Abstract The objective of this study was to determine whether a grape seed procyanidin extract (GSPE) exerts a triglyceride-lowering effect in a hyperlipidemic state using the fructose-fed rat model and to elucidate the underlying molecular mechanisms. Rats were fed either a starch control diet or a diet containing 65% fructose for 8 weeks to induce hypertriglyceridemia. During the 9th week of the study, rats were maintained on their respective diet and administered vehicle or GSPE via oral gavage for 7 days. Fructose increased serum triglyceride levels by 171% after 9 weeks, compared to control, while GSPE administration attenuated this effect, resulting in a 41% decrease. GSPE inhibited hepatic lipogenesis via down-regulation of sterol regulatory element binding protein 1c and stearoyl-CoA desaturase 1 in the fructose-fed animals. GSPE increased fecal bile acid and total lipid excretion, decreased serum bile acid levels and increased the expression of genes involved in cholesterol synthesis. However, bile acid biosynthetic gene expression was not increased in the presence of GSPE and fructose. Serum cholesterol levels remained constant, while hepatic cholesterol levels decreased. GSPE did not modulate expression of genes responsible for esterification or biliary export of the newly synthesized cholesterol, but did increase fecal cholesterol excretion, suggesting that in the presence of GSPE and fructose, the liver may secrete more free cholesterol into the plasma which may then be shunted to the proximal small intestine for direct basolateral to apical secretion and subsequent fecal excretion. Our results demonstrate that GSPE effectively lowers serum triglyceride levels in fructose-fed rats after one week administration. This study provides novel insight into the mechanistic actions of GSPE in treating hypertriglyceridemia and demonstrates that it targets hepatic de novo lipogenesis, bile acid homeostasis and non-biliary cholesterol excretion as important mechanisms for reducing hypertriglyceridemia and hepatic lipid accumulation in the presence of fructose.

Citation: Downing LE, Heidker RM, Caiozzi GC, Wong BS, Rodriguez K, Del Rey F, et al. (2015) A Grape Seed Procyanidin Extract Ameliorates Fructose-Induced Hypertriglyceridemia in Rats via Enhanced Fecal Bile Acid and Cholesterol Excretion and Inhibition of Hepatic Lipogenesis. PLoS ONE 10(10): e0140267. https://doi.org/10.1371/journal.pone.0140267 Editor: Marcia B. Aguila, State University of Rio de Janeiro, Biomedical Center, Institute of Biology, BRAZIL Received: June 9, 2015; Accepted: September 22, 2015; Published: October 12, 2015 Copyright: © 2015 Downing et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by start-up funding from the University of Nevada Reno and USDA Hatch (NEV 0738)/Multi-State project W-3122: Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety (http://nimss.umd.edu/lgu_v2/homepages/home.cfm?trackID=14436) to MLR. GCC was the recipient of a CONICYT Bicentennial Becas-Chile Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: BA, Bile acid; GSPE, Grape seed procyanidin extract; TG, Triglyceride

Introduction Rates of obesity, type 2 diabetes, non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD) have increased significantly in recent years, both in children and adults [1–4]. This surge has correlated with a significant increase in dietary fructose intake in the United States, due in large part to the rise in consumption of sugar-sweetened beverages [5]. Fructose is a highly lipogenic dietary factor [6] and increasing evidence points to an obesogenic role for fructose via the generation of substrates for de novo lipogenesis, resulting from rapid hepatic metabolism [7]. Since fructose metabolism is insulin-independent [2], there is less uptake and catabolism of triglyceride (TG)-rich lipoproteins by tissues, ultimately resulting in increased postprandial plasma TG levels [8]. Increased hepatic lipogenesis, combined with decreased uptake of TGs in peripheral tissues, is an important mechanism by which fructose induces steatosis and elevates serum TG levels [9]. In contrast to fructose-induced metabolic dysregulation, evidence indicates that diets rich in fruits and vegetables, e.g. the Mediterranean diet, exert protective effects against the development of the metabolic syndrome [10]. Such diets tend to be high in flavonoids, which exhibit cardioprotective effects in humans [11, 12]. Dietary procyanidins, a class of flavonoids commonly found in grapes, apples and red wine, have been shown to ameliorate risk factors associated with hypertriglyceridemia and steatosis [13–17]. We previously reported that a grape seed procyanidin extract (GSPE) exerts hypotriglyceridemic effects in vivo in a normolipidemic state [18–22]. We identified GSPE as a co-agonist ligand for the farnesoid x receptor (nuclear receptor subfamily 1, group H, member 4; FXR) [22], a transcription factor that regulates bile acid (BA), TG, cholesterol and glucose homeostasis [23–27]. Mechanistically, GSPE functions in conjunction with BAs, the endogenous ligands of FXR, to upregulate the expression of small heterodimer partner (nuclear receptor subfamily 0, group B, member 2; Shp), which represses sterol regulatory element binding protein 1c (Srebp-1c) expression, a key regulator of lipogenesis, resulting in the concomitant decrease in downstream lipogenic gene expression [19, 22]. Evidence also shows that GSPE administration increases fatty acid β-oxidation [19] and reduces VLDL-TG secretion [21]. Previous studies utilized a high-fat diet to examine the effects of GSPE [20, 28], however, since sugar intake is also a critical factor that can modulate metabolic homeostasis, particularly TG levels [29, 30], evaluation of the potential therapeutic impact of GSPE on animal models subjected to other dietary regimens, such as carbohydrate-induced hypertriglyceridemia, is warranted. Consequently, the aim of the current study was to more closely mimic a real-world scenario, by assessing whether GSPE can mitigate the effects of existing fructose-induced hypertriglyceridemia in vivo, and to determine the underlying molecular mechanisms. This investigation has important implications for further investigations in human subjects using GSPE as a potential natural therapy to counteract increased incidences of hypertriglyceridemia and steatosis. In the present study, we show that a high-fructose diet for 8 weeks significantly increases serum TG levels in rats, while also markedly inducing hepatic lipid accumulation (steatosis). Co-administration of GSPE with the high-fructose diet for one week only, during the 9th week of the study, effectively ameliorated the adverse consequential effects on serum TGs resulting from the high-fructose diet. This attenuation was achieved via enhanced fecal BA, total lipid, cholesterol and non-esterified fatty acid excretion, inhibition of hepatic de novo lipogenesis and increased TG catabolism. We evaluated the gene regulatory effects exerted by GSPE in the liver in the presence of fructose to gain a better insight and understanding regarding the molecular effects leading to the observed hypotriglyceridemic effect.

Materials and Methods Grape Seed Procyanidin Extract (GSPE) was obtained from Les Dérives Résiniques et Terpéniques (Dax, France). The extract was analyzed in-house using normal phase high performance liquid chromatography (HPLC), as previously described [31] to determine procyanidin composition based on the degree of polymerization. As shown in S1 Fig, GSPE is comprised of procyanidin monomers (68.68 ± 0.02%), dimers (26.16 ± 0.01%) and trimers (5.16 ± 0.02%). Animal feeding studies and diets Rats were housed under standard conditions and all experimental procedures were approved by the local Institutional Committee for Care and Use of Laboratory Animals (IACUC) at the University of Nevada, Reno (Protocol # 00502). Male Wistar rats, 7 weeks of age, were purchased from Charles River Laboratories. After one week of acclimation, rats were randomly assigned to either a control diet (n = 5) or fructose diet (n = 8) for 8 weeks (Harlan Teklad). As shown in Table 1, the starch control diet was a modification of AIN-93G (TD.94045) replacing all sucrose with starch, and was comprised of (% by weight) 17.7% protein, 58.9% carbohydrate and 7.2% fat, providing 3.7 Kcal/g (TD.110787). The fructose diet was a modification of AIN-93G replacing all sucrose and starch with fructose, and was comprised of 17.7% protein, 64.7% carbohydrate and 7.2% fat, providing 3.9 Kcal/g (TD.110786). The animals were maintained on the AIN-93G formulated diets throughout the 9-week study, consistent with previous reports [32, 33]. Food was replenished 3 times per week and food intake was estimated by subtracting the total amount of feed and the amount remaining in the box. Rats were weighed weekly. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Composition of the diets containing starch (control) and fructose as the major source of carbohydrate a . https://doi.org/10.1371/journal.pone.0140267.t001 Blood samples were collected at 0, 4 and 8 weeks to measure serum triglyceride levels. After 8 weeks on the diets, the rats were randomly assigned to receive either vehicle (water) or GSPE (250 mg/kg) via oral gavage for 7 days, while still consuming their assigned diets. The dose of GSPE used in this study is one-fifth of the no-observed-adverse-effect level (NOAEL) described for GSPE in male rats [34]. This dose is effective in reducing serum TG levels in normolipidemic C57BL/6 mice [19, 22] and rats [18], and was chosen to aid in the identification of the primary, short-term effects of grape seed procyanidins on lipid metabolism in the presence of fructose, in order to gain insight into the mechanisms that underlie their potential longer-term effects. Using a translation of animal to human doses based on metabolic body size [35] and estimating the food intake for a 60-kg human, the dose of GSPE used herein corresponds to ~1.8 g, which is less than a 2 g/day dose previously tested in human subjects [36]. On day 7, the rats were gavaged at 9am, food was then removed and they were sacrificed 5 hours later. Rats were anesthetized using isoflurane, blood was collected from the saphenous vein, after which the animals were euthanized using carbon dioxide, livers were excised and weighed, snap frozen in liquid nitrogen and stored at –80°C. For collection of the feces, rats were placed in clean cages 3 days prior to the end of the experiment and feces were manually collected at the end of the study, air-dried and weighed. Gene expression analysis Total RNA was extracted from liver using TRIzol (Life Technologies) according to the manufacturer’s instructions. Complimentary DNA (cDNA) was reverse transcribed using superscript III reverse transcriptase (Life Technologies), and real-time quantitative polymerase chain reaction (qPCR) was used to determine gene expression changes. qPCR was performed using a CFX96 Real-Time System (BioRad). Forward and reverse primers and probes were designed using Primer3Plus software [37] and purchased from Sigma-Aldrich or Integrated DNA Technologies. Expression of β-actin and TATA box binding protein (Tbp) were used as endogenous controls. All gene names, abbreviations and accession numbers can be found in S1 Table. Primer and probe sequences can be provided upon request. Biochemical Analysis Serum triglyceride and total cholesterol were measured enzymatically using InfinityTM kits (Thermo Scientific) according to the manufacturers’ instructions. Serum bile acid levels were measured using the Total Bile Acids Assay kit from Diazyme Laboratories, and serum free fatty acids were measured using a non-esterified fatty acid enzymatic colorimetric HR series NEFA-HR (2) assay from WAKO Chemicals USA Inc., performed according to the manufacturer’s instructions. Lipoprotein Lipase (LPL) activity was measured in serum using an ELISA kit (Cell Biolabs, Inc, San Diego, CA), and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Teco Diagnostic Kits (A526 and A561 respectively), according to the manufacturer’s instructions. Histology After removal from the rat, sections of excised liver were immediately immersed in 10% buffered formalin, and processed for hematoxylin and eosin (H&E) staining, which was performed by the Pathology Laboratory at University of Nevada, Reno. Evaluation of Steatosis by Image Analysis At least three non-consecutive microscopic fields per animal were randomly analyzed, by blindly moving the field of view, using an Olympus DP71 camera attached to a BX41 microscope, equipped with a UPlanFL 40X objective. Each image field was analyzed using a 94x70 point grid (P T ). The volume density of hepatic steatosis (V V [steatosis, liver]) was then estimated as the ratio of the points marking the vesicles of fat (P P ) compared to the number of test points using the following equation: V V [steatosis, liver] = P P [steatosis]/P T , as previously described [38]. Hepatic cholesterol measurement Hepatic cholesterol was extracted using the Folch extraction method and levels were measured as previously described [39, 40]. Briefly, lipids were extracted from 100 mg of liver using a chloroform/methanol mixture. Cholesterol was then determined using a colorimetric Infinity cholesterol assay kit, performed according to the manufacturers’ instructions. Extracted cholesterol contents were normalized to wet liver weight. Measurement of fecal bile acid, total lipid, cholesterol and free fatty acid excretion A modified version of the method reported by Modica et al., was used to measure fecal BA content [40]. Briefly, 0.2 g of dried feces was mixed with 2 ml of 2 mg/ml sodium borohydrate in ethanol and incubated at room temperature for 1 hour. Hydrochloric acid and sodium hydroxide were added and samples were vortexed