Cross-species fecal microbiota transplantation alters the carbohydrate metabolic phenotype: insights from Gansu zokor (Eospalax cansus)

Zhuohang Li; Maohong Yang; Yingying Zhang; Jianping He; Jingang Li

doi:10.1186/s12917-026-05469-w

. 2026 Apr 11;22:299. doi: 10.1186/s12917-026-05469-w

Cross-species fecal microbiota transplantation alters the carbohydrate metabolic phenotype: insights from Gansu zokor (Eospalax cansus)

Zhuohang Li ^1,^2,³, Maohong Yang ³, Yingying Zhang ³, Jianping He ^1,^2,^3,^✉, Jingang Li ^1,^2,^3,^✉

PMCID: PMC13195913 PMID: 41965671

Abstract

The Gansu zokor (Eospalax cansus), a subterranean rodent endemic to China, exhibits remarkable adaptability to hypoxic conditions. Although it is known that the gut microbiota of Gansu zokor undergoes changes under hypoxia, thereby influencing carbohydrate metabolism, it remains unclear whether such a hypoxia adaptation mechanism can be simulated in surface-dwelling animals. In this study, we established a cross-species fecal microbiota transplantation model from Gansu zokor to SD rats to investigate carbohydrate metabolic adaptations under hypoxia. Using 16 S rRNA sequencing, we characterized the features of gut microbiota. Furthermore, we examined molecular markers associated with glycolysis and the tricarboxylic acid cycle in the liver and brain of SD rats exposed to prolonged hypoxia. The results demonstrated that fecal microbiota transplantation altered the composition of gut microbiota in SD rats, leading to modifications in carbohydrate metabolic networks in the liver and brain under prolonged hypoxia. This shift rendered their carbohydrate metabolic patterns more similar to those observed in Gansu zokor. In conclusion, this study provides new evidence supporting the role of gut microbiota in hypoxia adaptation in subterranean rodent and offers a valuable animal model reference for the treatment of hypoxia-induced injuries via fecal microbiota transplantation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-026-05469-w.

Keywords: Carbohydrate metabolism, Fecal microbiota transplantation, Gansu zokor (Eospalax cansus), Hypoxia

Introduction

Hypoxia, as a prevalent physiological and pathological condition, extensively modulates diverse biological processes, ranging from adaptive evolution in high-altitude species [1] to ischemic tissue hypoxia [2] and metabolic remodeling in tumor microenvironments [3]. Variations in the dynamics of oxygen concentration influence cellular metabolisms through multilayered regulatory mechanisms, including adjustments in gene expression programs, activation of stress-responsive signaling pathways, and restructuring of energy metabolic networks [4]. As the core component of energy metabolism, carbohydrate metabolism plays a pivotal role in hypoxic responses. Prolonged hypoxic exposure induces multifaceted metabolic adaptations: enhances glycolytic pathway activity [5], activates gluconeogenic metabolism [6], and coordinates regulation of insulin signaling efficiency to meet dynamic energy demands [7].

The liver serves as the central hub for systemic metabolic homeostasis, functioning as the primary regulator of carbohydrate metabolism [8]. In the glucose-driven glycolytic pathway, glucose molecules enter liver cells via glucose transporters (GLUTs) and are finally catabolized to pyruvate in the cytoplasm by a multistep enzymatic reaction. The GLUTs specifically distributed in liver tissue are mainly glucose transporter 1 (GLUT1) and glucose transporter 2 (GLUT2) [5, 9, 10], and the aldolase isoforms are aldolase A (ALDOA), aldolase B (ALDOB), and aldolase C (ALDOC) [11]. Key regulatory enzymes include phosphofructokinase (PFK) and pyruvate kinase (PK), with PFK as the principal rate-limiting enzyme, and the liver-specific PK isoform pyruvate kinase liver and red blood cell (PKLR) controlling terminal glycolytic flux pyruvate [12]. In fructose-driven glycolysis, fructose enters hepatic cells through GLUT5, bypassing PFK, and is directly phosphorylated to fructose-1-phosphate (F1P) by ketohexokinase (KHK), subsequently entering the glycolytic pathway [13, 14]. Thus, KHK serves as the primary rate-limiting enzyme in fructose metabolism. Following glycolytic generation of pyruvate, mitochondrial oxidation converts pyruvate to acetyl-coenzyme A (Ac-CoA), which combines with oxaloacetate to initiate the tricarboxylic acid (TCA) cycle. Key regulatory enzymes in this cycle include citrate synthase (CS), isocitrate dehydrogenase (IDH), and α-ketoglutarate dehydrogenase (α-KGDHC) [15]. In comparison with liver, the glucose transporter involved in glucose metabolic pathways in brain tissue is exclusively GLUT1 [10], and PKM1 represents the predominant pyruvate kinase (PK) isoform expressed in the brain. The remaining metabolic pathways are consistent with those observed in hepatic tissue. Other glycolytic components remain conserved across both organs. Hypoxia exerts detrimental effects on hepatic and cerebral tissues while disrupting their energy metabolism. Studies have found that chronic intermittent hypoxia leads to reduced mitochondrial oxidative phosphorylation and poor metabolic recovery [16]. In the liver, hypoxia exposure upregulates inflammation-related genes and fibrosis-related metabolites, aggravating fibrosis and inflammation, thereby causing systemic energy metabolism disorders [17, 18]. In the brain, due to the imbalance between neuronal energy demands and limited energy reserves, hypoxia often acutely disrupts cerebral energy supply, resulting in irreversible damage [19]. Paradoxically, hypoxia-inducible factor-1α (HIF-1α) activation provides neuroprotection by shifting energy production from oxidative phosphorylation to glycolysis, maintaining ATP supply under oxygen constraints [20, 21].

