Dig Dis Sci DOI 10.1007/s10620-014-3128-0
ORIGINAL ARTICLE
Protective Effect of Grape Seed and Skin Extract Against High-Fat Diet-Induced Liver Steatosis and Zinc Depletion in Rat Kamel Charradi • Salem Elkahoui • Ines Karkouch • Ferid Limam • Fethy Ben Hassine Miche`le Veronique El May • Ezzedine Aouani
•
Received: 30 May 2013 / Accepted: 20 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Obesity is a tremendous public health problem, characterized by ectopic deposition of fat into non-adipose tissues as liver generating an oxidative stress that could lead to steato-hepatitis. Grape seed and skin extract (GSSE) is a complex mixture of polyphenolics exhibiting robust antioxidative properties. Aim We hypothesize that GSSE could protect the liver from fat-induced lipotoxicity and have a beneficial effect on liver function. Methods Hepatoprotective effect of GSSE was measured by using an experimental model of fat-induced rat liver steatosis. Male rats were fed a standard diet or a high-fat diet (HFD) during 6 weeks and treated or not with 500 mg/ kg bw GSSE. Lipid deposition into the liver was assessed by triglyceride, cholesterol and phospholipid measurements. Fat-induced lipoperoxidation, carbonylation, depletion of glutathione and of antioxidant enzyme K. Charradi (&) S. Elkahoui I. Karkouch F. Limam E. Aouani Laboratoire des Substances Bioactives (LSBA), Centre de Biotechnologie de Borj Cedria, BP-901, 2050 Hammam-Lif, Tunisia e-mail: [email protected] K. Charradi E. Aouani Faculte´ des Sciences de Bizerte, Universite´ de Carthage, 7021 Jarzouna, Tunisia F. Ben Hassine Laboratoire de Biochimie, Polyclinique de la CNSS d’El Khadra, Avenue Zobeir Ibn El Awam, 1003 Cite´ El Khadra Tunis, Tunisia M. V. El May Unite´ de recherche no 01/UR/07-08, Faculte´ de Medicine de Tunis, Tunis, Tunisia
activities were used as oxidative stress markers with a special emphasis on transition metal distribution. Results HFD induced liver hypertrophy and inflammation as assessed by high liver transaminases. HFD also induced an oxidative stress characterized by increased lipid and protein oxidation, a drop in glutathione and antioxidant enzyme activities as glutathione peroxidase and superoxide dismutase and a drastic depletion in liver zinc. Importantly, GSSE prevented all the deleterious effects of HFD treatment. Conclusions Data suggest that GSSE could be used as a safe preventive agent against fat-induced liver lipotoxicity which could also have potential applications in other nonalcoholic liver diseases. Keywords Zinc
Polyphenol Liver Fat Oxidative stress
Introduction Obesity is a serious worldwide health problem, implicated in various pathologies including type 2 diabetes, hypertension, cancer, cardiovascular dysfunction and non-alcoholic fatty liver diseases (NAFLD) which could eventually lead to hepatic cirrhosis and, ultimately, to hepatocellular carcinoma [1]. Obesity is a state of low-grade and chronic oxidative stress and inflammation in relation to reduced antioxidant defense and strong reactive oxygen species (ROS) production [2]. Oxidative stress plays a pivotal role in the development of NAFLD, particularly in the progression from steatosis to steatohepatitis [3]. The emerging hypothesis for the pathogenesis of non-alcoholic steatohepatitis is a ‘‘two-hit theory’’ consisting of a hepatic fat accumulation as the first hit and an oxidative stress as the
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second hit [4]. GSSE is a complex mixture of polyphenolics as oligomeric proanthocyanidins and unsaturated fatty acids largely used as a nutritional supplement [5]. GSSE received much attention due to its pleiotropic biological activities and health-promoting properties, such as antioxidant [6], free radical scavenging [7], lipid lowering [8], anti-tumor [9, 10] or neuroprotective effects [11]. All these beneficial health properties led to consider GSSE as a potential health food ingredient which received the GRAS (generally recognized as safe) certification from US FDA. The present study aimed to evaluate the effect of a specific and self-elaborated HFD from animal origin on liver steatosis and oxidative stress, with a special emphasis on transition metal distribution. We hypothesize that a treatment with a high-dose GSSE will prevent the HFDinduced hepatotoxicity and improve liver function.
