CideB Protein Is Required for the Biogenesis of Very Low Density Lipoprotein (VLDL) Transport Vesicle

Nascent very low density lipoprotein (VLDL) exits the endoplasmic reticulum (ER) in a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Similar to protein transport vesicles (PTVs), VTVs require coat complex II (COPII) proteins for their biogenesis from the ER membranes. Because the size of the VTV is large, we hypothesized that protein(s) in addition to COPII components might be required for VTV biogenesis. Our proteomic analysis, supported by Western blotting data, shows that a 26-kDa protein, CideB, is present in the VTV but not in other ER-derived vesicles such as PTV and pre-chylomicron transport vesicle. Western blotting and immunoelectron microscopy analyses suggest that CideB is concentrated in the VTV. Our co-immunoprecipitation data revealed that CideB specifically interacts with VLDL structural protein, apolipoprotein B100 (apoB100), but not with albumin, a PTV cargo protein. Confocal microscopic data indicate that CideB co-localizes with apoB100 in the ER. Additionally, CideB interacts with COPII components, Sar1 and Sec24. To investigate the role of CideB in VTV biogenesis, we performed an in vitro ER budding assay. We show that the blocking of CideB inhibits VTV budding, indicating a direct requirement of CideB in VTV formation. To confirm our findings, we knocked down CideB in primary hepatocytes and isolated ER and cytosol to examine whether they support VTV budding. Our data suggest that CideB knockdown significantly reduces VTV biogenesis. These findings suggest that CideB forms an intricate COPII coat and regulates the VTV biogenesis.     

Optimized negative-staining electron microscopy for lipoprotein studies

Background

Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents.

Scope of review

The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed.

Major conclusions

The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1 nm, but rarely better than 1 nm) method which has been used to discover the mechanics of a small protein, 53 kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14 Å-resolution IgG antibody three-dimensional map.

General significance

It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures.     

Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia

Monogenic hypobetalipoproteinemias include three disorders: abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) with recessive transmission and familial hypobetalipoproteinemia (FHBL) with dominant transmission. We investigated three unrelated Tunisian children born from consanguineous marriages, presenting hypobetalipoproteinemia associated with chronic diarrhea and retarded growth. Proband HBL-108 had a moderate hypobetalipoproteinemia, apparently transmitted as dominant trait, suggesting the diagnosis of FHBL. However, she had no mutations in FHBL candidate genes (APOB, PCSK9 and ANGPTL3). The analysis of MTTP gene was also negative, whereas SAR1B gene resequencing showed that the patient was homozygous for a novel mutation (c.184G>A), resulting in an amino acid substitution (p.Glu62Lys), located in a conserved region of Sar1b protein. In the HBL-103 and HBL-148 probands, the severity of hypobetalipoproteinemia and its recessive transmission suggested the diagnosis of ABL. The MTTP gene resequencing showed that probands HBL-103 and HBL-148 were homozygous for a nucleotide substitution in the donor splice site of intron 9 (c.1236+2T>G) and intron 16 (c.2342+1G>A) respectively. Both mutations were predicted in silico to abolish the function of the splice site. In vitro functional assay with splicing mutation reporter MTTP minigenes showed that the intron 9 mutation caused the skipping of exon 9, while the intron 16 mutation caused a partial retention of this intron in the mature mRNA. The predicted translation products of these mRNAs are non-functional truncated proteins.     

Association of leptin receptor gene Q223R polymorphism on lipid profiles in comparison study between obese and non-obese subjects

Objectives

Leptin is a hormone secreted from adipocytes. It regulates metabolism and energy homeostasis through the leptin receptor (LEPR) which is localized centrally in hypothalamus as well as in peripheral tissues. The aim of this study was to investigate the association of leptin receptor gene Q223R polymorphism on obesity in association with body mass index (BMI), lipid parameters, plasma leptin levels and homeostasis model assessment of insulin resistance (HOMA-IR).

Design and methods

The study included 110 obese and 90 non-obese subjects. The LEPR Q223R polymorphism was determined by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Plasma leptin levels, serum lipid and antropometric parameters were measured.

