Proteomic profiling of intestinal prechylomicron transport vesicle (PCTV)-associated proteins in an animal model of insulin resistance (94 char)

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Proteomic profiling of intestinal prechylomicron transport vesicle (PCTV)-associated proteins in an animal model of insulin resistance (94 char) Diana M. Wong a,b , Jennifer P. Webb a,b , Paul M. Malinowski c , Elaine Xu a , Joseph Macri c,d , Khosrow Adeli a,b,⁎ a

Molecular Structure and Function, Division of Clinical Biochemistry, Department of Pediatric Laboratory Medicine, Research Institute, The Hospital for Sick Children, 555 University Ave, Toronto, ON, Canada M5G 1X8 b Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 c Department of Laboratory Medicine, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, 1200 Main Street West, Hamilton, ON, Canada L8N 3Z5 d Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada L8L 2X2

AR TIC LE I N FO

ABS TR ACT

Article history:

Intestinal overproduction of apolipoprotein B (apoB)-48-containing chylomicrons is

Received 31 August 2009

increasingly recognized as an underlying factor in metabolic dyslipidemia commonly

Accepted 17 January 2010

observed in insulin-resistant states. Enhanced chylomicron assembly and secretion has been documented in animal models of insulin resistance, but the underlying mechanistic factors are unknown. Chylomicron assembly occurs through a series of complex vesicular

Keywords:

interactions involving prechylomicron transport vesicles (PCTVs), which transport lipids from

Metabolic dyslipidemia

the endoplasmic reticulum (ER) to the Golgi. We report proteomic profiles of PCTVs isolated

Microsome

from the enteric ER in the small intestine of the fructose-fed hamster, an established model of

Endoplasmic reticulum

diet-induced insulin resistance. Using 2D gel electrophoresis and tandem mass spectrometry,

Prechylomicron transport vesicle

PCTVs were characterized and proteomic profiles of PCTV-associated proteins from insulin-

(PCTV)

resistant and control enterocytes were developed, with the intention of identifying proteins

Proteomics

involved in insulin signaling attenuation and lipoprotein overproduction. A number of PCTV-

2D gel electrophoresis

associated proteins were found to be differentially expressed including microsomal

Mass spectrometry

triglyceride transfer protein (MTP), apoB-48, Sar1 and VAMP7. We postulate that altered expression of Sar1 and MTP may contribute to increased chylomicron assembly in the fructosefed hamster. These findings have increased our understanding of the intracellular assembly and transport of nascent chylomicrons and potential cellular factors responsible for lipoprotein overproduction in insulin-resistant states. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Metabolic dyslipidemia is defined as an abnormal lipid and lipoprotein profile commonly observed in insulin-resistant

states. It is the most common complication of insulin resistance and type 2 diabetes, and is an important risk factor for the development of atherosclerosis and cardiovascular disease. Metabolic dyslipidemia is characterized by hypertriglyceridemia,

⁎ Corresponding author. Division of Clinical Biochemistry, Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Ave, Toronto, ON, Canada M5G 1X8. Tel.: +1 416 813 8682; fax: +1 416 813 6257. E-mail address: [email protected] (K. Adeli). 1874-3919/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2010.01.010

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low HDL cholesterol, high LDL cholesterol, and elevated levels of free fatty acids (FFA) [1]. It is also well established that patients exhibiting these features are at an increased risk for atherosclerosis [2]. In insulin resistance, there is a decreased biological response to normal concentration of circulating insulin. Recent studies suggest that perturbations in key molecules of the insulin signaling cascade in the liver may underlie abnormal hepatic lipid and lipoprotein metabolism leading to metabolic dyslipidemia. Overexpression of key phosphatases, downregulation and/or activation of key protein kinase cascades lead to a state of mixed hepatic insulin resistance and sensitivity. The signaling changes, in turn, cause an increased expression of sterol regulatory element binding protein (SREBP) 1c, induction of de novo lipogenesis and a higher activity of microsomal triglyceride transfer protein (MTP). Altogether, these signaling changes, along with high exogenous FFA flux, collectively stimulate the hepatic overproduction of apoB-containing lipoprotein particles in the form of VLDL, the enteric overproduction of chylomicrons, the reduction in anti-atherogenic HDL cholesterol, and elevation of atherogenic LDL cholesterol in the plasma [3]. An increase in these highly atherogenic lipoproteins creates risk factors for coronary heart disease [4]. The dyslipidemia that accompanies insulin resistance is increasingly being considered a postprandial phenomenon. There is growing evidence that intestinal lipoprotein overproduction is a major contributor to the fasting and postprandial lipemia observed in insulin-resistant states [5]. Intestinal oversecretion of apoB-48-containing lipoproteins, accompanied by enhanced intestinal lipid synthesis in the form of cholesterol esters and TG, have been observed in insulinresistant states stimulated by chronic fructose feeding. Basal levels of lipoprotein secretion upon fructose feeding may be increased due to increased de novo lipogenesis, as well as increased MTP availability (reviewed in [1]). Insulin-resistant Syrian gold hamsters fed a high fructose diet, when compared to chow-fed hamsters, showed a significant shift toward secretion of larger, less dense chylomicrons [6]. Metabolic dyslipidemia is thus a common feature of insulin-resistant states and appears to arise from aberrant metabolism of apoBcontaining lipoproteins produced not only by the liver, but also the small intestine. The dysregulation of lipid absorption and metabolism by the small intestine in insulin-resistant states may underlie critical changes in lipid homeostasis that lead to the development of metabolic dyslipidemia. The rate-limiting step in the transport of dietary fat across the enterocyte is the generation of the prechylomicron transport vesicle (PCTV) from the ER. A prechylomicron particle has been recently discovered in the small intestine of the rat [7,8]. The particle is assembled in a specialized vesicular compartment containing a phospholipid coat, termed PCTV. The phospholipids coat is necessary for transport from the ER to the Golgi apparatus to protect from lipases. Once the vesicle reaches the Golgi, the coat is removed and a mature chylomicron is formed [10]. Although the PCTV has been studied extensively in cell biology systems to determine the mechanism of lipid transport [9–11], the proteins contained in the PCTV, or its proteome, and the profile of its lipid contents, have not been characterized. Recently, we have been able to isolate PCTVs from normal, chow-fed hamsters, and have performed proteomic analysis [12]. A number of proteins were identified

including proteins involved in the formation, transport, lipidation, and assembly of chylomicron particles. In the present study, we extend these findings by employing multiple proteomic techniques in an attempt to fully characterize the PCTV proteome in both normal and insulin-resistant states. We were able to successfully purify PCTVs from the enterocytes of normal and fructose-fed/insulin-resistant hamsters, perform proteomic analysis of intestinal PCTVs and characterize known and unknown proteins within the PCTV proteome in both fasting and postprandial states.

2.

Materials and methods

2.1.

Animal surgery and intestine collection

Male Syrian golden hamsters (Mesocricetus auratus) (Charles River, Montreal, QC, Canada) were maintained on a chow diet (C) or diet where 60% of calories come from fructose (F) (Dyets Inc, Bethlehem, PA) for two weeks, and fasted 8h overnight prior to sacrifice. The animals in the fed groups were maintained on a chow diet (CL) or fructose (FL) and given a bolus (200 μL) of olive oil 30 min prior to sacrifice. The lipolysis of olive oil results in many species of fatty acids and monoacylglycerol, and of these, oleic acid is the most predominant. In hamsters, we have shown that olive oil induces the formation and secretion of intestinal lipoproteins [13]. Therefore, we chose olive oil to represent the postprandial condition as it is physiologically relevant. The surgery was performed essentially as described in Haidari et al. [13]. In short, the animals were anesthetized with a continuous flow mixture of isoflurane, and oxygen (Baxter, Toronto, ON). An incision was made up in the middle of the animal's stomach and abdomen. The jejunum, approximately 10 cm of the proximal end of the small intestine, was excised, and rinsed several times with 1× PBS. The intestine was then longitudinally cut and then subsequently cut into 2 cm fragments.

