It has been previously shown that GB improves glucose and lipid metabolism by downregulating gluconeogenesis and liver steatosis in DIO mice (Kim et al., 2017). In this study, we demonstrated that global liver metabolites were altered by GB consumption in DIO mice and that changes in liver metabolites were highly correlated with metabolic phenotypes. Using CE-TOF/MS, metabolites were identified and categorized into 4 major metabolic pathways (Table 2; Fig. 1).
The mitochondrial membrane function, which was shown to be the most affected pathway, included 4 mechanisms (Fig. 1B). Mitochondria regulate energy homeostasis, hepatic lipid metabolism, and oxidative stress (Mansouri et al., 2018). Liver tissue from patients with liver steatosis, insulin resistance, and T2DM showed mitochondrial dysfunction (Koliaki et al., 2015). Choline plays various mechanistic roles in NAFLD, including VLDL export, ER stress, and mitochondrial function (Corbin & Zeisel, 2012). Choline and methionine deficiency decreases the composition of the mitochondrial membrane PC and phosphatidylethanolamine (PE), resulting in reduced activity of complex I in the respiratory system (Han et al., 2017; Petrosillo et al., 2007). GB consumption increased choline and methionine (Table 2; Fig. 2A, D), both of which were reduced by the HFD. This increase was expected to induce an increase in the amount of ethanolamine and ethanolamine phosphate by GB (Fig. 2B-C), which are precursors of PE (Patel & Witt, 2017). These change can be inferred to increase mitochondrial membrane formation. S-adenosyl methionine, which is produced from methionine and methyl donors in the process of PC formation from PE, decreased and SAH, with a methyl group removed from SAM, increased in GB-fed mice (Fig. 2E-F). These results suggest that increased PC composition in the mitochondrial membrane, which may have improved mitochondrial membrane function. Although choline metabolites increased in the HFD + GB group, glycerophosphocholine (GPC) showed a significant decrease (Table 2 (i)). This change can be interpreted as GPC acting as a choline donor. These metabolites were significantly correlated with serum HOMA-IR, determined using serum FBG and insulin levels. The correlation with HOMA-IR can also be interpreted as a correlation with serum FBG levels. Therefore, GB alters phosphatidylcholine and phosphatidylethanolamine metabolism in the liver of DIO mice, which may improve mitochondrial membrane functions and is, based on correlation analysis, highly related to the improvement of serum glucose levels and insulin resistance (Fig. 2A-F).
GB consumption altered metabolites related to beta-oxidation and energy homeostasis (Table 2; Fig. 1B). In nicotine and nicotinamide metabolism, NAD+, nicotinamide and NMN showed significantly different concentrations as a result of GB consumption (Table 2). NAD+ is a metabolite or coenzyme that plays a critical role in regulating various energy metabolism pathways, including β-oxidation and glycolysis, by assisting mitochondrial electron transport (Guarino & Dufour, 2019; Okabe et al., 2019). The increased flux of NAM, NMN, and NAD+ in GB-fed mice (Table 2 (ii); Fig. 2H-J) indicates that GB consumption improved energy homeostasis and ameliorated mitochondrial dysfunction in DIO mice, which is related to decreased serum glucose levels and insulin resistance. Carnitine is also essential metabolite for proper mitochondrial function, particularly transporting long-chain fatty acids to the inner mitochondrial membrane (Virmani & Cirulli, 2022) and its levels were significantly increased after GB consumption compared to that in HFD mice (Table 2; Fig. 2G). In addition, carnitine levels were significantly correlated with serum HOMA-IR (r = -0.7, p < 0.001) (Fig. 2G). These results indicate that GB may improve overall liver energy homeostasis by increasing NAD metabolism and beta-oxidation and might be related to the beneficial effects of hyperglycemia and insulin resistance in DIO mice.