Results

No association was found between LEPR gene Q223R polymorphism and BMI in both study and control groups. Strikingly study group with non-obese subjects and with the RR genotype (homozygous mutant) had significantly higher serum total cholesterol (p < 0.001) and low density lipoprotein cholesterol (LDL-cholesterol) levels (p < 0.05) than QR (heterozygous) and QQ (wild type) genotypes. In obese group, subjects with the RR genotypes had significantly higher triglycerides (p < 0.05) levels, waist (p < 0.05) and hip circumferences (p < 0.001) than the QQ and QR genotypes.

Conclusions

Our results suggest that the LEPR gene Q223R polymorphism has an association with waist and hip circumferences in obese group but no direct association with obesity although there is a significant influence on lipid profile both in obese and non-obese subjects.     

Endoplasmic reticulum-localized hepatic lipase decreases triacylglycerol storage and VLDL secretion

Hepatic triacylglycerol levels are governed through synthesis, degradation and export of this lipid. Here we demonstrate that enforced expression of hepatic lipase in the endoplasmic reticulum in McArdle RH7777 hepatocytes resulted in a significant decrease in the incorporation of fatty acids into cellular triacylglycerol and cholesteryl ester accompanied by attenuation of secretion of apolipoprotein B-containing lipoproteins. Hepatic lipase-mediated depletion of intracellular lipid storage increased the expression of peroxisome proliferator-activated receptor α and its target genes and augmented oxidation of fatty acids. These data show that 1) hepatic lipase is active in the endoplasmic reticulum and 2) intracellular hepatic lipase modulates cellular lipid metabolism and lipoprotein secretion.     

Apolipoprotein E: From lipid transport to neurobiology

Apolipoprotein (apo) E has a storied history as a lipid transport protein. The integral association between cholesterol homeostasis and lipoprotein clearance from circulation are intimately related to apoE’s function as a ligand for cell-surface receptors of the low-density lipoprotein receptor family. The receptor binding properties of apoE are strongly influenced by isoform specific amino acid differences as well as the lipidation state of the protein. As understanding of apoE as a structural component of circulating plasma lipoproteins has evolved, exciting developments in neurobiology have revitalized interest in apoE. The strong and enduring correlation between the apoE4 isoform and age of onset and increased risk of Alzheimer’s disease has catapulted apoE to the forefront of neurobiology. Using genetic tools generated for study of apoE lipoprotein metabolism, transgenic “knock-in” and gene-disrupted mice are now favored models for study of its role in a variety of neurodegenerative diseases. Key structural knowledge of apoE and isoform-specific differences is driving research activity designed to elucidate how a single amino acid change can manifest such profoundly significant pathological consequences. This review describes apoE through a lens of structure-based knowledge that leads to hypotheses that attempt to explain the functions of apoE and isoform-specific effects relating to disease mechanism.     

Differences in preterm and term milk fatty acid compositions may be caused by the different hormonal milieu of early parturition

Introduction

The hormonal milieus of pregnancy and lactation are driving forces of nutrient fluxes supporting infant growth and development. The decrease of insulin sensitivity with compensatory hyperinsulinemia with advancing gestation, causes adipose tissue lipolysis and hepatic de novo lipogenesis (DNL).

Subjects and methods

We compared fatty acid (FA) contents and FA-indices for enzyme activities between preterm (28–36 weeks) and term (37–42) milks, and between colostrum (2–5 days), transitional (6–15) and mature (16–56) milks. We interpreted FA differences between preterm and term milks, and their changes with lactation, in terms of the well known decrease of insulin sensitivity during gestation and its subsequent postpartum restoration, respectively.

Results

Compared with term colostrum, preterm colostrum contained higher indices of DNL in the breast (DNL-breast) and medium chain saturated-FA (MCSAFA), and lower DNL-liver and monounsaturated-FA (MUFA). Preterm milk also had higher docosahexaenoic acid (DHA) in colostrum and transitional milk and higher arachidonic acid (AA) in mature milk. Most preterm-term differences vanished with advancing lactation. In both preterm and term milks, DNL-breast and MCSAFA increased with advancing lactation, while DNL-liver, MUFA, long chain SAFA and AA decreased. DHA decreased in term milk. MUFA was inversely related to MCSAFA in all samples, correlated inversely with PUFA in colostrum and transitional milks, but positively in mature milk. MCSAFA correlated inversely with PUFA in mature milk.