2.2.

Primary enterocyte isolation

The intestine fragments were immersed in Cell Recovery Solution (BD Biosciences, Bedford, MA) for 1 h at 4 °C. The fragments were then washed with 1× PBS with agitation on an orbital shaker at 4 °C and gently tapped with a sterile dissection instrument. The PBS containing suspended villi was collected and pelleted with centrifugation at 100 g.

2.3.

Subcellular fractionation

The procedures for microsome and cytosol isolations were performed as described by Kumar and Mansbach [7] with slight modifications exactly as described in [12]. For radiolabeled microsomes, 20× 106 enterocytes were resuspended in Buffer B (137 mM NaCl, 1.5 mM EDTA, 11.5 mM KH2PO4, 8 mM Na2HPO4, 2.2 mM KCl, 0.5 mM DTT, and pH 7.2) supplemented with 10 mM glutamine and protease inhibitor cocktail (PI) (Roche, Mississauga, ON). In a separate tube, 50 μCi [3H]-oleic acid (PerkinElmer, Boston, MA) was added to a 10 mM BSA:oleate complex and then added to enterocytes. Cells were incubated at 37 °C for 30 min with occasional mixing, and centrifuged at 1000 g. Cell pellet was washed twice with 2% BSA in PBS. The pellet was resuspended in

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Buffer A (10 mM HEPES, pH 7.2, 0.25 M sucrose, and 2 mM EDTA) with PI. Non-radiolabeled cells were suspended in Buffer A directly. Cells were homogenized using the Parr bomb cell disruption vessel (Parr Instruments, Moline, IL) at 800 psi N2pressure. The homogenate was spun at 8500 g for 10 min at 4 °C in the Beckman Optima LE-80 ultracentrifuge (Palao Alto, CA). The post-nuclear supernatant (PNS) was then spun at 100 000 g for 3 h at 4 °C. Cytosolic supernatant was retained from nonradiolabeled microsomal isolations only. The cytosolic supernatant was dialyzed to Buffer C (25 mM HEPES, pH 7.2, 125 mM KCl, 2.5 mM MgCl2, 2 mM DTT, and 0.5 mM EDTA) and concentrated using an Amicon Ultra 15 Centrifugal tube (Millipore, Billerica, MA) at 4000 g until volume was less than 1 mL. The microsome pellet was resuspended in 3 mL of 1.22 M sucrose solution. In an ultracentrifuge tube, the microsomal suspension was carefully overlayed with 2.6 mL each of 1.15 M, 0.86 M and 0.25 M sucrose solutions in 10 mM HEPES, pH 7.2. The sucrose step gradient was spun at 82 000 g for 3 h at 4 °C (SW41Ti rotor). The gradient was fractionated by unloading 23×500 uL aliquots from the top of the tube. For highly purified ER, the density of the ER-containing fraction was adjusted to 1.22 M sucrose and spun again at 82 000 g for 3 h at 4 °C.

2.4.

Budding assay

The procedure for isolation of prechylomicron transport vesicles (PCTVs) was performed as described by Kumar and Mansbach [7]. In brief, 500 μg ER was incubated with 1000 μg cytosol and an ATP regenerating system (final concentrations 1 mM ATP, 5 mM phosphocreatine, 5 units phosphokinase, 1 mM MgCl2, 1 mM CaCl2, 0.5 mM DTT in 10 mM HEPES, and pH 7.2) at 35 °C for 35 min. The PCTV suspension was loaded onto a continuous gradient of 0.1–1.15 M sucrose in 10 mM HEPES, pH 7.2 and spun at 80 000 g for 95 min at 4 °C. The top 100 μLcontaining cytosolic proteins was discarded. The rest of the gradient was fractionated by unloading 23 × 500 μL aliquots from the top of the tube. Radiolabeled PCTVs could be detected by the incorporation of [3H]-oleic acid into [3H]-triglyceride in PCTVs. PCTVs could also be detected via immunoblot with anti-VAMP7 antibody, a marker of PCTVs [14].

2.5.

0.025% w/v deoxycholate, and 1.2 M KCl, PI) for 30 min on ice. BSA was added to a final concentration of 0.5% w/v. The chylomicron contents then underwent ultracentrifugation using the following gradient: 4 mL d 1.10 kg/L KBr overlayed with 3 mL d 1.065 kg/L KBr overlayed with 3 mL d 1.020 kg/L KBr overlayed with 2 mL d 1.006 kg/L KBr [13], to isolate large and small chylomicron, VLDL, LDL and HDL-sized particles. These methods are described in detail by Haidari et al. [13].

2.7.

2-D difference gel electrophoresis (2-DIGE)

PCTVs were concentrated to 10 mg/mL using Nanosep 10K Omega micro concentrators (Pall Corporation, Ann Arbor, MI). 50 μg of PCTVs from each diet condition, as well as a pool consisting of equal amounts of each sample used as an internal standard for quantitative comparisons [15] were labeled with Cy dyes according to the manufacturer's protocol (CyDye DIGE Fluor Labeling Kit, GE Healthcare, Piscataway, NJ). To avoid preferential labeling efficiency, Cy3 dye was used to label half of the samples from each group and the other half with Cy5 dye. Cy2 dye was used to label the internal standard sample. In brief, 400 pmol of Cy dye in 1 μL of anhydrous N,Ndimethylformamide was added to 50 μg of protein. After 30 min on ice in the dark, the reaction was quenched with 10 mmol/L lysine. The samples were then incubated for a further 10 min. Samples were combined according to the experimental design using 50 μg protein per Cy dye per gel, and solubilized in IEF sample buffer (9 M urea, 2 M thiourea, 1% w/v CHAPS, 2% DTT, 2% pharmalytes, and pH 3–10). The PCTV samples, together with an aliquot of the internal standard pool, were then separated by 2D-PAGE using reagents and equipment from GE Healthcare. Fluorescence images of the gels were obtained on a Typhoon 9400 scanner (GE Healthcare). Both image analysis and statistical quantification of protein levels were performed using DeCyderDIA (Difference In-gel analysis) 2D v.6.00.28 software (GE Healthcare). Protein spots were normalized to the internal standard and significant differences of 2-fold or more in-gel patterns using Student's T-test identified differentially expressed proteins. Spots of interest were excised from the gel using an automated spot picker and identified using MALDI-TOF.

Western blotting 2.8.

Equal volumes of samples and whole cell lysate as a positive control were prepared for mini-gel electrophoresis on 8% SDSPAGE and transferred to PVDF membrane (PerkinElmer, Boston, MA). The membrane was immunoblotted with an antibody against a protein of interest: anti-calnexin, antienolase, anti-LAMP1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) ; anti-GS28 and anti-Sar1 antibodies (Stressgen Bioreagents, Ann Arbor, MI). The anti-VAMP7 antibody was a gift from Dr. Charles Mansbach II at the University of Tennessee [8]. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ) were activated by ECL and developed to recording film.

2.6.

