The storage stability and concentration of acetoacetate differs between blood fractions.

BACKGROUND: Plasma concentrations of 3-hydroxybutyrate (3HB) are measured more often than acetoacetate (AcAc) which may be due to the reported storage instability of AcAc. The aims of the study were to compare the storage stability of AcAc in different blood fractions over time (90days) when stored at -80°C and to determine the postprandial concentration of AcAc in whole blood, plasma and red blood cells. METHODS: Blood was collected from fasting subjects (n=5): whole blood, plasma and red blood cells were isolated and deproteinised in perchloric acid, and supernatants were stored at -80°C until analysis. Postprandial concentrations of AcAc in whole blood, plasma and red blood cells were determined at regular intervals over 420min, after subjects (n=23) had consumed a mixed test meal. RESULTS: Storing deproteinised plasma at -80°C resulted in no significant change in AcAc concentration over 60days. In contrast, whole blood AcAc concentrations significantly decreased by 51% (p=0.018) within 30days. The concentration of AcAc in fasting and postprandial plasma was notably higher than that of whole blood and red blood cells. DISCUSSION: Our data demonstrates that plasma for AcAc analysis can be stored for longer than previously suggested provided that plasma is deproteinised and stored at -80°C

In vitro cellular models of human hepatic fatty acid metabolism: differences between Huh7 and HepG2 cell lines in human and fetal bovine culturing serum

Human primary hepatocytes are the gold standard for investigating lipid metabolism in non-alcoholic 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 mM glucose and either 2% HS or 10% fetal bovine serum. After three days insulin and insulin-like growth factor-1 signaling were measured and at seven days intracellular triacylglycerol (TAG), 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

In vitro cellular models of human hepatic fatty acid metabolism: differences between Huh7 and HepG2 cell lines in human and fetal bovine culturing serum

Human primary hepatocytes are the gold standard for investigating lipid metabolism in non-alcoholic 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 mM glucose and either 2% HS or 10% fetal bovine serum. After three days insulin and insulin-like growth factor-1 signaling were measured and at seven days intracellular triacylglycerol (TAG), 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

In vitro cellular models of human hepatic fatty acid metabolism: differences between Huh7 and HepG2 cell lines in human and fetal bovine culturing serum.

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

Effects on hepatic lipid metabolism in human hepatoma cells following overexpression of TGFβ induced factor homeobox 1 or 2

Transforming growth factor β induced factor homeobox (TGIF) 1 and 2 are two transcriptional repressors. Although TGIF1 has been found to be involved in lipid metabolism, no studies have yet investigated the role of TGIF2 in hepatic lipid metabolism. Here we aim to investigate effects on hepatic lipid metabolism following overexpression of the human and mouse TGIF1 and TGIF2 protein. We used modified mRNA molecules to transiently enhance the expression of these proteins in human hepatoma cells. We found all the mRNA molecules to be translated, except the one for human TGIF1. Transient transfection with the mouse TGIF1 mRNA molecules lowered levels of cholesterol (p < 0.001), triglycerides (p < 0.001), and apolipoprotein B (p < 0.05) in the cell media by ~40%, along with the mRNA levels of some key genes involved in lipid metabolism. In contrast, limited effects on these parameters were observed following transient transfection with the human and mouse TGIF2 mRNA molecules. To enable investigation of the effects following enhanced expression of the human TGIF1 protein, we stably overexpressed this protein in human hepatoma cells. In line with the above findings, we found cells stably overexpressing the human TGIF1 protein had lower levels of cholesterol (p < 0.05), triglycerides (p < 0.05) and apolipoprotein B (p < 0.05) in the cell media by ~30%. Hence, transient and stable overexpression of the TGIF1 protein appears to lead to an advantageous lipid profile

Effects on hepatic lipid metabolism in human hepatoma cells following overexpression of TGFβ induced factor homeobox 1 or 2

