In our study, we found that caffeine high-dose could reduce body weight and liver weight, and improve hyperlipidemia and insulin resistance in the HFD mice, demonstrating caffeine has a therapeutic effect on metabolic syndromes, which is consistent with previous studies [
15‐
17]. Moreover, we elucidate the mechanism of this anti- metabolic syndrome, which may involve gut microbiota, bile acid metabolism, lipid metabolism, and energy metabolism.
The nature of the intestinal microbiome has been shown to be closely related to IR in several human and animal model studies [
30,
31]. 16S rRNA sequencing was used to investigate how caffeine affects the composition of the gut microbiome. Compared to the NCD group, HFD mice have a substantial reduction in the abundance of
Bacteroidetes and a proportional increase in
Firmicutes, similar to previous studies [
20]. However, caffeine further reduced
Bacteroidetes, which seems likely that caffeine has its unique effect on the intestinal microbiome.
Bacteroidetes is a Gram-negative bacterium containing LPS, a potent activator of toll-like receptor 4(TLR4), which causes inflammatory responses and cytokine expression and secretion. In addition, Lower
Bacteroidetes may be associated with lower
Bacteroides at the genus level.
Bacteroides, Lactobacillus and
Lactococcus containing bile salt hydrolase (BSH) can hydrolyze conjugated bile acids into unconjugated bile acids [
37]. It has been reported that conjugated bile acid is a nuclear farnesoid X receptor (FXR) antagonist [
33]. Decreased BSH leads to increased conjugated bile acids, which increases the conversion of cholesterol to bile acids and reduces lipid accumulation. Likewise, in our plasma data, cholic acid, which was elevated in insulin-resistant mice and humans [
9,
34], was decreased by caffeine, while glycocholic acid was increased. This may suggest that the improvement of dyslipidemia by caffeine may be related to bile acid metabolism.
Bifidobacterium is a probiotic. In a human report, 16 healthy subjects drank a daily dose of 3 cups of coffee for three weeks, which raised the number of
Bifidobacterium compared with human faeces before coffee consumption [
35]. This bifidogenic effect of coffee was also discovered in animal models [
32]. Meanwhile, caffeine also increased this probiotic in the NAFLD patients [
36]. In accordance with our results, which hint the bifidogenic effect of coffee may be related to caffeine. Moreover,
Bifidobacterium and
Ruminococcaceae are short-chain fatty acid (SCFAs)-producing bacteria; SCFAs have been shown to increase gut barrier function [
37] and stimulate pancreatic islet beta-cell growth and proliferation, regulate the body's insulin sensitivity [
38].
Desulfovibrio, significantly elevated by caffeine, was shown to produce acetic acid and regulate liver lipid metabolism in mice to alleviate non-alcoholic fatty liver disease [
39]; to produce H2S, which maintains lipid metabolism balance [
40] and reduces systemic inflammation [
41]. In addition, the present study also shown
Bifidobacterium,
Ruminococcaceae and
Desulfovibrio were negatively associated with obesity indicators.
Serum metabolomics showed that the serum metabolites of the three groups were separated from each other. However, caffeine did not move the plasma metabolites of HFD mice closer to that of NCD mice, which may be the effect of caffeine as a foreign substance in the metabolism. Elevations of caffeine metabolites such as 1,7-Dimethylxanthine in plasma may illustrate this point. In our data, 1,7-Dimethylxanthine was positively correlated with
Dubosiella,
Alkaninndiges and
Faecalibaculum, possibly indicating that changes in the intestinal microbiome are associated with caffeine metabolism.
Dubosiella significantly elevated in response to caffeine, is not yet well studied. Still, this bacterium was negatively correlated with most inflammatory factors and indicators of obesity and positively associated with butyric acid [
42,
43]. Moreover,
Dubosiella is positively correlated with Adenosine 5'-monophosphate (AMP) and Linoleic acid, which Implies the relationship between
Dubosiella and energy metabolism. Metabolic pathway enrichment analysis showed that caffeine could regulate Steroid hormone biosynthesis, Primary bile acid biosynthesis, Biosynthesis of unsaturated fatty acids, Glycine, serine and threonine metabolism, Linoleic acid metabolism, Pyruvate metabolism and so on. The Biosynthesis of steroid hormones, the Biosynthesis of primary bile acids and the Biosynthesis of unsaturated fatty acids are closely related to lipid metabolism. Disturbances in lipid metabolism often accompany insulin resistance. In a study of the activity and function of the cytochrome P450 side-chain cleavage enzyme (CYP11A1), the rate-limiting enzyme for the conversion of cholesterol to pregnenolone, it was found that incubation of isolated mitochondria with the cholesterol analogue 22R-hydroxycholesterol resulted in the efficient formation of pregnenolone, the direct precursor for the synthesis of all steroid hormones [
44]. Estrogen has been reported to increase insulin sensitivity [
45]. Similarly, 22R-hydroxycholesterol is a natural ligand for the liver X receptor [LXR], and activation of LXR improves TNFα-induced insulin resistance in brown adipocytes [
46]. All of the above suggests that the improvement of insulin resistance by caffeine may be related to steroid hormone synthesis. Glycine, serine, threonine, linoleic acid and pyruvate acid ultimately produce energy metabolism through the tricarboxylic acid cycle. Caffeine is known to activate beta receptors, increase plasma cAMP concentrations and promote lipolysis and beta-oxidation [
47].