Empagliflozin (an SGLT2 inhibitor), alone or in combination with linagliptin (a DPP-4 inhibitor), prevents steatohepatitis in a novel mouse model of non-alcoholic steatohepatitis and diabetes

Abstract

Background: Sodium-glucose co-transporter-2 (SGLT2) inhibitors are new oral antidiabetic drugs that reduce hyperglycemia by promoting urinary glucose excretion. Glycosuria produced by SGLT2 inhibitors is associated with weight loss, mainly due to reduced fat volume. We investigated the effects of empagliflozin (selective SGLT2 inhibitor) and linagliptin (DPP-4 inhibitor) on steatohepatitis and fibrosis in a mouse model of non-alcoholic steatohepatitis (NASH) with diabetes.

Methods: A novel NASH model was generated by administration of streptozotocin to C57BL/6J mice at 2 days old, with a high-fat diet from 4 weeks. NASH mice aged 6 weeks were divided into four groups of 6 animals: vehicle, linagliptin (10 mg/kg), empagliflozin (10 mg/kg), and linagliptin + empagliflozin. The histological non-alcoholic fatty liver disease activity score was significantly lower in the empagliflozin and linagliptin + empagliflozin groups than in the vehicle or linagliptin groups. Hepatic expression of inflammatory genes (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) was decreased in the empagliflozin and linagliptin + empagliflozin groups compared with the vehicle group. The collagen deposition with Sirius red staining was significantly reduced in the linagliptin + empagliflozin group compared with the linagliptin or the empagliflozin group. Immunohistochemistry showed that expression of α-smooth muscle actin, a marker of myofibroblasts (fibrosis), was reduced in the linagliptin + empagliflozin group compared with the vehicle group, as was expression of type 1 and 3 collagen mRNA. Linagliptin + empagliflozin decreased expression of mRNAs for genes related to fatty acid synthesis, but did not increase mRNAs for β-oxidation-related genes.

Conclusions: While empagliflozin alone attenuates development of NASH showing anti-steatotic and anti-inflammatory effects, combined administration of empagliflozin and linagliptin can synergistically ameliorates NASH with stronger anti-fibrotic effects.

Keywords: Dipeptidyl peptidase- 4; Fibrosis; Inflammation; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Sodium-glucose co-transporter 2.

Fig. 1

Liver to body weight ratio (a) and liver triglyceride content (b) in the five groups. Data are mean ± SE. *P < 0.05, ^†P < 0.01, ^‡P < 0.001 vs. control; ^§P < 0.05, ^||P < 0.01, ^¶P < 0.001 vs. vehicle; ^#P < 0.05 vs. Linagliptin alone

Fig. 2

Representative microphotographs of liver sections stained with hematoxylin eosin and NAFLD activity score (NAS) in the five groups. Original magnification ×200. Data are mean ± SE. ^¶P < 0.001 vs. vehicle; **P < 0.01, ^††P < 0.001 vs. Linagliptin alone

Fig. 3

Representative microphotographs of immunohistochemical staining for F4/80 in liver sections (a) and percentage in area of positive immunostaining for F4/80 in the five groups (b). Normalized mRNA expression of F4/80 the liver of the five groups (c). Data are mean ± SE. *P < 0.05, ^‡P < 0.001 vs. control; ^§P < 0.05, ^¶P < 0.001 vs. vehicle

Fig. 4

Gene expression of inflammation in the liver of the five groups. Normalized mRNA expression tumor necrosis factor (TNF) α (a), monocyte chemoattractant protein (MCP)-1 (b), interleukin (IL)-6 (c), and suppressor of cytokine signaling (SOC)-3 (d). Data are mean ± SE. *P < 0.05 vs. control; ^§P < 0.05, ^||P < 0.01, ^¶P < 0.001 vs. vehicle; ^#P < 0.05, **P < 0.01 vs. Linagliptin alone

Fig. 5

Representative microphotographs of liver sections stained with Sirius red in the liver sections and percentage in area of positive staining for Sirius red in the five groups. Data are mean ± SD. ^§P < 0.05, ^¶P < 0.001 vs. vehicle; ^#P < 0.05 vs. Linagliptin alone; ^‡‡P < 0.05 vs. Empagliflozin alone

Fig. 6

Representative microphotographs of immunohistochemical staining for α-smooth muscle antigen (SMA) in liver sections and percentage in area of positive immunostaining for α-SMA in the five groups (a, b). Gene expression of fibrosis in the liver of the five groups. Normalized mRNA expression of collagen 1 and 3 (c, d). Data are mean ± SD. *P < 0.05 vs. control; ^§P < 0.05, ^¶P < 0.001 vs. vehicle

Fig. 7

Gene expression of lipid metabolism in the liver of the five groups. Normalized mRNA expression of fatty acid synthase (FAS) (a), acetyl-CoA carboxylase (ACC) 1 (b), peroxisome proliferator-activated receptor (PPAR) α (c), and acyl-coenzyme A oxidase (ACOX)1 (d). Data are mean ± SD. *P < 0.05, ^†P < 0.01, ^‡P < 0.001 vs. control; ^§P < 0.05, ^||P < 0.01, ^¶P < 0.001 vs. vehicle; ^#P < 0.05, **P < 0.01, ^††P < 0.001 vs. Linagliptin alone