Ahmed Bakillah

Amino Acids 430–570 in Apolipoprotein B Are Critical for Its Binding to Microsomal Triglyceride Transfer Protein

Several studies have demonstrated protein-protein interactions between microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). However, the binding sites involved in these interactions have not been elucidated. To identify an MTP binding site in apoB, we have expressed several apoB sequences as fusion proteins with the eight-amino acid FLAG peptide. The chimeras were transiently expressed in COS cells, and conditioned media were used to study the binding of these sequences to either immobilized or soluble MTP. A polypeptide containing amino acids 270–570 (B:270–570), but not 1–300, bound to MTP. AGI-S17, an antagonist of apoB-MTP binding, inhibited the binding of B:270–570 to MTP but not to M2, a monoclonal antibody that recognizes the FLAG peptide. These data indicated that B:270–570 contains an MTP binding site. Next, sequences within 270–570 were subjected to C-terminal truncations at natural proline residues. B:270–509 bound less efficiently than B:270–570, whereas, B:270–430 and other shorter chimeras did not bind to MTP. Furthermore, truncations at amino acids 502 and 509 decreased MTP binding by 73 and 42%, respectively. These data indicate that B:430–570 in the α1-globular domain of apoB plays a crucial role in MTP binding and presumably in the initiation and maturation of apoB-containing lipoproteins.

Nutrition & Metabolism: an impressive performance since inception

Nutrition & Metabolism was co-founded by Drs. Feinman and Hussain in 2004 as an open access, peer-reviewed, online journal to keep readers up-to-date on recent developments in important areas of nutrition, exercise physiology, clinical investigations, and molecular and cellular biochemistry of metabolism. The journal is dedicated to expedite rapid publication of manuscripts that significantly advance the knowledge of nutrition and metabolism related to exercise physiology, diabetes, obesity, lipidemias, metabolic syndrome, atherosclerosis, cancer etc. Nutrition & Metabolism also publishes manuscripts pertaining to biochemistry of metabolism, cell signaling, molecular and cellular biology of nutrients, nutrient gene interactions and other areas that have implications for human nutrition and medicine. Current, but not exclusive, interests are metabolic effects of diet composition, interactions of macronutrients, effect of nutrients on gene expression, metabolic control and compartment models, nutritional effect of hormones, and genomic analysis of dietary phenomena. Since, its inception, the journal has seen a steady growth in the number of papers submitted (Figure ​(Figure1).1). However, the number of papers published remained relatively small. This lead to a steady decline in the percentage of papers accepted for publication. Currently, Nutrition & Metabolism publishes approximately 36% of the submitted articles. The journal received an impressive first impact factor of 3.00 in 2009 indicating that published papers were highly cited soon after their publication (Figure ​(Figure2).2). It continues to maintain its impact factor. Furthermore, several of the published papers have been highly accessed. Figure 1 Number of articles submitted and published in the Nutrition & Metabolism (Lond) since inception. Figure 2 Impact Factor of Nutrition & Metabolism. Nutrition & Metabolism publishes two main types of articles; original research articles and brief communications, although various other forms of articles such as methods, perspectives, and letters are also considered for publication. All articles must report results of well-designed research investigations that try to answer a specific hypothesis in depth using multiple approaches. Usually, the published papers provide mechanistic insights into metabolic and signaling pathways as well as clinical outcomes related to various macro- and micro-nutrients, and compounds of therapeutic potential. Manuscripts dealing with therapeutic lifestyle changes involving exercise, macronutrient content etc. are also published to provide a forum for critical, scientific evaluation of these practices or recommendations. None of the scientific research papers are commissioned for publications. Authors elect to submit their papers for publications. Similarly, most of the reviews are submitted by investigators as they think Nutrition & Metabolism is the best forum for the publication of their reviews. Less than 1% of the published reviews were commissioned. The majority of solicited reviews were from editorial board members with the intention of providing guidelines to the prospective authors about the expertise of the editorial board and interest of the journal. These invited reviews were also subjected to extensive peer-review process in order to improve their quality. Hence, both invited and submitted reviews undergo extensive peer-review process. Every paper undergoes a two-tier, quality control, evaluation process. First, Editor-in-Chief, Managing Editor, or an Associate Editors (http://www.nutritionandmetabolism.com/about/edboard) determines the suitability of the manuscript for publication in Nutrition & Metabolism. Articles of low perceived importance or poor quality are immediately returned to authors with no further review. If the manuscript is deemed suitable for further evaluation, it is sent for peer-review. The first priority is given to anonymous, unbiased peer-reviewers suggested by the computer based on their publications related to the subject of the article. In addition, we solicit opinion from at least one member of the editorial board. Further, consideration is also given to the reviewers recommended by the authors. Hence, every paper undergoes a stringent peer review process to evaluate objectively the quality, originality, and scientific merit of the papers. After publication, the papers are open to comments by the scientific community. The comments are first reviewed by the editors for their validity and correctness and then sent to the authors for their response. Both comments and responses are published online and have open access. Besides the dedication and expertise of the Editorial Board members and reviewers, the success of the journal depends on the valuable contributions from investigators. Hence, we solicit comments from all the authors about the integrity and timeliness of the review process, authenticity, clarity and appropriateness of the reviews, and any other comments that would make Nutrition & Metabolism your first choice to disseminate your precious data. Nutrition & Metabolism is published by BioMed Central, part of Springer Science+Business Media. BioMed central is committed to ensuring open access peer-reviewed biomedical research. This means that published material is freely and universally accessible online. It is archived in at least one internationally recognized free access repository, and its authors retain copyright, allowing anyone to reproduce or disseminate articles, according to the BioMed Central copyright and license agreement. Nutrition & Metabolism’s articles are archived in PubMed Central, the US National Library of Medicine’s full-text repository of life science literature, and also at INIST in France and in e-Depot, the National Library of the Netherlands’ digital archive of all electronic publications. The journal is also participating in the British Library’s e-journals pilot project, and plans to deposit copies of all articles with the British Library. One of the major goals of Nutrition & Metabolism is the identification and publication of timely and important contributions to the field of nutrition & metabolism. We invite potential authors to visit the journal homepage http://www.nutritionandmetabolism.com, and submit their new and exciting papers online. Ahmed Bakillah, Managing Editor M. Mahmood Hussain, Editor-In-Chief January 1st, 2013

