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ObjectiveIncreased hepatic de novo lipogenesis (DNL) is suggested to be an underlying cause in the development of nonalcoholic fatty liver disease and/or insulin resistance. It is suggested that omega-3 fatty acids (FA) lower hepatic DNL. We investigated the effects of omega-3 FA supplementation on hepatic DNL and FA oxidation using a combination of human in vivo and in vitro studies.Research design and methodsThirty-eight healthy men were randomized to take either an omega-3 supplement (4 g/day eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) as ethyl esters) or placebo (4 g/day olive oil) and fasting measurements were made at baseline and 8 weeks. The metabolic effects of omega-3 FAs on intrahepatocellular triacylglycerol (IHTAG) content, hepatic DNL and FA oxidation were investigated using metabolic substrates labeled with stable-isotope tracers. In vitro studies, using a human liver cell-line was undertaken to gain insight into the intrahepatocellular effects of omega-3 FAs.ResultsFasting plasma TAG concentrations significantly decreased in the omega-3 group and remained unchanged in the placebo group. Eight weeks of omega-3 supplementation significantly decreased IHTAG, fasting and postprandial hepatic DNL while significantly increasing dietary FA oxidation and fasting and postprandial plasma glucose concentrations. In vitro studies supported the in vivo findings of omega-3 FAs (EPA+DHA) decreasing intracellular TAG through a shift in cellular metabolism away from FA esterification toward oxidation.ConclusionsOmega-3 supplementation had a potent effect on decreasing hepatic DNL and increasing FA oxidation and plasma glucose concentrations. Attenuation of hepatic DNL may be considered advantageous; however, consideration is required as to what the potential excess of nonlipid substrates (eg, glucose) will have on intrahepatic and extrahepatic metabolic pathways.Trial registration numberNCT01936779.

An increasing body of evidence now links estrogenic signalling with the metabolic syndrome (MS). Despite the beneficial estrogenic effects in reversing some of the MS symptoms, the underlying mechanisms remain largely undiscovered. We have previously shown that total estrogen receptor alpha (ERα) knockout (KO) mice exhibit hepatic insulin resistance. To determine whether liver-selective ablation of ERα recapitulates metabolic phenotypes of ERKO mice we generated a liver-selective ERαKO mouse model, LERKO. We demonstrate that LERKO mice have efficient reduction of ERα selectively within the liver. However, LERKO and wild type control mice do not differ in body weight, and have a comparable hormone profile as well as insulin and glucose response, even when challenged with a high fat diet. Furthermore, LERKO mice display very minor changes in their hepatic transcript profile. Collectively, our findings indicate that hepatic ERα action may not be the responsible factor for the previously identified hepatic insulin resistance in ERαKO mice.

[This corrects the article DOI: 10.1371/journal.pone.0057458.].

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. Exposing Huh7 cells to repeated doses of a physiological treatment media containing, human serum, fatty acids and sugar lead to the development of a mixed macrovesicular steatotic lipid droplet pattern that is similar to that found in isolated primary hepatocytes. This model displays several characteristics of early‐stage NAFLD in vivo therefore providing a strong basis of a physiological model that can be further manipulated to investigate in vitro intracellular TAG development and/or regression.

Human primary hepatocytes are the gold standard for investigating lipid metabolism in nonalcoholic fatty liver disease (NAFLD); however, due to limitations including availability and donor variability, the hepatoma cell lines Huh7 and HepG2 are commonly used. Culturing these cell lines in human serum (HS) has been reported to improve functionality; however, direct comparison of fatty acid (FA) metabolism in response to culturing in HS is lacking. The aim of this study was to compare FA metabolism between HepG2 and Huh7 cells in response to culturing in different sera. Both HepG2 and Huh7 cells were grown in media containing 11 mmol/L glucose and either 2% HS or 10% fetal bovine serum. After 3 days, insulin and insulin‐like growth factor‐1 signaling were measured. At 7 days, intracellular triacylglycerol (TAG) and media 3‐hydroxybutyrate, TAG and apolipoprotein B were measured, as was the FA composition of intracellular TAG and phospholipids. Both cell lines demonstrated higher levels of polyunsaturated fatty acid content, increased insulin sensitivity, higher media TAG levels and increased FA oxidation when cultured in HS. Notably, independent of serum type, Huh7 cells had higher intracellular TAG compared to HepG2 cells, which was in part attributable to a higher de novo lipogenesis. Our data demonstrate that intrahepatocellular FA metabolism is different between cell lines and influenced by culturing sera. As a result, when developing a physiologically‐relevant model of FA metabolism that could be developed for the study of NAFLD, consideration of both parameters is required. When cultured in media containing either 2% HS or 10% fetal bovine serum (FBS), we found both HepG2 and Huh7 cells had increased insulin sensitivity, higher media triacylglycerol (TAG) concentrations and increased FA oxidation when cultured in HS. Our data demonstrate that the serum that cells are cultured in and the cell line used can influence intrahepatocellular FA metabolism.

