| | Polyunsaturated fatty acid-enriched diets used for the treatment of canine chronic enteropathies decrease the abundance of selected genes of cholesterol homeostasis☆Received 23 May 2009; received in revised form 31 July 2009; accepted 2 August 2009. published online 07 September 2009. Abstract Lipids are important for cell function and survival, but abnormal concentrations may lead to various diseases. Cholesterol homeostasis is greatly dependent on the active transport by membrane proteins, whose activities coordinate lipid status with cellular function. Intestinal Niemann-Pick C1-Like 1 protein (NPC1L1) and scavenger receptor B1 (SR-B1) participate in the uptake of extracellular cholesterol, whereas ATP binding cassette A1 (ABCA1) mediates the efflux of excessive intracellular cholesterol. Caveolin-1 binds cholesterol and fatty acids (FA) and participates in cholesterol trafficking. Sterol response element binding protein-2 (SREBP-2) is a sensor that regulates intracellular cholesterol synthesis. Given that cholesterol is a constituent of chylomicrons, whose synthesis is enhanced with an increased FA supply, we tested the hypothesis that feeding polyunsaturated FA (PUFA)-enriched diets in treatment of canine chronic enteropathies alters the mRNA expression of genes involved in cholesterol homeostasis. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we compared the mRNA abundance of NPC1L1, SR-B1, ABCA1, caveolin-1, and SREBP-2 in duodenal mucosal biopsies of dogs with food-responsive diarrhea (FRD; n =14) and inflammatory bowel disease (IBD; n=7) before and after treatment with cholesterol-free PUFA-enriched diets and in healthy controls (n=14). The abundance of caveolin-1, ABCA1, and SREBP-2 were altered by PUFA-enriched diets (P<0.05), whereas that of NPC1L1 and SR-B1 mRNA remained unchanged. The gene expression of caveolin-1, ABCA1, and SREBP-2 was down-regulated (P<0.05) by PUFA-enriched diets in IBD dogs only. Our results suggest that feeding PUFA-enriched diets may alter cholesterol homeostasis in duodenal mucosal cells of dogs suffering from IBD. 1. Introduction  Inflammatory bowel disease (IBD) and food-response diarrhea (FRD) are the 2 most frequent phenotypes of chronic enteropathies in dogs [1]. They are often discriminated by their response to treatment with an elimination diet alone (in FRD) or combined with immunosuppressive therapy (in IBD). Long-chain polyunsaturated fatty acids (PUFA) exert nutritive and health-protective effects on intestinal cells and are often used in the treatment of inflammation and/or autoimmune diseases such as IBD [2]. The use of PUFA, and especially of n-3 PUFA, may be helpful in the treatment of IBD because n-3 PUFA have suppressive effects on the production of inflammatory cytokines by immune cells [3], [4]. The intestinal absorption of dietary fat implies increasing requirements of cholesterol and chylomicron formation [5]. Thus, intestinal chylomicron secretion is correlated with dietary fat content [6]. Cellular cholesterol homeostasis is influenced by the activity of several membrane proteins. Niemann-Pick C1-Like 1 protein (NPC1L1) is localized at the apical membrane of the small intestine and facilitates the uptake of dietary/biliary cholesterol [7]. Scavenger receptors (SR)-B1 are receptors for lipoproteins [8]. They mediate the uptake of extracellular cholesterol in the small intestine [9]. Caveolin-1 is a structural protein of membrane lipid rafts, which are rich in sphingolipids and cholesterol. This protein binds cholesterol with high specificity and participates in cholesterol homeostasis [10], [11]. Adenosine triphosphate-binding cassette A1 (ABCA1) is a cholesterol transporter that mediates the efflux of excessive amounts to high-density lipoproteins [12]. Sterol response element binding protein-2 (SREBP-2) is a cholesterol sensor that is proteolytically released from membranes of the endoplasmatic reticulum and acts as a regulator of intracellular cholesterol synthesis [13]. In the present study, dogs suffering from IBD and FRD were treated with cholesterol-free PUFA-enriched diets. We hypothesized that feeding cholesterol-free PUFA-enriched diets would lead to increased requirements for cholesterol and chylomicron formation, respectively, resulting in altered expression of selected genes involved in cholesterol homeostasis. In this study, we compared the mRNA abundance of NPC1L1, SR-B1, caveolin-1, SREBP-2, and ABCA1 in the duodenal mucosa of dogs with FRD and IBD before and after treatment with PUFA and in healthy controls. 