Apolipoprotein B100 quality control and the regulation of hepatic very low density lipoprotein secretion

Apolipoprotein B （apoB） is the main protein component of very low density lipoprotein （VLDL） and is necessary for the assembly and secretion of these triglyceride （TG）-rich particles. Following release from the liver, VLDL is converted to low density lipoprotein （LDL） in the plasma and increased production of VLDL can therefore play a detrimental role in cardiovascular disease. Increasing evidence has helped to establish VLDL assembly as a target for the treatment of dyslipidemias. Multiple factors are involved in the folding of the apoB protein and the formation of a secretion-competent VLDL particle. Failed VLDL assembly can initiate quality control mechanisms in the hepatocyte that target apoB for degradation. ApoB is a substrate for endoplasmic reticulum associated degradation （ERAD） by the ubiquitin proteasome system and for autophagy. Efficient targeting and disposal of apoB is a regu- lated process that modulates VLDL secretion and partitioning of TG. Emerging evidence suggests that significant overlap exists between these degradative pathways. For example, the insulin-mediated targeting of apoB to autop- hagy and postprandial activation of the unfolded protein response （UPR） may employ the same cellular machinery and regulatory cues. Changes in the quality control mechanisms for apoB impact hepatic physiology and pathology states, including insulin resistance and fatty liver. Insulin signaling, lipid metabolism and the hepatic UPR may impact VLDL production, particularly during the postprandial state. In this review we summarize our current understanding of VLDL assembly, apoB degradation, quality control mechanisms and the role of these processes in liver physiology and in pathologic states.

Expression of Monocyte Chemoattractant Protein-1 in Monocytes and Effects of Native and Oxidized Very Low Density Lipoproteins

Monocyte chemoattractant protein-1(MCP-1), a potent chemoattractant, is thought to play an important role in migration of monocytes into atherosclerotic lesions. The present study was designed to investigate the capacity of human peripheral blood monocytes to express MCP-1 and effects of native very low density lipoprotein (VLDL) and oxidized VLDL(OX-VLDL) on the expression. The total RNA was extracted from cultured monocytes, which were exposed to VLDL and OX-VLDL, and the media conditioned by monocytes were collected. MCP-1 mRNA expression was examined by Northern blot analysis. MCP-1 protein in conditioned media was determined by using sandwich ELISA. The results showed that monocytes can express MCP-1 after a 24 h incubation at 37℃，and the expression was markedly increased by a exposure to OX-VLDL, whereas the expression was slightly increased when exposed to VLDL. It suggests that the capacity of monocytes to produce MCP-1 that recruits and activates circulating monocytes may be of considerable importance in atherogenesis, and oxidation of VLDL enhances its potential to promote atherogenesis.

Function and Significance of Very Low Density Lipoprotein Receptor Subtype Ⅱ

To explore the functions of very low density lipoprotein receptor (VLDL-R) subtype II in lipoprotein metabolism and foam cells formation, the recombinant plasmid with the two subtypes cDNA was constructed respectively, the ldl-A7 cell lines were transfected and two cell lines expressing VLDL-R were obtained: one stably expressing the VLDLR with the O-linked sugar region (type I VLDLR) and the other without the O-linked sugar region (type II VLDLR). In the study on binding of VLDLR to their nuclein labeled natural ligands (VLDL and β-VLDL), it was found that surface binding of 125I-VLDL or 125I-β-VLDL of ldl-A7 cells transfected with type I VLDLR recombinant (ldl-A7-VRI) was more higher than that of ldl-A7 cells transfected with type II VLDLR recombinant (ldl-A7-VRII). After being incubated with VLDL for different time, the contents of triglyceride and total cholesterol in cells were mensurated, and the formation of foam cells and accumulation of lipid in cells was observed by oil-red O staining. The results showed that the contents of triglyceride and total cholesterol in ldl-A7-VR I were much higher than those in ldl-A7-VR II, and ldl-A7-VR I could transform into foam cells notably. It was suggested that type I VLDLR binds with relative higher affinity to VLDL and β-VLDL, and internalizes much more lipoprotein into cells. As a result, we can conclude that type I VLDLR plays a more important role in lipoprotein metabolism and foam cells formation than type II VLDLR.