New approaches to target microsomal triglyceride transfer protein

Purpose of review Microsomal triglyceride transfer protein (MTP), a chaperone for the biosynthesis of apolipoprotein B lipoproteins and CD1d, is a therapeutic candidate to decrease plasma lipids and to diminish inflammation. MTP inhibition increases plasma transaminases and tissue lipids, and therefore new approaches are needed to avoid them. Recent findings Inositol requiring enzyme1β has been identified as a novel intestine-specific regulator of MTP. A new function of MTP in cholesterol ester biosynthesis has been reported. The importance of the phospholipid transfer activity of MTP in the lipidation of apolipoprotein B and CD1d has been indicated. Diurnal variations in MTP expression and its induction by food availability have been observed. On the basis of these and other findings, we propose that upregulation of inositol requiring enzyme 1β, a combined reduction of cellular free cholesterol or triglyceride or both and MTP activity, specific inhibition of phospholipid or triglyceride transfer activities, and targeting of apolipoprotein B–MTP protein–protein interactions might be pursued to avoid some of the side effects associated with the inhibition of triglyceride transfer activity of MTP. We further speculate that short-lived MTP antagonists may be useful in controlling plasma and tissue lipids and in avoiding steatosis. Summary We have highlighted the importance of addressing the causal relationship between MTP inhibition and aberrant elevations in plasma liver enzymes. The proposed approaches may show that MTP targeting is a viable approach to lower plasma lipids.

MEASUREMENT OF APOLIPOPROTEIN B IN VARIOUS CELL LINES : CORRELATION BETWEEN INTRACELLULAR LEVELS AND RATES OF SECRETION

We have standardized simple but sensitive enzyme-linked immunoassays to understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibodies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts of low density lipoproteins to obtain a standard curve. Conditioned media were added to other wells in parallel, and the amount of apoB was quantitated from a linear regression curve. To standardize conditions for the measurement of intracellular apoB, cells were homogenized and solubilized with different concentrations of taurocholate. We found that 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2, Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular levels of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were positively correlated (r2=0.81, P=0.0009) in HepG2 cells. Furthermore, a positive correlation (r2=0.88, P<0.0001) was found between intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and apoB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but not the lipid-free apoB, was tightly controlled.