2. Materials and methods 2.1. Animals and diets The assay protocols were in accordance with the Swiss Law on Animal Protection. Sick dogs (n=21) with a history of chronic diarrhea for at least 6 wk were admitted for therapy at the Small Animal Clinic, University of Bern, Switzerland. Animals were clinically examined and scored using the Canine IBD Activity Index [14]. Based on the responsiveness of the dogs to dietary treatment within 14 d, animals were retrospectively grouped as suffering from FRD (n=14). In this group, dogs were on average 45 mo old (ranging from 9 to 134 mo), and their average body weight (BW) was 25.1kg (range 1.8–49kg). They were of different breeds (1 Yorkshire terrier, 1 French bulldog, 1 Weimaraner, 1 West Highland white terrier, 2 Labrador retrievers, 1 berger blanc suisse, 1 Pomeranian, 1 Newfoundland, 1 golden retriever, and 4 mongrels). All other dogs were additionally treated with corticosteroids and were grouped as suffering from IBD (n=7). Dogs of this group were on average 76 mo old (range 36-154 mo), and their average BW was 21.5kg (range 3-37kg). They were of different breeds (1 shar-pei, 1 papillon, 1 golden retriever, 1 bauceron, 1 American cocker spaniel, and 2 mongrels). Finally, 14 dogs, exclusively beagles, devoid of any gastrointestinal disorders, and euthanized in the frame of another experimental study, served as healthy controls. These dogs were on average 108 mo old (range 78-154 mo). Diets fed to dogs enrolled in this study were purchased from Biomill AG (Granges-près-Marnand, Switzerland). Except for PUFA, the composition of standard food (BIOMill Adult, fed to controls) and of PUFA-enriched formulations (fed to patients) were comparable. The PUFA content in BIOMill Adult was 0.63% for omega-3 and 3.5% for omega-6, whereas in PUFA-enriched diets the concentrations of omega-3 varied from 0.74% to 1.37% and of omega-6 from 3.12% to 3.86%. In addition, the amounts of crude fat were 15% and 16.5%, total protein 26% and 26.6%, carbohydrates 40.5% and 37.5%, and ash 7% and 7.7%, respectively, in standard diets and PUFA-enriched diets. Dogs with FRD received 0.14 and 0.48g/kg BW of n-3 and n-6 PUFA daily for 28 d, whereas IBD dogs received 0.16 and 0.48g/kg BW daily for 70d. The protein in the diets was from various sources. In standard diets, it was predominantly from chicken meal (64.8% of total protein, resp.), but also from corn gluten, wheat, soybeans, fish meal, corn, linseed, rice, wheat middlings, liquid, and beet pulp. However, in PUFA-enriched diets, the protein was predominantly from fish meal (54.7% of total protein, resp.), but also from potato protein, rice, whole egg meal, beet pulp, powder, and wheat middlings. On average, FRD dogs received about 14% less omega-3 PUFA daily than dogs suffering from IBD. Dogs with FRD were treated for 28 d and IBD dogs for 70 d. 2.2. Sampling Duodenal biopsies from sick dogs and tissue specimens collected within minutes after euthanasia of control dogs were processed and stored as previously described [15]. 