Proliferation of renal mesangial cells induced by very low density lipoprotein is mediated by p42/44 mitogen activated protein kinase

Background The plasma concentration of very low density lipoprotein （VLDL） is negatively correlated to renal function in glomerular diseases. Effects of VLDL on renal function have been partially attributed to the proliferation of mesangial cells. This study examined the potential role of the p42/44 mitogen activated protein kinase （MAPK） in mesangial cell proliferation induced by VLDL. Methods Mesangial cells were treated with VLDL at different concentrations or for different time. The cell cycle of the mesangial cells was analyzed by Xl-r assay and flow-cytometry; MAPK activity was also assayed. In some experiments, cells were treated with VLDL together with or without 0.1 pmol/L PD 98059. Results Ten to 500 μg/ml VLDL stimulated the proliferation of mesangial cells cultured in vitro in a concentration-dependent manner. The effect was associated with an increase in p42/44 MAPK activity. Increased proliferation of mesangial cells by VLDL was significantly attenuated by PD98059, a specific p42/44 MAPK inhibitor. Conclusion These results indicate that the p42/44 MAPK pathway is an important regulator of mesangial cell proliferation and of renal functions.

Effect of Divergent Selection for Plasma very Low Denstity Lipoprotein Concentration on Reproductive Performance in Broiler Chickens

The reproductive performance and abdominal fat of the high VLDL (H-VLDL) and low VLDL (L-VLDL) lines were studied after divergent selection for plasma very low density lipoprotein (VLDL) concentration in broiler chickens at 8 weeks of age. It was shown that the main reproductive traits were higher in L-VLDL line than H-VLDL line. The beneficial correlated responses of the reproductive traits were produced by the selection for plasma VLDL. 1) Plasma VLDL concentration was significantly correlated with abdominal fat (r=0.50, P<0.01), ahich was higher than that with plasma triglyceride concentration. There was a significant difference in abdominal fat between both lines but age increase or food restriction may decrease this difference. The H-VLDL line had about twice as much abdominal fat as had L-VLDL at 8 weeks of age, while its mean body weight was similar to that of the L-VLDL line from birtl to the end of laying. 2) Mortality from apparently stress-related causes by food restriction for H-VLDL line females began to increase from 15 weeks of age and was 21% (38/180) higher compared with 13% (23/180) for L-VLDL line females. 3) Age of 5% Lying for L-VLDL line was 3 days less than that of H-VLDL line, age of 50% Lying was 37 days less for L-VLDL line than H-VLDL line, and the age of laying peak was similiar in both lines. Rate of lay at peak in L-VLDL and H-VLDL lines were 70.3%, 66.4%, respectively. 4) Fertility rate in L-VLDL line was significantly higher than that of H-VLDL line at 27 weeks of age (P=0.01) and there was a little difference in fertility rates between H-VLDL and L-VLDL line during late laying period. There were no significant differences between H-VLDL and L-VLDL lines in of day-old healthy the numbers chicks and hatchabilities of fertile and total eggs wheres mean numbers of healthy day-old chicks per hen were more is L-VLDL line than in H-VLDL line. 5) Under food restriction, egg production was higher in L-VLDL line than in H-VLDL line. It should be emphasized that the rate and amount of egg production in L-VLDL line were both significant higher than in H-VLDL line during early laying period (from 23 to 43 weeks of age, P<0.01). The mean egg production of LF line hens was ll eggs more than that of HF line ones throughtout the laying period (from 23 to 62 weeks of age). 6) Eggs in L-VLDL line were slightly heavier than those in H-VLDL line. Feed conversion (FC) ratio for L-VLDL line was lower than that in H-VLDL line betwen 23 and 62 weeks of age (5.01 and 5.46, respectively), especially FC ratio of L-VLDL line was significantly lower than that of HF line during 23 and 43 weeks of age (P<0.01). Also FC efficiency of breeding hens was higher in L-VLDL line than in H-VLDL line.

Lipoprotein metabolism in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease （NAFLD）, an pathologies characterized by fatty accumulation in escalating health problem worldwide, covers a spectrum of hepatocytes in early stages, with potential progression to liver inflammation, fibrosis, and failure. A close, yet poorly understood link exists between NAFLD and dyslipidemia, a constellation of abnormalities in plasma lipoproteins including triglyceride-rich very low density lipoproteins. Apolipoproteins are a group of primarily liver-derived proteins found in serum lipoproteins; they not only play an extracellular role in lipid transport between vital organs through circulation, but also play an important intracellu- lar role in hepatic lipoprotein assembly and secretion. The liver functions as the central hub for lipoprotein metab- olism, as it dictates lipoprotein production and to a significant extent modulates lipoprotein clearance. Lipoprotein metabolism is an integral component of hepatocellular lipid homeostasis and is implicated in the pathogenesis, potential diagnosis, and treatment of NAFLD.

