Diabetes poses a significant threat to human health. Exocrine pancreatic dysfunction is related to diabetes, but the exact mechanism is not fully understood. This study aimed to describe the pathological phenotype and pathological mechanisms of the pancreas of transgenic pigs (PIGinH11) that was constructed in our laboratory and to compare it with humans. We established diabetes-susceptible transgenic pigs and subjected them to high-fat and high-sucrose dietary interventions. The damage to the pancreatic endocrine and exocrine was evaluated using histopathology and the involved molecular mechanisms were analyzed using single-nucleus RNA-sequencing (SnRNA-seq). Compared to wild-type (WT) pigs, PIGinH11 pigs showed similar pathological manifestations to type 2 diabetes patients, such as insulin deficiency, fatty deposition, inflammatory infiltration, fibrosis tissue necrosis, double positive cells, endoplasmic reticulum (ER) and mitochondria damage. SnRNA-seq analysis revealed 16 clusters and cell-type-specific gene expression characterization in the pig pancreas. Notably, clusters of Ainar-M and Endocrine-U were observed at the intermediate state between the exocrine and endocrine pancreas. Beta cells of the PIGinH11 group demonstrated the dysfunction with insulin produced and secret decreased and ER stress. Moreover, like clinic patients, acinar cells expressed fewer digestive enzymes and showed organelle damage. We hypothesize that TXNIP that is upregulated by high glucose might play an important role in the dysfunction of endocrine to exocrine cells in PIGinH11 pigs.

Background: This research aims to reveal the cellular cross talk in fibrosis liver using transgenic pigs (TG) expressing humanized risk genes (PNPLA3I148M-GIPRdn-hIAPP) as a metabolic dysfunction–associated steatotic liver disease (MASLD) model. Methods and Results: The study uses single-nucleus sequencing to reveal the differentiation and interaction characteristics of various cell populations in the liver during the development of MASLD. After 6 months of high-fat, high-sucrose diet induction, the model pigs exhibited obvious liver pathological features, including fat deposition, inflammatory cell aggregation, fibrosis, and blocked insulin signaling pathways, similar to PNPLA3 rs738409 C>G carriers. Single-nucleus RNA sequencing showed that pigs share a high correlation with human hepatic cell types and zonation. HSCs in TG pigs are more activated, showing enhancing fibrosis-related pathways and declining retinol metabolism. Pseudo-trajectory analysis revealed that over 90% of macrophages in TG liver differentiated to Fate 1 (CD68hi) with higher expression of major histocompatibility complex-II molecules, proinflammatory cytokines, phagosomes, and lysosomal-related genes. Active cell interactions were found between HSCs, endothelial cells, and Fate1 macrophages. Ligand and receptor interactions, including FGF23-FGFR, PDGFs-PDGFRs, EFNA1-EPHRs, and CXCL12-CXCR4/CXCR7, were predicted to involve in hepatic fibrosis in model pigs. Conclusions: The transgenic pig model of MASLD exhibits liver pathological features consistent with patients with MASLD. Our data supplemented the mechanism by which PNPLA3 mutations lead to hepatic steatosis, depicted a detailed atlas of hepatic profibrosis cellular network, and provided a reliable large animal model and data reference for MASLD drug development and precision treatment. Export