2.3. Total RNA extraction and quantitative reverse transcriptase polymerase chain reaction Following extraction with the SV (spin or vacuum) Total RNA Isolation System (Promega, Madison, WI, USA; cat. #Z 3100), 3μg of total RNA were reverse-transcribed with the High Capacity cDNA Reverse Transcription kit (# 0806057, ABI, WI) according to the manufacturer's instructions. Specific primers were designed using canine sequences indicated at http://www.ensembl.org/index.html. They produced amplicons that span at least 2 distinct exons in the coding sequence. Gene references, primer sequences, and amplicon lengths were: NPC1L1 [DQ897676; forward: 5′-ttccccgaggagtcttacct-3′; reverse: 5′-agcaggctagggagttgaca-3′; 151bp], SR-B1 [ENSCAFT00000011179; forward: 5′-agggcaagtttgggctattt-3′, reverse: 5′-gaattccagcgaggtctcag-3′; 218bp], caveolin-1 [ENSCAFT00000005462; forward: 5′-acaccaaggaaatcgacctg-3′; reverse: 5′-ggcaaagtaaatgccccata-3′; 227bp], SREBP-2 [ENSCAFG00000001013; forward: 5′-ccttcctctgcctctccttt-3′; reverse: 5′-cacaaagacgctcaggacaa-3′; 200bp], and of cyclophilin B [ENSCAFT00000027006; forward: 5′-ggtcatcggtctctttggaa-3′; reverse: 5′-gatgctctttcctccagtgc-3′; 175bp]. Primers for amplification of ABCA1 and ubiquitin were obtained from studies by Farke et al. [16] and Gropp et al. [17], respectively. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in duplicate on the Rotor-Gene 6000 (Corbett Life Science, Mortlake, Australia) in a final volume of 10μL PCR mixture containing 10ng cDNA, 5mmol/L of gene-specific forward and reverse primers, and 1×sensimix NoRef PCR Master Mix (Quantace, London, UK; # QT505-05). The gene expression of targets was related to the mean expression of ubiquitin and cyclophillin B, as previously described [15]. The efficiency of PCR ranging from 0.82–0.89 was determined in serially diluted cDNA using Rotor-Gene analysis software. Intra- and interassay coefficients of variation of replicates were determined, and values varied between 1.9% and 6.8%, respectively. 2.4. Statistical analysis To identify the differences before and after treatment, mRNA data of NPC1L1, SR-B1, SREBP-2, caveolin-1, and ABCA1 were evaluated with the Wilcoxon signed rank test. Kruskal-Wallis 1-way analysis of variance (ANOVA) on ranks was used to identify differences among patients (before and after treatment) and healthy controls. Significance was set at P<0.05. 3. Results In FRD dogs, mRNA concentrations of NPC1L1, SR-B1, caveolin-1, SREBP-2, and ABCA1 were not altered by treatment (Fig. 1A-E). In addition, compared to controls, mRNA concentrations of NPC1L1 (Fig. 1A) and SREBP-2 (Fig. 1D) were higher (P<0.05), whereas SR-B1 expression (Fig. 1B) was lower (P<0.05). The mRNA abundance of ABCA1 (Fig. 1E) was similar to that of controls. In IBD dogs, mRNA concentrations of caveolin-1 (Fig. 1C), ABCA1 (Fig. 1E), and SREBP-2 (Fig. 1D) were down-regulated by treatment (P<0.05), whereas mRNA concentrations of NPC1L1 and SR-B1 were unchanged (P>0.05). In these animals, mRNA concentrations of NPC1L1, SREBP-2, and ABCA1 before treatment were higher (P<0.05), whereas concentrations of caveolin-1 and SR-B1 were comparable (P>0.05) to those of controls. 4. Discussion Our investigations demonstrated that PUFA-enriched diets did not have a significant impact on the gene expression of NPC1L1 in patients. The similar NPC1L1 mRNA concentrations before and after treatment in FRD dogs are in contrast with studies in rats [18] and mice [19] reporting suppressive effects of PUFA on NPC1L1 gene expression. Contrary to FRD, mRNA concentrations of NPC1L1 in IBD dogs decreased after treatment to values comparable to those in controls. Regardless of how NPC1L1 facilitates cholesterol absorption, the lack of a significant increase of NPC1L1 mRNA suggests its minor role in mediating increasing cholesterol requirements relative to dietary PUFA absorption [5]. In this study, PUFA-enriched diets did not affect SR-B1 gene expression in patients. The lack of any effect on SR-B1 gene expression, especially in IBD dogs, implies that SR-B1, similarly to NPC1L1, is not essential for cholesterol uptake relative to PUFA absorption. In the present study, mRNA concentrations of caveolin-1 decreased only in dogs suffering from IBD. The discrepancy with FRD dogs is possibly because of the difference in treatment courses and subsequent amounts of PUFA ingested. The down-regulation of caveolin-1 in dogs with IBD after treatment suggests that this gene is a PUFA-responsive gene in canine enterocytes. It is known that caveolin-1 forms a high-affinity complex with cholesterol [10] and has the potential to translocate from the plasma membrane toward intracellular compartments in response to