The gut microbiota, often called the “second genome” of mammals, exerts profound regulatory effects on host carbohydrate metabolism by establishing a complex metabolic and signaling networks [22, 23]. This complex ecosystem, comprising 10¹³ to 10¹⁴ microbial cells, participates in not only direct fermentation of dietary fibers [24], but also influence host metabolism by regulating the gut-liver axis [25]. Dysbiosis of the gut microbiota has been mechanistically linked to metabolic disorders, particularly obesity [26] and type 2 diabetes [27]. Moreover, hypoxia induces gut microbiota dysbiosis, leading to a reduced abundance of Lactobacillus, which in turn accelerates the senescence of bone marrow mesenchymal stem cells. This dysbiosis is also associated with an increased abundance of Bacteroides and decreased abundances of Bifidobacterium, Helicobacter, and Prevotella, along with the disruption of short-chain fatty acid (SCFA)-producing bacteria [28, 29]. Fecal microbiota transplantation (FMT) is a widely used method to study causal relationships between gut microbiota and diseases. Some studies have shown that FMT affects nervous system function through the microbiota-gut-brain axis [30]. In recent years, the application of FMT in the field of metabolic diseases has attracted considerable attention [29]. Its efficacy in treating glucose metabolism disorders, in particular, has been corroborated by multiple studies. In type 2 diabetic mice, FMT can restore the gut microbiota and ameliorate insulin resistance [31]. This finding is further supported by clinical evidence. For instance, research by Anne et al. demonstrated that transplanting gut microbiota from lean donors to male recipients with metabolic syndrome successfully restored microbial composition and reversed insulin resistance [32]. Furthermore, FMT may alleviate glucose metabolic disturbances through mechanisms such as activation of SCFA receptors and promotion of glucagon-like peptide-1 (GLP-1) secretion [33]. It may also facilitate the regeneration of pancreatic β-cells, thereby aiding in the repair of tissue damage caused by type 2 diabetes [34]. Additionally, FMT shows therapeutic potential for metabolic complications such as D-lactic acidosis [35].

Subterranean mammals face persistent environmental challenges [36]. Several studies demonstrate, compared to surface-dwelling animals, subterranean rodents exhibit greater tolerance to hypoxic conditions in order to maintain their metabolic rates [37, 38]. Gansu zokor (Eospalax cansus), representative subterranean rodents, have evolved distinctive hypoxic adaptation mechanisms through long-term evolutionary selection. Gansu zokor can survive over 4 h survive in environments with less than 10% oxygen [39], and their carbohydrate metabolism also changes its adaptive strategy under hypoxic conditions [40]. Furthermore, hypoxia-adapted subterranean species like plateau zokor (Eospalax baileyi) exhibit unique gut microbial profiles enriched in SCFA-producing and energy-metabolizing taxa, suggesting co-evolved microbial support for hypoxic endurance [41]. Preliminary studies by our laboratory revealed under different oxygen concentrations, the intestinal gut microbiota of the Gansu zokor undergo various changes, thereby affecting carbohydrate metabolism and lipid metabolism [42, 43]. However, the question of whether this amazing hypoxic carbohydrate metabolism mechanism of the Gansu zokor can be translated to surface animals. This question remains to be addressed in order to better understand and even apply this model of hypoxic adaptation.

To further investigate whether the hypoxia-adapted metabolic traits mediated by the gut microbiota of the Gansu zokor can influence surface rats, this study integrates the hypoxia metabolic characteristics of the Gansu zokor with FMT to establish a hypoxic metabolism research model involving cross-species FMT. By transplanting Gansu zokor fecal microbiota into Sprague-Dawley (SD) rats and validating microbial colonization through 16 S rRNA sequencing, we subsequently exposed the recipients to hypoxia (10.5% O₂, 4w). After that, systemic evaluations were then conducted on glucose and fructose metabolic pathways as well as TCA cycle components. These findings provide new evidence to study the mechanism of hypoxic carbohydrate metabolism adaptation in subterranean rodent gut microbiota and provide an important animal model reference for hypoxic injury treatment based on FMT.

Materials and methods

Animal administration and ethics statement

The study utilized two animal species: Gansu zokor and the SD rats. Adult Gansu zokor (230 ± 25 g) were captured their natural habitat in Tongchuan City, Shaanxi Province, China (35°12′N, 109°11′E). Captured individuals were housed individually in cages (47.5 × 35.0 × 20.0 cm) at 25 °C. Adult Gansu zokor were selected based on the age determination method [44].

3-week-old SD rats (61 ± 10 g) were obtained from the School of Medicine, Xi’an Jiaotong University, and housed under identical environmental conditions in a group-specific manner, with up to 2 animals per cage. All animals were adapted to laboratory conditions for at least 4 weeks before hypoxia treatment, during which they were allowed to freely feed (Gansu zokor, carrots; SD rats, standard rat food and water). All experimental animals belong to our research team. All animal procedures were reviewed and approved by the Animal Management Committee and Ethical Review Committee of Experimental Animal Welfare, Shaanxi Normal University, and were conducted in accordance with the guidelines of the China Wildlife Conservation Association.

Animal models and intervention strategies

This study included a total of twenty experimental animals: ten Gansu zokor and ten SD rats. Ten healthy Gansu zokor were randomly divided into two groups: (a) normoxic group (Tn): environmental oxygen concentration; (b) hypoxic group (Tl): 10.5% O₂, 4 weeks. Ten healthy SD rats were randomly divided into two pre-treatment groups: (a) normoxic control group (Rn): environmental oxygen concentration without FMT; (b) normoxic FMT group (Rf): environmental oxygen concentration with FMT from Tn. After FMT, the 10 SD rats from the normoxic groups (Rn and Rf) were placed under hypoxic conditions (10.5% O₂, 4 weeks). The original control and experimental group structures were retained, with no changes in animal count or individual assignment. The hypoxia-exposed Rn and Rf groups were then renamed accordingly: (c) hypoxic control group (Rlc): Rn group exposed to 10.5% O₂ for 4 weeks; (d) hypoxic FMT group (Rlf): Rf group exposed to 10.5% O₂ for 4 weeks. Oxygen concentration in this experiment was referred to the previous laboratory study [43].

Pre-treatment of fecal samples

Fresh feces were collected daily from the Gansu zokor normoxic group (Tn) between 9:00–11:00 a.m [45]., and the feces were diluted with sterile PBS (1:10, m: v), centrifuged (600 g, 15 min, 4 °C), and the supernatant was taken. The resulting supernatant was mixed with 10% sterile glycerol to make the fecal suspension, and immediately stored at −80 °C [46].