Methods and Materials
Table 1 LC-MS/MS data of some phenolic compounds found in carignan GSSE Compounds
Relative abundance (%)
Catechin
1.31
Epicatechin
1.61
Procyanidin dimer Procyanidin trimer
0.23 ND
Quercetin
0.55
Resveratrol
0.07
Rutin
1.00
Vanillin
9.21
Gallic acid
41.53
P-coumaric acid
0.19
Rosmarinic acid
0.37
2,5-Dihydroxybenzoı¨c acid
41.26
Caffeic acid
1.40
Chlorogenic acid
0.17
Ferulic acid
1.00
Reagents and Diets Grape seed and skin extract was processed from a grape cultivar (Carignan) of Vitis vinifera from northern Tunisia. Seeds were manually separated from skin, air-dried and grounded separately with a coffee grinder (FP 3121 Moulinex) until a fine powder was obtained. Both powders were then mixed at 50:50 ratio on a dry weight basis in 10 % ethanol (v/v) at dark. After vigorous stirring and centrifugation at 10,000g for 15 min at 4 °C, supernatant containing soluble polyphenols was administered to animals by intraperitoneal (i.p) route. Total phenolic content and GSSE composition (Table 1) were determined according to [13]. Standard diet (SD) for rodent in pelleted form was purchased from ALMAS Bizerta (Tunisia). Highfat diet (HFD) was prepared by soaking commercial food pellets into warmed (100 °C) and liquefied abdominal fat from animal origin (sheep) during 15 min and allowed to dry at room temperature. Sheep abdominal fat was used to feed the rats in order to mimic the local Tunisian feeding habits. Composition of SD and HFD is shown in Table 2. Animals and Experimental Design Twenty-four male Wistar rats (210–230 g) from Pasteur Institute (Tunis) were used in this study, in conformity with the local ethics committee of Carthage University and in agreement with the NIH guidelines [12]. They were maintained in animal facility at controlled temperature (22 ± 2 °C), a 12-h light/dark cycle, and divided into 4 groups of 6 animals each, fed with standard diet (SD) or HFD for 6 weeks. Rats received by daily i.p injection (8.00–9.00 am) either 10 % ethanol as vehicle (control SD and HFD) or
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500 mg/kg bw GSSE (SD ? GSSE and HFD ? GSSE). Parenteral administration of GSSE avoided the gastric barrier, the poor absorption of polyphenols through the intestine as well as hepatic and microflora transformation and inactivation of polyphenols. At the end of the treatment, rats were killed by decapitation, their blood collected using heparin as anticoagulant and plasma processed for lipids and oxidative stress assessment (not shown). Liver was isolated, weighed, homogenized in phosphate saline buffer pH 7.4 with an ultrathurax T25 homogenizator, centrifuged at 10,000g for 15 min at 4 °C and supernatant stored at -80 °C. After thawing, homogenates were used for the determination of transaminases, alcohol dehydrogenase activity, lipase activity, ectopic lipid content, oxidative stress status, metal ions distribution as well as intracellular mediators as calcium, nitric monoxide and hydrogen peroxide. The relative liver weight per 100 g body weight was expressed as [liver weight (g)/body weight on day 45 (g)] 9 100. Liver Analyses Lipids were extracted from whole liver according to [13]. Triglyceride and total cholesterol were determined using commercially available kits from Biomaghreb (Tunisia) according to [14] and [15], respectively. LDL-cholesterol was determined using a commercial kit from Biolabo, SA (France), according to [16]. Phospholipid (PL) and HDLPL were determined using assay kits purchased from BioMe´rieux, SA, Marcy-l’etoile, (France) according to [17]. Apo B and apo AI were determined using an assay kit from Konelab clinical chemistry analyzer (Thermo Clinical Labsystems, Espoo, Finland), according to [17].