Transforming growth factor β induced factor homeobox (TGIF) 1 and 2 are two transcriptional repressors. Although TGIF1 has been found to be involved in lipid metabolism, no studies have yet investigated the role of TGIF2 in hepatic lipid metabolism. Here we aim to investigate effects on hepatic lipid metabolism following overexpression of the human and mouse TGIF1 and TGIF2 protein. We used modified mRNA molecules to transiently enhance the expression of these proteins in human hepatoma cells. We found all the mRNA molecules to be translated, except the one for human TGIF1. Transient transfection with the mouse TGIF1 mRNA molecules lowered levels of cholesterol (p < 0.001), triglycerides (p < 0.001), and apolipoprotein B (p < 0.05) in the cell media by ~40%, along with the mRNA levels of some key genes involved in lipid metabolism. In contrast, limited effects on these parameters were observed following transient transfection with the human and mouse TGIF2 mRNA molecules. To enable investigation of the effects following enhanced expression of the human TGIF1 protein, we stably overexpressed this protein in human hepatoma cells. In line with the above findings, we found cells stably overexpressing the human TGIF1 protein had lower levels of cholesterol (p < 0.05), triglycerides (p < 0.05) and apolipoprotein B (p < 0.05) in the cell media by ~30%. Hence, transient and stable overexpression of the TGIF1 protein appears to lead to an advantageous lipid profile

Sex-specific differences in hepatic fat oxidation and synthesis may explain the higher propensity for NAFLD in men

Context and Objective: In most populations a greater proportion of men have hepatic steatosis than women. Sex-specific differences in hepatic dietary fatty acid (FA) metabolism have not been well characterized. We compared fasting and postprandial hepatic FA synthesis (de novo lipogenesis [DNL]) and oxidation in men and women. Participants and Methods: Fasting and postprandial hepatic FA metabolism was studied in 22 healthy men (n = 11) and women with similar age, body mass index, and liver fat content using metabolic substrates labeled with stable-isotope tracers (2H2O and [U13C]palmitate). Dietary FA oxidation was assessed by appearance of 13C into plasma 3-hydroxybutyrate and breath CO2 as markers of liver and whole-body FA oxidation, respectively. Results: Despite similar liver fat content, fasting and postprandial plasma triacylglycerol (TG) concentrations were significantly (P &lt; .05) higher in men compared with women. The appearance of 13C from dietary FA into plasma 3-hydroxybutyrate and breath CO2 was greater (P &lt; .05) in women compared with men. Although the contribution of DNL into very low-density lipoprotein (VLDL)-TG was similar (∼10%) in the fasting state, there was a divergence in pattern over the course of the study, with men maintaining a higher contribution of DNL to VLDL-TG than women (P = .006 time x sex interaction). Conclusions: The combination of lower dietary FA oxidation and a prolonged increase in DNL observed in men may represent partitioning of FA into esterification and storage pathways within the liver, leading to greater VLDL-TG production, and predispose to the sex difference in hepatic steatosis.</p

Hepatic de novo lipogenesis is suppressed and fat oxidation is increased by omega-3 fatty acids at the expense of glucose metabolism

Objective: Increased hepatic de novo lipogenesis (DNL) is suggested to be an underlying cause in the development of non-alcoholic fatty liver disease (NAFLD) and/or insulin resistance. It is suggested that the 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 methods: Thirty-eight healthy males were randomised to take either an omega-3 supplement (4g/d EPA+DHA as ethyl esters) or placebo (4g/d olive oil) and fasting measurements were made at baseline and 8 weeks. The metabolic effects of omega-3 FAs on intra-hepatocellular triacylglycerol (IHTAG) content, hepatic DNL and FA oxidation were investigated using metabolic substrates labelled with stable-isotope tracers. In vitro studies, using a human liver cell-line were undertaken to gain insight into the intra-hepatocellular effects of omega-3 FAs. Results: Fasting plasma triacylglycerol 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 whilst significantly increasing dietary FA oxidation and fasting and postprandial plasma glucose concentrations. In vitro studies supported the in vivo findings of omega-3 fatty acids (EPA+DHA) decreasing intracellular TAG through a shift in cellular metabolism away from FA esterification towards oxidation. Conclusions: Omega-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 non-lipid substrates (e.g. glucose) will have on intra- and extra-hepatic metabolic pathways.</p