Antibiotic cocktail pretreatment

The normoxic FMT group (Rf) was pretreated with the antibiotic cocktail under normoxia to establish sterile rat models. 1 g/L ampicillin solution was feed for 7 days; followed by oral gavage with a cocktail of metronidazole (100 mg/kg), neomycin (100 mg/kg), and vancomycin (50 mg/kg) once daily for 3 days [47]. Normoxic control group (Rn) were given free access to water for 7 days, followed by sterile PBS gavage for the last 3 days. Oral gavage treatments were administered once daily under normoxia between 08:00–10:00 a.m., with 10 mL/kg of solution per rat at one time.

FMT treatment

SD rats (Rn and Rf) were suspended for 24 h and treated with FMT and control treatment. The normoxic FMT group (Rf) was treated by gavage once daily morning with 10 mL/kg of fecal suspension once for 14 days. In contrast, the normoxic control group (Rn) was treated with 10 mL/kg of 10% sterile PBS per rat by gavage [48]. After 14 days, fecal samples were collected from all subjects in both groups prior to hypoxia exposure. During the subsequent 4-week hypoxia phase, gavage administration was maintained at weekly intervals.

Details of the experimental groups and treatments are provided in Supplementary Information for Materials and Methods Figure S1.

Animal sample collection and treatment

At the end of the hypoxic protocol, fresh fecal samples were collected and then anesthetized with pentobarbital sodium. Subsequently, venous blood samples were collected and centrifuged (3,000 rpm, 10 min, 4 °C) to separate plasma. At the same time, liver and brain tissues were collected and immediately preserved in liquid nitrogen and later stored at −80 °C for subsequent experiments.

16 S rRNA sequencing and analysis

Fecal microbial genomic DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN, cat#51504), and the success of DNA extraction was verified by agarose gel electrophoresis. The hypervariable V3-V4 region of the 16 S rRNA gene was amplified through PCR, followed by sequencing on the Illumina NovaSeq platform (Shanghai Personalbio Technology Co., Ltd., Shanghai, China) according to established protocols. Raw sequencing data were processed using Quantitative Insights into Microbial Ecology (QIIME2 2019.4) software. Sequence processing, including quality control, denoising, read merging, and chimera removal, was conducted using DADA2 (v1.1), with the range of sequences per sample provided in Supplementary Information Table S2. Subsequent amplicon sequence variant (ASVs) analysis and read processing were conducted through the Genescloud platform (https://www.genescloud.cn/). Details of the 16 S rRNA analysis are provided in the Supplementary Information.

RNA isolation and real-time quantitative PCR

Total RNA was extracted from liver and brain tissues using RNAiso Plus (TAKARA, Kusatsu, Japan). Reverse transcription of isolated RNA was performed using the Reverse Transcription Kit (TransGenBiotech, Beijing, China), and primers were designed and synthesized by Sangon Biotech (Shanghai, China) with the primer sequences shown in Supplementary Information Table S1. Real-time quantitative PCR was conducted on the Reaction Optics module (Bio-Rad Laboratories, Hercules, CA, USA) using the CFX Connect system, according to the instructions. Gene expression quantification utilized the 2^−ΔΔCt method, with β-tubulin serving as the endogenous normalization control.

Protein extraction and western blot analysis

Protein extraction of liver and brain tissues was performed using the ProteinExt Mammalian Total Protein Extraction Kit (TransGen Biotech, Beijing, China), and protein samples (40 µg per lane) were separated by electrophoresis using 10% SDS-polyacrylamide gel electrophoresis (PAGE), and later transferred onto polyvinylidene difluoride (PVDF) membranes. After 5% skimmed milk blocking and incubation with primary and secondary antibodies, the protein bands were visualized on a ChemiScope 6100 Chemiluminescence Imaging System (CLiNX, Shanghai, China) using chemiluminescent solution (dongxisw, Xi’an, China). And protein quantification was performed using ImageJ software, normalized to β-tubulin. Detailed experimental procedures and antibody information are provided in the Supporting Information.

Tissue homogenization was performed in phosphate-buffered saline (10× tissue volume) and centrifuged (5,000 rpm, 15 min, 4 °C). Supernatants were subjected to quantification of citrate synthase (CS), isocitrate dehydrogenase (IDH), and α-ketoglutarate dehydrogenase (α-KGDHC) using specific enzyme-linked immunosorbent assay (ELISA) kits. (CS kit, Meimian, Yancheng, China; IDH kit, Meimian, Yancheng, China; α-KGDHC kit, Meimian, Yancheng, China).

Glucose and fructose content determination

The glucose and fructose content kits (Feiya, Yancheng, China) were used to determine the glucose and fructose content in the liver and brain tissues of all animals according to the manufacturer’s instructions.

Statistical analysis

All experimental data are expressed as mean ± standard error of the mean (Mean ± SEM). Statistical analysis was performed using independent Student’s t-tests in SPSS 20.0 (IBM Corp., Armonk, NY, USA) to assess between-group differences. All bar graphs were generated by GraphPad Prism 9.0 (GraphPad Software, San Diego, USA).

Result

FMT alters gut microbial composition and diversity in SD rats

To examine the long-term hypoxic effects on gut microbiota structure in Gansu zokor and SD rats, and validate FMT efficacy, we performed fecal 16 S rRNA sequencing. To better present the results, comparative analyses were separated by oxygenation status: normoxic groups (Tn, Rn, Rf) versus hypoxic groups (Tl, Rlc, Rlf).

Venn diagram (Fig. 1A) presented us with the number of shared sequences between groups, the FMT-treated SD rats (Rf) had an increase in shared sequences with the Gansu zokor and retained 4 sequences after hypoxic treatment (Rlf). Microbial composition analysis (Fig. 1B) revealed that neither FMT nor prolonged hypoxia altered the two phyla with the highest relative abundances in either group—Firmicutes and Bacteroidetes.

