Diabetes accelerates steatohepatitis in mice: liver pathology and single-cell gene expression signatures

Glucose lowering independently reduces liver fibrosis in human NAFLD. We 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 displayed more severe liver steatosis, hepatocyte ballooning, and regenerative nodules compared to other groups. Diabetes upregulated inflammatory cytokine-associated genes and elevated the M1/M2 macrophage ratios in the liver. Single-cell RNA sequencing (scRNAseq) analysis of non-parenchymal cells in the liver showed that diabetes reduced the Kupffer cells and increased bone marrow-derived recruited inflammatory macrophages, such as Ly6Chi-RM. Diabetes globally reduced liver sinusoidal endothelial cells (LSECs). Furthermore, the upregulation of genes related to the receptor for advanced glycation end products (RAGE)/toll-like receptor 4 (TLR4) was observed in Ly6Chi-RM and LSECs under diabetes, suggesting a possible role of the RAGE/TLR4 signaling in the interaction between inflammatory macrophages and LSECs.The 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. scRNAseq analysis indicates that diabetes activates inflammatory macrophages and impairs LSECs through the RAGE/TLR4 signaling pathway. These findings open avenues for discovering novel therapeutic targets for diabetic steatohepatitis.