FA supply [20]. Thus, it is questionable whether cholesterol may co-translocate with caveolin-1 in response to PUFA-enriched diets. The decreased caveolin-1mRNA concentration in the dogs with IBD strongly suggests low intracellular cholesterol concentrations, since caveolin-1mRNA expression is transcriptionally regulated by cholesterol [11]. In this context, down-regulation of ABCA1 in dogs with IBD is not surprising, because this gene is necessary for efflux of excessive cholesterol [12]. The decrease of ABCA1 may result from the suppressive effect of PUFA [19] and/or, at least partly, from an insufficient regulation by caveolin-1 [21]. Previous in vitro investigations on inflammatory processes in hepatocytes and kidney cells reported intracellular accumulation of cholesterol, since interleukin 1β suppressed ABCA1 expression and subsequent cholesterol efflux [22], [23]. In the present ex vivo study duodenal mucosal mRNA concentrations of interleukin 1β in dogs with FRD were unchanged, whereas concentrations in dogs with IBD were higher as compared to healthy dogs (data not shown). Interestingly, mRNA concentrations of ABCA1 followed a very similar pattern; ABCA1 in FRD was unchanged, whereas concentrations in IBD were significantly higher as compared with controls. These results may suggest the existence of ABCA1-responsive and ABCA1-independent inflammatory pathways in the development of the 2 forms of canine enteropathies. A decrease of SREBP-2 in IBD dogs is intriguing, because intracellular cholesterol is supposedly low in these animals. Because SREBP-2 is a cholesterol sensor that is proteolytically released from membranes of the endoplasmatic reticulum when cholesterol concentrations are low [13], an increased gene expression could have been expected. The reasons for reduced SREBP-2mRNA concentrations are unknown, but an interference of ingested PUFA on the posttranslational maturation of SREBP-2 is possible [24]. 5. Conclusions The lack of significant changes in gene expression of NPC1L1 and SR-B1 suggests that these genes do not represent key pathways for cholesterol supply related to PUFA absorption in the canine intestine. In turn, the significant alterations in mRNA abundance of caveolin-1, SREBP-2, and ABCA1 suggest their significant involvement in processes related to cholesterol supply and homeostasis in dogs suffering from IBD and treated with PUFA-enriched diets. The gene expression and functional activity of caveolin-1, SREBP-2, and ABCA1 in IBD dogs possibly contribute to adequate cholesterol use in intestinal cells and thereby potentially prevent intracellular cholesterol accumulation, a risk factor for cell inflammation. Acknowledgements The authors thank Maria Feher at the Institute of Biochemistry and Molecular Medicine of the University of Bern for her contribution to RNA preparation, and Biomill SA (Granges-près-Marnand, Switzerland) for providing the standard and PUFA-enriched diets. References [1]. [1]Allenspach K, Gaschen F. Canine chronic enteropathies: a review. Schweizer Archiv für Tierheilkunde. 2003;145:209–222. MEDLINE [2]. [2]Simopoulos A. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr. 2002;21:495–505. MEDLINE [3]. [3]Endres S, Ghorbani R, Kelley VE, Georgilis K, Lonnemann G, van der Meer JW, et al The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med. 1989;320:265–271. MEDLINE |
CrossRef
[4]. [4]Robinson DR, Urakaze M, Huang R, Taki H, Sugiyama E, Knoell CT, et al. Dietary marine lipids suppress continous expression of interleukin-1 beta gene transcription. Lipids. 1996;31:S23–S31.
CrossRef
[5]. [5]Wood P, Imaichi K, Knowles J, Michaels G, Kinsell L. The lipid composition of human plasma chylomicrons. J Lipid Res. 1964;5:225–231. MEDLINE [6]. [6]Kolopissis AD, Griglio S, Le Liepvre X. Intestinal very low density lipoprotein secretion in rats fed various amounts of fat. Biochim Biophys Acta. 1982;711:33–39. MEDLINE [7]. [7]Altman SW, Davis HR, Zhu LJ, Yao X, Hoos LM, Tetzloff G, et al. Niemann-Pick C1-Like 1 protein is critical for intestinal cholesterol absorption. Science. 2004;303:1201–1204.
