s2fieldsofstudy: Biology Chemistry

text:
Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study

The use of gene therapeutics, including short interfering RNA (siRNA), is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs) which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules.

Introduction
Recently, therapeutics have shown a vast diversification from small molecule drugs. Peptide and nucleic acid based therapeutics are among these alternatives and have been developed tremendously, to the point of clinical trials [1][2][3]. Short interfering RNA (siRNA) is among the therapeutics which have captured the interest of researchers [1,[4][5][6][7]. Once introduced into cells these oligonucleotides drive the RNA interference (RNAi) process [8], in which the expression of a target protein is suppressed by stimulating the specific degradation of messenger RNA. This mechanism leads to high specificity and large number of possible targets.
This paper focuses on cell penetrating peptides (CPPs) with respect to their delivery of siRNA to cells through the use of computer modeling and simulation. We studied the structural features of seventeen CPPs and their capacity to form noncovalent complexes with siRNA.  machinery of RNAi [4]. Usually, the siRNA is generated by Rnase III, the "Dicer" enzyme which cleaves long dsRNA into siRNA. The siRNAs can then bind to the RISC (RNAinduced silencing complex). Within the RISC, the siRNA is unwound and the sense strand is removed for degradation by nucleases present in the cell. The antisense strand specifically targets certain sequences of mRNA and directs them to the RISC. Then, it anneals with the complementary base pair. Finally, a rapid degradation of the target mRNA takes place, and consequently decreased protein expression results. For siRNA to become a viable therapeutic, enzyme and environmental degradation have to be overcome with a proper delivery system. Therefore, extensive attention has been given to noninvasive peptide based delivery of siRNA into mammalian cells.

Cell Penetrating Peptides (CPPs).
Cell penetrating peptides are a distinct class of small peptidic molecules, having a special ability to trigger the movement of molecules across the cell membrane. CPPs have been shown to penetrate the cell membrane as well as mitochondrial and nuclear membranes without damaging them [25]. Known CPPs share many structural features and physical properties. Firstly, they are all water soluble. Secondly, CPPs are relatively small in size, not having more than 35 amino acid residues. Moreover, the cytotoxicities of these molecules are very low [26]. There is no unique classification for CPPs. They can be categorized according to their origin, according to their ability to link with the cargo, and according to their structure. Classification of CPPs by their structure separates peptides as (i) cationic CPPs, (ii) amphipathic CPPs, and (iii) hydrophobic CPPs.

CPP-siRNA Complexes.
CPPs of all three classes have been used for the delivery of siRNA with success. Here several peptides adsorb on the surface of siRNA. Interactions driving peptide adsorption can be nucleotide specific, or nonspecific. Protein-nucleic acid complexation is primarily governed by electrostatic interactions where the negatively charged backbone of the polynucleotide is the key acceptor of charged species.
The secondary structure and shape complementarity also plays a role in the binding of peptides to nucleotides.  Previously Schleif et al. speculated that helical peptides would show high binding affinities with double stranded RNA. The authors suggested that when the alpha-helix CPP is tilted, it can fit into the major groove of the dsRNA via hydrogen bonds and van der Waals interactions [27][28][29]. Furthermore, nitrogen base pairs which are exposed grooves of the RNA can become involved in sequence specific H bonds [28,30]. It follows that the conformation of dsRNA influences the binding of peptides due to the presence of 2 -OH groups in RNA; it naturally exists mostly in the thermodynamically stable A-form [31]. Thus DNA or B-formed RNA (see Figure 2) will not trigger the RNAi pathway. However, it is important to look into both conformations of RNA to get a broader idea about binding of CPPs to comprehend if there is a significant difference in between the bindings and thereby analyze any alternative means to fine-tune the siRNA-CPP complex.
As a result of inherent peptide-nucleic acid interaction, complexation of siRNA with multiple peptides usually occurs. This leads to the formation of complex, with a net positive charge. Further aggregation of these complexes forms dense nanoparticles, with a size of 10 2 nm that may be internalized via endosomes [12,32,33]. This formation of a positively charged nanoparticle is important for translocation Journal of Biophysics 3 into the cell membrane and delivering the therapeutics. Here, our main focus is to study the formation of the positively charged complex which is the initial step of internalization [34].
In this research, we study the initial binding of CPPs to siRNA and focus on the interactions governing the complexation. To achieve a complete understanding about the binding of CPPs to siRNA, we performed docking calculations for 17 different peptides from all the structural classes. Calculations were performed for binding of CPPs for both A-and Bforms of the same GAPDH siRNA (see Figure 2). This siRNA downregulates the translation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) enzyme. GAPDH catalyses the conversion of glyceraldehyde-3-phosphate to Dglycerate 1,3-bisphosphate in the glycolytic breakdown of glucose. Furthermore, best scored CPPs from three categories were docked with another different siRNA, HPV si16E6, to confirm the binding pattern. To generalize the findings of the study, siRNA downregulating Human Papillomavirus (type 16) E6 oncoprotein, si16E6, was used for comparison.

Generating Coordinates of siRNA.
For the study, two siR-NA molecules were arbitrarily chosen, namely, the GAPDH and si16E6. Ideal A-and B-forms of helical structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with a sense strand 5 -GACGUAAACGGCCACAAGUUC-3 and antisense strand of 5 -ACUUGUGGCCGUUUACGUCGC-3 , were generated using the make-na online server (NAB [35], Nucleic Acid Builder based application with Generalized Born, Poisson-Boltzmann, or 3D-RISM implicit solvent models). Ideal A-form of si16E6 with sense strand 5 -GCA-ACAGUUACUGCGACGUUU-3 and an antisense strand of 5 -ACGUCGCAGUAACUGUUGCUU-3 was also generated similarly with the same procedure. Once created, the stability of siRNA in aqueous medium was checked through molecular dynamics simulations using explicit (TIP3P) water at physiological temperature (310 K), for 24 ns using NAMD code and CHARMM v27 force field with time step of 2 fs/ts. The particle-particle mesh Ewald method was used for the long-ranged Coulombic interactions. A cutoff of 12Å was used for the van der Waals interactions and shortranged Coulombic interactions. Neighbor atom pair-lists were truncated at a distance of 13.5Å and were updated every 10 time steps. RATTLE/SHAKE algorithms were used to constrain the bond length of the hydrogen-heavy atom bonds.

Generating Coordinates of Cell Penetrating Peptides.
Generation and structure prediction of CPPs were carried out using PEP-FOLD [36][37][38] 2011 online server. All peptides were subjected to energy minimization via the NAMD code [39] and minimized structures were used for further calculations. For the minimization, the CHARMM v27 force field was used with the conjugate gradient minimization scheme.