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Chylomicron density centrifugation

Chylomicrons were released from PCTVs by carbonate treatment to open up membranes (1 mL sodium carbonate, pH 11.5,

MALDI-TOF mass spectrometry (MS)

The in-gel trypsin digestion and C18 ZipTip (Millipore) sample cleanup followed the protocol described in Bagshaw et al. [16]. Protein mass spectra were acquired using an Ettan MALDITOF/Pro mass spectrometer (Amersham Biosciences) using 0.4 μL out of 5 μL of the digest mixture of alpha-cyano 4 hyroxy-cinnamic acid (4-CHCA) matrix. Data were obtained in reflectron mode using conditions described previously by Macri et al. [17]. For spectrum calibration, trypsin peaks were used (one mostly detected is 842.52 Da peak). The parameters for calibrant detection were: tolerance 1 m/z S/N 3 with minimum quality value of 0.6. The peptides were detected using the centroid algorithm, with mass range of 800–3500 Da. The mass tolerance was 0.2 m/z for Mono and 1 m/z for Average masses with minimum quality value of 0.6 and S/N of 3. Peaks used for analysis were the ones automatically generated by the software so the rejection parameters used

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would be the ones applied by the software. The resulting peptide map was searched using Profound (version 2006.01.03) protein software against the NCBInr database.

2.9.

Direct liquid chromatography tandem MS (LC-MS/MS)

50 μg total PCTV protein was dissolved in 6 M urea/2 M thiourea solution with PI. The PCTVs were lysed and DTT was added to sample to give a final concentration of 40 mM DTT. Following a 30 min incubation, iodoacetamide was added to give a final concentration of 150 mM iodacetamine. LysC (Promega, Madison, WI) was prepared according to the manufacturer's protocol and incubated with sample for 3 h. Sample was also digested with trypsin overnight at room temperature. Sample was subject to ZipTip cleanup and eluted sample in 0.1% TFA/50% acetonitrile solution was injected directly into a Thermo Electron LCQ Deca XP (Thermo Scientific, San Jose, CA) coupled to an Agilent capillary HPLC 1100 series (Agilent Technologies, Palo Alto, CA, USA) and C18 column (150 mm × 4.6 mm, 5 μm, Zorbax Extend). Sequencing data was analyzed using the ProteinProspector MS-Tag database search engine.

2.10.

Database searching

The LC-MS/MS spectra were extracted by using Bioworks version 2.0. All MS/MS samples were analyzed using X! Tandem (http://www.thegpm.org; version 2006.09.15.5). X! Tandem was set up to search the International Protein Index (v.3.22, subset rodent, 92 866 entries) assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of +/−3 Da. Iodoacetamide derivative of cysteine was specified in X! Tandem as a fixed modification. Pyro-glu from E of glutamic acid, pyro-glu from Q of glutamine, deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, sulphone of methionine, tryptophan oxidation to formylkynurenin of tryptophan and acetylation of lysine and the N-terminus were specified in X! Tandem as variable modifications. The maximum number of missed cleavages allowed was 1.

2.11.

Criteria for protein identification

Scaffold (version Scaffold_2_00_06, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability as specified by the Peptide Prophet algorithm [18]. Protein identifications were accepted if they could be established at greater than 50.0% probability and contained at least 1 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [19]. Proteins that contained similar peptides and could not be differentiated based on MS/ MS analysis alone were grouped to satisfy the principles of parsimony.

2.12.

manufacturer's protocol (Applied Biosystems, Foster City, CA). In brief, 100 μg protein of each condition was added to 20 μL of dissolution buffer (25 mM triethylammonium bicarbonate [TEAB], pH 8.5, 8 M urea, 2% Triton X-100, and 0.1% SDS). Samples were reduced with 4 mM Tris[2-carboxyethyl] phosphine [TCEP] at 60 °C for 1 h and then cysteines were blocked with 8 mM methyl methanethiosulfonate at 20 °C for 10 min. The samples were diluted with 50 mM TEAB, pH 8.5, so that the urea concentration was less than 1 M. The samples were individually digested with 5 μg trypsin with 1 mM CaCl2 overnight at 37 °C. One unit of each iTRAQ label was dissolved in 75% ethanol and added to the samples as follows: 114-C, 115-CL, 116-F, 117-FL. The sample and label were incubated for 1 h at room temperature. The labeled peptides were pooled and subject to sample cleanup via strong cationic exchange chromatography as well as reverse-phase chromatography before direct LC-MS/MS. The combined iTRAQ sample was injected directly into Applied Biosystems/MDS Sciex API QSTAR XL Pulsar coupled with an Agilent nano HPLC 1100 series and C18 column (150 mm × 4.6 mm, 5 μm, Zorbax Extend) for reverse-phase liquid chromatographic separation of peptides. The obtained MS/MS fragmentation data were used to search the NCBI database using the MASCOT search engine for identification and proQUANT searching for peak quantitation.

2.13.

Transmission electron microscopy (TEM)

Isolated PCTVs from a budding assay were fixed onto a Formvar-coated grid and negatively stained with 1% phosphotungstic acid or 2% uranyl acetate. A JEM 1230 transmission electron microscope operated at 80 kV b was used. Digital images of 1024 × 1024 pixels were acquired with a CCD camera attached to a microscope. Representative samples of small intestine were immersed in the perfusing fixative (1% glutaraldehyde, 4% paraformaldehyde) for ultrastructural morphology by electron microscopy. Further processing included postfixation in 1% osmium tetroxide, a positive stain. The specimens were then dehydrated in a graded series of acetone from 50% to 100% and subsequently infiltrated and embedded in Epon-Araldite epoxy resin. The processing steps from post fixation to polymerization of resin blocks were carried out in a microwave oven, Pelco BioWave 34770 using similar procedures, with slight modification, as recommended by the manufacturer. Ultrathin sections were cut with a diamond knife on the Reichert Ultracut E. Sections were stained with uranyl acetate and lead citrate before being examined in the electron microscope (JEM-1230). Digital electron micrographs were acquired directly with a 1024 × 1024 pixels CCD camera attached to the microscope.

3.

Results

3.1. Isolation and visualization of PCTVs and whole intestine from chow-fed and fructose-fed hamsters

ITRAQ labeling

PCTV protein from each condition of the 4 diet conditions was labeled with one iTRAQ label (114–117) according to the

PCTVs from four conditions, control, chow-fed animals under fasted (C) or fed (CL), or fructose-fed under fasted conditions (F) or fed (FL) conditions, were viewed via transmission

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electron microscopy (TEM) using a phosphotungstic acid stain, as shown in Fig. 1. The outer phospholipid layer can be seen circling outside of the PCTV. PCTVs were found to range from 100 to 500 nm in diameter; consistent with the size of chylomicrons. PCTVs from the CL, F and FL conditions appeared very similar to the control (C), as seen in Fig. 1. There were some minor differences but no major size shift was observed. In addition, whole small intestinal pieces of the Syrian golden hamster were visualized to examine PCTV formation in the enterocyte. Pieces of intestine were immediately fixed with formaldehyde, post-surgery, to visualize PCTV formation in the resting (no fast) and postprandial (fat-loaded) states. Intestines from solely fasted hamsters were not used due to a high level of cell death. The chow-fed intestine can be seen in Fig. 2A, where PCTV formation appeared to be minimal. However, in Fig. 2B, in fat-loaded, CL intestine, numerous PCTVs were visible. The large circles >500 nm without a membrane outer layer appear to be fat droplets in Fig. 2B. Similar to the chow-fed animals, the fructose-fed (fasted; no fat load) intestine had almost no PCTVs visible, as seen in Fig. 2C. There appeared to be more PCTVs in the FL intestine compared to the F intestine, and a greater abundance of fat droplets in the FL intestine (2D), compared to the CL intestine (2B).

[3H]-triglyceride counts than fat-loaded hamsters, likely due to an extremely large pool of unlabeled fatty acid in the intestine of fat-loaded hamsters leading to considerable dilution of labeled fatty acid pool and thus a reduced amount of label incorporation into PCTV triglyceride (see discussion below).