The alpha-diversity metrics (Shannon index and observed species; Fig. 1C) illustrated that the FMT-treated SD rats had lower diversity and richness of microbial communities. Principal coordinate analysis (PCoA) of β-diversity (Fig. 1D) and permutational multivariate analysis of variance (PERMANOVA) (Fig. 1E), together with hierarchical clustering of samples at the genus level (Fig. 1F), demonstrated that the grouping of samples largely reflected the underlying characteristics of the microbial communities. Inter-group differences were statistically significant (p < 0.05), indicating distinct microbial community structures among the six experimental groups. Linear discriminant analysis Effect Size (LEfSe) (Fig. 1G) further identified group-specific biomarkers. Normoxic group: Rn (Turicibacteraceae), Rf (Actinobacteria, Bifidobacteriaceae), Tn (S24-7). Hypoxic group: Rlc (Bacteroidaceae), Rlf (Lactobacillaceae, Bacilli), Tl (Allobaculum, Erysipelotrichaceae).

Collectively, these results confirm the validity of the experimental grouping. Following FMT, a significant difference in gut microbiota composition was observed between the Rn and Rf groups (p < 0.01), demonstrating that FMT altered the compositional and structural characteristics of the gut microbiota in SD rats.

Changes in hepatic carbohydrate metabolism following FMT in SD rats under prolonged hypoxia

Hepatic glucose metabolism tends to increase after FMT in SD rats

To explore the impact of FMT on hepatic glucose metabolism in SD rats under prolonged hypoxia (10.5% O₂, 4 w), we analyzed glucose metabolism pathway components at transcriptional and translational levels via RT-qPCR and Western blot. As shown in Fig. 2A, the Rlf group showed elevated mRNA expression levels of three aldolases involved in glucose metabolism compared to the Rlc group, with Aldob and Aldoc exhibiting statistically significant upregulation (p < 0.05). The mRNA expression of the glucose transporter Glut2 and the rate-limiting enzyme Pfk were also elevated. Notably, this expression pattern showed species-specific differences: In the Tl group, all examined genes except Aldoc and Glut1 demonstrated significantly lower mRNA expression levels relative to the Rlc group (p < 0.05).

As shown in Fig. 2B, the protein levels of ALDOC (1.193) and PKLR (0.735) in the Rlf group were increased compared with those in the Rlc group (ALDOC: 0.813; PKLR: 0.451), and GLUT1 protein expression was significantly upregulated (p < 0.01). In the Tl group, related proteins levels of most glucose metabolism—with the exception of ALDOB and transporters—were also significantly higher than those in the Rlc group (p < 0.01). Together with the hepatic glucose content data shown in Fig. 2C, these findings suggest that under prolonged hypoxia, hepatic glucose metabolism is more active in Gansu zokor than in SD rats. Moreover, SD rats that received FMT from Gansu zokor exhibited altered expression of genes and proteins involved in hepatic glucose metabolism, reflecting a trend toward enhanced glucose metabolic activity.

Hepatic fructose metabolism network is altered after FMT in SD rats

In the fructose metabolic pathway, GLUT5 and KHK serve as key biomarkers for assessing fructose utilization intensity. As shown in Fig. 3A, prolonged hypoxic exposure (10.5% O₂, 4 w) induced differential regulation of these biomarkers at transcriptional and translational levels. The Rlf group exhibited significantly elevated mRNA expression of Khk and Glut5 compared to the Rlc group, with Khk showing more pronounced upregulation (p < 0.05). In the Tl group, the mRNA expression levels of Khk and Glut5 were significantly lower than those in the Rlc group (p < 0.01).

At the protein level, as shown in Fig. 3B, the GLUT5 protein content in the Rlf group was significantly higher than that in the Rlc group (p < 0.05), while KHK protein levels showed no significant change—a pattern consistent with that observed in the Tl group relative to the Rlc group. Hepatic fructose content, presented in Fig. 3C, did not substantially differ among the three groups. Collectively, these findings suggest that the hepatic fructose metabolic network is remodeled in Gansu zokor compared with control SD rats, and that FMT-treated SD rats subjected to exhibit a fructose metabolic profile relatively similar to that of Gansu zokor.

Hepatic TCA cycle intensity tends to decline after FMT in SD rats

To investigate TCA cycle intensity under prolonged hypoxia (10.5% O₂, 4 w), we analyzed three rate-limiting enzymes CS, IDH and α-KGDHC. The ELISA results in Fig. 4 showed that, in the Rlf group, IDH and α-KGDHC levels decreased significantly compared to Rlc (p < 0.05), whereas CS level remained stable. Strikingly, the concentrations of the three rate-limiting enzymes in Tl were relatively similar to those in the Rlf group. These findings suggest that, compared with control SD rats, the hepatic TCA cycle intensity in Gansu zokor exhibits a decreasing trend, and that the concentration profiles of these three enzymes in FMT-treated SD rats follow a comparable trend to that observed in Gansu zokor, also showing a tendency toward reduced TCA cycle intensity.

Fig. 4 — Analysis of key enzymes levels in TCA cycle of Gansu zokor and SD rats livers. A CS level; B IDH level; C α-KGDHC level. All data are shown as Mean ± SEM. Statistical symbols: *p < 0.05 and **p < 0.01 between same species; #p < 0.05 and ##p < 0.01 between different species. Rlc: hypoxic control group; Rlf: hypoxic FMT group; Tl: hypoxic Gansu zokor group

Changes in cerebral carbohydrate metabolism following FMT in SD rats under prolonged hypoxia

Cerebral glucose metabolism tends to increase after FMT in SD rats

To explore potential organ-specific variations in FMT-mediated regulation of the glucose metabolism pathway during prolonged hypoxia exposure (10.5% O₂, 4 w) in SD rats, we focused on cerebral metabolic responses given the brain’s high sensitivity to glucose metabolism. As shown in Fig. 5A, compared with the Rlc group, the Rlf group exhibited elevated the mRNA expression of Pfk, while other measured glucose metabolism components showed significant downregulation (p < 0.05). The Tl group displayed markedly higher transcriptional levels of key regulators (Aldoa, Aldob, Pfk, and Glut1) relative to the Rlc group, with all comparisons reaching statistical significance (p < 0.01).