Molecular Docking Calculations. Docking calculations
were performed by the ClusPro [40,41] online server with default (balanced weight) configurations. For docking simulations of CPP with siRNA, the siRNA was taken as the receptor while the CPP was regarded as the ligand. Docking calculations were submitted and the resulting output with minimum energy and highest clusters was taken for further calculations. These structures were minimized as before using the NAMD code [39] and GROMACS [42] v5 with the CHARMM [43] v27 force field and used for subsequent docking calculations. In the next step, the CPP-siRNA complex was taken as the receptor, and another CPP was taken as the ligand. This process was carried out up to 30 CPPs and the generated binding scores and coordinates were recorded. This docking process was carried out for GAPDH (A-and B-forms) and HPV si16E6 (A-forms). For A-form of GAPDH minimization was done using NAMD code and the rest were carried out in GROMACS version 5. From the amphipathic, cationic, and hydrophobic classes, the three best scoring CPPs were used and docking studies carried out. However, for HPV si16E6, docking was carried out for three best scoring CPPs in three groups.
A similar procedure was used to evaluate the dimerization docking energy scores, but here only two peptides were considered.
where denotes the docking score, while shape , elec , and pair denote shape complementarity, electrostatic, and desolvation contributions, respectively. The shape complementarity term shape accounts for both attractive ( attr ) and repulsive ( rep ) interactions. ClusPro provides four variations of energy functions by varying 1 , 2 , and 3 coefficients. Docking scores reported use the "balanced" energy function, where the weightage of the components is = 0.40 rep − 0.40 att + 600 elec + 1.00 DARS . (2) Both attraction and repulsion terms were considered in the energy function with the same weight. Electrostatic interactions were considered between the two proteins surrounded by solvent using simplified Generalized Born (GB) theory, with constant radii. More importantly, ClusPro introduces new structure-based, pairwise intermolecular potential DARS (Decoys as the Reference State). Detailed information on these terms can be found in Kozakov et al. (2006).

Explicit Solvent Molecular Dynamics Simulations.
Both NAMD and GROMACS code were used for molecular dynamics simulations with a time step of 2 fs/ts. All the simulations of involving GAPDH B-form siRNA were carried out in NAMD, while simulations involving GAPDH Aform siRNA and siR16E6 siRNA were run in GROMACS. To maintain the same environment CHARMM v27 force field was used along with a cutoff of 12Å for the van der Waals interactions and short-ranged Coulombic interactions, while the particle-particle mesh Ewald method was used for the long-ranged Coulombic interactions. Neighbor atom pair-lists were truncated at a distance of 13.5Å and were updated every 10 time steps. CHARMM compatible TIP3P explicit waters were used for all simulations. To constrain the bond length of the hydrogen-heavy atom bonds the LINCS algorithm and RATTLE/SHAKE algorithms were used in GROMACS and NAMD, respectively.

Results and Discussion
In this study, we investigated the binding of cell penetrating peptides (CPPs) onto short interfering RNA (siRNA). Since this delivery route is under vigorous development, structural features of peptides which increase the effectivity of siRNA delivery are of prime interest. Specifically, discovering features which enhance binding of peptides to siRNA or enhance the cellular uptake would be advantageous in future design of de novo peptides.