Conclusion

Higher maternal insulin sensitivity at preterm birth may be the cause of lower MUFA (a proxy for DNL-liver) and higher MCSAFA (a proxy for DNL-breast) in preterm colostrum, compared with term colostrum. Restoring insulin sensitivity after delivery may be an important driving force for milk FA-changes in early lactation.     

Lipid metabolism in mammalian tissues and its control by retinoic acid

Evidence has accumulated that specific retinoids impact on developmental and biochemical processes influencing mammalian adiposity including adipogenesis, lipogenesis, adaptive thermogenesis, lipolysis and fatty acid oxidation in tissues. Treatment with retinoic acid, in particular, has been shown to reduce body fat and improve insulin sensitivity in lean and obese rodents by enhancing fat mobilization and energy utilization systemically, in tissues including brown and white adipose tissues, skeletal muscle and the liver. Nevertheless, controversial data have been reported, particularly regarding retinoids' effects on hepatic lipid and lipoprotein metabolism and blood lipid profile. Moreover, the molecular mechanisms underlying retinoid effects on lipid metabolism are complex and remain incompletely understood. Here, we present a brief overview of mammalian lipid metabolism and its control, introduce mechanisms through which retinoids can impact on lipid metabolism, and review reported activities of retinoids on different aspects of lipid metabolism in key tissues, focusing on retinoic acid. Possible implications of this knowledge in the context of the management of obesity and the metabolic syndrome are also addressed. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Association of MTHFR and PPARγ2 gene polymorphisms in relation to type 2 diabetes mellitus cases among north Indian population

Background

Type 2 diabetes mellitus is a multifactorial and polygenic disease, which is considered as a major life threatening problem all over the world. There has been a worldwide effort in the identification of susceptibility genes for type 2 diabetes mellitus and its complications. At present, adequate data is not available dealing with MTHFR (rs1801133) and PPARγ2 (rs1801282) gene polymorphisms and its association with type 2 diabetes mellitus cases among north Indian populations. Thus, we conceived the need for further studies to investigate MTHFR and PPARγ2 gene polymorphisms and their susceptibility to type 2 diabetes mellitus in north Indian population.

Materials and methods

In this study, a total 175 subjects including 87 type 2 diabetes mellitus cases and 88 controls were enrolled. MTHFR and PPARγ2 gene polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP).

Results

The MTHFR gene CC, CT, TT genotype frequencies obtained were 40%, 43%, and 17% in type 2 diabetes mellitus cases and 56%, 29%, and 15% in healthy controls respectively. The OR for CC was 0.54 (95%CI 0.29–0.98, P = 0.041, χ2 = 4.18, power = 0.98), for CT 1.76 (95%CI 0.94–3.30, P = 0.07, χ2 = 3.2, power = 0.96), and for TT 1.2 (95%CI 0.53–2.70, P = 0.66, χ2 = 0.198, power = 0.76). The PPARγ2 gene GG CG, CC genotype frequencies obtained were 28%, 41%, and 31% in cases and 40%, 39%, and 21% in healthy controls respectively. OR for GG was 0.58 (95%CI 0.30–1.09, P = 0.08, χ2 = 2.9, power = 0.96), for CG 1.12 (95%CI 0.61–2.05, P = 0.71, χ2 = 0.137, power = 0.778), and for CC 1.63 (95%CI 0.82–3.23, P = 0.156, χ2 = 2.01, power = 0.92).

Conclusion

It might be recommended that MTHFR CC genotype seems to be a good marker for the early identification of population at risk of type 2 diabetes mellitus. While we have detected significant difference in allelic frequencies of PPARγ2 C (Proline) and G (Alanine), but at genotypic level significant difference was not detected in this case–control study. Further study with larger groups may be required to validate the study.