The protective effects of the β3 adrenergic receptor agonist BRL37344 against liver steatosis and inflammation in a rat model of high-fat diet-induced nonalcoholic fatty liver disease (NAFLD)

Abstract

Background: Our objective was to investigate the efficacy of the beta-3 adrenergic receptor (β3-AR) agonist BRL37344 for the prevention of liver steatosis and inflammation associated with nonalcoholic fatty liver disease (NAFLD).

Methods: Four groups were established: a control group (given a standard diet), a high-fat diet (HFD) group, an HFD + β3-AR agonist (β3-AGO) group, and an HFD + β3-AR antagonist (β3-ANT) group. All rats were fed for 12 weeks. The β3-AR agonist BRL37344 and the antagonist L748337 were administered for the last 4 weeks with Alzet micro-osmotic pumps. The rat body weights (g) were measured at the end of the 4th, 8th, and 12th weeks. At the end of the 12th week, the liver weights were measured. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed with a Hitachi automatic analyzer. The lipid levels of the triglycerides (TGs), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) and the concentrations of free fatty acids (FFAs) were also measured. An oil red O kit was used to detect lipid droplet accumulation in hepatocytes. Steatosis, ballooning degeneration and inflammation were histopathologically determined. The protein and mRNA expression levels of β3-AR, peroxisome proliferator-activated receptor-alpha (PPAR-α), peroxisome proliferator-activated receptor-gamma (PPAR-γ), mitochondrial carnitine palmitoyltransferase-1 (mCPT-1), and fatty acid translocase (FAT)/CD36 were measured by western blot analysis and RT-qPCR, respectively.

Results: After treatment with the β3-AR agonist BRL37344 for 4 weeks, the levels of ALT, AST, TGs, TC, LDL-C and FFAs were decreased in the NAFLD model group compared with the HFD group. Body and liver weights, liver index values and lipid droplet accumulation were lower in the HFD + β3-AGO group than in the HFD group. Decreased NAFLD activity scores (NASs) also showed that liver steatosis and inflammation were ameliorated after treatment with BRL37344. Moreover, the β3-AR antagonist L748337 reversed these effects. Additionally, the protein and gene expression levels of β3-AR, PPAR-α, and mCPT-1 were increased in the HFD + β3-AGO group, whereas those of PPAR-γ and FAT/CD36 were decreased.

Conclusion: The β3-AR agonist BRL37344 is beneficial for reducing liver fat accumulation and for ameliorating liver steatosis and inflammation in NAFLD. These effects may be associated with PPARs/mCPT-1 and FAT/CD36.

Keywords: Inflammation; Liver steatosis; NAFLD; β3-adrenergic receptor.

Fig. 1

Protein and mRNA expression of β3-AR in the liver in the four groups. a Quantitative analysis of β3-AR protein levels relative to GAPDH levels (each group, n = 8). b Western blot analysis of β3-AR protein levels using specific β3-AR antibodies. GAPDH was used as a loading control. c β3-AR mRNA expression in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups (each group, n = 8). β3-AGO refers to the β3-AR agonist BRL37344, and β3-ANT refers to the β3-AR antagonist L748337. *P < 0.05 vs the control group, #P < 0.05 vs the HFD group

Fig. 2

Histological changes in the liver in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups (each group, n = 8). a The control group showed normal liver histological features, and the hepatocytes were arranged in an orderly manner. b In the HFD group, prominent hepatic steatosis, inflammation and ballooning degeneration were observed. c In the HFD + β3-AGO group, significantly improved structural changes with limited hepatic steatosis, inflammation and ballooning degeneration were observed. d The image for the HFD + β3-ANT group shows several scattered balloon cells that are much larger than the surrounding steatotic hepatocytes, in sharp contrast to the normal hepatocytes in the image in (a). The magnification is 200 ×

Fig. 3

The β3-AR agonist BRL37344 ameliorated HFD-fed rat liver steatosis, exerting a protective effect. a Rat body weight at 0, 4, 8, and 12 weeks. b Liver weight at the 12th week in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups. c Liver index values at the 12th week in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups. d Liver homogenate TG levels (mmol/g). e Liver homogenate TC levels (mmol/g). f Liver homogenate FFA levels (μmol/g). g Oil red O was used to visualize lipid droplets in the hepatocytes. The magnification is 200×. Lipid droplets are stained light red, and nuclei are stained blue. h Quantitative analysis of lipid droplets in hepatocytes. TG: triglyceride. TC: total cholesterol. FFA: free fatty acid. *P < 0.05 vs the control group, #P < 0.05 vs the HFD group; n = 8 per group

Fig. 4

The β3-AR agonist BRL37344 modulated key proteins and genes for FFA β-oxidation. a Quantitative analysis of the protein levels of PPAR-α and PPAR-γ in the livers of rats in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups (each group, n = 8). b Representative western blot bands of the PPAR-α and PPAR-γ proteins in the livers of rats in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups. c Quantitative analysis of the expression of the PPAR-α and PPAR-γ genes in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups (each group, n = 8). d Quantitative analysis of the protein levels of mCPT-1 and CD36 in the livers of rats in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups (each group, n = 8). e Representative western blot bands of the mCPT-1 and CD36 proteins in the livers of rats in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups. f Quantitative analysis of the expression of the mCPT-1 and CD36 genes in the control, HFD, HFD + β3-AGO and HFD + β3-ANT groups (each group, n = 8). *P < 0.05 vs the control group, #P < 0.05 vs the HFD group

Fig. 5

β3-AR stimulation plays a role in alleviating liver steatosis associated with NAFLD. Treatment with the β3-AR agonist BRL37344 for 4 weeks decreased CD36 in NAFLD rats, thus decreasing FAs entry into hepatocytes. On the other hand, increases in mCPT-1 led to increased FAs entry into mitochondria. These two processes together caused a decrease in FA levels in hepatocytes. In addition, the increased FA levels in mitochondria and the increased PPAR-ɑ levels were beneficial for mitochondrial β-oxidation. BRL37344 also decreased PPAR-γ expression. All of the above factors contributed to the relief of liver steatosis in NAFLD rats. β3-AR: beta-3 adrenergic receptor; FAs: fatty acids; mCPT-1: mitochondrial carnitine palmitoyltransferase-1; PPAR-ɑ: peroxisome proliferator-activated receptor-alpha; PPAR-γ: peroxisome proliferator-activated receptor-gamma

Fig. 6

Typical mitochondria changes of liver under transmission electron microscopy. a The size of mitochondria was uniformly sized in the control group. b Mitochondrial matrix was swollen in the HFD group. c Compared with the HFD group, mitochondrial ultrastructural damage was reversed by BRL37344 in the HFD + β3-AGO group. d The HFD + β3-ANT group showed more severe mitochondrial swelling and increased lysosomes than that of the HFD group. The magnification was 15,000× in each image (each group, n = 8)