Mice subjected to aP2-Cre mediated ablation of microsomal triglyceride transfer protein are resistant to high fat diet induced obesity

BackgroundMicrosomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. MTP has been shown on the surface of lipid droplets of adipocytes; however its function in adipose tissue is not well defined. We hypothesized that MTP may play critical role in adipose lipid droplet formation and expansion.MethodsPlasmids mediated overexpression and siRNA mediated knockdown of Mttp gene were performed in 3T3-L1 pre-adipocytes to evaluate the effects of MTP on cell differentiation and triglyceride accumulation. Adipose-specific knockdown of MTP was achieved in mice bybreeding MTP floxed (Mttpfl/fl) mice with aP2-Cre recombinase transgenic mice. Adipose-specific MTP deficient (A-Mttp-/-) mice were fed 60 % high-fat diet (HFD), and the effects of MTP knockdown on body weight, body fat composition, plasma and tissues lipid composition, glucose metabolism, lipogenesis and intestinal absorption was studied. Lipids were measured in total fasting plasma and size fractionated plasma using colorimetric assays. Gene expression was investigated by Real-Time quantitative PCR. All data was assessed using t-test, ANOVA.ResultsMTP expression increased during early differentiation in 3T3-L1 cells, and declined later. The increases in MTP expression preceded PPARγ expression. MTP overexpression enhanced lipid droplets formation, and knockdown attenuated cellular lipid accumulation. These studies indicated that MTP positively affects adipogenesis.The ablation of the Mttp gene using aP2-Cre (A-Mttp-/-) in mice resulted in a lean phenotype when fed a HFD. These mice had reduced white adipose tissue compared with wild-type Mttpfl/fl mice. The adipose tissue of A-Mttp-/- mice had increased number of smaller size adipocytes and less macrophage infiltration. Further, these mice were protected from HFD-induced fatty liver. The A-Mttp-/- mice had moderate increase in plasma triglyceride, but normal cholesterol, glucose and insulin levels. Gene expression analysis showed that the adipose tissue of the A-Mttp-/- mice had significantly lower mRNA levels of PPARγ and its downstream targets.ConclusionThese data suggest that MTP might modulate adipogenesis by influencing PPARγ expression, and play a role in the accretion of lipids to form larger lipid droplets. Thus, agents that inactivate adipose MTP may be useful anti-obesity drugs.

Assembly and Secretion of VLDL in Nondifferentiated Caco-2 Cells Stably Transfected With Human Recombinant ApoB48 cDNA

Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (> 50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (> 80%) by triacsin C due to > 60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.

Lysophosphatidylcholine increases apolipoprotein B secretion by enhancing lipid synthesis and decreasing its intracellular degradation in HepG2 cells.

Free fatty acids and lysophosphatidylcholine (lysoPC) are the major lipids bound to human plasma albumin. The effects of fatty acids on the hepatic production of Apolipoprotein B (apo B) have been studied but those of lysoPC have not. In HepG2 cells, lysoPC increased apo B secretion in different experiments by 50-120%, but did not affect the flotation properties of secreted lipoproteins. LysoPC affected neither the cellular protein levels nor apo A-I secretion suggesting that its effect was specific to apo B. Apo B secretion was maximum after incubating cells for 6 h with 0.2 mM lysoPC as equimolar fatty acid free bovine serum albumin (BSA) complexes. LysoPC was metabolized by cells and its fatty acids were used for the synthesis of phosphatidylcholine and triglycerides (TG). Experiments were performed to understand the mechanism of lysoPC action. LysoPC increased the incorporation of 3H-glycerol into newly synthesized cellular (3-fold) and secreted (4-fold) triglycerides, and increased the synthesis (40%) and secretion (4-fold) of phospholipids. LysoPC did not affect apo B synthesis, but inhibited the intracellular degradation of apo B and increased its secretion. Triacsin C (5 microM), an inhibitor of long chain acyl-CoA synthase, completely inhibited the induction of lipid synthesis and abolished the effect of lysoPC on apo B secretion. These studies indicated that lysoPC increased apo B secretion by inducing lipid synthesis; newly synthesized lipids probably protected apo B from intracellular degradation and enhanced secretion. These studies are consistent with the hypothesis that physiologic concentrations of lysoPC can be an important modulator for hepatic apo B secretion.