and allowed to digest for 12 hours under reflux. The samples were then filtered and dried under nitrogen. Samples were re-suspended in milli-Q water and filtered through Sep-Pak C18 cartridges (Thermo Scientific), washed and eluted with methanol and dried under nitrogen. Samples were re-dissolved in 1 ml methanol and BA concentrations were measured enzymatically, using the Total Bile Acids Assay kit from Diazyme Laboratories. Fecal cholesterol and non-esterified fatty acids were extracted as previously described [40], and cholesterol levels were measured using a colorimetric Infinity cholesterol assay kit and non-esterified fatty acids were quantified using a Wako diagnostics HR Series NEFA-HR (2) assay. Total fecal lipids were assessed via gravimetric analysis following Folch extraction. Briefly, 0.5 ml aliquots of the chloroform layer were placed into pre-weighed glass tubes (three tubes per sample) and allowed to evaporate in a fume hood overnight. The next day, the weights were recorded and converted to percent lipids (mg lipid/mg dry fecal weight). Statistical Analysis Data represents the mean ± SEM for the fold change relative to control (or endogenous gene expression) (n = 4 or 5 per treatment, per group, analyzed in triplicate). One-way analysis of variance (ANOVA) followed by Holm-Sidak post-hoc tests was employed to detect significant differences between groups. Treatment differences were considered statistically significant at p<0.05. All statistical analyses were performed using GraphPad Prism version 6.05 for Windows, GraphPad Software (San Diego, CA).

Discussion The study presented herein provides new evidence establishing that one week administration with GSPE effectively lowers serum TG levels in a model of fructose-induced hypertriglyceridemia. Previous reports showed that GSPE decreases serum TGs in a normolipidemic state in several animal models, including rats [18, 19, 22]. We now show that, under conditions of severe hypertriglyceridemia, GSPE causes a significant 41% reduction in serum TG levels. This is comparable to that seen with fenofibrate, one of the most commonly prescribed lipid-lowering agents in the world [47], which reduces serum TGs by 36% [48]. Cholesterol synthesis gene expression was increased in the fructose-GSPE treated animals, and was accompanied by a concomitant increase in fecal excretion of cholesterol, which could occur via transintestinal cholesterol efflux (TICE) [49]. Additionally, compared to the control diet, fructose caused a marked increase in hepatic lipid droplet accumulation, indicative of steatosis (>5% lipid volume), which was significantly attenuated following co-administration with GSPE. It is well established that high-fructose feeding causes diet-induced alterations in lipid metabolism [50]. In the present study, we observed a significant 171% increase in serum TGs in the rats after 9 weeks on the fructose diet. Increased expression of Srebp-1c was previously shown to be one mechanism underlying fructose-induced hypertriglyceridemia, however, it is not the only mechanism involved [8]. Pparα serves as an essential regulator of lipid metabolism, with gene ablation disrupting normal lipid homeostasis [51]. Hepatic suppression of Pparα was also identified as a mechanism contributing to serum hypertriglyceridemia induced by a high-fructose diet [50]. Additionally, reduced Fgf21 levels have been shown to decrease lipolysis [43]. In the current study, GSPE administration in the fructose-fed rats significantly decreased the expression of both Srebp1c and Scd1, indicating decreased TG synthesis, thereby contributing to reduced serum TG levels. We did not observe an increase in Srebp-1c following fructose consumption, likely due to the fact that the rats were fasted for 5 hours prior to sacrifice. It is well known that Srebp-1c is sensitively suppressed by fasting or nutritional deprivation [52, 53]. Therefore, fasting could have resulted in the lack of induction in the expression of Srebp-1c and its downstream targets, including Pgc-1β and Fasn, in addition to Mlxipl, in the fructose-fed animals. Despite no effect on Srebp1c, Fgf21 expression was reduced in the fructose group, indicating decreased lipolysis [43]. Therefore, it is possible that repression of Fgf21 is the underlying mechanism by which fructose induced hypertriglyceridemia in these rats. Reduced serum BA levels observed in the fructose-GSPE treated rats correlate with increased fecal BA output. Indeed, decreased intestinal BA absorption, combined with reduced hepatic lipogenesis, may be linked to the observed reduction in serum TG levels. Reduced