CrossRef
[8]. [8]Trigatti B, Rigotti A, Krieger M. The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism. Curr Opin Lipidol. 2000;11:123–131. MEDLINE |
CrossRef
[9]. [9]Werder M, Han CH, Wehrli E, Bimmler D, Schulthess G, Hauser H. Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine. Biochemistry. 2001;40:11643–11650. [10]. [10]Murata M, Peranen J, Schreiner R, Wieland F, Kurzchalia TV, Simons K. VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci U S A. 1995;92:10339–10343. MEDLINE |
CrossRef
[11]. [11]Fielding CJ, Bist A, Fielding PE. Caveolin mRNA levels are upregulated by free cholesterol and downregulated by oxysterols in fibroblast monolayers. Proc Natl Acad Sci U S A. 1997;94:3753–3758. MEDLINE |
CrossRef
[12]. [12]Oram JF, Heinecke JW. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease. Physiol Rev. 2005;85:1343–1372. MEDLINE |
CrossRef
[13]. [13]Brown MS, Goldstein JL. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci U S A. 1999;96:11041–11048. MEDLINE |
CrossRef
[14]. [14]Jergens AE, Schreiner CA, Frank DE, Niyo Y, Ahrens FE, Eckersall PD, et al. A scoring index for disease activity in canine inflammatory bowel disease. J Vet Intern Med. 2003;17:291–297. MEDLINE |
CrossRef
[15]. [15]Ontsouka EC, Bruckmaier RM, Steiner A, Blum JW, Meylan M. Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows. J Recept Signal Transduct Res. 2007;27:147–166.
CrossRef
[16]. [16]Farke C, Viturro E, Meyer HH, Albrecht C. Identification of the bovine cholesterol efflux regulatory protein ABCA1 and its expression in various tissues. J Anim Sci. 2006;84:2887–2894.
CrossRef
[17]. [17]Gropp FN, Greger DL, Morel C, Sauter S, Blum JW. Nuclear receptor and nuclear receptor target gene messenger ribonucleic acid levels at different sites of the gastrointestinal tract and in liver of healthy dogs. J Anim Sci. 2006;84:2684–2691.
CrossRef
[18]. [18]Mathur SN, Watt KR, Field FJ. Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid. J Lipid Res. 2007;48:395–404. MEDLINE |
CrossRef
[19]. [19]de Vogel-van den Bosch HM, de Wit NJ, Hooiveld GJ, Vermeulen H, van der Veen JN, Houten SM, et al. A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine. Am J Physiol Gastrointest Liver Physiol. 2008;294:G1171–1180.
CrossRef
[20]. [20]Pol A, Martin S, Fernández MA, Ingelmo-Torres M, Ferguson C, Enrich C, et al. Cholesterol and fatty acids regulate dynamic caveolin trafficking through the Golgi complex and between the cell surface and lipid bodies. Mol Biol Cell. 2005;16:2091–2105. MEDLINE |
CrossRef
[21]. [21]Lin YC, Ma C, Hsu WC, Lo HF, Yang VC. Molecular interaction between caveolin-1 and ABCA1 on high-density lipoprotein-mediated cholesterol efflux in aortic endothelial cells. Cardiovasc Res. 2007;75:575–583.
CrossRef
[22]. [22]Ma KL, Ruan XZ, Powis SH, Chen Y, Moorhead JF, Varghese Z. Inflammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of apolipoprotein E knockout mice. Hepatology. 2008;48:770–781.
CrossRef
[23]. [23]Ruan XZ, Moorhead JF, Fernando R, Wheeler DC, Powis SH, Varghese Z. Regulation of lipoprotein trafficking in the kidney: role of inflammatory mediators and transcription factors. Biochem Soc Trans. 2004;32:88–91. MEDLINE |
CrossRef
[24]. [24]Worgall TS, Sturley SL, Seo T, Osborne TF, Deckelbaum RJ. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing concentrations of mature sterol regulatory element-binding protein. J Biol Chem. 1998;273:25537–25540. MEDLINE |
CrossRef
a Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Buehlstrasse 28, CH 3012, Switzerland b Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, CH 3012 Switzerland  Corresponding author. Tel.: +41 31 631 4108; fax: +41 631 3410.
☆ Cholesterol homeostasis in canine chronic enteropathies/Ontsouka et al. PII: S0739-7240(09)00091-5 doi:10.1016/j.domaniend.2009.08.001 © 2009 Elsevier Inc. All rights reserved. | |
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