3.2. Differential incorporation of [3H]-oleic acid into PCTVs from chow-fed and fructose-fed/insulin-resistant hamsters

3.4. Comparative proteomic analysis of PCTVs by direct LC-MS/MS, 2DIGE, and iTRAQ labeling

A major goal of this project was to determine the differential expression of PCTV-associated proteins in normal and insulin-resistant states, under both fasting and postprandial conditions. The fructose-fed hamster model of insulin resistance was employed for these studies. Radiolabeled PCTVs were isolated from enterocytes of hamsters under the following conditions: chow-fed, fasted (C); chow-fed, fatloaded (CL); fructose-fed fasted (F); and fructose-fed, fatloaded (FL). The animals receiving a fat load (200 μL body weight olive oil) were sacrificed 30 min post intragastric gavage. The PCTVs were budded and isolated as described in Materials and methods. The top of the gradient (fractions 1–5) contains PCTVs (Fig. 3), and the bottom of the gradient contains ER (fraction 21). Interestingly, fructose-fed hamsters had PCTVs with higher [3H]-triglyceride counts than chow-fed hamsters (Fig. 3). Surprisingly, however, incorporation of [3H]oleic acid into PCTVs budded from fasted hamsters gave more

PCTVs from all four dietary conditions were analyzed using direct LC-MS/MS. Sequencing data was analyzed using the ProteinProspector MS-Tag database search engine to identify proteins, as seen in Table 2. Several groups of proteins were found to be associated with PCTVs under all four dietary conditions, namely chaperones, vesicular transport proteins, cytosolic proteins and lipid related proteins. However, PCTVs from fructose-fed hamsters expressed proteolytic proteins, which were not expressed in chow-fed hamsters. Fructose-fed hamsters also expressed more chaperone proteins than chowfed hamsters. 2-D difference gel electrophoresis (DIGE) was conducted to examine the differential expression of proteins under the four conditions. Sample 2-DIGE gels can be seen in Figs. 5 and 6 for comparison of chow and fructose diets, respectively. The higher expression of proteins in the C and F conditions can be seen by the abundance of green, and vice versa for the CL and

3.3. Differential protein expression in PCTVs from chow-fed and fructose-fed hamsters Specific proteins were examined in the C, CL, F and FL conditions using immunoblot to determine the differential expression of Sar1, VAMP7, MTP and apoB-48. Densitometry was performed on the exposed films for each protein blot and graphed, as seen in Fig. 4. A paired 2-tailed Student's T-test was used to determine whether CL, F, and FL dietary conditions were significantly changed from C, considered the control. For the CL condition, apoB-48, MTP, Sar1 and VAMP7 were all significantly changed from C. For the F condition, apoB-48, MTP and Sar1 were significantly changed from C. According to a 2-way ANOVA, the fructose feeding induced significant changes in Sar1 and VAMP7 (proteins associated with vesicular transport), and the postprandial state induced significant changes in MTP and apoB-48 (proteins associated with chylomicron assembly). A summary of the data is shown in Table 1.

Fig. 1 – Visualization of PCTVs using TEM. A 5 μL drop of PCTV from C (A), CL (B), F (C), and FL (D) dietary conditions was pipetted onto a Formvar-coated grid and stained with phosphotungstic acid. The samples were analyzed in a JEM 1230 transmission electron microscope (JEOL USA, Inc.) operated at 80 kV. Digital images of 1024 × 1024 pixels were acquired with a CCD camera (AMT Advantage HR camera system, AMT) attached to a microscope.

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Fig. 2 – Visualization of PCTVs in intact enterocytes. A piece of small intestine from chow-fed (A), chow-fed fat-loaded (B), fructose-fed (C) or fructose-fed fat-loaded (D) hamsters was fixed in 10% formalin and sliced into small sections under light microscope. Small sections were immersed in perfusing fixative and stained with osmium. Following thin layer sectioning, the intestine was stained with phosphotungstic acid and viewed under TEM. The arrows show representative PCTVs. The black bar represents 500 nm.

FL conditions by an abundance of blue. Comparative analysis of the differential expression of proteins in all four dietary conditions was performed by DeCyder-DIA (Difference In-gel analysis) 2D software to give a quantitative result. A minimum 2-fold change was used to compare C vs. CL, F vs. FL, C vs. F, and CL vs. FL. The differentially expressed proteins were identified using a separate gel, containing 750 μg total protein of all four conditions, which was overlayed with the 2-DIGE gels. The spots from the ID gel were cut out and applied to MALDI-TOF to identify the proteins. The identification of proteins and their differential expression under the four conditions can be seen in Table 3. A Student's T-test of p < 0.05 was used to determine the significance of each condition based on four gels. Over 130 proteins were differentially expressed; however, not all proteins could be identified based on protein abundance. Many groups of proteins were found to be differentially expressed including vesicular

transport proteins, intestinal resident proteins, chaperones, proteins involved in fructose metabolism, and proteins associated with chylomicron assembly. Vesicular transport proteins appeared upregulated in the CL condition compared to C. The onset of insulin resistance did not yield significant protein changes more than 2-fold in the fasting states. Interestingly, there was an upregulation of chaperones in the FL condition compared to F. A more sensitive method to detect differential expression of proteins in PCTVs of different diet conditions was by iTRAQ labeling. PCTV protein from C, CL, F and FL was labeled with iTRAQ labels 114, 115, 116 and 117, respectively. Sample mixture was applied to direct LC-MS/MS. ProQUANT software determined the relative amount of each protein in the mixture as well as significant changes between two conditions. The peptide sequences were searched through both mouse and rat databases. Although over 196 distinct peptides were found

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Fig. 3 – Radiolabeled PCTV fractionation of budding assays of ER isolated from hamster enterocytes. 500 μg ER protein in buffer (30 mM HEPES, 2.5 mM Mg acetate, 30 mM KCl, and pH 7.3) containing an ATP regenerating system [final concentrations, 1 mM ATP, 5 mM phosphocreatine, 5 units creatine phosphokinase, 1 mM CaCl2, and 0.5 mM DTT in 10 mM HEPES, pH 7.2], and 1000 μg cytosolic protein at 37 °C for 30 min. The suspension was then loaded onto a continuous sucrose gradient (0.1–1.15 M sucrose, 10 mM HEPES, and pH 7.3) and centrifuged for 80 000 g for 95 min at 4 °C (n = 3). Fraction 1 represents the top (least dense) and fraction 21 represents the bottom (most dense) of the gradient.

and 54 unique proteins identified from a 400 μg total protein, less than 20 proteins were found to be significantly changed, according to a paired 2-tailed Student's T-test performed by

the proQUANT software. Out of these significantly changed proteins, only 8 had a 2-fold or greater change, as seen in Table 4. The group of proteins that was significantly differentially expressed coincided with the results of 2DIGE. Similar to the 2DIGE results, there were few proteins that were significantly different when comparing the F and C conditions. New proteins that were not detected to be differentially expressed in 2DIGE included lactate dehydrogenase, keratin complex 2, and hemoglobin beta subunit. In addition, though not exactly the same, some iTRAQ results were very close to the 2DIGE results: when comparing FL to F condition, prolyl 4-hydroxylase increased 1.97 fold as determined by iTRAQ versus 2.38 fold as determined by 2DIGE. Similarly, when comparing FL and CL conditions, aldolase 2 increased 2.90 fold with iTRAQ versus 2.13 fold with 2DIGE.

4.

Discussion

Insulin resistance was induced in male Syrian Golden hamsters by feeding a high fructose diet for two weeks.

Fig. 4 – Differential expression of apoB-48, MTP, Sar1 and VAMP7 in PCTVs. (A) 10 μg total protein was analyzed using SDS-PAGE (n = 3). Densitometry was performed on films exposed from immunoblots for apoB-48, MTP, Sar1 and VAMP7 in PCTV protein. Significant changes in each condition from the control (C) are indicated by *, as determined by p < 0.05 using a one-way ANOVA. (B) Representative immunoblots for apoB-48, MTP, Sar1 and VAMP7.