BA absorption necessitates increased conversion of cholesterol into BAs. Since the rats consumed a cholesterol-free diet, increased endogenous cholesterol synthesis is necessary for the production of BAs. This is consistent with the observed increase in cholesterol synthesis gene expression observed in this study. Interestingly, there was no effect on Cyp7a1, which encodes cholesterol 7α-hydroxylase, the rate-limiting enzyme in BA biosynthesis. Cyp8b1, responsible for canonical BA synthesis, was decreased, with no changes in alternative BA biosynthetic gene expression, indicating that the newly synthesized cholesterol is not then shuttled into the pathway for BA production. In addition, the most readily available source of acetyl CoA for use in cholesterol synthesis would be from catabolized TGs, which provides an elegant explanation for the observed reduction in serum TGs, even in the presence of fructose. GSPE administration profoundly increased total fecal lipid excretion, as well as BA excretion. It is known that hepatobiliary cholesterol excretion is not the only way to remove cholesterol from the body. The proximal part of the small intestine is now known to actively secrete cholesterol, via a pathway called transintestinal cholesterol efflux (TICE) [49]. The rate of TICE strongly depends on the presence of a cholesterol acceptor, and increased levels of BAs in the intestinal lumen are known to increase TICE [54]. When bile salts are combined with phospholipid, the TICE pathway is strongly stimulated [55]. Therefore, increased levels of both BA and total lipid within the intestine could stimulate TICE and therefore contribute to enhanced fecal cholesterol excretion. Consequently, serum cholesterol levels remained the same due to an equilibrium being achieved between the rate of endogenous cholesterol synthesis and the amount secreted from the liver into the blood to be subsequently excreted via TICE. Importantly, and in agreement with this notion, the amount of cholesterol excreted in the feces exceeds the levels that could have originated from dietary intake. In addition to decreased serum TG levels, GSPE treatment significantly decreased hepatic steatosis induced by fructose, as evidenced by the reduction in hepatic lipid droplet accumulation and cholesterol content. Steatosis by itself is considered to be a relatively benign and reversible condition [56]. However, transition from steatosis into NASH represents a key step in pathogenesis, since it sets the stage for further liver damage, including development of fibrosis, cirrhosis and eventually hepatocellular carcinoma [56]. Progression from simple steatosis to NASH usually involves a “second hit”, e.g. oxidative stress and inflammation, and previous studies have shown that accumulation of cholesterol, rather than fatty acids or triglycerides, is critical for this progression [57]. Reduction in hepatic cholesterol content has been proposed as a fundamental treatment strategy for NAFLD [58]. It may be speculated that one reason why the newly synthesized cholesterol is not converted to BAs, esterified or moved to the plasma membrane for export via the apolipoprotein/Abca1 pathway by GSPE in the presence of fructose, is because it is removed from the liver as a protective mechanism against further progression of steatosis. In conclusion, this study provides valuable and innovative insight into the molecular regulatory actions of GSPE in treating hypertriglyceridemia in the fructose-fed rat model. This is achieved at the molecular level via GSPE-induced down-regulation in the hepatic expression of Srebp1c and Scd1, to decrease lipogenesis, combined with increased endogenous hepatic cholesterol synthesis, without any corresponding increase in de novo BA biosynthesis from the newly synthesized cholesterol in the fructose-fed rats. The novel observations resulting from this study demonstrate that, in the presence of fructose, GSPE alters the conversion of endogenously synthesized cholesterol, directing it through TICE for export via the feces. These results, combined with the decreased levels of steatosis, indicate that GSPE warrants further investigation as a treatment strategy against metabolic dysregulation and potential for amelioration of hepatic steatosis.

Acknowledgments The authors would like to thank Dr. Mike Teglas, DVM, Ph.D., Department of ANVS, for assistance with histological analysis of rat liver sections.

Author Contributions Conceived and designed the experiments: MLR. Performed the experiments: LED RMH GCC BSW KR FDR MLR. Analyzed the data: LED RMH GCC MLR. Wrote the paper: LED GCC MLR.