The Effects of Butyrate on Induced Metabolic-Associated Fatty Liver Disease in Precision-Cut Liver Slices

Abstract

Metabolic-associated fatty liver disease (MAFLD) starts with hepatic triglyceride accumulation (steatosis) and can progress to more severe stages such as non-alcoholic steatohepatitis (NASH) and even cirrhosis. Butyrate, and butyrate-producing bacteria, have been suggested to reduce liver steatosis directly and systemically by increasing liver β-oxidation. This study aimed to examine the influence of butyrate directly on the liver in an ex vivo induced MAFLD model. To maintain essential intercellular interactions, precision-cut liver slices (PCLSs) were used. These PCLSs were prepared from male C57BL/6J mice and cultured in varying concentrations of fructose, insulin, palmitic acid and oleic acid, to mimic metabolic syndrome. Dose-dependent triglyceride accumulation was measured after 24 and 48 h of incubation with the different medium compositions. PCLSs viability, as indicated by ATP content, was not affected by medium composition or the butyrate concentration used. Under induced steatotic conditions, butyrate did not prevent triglyceride accumulation. Moreover, it lowered the expression of genes encoding for fatty acid oxidation and only increased C4 related carnitines, which indicate butyrate oxidation. Nevertheless, butyrate lowered the fibrotic response of PCLSs, as shown by reduced gene expression of fibronectin, alpha-smooth muscle actin and osteopontin, and protein levels of type I collagen. These results suggest that in the liver, butyrate alone does not increase lipid β-oxidation directly but might aid in the prevention of MAFLD progression to NASH and cirrhosis.

Keywords: MAFLD; NAFLD; SCFA; butyrate; fatty acid oxidation; fibrosis; liver; steatosis.

Figure 1

Induction and characterisation of steatosis in PCLS. (A) TG levels and (B) ATP concentration after 48 h incubation with GFI or GFIPO medium, compared to CTR. Data are presented as mean percentage ± SEM. (C) Heat map of mRNA levels of genes encoding transcription factors, transporters (1) and enzymes (2: triglyceride assembly, 3: lipid synthesis, 4: FA efflux, 5: FA oxidation) involved in glucose and fatty acid metabolism, inflammation and fibrosis after 48 h incubation in CTR, GFI or GFIPO medium. Medium compositions were abbreviated according to Table 1 (CTR = control; GFI = glucose, fructose, and insulin; and GFIPO = GFI plus palmitic acid and oleic acid). Differential expression is presented using Z-scores (red: high Z-score, upregulated; blue: low Z-score, downregulated). * = p < 0.05, ** = p < 0.01 compared to CTR, # = p < 0.05 compared between GFI and GFIPO. Individual values are shown as circles.

Figure 2

Effects of butyrate treatment on triglyceride accumulation and metabolism. (A) Relative TG levels in the three different media with 1 mM NaCl (control) or 1mM NaB (sodium butyrate). Data are presented as mean percentage of control ± SEM. (B–D) Heat maps of measured mRNA expression levels of genes related to lipid and glucose metabolism after culture in (B) CTR medium, (C) GFI medium, (D) GFIPO medium with 1 mM NaCl or 1mM NaB. Medium compositions were abbreviated according to Table 1 (CTR = control; GFI = glucose, fructose, and insulin; and GFIPO = GFI plus palmitic acid and oleic acid). Differential expression is presented using Z-scores (red: high Z-score, upregulated; blue: low Z-score, downregulated). * = p < 0.05 compared to NaCl. Individual values are shown as circles.

Figure 3

Effects of butyrate treatment on clearance and oxidation of fatty acids. Heat map of measured expression levels of genes related to lipid efflux (Apob, Apoe and Angptl4) and genes related to fatty acid oxidation (Slc25a20, Pdk4, Cyp4a11, Crat, Acox1, Acadm and Cpt1) after culture in (A) CTR medium, (B) GFI medium, (C) GFIPO medium with 1 mM NaCl (control) or 1 mM NaB (butyrate). Medium compositions were abbreviated according to Table 1 (CTR = control; GFI = glucose, fructose, and insulin; and GFIPO = GFI plus palmitic acid and oleic acid). Differential expression is presented using Z-scores (red: high Z-score, upregulated; blue: low Z-score, downregulated). (D) Intracellular butyrylcarnitine (C4) and hydroxybutyrylcarnitine (C4OH) quantification in CTR, GFI and GFIPO with 1 mM NaCl or 1 mM NaB. Data are presented as mean ± SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to NaCl. Individual values are shown as circles.

Figure 4

Effects of butyrate on inflammation and tissue remodelling. (A–C) Heat maps displaying mRNA expression of genes indicative of inflammation (Il1b, Il6, Ucp2, Fas, Sod1) and genes related to tissue remodelling (Tgfb1, Fn1, Spp1, Acta2) after culture in (A) CTR medium, (B) GFI medium, (C) GFIPO medium with 1 mM NaCl (control) or 1 mM NaB (butyrate). Differential expression is presented using Z-scores (red: high Z-score, upregulated; blue: low Z-score, downregulated). (D) Excreted pro-collagen Iα1 after culture in CTR, GFI and GFIPO medium with 1mM NaCl or 1mM NaB. (E) Difference in intracellular ATP content after culture with NaB as compared to NaCl. Medium compositions were abbreviated according to Table 1 (CTR = control; GFI = glucose, fructose, and insulin; and GFIPO = GFI plus palmitic acid and oleic acid). Data are presented as mean ± SEM.* = p < 0.05, ** = p < 0.01 compared to NaCl. Individual values are shown as circles.