Table 1 – Proteins differentially expressed in PCTVs from chow-fed hamsters in the fed state (CF) compared to chow-fed hamsters under fasted conditions (C), and from fructose-fed hamsters compared to chow-fed hamsters under ambient conditions, as determined by immunoblotting and densitometry (n = 3). Significant fold changes are shown, as determined by p ≤ 0.05 using a one-way ANOVA. Protein identity

CL/C

F/C

Sar1 VAMP7 ApoB-48 MTP

2.13 2.00 2.75 2.00

2.33 2.20 2.11

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Table 2 – Proteins positively identified in the washed PCTV fraction from chow-fed hamsters under fasted conditions (C), chow-fed hamsters that were fat-loaded (CL), fructose-fed animals under fasted conditions (F) and fructose-fed hamsters that were fat-loaded (FL) using direct LC-MS/MS (n = 3). PCTV-associated proteins

NCBI database Mr accession no. (kDa)

Sequence coverage

Cytoskeleton proteins Adenylyl cyclase-associated protein 1 (CAP 1) Actin, beta Actin, gamma 1 Alpha actinin 4 Cofilin 1 Profilin Tubulin alpha-3 chain Tubulin, beta-2

gi|59709467 gi|1351867 gi|94373495 gi|11230802 gi|8393101 gi|42476144 gi|27762594 gi|22165384

51.86 42.03 42.11 105.31 25.29 15.15 50.75 50.24

Vesicular transport proteins Annexin A4 Clathrin, heavy polypeptide (Hc)

gi|7304889 gi|51491845

36.21 16/319 (5%) 193.19 22/1765 (1%)

Lipid-binding proteins FABP1, liver FABP2, intestinal FABP3, heart FABP4, adipocyte FABP6, ileal/Gastrotropin Retinol binding protein 2, cellular Microsomal triglyceride transfer protein

gi|54306424 gi|6679737 gi|13162363 gi|54306428 gi|4033695 gi|78126163 gi|1709168

14.29 15.06 10.94 14.52 14.59 15.77 99.90

Chaperones HSP1/chaperonin Heat shock protein cognate 71 kDa HSP 70/78 kDa glucose-regulated protein (GRP) /BIP HSP 90-alpha (HSP 86) Prolyl 4-hydroxylase, beta polypeptide Peptidylprolyl isomerase A Valosin-containing protein/p97 Intestinal proteins Aconitase 1 / iron response element binding protein Keratin complex 1 Keratin, type II cytoskeletal 8 Plastin 1, intestinal Villin 1 Enzymes Glycolysis Aldolase B Triosephosphate isomerase Glyceraldehyde 3-phosphate dehydrogenase GAPDH Glycerol-3-phosphate dehydrogenase Phosphoglycerase kinase 1 Phosphoglyceromutase 2 Enolase Fructose metabolism Ketohexokinase Citric acid cycle Isocitrate dehydrogenase, cytoplasmic Malate dehydrogenase 1, NAD Malate dehydrogenase 2, NAD Nucleoside diphosphate kinase A Nucleoside diphosphate kinase B/expressed in nonmetastic cells 2 Methionine metabolism S-adenosylhomocysteine hydrolase Spermine synthase Other enzymes Aldehyde dehydrogenase family 1, subfamily A2

65/474 253/375 253/402 109/192 40/166 30/140 80/450 116/445

No. sequences of matched peptides

X X X X X X X X

X X X X X X X X

X X X X X X X X

X X X X X X X X

3 1 19 7 2 3 5 5

X X

X X

X X X X

1 1

(23%) (10%) (8%) (20%) (11%) (23%) (1%)

X X X X X X X

X X X X X X

X X X X X X X X X X X X

2 1 1 1 1 3 1

gi|40556608 gi|123647 gi|121570 gi|14270366 gi|19880309 gi|118098 gi|17865351

83.57 109/724 (15%) 70.87 102/646 (16%) 68.57 12/654 (2%) 85.20 29/733 (4%) 57.51 50/509 (10% 18.13 54/164 (33%) 89.99 102/806 (13%)

X X X X X X X

X X X X X X X

X X X X X X X

X X X X X X

7 7 1 4 3 3 5

gi|110347487 gi|27465585 gi|13624315 gi|34865784 gi|6678573

98.13 55/189 44.54 36/429 35.41 66/487 70.76 55/630 93.26 110/827

(6%) (8%) (14%) (9%) (13%)

X X X X X

X X X X X

X X X X X

X X X X X

2 4 4 5 9

gi|21450291 gi|6678413 gi|89573925 gi|57527919 gi|1730519 gi|8393948 gi|6679651

40.04 27.02 23.70 37.45 44.91 70.76 51.74

(24%) (28%) (24%) (11%) (19%) (16%) (12%)

X X X X X X X

X X X X X X X

X X X X X X X

X X X X X X X

5 5 4 3 4 3 1

gi|31982229

33.30 19/298 (6%)

X

X

X X

1

gi|89573969 gi|15100179 gi|31982186 gi|462690 gi|55926145

41.10 36.63 36.09 17.21 17.28

49/365 29/334 19/338 26/152 36/152

X X X X X

X X X X

X X X X X

X

3 2 1 2 2

gi|8392878 gi|76559933

36.42 32.39

11/432 (3%) 32/366 (9%)

X X

X X

X X X X

1 2

gi|14192935

55.22

14/501 (3%)

X

X

X X

1

23/98 13/132 10/133 21/103 14/127 31/124 13/895

86/364 69/249 53/222 39/349 78/417 41/253 50/434

(19%) (67%) (63%) (12%) (24%) (21%) (18%) (26%)

C CL F FL

(14%) (9%) (6%) (17%) (24%)

X X X

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Table 2 (continued) PCTV-associated proteins

NCBI database Mr accession no. (kDa)

Sequence coverage

C CL F FL

No. sequences of matched peptides

Enzymes Carboxylesterase 5 CNDP dipeptidase 2 (metallopeptidase) Lactate dehydrogenase A Serine (or cysteine) proteinase inhibitor, clade B, member 1a Creatine kinase M-type (Creatine kinase, M chain) (M-CK)

gi|2641986 gi|31981273 gi|8393706 gi|72255515 gi|6978661

62.38 59/559 (11%) 52.77 91/475 (19%) 95.90 114/332 (34%) 41.69 21/379 (6%) 43.31 151/381 (40%)

X X X X X

X X X X X

X X X X X

X X X X X

4 5 1 1 11

Translation factors EF1a/Eukaryotic translation elongation factor 1 alpha 1 EF2/Eukaryotic translation elongation factor 2 (EeF2) Heterogeneous nuclear ribonucleoprotein A2/B1 isoform 1 Poly(A) binding protein, cytoplasmic 4, isoform 1 Major vault protein Laminin receptor/40 S ribosomal SA (p40)

gi|15805031 gi|119168 gi|109134362 gi|27682273 gi|41055865 gi|228997

50.47 95.40 36.03 72.51 95.95 32.84

89/462 54/858 39/349 62/630 33/864 33/295

(19%) (6%) (11%) (10%) (4%) (11%)

X X X X X X

X X X X X X

X X X X X X

X X X X X X

5 3 2 4 1 2

Miscellaneous proteins Hemoglobin alpha subunit Hemoglobin beta subunit Selenium-binding protein 2

gi|122440 gi|232232 gi|18266692

15.30 16.04 53.07

40/141 (28%) 76/147 (22%) 59/472 (13%)

X X X

X X X

X X X X X X

3 6 4

Fructose-fed hamsters have previously been extensively characterized and shown to exhibit whole-body insulin resistance as well as intestinal chylomicron overproduction (reviewed in

[1]). PCTV formation in fructose-fed/insulin-resistant hamster intestinal enterocytes was compared to that of chow-fed hamsters by examining the amount of radiolabeled [3H]-oleic

