Glucose plays a pivotal role in cell synthesis and function, serving as an essential energy source for maintaining animal physiological metabolism. Microorganisms in the rumen of ruminants can break down feed and produce metabolites such as volatile fatty acids (VFAs), proteins, and vitamins. These metabolites are transported from the rumen to the liver through the gut–liver axis to synthesize glucose via gluconeogenesis in hepatocytes (Wang et al., 2023; Yang et al., 2024). Enhanced gluconeogenesis can bolster feed utilization efficiency (Oliver, 1975) and foster the growth and development of ruminants (Hammon et al., 2013). A deficiency in glucose and precursors for gluconeogenesis during the perinatal phase can culminate in conditions like ovine pregnancy toxemia (Schlumbohm and Harmeyer, 2004). To improve the production performance of ruminants, the key is to improve the utilization rate of feed. The main method is to increase glucose production, that is, to adjust the gluconeogenesis process.

The regulation of gluconeogenesis involves many aspects, including the substrates (e.g., lactic acid, propionic acid, and amino acid), the activities of related enzymes (e.g., PEPCK and G6Pase), and the hormones (e.g., insulin and glucagon) (Bougouin et al., 2018; Caton et al., 2010; Dashty, 2013). The influence of short-chain fatty acid (SCFA) content on gluconeogenesis is the most critical. SCFAs are important gluconeogenesis substrates in ruminants, accounting for 60% of the carbon source required for gluconeogenesis (Larsen et al., 2013). Studies have shown that SCFAs can reach the liver through portal vein circulation in sheep, participate in hepatic gluconeogenesis, and regulate enzyme activity and gene expression related to energy metabolism (Yang et al., 2024). The experimental results of Zhang et al. also showed that injecting propionic acid into cattle can increase serum insulin levels and the mRNA expression levels of key gluconeogenesis enzymes PCK1 and G6PC (Zhang et al., 2015).

Inulin is a naturally occurring fructose-type plant polysaccharide, and its basic unit is β-D-fructopyran, which forms a stable chemical structure through β-(1-2)-glycosidic bonds. As a dietary fiber, inulin has many biological functions, such as enhancing immunity, improving growth performance, promoting intestinal digestion and absorption, and improving metabolic disorders in animals (Tawfick et al., 2022). Inulin is widely used in the diets of humans (Chambers et al., 2019), rats (Zhang et al., 2018), and sows (Li et al., 2020) to modulate gut microbiota, increase insulin sensitivity, and improve glucose metabolism. In ruminant studies, inulin has been shown to improve the gastrointestinal flora of dairy cows, increase the VFA levels in ruminal fluid, and improve immunity and lactation performance (Wang et al., 2021b, 2022). Another study indicated that dietary inulin supplementation enhances the body weight and feed efficiency of beef cattle and changes the rumen fermentation mode from acetic acid fermentation to propionic acid fermentation (Tian et al., 2019). Our previous study also indicated that inulin as a feed additive can effectively improve the intestinal immunity and feed efficiency of goats and promote the growth of ruminants (Yuan et al., 2022).

The regulatory effect of inulin on glucose metabolism in monogastric animals has sparked great interest in its use as a feed additive to improve the efficiency and growth performance of ruminants by regulating metabolism. Previous studies have not investigated inulin’s role in hepatic gluconeogenesis using a systems biology approach. Therefore, this study builds on the premise that inulin regulates rumen microbiota. The molecular mechanism of inulin regulating gluconeogenesis in ruminants can be elucidated by analyzing the gene expression of ruminal microorganisms, ruminal epithelial transporters, liver metabolites, and gluconeogenesis-related enzymes. The aim of this study is to provide a theoretical basis for inulin as a feed additive to improve ruminant glucose metabolism and enhance feed efficiency.

As a feed additive, inulin can effectively improve the feed conversion rate and weight gain of animals, thereby enhancing production efficiency. This phenomenon has attracted extensive attention (Grand et al., 2013; Li et al., 2019). In our previous study (data have been published), the average daily weight gain of goats in the inulin group was increased (Yuan et al., 2022). These results demonstrate the potential of inulin as a feed additive in improving the growth performance of goats. However, the specific mechanism by which inulin improves the growth performance of ruminants remains unclear. Adequate energy is essential to improve the growth performance of ruminants. Studies have shown a positive correlation between blood glucose levels and average daily weight gain (Wang et al., 2020b). We found that goat rumen microbiota, liver metabolites, and hormones were significantly altered after dietary inulin supplementation. These results confirm that dietary inulin supplementation has an impact on goat energy metabolism.