3.1.
Binding of CPPs to siRNA. As described in the methodology, docking calculations were performed to investigate the binding of CPPs to the siRNA, particularly as a function of siRNA : CPP ratio. Calculations were carried out through the ClusPro server, which uses a rigid body docking algorithm in generating the favorable orientations of docking, based on clustering. Docking scores are an approximate analog to binding free energies calculated by more thorough computational techniques such as molecular dynamics simulations. However, due to the expense of free energy calculations, and the number of different binding energies which are required to exhaustively analyze this problem, we have used docking as a realistic alternative. Furthermore, we focus more on the molecular geometries produced, and trends in the binding scores are observed. To generalize our results, cell penetrating peptides over a wide range of physicochemical characters were studied: cationic, amphipathic, and hydrophobic. Details on the CPP used in the study are given in Table 1, including the amino acid sequences, trivial name, nominal charge, and nominal charge per residue.
The complete set of binding scores obtained by docking calculated are available in the supplementary data in Supplementary Material available online at https://doi.org/10.1155/ 2017/1059216. However, since we are more concerned about the trends in the sequential binding events rather than the absolute scores, a concise graphical representation of docking results is shown in Figures 3 and 4.
From the binding energies (shown in Figures 3 and 4) the general trend is apparent; initially the binding of CPP onto  Table 1) with GAPDH (A-form).
the siRNA is the most favorable, while the magnitude of the binding scores generally decreases with the number of CPPs complexed with the siRNA. To illustrate the trends specific to each class of peptide, we will discuss them separately.
Cationic CPPs. Cationic peptides are, by nature, rich in positively charged residues, mostly arginine and lysine. For example, R10 consists of only arginine. As a result of having these residues present, the number of possible intramolecular hydrogen bonds is decreased [71]. Therefore, the secondary structure presents predominantly coils and tubes, not helices [72]. The same phenomena can be seen with HIV-TAT (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57), which is predicted to have a random coil structure. All cationic peptides have a nominal charge between +7 and +10. Due to Coulombic attraction between the positively charged CPP and the negatively charged siRNA, peptides with higher positive charge density would bind more strongly. This is also supported by the binding scores for the complexation of the cationic peptides with siRNA (see Figures  3 and 4). Among the cationic peptides, R10 shows the maximum charge density (nominal charge density of 1.00) and consequently it displays relatively high affinity for the  Table 1) with GAPDH (B-form). initial binding of peptides to the siRNA. Complexation driven by Coulombic interactions would be expected to decrease in magnitude due to progressively diminishing attractive force. This is illustrated through R10, which shows a drastic and monotonous lowering (in magnitude) of binding energy score at higher CPP : siRNA ratios, plateauing at around scores of −100 (Figures 3(a) and 4(a)). This trend is observed to a lesser extent in all of the cationic CPPs. Among them the highest gradient of binding energy score and largest initial binding energy score is displayed in CCMV-Gag and R10 with both forms of siRNA. Considering all cationic CPPs, it can be seen that Penetratin has low binding scores at high CPP : siRNA ratios. It stabilizes after binding of ∼15th CPP at ∼ −700 binding score.
When inspecting the orientation and placement of the cationic CPP on the siRNA (in both A-and B-forms), in all considered cases the initial CPP complexation occurs at the major groove of the siRNA (see Figures 5A1 and 5B1). However, unexpectedly, this is seen for both helical and random coil peptides. After the addition of a couple (two to three) of CPPs, the major groove is filled, and peptides preferentially bind to the minor groove of the siRNA, as seen in Figures 5A2 and 5B2. However, beyond 5-7 peptides bound (depending on the specific peptide), the peptides bind in a random orientation, seemingly driven by non-Coulombic forces. At high CPP : siRNA ratios, the peptides seem not to bind directly onto the siRNA but rather interact with the peptide already bound onto the nucleotide. However, in the case of the cationic peptides, the ionic groups favor exposure to water and therefore the complex acquires a very open structure with high solvent accessible surface areas (SASAs) (see Figures  5A3 and 5B3).
Hydrophobic CPPs. These peptides contain hydrophobic amino acid residues and display little or no charge at all. Due to the absence of charged residues, these peptides show diminished electrostatic interactions; binding to the siRNA is driven by van der Waals and hydrophobic forces. Consequently, the hydrophobic peptides show comparatively low magnitude in their binding scores, ranging from 600 to 1200. Furthermore, unlike the Coulombic interactions, the magnitudes of binding do not decrease in magnitude with the binding of CPP because the strength of hydrophobic and van der Waals interactions remains relatively constant. As a result, the binding scores of the hydrophobic peptides generally do not fall to values observed with the cationic peptide class.
Due to the weakness of van der Waals interactions, their total interactions tend to be nondirectional. Therefore, unlike cationic and amphipathic CPPs, the hydrophobic peptides do not follow specific orientations in binding to siRNA. Initially the hydrophobic peptides were predicted to bind to either the major groove or the minor groove. Figures 6B1 and 6B2 show the first two HIV-1 gp41 (1-23) peptides binding to the minor groove of the siRNA. However, in the A-form, HIV-1 gp41 (1-23) has randomly bound to the major groove, resulting in a significantly low binding energy score. Upon further binding of peptides, they show no orientational bias and complex in a random manner. Furthermore, because of the hydrophobic composition of the peptides, the peptide-siRNA complex arranges to minimize the surface area, as evidenced by the low solvent SASA values that were observed (see supplementary data). This leads to the formation of a dense, tightly packed peptide-siRNA particle ( Figures 6A3  and 6B3).
Amphipathic CPPs. These peptides exhibit both hydrophobic and hydrophilic behavior and/or regions in their structure. Usually, this capability comes with the presence of lysine as a component in the peptide [73]. All amphipathic peptides considered in the study are rich in lysine and have nominal charges between +4 and +8. Having both the positive charge of the cationic class and the nonpolar groups characterizing the hydrophobic peptides, this class shows the advantages of both. They show the initially strong binding of cationic peptides, a gradual decline, and finally plateauing (see Figures  3 and 4). As with the cationic CPPs, the amphipathic CPPs also bind initially to the major groove ( Figures 7A1 and  7B1) with negative binding energy scores ranging from −1200 to −1800. However, when the number of CPPs is increased, binding energies rise sharply within 1-5 CPPs and becomes steady. Due to the presence of nonpolar groups, . Surprisingly, there is no clear trend between the predicted secondary structure (which is used in the docking) and the binding scores generated, where helical, beta-sheet, and random coil structures all exhibited similar binding scores. From the amphipathic peptides, sC18 and CADY indicate slight deviations. sC18 shows comparatively larger elevation of binding energies; CADY shows less. This indicates a favorable complexation of CADY and less favorable complexation of sC18 with siRNA. Considering the results collectively, cell penetrating peptides with helical structures show higher binding affinity to siRNA such as CADY, C6, and sequence HIV-1 gp41 (1-23). By nature, most relatively small (<40 residue) peptides tend to arrange in helical structures; helices maximize intramolecular hydrogen bonding and provide better distribution of residues throughout the structure, which enables higher interacting power with the external environment. Spatial arrangement of cell penetrating peptides also provides less steric hindrance to bind to the major or the minor groove. Furthermore, having larger side chains on the peptide discourages the binding, thus showing higher binding energies. Particularly, peptides containing tryptophan and tyrosine exhibit slightly attenuated binding.
Since almost all siRNAs show identical structures, the same binding pattern was observed as shown in Figure 8. However, due to structural differences in major grooves and minor grooves, slight lowering of binding energy was observed in A-forms (for both GAPDH and HPV-si16E6).
Considering the results, the following stages of peptide binding can be extracted. (1) Initially electrostatic and shape complementarity drive the binding of cell penetrating peptides into the major groove of siRNA, where the maximum salt bridges may be formed. At this stage positive charge on the peptide is beneficial. Due to the small size of siRNA, only 2-3 CPPs can be accommodated into the major groove.
(2) Subsequent peptides tend to bind to minor groove of siRNA while some get arranged perpendicular to the siRNA. Increased charge on the peptide tends to favor binding of the peptides to the minor groove. However, with the increasing number of peptides, more peptide-peptide interactions are observed. With further increase of the CPP to siRNA ratio, the peptides are observed to bind either onto the siRNA or to previously bound CPPs. Here, the competition of the peptide-siRNA and the peptide-peptide interaction play a major role.
(3) Finally, once the surface charge on the siRNA has been screened, and a complete coating of the siRNA has taken place, further aggregation of peptides may occur, where the size of the siRNA-peptide complex is increased.
The docking scores for binding CPP initially to the Aand B-form show slight differences. The main reason that can be identified is the shape of the major groove of siRNA. The A-form helix is more coiled than the B-form helix, creating narrow, deep major groove and shallow, wide minor groove. Thus CPPs can interact with deeper major groove and thereby create a stable complex. This phenomenon is often evident in cationic CPPs since the positive charge drives Coulombic attraction with negative charges in nucleotide. Moreover, whenever a CPP is bound to major groove, it shows negatively larger binding score regardless of the type of CPP. One such example is initial binding of HIV1gp41 and C105Y to A-form of siRNA which is significantly lower than that of others since they are bound to major groove.

Aggregation and General
Outlook. It goes without saying that the aggregation behavior of the peptides is essential information. To better evaluate this quantity, docking simulations of CPP dimerization were also carried out. Although approximate, the resultant scores agree with the expected trend; cationic peptides showed the lowest binding, while the amphipathic and hydrophobic peptides showed more favorable binding scores (see supplementary information for values). The balance between the peptide-siRNA and peptide-peptide interactions also affects the formation of nanoparticles and release of the therapeutic molecule(s) to the cytoplasm. Having a highly stable complex might be problematic when releasing the therapeutic molecule. On the other hand, if peptide-peptide interactions are too high, proteins will aggregate prior to complexing with siRNA. The size and nature of the aggregate are also important in the cell membrane translocation of siRNA-CPP complexes.
Experimental results suggest that aggregates of several siRNA-CPP complexes will interact with each other and produce larger particles (which reside in the 10 2 nm size scale), which have the ability to overcome the cell membrane barrier. Here, the siRNA : CPP 1 : 30 complexes are ∼10 nm in size. However, among the mentioned three categories, a significant variation in the SASA can be seen (see supplementary information). For the cationic peptides, for siRNA : CPP of 1 : 30, the SASA values are in the range of 402-550 nm 2 , while for the hydrophobic and amphipathic peptide the ranges were 185-265 nm 2 and 395-472 nm 2 , respectively. The size and the physicochemical nature of the exposed surface and the size of final multicomplex nanoparticle are known to have a large impact on the internalization. However, we cannot predict the final size of the aggregates using the techniques used in this research but only suggest that the highly charged complexes are unlikely to aggregate in large numbers due to the unfavorable charge and solvation effects.
The structures and associated binding scores calculated here should only be taken as representative. Although the docking algorithm used samples over many of the possible locations and orientations of binding, only limited flexibility is allowed for the peptide when binding. Therefore, firstly the initial predicted structure plays a large role in the predictions, and secondly the process does not sample the free energy of the binding processes with the correct weights. So there will be a significant error in the associated results. Given this limitation, we have taken care not to use the absolute binding scores in interpreting the results and use only the relative differences between the peptides, and the variation in binding scores for a single type of peptide, to minimize systematic errors. However, due to the sensitivity of peptide structure to environmental conditions, this is a large source of uncertainty in the study. We hope future studies of chosen peptides and structures may allow more quantitative information about the binding process, which will allow future experimentalists and peptide designers to further progress in this field. Because much of the mechanisms involved in this delivery mechanism are not fully understood, our knowledge in properties which enhance cellular uptake is largely empirical. More pedagogical investigation of each step in the pathway of complexation, internalization, release, and metabolism is required to fully understand and manipulate this attractive technology.


