Ablation of gut microbiota alleviates obesity-induced hepatic steatosis and glucose intolerance by modulating bile acid metabolism in hamsters

Abstract

Since metabolic process differs between humans and mice, studies were performed in hamsters, which are generally considered to be a more appropriate animal model for studies of obesity-related metabolic disorders. The modulation of gut microbiota, bile acids and the farnesoid X receptor (FXR) axis is correlated with obesity-induced insulin resistance and hepatic steatosis in mice. However, the interactions among the gut microbiota, bile acids and FXR in metabolic disorders remained largely unexplored in hamsters. In the current study, hamsters fed a 60% high-fat diet (HFD) were administered vehicle or an antibiotic cocktail by gavage twice a week for four weeks. Antibiotic treatment alleviated HFD-induced glucose intolerance, hepatic steatosis and inflammation accompanied with decreased hepatic lipogenesis and elevated thermogenesis in subcutaneous white adipose tissue (sWAT). In the livers of antibiotic-treated hamsters, cytochrome P450 family 7 subfamily B member 1 (CYP7B1) in the alternative bile acid synthesis pathway was upregulated, contributing to a more hydrophilic bile acid profile with increased tauro-β-muricholic acid (TβMCA). The intestinal FXR signaling was suppressed but remained unchanged in the liver. This study is of potential translational significance in determining the role of gut microbiota-mediated bile acid metabolism in modulating diet-induced glucose intolerance and hepatic steatosis in the hamster.

Keywords: ALT, alanine amino-transferase; AST, aspartate transaminase; AUC, area under curve; ApoB, apolipoprotein B; BAs, bile acids; BSH, bile acid hydrolase; CA, cholic acid; CAPE, caffeic acid phenethyl ester; CDCA, chenodeoxycholic acid; CETP, cholesterol ester transfer protein; CYP27A1, cytochrome P450 family 27 subfamily A member 1; CYP7A1, cytochrome P450 family 7 subfamily A member 1; CYP7B1; CYP7B1, cytochrome P450 family 7 subfamily B member 1; CYP8B1, cytochrome P450 family 8 subfamily B member 1; DCA, deoxycholic acid; FGF15/19, fibroblast growth factor 15/19; FXR; FXR, farnesoid X receptor; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GTT, glucose tolerance test; Gut microbiota; H&E, hematoxylin and eosin; HFD, high fat diet; ITT, insulin tolerance test; LCA, lithocholic acid; Metabolic disorders; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PBA/SBA, primary bile acids to secondary bile acids; T2D, type 2 diabetes; TC, total cholesterol; TCA, taurocholic acid; TG, triglycerides; TβMCA; TβMCA, tauro-β-muricholic acid; UDCA, ursodeoxycholic acid; UPLC–MS/MS, ultra performance liquid chromatography–tandem mass spectrometry; VLDL, very low-density lipoprotein; eWAT, epididymal white adipose tissue; sWAT, subcutaneous white adipose tissue.

Graphical abstract

Figure 1

Ablation of gut microbiota protects from obesity-induced hepatic steatosis and liver injury in hamsters. The hamsters were fed a 60% high-fat diet and given vehicle or antibiotics for 4 weeks (n=5 hamsters/group). (A) The liver weight. (B) The ratio of liver weight to body weight. (C) Representative H&E (upper panel) and oil red O (lower panel) staining of liver sections of the two groups. Scale bars: 200 μm (3 images/hamster). (D) Steatosis degree (the ratio of vacuolar area to whole area in one image, 3 images/hamster). (E) Hepatic triglycerides. (F) Plasma triglycerides. (G) The relative expression of genes involved in fatty acid synthesis, uptake and oxidation in the liver. (H) Plasma ALT levels. (I) Plasma AST levels. Data are presented as the mean±SEM. ^*P<0.05, ^**P<0.01 versus vehicle by two-tailed Student׳s t-test.

Figure 2

Ablation of gut microbiota improves obesity-induced insulin resistance in hamsters. The hamsters were fed a 60% high-fat diet and given vehicle or antibiotics for 4 weeks (n=5 hamsters/group). On the fourth week, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed. (A) GTT; (B) Area under the curve (AUC); (C) ITT. (D) Fasting glucose levels; (E) Fasting insulin levels; (F) HOMA-IR; (G) The ratios of fat mass to body weight in subcutaneous adipose tissue (sWAT), epididymal adipose tissue (eWAT) and brown adipose tissue (BAT); (H) The relative expression of thermogenic gene mRNAs in the sWAT. Data are presented as the mean±SEM, n=5 hamsters/group. ^*P<0.05, ^**P<0.01 versus vehicle by two-tailed Student׳s t-test.

Figure 3

Alternative bile acid synthesis in the liver is elevated after antibiotic treatment. The hamsters were fed a 60% high-fat diet and given vehicle or antibiotics for 4 weeks (n=5 hamsters/group). (A) Bile acid synthesis in the liver; (B) Hepatic cholesterol; (C) Total primary bile acids in the liver; (D) Primary bile acid profiles in the liver; (E) Primary bile acid composition in the liver; (F) The ratio of 12α-OH bile acids to non-12α-OH bile acids in the liver [12α-OH/non12α-OH BA=(GCA+CA+TCA+GDCA+DCA+TDCA)/(LCA+HDCA+CDCA+UDCA+βMCA+GCDCA+GUDCA+TLCA+TUDCA+THDCA+TβMCA)]; (G) The relative expression of genes involved in bile acid synthesis. Data are presented as the mean±SEM, n=5 hamsters/group. ^*P<0.05, ^**P<0.01 versus vehicle by two-tailed Student׳s t-test.

Figure 4

Microbial modulation of bile acid metabolism in the gut. (A) Total ileal bile acids; (B) Bile acid levels in the ileum; (C) Bile acid composition in the ileum; (D) The ratio of primary bile acids to secondary bile acids in the ileum; (E) The ratio of unconjugated bile acids to conjugated bile acids in the ileum. Data are presented as the mean±SEM, n=5 hamsters/group. ^*P<0.05, ^**P<0.01 versus vehicle by two-tailed Student׳s t-test.

Figure 5

Intestinal FXR is suppressed in the absence of gut microbiota. (A) The relative abundance of intestinal FXR and its target genes; (B) The mRNA levels of genes in the hepatic FXR signaling. Data are presented as the mean±SEM, n=5 hamsters/group. ^*P<0.05, ^**P<0.01 versus vehicle by two-tailed Student׳s t-test.