Inulin is a dietary fiber that regulates glucose metabolism (Hughes et al., 2022). Miao et al. reported that 3.33 and 1.67 g/kg/day of inulin supplementation significantly reduced blood glucose and insulin levels in gestational diabetes mellitus mice (Miao et al., 2021). Similar results were reported by Zhang et al. (2021), where blood glucose and insulin levels in high-fat mice were significantly reduced when supplemented with 2 g/kg of inulin. The addition of inulin in this study did not cause changes in blood glucose but reduced blood insulin levels. Our results are different, probably because, first of all, the high-fat diet in the above study led to changes in the health status of the animals, such as insulin resistance and reduced glucose tolerance. However, Zhang et al. did not show any changes in blood glucose and insulin levels after feeding normal feed with added inulin (Zhang et al., 2021). Secondly, the above studies are all aimed at monogastric animals, but ruminants can degrade inulin by rumen microorganisms and produce a large amount of gluconeogenetic precursors, such as propionic acid, lactic acid, and amino acids (Zhao et al., 2014; Wang et al., 2021b). These SCFAs are transported into the bloodstream through a transporter protein in the rumen epithelium and then into the liver as a gluconeogenesis substrate (Dijkstra, 1994). Hepatic gluconeogenesis is the most important pathway for ruminants to obtain glucose (Wang et al., 2023), and the decrease in insulin promotes gluconeogenesis (Hatting et al., 2018). Insulin promotes glycogen synthesis, which reduces blood sugar levels, and the liver is more sensitive to insulin signaling (Lawrence and Roach, 1997; Zhao et al., 2016). This might explain the significant increase in liver glycogen in the inulin group, but no change in muscle glycogen.

As the main digestive organ of ruminants, the function of the rumen is to convert the feed into VFAs through the microbial fermentation process (Hoover and Miller, 1991). Different diets can cause changes in VFA content (Wanapat et al., 2015). Zhao et al. used in-vitro fermentation to simulate the rumen environment and found that the content of TVFA, propionic acid, and butyric acid in the inulin group increased compared to the starch group, but the molar percentage of acetic acid and the A/P ratio decreased (Zhao et al., 2014). In addition, it was also observed that after adding inulin to the diet, the content of TVFA and the ratio of propionic acid and butyric acid in the rumen of dairy cows were increased, and the ratio of A/P was decreased (Wang et al., 2021b). In the present study, we found that in the inulin group, the content of TVFA and the ratio of propionic acid were higher, and the ratio of acetic acid and A/P decreased, which means that rumen fermentation mode changed from acetic acid to propionic acid. Propionic acid is an important substrate for gluconeogenesis and provides more than 70% of the carbon source for glucose production. Cui et al. have shown that increasing propionic acid content in the sheep rumen enhanced energy conversion efficiency and promoted weight gain in ruminants (Cui et al., 2023). GPR41 is a member of the G-protein-coupled receptor family and is expressed in animal gastrointestinal tissue. As a receptor for VFAs, GPR41 can bind to propionic acid and regulate insulin secretion to regulate energy metabolism (Kimura et al., 2020). Previous studies have shown that increased concentrations of propionic acid and butyric acid in the intestines of mice fed a dietary fiber diet, as well as increased expression of GPR41, promoted gluconeogenesis (De Vadder et al., 2014). The reduction in goat insulin levels observed in the inulin group in this study might be related to increased propionic acid concentration and increased expression of GPR41. The rumen epithelium contains a VFA transport system, and more than 75% of rumen VFAs enter the liver through the rumen epithelium transport system to provide energy for ruminants (Na and Guan, 2022). The rumen epithelial transport system is regulated by transporters (e.g., MCT1, AE2, DRA, PAT1). MCT1 is located in the basal layer of rumen epithelium and mediates the electroneutral transport of SCFA (Sivaprakasam et al., 2011). DRA, AE2, and PAT1 are mainly distributed outside the basement membrane and are responsible for exchanging SCFA⁻ and HCO₃ ⁻ inside and outside the cell (Wang et al., 2021a; Yu, 2021; Husain et al., 2025). By adjusting the diet to increase the mRNA expression levels of these SCFA transporters, the VFAs’ absorption capacity can be enhanced. Consequently, the increased expression of SCFA transporters in goats in the inulin group suggests that inulin not only promoted the production of large quantities of propionic acid in the goat rumen but also enhanced the overall absorption capacity of SCFA, allowing more propionic acid to enter the liver, providing a sufficient substrate for gluconeogenesis.