Fig. 5 – The differential expression of proteins in the C (green) and CL (red) conditions using 2-D difference gel electrophoresis. 150 μg protein of each condition was labeled with commercially available cydye (green or red) according to the manufacturer's protocol (Amersham Biosciences) for a total of 750 μg PCTV protein pooled from 20 hamsters. Protein was solubilized in 9 M urea, 2 M thiourea, 1% CHAPS and 15 mM DTT. The addition of rehydration buffer allowed the protein solution to soak into 14 cm Immobiline Drystrips. The first dimension was run on Amersham's IPGphore isoelectric focusing (IEF) unit for 9 h. Following IEF, the Drystrip was soaked in equilibration buffer and loaded into an 8% SDS-PAGE gel, and run overnight. The PAGE gel was viewed on a Typhoon imager at different wavelengths to view different conditions (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6 – The differential expression of proteins in the F (red) and FL (green) fed conditions using 2-D difference gel electrophoresis. 150 μg protein of each condition was labeled with commercially available cydye (green or red) according to the manufacturer's protocol (Amersham Biosciences) for a total of 750 μg PCTV protein pooled from 20 hamsters. Protein was solubilized in 9 M urea, 2 M thiourea, 1% CHAPS and 15 mM DTT. The addition of rehydration buffer allowed the protein solution to soak into 14 cm Immobiline Drystrips. The first dimension was run on Amersham's IPGphore isoelectric focusing (IEF) unit for 9 h. Following IEF, the Drystrip was soaked in equilibration buffer and loaded into an 8% SDS-PAGE gel, and run overnight. The PAGE gel was viewed on a Typhoon imager at different wavelengths to view different conditions (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

acid incorporated into PCTV triglyceride. Predictably, fructosefed hamsters had PCTVs with higher [3H]-triglyceride counts than chow-fed hamsters. Our laboratory has previously shown that basal levels of intestinal lipoprotein secretion are increased in enterocytes from fructose-fed/insulin-resistant hamsters [13]. We have found that increased stability of apoB-48, increased de novo lipogenesis, as well as increased MTP mass and activity were the factors contributing to these observations [13]. Surprisingly, however, although all conditions were labeled with equal amounts of [3H]-oleic acid, PCTVs from fasted hamsters had higher [3H]-triglyceride counts than fat-loaded hamsters. This phenomenon is likely due to an extremely large pool of unlabeled fatty acid in the intestine of fat-loaded hamsters. Fat loading is expected to cause a substantial increase in the pool of unlabeled fatty acid in the enterocyte leading to considerable dilution of labeled fatty acid pool and thus a reduced amount of label incorporation into PCTV triglyceride. In summary, the comparison of radiolabeled PCTV triglyceride under these conditions suggests that under basal (fasted) state, fructose-fed hamster enterocytes assemble a larger pool of PCTVs than the chow-fed controls. However, comparison in the

fat-loaded conditions was not possible using this approach due to interference from a large pool of free fatty acids released from the fat load. PCTVs isolated from chow- and fructose-fed hamsters, under fasted and postprandial conditions were examined using a number of approaches, including immunoblotting, direct LC-MS/MS, 2-DIGE and MALDI-TOF, as well as iTRAQ, to evaluate the differential expression of proteins following the induction of insulin resistance. Utilizing distinct platforms to analyze differential expression serves to validate the data obtained from any one specific approach. In addition, as these methods are distinct and complimentary, it serves to provide a more comprehensive analysis of the PCTV proteome. Proteins of particular interest were examined in PCTVs by direct immunoblot to determine their differential expression. Sar1 and VAMP7 were examined due to their role in vesicular transport, and MTP and apoB-48 due to their role in chylomicron assembly. Fig. 4 represents the relative fold change in protein abundance compared to the control (chow-fed/ fasted condition), whereas Table 3 shows the significant changes observed under specific conditions. In the normal,

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Table 3 – Differentially expressed primary enteric PCTV proteins following the induction of insulin resistance and oral fat load determined by 2DIGE. Chow-fed fat-loaded (CL) hamsters were compared to chow-fed (C) hamsters under fasted conditions. Fructose-fed fat- loaded hamsters were compared to fructose-fed (F) hamster. F were compared to C, and FL were compared to CL (n = 3). Significant changes are shown, as determined by p < 0.05 using a paired 2-tailed Student's T-test, where 2-fold changes or more are in bold emphasis. Spot no. Cytoskeleton 1398 1644 1673 1677 1785

Protein identity proteins Tubulin beta Gamma actin Gamma actin Gamma actin Gamma actin

MALDI-TOF coverage

pI (actual)

Mr (kDa)

CL/C

FL/F

F/C

FL/CL

43% 35.3 35.3% 32% 23%

4.8 5.6 5.6 5.6 7

50 42 42 42 62

2.48 2.40 2.91 3.00 2.32

1.46 − 1.04 − 1.19 − 1.05 −2.65

1.69 1.47 1.68 1.3 1.12

− 1.00 − 1.71 − 2.06 − 2.21 − 1.02

Vesicular transport proteins 926 Eea1 protein

11.8%

5.6

162

− 2.66

−3.95

− 1.06

− 1.57

Intestinal-specific proteins 825 Villin 1 837 Villin 1

20.4% 20.4%

5.7 5.7

93 93

2.26 2.09

1.52 1.46

1.61 1.56

1.08 1.10

Chaperones 765 767 783 1029 2412

Valosin-containing protein Valosin-containing protein GRP94/GP96 GRP78/BiP Prolyl 4-hydroxylase (beta polypeptide)

48% 51% 19.5% 36% 20.6%

5.1 5.1 4.7 5.1 4.8

90 90 92 72 57

1.60 1.58 3.29 1.63 1.46

2.83 2.05 2.38 2.02 2.38

1.22 1.34 1.64 − 1.09 − 1.14

2.14 1.73 1.18 1.13 1.42

Enzymes 1742 1751 1752 1767 1634 1646 1647 1648 1649 1656 1657 2409

Aldolase 2, B isoform Aldolase 2, B isoform Aldolase 2, B isoform Aldolase 2, B isoform Muscle creatine kinase Muscle creatine kinase Muscle creatine kinase Muscle creatine kinase Muscle creatine kinase Muscle creatine kinase Muscle creatine kinase Muscle creatine kinase

30% 30% 30% 30% 27% 25% 36% 36% 25% 36% 36% 23%

9 9 9 9 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6

40 40 40 40 43 43 43 43 43 43 43 43

1.29 1.49 1.33 1.40 1.10 1.19 −1.13 −1.05 1.26 1.04 1.27 1.05

1.71 1.70 1.70 1.70 −1.95 −2.02 −2.49 −2.41 − 1.91 −2.49 −2.12 −2.51

1.54 1.88 1.63 1.85 − 1.32 − 1.05 − 1.17 − 1.18 − 1.01 − 1.11 − 1.03 − 1.42

2.04 2.15 2.09 2.24 − 2.8 − 2.53 − 2.58 − 2.69 − 2.42 − 2.88 − 2.78 − 2.69

20.9% 54% 18%

5.4 6.3–6.8 6.5

96 96 74

1.47 2.46 2.01

3.34 1.89 1.32

1.58 1.71 2.32

3.58 1.31 1.52

18.6%

5.8

53

−1.81 −1.72 −1.46 −1.47

−2.24 3.25 8.48 7.6

1.24 − 1.86 − 1.24 − 1.15

1.00 3.01 9.98 9.75

Translation factors 651 Major vault protein 724 Unnamed protein product (EF2-like) 1243 Lamin A Miscellaneous 1447 Selenium-binding protein 2 1104 Albumin 1107 Albumin 1114 Albumin

chow-fed hamsters, MTP, Sar1 and VAMP7 all approximately doubled following the induction of postprandial state (fat load). Also, apoB-48 was found to be upregulated 2.75-fold postprandially. The increases in both chylomicron assembly and vesicular transport proteins suggest that, many more chylomicrons are being assembled, and much more PCTVs are being budded in the postprandial compared to fasted state. Upon the induction of insulin resistance, the expression of Sar1, apoB-48 and MTP in the fasting state was all significantly upregulated, more than 2-fold, compared to the control. Upregulation in protein expression in intestinal PCTVs of

insulin-resistant hamsters is consistent with our previous observations [13]. Chylomicron secretion was increased under basal conditions and MTP was increased in fructose-fed/ insulin-resistant hamster intestines [13]. Although a similar level of upregulation was observed, upregulation of protein expression in the postprandial state is probably a transient effect induced by the fat load. Whereas in insulin-resistant hamsters, upregulation of protein expression is most likely to be chronic. Interestingly, although not statistically significant, Sar1 and VAMP7 expression slightly decreased during insulin resistance in the postprandial compared to the fasted