Protein quantification analyses presented in Fig. 5B, the three key proteins related to glucose metabolism, PFK, GLUT1 and PKM1, were all increased in the Rlf group relative to the Rlc group, especially PFK (p < 0.01). The Tl group showed basically the same results as the Rlf group, and in particular, the Tl group had higher levels of all three proteins than the Rlf group. Discrepancies between some RT-qPCR and Western blot results may be attributed to post-transcriptional regulation. Taken together with the cerebral glucose content shown in Fig. 5C, we hypothesize that, under prolonged hypoxia, both Gansu zokor and SD rats transplanted with Gansu zokor gut microbiota exhibit a trend toward enhanced brain glucose metabolism compared with control SD rats.

Cerebral fructose metabolism network is altered after FMT in SD rats

As shown in Fig. 6, under prolonged hypoxia (10.5% O₂, 4 w), qPCR analyses revealed transcriptional upregulation of Khk and Glut5 in the Rlf group compared to the Rlc group. However, the GLUT5 protein level in the Rlf group was significantly lower relative to the Rlc group (p < 0.01), and there was no significant change in KHK protein. This transcriptional-protein discordance was accentuated in the Rlc group. The mRNA expression of Khk and Glut5 in Gansu zokor was significantly higher than that in the Rlc group (p < 0.01), while the WB results showed that, on the contrary, the GLUT5 and KHK protein levels were significantly lower than those in the Rlc group (p < 0.01). These comparative analyses demonstrated that these comparative analyses indicate that the mismatch between transcriptional activation and post-translational regulation in the Rlf group is similar to that observed in the Tl group.

Figure 6C showed the fructose level in the brain, and it was obvious that the fructose level in both the Rlf and Tl groups was significantly higher than that in the Rlc group (p < 0.01). Based on the above results, we hypothesized that under prolonged hypoxia, cerebral fructose metabolism in Gansu zokor exhibited a lower intensity compared with that in SD rats. In FMT-treated SD rats, however, this alteration was reflected only as a decrease in GLUT5 protein expression, suggesting that the cerebral fructose metabolic network in these rats has been remodeled to approach, but not fully recapitulate, the metabolic profile observed in Gansu zokor.

Cerebral TCA cycle intensity tends to decline after FMT in SD rats

Figure 7 revealed hypoxia-induced remodeling of TCA cycle regulators. Under prolonged hypoxia (10.5% O₂, 4 w), the levels of IDH and α-KGDHC enzymes decreased, and the level of CS enzymes increased in the Rlf group compared to the Rlc group. Gansu zokor exhibited analogous enzymatic patterns to the Rlf group. Based on these findings, both Gansu zokor and FMT-treated SD rats displayed a trend toward reduced TCA cycle intensity in the brain compared with control SD rats, characterized by suppressed dehydrogenase levels (IDH, α-KGDHC) with compensatory CS level elevation.

Fig. 7 — Analysis of key enzymes levels in TCA cycle of Gansu zokor and SD rats brains. A CS level; B IDH level; C α-KGDHC level. All data are shown as Mean ± SEM. Statistical symbols: *p < 0.05 and **p < 0.01 between same species; #p < 0.05 and ##p < 0.01 between different species. Rlc: hypoxic control group; Rlf: hypoxic FMT group; Tl: hypoxic Gansu zokor group

Discussion

In recent years, the gut microbiota has garnered increasing scientific recognition for its profound influence on host physiology, with emerging research continually expanding our understanding of its functional roles [49, 50]. FMT, a therapeutic modality tracing back to 4th-century treatments for gastrointestinal disorders [51]. Nowadays, FMT has become a common tool in hospitals and has shown positive responses in the treatment of many diseases. In this study, we transplanted the gut microbiota of the hypoxia-adapted Gansu zokor into ground-living SD rats by FMT. Through 16 S rRNA sequencing and biochemical molecular assays, we investigated the microbial community restructuring in recipient rats under hypoxia, as well as the changes in hepatic and cerebral glycolysis and the TCA circulating fluxes. Through research, we have obtained the following three findings: (1) FMT altered the gut microbial composition and diversity in SD rats. (2) FMT-treated SD rats exhibited organ-dependent differences in hepatic and cerebral carbohydrate metabolism under prolonged hypoxia. Interestingly, this pattern relatively resembled the carbohydrate metabolic profile observed in Gansu zokor. (3) Compared with control SD rats, FMT-treated SD rats exhibited a trend toward suppressed TCA cycle activity in both the liver and brain under prolonged hypoxia—a pattern broadly convergent with that observed in Gansu zokor. Based on these findings, we hypothesize that FMT alters the composition and structure of the gut microbiota in SD rats, thereby modulating the systemic carbohydrate metabolic phenotype of SD rats under prolonged hypoxia, rendering it more similar to that of Gansu zokor.

FMT alters gut microbiota composition and structure in SD rats

The 16 S rRNA sequencing results revealed that the number of shared sequences by SD rats receiving FMT and Gansu zokor increased in normoxia, and four shared sequences were also retained under hypoxia. The alpha-diversity index demonstrated that the diversity and richness of the gut microbiota in recipient SD rats were lower, which may be due to the fact that some flora of Gansu zokor could not survive stably in SD rats, coupled with the fact that hypoxia also destabilizes the gut microbiota, which led to this result. Based on β-diversity analysis, significant differences in gut microbiota were observed between the control SD rats and FMT-treated SD rats after FMT (p < 0.01), indicating that FMT altered the composition and structure of the gut microbiota in SD rats.

At the phylum level, Firmicutes and Bacteroidetes were the two phyla with the highest relative abundances in both SD rats and Gansu zokor. This compositional feature remained unchanged in both species after four weeks of hypoxic exposure. This is consistent with previous laboratory studies on the gut microbiota of Gansu zokor [42, 43]. The two main phylums in mammalian gut microbiota are Firmicutes and Bacteroidetes [52–54]. These two phylums are primarily responsible for food fermentation in the gut and are associated with carbohydrate diets [55, 56]. Literature reports indicate that the Firmicutes/Bacteroidetes ratio not only affects carbohydrate metabolism but may also induce insulin resistance, potentially leading to diabetes [57–59]. This study found that under prolonged hypoxia, compared to the control SD rats (F/B ratio: 3.50), FMT-treated SD rats and Gansu zokor had higher Firmicutes/Bacteroidetes ratios (4.33 and 6.81, respectively), along with enhanced hepatic glycolysis. It is thus hypothesized that the Firmicutes/Bacteroidetes ratio positively correlates with hepatic glycolytic intensity.