extfieldsofstudy: Chemistry Medicine

text:
Bisphenol A promotes cholesterol absorption in Caco-2 cells by up-regulation of NPC1L1 expression

Background Bisphenol A (BPA), an commonly exposed environmental chemicals in humans, has been shown to have a hypercholesterolemic effect with molecular mechanism not clear. Since intestinal cholesterol absorption plays a major role in maintaining total body cholesterol homeostasis, the present study is to investigate whether BPA affects cholesterol absorption in the intestinal Caco-2 cells. Methods: The Caco-2 cells were pretreated with BPA at different concentrations for 24 h and then incubated with radioactive micellar cholesterol for 2 h. The absorption of radioactive cholesterol was quantified by liquid scintillation. The expression of Niemann-Pick C1-like 1 (NPC1L1) and sterol regulatory element binding protein-2 (SREBP-2) was analyzed by Western blot and qPCR. Results We found that confluent Caco-2 cells expressed NPC1L1, and the absorption of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. We then pretreated the cells with 0.1–10 nM BPA for 24 h and found that BPA at 1 and 10 nM doses promoted cholesterol absorption. In addition, we found that the BPA-induced promotion of cholesterol absorption was associated with significant increase in the levels of NPC1L1 protein and NPC1L1 mRNA. Moreover, the stimulatory effects of BPA on cholesterol absorption and NPC1L1 expression could be prevented by blockade of the SREBP-2 pathway. Conclusions This study provides the first evidence that BPA promotes cholesterol absorption in the intestinal cells and the stimulatory effect of BPA is mediated, at least in part, by SREBP-2-NPC1L1 signaling pathway.

Background
Hypercholesterolemia is a significant risk factor for cardiovascular disease [1]. In addition to denovo cholesterol synthesis, cholesterol derived from the dietary and biliary cholesterol absorption also contributes to the amount of cholesterol circulating in plasma [2]. In fact, the absorption of cholesterol from the intestine is an important determinant of plasma cholesterol levels [3], those individuals with high absorption rates are hypercholesterolemic and generally have an increased number of cardiovascular events [4]. Moreover, inhibition of cholesterol intestinal absorption leads to lower cholesterol levels [5].
Cholesterol absorption in intestine is a multi-step process and this process is mediated by a specific membrane protein named Niemann-Pick C1-like 1 (NPC1L1) protein [6], which can be specifically inhibited by ezetimibe [7]. NPC1L1 is highly expressed in the small intestine and it is required for intestinal cholesterol absorption. NPC1L1 knockout mice exhibited an 70% reduction in cholesterol absorption and were resistant to diet-induced hypercholesterolemia [6,8]. Furthermore, NPC1L1 has been recently identified as a novel target gene of sterol regulatory element binding protein-2 (SREBP-2) [9]. There are two putative sterol regulatory elements in the human NPC1L1 promoter and they are essential for mediating the effects of cholesterol on promoter activity [9].
Bisphenol A (BPA) is a man-made compound highly prevalent in our environment and suspected to act as an endocrine disruptor [10]. It is intensively produced and used as a monomer of polycarbonate plastics and epoxy resins [11]. The human population is widely exposed to low levels of BPA, primarily by way of the diet by migration from food and beverage containers [11]. Biomonitoring surveys have demonstrated that detectable levels of BPA are present in the urine of nearly all sampled adults in U.S. population [12]. The ability of BPA to cause adverse human health effects is highly documented [13][14][15]. Many experimental and epidemiological studies highlighted potential links between BPA exposure and the development of cancer and disorders of reproductive, neuroendocrine, and immune systems [14,16]. Recent epidemiological and animal studies have suggested that circulating BPA levels are associated with prevalence of coronary heart diseases and a number of cardiovascular risk factors such as hypercholesterolemia [17][18][19]. Several in vivo studies have shown that BPA exposure increased plasma total cholesterol and low density lipoprotein cholesterol levels [20,21]. However, the molecular mechanism underlying the hypercholesterolemic effects are still unknown. Since the intestine plays a major role in maintaining total body cholesterol homeostasis, we, in the present study, addressed a question whether BPA affects the cholesterol absorption in the enterocytes.

Reagents
BPA and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Dulbecco's modified Eagle's medium (DMEM), M199 medium, heatinactivated fetal bovine serum (FBS),1% non-essential amino acids, and other materials were purchased from either Invitrogen (Carlsbad, CA, USA) or Sigma-Aldrich (St. Louis, MO, USA). [ 14 C]cholesterol (50 mCi/mmol) was purchased from American Radiolabeled Chemicals Inc (St. Louis, MO, USA) and NPC1L1 antibody from Santa Cruz (Santa Cruz, CA, USA). cDNA synthesis kit was purchased from Invitrogen Life Technology (Carlsbad, CA, USA) and SYBR Green-based real-time PCR kit was obtained from Applied Biosystems (Foster City, CA,USA). All other chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture and cholesterol absorption assay
Caco-2 cells were obtained from American Tissue Culture Collection (Virginia, USA) and were cultured in DMEM, containing 10% FBS, 1% penicillin-streptomycin, 2 mM Lglutamate, and 1% non-essential-amino acids to 100% confluence. Before the experiment of cholesterol absorption, the cells were firstly cultured in medium containing the delipidized FBS for 24 h as described [22]. Then the cells were washed three times with M199 buffer and incubated with fresh medium containing the cholesterol micelles for 2 h. The micellar cholesterol solutions were prepared as in our previous publication [22]. After incubation, the medium was removed, and the cells were washed three times with ice-cold PBS. The cell pellets were dissolved in 0.1 M NaOH, and an aliquot of 0.1 ml of the lysate was taken for liquid scintillation counting.

Effects of BPA on cholesterol absorption
To investigate the effects of BPA on cholesterol absorption, the cells were pretreated with 0.1-10 nM BPA for 24 h [23]. After the pretreatment, the medium was removed and the cells were washed three times with icecold PBS, followed by incubation with the cholesterol micelles as described above. BPA was delivered to the cells using DMSO solvent, the amount of DMSO added to the cells was not greater than 0.1% (v/v). The cells treated with the equal amount of DMSO alone were taken as controls.

Real-time quantitative PCR
Methods for RNA extraction and real-time quantitative PCR (qPCR) have been previously described in our previous publications [22]. The primers used in qPCR to quantify the mRNA of NPC1L1 and SREBP-2 were shown in Table 1. GAPDH was amplified as an internal control.

