Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver condition with potential progression to cirrhosis and impaired regeneration post-resection. A key mechanism underlying lipotoxicity is endoplasmic reticulum (ER) stress, particularly the activation of the unfolded protein response (UPR). This study investigates the interplay between lipid accumulation, endoplasmic reticulum (ER) stress, and cellular outcomes, focusing on the balance between autophagy and apoptosis. We cultured primary human hepatocytes (PHH) in a free fatty acid (FFA)-enriched medium for 120 h, assessing lipid accumulation, metabolism, and the expression of selected UPR markers. Additionally, we investigated the effects of lipid load on cell activity and growth in proliferating HepG2 cells. We observed that FFA uptake consistently induced ER stress, shifting cellular responses toward apoptosis under high lipid loads. Donor-specific differences were evident, particularly in lipid storage, excretion, and sensitivity to lipotoxicity. Some donors exhibited limited triglyceride (TAG) storage and excretion, leading to an excess of FFA whose metabolic fate remains unclear. Proliferation was more sensitive to lipid accumulation than overall cell activity, with even low FFA concentrations impairing growth, highlighting the vulnerability of regenerative processes to steatosis. The study elucidates how ER stress pathways, such as PERK-CHOP and IRE1α-JNK, are differentially activated in response to lipid overload, tipping the balance toward apoptosis in severe cases. The limited activation of repair mechanisms, such as autophagy, further emphasizes the critical role of ER stress in determining hepatocyte fate. The donor-dependent variability highlights the need for personalized strategies to mitigate lipotoxic effects and enhance liver regeneration in steatosis-related conditions.

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a sexually dimorphic condition, with higher prevalence in men than in women. Sex differences in hepatic lipid metabolism and the modulatory role of sex hormones have been described but are still insufficiently understood. The aim of this study was to introduce the variables sex and sex hormones into a human in vitro model of hepatic steatosis. Methods: Primary human hepatocytes (PHHs) were isolated from male and female donors, treated with free fatty acids (FFA) to induce steatosis, and further exposed to physiological concentrations of estrogen, progesterone, or testosterone. Intracellular triacylglyceride (TAG) content, lipid droplet (LD) formation, FFA uptake, and very-low-density lipoprotein (VLDL) excretion were assessed. In parallel, the expression of lipid metabolism-related genes was quantified by qPCR. Results: FFA treatment induced comparable uptake and intracellular TAG storage in both sexes. However, female PHHs secreted approximately twice as many VLDL particles as male cells. Steatosis significantly increased expression of LDLR, CPT2, and PLA1A only in male PHHs. Sex hormones exerted distinct, sex-specific effects: estrogen reduced TAG accumulation in female PHHs; whereas testosterone reduced TAG in male but increased it in female PHHs after prolonged treatment. LD characterization confirmed sex- and hormone-dependent differences in lipid storage patterns. In male PHHs, progesterone promoted lipid storage and increased apoB-100 secretion, accompanied by reduced LDLR and APOA5 expression, and testosterone increased the FFA-mediated CPT2 even further. Conclusions: Sex and sex hormones distinctly shape hepatocellular lipid handling under steatotic conditions. While female PHHs demonstrated greater lipid excretion capacity, male PHHs exhibited stronger transcriptional responses. Sex-specific responses to estrogen and testosterone resembled clinical observations on sex hormone effects. These findings highlight the need to account for sex-specific differences in MASLD pathophysiology and therapeutic strategies.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver condition with potential progression to cirrhosis and impaired regeneration post-resection. A key mechanism underlying lipotoxicity is endoplasmic reticulum (ER) stress, particularly the activation of the unfolded protein response (UPR). This study investigates the interplay between lipid accumulation, endoplasmic reticulum (ER) stress, and cellular outcomes, focusing on the balance between autophagy and apoptosis. We cultured primary human hepatocytes (PHH) in a free fatty acid (FFA)-enriched medium for 120 hours, assessing lipid accumulation, metabolism, and the expression of selected UPR markers. Additionally, we investigated the effects of lipid load on cell activity and growth in proliferating HepG2 cells. We observed that FFA uptake consistently induced ER stress, shifting cellular responses toward apoptosis under high lipid loads. Donor-specific differences were evident, particularly in lipid storage, excretion, and sensitivity to lipotoxicity. Some donors exhibited limited triglyceride (TAG) storage and excretion, leading to an excess of FFA whose metabolic fate remains unclear. Proliferation was more sensitive to lipid accumulation than overall cell activity, with even low FFA concentrations impairing growth, highlighting the vulnerability of regenerative processes to steatosis. The study elucidates how ER stress pathways, such as PERK-CHOP and IRE1α-JNK, are differentially activated in response to lipid overload, tipping the balance toward apoptosis in severe cases. Limited activation of repair mechanisms, such as autophagy, further emphasizes the critical role of ER stress in determining hepatocyte fate. The donor-dependent variability highlights the need for personalized strategies to mitigate lipotoxic effects and enhance liver regeneration in steatosis-related conditions.