LEfSe analysis revealed that under normoxia, the biomarker enriched in control SD rats was Turicibacteraceae, whereas FMT-treated SD rats exhibited Actinobacteria and Bifidobacteriaceae as biomarkers, indicating that FMT altered the gut microbial biomarkers and, to some extent, reshaped the microbial community structure in SD rats. Of note, the biomarker identified in normoxic Gansu zokor was the S24-7 family (within Bacteroidota), which was not detected as a biomarker in FMT-treated SD rats—presumably due to its low colonization abundance failing to reach the threshold for biomarker identification. Under hypoxia, the biomarker in control SD rats was Bacteroidaceae, while FMT-treated SD rats were characterized by Lactobacillaceae. In contrast, Gansu zokor under hypoxia showed Erysipelotrichaceae and Allobaculum as their biomarkers. Collectively, FMT induced shifts in the biomarker profiles of SD rats, although these profiles remained distinct from those of Gansu zokor. These findings further support the regulatory role of FMT in modulating host gut microbiota composition.

From the genus-level compositional profile, Lactobacillus exhibited the highest relative abundance in SD rats after FMT, which is a common Firmicutes phylum bacterium capable of fermenting various carbohydrates. Studies demonstrate positive correlations between Lactobacillus abundance and differential metabolites involved in amino acid and carbohydrate metabolism, indicating its potential role in biosynthetic pathway modulation [60, 61]. Our findings align with these observations: under prolonged hypoxia, FMT-treated SD rats with stronger glycolytic intensity had a higher relative abundance of Lactobacillus compared to control SD rats. Genus-level analysis revealed that FMT led to a tendency toward higher Allobaculum abundance in SD rats under normoxia. Given that Allobaculum constitutes the most dominant genus and serves as a hypoxia-associated biomarker in Gansu zokor, this observation points to partial engraftment of this biomarker in the SD rats gut after FMT. However, after prolonged hypoxia, the relative abundance of Allobaculum in FMT-treated SD rats instead decreased. In contrast, the genus that displayed a more pronounced increasing trend remained Lactobacillaceae—the most abundant genus in SD rats—which also served as the biomarker for FMT-treated SD rats under hypoxia. Research shows that Lycium barbarum polysaccharides can enhance Lactobacillus and Allobaculum proliferation, improving lipid metabolism [62]. Additionally, murine Allobaculum strains may regulate autoimmunity [63]. Based on these observations, we speculate that the relatively high abundance of Allobaculum in Gansu zokor may represent a distinctive feature of its adaptation to hypoxic environments. This inference is consistent with previous findings from our laboratory [42]. Allobaculum may synergized with Lactobacillus in carbohydrate metabolism and probably modulating immune homeostasis. However, FMT alone failed to fully replicate this feature in SD rats. Under prolonged hypoxia, SD rats preferentially relied on Lactobacillus rather than Allobaculum to regulate carbohydrate metabolism.

FMT alters hepatic and cerebral carbohydrate metabolic phenotypes in SD rats under prolonged hypoxia

Carbohydrates are one of primary energy substrates for biological functions, with dysregulation of their metabolic networks contributing to the pathogenesis of metabolic disorders. Both glucose and fructose are substrates of the glycolytic pathway and play important roles in carbohydrate metabolism [64]. Glucose participates in neuronal signaling pathways governing feeding behavior, energy balance, and systemic glucose homeostasis [65, 66]. Fructose is primarily metabolized by the livers and kidneys [67, 68]. It can enhances enzymatic activity related to lipogenesis and glycolysis [69]. However, excessive fructose may lead to metabolic syndrome and impaired skeletal development [70, 71].

The liver is one of the main places for carbohydrate conversion in the human body [72]. While earlier work indicated that the liver of Gansu zokor exhibits high metabolic flexibility, showing lower carbohydrate metabolism intensity under prolonged hypoxia than under normoxia [43]. Building upon our previous work, the present study provides preliminary insights into the regulatory role of gut microbiota in carbohydrate metabolism. Our findings indicate that under prolonged hypoxic conditions, both FMT-treated SD rats and Gansu zokor exhibited a trend toward enhanced hepatic glucose metabolism relative to control SD rats, as evidenced by increased protein activities of the key glucose metabolism enzymes PFK and PK. This phenomenon may be associated with microbiota—derived SCFAs under hypoxic conditions, previous studies have demonstrated that SCFAs can upregulate fibroblast growth factor 21 (FGF21) expression, thereby enhancing hepatic glucose uptake [73, 74]. Of note, the observed changes in hepatic glycolytic enzyme activities were more pronounced in Gansu zokor than in FMT-treated SD rats. This result suggests that although FMT-treated SD rats displayed a tendency toward a hepatic glucose metabolic profile approaching that of Gansu zokor, the degree of convergence remained modest. It can thus be inferred that FMT may exert modulatory effects on the hepatic metabolic phenotype of SD rats, presumably by altering gut microbial composition and structure. However, possibly due to interspecies differences in organ-specific metabolic characteristics and genetic background, SD rats under the current experimental conditions did not fully recapitulate the metabolic pattern observed in Gansu zokor. The underlying mechanisms warrant further investigation.

