Diabetes Accelerates Steatohepatitis in Mice: Liver Pathology and Single-Cell Gene Expression Signatures.

Glucose lowering independently reduces liver fibrosis in human nonalcoholic fatty liver disease. This study investigated the impact of diabetes on steatohepatitis and established a novel mouse model for diabetic steatohepatitis. Male C57BL/6J mice were fed a 60% high-fat diet (HFD) and injected with carbon tetrachloride (CCl4) and streptozotocin (STZ) to induce diabetes. The HFD+CCl4+STZ group showed more severe liver steatosis, hepatocyte ballooning, and regenerative nodules compared with other groups. Diabetes up-regulated inflammatory cytokine-associated genes and increased the M1/M2 macrophage ratios in the liver. Single-cell RNA sequencing analysis of nonparenchymal cells in the liver showed that diabetes reduced Kupffer cells and increased bone marrow-derived recruited inflammatory macrophages, such as Ly6Chi-RM. Diabetes globally reduced liver sinusoidal endothelial cells (LSECs). Furthermore, the up-regulation of genes related to the receptor for advanced glycation end products (RAGE)/Toll-like receptor 4 was observed in Ly6Chi-RM and LSECs in mice with diabetes, suggesting a possible role of RAGE/Toll-like receptor 4 signaling in the interaction between inflammatory macrophages and LSECs. This study established a novel diabetic steatohepatitis model using a combination of HFD, CCl4, and STZ. Diabetes exacerbates steatosis, hepatocyte ballooning, fibrosis, regenerative nodule formation, and the macrophage M1/M2 ratios triggered by HFD and CCl4. Single-cell RNA sequencing analysis indicated that diabetes activates inflammatory macrophages and impairs LSECs through the RAGE/Toll-like receptor 4 signaling pathway. These findings open avenues for discovering novel therapeutic targets for diabetic steatohepatitis.