Western blot analysis
Methods for cell free extract and Western blotting has been previously described [22,24]. In brief, 40 μg of proteins in cell lysate was subjected to 7.5% SDS-PAGE and transferred to a nitrocellulose membrane electrophoretically overnight. The membranes were incubated with anti-NPC1L1 antibody (1:5000) and then with second antibody (1:50000) conjugated with horseradish peroxidase. The specific NPC1L1 bands (145 kD) were identified by enhanced chemiluminescence advance reagent. The membranes were then stripped and reprobed with anti-actin antibody.

Table 1 Primers used in this study
Primer Name Sequence

RNA interference
SREBP-2 expression was knocked down by transfection of human SREBP-2 small interfering RNA (siRNA) with Caco-2 cells. A nonrelated, scrambled siRNA was used as a control. Transfection reagent and all siRNA oligos were designed and synthesized by Dharmacon. Transfections were carried out according to the manufacturer's protocol using the DharmaFECT Reagent 4.

Statistical analysis
The results are presented as mean ± S.E.M. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Bonferroni posttest for multiple comparisons. Differences were considered significant at P < 0.05.

Absorption of cholesterol in Caco-2 cells was mediated by NPC1L1
Because the cholesterol absorption in intestine was mediated by NPC1L1 and ezetimibe is a specific inhibitor for NPC1L1, we first examined whether the Caco-2 cells express NPC1L1 and whether cholesterol absorption in Caco-2 cells under the experimental conditions was related to the functions of NPC1L1. As shown in Fig. 1a, Western blot analysis clearly demonstrated the expression of NPC1L1 protein in these cells. In addition, the cholesterol absorption was dose-dependently inhibited by ezetimibe (Fig. 1b).

BPA promoted cholesterol absorption in Caco-2 cells
We then addressed a question whether BPA can promote cholesterol absorption. Caco-2 cells were pre-

BPA promoted NPC1L1 protein and mRNA expression
To elucidate the molecular mechanism by which BPA promoted cholesterol absorption in Caco-2 cells, we then further analyzed the impact of BPA on NPC1L1 expression in the intestinal cells. After pretreating the cells with different concentrations of BPA for 24 h, the protein and mRNA expression of NPC1L1 were evaluated by Western blot and real-time PCR, respectively. As shown in Fig. 3a, pretreatment of the cells with BPA significantly increased NPC1L1 protein expression. No similar changes could be identified for the levels of β-actin. Simultaneously, the mRNA expression of NPC1L1 was dose-dependently increased by BPA, as normalized with that of control gene GAPDH (Fig. 3b). Furthermore, to address that the stimulatory effect of bisphenol A on cholesterol absorption is solely mediated by NPC1L1, we used NPCIL1 inhibitor ezetimibe to block NPC1L1 and found that the enhanced cholesterol absorption induced by bisphenol A was markedly attenuated (Fig. 3c).
The SREBP-2 may be involved in the BPA-induced promotion of NPC1L1 expression and cholesterol absorption Since the expression of NPC1L1 is regulated by SREBP-2, we then wanted to know whether SREBP-2 could be involved in our observed NPC1L1 up-regulation. After pretreating the cells with BPA for 24 h, we found that the expression of SREBP-2 was dose-dependently upregulated (Fig. 4a). We next examined the effects of the knockdown of SREBP-2 by small interfering RNA transfection on BPA-mediated NPC1L1 expression and cholesterol absorption. When SREBP-2 was suppressed by the specific RNA interference, no effect of BPA on NPC1L1 expression could be detected (Fig. 4b), and no significant increase of micellar cholesterol absorption by BPA in Caco-2 cells could be identified (Fig. 4c).