Unlike glucose, fructose catabolism provides critical advantages by bypassing PFK-mediated rate-limiting steps, enabling rapid ATP generation [75]. The present study revealed that both FMT-treated SD rats and Gansu zokor exhibited distinct fructose metabolic networks compared with control SD rats, as demonstrated by the significantly upregulated expression of the fructose-metabolizing protein GLUT5. Studies show that hypoxia eliminates metabolic constraints on fructose in organs like the liver and kidneys [76]. Under prolonged hypoxia, the hepatic protein level of the fructose transporter GLUT5 was significantly higher in both FMT-treated SD rats and Gansu zokor than in control SD rats, whereas no significant difference was observed in the expression of KHK. These findings suggest that FMT may have shifted, to some extent, the fructose metabolic pattern of SD rats toward that of Gansu zokor—that is, under prolonged hypoxia, their fructose metabolic profile exhibited a certain degree of alteration relative to control SD rats. High GLUT5 protein expression enhances hepatic fructose uptake capacity, preventing blood fructose accumulation. Stable KHK levels relatively may be maintained through post-translational modifications or substrate inhibition due to negative feedback mechanisms.

In brain tissue, FMT-treated SD rats exhibited a trend toward enhanced glucose metabolism compared with control SD rats under prolonged hypoxia, as evidenced by significantly upregulated PFK protein expression, while PKM1 showed an increasing tendency that did not reach statistical significance. This metabolic profile shared certain similarities with, yet remained not fully consistent with, the cerebral glucose metabolic pattern observed in Gansu zokor under prolonged hypoxic exposure: in Gansu zokor, PKM1 protein levels were also significantly elevated, suggesting a more fully activated glycolytic pathway. A similar but discrepant pattern was also observed in fructose metabolic flux. In FMT-treated SD rats, GLUT5 expression in the brain was significantly decreased, whereas KHK expression showed no appreciable change. In contrast, both GLUT5 and KHK were significantly downregulated in the brain of Gansu zokor, indicating an overall lower fructose metabolic flux relative to control SD rats. These findings suggest that although FMT was able to modify, to a certain extent, the cerebral fructose metabolic profile of SD rats, it was insufficient to fully recapitulate the reduced fructose metabolic state characteristic of Gansu zokor. Collectively, FMT exerted a measurable influence on the systemic carbohydrate metabolic phenotype in the brain of SD rats. However, under the present experimental conditions, the metabolic pattern did not fully converge with that of Gansu zokor. It is worth noting that in contrast to the liver, the brain of Gansu zokor adopted a more pronounced strategy of fructose metabolic suppression under prolonged hypoxia. This organ-dependent divergence in metabolic regulation may reflect an adaptive mechanism for optimizing energy allocation and maintaining physiological homeostasis under hypoxic stress. The underlying mechanisms warrant further investigation.

Previous studies indicated that Gansu zokor, like naked mole-rats, can utilize fructose-driven glycolysis to supply energy to the brain [40, 75]. The low fructose metabolism in Gansu zokor brains under prolonged hypoxia might be a unique adaptive mechanis. In response to acute severe hypoxia (6.5% O₂ 6 h), the fructose-driven glycolysis in the brain of Gansu zokor is enhanced, which is likely a survival mechanism under such extreme conditions [43]. In contrast, during prolonged hypoxia (10.5% O₂ 4 w), the level of fructose glycolysis is reduced. This attenuation may represent a metabolic adaptation to prolonged hypoxia, as Gansu zokor no longer requires rapid fructose metabolism to generate emergency energy. Additionally, this could prevent excessive accumulation of advanced glycation end products (AGEs), which may lead to neuronal degeneration and inflammatory responses [77, 78]. This aligns with Brooks’ findings in turtles (Pseudemys scripta). The ability of turtle brains to survive prolonged hypoxia is also a result of metabolic rate suppression [79]. Interestingly, FMT was found to partially induce metabolic adaptations in SD rats that paralleled, but did not fully replicate, those observed in Gansu zokor. The slightly lower KHK protein level in FMT-treated SD rats compared with Gansu zokor further underscores the differences in their metabolic regulatory profiles.

In summary, FMT exerted a measurable influence on the global carbohydrate metabolic phenotypes in the liver and brain of SD rats by altering the composition and structure of their gut microbiota. However, under the present experimental conditions, the metabolic patterns of SD rats did not fully converge with those of Gansu zokor. This phenomenon may involve multiple factors and, given the limitations of the current study design, cannot be definitively attributed to any single cause—an issue that warrants further investigation.

FMT-Treated SD rats exhibit a trend toward decreased hepatic and cerebral TCA cycle under prolonged hypoxia

Substantial evidence confirms that hypoxia suppresses TCA cycle activity, as observed in acute-on-chronic liver failure and neoplastic cells [73, 80]. Under prolonged hypoxia, cross-species FMT altered the concentrations of key rate-limiting enzymes of the TCA cycle in the liver and brain of SD rats, thereby exerting an influence on TCA cycle flux and shifting it toward the metabolic pattern characteristic of the hypoxia-adapted Gansu zokor. Specifically, significantly lower concentrations of IDH and α-KGDHC in the livers of FMT-treated SD rats and Gansu zokor directly constrains carbon flux from isocitrate to succinyl-CoA in the TCA cycle. CS levels remained stable, possibly because CS enhances citrate synthesis, and citrate has beneficial effects on brain oxidative stress, neuronal damage, liver and DNA damage [74]. In the cerebral TCA cycle, low expression of IDH and α-KGDHC accompanied by high expression of CS was observed in both FMT-treated SD rats and Gansu zokor. As previously discussed, elevated CS enhances citrate synthesis, which can alleviate neuronal injury [74]. The low expression of IDH and α-KGDHC may restrict subsequent carbon flow, reduce TCA cycle rate to prioritize synaptic function over maximal ATP output. Given that these three enzymes represent the most critical rate-limiting steps in the TCA cycle, their collective modulation plays a particularly dominant role in the overall regulation of cycle flux. The present results show that SD rats subjected to FMT exhibited a trend toward reduced TCA cycle activity compared with controls—a pattern that paralleled the TCA cycle characteristics observed in Gansu zokor under hypoxic conditions. This finding offers experimental evidence that may inform future investigations into the role of gut microbiota in modulating mitochondrial oxidative metabolism under hypoxic stress.

