Modifying nutritional substrates induces macrovesicular lipid droplet accumulation and metabolic alterations in a cellular model of hepatic steatosis

Abstract

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.

FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells

“…A major question is how do these cells generate energy? Liver cell lines like HepG2 cells have been widely used for human liver metabolism and xenobiotic metabolism [81][82][83][84][85][86] . In previous studies of HepG2 metabolism, a "no glucose" condition exhibits very low glycolysis, which may also model blocked glycolysis 81 .…”

Physiological and pathophysiological concentrations of fatty acids induce lipid droplet accumulation and impair functional performance of tissue engineered skeletal muscle

“…After a total of 14 days in culture, TESkM constructs were treated with 0.25% FA‐free bovine serum albumin (FAF‐BSA) alone or conjugated to oleic, palmitic, linoleic, and α‐linoleic acid (OPLA) (physiological ratio 45:30:24:1%) at a concentration of 200 and 800 µM, as used previously to induce lipid droplet accumulation in a non‐adipose cell line (Gunn et al, 2020). OPLA was supplemented into high glucose, phenol red free DMEM with 2% horse serum, 4 mM l ‐glutamine, 1 mM sodium pyruvate, and 1% P/S and were in addition to the concentrations of albumin and triglycerides contained in the horse serum.…”

“…Despite the importance of FA in skeletal muscle metabolism (Badin et al, 2013), quite how physiological and pathophysiological concentrations of FA influence TESkM function is yet to be defined. In order to offer some insight into these mechanisms, the TESkM model developed by our research group (Capel et al, 2019), was exposed to physiological and pathophysiological concentrations of FA which have previously been shown to induce lipid accumulation in non‐adipose cell lines (Green et al, 2015; Gunn et al, 2020; Hodson et al, 2008). This allowed investigation of the impact of FA overload upon LD formation, gene expression, insulin sensitivity, and functional capacity in TESkM.…”

Cell Models and Omics Techniques for the Study of Nonalcoholic Fatty Liver Disease: Focusing on Stem Cell-Derived Cell Models

“…The application of combined strategies with two omics technologies or more will lead to a more exhaustive and global NAFLD characterization and will contribute to a better understanding of all the involved mechanisms. Along these lines, a few reports illustrate the application of this integrative strategy to study NAFLD in diverse cell liver models, such as PHH, hepatoma cell lines, or HLCs [ 70 , 97 , 125 , 126 ]. However, the interpretation and integration of data from multiple omics are difficult because alterations in gene or protein expression and metabolite levels occur on different time scales.…”

“…Flow cytometry is the main technique followed in cytomics studies, but some microscopy-based technologies such as high-content screening (HCS) have also been applied to study NAFLD in vitro. For instance, different fluorescent probes can be used to stain and quantify lipids [ 126 ]. Flow cytometry has been applied and/or HCS assays have been run to study drug-induced steatosis in HepG2, HepaRG, or Upcyte cells [ 50 , 51 , 54 , 81 ].…”