111 research outputs found
Insulin receptor activation and down-regulation by cationic lipid transfection reagents
BACKGROUND: Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000â„¢ and FuGENE-6â„¢. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types RESULTS: We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000â„¢ on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000â„¢ can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. CONCLUSION: Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process
TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1β/Derlin-1 pathway
Oligodendroglial Process Formation is Differentially Affected by Modulating the Intra- and Extracellular Cholesterol Content
Regulation of the hepatic ACAT2 expression and roles of HNF1alhpa and HNF4alpha in cholesterol 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 apoB-containing lipoproteins and
secreted into plasma, whereas ACAT1 is involved in esterification of
cholesterol in other cells (e.g. macrophages) and thereby prevents
apoptosis. 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
HNF1alpha and HNF4alpha 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 ACAT2
mRNA expression, an increased enzymatic activity of ACAT2, and increased
esterified cholesterol mass upon cholesterol loading. These results
suggested that ACAT2, but not ACAT1, is transcriptionally regulated by
cholesterol in humans. Additionally, we showed that cell differentiation
affects the mRNA expression of ACAT1 and ACAT2 in HuH7, but not in HepG2
cells. 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 HNF1alpha and
HNF1beta, which binds to this site, play an important part in the
regulation 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 onset
diabetes, and beta-cell dysfunction. Mutations of the genes encoding
HNF1alpha and HNF4alpha 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 HNF1alpha as an
important regulator of ACAT2. HNF4alpha is an upstream regulator of
HNF1alpha. 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
HNF4alpha in the transcriptional regulation of ACAT2 and identified
HNF4alpha 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.
Paper IV: Niemann-Pick C1 like 1 (NPC1L1) is highly expressed in human
liver and intestine. NPC1L1 is a key regulator of intestinal cholesterol
absorption but its hepatic function is not well defined. Thus, we aimed
to gain more insight into the hepatic expression of the human NPC1L1
gene. Gene expression analyses were performed in liver samples from
Chinese patients with or without cholesterol gallstone disease. Strong
positive correlations between NPC1L1 and sterol regulatory element
binding protein 2 (SREBP2) and between NPC1L1 and HNF4alpha were
observed. HNF4alpha is an upstream regulator of HNF1alpha. Thus, we
further investigated possible roles of SREBP2, HNF4alpha, and HNF1alpha
in the hepatic regulation of NPC1L1. We identified an important HNF1
binding site located -158 to -144 bp upstream of the transcription start
site of the human NPC1L1 promoter. Also, we showed that SREBP2 and
HNF1alpha are important transcription factors for the hepatic NPC1L1
promoter activity that can bind to and regulate its expression in humans.
Moreover, it is possible that HNF4alpha may function by transactivating
NPC1L1 via binding to other transcription factors, including HNF1alpha.
Collectively, these studies imply that ACAT2 is under metabolic control
and that HNF1alpha and HNF4alpha participates in several important
processes in cholesterol metabolism. HNF1alpha may participate in hepatic
cholesterol esterification, uptake of free cholesterol (FC) in
hepatocytes, and in bile acid synthesis. HNF4alpha may participate in
esterification of cholesterol in high density lipoprotein (HDL), affect
plasma levels of esterified cholesterol and triglycerides in VLDL- and
LDL-particles, and indirectly participate in the regulation of uptake of
FC in hepatocytes
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
Alterations in bile acid synthesis in carriers of HNF1α mutations.
OBJECTIVES: Heterozygous mutations in hepatocyte nuclear factor 1α (HNF1α) cause maturity onset diabetes of the young 3 (MODY3), an autosomal dominant form of diabetes. Deficiency of HNF1α in mice results in diabetes, hypercholesterolaemia and increased bile acid (BA) and cholesterol synthesis. Little is known about alterations in lipid metabolism in patients with MODY3. The aim of this study was to investigate whether MODY3 patients have altered cholesterol and BA synthesis and intestinal cholesterol absorption. A secondary aim was to investigate the effects of HNF1α mutations on the transcriptional regulation of BA metabolism. METHODS: Plasma biomarkers of BA and cholesterol synthesis and intestinal cholesterol absorption were measured in patients with MODY3 (n = 19) and in matched healthy control subjects (n = 15). Co-transfection experiments were performed with several promoters involved in BA metabolism along with expression vectors carrying the mutations found in these patients. RESULTS: Plasma analysis showed higher levels of BA synthesis in MODY3 patients. No differences were observed in cholesterol synthesis or intestinal cholesterol absorption. Co-transfection experiments showed that one of the mutations (P379A) increased the induction of the cholesterol 7α-hydroxylase promoter compared to HNF1α, without further differences in other studied promoters. By contrast, the other four mutations (L107I, T260M, P291fsinsC and R131Q) reduced the induction of the farnesoid X receptor promoter, which was followed by reduced repression of the small heterodimer partner promoter. In addition, these mutations also reduced the induction of the apical sodium-dependent bile salt transporter promoter. CONCLUSIONS: BA synthesis is increased in patients with MODY3 compared with control subjects. Mutations in HNF1α affect promoters involved in BA metabolism. This article is protected by copyright. All rights reserved
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 11Â mmol/L glucose and either 2% HS or 10% fetal bovine serum. After 3Â days, insulin and insulin-like growth factor-1 signaling were measured. At 7Â 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
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