Type 2 diabetes (T2D) is partly caused by reduced functional β-cell mass, with increased saturated fat intake potentially contributing to β-cell dysfunction. Rodent studies show that long-chain saturated fatty acids (LC-SFA), particularly palmitate, induce "lipotoxicity" in β-cells, a process not fully understood or universally accepted in human cells. While some mechanisms like perturbed lipid homeostasis, reactive oxygen species, ER stress, mitochondrial dysfunction, and altered autophagy have been proposed, the reasons for LC-SFA-induced β-cell death and the protective effects of monounsaturated fatty acids (LC-MUFA) like oleate remain unclear. The human EndoC-BH1 β-cell line shows significant resistance to palmitate toxicity, making it crucial to compare palmitate handling in human and rodent cells. This study aims to understand the intracellular distribution of LC-SFA and its effects on subcellular morphology to elucidate the factors influencing β-cell sensitivity to palmitate.

Numerous studies have investigated the mechanisms behind β-cell lipotoxicity, utilizing various multi-omics and molecular biology techniques. These studies have identified several intracellular signaling pathways involved, including perturbations in lipid homeostasis, reactive oxygen species production, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and altered autophagy. However, the findings are often conflicting, and few studies have elucidated the mechanisms underlying the protective effects of unsaturated fatty acids. Many mechanisms are compartmentalized within subcellular organelles like the ER, lipid droplets, and mitochondria, suggesting a need for broader investigation into the intracellular distribution of LC-SFAs and their impact on subcellular morphology.

The study used INS-1E and INS-1 823/13 rodent β-cells and the human EndoC-BH1 β-cell line. Cells were treated with palmitate (C16:0), oleate (C18:1), or both, and the subcellular distribution of palmitate was studied using confocal fluorescence microscopy with the fluorescent palmitate analogue BODIPY FL C16. Electron microscopy (EM) was used to visualize subcellular morphology. Western blotting assessed protein expression levels, and cell viability was determined using vital dye staining. Immunofluorescence with anti-PLIN2 antibody and CellLight® Golgi-RFP marker were used for co-localization studies. Statistical analysis was performed using GraphPad Prism with ANOVA and Tukey's post hoc test.

Palmitate exposure caused dose-dependent INS-1E cell death, which was attenuated by oleate. EndoC-BH1 cells showed remarkable resistance to palmitate-induced death, even at high concentrations. In INS-1E cells, BODIPY FL C16 initially localized to the Golgi apparatus, correlating with distended intracellular membranes observed under EM. However, this did not activate the PERK-dependent ER stress pathway. In EndoC-BH1 cells, BODIPY FL C16 co-localized with PLIN2, indicating preferential routing into lipid droplets. Co-treatment with palmitate and oleate in INS-1E cells directed BODIPY FL C16 to lipid droplets and reduced cell death. Electron microscopy revealed Golgi apparatus swelling in palmitate-treated INS-1 cells, absent in EndoC-BH1 cells or INS-1 cells co-treated with oleate. Palmitate did not activate the PERK-dependent ER stress pathway in either cell type, even though tunicamycin induced ER stress in INS-1E but not EndoC-BH1 cells. The study observed a clear colocalization of BODIPY FL C16 with PLIN2 in EndoC-BH1 cells exposed to palmitate and in INS-1E cells co-treated with palmitate and oleate, but not in INS-1E cells treated with palmitate alone. The formation of lipid droplets was time-dependent (2h,6h,24h) and was more rapid in EndoC-BH1 than in INS-1E cells, except when the latter was co-treated with oleate.

The study reveals distinct differences in palmitate handling between rodent and human β-cells, supporting the idea that lipotoxicity might be primarily a rodent phenomenon. The resistance of EndoC-BH1 cells may be due to high stearoyl CoA desaturase (SCD) expression, converting LC-SFA to less toxic monounsaturated forms. The discrepancy between the results obtained for EndoC-BH1 cells in vitro and the reported sensitivity of primary human islets to palmitate warrants further research, to establish whether the response to palmitate in human EndoC-BH1 cells is a cell line artifact or accurately represents in vivo conditions. The maintenance of viability in both INS-1 and EndoC-BH1 cells when palmitate is routed to lipid droplets suggests that this trafficking pathway offers protection against lipotoxicity. This finding is supported by previous research demonstrating the protective role of lipid droplet-associated proteins like PLIN1 and PLIN5. However, the role of lipid droplets in protecting against lipotoxicity requires further investigation.

This study demonstrates that rodent β-cells rapidly incorporate palmitate into the Golgi apparatus, leading to morphological changes and cell death, while human β-cells preferentially route palmitate to lipid droplets, maintaining viability. The redirection of palmitate towards lipid droplets, achieved by co-treatment with oleate in rodent cells or naturally in human cells, appears crucial for maintaining β-cell survival. Future studies should focus on the lipid composition of intracellular membranes, the specific organelles involved, and the factors determining palmitate routing within cells.

The study used cell lines rather than primary human islets. While EndoC-BH1 cells are considered a good model of human β-cells, they might not fully capture the complexity of in vivo conditions. The use of BODIPY FL C16 as a palmitate analogue might not perfectly reflect the behavior of endogenous palmitate. Further research should also focus on the relationship between ER stress and lipotoxicity; the exact role of ER stress in lipotoxicity, even in rodent cells, needs to be further investigated in relation to palmitate's influence on the Golgi apparatus.