Acyl-Coenzyme A:cholesterol acyltransferases (ACATs) 1 and 2 are integral membrane proteins located in rough endoplasmatic reticulum that catalyzes the formation of cholesteryl esters (CEs) from cholesterol and long-chain fatty acids. ACAT1 is present in most tissues, whereas ACAT2 is confined to enterocytes and hepatocytes. Disparities in tissue expressions, together with animal studies, suggests that ACAT2-derived CEs are incorporated into hepatic and intestinal apo B-containing lipoproteins and secreted into plasma, whereas ACAT1 is involved in esterification in other cells (e.g. macrophages) and thereby Hepatic nuclear factors (HNFs) 1 and 4 are involved in diverse metabolic pathways (e.g. glucose, cholesterol, and fatty acid metabolism)and are highly expressed in liver, pancreas, and kidney. The overall aim of this thesis was to gain more insight into the molecular mechanisms that participate in the hepatic regulation of ACAT2 and the roles of HNF1α and HNF4α in cholesterol metabolism.In Paper I we aimed to investigate a possible transcriptional regulation by cholesterol of the human ACAT2 gene. In addition, we aimed to appraise the use of two human hepatoma cell lines, HuH7 and HepG2, as model systems in studies of ACAT. We showed a dose-dependent increase of ACAT2mRNA expression, an increased enzymatic activity of ACAT2, and increase desterified cholesterol mass upon cholesterol loading. These results suggested that ACAT2, but not ACAT1, is transcriptionally regulated bycholesterol in humans. Additionally, we showed that cell differentiation affects the mRNA expression of ACAT1 and ACAT2 in HuH7, but not in HepG2cells. Since HuH7 cells required much lower concentrations of cholesterol to obtain similar results as HepG2 cells, and were more sensitive to cholesterol depletion, HuH7 cells may represent a better system for sterol-studies of ACAT.In Paper II we aimed to characterize mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified an important HNF1 binding site, located -871 to -866 bp upstream of the transcription start site, which serves as a positive regulator of the ACAT2 gene expression and showed that this site is functionally active both in vitro and in vivo. The transcription factors HNF1α and HNF1β, which binds to this site, play an important part in there gulation of the human ACAT2 promoter. Paper III: Maturity onset diabetes of the young (MODY) is a group of syndromes characterized by autosomal dominant inheritance, early on set diabetes, and β-cell dysfunction. Mutations of the genes encodingHNF1α and HNF4α cause MODY3 and MODY1, respectively. ACAT2 is thought to be responsible for production of CEs in hepatic very low density lipoprotein (VLDL) assembly. We identified HNF1α as an important regulator of ACAT2. HNF4α is an upstream regulator of HNF1α. Thus we hypothesized that MODY3 and possibly MODY1 subjects may have lower VLDL esterified cholesterol. Unexpectedly, we found that MODY1 subjects had lower VLDL and low density lipoprotein (LDL)esterified cholesterol levels, whereas MODY3 subjects had similar lipoprotein composition as controls. Hence, we characterized the role of HNF4α in the transcriptional regulation of ACAT2 and identified HNF4α as an important regulator of the hepatocyte-specific expression of ACAT2. These studies suggested that the lower levels of esterified cholesterol in VLDL- and LDL-particles in MODY1 subjects may at least in part be due to lower ACAT2 activity in these patients.