| Résumé: | <p>Pathological accumulation of intrahepatocellular triacylglycerol (IHTAG) is the first stage of metabolic dysfunction-associated steatotic liver disease (MASLD). Intervention studies have shown that diets rich in saturated fatty acids (SFA) compared to unsaturated fatty acids (UNSFA) increase IHTAG accumulation to a greater extent. Studying the underlying metabolic mechanisms driving these differences in IHTAG accumulation in vivo in humans remains challenging therefore, in vitro cellular models which recapitulate the effects of dietary alterations in hepatic metabolism are needed.</p>
<p>Huh7 cells, cultured with fatty acid (FA) treatments, enriched in either SFA or MUFA and given at either a high (800uM) or low (200uM) concentration, were used in combination with a low glucose (5.5mM) media to explore hepatic lipid metabolism: IHTAG composition reflected that of the provided treatments suggesting a lack of preferential uptake. Consequently, there were notable disparities in glucose production and cellular oxidation. Additionally, differences were seen in lipid droplet morphology with Huh7 cells treated with SFA accumulating small homogenous lipid droplets, compared to those treated with UNSFA which accumulated a heterogeneous pattern of large and small lipid droplets.</p>
<p>The response of primary human hepatocytes (PHH) to FA treatments enriched in SFA or UNSFA was also investigated, however, these cells proved challenging to study as results were confounded by pre-existing IHTAG accumulation and blunted metabolic responses. RNA sequencing showed divergence in the differentially expressed genes between Huh7 and PHH cells, however, both cells implicated SFA enriched treatment as being the more detrimental of the two FA compositions. Overall, the addition of FA compared to controls treated with no-fat showed a greater effect on the transcriptional profile when compared to FA composition or concentration changes in Huh7 cells and PHH.</p>
<p>Lifestyle changes are the primary suggested treatment for patients presenting with MASLD and involve the changing of both dietary quantity and composition. To attempt to understand these changes at an cellular level, cellular responses to changes in either the provided FA concentration or composition were analyzed. Although Huh7 cells continued to non-selectively take up the FA provided, despite significant previous FA accrual, results showed blunted responses to hormonal stimulation. Of interest, those Huh7 cells previous treated with FA enriched in SFA, which had displayed small homogeneous lipid droplet patterns, when treated with FA enriched in UNSFA took on the morphology of those cells treated with UNSFA for the whole treatment period.</p>
<p>Human derived TAG-rich remnants, an under-studied source of hepatic IHTAG, enriched in either SFA or UNSFA were labelled using a novel protocol with stable isotopes and Huh7 cells were found to take up these remnants non-selectively. TAG-rich remnants FA composition significantly effected glucose uptake and urea production although further work is required to explore the intrahepatic metabolic changes.</p>
<p>Overall, I developed physiologically-relevant models of hepatocellular metabolism and steatosis, reflecting differences in dietary composition. Data showed that although FA composition did not alter IHTAG accumulation, it did alter a number of metabolic pathways, including oxidation, glucose handling, and significantly it had a strong effect on lipid droplet morphology.</p>
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