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Table 4 – Differentially expressed primary enteric PCTV proteins following the induction of insulin resistance and oral fat load determined by iTRAQ labeling. Chow-fed fat-loaded (CL) hamsters were compared to chow-fed (C) hamsters under fasted conditions. Fructose-fed fat-loaded hamsters were compared to fructose-fed (F) hamster. F were compared to C, and FL were compared to CL. n = X, significant changes are shown, as determined by p < 0.05 using a paired 2-tailed Student's T-test, where 2-fold changes or more are in bold emphasis. Protein identity

Cytoskeleton proteins Profilin 1

Fold change CL/C

FL/F

−1.64

−1.86

Vesicular transport proteins Annexin A6 Lipid-binding proteins FABP1, liver FABP2, intestinal Microsomal triglyceride transfer protein (MTP) Chaperones Prolyl 4-hydroxylase, beta polypeptide (PDI) Valosin-containing protein (p97) Peptidylprolyl isomerase A

F/C FL/CL

−1.30

−1.92 − 2.54

−1.34 −1.88

1.44 1.62

−1.50

2.27

1.50

1.64 −1.85

Intestine-specific proteins Keratin complex 1 Keratin complex 2

1.73 1.57

Enzymes Aldolase 2, B isoform 1.50 Lactate dehydrogenase A −1.91 Creatine kinase, muscle 1.36 Aldehyde dehydrogenase −1.82 Glycerol-3-phosphate dehydrogenase 1 (soluble) Expressed in non-metastatic cells 2 Acyl-Coenzyme A oxidase 1, palmitoyl

−1.40 1.54 −2.18 2.29 −1.58

2.97 2.46

1.85 1.42 3.46

Transcription/translation factors Poly A binding protein, cytoplasmic 2 Miscellaneous Selenium-binding protein 2 Hemoglobin beta subunit Spetex-2C protein

2.49

−1.62

−1.71

−1.70

1.57 2.23

2.74

condition. Finally, statistical analysis using a two-way ANOVA indicates that insulin resistance causes significant changes in vesicular transport proteins. The postprandial state appears to cause significant changes to proteins involved in chylomicron assembly. Since the hamster database is still incomplete, there are obstacles to identifying some of the previously identified PCTV proteins including apoB48, Sar1, and VAMP7 by proteomic methods. Moreover, there may be differences in sequence between hamster and other rodent sequences of these proteins.

4.1. Differential expression following a fat load (postprandial state) in normal hamsters The postprandial state was found to induce differential expression of specific proteins compared to the control. The fat load in normal hamsters caused an increase in GRP94, a stress-induced chaperone supporting the hypothesis that the postprandial state may induce ER stress in intestinal enterocytes. Upregulation of particular cytoskeletal proteins, up to 3-fold in the postprandial state, suggests that stress may be associated with an increase in PCTV formation and transport. Induction of chylomicron assembly may be contributing to these observations. Also, upregulation of PCTV-associated villin is consistent with these observations. Villin is a structural protein in the intestine that binds actin; villin could be involved in providing a structural framework to guide the PCTV across the cell. Surprisingly, changes in key vesicular transport proteins, such as COPII proteins, were not detected using either 2-DIGE or iTRAQ labeling to verify this hypothesis. Interestingly, FABP2 was found to be downregulated in the postprandial state by more than 2.5-fold compared to the control using iTRAQ labeling. The abundance of fatty acids available at the brush border membrane (BBM) after hydrolysis of the olive oil fat load may explain these observations. Therefore, the majority of the FABP2 molecules may be localized at the BBM for fatty acid absorption, rather than in the PCTV. Several other proteins were found to be differentially expressed in the postprandial state, however, their function in the PCTV remain unknown. First, an unnamed protein product was found to be upregulated in the postprandial state by almost 2.5-fold. The unnamed protein product shares some sequence similarity with translation factor Elongation Factor (EF) 2. However, the molecular weight was not consistent with EF2. It could be a hamster homolog of EF2 or a protein with a domain similar to EF2 with a completely different function. Lastly, Eea1 was downregulated more than 2.6-fold in the postprandial state. Like clathrin, Eea1 is involved in endocytosis. Eea1 interacts with phosphatidylinositol 3-phosphate (PI3P) to be recruited to endosomal membranes. PI3P is a marker of human vacuolar protein sorting 34 (hVps34) activity, a class III phosphoinositide 3-kinase (PI3K) that drives S6 kinase 1 (S6K1) activation [20]. A nutrient overload by a high fat diet in mice can induce constitutive S6K1 activation, which can lead to insulin resistance by suppressing insulin-induced class I phosphoinositide 3-kinase (PI3K) signaling [21]. The relevance of the downregulation of Eea1 in the PCTV is unknown; however, this observation could give insight to the cellular processes occurring during the postprandial state.

4.2. Alterations in PCTV proteome in response to fructose feeding and induction of an insulin-resistant state The induction of insulin resistance resulted in differential expression of PCTV-associated chaperone proteins, in both fasting and postprandial states, as shown in Table 2. Additional chaperones were expressed in PCTVs from insulinresistant hamsters of both the fasted and postprandial states. Overall, the induction of HSPs in PCTVs from fructose-fed hamsters may be a response to the development of insulin

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resistance and ER stress. ER stress associated with obesity, insulin action and type 2 diabetes has been well documented [22]. HSPs have the potential to reduce ER stress and prevent the suppression of insulin receptor signaling caused by the activation of IkappaB kinase-beta (IKK-beta) and c-Jun N-terminal kinase (JNK), kinases involved in the molecular mechanism of obesity-induced insulin resistance [22,23]. HSP1 and HSP70, both found in PCTVs, have the potential to prevent the activation of IKK-beta and JNK, respectively [24]. As shown in Fig. 6, in the fructose-fed hamster, proteins associated with chylomicron assembly were found to be upregulated compared to the normal, chow-fed hamster, reflecting the overproduction of intestinal lipoproteins in insulin resistance. However, vesicular transport proteins were not significantly upregulated to the same extent as chylomicron assembly proteins; especially apparent in the fructose-fed/postprandial condition. Even though there were more chylomicrons being produced in the enteric ER under these conditions, these data suggest that the rate of vesicle budding may not be sufficient to rapidly transport the overproduced lipoprotein cargo. Overall, these observations imply that the enterocyte may control the overproduction of chylomicrons, as well as their transport through the cell, by limiting the amount of PCTV budding, possibly through a degradative pathway. The induction of insulin resistance was also found to be associated with differential expression of specific proteins in PCTVs in both fasting and postprandial states. An upregulation of chaperones in the postprandial state may indicate an attempt by the cell to accommodate the large increase in protein cargo and the increase in apoB-48-containing lipoprotein overproduction, a phenomenon previously documented in enterocytes from the fructose-fed hamster intestine [13]. Among the proteins differentially expressed, GRP78 is part of the ER protein folding machinery [25], and acts to prevent the aggregation of newly synthesized proteins [26]. GRP78 and other ER chaperones are known to be associated with newly synthesized apoB during its biogenesis in the ER [27]. Our laboratory has also reported a strong association between apoB-100 and GRP78 in the ER of HepG2 cells [28]. GRP78 could be upregulated in PCTVs during insulin resistance to prevent newly synthesized proteins, particularly the highly hydrophobic stretches of the apoB48 polypeptide, from aggregating during chylomicron assembly since chylomicron overproduction could be further aggravated by the postprandial state. Another differentially expressed protein was PDI. PDI is a known ER chaperone and also acts as a subunit of MTP, which is responsible for adding neutral lipids to the nascent apoB [29]. PDI is known to be induced with ER stress [30], and has been found to be upregulated with experimentally-induced diabetes [31]. PDI is upregulated in the postprandial condition more than 2-fold, probably due to its association with MTP. MTP, which is known to have increased mass and activity in fructose-fed hamsters [13], was also found to be upregulated in the insulin-resistant, postprandial condition in the present study. The induction of insulin resistance caused significant upregulation of both PDI and MTP, at 2.27- and 2.49-fold, respectively. Lastly, p97 was upregulated more than 2-fold in the postprandial state. Since p97 is a multifunctional protein, its upregulation in the insulin-resistant, postprandial state

