apoB Secretion In HepG2 Cells Research Articles

Screening of soy protein-derived hypotriglyceridemic di-peptides in vitro and in vivo

BackgroundSoy protein and soy peptides have attracted considerable attention because of their potentially beneficial biological properties, including antihypertensive, anticarcinogenic, and hypolipidemic effects. Although soy protein isolate contains several bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptide sequences are responsible for the triglyceride (TG)-lowering effects. In the present study, we investigated the effects of soy protein-derived peptides on lipid metabolism, especially TG metabolism, in HepG2 cells and obese Otsuka Long-Evans Tokushima fatty (OLETF) rats.ResultsIn the first experiment, we found that soy crude peptide (SCP)-LD3, which was prepared by hydrolyze of soy protein isolate with endo-type protease, showed hypolipidemic effects in HepG2 cells and OLETF rats. In the second experiment, we found that hydrophilic fraction, separated from SCP-LD3 with hydrophobic synthetic absorbent, revealed lipid-lowering effects in HepG2 cells and OLETF rats. In the third experiment, we found that Fraction-C (Frc-C) peptides, fractionated from hydrophilic peptides by gel permeation chromatography-high performance liquid chromatography, significantly reduced TG synthesis and apolipoprotein B (apoB) secretion in HepG2 cells. In the fourth experiment, we found that the fraction with 0.1% trifluoroacetic acid, isolated from Frc-C peptides by octadecylsilyl column chromatography, showed hypolipidemic effects in HepG2 cells. In the final experiment, we found that 3 di-peptides, Lys-Ala, Val-Lys, and Ser-Tyr, reduced TG synthesis, and Ser-Tyr additionally reduced apoB secretion in HepG2 cells.ConclusionNovel active peptides with TG-lowering effects from soy protein have been isolated.

Lignophenols Decrease Oleate‐Induced Apolipoprotein‐B Secretion in HepG2 Cells

Lignin is one of the components in the plant cell wall, after cellulose, making up 20-30% of the global plant biomass. Lignophenols (LPs) are derivatives of lignin with high phenolic function and antioxidant properties. However, their medicinal property is not well characterised. Apolipoprotein-B (apo-B) is an essential component in very low-density lipoprotein, and high levels of serum apolipoprotein-B (apo-B) are a major factor for coronary heart disease. In this study, we examined the effect of lignophenols on apo-B secretion in HepG2 cells. HepG2 cells were treated with varying concentrations of LPs and 0.8 mm sodium oleate. LPs decreased oleate-induced apo-B secretion in a dose-dependent manner. LPs also decreased oleate-induced microsomal triglyceride transfer protein (MTTP) mRNA expression and cellular total cholesterol, suggesting that lipid bioavailability and lipidation of lipoprotein are likely involved in the decreased secretion of apo-B. Furthermore, LPs decreased oleate-induced mature sterol regulatory element binding protein 2 (SREBP-2), a transcription factor that activates cholesterol biosynthesis. This is the first study to show that LPs can decrease oleate-induced apo-B secretion in HepG2 cells. The modulations of MTTP mRNA expression, cellular total cholesterol metabolism and mature SREBP-2 expression may be important factors in the regulation of apo-B secretion by LPs.

Molecular structures of citrus polymethoxylated flavonoids (PMFs) determine their inhibitory effect on apoB secretion in HepG2 cells

BackgroundCitrus PMFs, such as nobiletin and tangeretin, have been shown to modulate cholesterol (CH) homeostasis in vitro and in vivo, although the underlying mechanism of action has not yet been fully elucidated. We explored the structure‐activity relationship of PMFs regarding apoB secretion.MethodsHepG2 cells were incubated with 20 natural and synthetic PMFs (0–100 μM), while lovastatin (3 μM) and mevalonate (10 mM) were used as controls to modulate cellular CH‐metabolism. Apolipoprotein B (apoB) secretion was determined by using ELISA and 14C‐acetate and 14C‐glycerol were used to monitor cellular CH and triglyceride (TG) synthesis, respectively. LDL‐receptor expression was measured by ELISA and its activity by monitoring fluorescent‐labelled LDL uptake.ResultsCitrus PMFs and their natural 4′‐demethylated metabolites most strongly reduced apoB secretion (IC50 10–60μM). The most active PMFs have a fully methoxylated A‐ring (see structure), while the methoxylation of the B‐ring seemed less important for apoB‐secretion. In addition, PMFs with a fully methoxylated A‐ring more strongly inhibited TG‐ and CH‐synthesis compared to other citrus flavonoids. PMFs did not up‐regulate LDL‐receptor expression or activity.ConclusionCitrus PMFs (nobiletin, tangeretin and their metabolites) with a fully methoxylated Aring have the strongest inhibitory effect on apoB‐secretion in HepG2 cells.

Pharmacological characterization in vitro of EP2306 and EP2302, potent inhibitors of squalene synthase and lipid biosynthesis

We investigated the effects of EP2306 and EP2302, two novel 2-biphenylmorpholine derivatives, on squalene synthase activity in rabbit and human liver microsomes, lipid biosynthesis, low-density lipoprotein (LDL) receptor expression and LDL protein uptake as well as apoB secretion in HepG2 cells. Both EP2306 and EP2302 inhibited squalene synthase activity dose-dependently. In rabbit liver microsomes, the IC 50 values were 33 μM for EP2306 and 0.6 μM for EP2302 whereas in human liver microsomes, they were 63 μM for EP2306 and 1 μM for EP2302. Both EP2300 compounds inhibited cholesterol production by HepG2 cells dose dependently with IC 50 values of 13.3 μM for EP2306 and 3 μM for EP2302. Furthermore, both EP2300 compounds and simvastatin significantly reduced triglyceride synthesis and apoB secretion and increased LDL receptor expression and LDL uptake in HepG2 cells. In summary, we have shown that EP2300 compounds are potent inhibitors of squalene synthase activity in rabbit and human liver microsomes and also they are effective inhibitors of cholesterol and triglyceride biosynthesis in HepG2 cells. These results suggest that EP2306 and EP2302 might prove to be useful for lipid-lowering and treatment of atherosclerosis in vivo.