Discussion
The findings of our present study demonstrate for the first time that BPA at environmentally relevant doses promotes cholesterol absorption in the intestinal cells and the stimulatory effect of BPA is mediated, at least in part, by SREBP-2-NPC1L1 signaling pathway. BPA is an environmental chemical used as a constituent monomer in polycarbonate plastics, which are used extensively in drinks containers and food packaging, and in the production of oxidants used in the lining of canned goods [11]. Exposure to BPA is believed to be  Fig. 3 The effect of BPA on NPC1L1 expression in Caco-2 cells. a The cells were treated with BPA at different concentrations for 24 h, and the whole-cell lysates were analyzed by Western blot. The results are representative of three independent experiments. b NPC1L1 mRNA abundance was determined by real-time RT-PCR as described in Methods. Expression values were normalized to housekeeping genes, and expression in untreated cells was set to 1. Values shown represent means ± SEM of three independent experiments, *P < 0.05, **P < 0.01, compared to untreated cells. c After the blockade of NPC1L1 expression by ezetimibe(EZE), Caco-2 cells were incubated with BPA for 24 h, then incubated with radioactive micellar cholesterol for additional 2 h. The absorption of cholesterol in the absence of BPA was normalized to 100%. Results are mean ± SEM from triplicate determinations in three separate experiments. *P < 0.05 compared to untreated cell mainly through dietary intake with additional exposure through water, dental sealants, inhalation of household dusts, and exposure through skin [11]. A lot of studies have documented widespread human exposure to BPA [12]. Levels of BPA ranging from 0.3 to 5 ng/mL (~1-20 nM) are present in adult and fetal human plasma, urine, and breast milk [25]. BPA, a lipophilic compound, can accumulate in fat, with detectable levels found in 50% of breast adipose tissue samples from women [26]. BPA expoure has been reported to cause adverse human health effects such as cancer, reproductive, neuroendocrine, and immune systems disorders [13,14,16]. Additionally, several lines of recent evidence suggest that an association between circulating BPA levels and hypercholesterolemia, studies in animals and humans have also shown that BPA exposure increased cholesterol levels in blood [19][20][21], but the molecular mechanisms underlying the hypercholesterolemic effects of BPA are still unkown. Since the intestine plays a major role in maintaining total body cholesterol homeostasis, and human exposure to BPA is primarily through dietary intake, whether absorption of cholesterol in the enterocytes can be affected by BPA is therefore an important question to be studied. Our results for the first time show that BPA at environmentally relevant doses has stimulatory effects on cholesterol absorption in the intestinal cells. The conclusion is supported by the following evidence. First, the Caco-2 cells used in our experiments expressed NPC1L1 and the absorption of cholesterol in our system was mainly mediated by NPC1L1. Second, pretreatment of the cells with BPA significantly promoted cholesterol absorption, which was accompanied by an increase of NPC1L1 protein and mRNA expression.
Exposure of humans to BPA is ubiquitous at low levels(~1-20 nM), the dose used in the present study is environmentally relevant and our research may have clinical implication. Several studies have revealed that exposure to environmentally relevant BPA doses could alter various biological functions, including reproductive, behavioral, metabolic, and immune systems [16]. Hugo et al. has shown that BPA at environmentally relevant doses inhibits the release of a key adipokine that protects humans from metabolic syndrome [23], which may be one mechanism that is responsible for the metabolic disorders and obesity caused by BPA. Furthermore, our results also potentially suggest a new mechanism that is responsible for the hypercholesterolemic effects of BPA. It is noted that the liver is also important for systemic Fig. 4 The promotion of BPA on NPC1L1 expression and cholesterol absorption may be mediated through SREBP-2. a Expression analysis of SREBP-2 in Caco-2 cells after 24 h of stimulation with increasing concentrations of BPA. Data are presented as means ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 compared to untreated cells. b Up-regulation of NPC1L1 expression in Caco-2 cells after 24 h incubation with BPA, by transfecting short interfering RNA (siRNA) for SREBP-2 compared with scramble control transfections. Data are presented as means ± SEM of three independent experiments. *P < 0.05 compared to untreated cells. c After the inhibition of SREBP-2 expression using siRNA, Caco-2 cells were incubated with BPA for 24 h, then incubated with radioactive micellar cholesterol for additional 2 h. The absorption of cholesterol in the absence of BPA was normalized to 100%. Results are mean ± SEM from triplicate determinations in three separate experiments. **P < 0.01 compared to untreated cell lipid metabolism and is the primary target organ of BPA. A recent study in mice has shown that BPA exposure significantly increased the expression levels of genes related to lipid synthesis such as HMG-CoA reducase, the key enzyme responsible for cholesterol synthesis in liver [27]. Take together, there results indicate that BPA can increase the cholesterol levels by two mechanisms, one in the intestine to enhance the absorption via NPC1L1 transporter, and the other in the liver to increase the synthesis by HMG-CoA reducase.
NPC1L1 is a crucial transporter for cholesterol absorption [6]. Understanding the regulation of its expression is of importance in human health and disease. Recent studies have shown the increase in NPC1L1 expression in hypercholesterolemia associated with diseases such as diabetes mellitus [28,29]. Moreover, NPC1L1 inhibition or deficiency was shown to protect against diet-induced hypercholesterolemia and hepatic steatosis [30,31]. Since NPC1L1 plays a pivotal role in mediating intestinal cholesterol absorption and the processes of cholesterol homeostasis, we argued that BPA may directly influence its function. In our studies, we found that pretreatment of the cells with BPA significantly increased NPC1L1 mRNA and protein levels, and the changes in cholesterol absorption consistently corresponded to the changes in NPC1L1 mRNA and protein expression. Moreover, to address that the stimulatory effect of bisphenol A on cholesterol absorption is solely mediated by NPC1L1, we further used NPCIL1 inhibitor ezetimibe to block NPC1L1 and found that the enhanced cholesterol absorption induced by bisphenol A was markedly attenuated. These results suggest the mechanism may involve an increase in cholesterol absorption due to a up-regulation of NPC1L1, thus contributing to the hypercholesterolemic effect of BPA.
Regarding the molecular signals regulating the intestinal expression of NPC1L1, we found that expression of the transcription factor SREBP-2 is involved in BPAinduced up-regulation of NPC1L1. It is known that the expression of NPC1L1 is mainly regulated by transcription factor SREBP-2. There are two putative sterol regulatory elements in the human NPC1L1 promoter [9], and the expression of NPC1L1 gene can be up-regulated by SREBP-2 activation in the intestine [9]. Moreover, BPA has been found to induce liver SREBP-2 expression in mice [20,32]. Thus, we hypothesized that BPA influenced NPC1L1 expression presumably through the SREBP-2-mediated signal transduction pathway. In supporting this hypothesis, when Caco-2 cells were stimulated with BPA, we did find significantly increased expression of the SREBP-2 in the cells, and when SREBP-2 was depleted by siRNA, NPC1L1 expression was no longer increased by BPA, and no increase in micellar cholesterol absorption was observed. Our studies demonstrate that BPA modulates intestinal NPC1L1 expression at both transcriptional and translational regulations and the SREBP-2 transcription factor may be critically involved in such regulations. Furthermore, with regard to the mechanism by which BPA up-regulated the expression of SREBP-2, the epigenetic regulation may be involved. It is known that promoter methylation levels tend to relate negatively to gene expression. Ke et al. has recently shown that the promoters of SREBP-2 and HMG-CoA reducase were hypomethylated in the BPA-exposed mice, which is very likely to contribute to the promoted transcription of SREBP-2 and its targets. Because DNA methyltransferase knockdown led to the promoter hypomethylation and increased mRNA expression of SREBP-2 and HMG-CoA reducase [27]. These results indicated that SREBP-2 was up-regulated by BPA via reprogramming the DNA methylation patterns.

Conclusions
In conclusion, our current results imply a potential role of BPA, at environmentally relevant concentrations that can be detected in vivo, in stimulating intestinal cholesterol absorption and in promoting hypercholesterolaemia. The mechanisms may be involved in SREBP-2 activation and subsequent up-regulation of NPC1L1 expression.


s2fieldsofstudy: Biology Medicine

text:
The effect of weight loss on HDL subfractions and LCAT activity in two genotypes of APOA-II -265T>C polymorphism

Background People may have different responses to the same environmental changes. It has been reported that genome variations may be responsible for these differences. Also, HDL subfractions may be influenced by different genetic variations. The aim of the present study was to determine gene-diet interactions and to evaluate the influence of weight loss on HDL subfractions between two genotypes of -265 T>C APOA-II polymorphism. Methods In the present study, 56 overweight and obese patients with type 2 diabetes mellitus were selected from 697 genotype-specified subjects. After matching for gender, age and BMI at the beginning of the study, an equal number of patients remained on each genotype of APOA-II (TT/TC and CC group). After a 6-week calorie restriction program, 44 patients completed the study. Serum HDL subfractions, including HDL2 and HDL3 and LCAT activity, were compared between the two genotypes and, before and after the intervention, were separated in each genotype. Results Serum concentration of HDL and its subfractions decreased significantly due to the weight loss. A comparison of the mean changes between the genotypes showed that HDL3 significantly decreased in the CC genotype while, in the TT/TC group, the serum concentration of HDL2 was significantly reduced. However, the increase of LCAT activity was not significant among the two genotypes. Conclusion A comparison of mean changes of variables within two genotype groups showed that C homozygote carriers lead to a general shift toward larger size HDL subfractions and T allele carriers shift toward smaller size HDL subfractions after weight loss.