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could be for several reasons. First, p97 may appear in the PCTVs for its ubiquitin-dependent protein degradation role [32] to degrade proteins in response to induction of ER stress. The hypothesis coincides with the apparent induced expression of many proteosomal subunits in PCTVs during insulin resistance. Second, p97 also plays a role in membrane fusion [33], so its upregulation could be due to an alternative mechanism for PCTV budding, in conjunction with clathrin. Since there is an overproduction of chylomicrons in insulin resistance, perhaps the cell cannot accommodate the transport of more PCTVs induced by the fat load using the traditional COPII mechanism. This may explain the slight downregulation of Sar1 and VAMP7 in the postprandial state during insulin resistance. Few significant changes were observed upon the induction of insulin resistance in the fasting state, using 2-DIGE and iTRAQ. Protein abundance problems using 2-DIGE could be a reason why no significant changes were observed when using 2-DIGE and iTRAQ. iTRAQ labeling only showed significant changes more than 2-fold for 2 proteins: creatine kinase and hemoglobin beta subunit. Since there is an upregulation in the basal level of apoB-48-containing lipoprotein production in the enterocyte [13], an upregulation of creatine kinase for PCTV budding would be expected. Moreover, the upregulation of hemoglobin in the fructose-fed condition could be due to different iron requirements in insulin-resistant versus normal hamsters. Clinical studies show that obese women who are insulin-resistant have increased body iron stores [34]. Both 2-DIGE and iTRAQ labeling detected more differentially expressed proteins in the postprandial states rather than the fasted states upon the onset of insulin resistance. Actin and creatine kinase were both found to be downregulated, and major vault protein (MVP) and p97 were all found to be upregulated. Again, this pattern of protein expression could be indicative of less PCTVs budding using COPII proteins and the use of alternative budding mechanisms due to excessive lipoprotein overproduction in the insulin-resistant state [13]. Other interesting proteins found to be differentially expressed in the insulin-resistant, postprandial condition were major vault protein, selenium-binding protein 2 (SBP2) and Spetex2C. First, although the expression of major vault protein was upregulated more than 3-fold, its function in PCTVs is unknown. MVP is the major constituent of the vault particle, the largest known ribonuclear protein complex, and has mainly been studied in tumours [35]. However, MVP was found to be a dominant phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-binding protein in a yeast two-hybrid screen [36]. PTEN is a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway, insulin signaling and insulin sensitivity in adipose tissue [377]. PTEN has also been found to contribute to the development of insulin resistance in skeletal muscle [38]. Therefore, in insulinresistant states, if PTEN is upregulated, then MVP may also be upregulated since it is a dominant PTEN binding partner. Next, SBP2 was found to be downregulated more than 2-fold in the insulin-resistant, postprandial state. There is direct evidence that micronutrients such as zinc, selenium and vitamin E has a beneficial effect on insulin sensitivity and some components of the antioxidant defense system in an animal model of insulin resistance [39]. Since selenium

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promotes insulin sensitivity, SBP2 downregulation may be an indicator of changes in selenium transport in insulin resistance. Finally, Spetex-2C protein was upregulated in the insulin-resistant, postprandial condition 2.74-fold. The expression of Spetex-2C protein has been correlated with cell differentiation of spermaytids in rat testis. Since sperm contamination in the intestinally-derived PCTVs was highly unlikely, the hamster protein sequence detected likely shares homology and similarity to Spetex-2C, but probably has a different function in the PCTV entirely.

mechanism for PCTV budding alongside the traditional COPII mechanism. Overall, the results of this study support the notion that differential expression of specific proteins within the PCTV in the insulin-resistant state may signify the marked overproduction of apoB-48 chylomicron particles. Further research into PCTV-associated proteins and their function will help elucidate the cellular and molecular basis of intestinal lipoprotein abnormalities observed in insulin-resistant states.

Acknowledgements 5.

Conclusions

We have previously shown that the Syrian golden hamsters on a fructose-fed dietary background have a marked increase in fasting and postprandial intestinal lipoprotein production [3,13]. We have shown that this was due to increased stability of apoB-48, increased de novo lipogenesis, as well as increased MTP mass and activity [13], however, there may be other mechanisms contributing to the lipoprotein overproduction observed in these animals under fasted conditions or after being challenged with a gavage of olive oil. The current study shows (for the first time) that enhanced prechylomicron particle assembly may also be contributing to intestinal dyslipidemia observed in obesity and insulin-resistant states. The formation of PCTV in intestinal enterocytes and its role in intestinal fat absorption and chylomicron secretion has received little attention in the field. Pioneering work in the laboratory of Dr. Charles Mansbach II in the rat model has begun to unravel the critical role of this “VLDL-like” transport vesicle in chylomicron assembly and secretion. After eight years of research, the PCTV is finally becoming accepted in the scientific community as a bona fide entity. Recent animal model studies have not only determined the normal transport of the PCTV in the rat intestine, but also discovered two alternative mechanisms of transport that will be useful in studying specific disease states. Despite these advances, only limited data is available on the PCTV and its associated protein factors. Identification of PCTV-associated proteins (the PCTV proteome) is a crucial first step in discovering the mechanisms involved in PCTV formation, its vesicular transport and its role in chylomicron assembly and secretion. The induction of insulin resistance was associated with an upregulation of proteins involved in chylomicron assembly, vesicular transport, protein folding, and proteolysis in the PCTV. Previous proteomic studies using the Syrian golden hamster showed a significant change in the expression of ER proteins upon the induction of insulin resistance in the liver [40]. Therefore, the up- or downregulation of PCTV-specific proteins could be specific to the PCTV or affected by changes in ER proteins that are related to other functions besides PCTV transport. Based on these data, we postulate that insulin resistance induces ER stress in the enterocyte, which causes chylomicron overproduction and a possible increase in PCTV budding. Moreover, there is an increase in chaperones due to the unfolded protein response and an upregulation in proteolytic proteins, which may be used as a method to regulate the amount of PCTV budding. In addition, increased chylomicron assembly by insulin resistance may require the use of clathrin as an alternative

The authors would like to thank Dr. Charles M. Mansbach II and Dr. Shadab A. Siddiqi for their technical guidance and training of DMW, particularly in the isolation of intestinal PCTVs. This work was supported by operating grants from the Canadian Institutes for Health Research Heart (CIHR) to KA. DMW holds a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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