New predictors of in-stent restenosis in patients with diabetes mellitus undergoing percutaneous coronary intervention with drug-eluting stent

Background Percutaneous coronary intervention （PCI） had become the major therapeutic procedure for coronary artery disease （CAD）, but the high rate of in-stent restenosis （ISR） still remained an unsolved clinical problem in clinical practice. Increasing evidences suggested that diabetes mellitus （DM） was a major risk factor for ISR, but the risk predictors of ISR in CAD patients with DM had not been well characterized. The aim of this study was to investigate the clinical and angiographic characteristic predictors significantly associated with the occurrence of ISR in diabetic patients following coronary stenting with drug-eluting stent （DES）. Methods A total of 920 patients with diabetes who diagnosed CAD and underwent coronary DES implantation at Beijing Anzhen Hospital in China were consecutively enrolled from January 2012 to December 2012. Of these, 440 patients underwent the second angiography within ≥ 6 months due to the progression of treated target lesions. Finally, 368 of these patients who met the inclusion and exclusion criteria were followed up by angiography after baseline PCI. According to whether ISR was detected at follow-up angiography, patients were divided into the ISR group （n = 74） and the non-ISR group （n = 294）. The independent predictors of ISR in patients with DM were explored by multivariate Cox＇s proportional hazards regression models. Results A total of 368 patients （260 women and 108 men） with a mean ages of 58.71 ± 10.25 years were finally enrolled in this study. Of these, ISR occurred in 74/368 diabetic patients （20.11%） by follow-up angiography. Univariate analysis showed that most baseline characteristics of the ISR and non-ISR group were similar. Patients in the ISR group had significantly higher serum very low density lipoprotein cholesterol （VLDL-C）, triglyceride （TG） and uric acid （UA） levels, more numbers of target vessel lesions, higher prevalence of multi-vessel disease, higher SYNTAX score, higher rate of previous but lower rate of drinking compared with patients in the non-ISR group. The independent predictors of ISR in patients with DM after DES implantation included VLDL-C （HR = 1.85, 95% CI： 1.24-2.77, P = 0.002）, UA （per 50 μmol/L increments, HR = 1.19, 95% CI： 1.05 1.34, P = 0.006）, SYNTAX score （per 5 increments, HR = 1.34, 95% CI： 1.03-1.74, P = 0.031） and the history ofPCI （HR = 3.43, 95% CI： 1.57-7.80, P = 0.003） by the multivariate Cox＇s proportional hazards regression analysis. Conclusions The increased serum VLDL-C and UA level, higher SYNTAX score and the history of previous PCI were independent predictors of ISR in patients with DM after coronary DES implantation. It provided new evidence for physi- cians to take measures to lower the risk oflSR for the better management of diabetic patients after PCI.

Role of VLDL Receptor in the Process of Foam Cell Formation

The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, β VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (LRP) and VLDLR were detected by semi quantitative RT PCR. The results demonstrated that VLDL, β VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or β VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl A7 VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.

Effect of Insulin on the Differential Expression of VLDL Receptor Isoforms of SGC7901 Cell and Its Biological Implication

This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication.In vitro, moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively.The results showed that, at certain time interval, insulin could down-regulate expression of type Ⅰ VLDLR and up-regulate the expression of type Ⅱ VLDLR in SGC7901 cells, at both protein and RNA level.We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.

Type Ⅱ VLDLR promotes cell migration by up-regulation of VEGF, MMP2 and MMP7 in breast cancer cells

Objective： Very low density lipoprotein receptor （VLDLR） has been considered as a multiple function receptor due to binding numerous ligands, causing endocytosis and regulating cellular signaling. Our group previously reported that type II VLDLR overexpression in breast cancer tissues. The purpose of this study is to characterize type II VLDLR activities during cell migration using breast cancer cell lines. Methods： Western blotting was used to test protein expression. Cell migration was analyzed by Scratch wound assay. The mRNA expression was tested by realtime-PCR. Reporter assay was to test the transcription activity. Results： Scratch wound and Report assay indicated up-regulated VLDLR II expression promotes cell migration via activating Wnt/β-catenin pathway. The target genes such as VEGF, MMP2 and MMP7 were upregulated in VLDLR II overexpressed cells. On the contrary, cells treated with TFPI had an inhibition effect of cell migration response to down-regulation of VLDLR Ⅱ. Conclusion： Type Ⅱ VLDLR conferred a migration and invasion advantage by activating Wnt/β- catenin pathway, then up-regulating VEGF, MMP2 and MMP7 in breast cancer cells.

Rapid Isolation of Large Amount of Plasma VLDL and LDL by a Two Step Ultracentrifugation

A rapid method to obtain large amount of VLDL and LDL by ultracentrifugation is described. The mixture of VLDL and LDL was isolated and concentrated from plasma by an ultracentrifugation at 265 000 g for 2h. VLDL and LDL were separated and purified by a further ultracentrifugation at 265 000g for 3h.This method combines the advantages of both sequential flotation ultracentri-fugation and density gradient ultracentrifugation. It can process a large volume of plasma in a short time. The purity of isolated VLDL and LDL was confirmed by the lipoprotein electrophoresis on agarose gel and PAGE and by the apolipoprotein electrophoresis on SDS-PAGE. This rapid.economical method is of great value in practical application.