Background
The growing prevalence of obesity and type 2 diabetes mellitus (T2DM) in the world makes these conditions a global public health problem [1]. Moreover, the incidence of T2DM is linked to obesity, and Asian people develop diabetes at a lower degree of obesity and at a younger age [2]. Both obesity and T2DM mostly result from a changing lifestyle, such as increased daily energy intake and a sedentary lifestyle, yet both are preventable by lifestyle modification [1]. The primary goal for overweight or obese people who have T2DM is to lose weight. A variety of diets have been suggested for treating obesity; however, in general, a calorie restriction diet is recommended for these patients [3]. However, it has reported that people show different responses to similar environmental changes, including dietary modifications. It is suggested that genetic differences may, therefore, be the reason for these variations [4]. One of the candidate genes that is involved here is apolipoprotein A-II (APOA-II). APOA-II is the second most common protein in high-density lipoprotein (HDL) [5].
HDL is secreted from the liver as discoidal particles, which consist of two or more apolipoprotein molecules, phospholipids and unesterified cholesterol [6]. Unesterified cholesterols are substrates for Lecithin: cholesterol acyltransferase (LCAT). The act of LCAT converts discoidal HDL into the large spherical HDL particles [6]. Spherical HDL contain a core of neutral lipids such as triglyceride and cholesteryl esters surrounded by phospholipids, unesterified cholesterol and apolipoproteins, which can be separated by ultracentrifugation into two major subfractions consisting of HDL2 and HDL3 [7]. HDL2 particles are generated when LCAT esterified free cholesterol on the surface of HDL3. Conversely, HDL3 is formed when HDL2 particles are hydrolyzed by hepatic lipase (HL) enzyme [8]. HDL2 particles are larger and less dense than HDL3 [6,8].
It has been found that APOA-II can modulate the activity of enzymes implicated in HDL metabolism such as LCAT and, consequently, affect the HDL particle size [5]. It has been proven that APOA-I is a potent catalytic activator of LCAT [9]. In vitro studies have shown that APOA-II has more affinity with HDL surface and can displace APOA-I from HDL. This process may impair the function of APOA-I involved in the reverse cholesterol transfer (RCT) [10]. As some studies have shown, in human APOA-II transgenic mice the capacity of HDL to promote cellular cholesterol efflux decreases [11]. In APOA-II transgenic mice, overexpression of APOA-II can reduce LCAT activity through the displacement of APOA-I from the surface of HDL [12].
There are limited studies on the relationship between plasma APOA-II levels and HDL subfractions or LCAT activity in humans. In APOA-II -265 T>C polymorphism, there is a substitution of T-to-C at the 265 position of this gene. This replacement results in a decreased APOA-II expression in the liver and, as a consequence, different serum APOA-II concentrations in two genotypes [13]. It was proposed that -265 T>C APOAII polymorphism is related to anthropometric variables [13,14], while daily food intake such as saturated fatty acids [15,16] and similar behaviors have an effect on weight loss [17].
Due to different APOA-II expressions in the liver and, consequently, different serum APOA-II levels in APOA-II -265 T > C polymorphism, we hypothesize that APOA-II genotype might be associated with different levels of LCAT activity and HDL subfractions. Therefore, to test this hypothesis, this study was designed to determine the HDL subfraction changes of two different genotypes after the same lifestyle modification in order to gain a better understanding of the gene-diet interaction.

Study design and diet intervention
In this study, 56 overweight (BMI = 25-29.9 kg/m 2 ) and obese (BMI ≥ 30 kg/m 2 ) type 2 diabetes (T2D) patients with a mean age of 56.68 ± 5.95 were selected from the 697 genotype-specified subjects in the earlier study [18]. The patients were matched for gender, age, and BMI and, at the baseline of study, an equal number of patients remained on each genotype of APOA-II (28 people in the TT/TC group and 28 people in the CC group). Exclusion criteria were smoking, alcohol consumption, pregnancy, lactation, having certain diseases such as liver and thyroid disease, kidney failure, cancer and use of some medications, including anti-inflammatory agents, nutrient supplements and insulin injection.
Weight, height and waist circumference were measured by a trained dietitian using standard protocols [19]. The total energy requirements were calculated using Mifflin-St. Jeor formula for basal energy expenditure (BEE), then the thermic effect of food (TEF) and activity thermogenesis (AT) were added to the BEE to obtain the total energy expenditure (TEE) for each patient [19]. This dietary program was based on a caloric restriction of 500-1000 kcal/d [3]. In the present study, the diet prescribed provided 750 kcal/d less than their calorific needs. Only two patients in each group received 500 kcal/d less than their daily calorific needs. A food menu was designed with a specified energy and fiber intake (30 gr/d) [20]. The percentage of energy intake was 55-65% of energy from carbohydrates, 20-25% from fats and 15-20% from proteins [21]. The recipes were set on a daily basis for each individual for a period of six weeks by a trained dietitian. Every two weeks, seven menus with the same daily calories were given to the patients. Therefore, patients could choose the desired menu, or even repeat a menu in one specific week. Also, the same amount of calories was calculated for every meal; for example, there were seven lunch menus with the same calorific amounts, enabling the patients to choose their favorite.
The compliance was checked using weekly-based phone calls and, every second week, patients were invited to the clinic in order to measure weight and waist circumference and estimate dietary intakes. A trained dietitian taught the patients how to complete food diaries and, if participants exchanged their food items, they had to write this in their food diaries. Participants who completed ≥ 80% of the planned diets were encouraged to continue for the subsequent weeks, while those subjects who did not complete ≥ 80% of the prescribed diet for one successive week were excluded from the study [22].
Physical activity was evaluated using an international physical activity questionnaire (IPAQ) [23]. The patients were requested to maintain their usual physical activity during the intervention.

Sample collection and measurement of serum HDL2, HDL3 and LCAT activity
At the baseline of the study and after six weeks intervention, 15-20 ml of fasting blood samples (after 10-12 hours fasting) were taken from the patients and allocated into separate tubes with and without anticoagulant Ethylenediaminetetraacetic acid (EDTA).
The blood samples without EDTA were centrifuged at 3000 rpm for 10 min at 4°C and serum samples were stored at −80°C until the analysis. A concentration of serum HDL2 and HDL3 was measured using a commercial ELISA kit (Crystal Day, China; Cat. No: E2188Hu and E2189Hu, respectively) according to the instructions provided by the manufacturer. The serum LCAT activity was measured using a commercial fluorimetric activity assay kit (Roar Biomedical, NY, USA; Cat. No: MAK107) according to the manufacturer's instructions. LCAT activity was calculated by the changes in 470/390 nm emission intensity. The emission intensities of 390 nm indicate that the substrate hydrolyzed and 470 nm show the substrate is not hydrolyzed.

Statistical analysis
Statistical analysis was carried out using SPSS 16.0 for Windows 7. The characteristic of the study population is expressed as mean ± standard deviation. The normality of distribution variables was assessed using the Kolmogorov-Smirnov test. The serum HDL2 and HDL3 concentration was log-transformed to normalize the error distribution. The independent t-test and chi-square test were used to compare continuous and categorical variables, respectively, between the two genotypes. A covariance test was used to adjust the confounders of each dependent variable. A paired sample t-test was used to compare the changes within the groups. The APOAII genotypes were analyzed using a comparison of homozygote of CC with the combined homozygote of TT and heterozygote TC (TT + TC). The mean difference was used to distinguish any association between parameters and the genotype groups. The significance level was defined as P < 0.05 for all statistical tests.