Potential Underlying Mechanisms

Although the present study preliminarily reveals the impact of cross-species FMT on host carbohydrate metabolism, several limitations should be acknowledged. First, our investigation focused primarily on alterations in global metabolic phenotypes without delving into the underlying regulatory mechanisms, future studies incorporating single-bacterial colonization and gene knockout models may help clarify the molecular pathways through which specific gut microbes influence host glucose metabolism. Second, the donor microbiota used here was derived from Gansu zokor under normoxia, whether FMT from hypoxia-conditioned donors would produce different metabolic outcomes merits further exploration. Third, due to methodological constraints, RT-qPCR and Western blot analyses are subject to inherent limitations in cross-species comparisons, and while our primary aim was to assess changes in FMT-treated SD rats, the unique hypoxia-adaptive mechanisms of Gansu zokor require validation through more rigorous approaches. Finally, sex was not incorporated as a variable in the experimental design or analysis, future work should systematically evaluate the role of sex in modulating the effects of FMT.

Conclusion

In summary, this study significantly advances our understanding of gut microbiota-mediated regulation of hepatic and cerebral carbohydrate metabolism in Gansu zokor during prolonged hypoxia. For the first time, we integrated the hypoxia adaptation characteristics of Gansu zokor with FMT, establishing a cross-species transplantation model for hypoxic metabolism research. The results demonstrate that hypoxic adaptation in Gansu zokor involves gut microbiota-mediated regulation of carbohydrate metabolism. Importantly, modulation of the gut microbiota in SD rats via FMT was able to exert a influence on their global carbohydrate metabolic phenotype. This provides new evidence for studying the hypoxic adaptation mechanisms of gut microbiota in subterranean rodents, and offers an important animal model reference for hypoxic injury treatment based on gut microbiota transplantation.

Supplementary Information

Supplementary Material 1.^{(5.4MB, docx)}

Supplementary Material 2.^{(898KB, tif)}

Supplementary Material 3.^{(1.9MB, docx)}

Supplementary Material 4.^{(175.7KB, pdf)}

Acknowledgements

This work was financially supported by the Natural Science Basic Research Program of Shaanxi (Program No. 2024JC-ZDXM-14).

Authors’ contributions

JL, JH and ZL conceived and designed the study. ZL wrote the manuscript with the help of JL and JH. ZL, MY and YZ collected the samples and ZL performed the experimental work. ZL analyzed the data. All authors contributed to the revision of the manuscript and approved the final manuscript.

Funding

This work was financially supported by the Natural Science Basic Research Program of Shaanxi (Program No. 2024JC-ZDXM-14).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The experimental procedures were reviewed and approved by the Animal Management Committee and the Ethical Review Committee of Experimental Animal Welfare of Shaanxi Normal University, and the animals were treated in accordance with the regulations of the China Wildlife Conservation Association.

Consent for publication

Not applicable. This study did not include any form of personal data (including any personal details, images, or videos).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Jianping He, Email: hejianping@snnu.edu.cn.

Jingang Li, Email: jingang@snnu.edu.cn.

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75.Park TJ, Reznick J, Peterson BL, Blass G, Omerbašić D, Bennett NC, et al. Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat. Science. 2017;356(6335):307–11. [DOI] [PubMed] [Google Scholar]
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77.Ooi H, Furukawa A, Takeuchi M, Koriyama Y. Toxic advanced glycation end-products inhibit axonal elongation mediated by β-tubulin aggregation in mice optic nerves. Int J Mol Sci. 2024; 25(13):7409. 10.3390/ijms25137409 [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Gugliucci A. Formation of fructose-mediated advanced glycation end products and their roles in metabolic and inflammatory diseases. Adv Nutr. 2017;8(1):54–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Brooks SP, Storey KB. Anoxic brain function: molecular mechanisms of metabolic depression. FEBS Lett. 1988;232(1):214–6. [DOI] [PubMed] [Google Scholar]
80.Yu Z, Li J, Ren Z, Sun R, Zhou Y, Zhang Q, Wang Q, Cui G, Li J, Li A, et al. Switching from Fatty Acid Oxidation to Glycolysis Improves the Outcome of Acute-On-Chronic Liver Failure. Adv Sci (Weinheim Baden-Wurttemberg Germany). 2020;7(7):1902996. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(5.4MB, docx)}

Supplementary Material 2.^{(898KB, tif)}

Supplementary Material 3.^{(1.9MB, docx)}

Supplementary Material 4.^{(175.7KB, pdf)}

Data Availability Statement

No datasets were generated or analysed during the current study.

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PERMALINK

Cross-species fecal microbiota transplantation alters the carbohydrate metabolic phenotype: insights from Gansu zokor (Eospalax cansus)

Zhuohang Li

Maohong Yang

Yingying Zhang

Jianping He

Jingang Li

Abstract

Supplementary Information

Introduction

Materials and methods

Animal administration and ethics statement

Animal models and intervention strategies

Pre-treatment of fecal samples

Antibiotic cocktail pretreatment

FMT treatment

Animal sample collection and treatment

16 S rRNA sequencing and analysis

RNA isolation and real-time quantitative PCR

Protein extraction and western blot analysis

Glucose and fructose content determination

Statistical analysis

Result

FMT alters gut microbial composition and diversity in SD rats

Fig. 1.

Changes in hepatic carbohydrate metabolism following FMT in SD rats under prolonged hypoxia

Hepatic glucose metabolism tends to increase after FMT in SD rats

Fig. 2.

Hepatic fructose metabolism network is altered after FMT in SD rats

Fig. 3.

Hepatic TCA cycle intensity tends to decline after FMT in SD rats

Fig. 4.

Changes in cerebral carbohydrate metabolism following FMT in SD rats under prolonged hypoxia

Cerebral glucose metabolism tends to increase after FMT in SD rats

Fig. 5.

Cerebral fructose metabolism network is altered after FMT in SD rats

Fig. 6.

Cerebral TCA cycle intensity tends to decline after FMT in SD rats

Fig. 7.

Discussion

FMT alters gut microbiota composition and structure in SD rats

FMT alters hepatic and cerebral carbohydrate metabolic phenotypes in SD rats under prolonged hypoxia

FMT-Treated SD rats exhibit a trend toward decreased hepatic and cerebral TCA cycle under prolonged hypoxia

Potential Underlying Mechanisms

Conclusion

Supplementary Information

Acknowledgements

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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