Precipitating factors and targeted therapies in combating the perils of sickle cell disease--- A special nutritional consideration

Nutritional research in sickle cell disease has been the focus in recent times owing to not only specific nutritional deficiencies, but also the improvements associated with less painful episodes. Though hydroxyurea remains the drug of choice, certain adverse health effects on long term supplementation makes room for researches of different compounds. Macro and micro nutrient deficiencies, along with vitamins, play an important role in not only meeting the calorific needs, but also reducing clinical complications and growth abnormalities. Symptoms of hyper protein metabolism, increased cell turnover, increased cardiac output, and appetite suppression due to enhanced cytokine production, might give us leads for better understanding of the mechanisms involved. Different nutritional approaches comprising of traditional herbal therapies, antioxidants, flavonoids, vitamins, minerals etc., reducing oxidative stress and blood aggregation, have been tried out to increase the health potential. Nutritional therapies may also serve complementary to the newer therapies using ozone, hematopoietic stem cell transplantation, antifungal medications, erythropoietin etc. Herein we try to present a holistic picture of the different patho-physiological mechanisms, and nutritional strategies adopted.

Nitrated apolipoprotein AI/apolipoprotein AI ratio is increased in diabetic patients with coronary artery disease.

AIMS/HYPOTHESIS Recent studies have suggested that determination of HDL function may be more informative than its concentration in predicting its protective role in coronary artery disease (CAD). Apolipoprotein AI (apoAI), the major protein of HDL, is nitrosylated in vivo to nitrated apoAI (NT-apoAI) that might cause dysfunction. We hypothesized that NT-apoAI/apoAI ratio might be associated with diabetes mellitus (DM) in CAD patients. METHODS We measured plasma NT-apoAI and apoAI levels in 777 patients with coronary artery disease (CAD) by ELISA. Further, we measured plasma cholesterol efflux potential in subjects with similar apoAI but different NT-apoAI levels. RESULTS We found that median NT-apoAI/apoAI ratio was significantly higher in diabetes mellitus (DM) (n = 327) versus non-diabetic patients (n = 450). Further analysis indicated that DM, thiobarbituric acid-reactive substances and C-reactive protein levels were independent predictors of higher NT-apoAI/apoAI ratio. There was negative correlation between NT-apoAI/apoAI and use of anti-platelet and lipid lowering drugs. The cholesterol efflux capacity of plasma from 67 individuals with differing NT-apoAI but similar apoAI levels from macrophages in vitro was negatively correlated with NT-apoAI/apoAI ratio. CONCLUSIONS Higher NT-apoAI/apoAI ratio is significantly associated with DM in this relatively large German cohort with CAD and may contribute to associated complications by reducing cholesterol efflux capacity.

Tetradecylthioacetic acid (a 3-thia fatty acid) impairs secretion of oleic acid-induced triacylglycerol-rich lipoproteins in CaCo-2 cells.

The fatty acid analogue tetradecylthioacetic acid (TTA) has previously been shown to decrease triacylglycerol secretion in CaCo-2 cells (Gedde-Dahl et al., J. Lipid Res. 36 (1995) 535-543). The present study was designed to further elucidate the effect of TTA on lipoprotein production in CaCo-2 cells. TTA did not affect oleic acid-induced triacylglycerol synthesis, but it significantly decreased secretion of newly synthesized triacylglycerol when compared to cells incubated with oleic acid alone or oleic acid in combination with palmitic acid. In contrast, pulse-chase experiments showed no difference in the amount of labeled triacylglycerol secreted from cells exposed to either fatty acid combination during the chase period, indicating that TTA did not affect the secretory process in general. Cells incubated with TTA alone secreted triacylglycerol present at 1.025<rho<1.073 g/ml, corresponding to the low density lipoprotein/intermediate density lipoprotein density range. In contrast, cells supplemented with oleic acid or oleic acid in combination with TTA secreted triacylglycerol mainly in the very low density lipoprotein/chylomicron density range (rho<1.006 g/ml). Despite a marked decrease in triacylglycerol secretion, TTA treatment did not change secretion of apolipoprotein B nor the activity of microsomal triacylglycerol transfer protein (MTP) in the cells. Furthermore, the presence of TTA in cellular triacylglycerol had no effect on the ability of purified MTP to transfer triacylglycerol from donor to acceptor vesicles. Together, the above observations suggest that TTA interferes with other MTP-independent factors that regulate the intestinal lipoprotein secretion.