopathological analysis indicated that even upon consumption of a 40% crude fat diet, the lipid droplet area within the liver remained lower in Arctic foxes than in silver foxes (Figure 2A). Accordingly, consumption of a 40% crude fat diet did not increase the hepatic TAG content in the Arctic fox, nor did it result in elevated serum levels of AST and ALT (Supplementary Figure S2A). Transcriptomic analysis revealed significant differences in global gene expression profiles between LFA and LFS groups as well as between the LFA and HFA groups (p < 0.01; Figure 2B). This analysis led to the identification of DEGs in the following comparisons: LFA versus LFS and HFA versus LFA. A total of 5,504 DEGs were significantly upregulated and 1,719 DEGs were downregulated between the LFA and LFS groups (Figure 2C). In contrast, the comparison between the HFA and LFA groups revealed 30 DEGs, 15 of which were upregulated and 15 of which were downregulated (Supplementary Figure S2B), suggesting that consumption of a 40% crude fat diet does not induce substantial alterations in the liver function of Arctic foxes.

The upregulated DEGs in LFA versus LFS were significantly enriched in the PI3K-Akt signaling pathway (Igf1, Igf1r, Irs1), purine metabolism pathway (Nme2, Cant1, and Adcy6), insulin signaling pathway and pyrimidine metabolism pathway (Nme3, Cant1, and Nt5e) (Figure 2D; Supplementary Figure S2C). These DEGs were also significantly enriched in the following biological process terms: purine-containing compound metabolic process, glucose homeostasis, fatty acid oxidation (Acox1, Acadm, Ppara), pyrimidine-containing compound metabolic process and neutral lipid biosynthetic process (Gpat3, Lpin3, Dgat1, Figure 2E; Supplementary Figure S2C). In addition, insulin-like growth factor binding and insulin receptor substrate binding were noted as enriched molecular function terms (Figure 2E). The downregulated DEGs were enriched in the biosynthesis of cofactors, carbon metabolism, and amino sugar and nucleotide sugar metabolism pathways (Figure 2D); the biological process terms positive regulation of proteolysis and cellular amino acid metabolic process; the cellular component terms organelle outer membrane and sarcoplasmic reticulum; and the molecular function terms structural constituent of ribosome and catalytic activity (Figure 2E).

WGCNA was used to explore the associations between the DEGs and serum biochemical parameters, and 17 modules containing between 45 and 3,084 genes per module were identified (Figure 2F). Module 13 showed a significant positive correlation with the serum concentrations of CHO, HDL-C and VLDL (Figure 2F). The genes within module 13 were enriched in pathways such as ABC transporters, pyrimidine metabolism, PPAR signaling pathway and purine metabolism (Figure 2G). Triglyceride biosynthetic process, pyrimidine-containing compound metabolic process, lipid oxidation, regulation of glucose metabolic process, and response to purine-containing compound were identified as enriched biological process terms. Insulin-like growth factor 1 binding and UDP-xylosyltransferase activity were identified as enriched molecular function terms (Figure 2G). Taken together, these findings suggest that pyrimidine, purine, and glucose metabolism, along with insulin-like growth factor and insulin receptor responses, are likely enhanced in the liver of Arctic foxes. Further analysis revealed significantly greater hepatic concentrations of glycogen, cAMP, uridine, IGF1R and IRS1 in Arctic foxes than in silver foxes (p < 0.05); in contrast, the hepatic concentration of glucose was lower in Arctic foxes than in silver foxes, although the difference was nonsignificant (Figures 3A–C). Additionally, the serum concentration of IGF-1 was significantly greater in Arctic foxes than in silver foxes, although the serum concentration of GH was not significantly different between Arctic and silver foxes (Figure 3D).

Given the significant changes in body mass, serum cholesterol and liver transcriptome profiles in Arctic foxes (LFA group) compared with silver foxes (LFS group), we explored the lipid composition and concentration in adipose tissue and identified 443 TAGs, 46 diacylglycerols (DAGs) and 26 phosphatidylethanolamines (PEs) (Supplementary Figure S3). TAGs constituted more than 99% of the lipid content in all the samples (Figure 4A). The total concentrations of TAG (p < 0.05) and PE (p < 0.01) in Arctic foxes were significantly greater than those in silver foxes, while the total concentration of free fatty acids (FFAs) was not significantly different (Figure 4B). The PCA results revealed that the lipid profiles were significantly different between the LFA and LFS groups (p < 0.05; Figure 4C). The abundances of 215 lipids were significantly increased in Arctic foxes compared with silver foxes, and the numbers of significantly increased TAGs, PEs and DAGs were 160, 14 and 12, respectively (Figure 4D; Supplementary Tables S3, S4). Further comparison revealed that 8 of the 10 lipids with the greatest log₂ fold change (FC) values were TAGs and the other 2 were PEs (Figure 4E), indicating likely increases in TAG and PE synthesis. We thus measured the concentrations of LPCAT4 and FAS that catalyze the synthesis of PEs and fatty acids in adipose tissue. The concentration of LPCAT4 (p < 0.01) in Arctic foxes was significantly greater than that in silver foxes, while the concentration of FAS in Arctic foxes was significantly less than that in silver foxes (p < 0.05) (Figure 4F), suggesting the PE synthesis is increased and fatty acid synthesis is decreased in Arctic foxes.