Different diets often lead to changes in the composition of rumen bacteria, and VFA concentrations are closely related to rumen bacteria. The PCoA analysis suggested that inulin addition altered the microbial composition. This result was similar to the report that dietary supplementation of inulin altered the composition of rumen microorganisms in beef steers (Louis et al., 2007). The reason for this result might be that inulin could be fermented and metabolized by rumen microorganisms (Tian et al., 2019; Zhao et al., 2014). Furthermore, the decreased OTU and ACE indexes in the inulin group and the similar Chao1, Shannon, and Simpson indices between the two groups indicated that dietary inulin supplementation decreased microbial diversity and did not change the richness of rumen microorganisms in the ruminal fluid of goats. At the phylum level, the increased abundance of Firmicutes was noted in the inulin group. Firmicutes can regulate the absorption of carbohydrates (Huang et al., 2024). Moreover, at the genus level, we observed higher abundance levels of Lachnospiracea, Blautia, Prevotella-1, and Pseudobotyrivibrio in the inulin group compared with the control group in this study. Lachnospiracea, which belongs to the phylum Firmicutes, can degrade fructose and cellobiose to promote propionic acid production in the rumen (Engels et al., 2016). Blautia is a probiotic that can generate large amounts of SCFA (Liu et al., 2021). Similarly, adding resistant starch to the diet of high-fat mice increased the abundance of Blautia in the intestine (Yang et al., 2024). Meanwhile, a similar result was observed by Sun et al., where inulin addition could stimulate the growth of Preverella-1 in human intestines. Prevotella-1 can participate in the metabolism and utilization of plant polysaccharides and can promote propionic acid fermentation in the bovine rumen (Sun et al., 2020; Wu et al., 2022). Pseudobotyrivibrio is positively correlated with propionic acid production in high-fat mice (Henning et al., 2018). Therefore, the higher abundance levels of Lachnospiracea, Blautia, Prevotella-1, and Pseudobotyrivibrio were consistent with the increased propionic acid production in the inulin group in this study. It was likely that inulin supplementation promoted the production of propionic acid by increasing the abundance levels of propionic acid-producing bacteria.

Changes in the gut microbiome can cause changes in the metabolic characteristics of the liver. This is due to the transport of intestinal microbiota metabolites (including carbohydrates, VFAs, and amino acids) into the liver through the gut–hepatic axial portal vein system, thereby affecting metabolic processes (Bauer et al., 2022). Studies report that as populations of VFA and amino acid-producing bacteria increase in the ileum of mice with non-alcoholic fatty liver disease, the concentrations of metabolites such as alanine, aspartate, and glucose also increase in the liver (Qiu et al., 2023). This suggests that dysregulation of intestinal flora may influence hepatic metabolic pathways. We applied metabolomics to analyze the effect of dietary inulin addition on the metabolic profiles in the livers of goats. The 95% confidence complete interval separation for PCA score maps was observed in the livers of goats between the two groups, suggesting that dietary inulin supplementation altered the metabolic profiles of the livers. The contents of arachidonic acid, FA 18_2, 9-Oxo-ODE, FA 18_2;O, and 8(R)-hydroperoxylinoleic acid in the inulin group increased significantly, and linoleic acid metabolism was significantly upregulated. Studies have reported that a significant increase in arachidonic acid content was found in sheep fed high-fiber diets, because Prevotella can activate the activity of phospholipase A2 to stimulate arachidonic acid production (Wu et al., 2020). Linoleic acid, also known as unsaturated fatty acid, can increase NR4A1 expression (de Vera et al., 2016), which promotes gluconeogenesis through the AKT–FOXO1–PEPCK/G6Pase pathway (Miao et al., 2019). We observed a notable elevation in L-arginine and 5-phosphoribosylamine levels in the hepatic tissues of goats subjected to inulin supplementation, accompanied by significant alterations in amino acid metabolic pathways. Dietary inulin supplementation has been shown to improve the rumen microbiota of dairy cows and increase the degradation of microbial proteins, which are converted into amino acids to improve amino acid metabolism (Wang et al., 2021). This might explain why amino acid metabolism was enhanced in this study. Arginine, a non-essential amino acid, is converted to glutamine by arginine succinyltransferase. 5-Phosphoribosylamine is produced from glutamine catalyzed by ribose phosphate pyrophosphokinase. Glutamine can enter the TCA cycle to yield oxaloacetic acid as a substrate for gluconeogenesis (Simões et al., 2024). Furthermore, the supplementation of arginine in the rumen enhanced the expression of gluconeogenetic genes (PEPCK and G6Pase) in cows (Souza Simões et al., 2024). The process of gluconeogenesis is the conversion of propionic acid, glycogenic amino acid, lactic acid, glycerol, and other substances into pyruvate as the initial reaction of gluconeogenesis. Pyruvate generates oxaloacetic acid through pyruvate carboxylase. However, since the mitochondria cannot transport oxaloacetic acid, oxaloacetic acid can only be transported into the cytoplasm through the tricarboxylic acid cycle into malic acid (Chandel, 2021). Increased propionic acid and glycoamino acid contents in the inulin group may be an important reason for higher levels of malic acid and citric acid than in the control group. The above results showed that inulin supplementation caused significant changes in goat rumen microbiota, which in turn caused changes in goat liver metabolites. Inulin supplementation improved linoleic acid metabolism, amino acid metabolism, and the TCA cycle and provided sufficient substrates for liver gluconeogenesis, offering strong evidence that inulin supplementation could improve gluconeogenesis in ruminants.