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-β), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-β may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-β, as well as TGF-α, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-β significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-β were dose-dependent, i.e. 1 ng/ml of TGF-β decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-β decreased apoM mRNA levels more than 65%. The effect of TGF-β on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-β where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-β on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-α, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-β. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-β), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-β may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-β, as well as TGF-α, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-β significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-β were dose-dependent, i.e. 1 ng/ml of TGF-β decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-β decreased apoM mRNA levels more than 65%. The effect of TGF-β on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-β where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-β on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-α, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-β. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.

Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.

Microsomal triglyceride transfer protein (MTP) regulation in HepG2 cells: insulin negatively regulates MTP gene expression.

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apoB-containing lipoproteins. In this study, four factors that modulate lipid and lipoprotein metabolism were tested for their ability to regulate MTP levels in HepG2 cells. Of the factors tested, only insulin (> or = 10(-9) M), and high concentrations of glucose (> 30 mM) were found to decrease MTP large subunit mRNA levels. Oleate and glucagon had no effect on MTP mRNA levels. The insulin effect was dose- and time-dependent and was mediated through the insulin receptor. In addition, insulin also decreased protein disulfide isomerase (the small subunit of MTP) mRNA levels, although to a lesser extent. Due to the slow turnover rate of MTP (t1/2 = 4.4 days), short-term insulin treatment (24 h) did not change MTP activity levels, indicating that the regulation of MTP mRNA levels by insulin is unrelated to insulin's acute inhibition of apoB secretion in HepG2 cells. In summary, MTP mRNA levels are acutely regulated by insulin in HepG2 cells; however, sustained changes in MTP mRNA levels would be required to affect MTP protein levels.

Exogenous VLDL stimulates apolipoprotein B secretion from HepG2 cells by both pre- and post-translational mechanisms.

Previous studies have suggested that oleic acid (OA), by increasing availability of triglyceride (TG) and/or cholesteryl ester (CE), increases the secretion of apolipoprotein B (apoB) from HepG2 cells. The present studies were conducted to determine the effect of exogenous very low density lipoproteins (VLDL), which can provide TG and cholesterol to cells and be returned to the liver after secretion, in the regulation of hepatic apoB secretion. Addition of exogenous VLDL (50 micrograms protein/ml) to culture media was found to significantly stimulate apoB secretion from HepG2 cells. This effect was observed consistently with VLDL isolated from either normal or hyperlipidemic subjects. The effects of addition of VLDL were compared to that of OA, which was previously shown to stimulate apoB secretion by a posttranslational mechanism. VLDL appeared to increase apoB secretion by two mechanisms; the dominant one being posttranslational. Thus, VLDL protected newly synthesized apoB from rapid intracellular degradation in a manner similar to OA. Although treatment with VLDL increased the mass of both TG and CE, 3-fold and 2.6-fold, respectively, it appeared that the increase in TG was the critical factor associated with increased apoB secretion. Triacsin D, which is a potent inhibitor of TG synthesis, significantly inhibited the VLDL-induced stimulation of apoB secretion. Inhibition of apoB secretion by Triacsin D was associated with the loss of the protective effect of VLDL on newly synthesized apoB. In addition to its posttranslational effects, exogenous VLDL also regulated apoB secretion at the pretranslational level. Thus, we also observed that VLDL treatment consistently increased synthesis of apoB protein by 20-30%, an effect that is not observed after treatment of HepG2 cells with OA. A sensitive solution hybridization/RNase protection assay indicated that the increased apoB synthesis was associated with a 20-30% increase in apoB mRNA in VLDL-treated HepG2 cells. OA treatment had no effect on apoB mRNA levels. We conclude that VLDL treatment stimulates apoB secretion in HepG2 cells primarily by supplying fatty acids for TG synthesis. However, 20-30% of the stimulatory effect was due to a second mechanism that appeared to be pretranslational. Based on studies with OA, it appears that some component of VLDL other than TG-derived fatty acid was responsible for this effect.

Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells.

A major theme of this review is that apoB secretion is regulated post-translationally, and that apoB secretion reacts rapidly to the current state of lipid metabolism in the cell. Therefore, as discussed by Fungwe et al. (122), the metabolism of triglyceride and of cholesteryl ester, in so far as both can be used as core lipids for apoB-containing LPs, are inextricably linked, and the shortage of one or both of these lipids could, by "allowing" increased intracellular degradation in the ER, inhibit the secretion of apoB. Another theme in this review is that the regulation of apoB secretion may be quite different in rat hepatocytes compared to cultured cells (HepG2) used as a model for human hepatocytes. Exogenous fatty acids appear to modulate the rate of apoB secretion in HepG2 cells, whereas they have only minimal effects on apoB secretion in rat hepatocytes or liver. Increased dietary cholesterol, on the other hand, appears to be an important modulator of apoB secretion in rats, but the evidence for effects of cholesterol on apoB secretion in HepG2 cells is less convincing. Finally, because HepG2 cells are an immortalized cell line, there could be many differences between these cells and human hepatocytes in vivo. Therefore, many of the results obtained with HepG2 cells should be corroborated in primary cultures of human hepatocytes. However, investigators utilizing primary human hepatocytes should be sure that the culture conditions are adequate to maintain the continued transcription of liver specific genes and to prevent the dedifferentiation of these cells in culture (85, 86).