Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterised by the pathological accumulation of intrahepatocellular triglyceride (IHCTG). Although initially benign, IHCTG may lead to inflammatory activation and progression to steatohepatitis and more severe liver disease. Hyperinsulinemia and dietary fat quantity and composition have been associated with MASLD development and progression which may be mediated through an aberrant decrease in autophagy. The aim of this work was to determine the effects of insulin concentration and fatty acid (FA) quantity and composition on hepatocellular autophagic flux and inflammatory activation in an in vitro model of IHCTG accumulation.

Methodology: Huh7 hepatocyte cells were cultured for 7 days in low fat-low sugar (LFLS) or high fat-high sugar (HFHS) media with either predominantly unsaturated or saturated FA compositions and at 0.1 or 100 nmol insulin concentrations. IHCTG was extracted from cells and analysed using gas chromatography. Autophagic flux was quantified by Western Blotting through accumulation of LC3-II with and without autophagic inhibition and media cytokines were measured using Olink® Target 48 Cytokine Panel. Results from six biological repeats and analysed with two-way ANOVA followed by pairwise t-tests with BH correction for multiple comparisons.

Results: Cells cultured in HFHS compared to LFLS media had higher IHCTG concentration (p<0.0001) and the IHCTG composition reflected the FAs cells were cultured in. Compared to cells cultured in LFLS media, cells cultured in HFHS media (saturated or unsaturated FAs) had higher secretion of chemokines (CXCL9, CCL3, and CCL4) but cells cultured in HFHS media with unsaturated FAs also had a higher secretion of CXCL10, IL6 and CXCL8 (p<0.01) and tended (p=0.074) to have a lower autophagic flux. There were no other differences in autophagic flux between conditions and insulin had no effect on either IHCTG accumulation, composition or cytokine secretion.

Conclusions: Higher FA quantity increased inflammatory activation, especially with unsaturated FAs which may have been mediated by a decrease in autophagy. Hyperinsulinemia had limited effects on this model which may suggest that hepatocytes adapt better to changes in insulin concentration than to changes in FA quantity or quality.