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Since in vitro models recapitulating this are limited, the aims of this study were to develop mixed macrovesicular steatosis in immortalized hepatocytes and investigate effects on intracellular metabolism by altering nutritional substrates.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Huh7 cells were cultured in 11\u00a0mM glucose and 2% human serum (HS) for 7\u00a0days before additional sugars and fatty acids (FAs), either with 200\u00a0\u00b5M FAs (low fat low sugar; LFLS), 5.5\u00a0mM fructose\u00a0+\u00a0200\u00a0\u00b5M FAs (low fat high sugar; LFHS), or 5.5\u00a0mM fructose\u00a0+\u00a0800\u00a0\u00b5M FAs (high fat high sugar; HFHS), were added for 7\u00a0days. FA metabolism, lipid droplet characteristics, and transcriptomic signatures were investigated.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Between the LFLS and LFHS conditions, there were few notable differences. In the HFHS condition, intracellular triacylglycerol (TAG) was increased and the LD pattern and distribution was similar to that found in primary steatotic hepatocytes. HFHS\u2010treated cells had lower levels of de novo\u2010derived FAs and secreted larger, TAG\u2010rich lipoprotein particles. RNA sequencing and gene set enrichment analysis showed changes in several pathways including those involved in metabolism and cell cycle.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Repeated doses of HFHS treatment resulted in a cellular model of NAFLD with a mixed macrovesicular LD pattern and metabolic dysfunction. Since these nutrients have been implicated in the development of NAFLD in humans, the model provides a good physiological basis for studying NAFLD development or regression in vitro.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.14814\/phy2.14482","type":"journal-article","created":{"date-parts":[[2020,7,8]],"date-time":"2020-07-08T22:01:14Z","timestamp":1594245674000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Modifying nutritional substrates induces macrovesicular lipid droplet accumulation and metabolic alterations in a cellular model of hepatic steatosis"],"prefix":"10.14814","volume":"8","author":[{"given":"Pippa J.","family":"Gunn","sequence":"first","affiliation":[{"name":"Oxford Centre for Diabetes, Endocrinology and Metabolism Radcliffe Department of Medicine University of Oxford Oxford 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