Background and Aims Nonalcoholic fatty liver disease (NAFLD) begins with steatosis, where a mixed macrovesicular pattern of large and small lipid droplets (LDs) develops. 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. Methods Huh7 cells were cultured in 11 mM glucose and 2% human serum (HS) for 7 days before additional sugars and fatty acids (FAs), either with 200 µM FAs (low fat low sugar; LFLS), 5.5 mM fructose + 200 µM FAs (low fat high sugar; LFHS), or 5.5 mM fructose + 800 µM FAs (high fat high sugar; HFHS), were added for 7 days. FA metabolism, lipid droplet characteristics, and transcriptomic signatures were investigated. Results 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‐treated cells had lower levels of de novo‐derived FAs and secreted larger, TAG‐rich lipoprotein particles. RNA sequencing and gene set enrichment analysis showed changes in several pathways including those involved in metabolism and cell cycle. Conclusions 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.

Context Genetic variants affecting the nuclear hormone receptor coactivator Steroid Receptor Coactivator, SRC-1, have been identified in people with severe obesity and impair melanocortin signaling in cells and mice. As a result, obese patients with SRC-1 deficiency are being treated with a Melanocortin 4 receptor agonist in clinical trials. Objective Here, our aim was to comprehensively describe and characterise the clinical phenotype of SRC-1 variant carriers to facilitate diagnosis and clinical management. Design In genetic studies of 2,462 people with severe obesity, we identified 23 rare heterozygous variants in SRC-1. We studied 29 adults and 18 children who were SRC-1 variant carriers and performed measurements of metabolic and endocrine function, liver imaging and adipose tissue biopsies. Findings in adult SRC-1 variant carriers were compared to 30 age and BMI-matched controls. Results The clinical spectrum of SRC-1 variant carriers included increased food intake in children, normal basal metabolic rate, multiple fractures with minimal trauma (40%), persistent diarrhea, partial thyroid hormone resistance and menorrhagia. Compared to age, sex and BMI matched controls, adult SRC-1 variant carriers had more severe adipose tissue fibrosis (46.2% v 7.1% respectively, P=0.03) and a suggestion of increased liver fibrosis (5/13 cases versus 2/13 in controls, odds ratio 3.4), although this was not statistically significant. Conclusions SRC-1 variant carriers exhibit hyperphagia in childhood, severe obesity and clinical features of partial hormone resistance. The presence of adipose tissue fibrosis and hepatic fibrosis in young patients suggests that close monitoring for the early development of obesity-associated metabolic complications is warranted.