Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis-associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects

Abstract

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development.

Figure 1

Anagliptin ameliorates NASH-like liver phenotypes in MC4R-KO mice. (a) Experimental protocol for examination of the preventive effect of anagliptin (Ana) on the development of NASH in melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet (KO/WD). Wildtype (WT) mice on standard diet (WT/SD) were used as the control group. (b) Growth curve of WT and MC4R-KO mice. Open circle, WT/SD treated with the vehicle (n = 6); closed triangle, MC4R-KO/WD treated with the vehicle (n = 15); gray square, MC4R-KO/WD treated with anagliptin (n = 15), respectively. (c) Tissue weights of liver and epididymal fat after 10 and 20 weeks of treatment with anagliptin. (d) Liver cholesterol and triglyceride (TG) contents at 10 and 20 weeks. (e) Effect of anagliptin treatment on glucose metabolism at 10 weeks. (f) Effect of anagliptin treatment on the dipeptidyl peptidase-4 (DPP-4) activity and the active GLP-1 concentrations in the plasma at 10 weeks. (g) Hematoxylin and eosin staining of the liver at 20 weeks. Histological analysis using NASH activity score (NAS). *p < 0.05, **p < 0.01; N.S. not significant. WT/SD treated with vehicle (n = 6), KO/WD treated with anagliptin or vehicle (n = 12 and 15 at 10 and 20 weeks, respectively).

Figure 2

Anagliptin suppresses hepatic inflammation in MC4R-KO mice. Hepatic mRNA expression of inflammation-related genes after 10 (a) and 20 (b) weeks of treatment with anagliptin. (c) F4/80 immunostaining of the liver at 10 weeks. Insets indicate representative images of hepatic crown-like structure (hCLS). Scale bars, 100 µm. **p < 0.01; N.S. not significant. WT/SD treated with vehicle (n = 6), KO/WD treated with anagliptin or vehicle (n = 12 and 15 at 10 and 20 weeks, respectively).

Figure 3

Anagliptin suppresses liver fibrosis in MC4R-KO mice. Hepatic mRNA expression of fibrosis-related genes after 10 (a) and 20 (b) weeks of treatment with anagliptin. (c) Representative images of Sirius Red staining of the liver at 20 weeks. Scale bars, 200 µm. (d) Quantification of the collagen type I-positive area and the hydroxyproline content of the liver at 20 weeks. *p < 0.05, **p < 0.01; N.S. not significant. WT/SD treated with vehicle (n = 6 at 20 weeks), KO/WD treated with anagliptin or vehicle (n = 12 and 15 at 10 and 20 weeks, respectively).

Figure 4

Anagliptin does not affect hepatocyte death in MC4R-KO mice. (a) TUNEL staining of the liver after 10-week treatment with anagliptin. Insets indicate representative images of TUNEL-positive cells. Scale bars, 100 µm. (b) Caspase-3/7 activity of the liver at 10 weeks. *p < 0.05, **p < 0.01; N.S. not significant (n = 6 WT/SD, n = 12 KO/WD).

Figure 5

Anagliptin does not affect adipose tissue inflammation in MC4R-KO mice. Adipose tissue mRNA expression of inflammation-related genes after 10-week treatment with anagliptin. **p < 0.01; N.S. not significant (n = 6 WT/SD, n = 12 KO/WD).

Figure 6

GLP-1 analogue suppresses macrophage inflammatory phenotypes in vitro. (a) mRNA expression levels of GLP-1 receptor and inflammation- and fibrosis-related genes in hepatic macrophages using microarray database (accession GSE104901). **p < 0.01 (n = 5). (b) mRNA expression levels of GLP-1 receptor in lipopolysaccharide (LPS)-stimulated macrophages. RAW264 cells or peritoneal macrophages were stimulated with LPS (10 ng/ml) for 6 h. (n = 4–6). (c) mRNA expression levels of inflammation- and fibrosis-related genes in LPS-stimulated cultured macrophages. RAW264 cells were pretreated with Exendin-4 (Ex-4, 4 ng/ml), a GLP-1 analogue, for 24 h, and then treated with LPS (10 ng/ml) for additional 6 h. *p < 0.05, **p < 0.01 (n = 6). (d) mRNA expression levels of inflammation- and fibrosis-related genes in palmitate-stimulated cultured macrophages. RAW264 cells were pretreated with Ex-4 (4 ng/ml) for 24 h, and then treated with palmitate (200 µM) for additional 24 h. *p < 0.05, **p < 0.01 (n = 4). (e) mRNA expression levels of inflammation- and fibrosis-related genes in dead cell-stimulated cultured macrophages. RAW264 cells were pretreated with Ex-4 (4 ng/ml) for 24 h, and then treated with dead hepatocytes (1 × 10⁵ cells/well in 24 well plate) for additional 8 h. *p < 0.05, **p < 0.01 (n = 4).

Figure 7

Anagliptin attenuates liver carcinogenesis in MC4R-KO mice. (a) Experimental protocol for examination of the preventive effect of anagliptin on the development of liver tumors in MC4R-KO mice. (b) Representative images of gross appearance and computed tomography of the liver. Yellow arrows indicate liver tumors and yellow arrowheads indicate preneoplastic foci. (c) Representative images of hematoxylin and eosin staining of the liver at 47 weeks. Scale bars, 100 µm. (d) Number of foci and tumors and the size of tumors in the liver. *p < 0.05 (Vehicle, n = 11; Anagliptin, n = 12). (e) mRNA expression levels of tumor-related genes in non-tumor (NT) and tumor (T) lesions in the liver. *p < 0.05, **p < 0.01 (n = 4).