PEPCK and G6Pase are rate-limiting enzymes that regulate gluconeogenesis, and their activity reflects the situation of gluconeogenesis in animals (Agca et al., 2002). G6PDH is the main rate-limiting enzyme in the pentose phosphate pathway. Gluconeogenesis can provide energy for the pentose phosphate pathway (Allmann et al., 2013; Liang et al., 2017). HK, a rate-limiting enzyme for glycolysis, oversees glucose decomposition to produce pyruvate, essentially countering gluconeogenesis (De Jesus et al., 2022). The increase in PEPCK and G6Pase expression and the decrease in HK expression in the inulin group proved that inulin promoted gluconeogenesis while inhibiting glycolysis. In addition, we found that FoxO1 mRNA expression in goat liver increased in the inulin group. FoxO1 is a key gene that regulates gluconeogenesis. Earlier studies have shown that constitutively active FoxO1-expressing transgenic mice manifested upregulated expression of genes linked to gluconeogenesis and downregulated glycolysis-related genes (Zhang et al., 2006). In contrast, FoxO1 gene knockout mice exhibited reduced hepatic gluconeogenesis (Kamagate et al., 2010). Kawasoe et al. (2022) have reported a higher expression level of FoxO1 in the liver of mice fed with a diet containing 5% inulin, which was consistent with our results. The effect of FoxO1 on promoting gluconeogenesis is achieved by increasing the activities of PEPCK and G6Pase (Kamagate et al., 2010). The present study investigated the effects of dietary inulin supplementation on gluconeogenesis-related enzyme activity and gene expression. We speculate that upregulation of gluconeogenesis-related gene enzyme activity may be related to the alteration of insulin secretion and metabolites by inulin in goats. It has been reported that insulin can downregulate the expression of FoxO1 by activating the PI3K/AKT signaling pathway (Zhang et al., 2019). The maternal insulin infusion significantly reduced the expression of FoxO1 and PEPCK/G6Pase in the liver of hypoglycemic fetuses (Thorn et al., 2012). We speculate that the upregulation of gluconeogenesis-related gene enzyme activity may be related to hormone metabolism, TCA cycle, linoleic acid metabolism, and amino acid metabolism regulation. Although the addition of 18.9% inulin in this study showed a positive regulatory effect on rumen microorganisms and liver metabolism in goats, excessive inulin addition may lead to a decrease in the regulatory efficiency of ruminant microbiota and production performance, which may be related to the degradation efficiency of inulin in the rumen (Wang et al., 2021b). Given the current paucity of systematic research on optimal inulin additive levels in ruminant feed, future studies should further establish the dose–response relationship for inulin supplementation. This will help optimize ruminant feed formulations.

Inulin supplementation can reduce serum insulin levels and improve gluconeogenesis enzyme activity (PEPCK, G6Pase). It indicates that the addition of inulin to the diet promotes goat gluconeogenesis. In-depth investigation through omics analysis found that the promotion of inulin on goat gluconeogenesis was achieved by increasing gluconeogenetic substrates. Specifically, inulin altered the composition of goat rumen microorganisms and enhanced the gene expression of rumen epithelial transporters. The goat liver metabolome showed that inulin addition increased the levels of TCA intermediates and amino acids. This study confirms the efficacy of inulin as a feed additive for enhancing ruminant glucose metabolism, providing a foundation for future research on optimizing ruminant feed efficiency through inulin supplementation.