In DIO mice, glycolysis and gluconeogenesis-related metabolite, including glucose-6-phosphate, fructose-6-phosphate, and dihydroxyacetone phosphate, were lower than those of the NC and HFD + GB groups, but 3-phosphoglyceric acid levels increased in GB-fed mice (Fig. 3A-D). Additionally, our previous study indicates that GB regulates gluconeogenesis by regulating PEPCK, G6Pase, and upstream transcription factors (Kim et al., 2017). As an alternative glucose metabolism, pentose phosphate pathway (PPP) play an important roles in glucose metabolism (Ge et al., 2020) and PPP branches off after the first step of glycolysis and consumes the intermediate glucose 6-phosphate (G6P) to generate fructose 6-phosphate (F6P) and glyceraldehyde 3-phosphate (G3P) (Patra & Hay, 2014). GB increased 6-phosphogluconic acid and ribose-5-phosphate levels, which are known to be in an oxidative state in PPP (Fig. 3F-H). Increased oxidative state metabolites in PPP can be referred to as increased NADPH (Yen et al., 2020). However, NADPH also induces the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is important for cellular antioxidant effects (Wamelink et al., 2008). The metabolite data in this study showed increased GSH levels in GB-fed mice compared with HFD mice, which can be inferred as an increased antioxidant effect (data not shown). These result suggested that increase of oxidative states in PPP by GB leads to conversion of GSH, which can prevent oxidative modifications that lead to mitochondrial dysfunction.
Fructose 6-phosphate is converted to glucosamine-6-phosphate through a glutamine fructose-6-phosphate aminotransferase (GFAT) gene and can produce GlcNAc-6-P by binding with acetyl CoA by glucosamine-6-phosphate N-acetyltransferase (Akella et al., 2019). The levels of both GlcNAc-6-P and GlcNAc-1-P were increased in the livers of mice fed GB (Table 2 (iii); Fig. 3I-J), which could be expected to promote the consumption of acetyl CoA, which is a precursor of fatty acid synthesis. GlcNAc-6-P and GlcNAc-1-P were significantly correlated with serum glucose and insulin resistance phenotypes (Fig. 3L).
We previously demonstrated that GB improves hepatic steatosis by regulating transcription factors, such as mature sterol regulatory element-binding transcription factor 1 (mSREBP1) and downstream proteins related to lipid metabolism (Kim et al., 2017). In the metabolite analysis, 4 metabolites related to lipid metabolism were detected in the liver. Decanoic acid (C10:0) and dodecanoic acid (C12:0), as MCFA, decreased in the HFD group, but increased in the HFD + GB group (Table 3; Fig. S1A-B). Increased MCFA downregulates transcription factors, such as liver X receptor α (LXRα), carbohydrate response element-binding protein (ChREBP), and long-chain fatty acid elongase 9 (Elovl6), and ameliorates insulin resistance induced by long-chain fatty acid (LCFA)-rich diets (Sun et al., 2013). Therefore, the increase of MCFA by GB intake in DIO mice, influence liver steatosis and insulin signaling by regulating liver transcription factors. Cholic acid and taurine are related to cholesterol metabolism as they convert cholesterol to primary bile acid, and high-cholesterol-fed rats showed improvement in cholesterol metabolism through taurine supplementation (Murakami et al., 2016). Cholic acid and taurine levels were significantly decreased in HFD-fed mice, but the levels of metabolites were significantly increased in the HFD + GB group (Fig. S1C-D), indicating that the conversion of cholesterol to bile acid increased, which improved liver steatosis because of the increased excretion of cholesterol in the liver tissue. In this study, metabolites such as LCFA and LysoPCs were not detected, which can be inferred through the CE-TOF/MS metabolite analysis.
Several amino acids, including BCAA, were altered after GB intake (Table 1). Analyzing metabolites in the serum of humans who had NAFLD and NASH revealed that the level of amino acids changed. In particular, serine, glycine, and aspartate levels showed significant decreases in the NAFLD-induced groups (Hasegawa et al., 2020). In the case of BCAA, the plasma levels of metabolites were increased in NAFLD (Gaggini et al., 2018). However, BCAA alleviates hepatic steatosis and liver injury by suppressing fatty acid synthase (FAS) (Honda et al., 2017). In this study, isoleucine, a BCAA, was increased by GB intake; however, the effect of the increase in isoleucine on liver metabolism needs further research. These amino acids can be used as biomarkers to investigate the effects of GB on the liver.