Results
At the baseline of the study, 56 patients with T2D were matched by means of gender, age and BMI. In total, 44 patients completed the study (7 men and 15 women in each group), while 12 patients did not complete the study. In the CC genotype group, cancer was diagnosed in a patient during intervention and a patient traveling abroad; in the TT/TC genotype group, a patient caught a severe cold and used corticosteroid drugs, while a patient was dropped to match with the CC group. The other patients were withdrawn because of poor compliance. Finally, 22 patients (7 men and 15 women in each group) completed the study.
The characteristics of the study population based on the genotype groups are shown in Table 1. When the subjects were matched at the baseline of the study, no significant differences were recorded in BMI and waist to hip ratio between the two genotype groups. Also, serum HDL and HDL subfraction levels were not significantly different between the two genotypes at the baseline of the study.
After six weeks of calorie restriction, the mean BMI was reduced from 31.07 (±4.39) kg/m 2 to 29.76 (±4.2) kg/m 2 and the mean weight was reduced from 81.68 (±13.11) kg to 78.41 (±12.96) kg. The effects of the six weeks intervention on serum HDL and HDL subfractions concentration and LCAT activity in the total population, as well as in the two genotype groups, are summarized in Table 2. In the total population, without considering the genotype, the mean concentration of HDL and HDL2 decreased significantly (P < 0.001 and P = 0.03, respectively), while LCAT activity increased, but not significantly, after weight loss. However, when compared within the groups, the serum HDL2 level significantly reduced only in the TT/TC group (P = 0.005), while within the CC group the reduction of HDL3 was significant (P = 0.04). The level of HDL decreased significantly in both groups (P = 0.003 in TT/TC group and P = 0.04 in CC group). The mean difference of variables between the two genotypes before and after adjusting for physical activity and changes of BMI has been shown in Table 3. There are statistically significant differences in the TT/TC genotype group as compared to the CC genotype group. There were inverse correlations between the mean difference values of serum APOA-II, ratio of APOA-II to APOA-I and HDL3 in the total population and CC genotype. In contrast, these correlations are positive in the TT/TC genotype group (Table 4).

Discussion
To the best of our knowledge, this study is the first attempt to investigate the effects of weight loss in -256 T>C polymorphism and to gain a better understanding of the differences between genotype in response to the same modification. In the present study, HDL concentration decreased significantly in both groups after intervention; however, the comparison of HDL subfractions showed that HDL2 decreased significantly in the TT/TC group, while HDL3 decreased significantly in the CC genotype group. Weight loss is an important regulator of dyslipidemia [24]. When analyzing the effect of weight loss by calorie restriction on the HDL level, a meta-analysis of 70  [25]. The responsible mechanism for reduced HDL during calorie restriction may consist of an increased degradation and/or decreased production of HDL particles, as well as calorie restriction, causing a decrease in the production of chylomicron-derived HDL particles [26]. The result of the Genetics of Coronary Artery Diseases in Alaska Natives (GOCADAN) study indicated that individual HDL subfractions may be influenced by different genetic variations [27]. As mentioned earlier, the replacement of T with C at the position −256 of APOA-II resulted in a decreased expression of APOA-II in the liver, while different genotypes of APOA-II have different serum APOA-II levels [13]. The effects of APOA-II on HDL size for the first time were observed in studies of inbreed strains of mice. In these studies, as the level of APOA-II increased, the size of HDL increased continuously [28,29]. In addition, the studies of APOA-II transgenic mice confirmed the positive relation between higher levels of APOA-II and increased HDL size [30,31].
Although these studies used other methods such as Electrophoresis [30] and Gel-filtration chromatography [31], their results are in agreement with the present study. We observed that, after weight loss, HDL3 was reduced significantly in the CC genotype group; on the other hand, the reduction of HDL2 in the CC genotype group (P = 0.95) in comparison with the TT/TC genotype group (P = 0.005) was much less. This shows that, after weight loss, the CC genotype shifts toward largersized HDL ( Table 2). The ratio of APOA-II to APOA-I reduced significantly in the TT/TC group, while it did not change in the CC genotype (Table 2). Also, a negative correlation between APOA-II, ratio of APOA-II/ APOA-I and HDL3 was seen in the CC genotype group (Table 4). It seems that APOA-II effects the HDL size by reducing the HDL ability to act as a substrate for HL [32]. In addition, the ratio of APOA-II to APOA-I is one of the important parameters of HDL function. Hedrich et al. claimed that, when the ratio of APOA-II to APOA-I increases, the HDL size becomes larger due to the inhibition of HL [32]. They have proposed that, in HDL with higher ratios of APOA-II to APOA-I, hydrolysis of phospholipid triglyceride by HL is reduced [32]. In addition, LCAT esterified cholesterol on the surface of HDL3 and the cholesterol esters are more hydrophobic than free cholesterol and they immigrate into the core of   the lipoprotein, with the resulting of generation HDL2 particles in the plasma [8]. In the present study, LCAT activity increased after the intervention, but this was not significant. The results of other studies demonstrated increased LCAT activity after weight loss in dogs [33], and decreased significantly LCAT mass and activity at one month after weight loss by gastric bypass surgery in obese women [34]. The difference between the results might be due to different methods for measuring LCAT activity, where they used radioactivity [33] or measuring increased cholesterol ester in plasma as a result of LCAT activity [34]. As is shown in Table 4, there is a nonsignificant negative correlation between APOA-II and LCAT activity. APOA-I is an activator of LCAT, whereas APOA-II neither activates nor inhibits LCAT activity [35]. In addition, APOA-II is more hydrophilic than APOA-I and can inhibit LCAT activity indirectly by displacing APOA-I from the surface of HDL, or by hindering LCAT binding to APOA-I on HDL and inhibit cholesterol esterification [36]. However, to justify the result of HDL subfractions in this study, there is a need to measure other enzymes related to HDL metabolism. It has been proposed in other studies that APOA-II can alter other enzymes related to HDL, including the inhibition of cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP) and Endothelia lipase (EL) [37], which can have an influence on the maturation process of HDL subclasses. However, it was not possible for us to measure these enzymes. The evidences regarding the pro-atherogenic or antiatherogenic effect of HDL subfractions in humans are conflicting and confusing, and it is not clear whether it is related to HDL size or apolipoprotein composition, density, mobility or a combination of their properties [38]. In this regard, some studies indicate that smaller, dense HDLs are associated positively with CVD [39]. In contrast, Kontush et al. demonstrated that small, dense HDL has a potential role in protecting LDL from oxidation [40]. In another study, it was demonstrated that both HDL2 and HDL3 protect against CVD [41]. However, most studies that investigate the therapeutic modulation of HDL subfraction demonstrate that larger HDL particles are more protective against CVD [42]. In sum, according to a review article by Rizzo et al., further clinical investigations are required to clarify the physicochemical and functional heterogeneity of HDL subfractions [38].

Conclusion
A comparison of mean changes of variables within two genotype groups showed that weight loss resulted from a reduction of HDL in both groups. However, in C homozygote carriers, it was shown that HDL3 reduced significantly and leads to a general shift toward larger size HDL subfractions after intervention, while in T allele carriers HDL2 decreased significantly and weight loss leads to shift toward smaller size HDL subfractions.

Study limitation
The present study has some limitations. First, randomization was not carried out when selecting the patients and placing them in the two genotype groups, which might have had an influence the results. Second, due to the limitation in patients in the CC genotype group and exclusion criteria for selecting patients, there was no control group in each genotype. Third, due to financial limitations, we did not measure other enzymes related to HDL size such as CETP, HL, EL and PLTP. Finally, we have examined a short-term weight loss during a six-week period; therefore, a more prolonged course of weight maintenance could show the full benefits of weight loss and corroborate the findings.