Dig Dis Sci Table 2 Composition of SD and HFD Parameter
SD
HFD
Lipid % (w/w)
3.00
28.00
Energetic contribution
5.00
39.00
% (w/w)
40.00
32.00
Energetic contribution
70.00
45.00
% (w/w)
14.00
12.00
Energetic contribution
25.00
16.00
Carbohydrate
Protein
Fiber % (w/w)
7.50
6.00
14.00 9.00
12.00 7.20
Methionine % (w/w)
0.46
0.37
Cystein % (w/w)
0.34
0.27
Threonine % (w/w)
0.78
0.62
Tryptophane % (w/w)
0.25
0.20
Moisture % (w/w) Ash % (w/w)
Copper (mg/kg)
10.00
8.00
Manganese (mg/kg)
50.00
40.00
Zinc (mg/kg)
50.00
40.00
Iron (mg/kg)
40.00
32.00
Calcium (mg/kg)
1.20
0.94
Magnesium (mg/kg)
0.05
0.04
Phosphorus (mg/kg)
0.50
0.40
Selenium (mg/kg)
0.10
0.08
Iodide (mg/kg)
0.70
0.56
Cobalt (mg/kg)
0.10
0.08
Liver alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) were determined using assay kits from Biomaghreb (Tunisia), according to [18]. Lipoperoxidation was evaluated by malondialdehyde (MDA) measurement [19]. Briefly, liver homogenates were precipitated with 20 % trichloroacetic acid (TCA) and MDA from supernatant was allowed to react with thiobarbituric acid (TBA). Spectrophotometric measurement was done at 532 nm and MDA concentration calculated using the absorbance coefficient of MDA–TBA complex 1.56 9 105 cm-1 M-1. Oxidative damage to proteins was evaluated by quantifying protein carbonylation in liver homogenates according to [20]. Briefly, after proteins precipitation with 20 % TCA and dissolution in 2,4 dinitrophenylhydrazine (DNPH)containing buffer, absorbance was measured at 366 nm and results expressed as nmol carbonyl/mg protein. Total protein was determined according to [21]. Reduced (GSH) and oxidized glutathione (GSSG) were determined according to [22]. Liver homogenates were also used for the determination of antioxidant enzyme activities. Glutathione peroxidase
(GPx; E.C.1.11.1.9.) was measured according to [23]. Briefly, liver homogenates (2 mg protein) were mixed with phosphate buffer 100 mmol pH 7,4 containing 4 mmol GSH and 5 mmol H2O2 in 1 mL final volume. The mixture was incubated at 37 °C for 1 min; then, 0.5 mL TCA (5 %, w/v) was added to stop the reaction. After centrifugation at 1,500g for 5 min, 0.2 mL supernatant was mixed with phosphate buffer 100 mmol pH 7.4 containing 10 mmol 2-nitrobenzoı¨c acid (DTNB). GPx activity was measured at 412 nm. Catalase (CAT; E.C.1.11.1.6.) was assayed by measuring the initial rate of H2O2 disappearance at 240 nm [24]. The reaction mixture contained 33 mmol H2O2 in 50 mmol phosphate buffer pH 7 and 5 lL extract in 1 mL final volume and CAT activity calculated using an extinction coefficient of 40 mmol-1 cm-1 for H2O2. Superoxide dismutase (SOD; E.C.1.15.1.1.) was determined using a modified epinephrine assay [25]. At alkaline pH, superoxide anion O2- causes the autoxidation of epinephrine to adenochrome. Liver extract was added to a 2 mL reaction mixture containing 4U bovine catalase, 100 lg epinephrine and 62.5 mmol sodium bicarbonate buffer pH 10.2, and changes in absorbance were recorded at 480 nm. Characterization of SOD isoforms was performed using KCN (3 mmol) as Cu–Zn inhibitor or H2O2 (3 mmol) which affects Cu/Zn and Fe-SOD, whereas MnSOD is insensitive to both inhibitors. Alcohol dehydrogenase activity (E.C.1.1.1.1) was evaluated by following NADH formation at 340 nm and 37 °C according to [26]. Lipase activity was determined according to [27] using p-nitrophenol dodecanoate in dimethylsulfoxide (DMSO) and ethanol as substrate. The reaction mixture containing 5 mmol p-nitrophenol dodecanoate, 50 mmol Tris–HCl buffer pH 8.5 and 50 lL of sample was incubated at 37 °C for 1 h, and the reaction stopped with 60 mmol EDTA. After centrifugation at 10,000g for 5 min, absorbance was measured at 412 nm. One unit is defined as the amount of enzyme catalyzing the release of 1 lmol p-nitrophenol (e = 18.3 mmol-1 cm-1). Ionizable calcium was determined according to [28] using an assay kit from Biomaghreb, Tunisia. At basic pH, calcium constitutes cresolphtalein a purple complex measurable at 570 nm. Briefly, 50 lL liver extract was added to 650 lL of reaction mixture containing 2-amino-2-methyl-1-propanolol buffer (500 mmol L-1), cresolphtalein (0.62 mmol L-1) and hydroxyl-8 quinoleine (69 mmol L-1). Incubation was carried out at room temperature for 5 min assuming the complex was stable for 1 h. H2O2 was determined according to [29] using an assay kit from Biomaghreb, Tunisia. Briefly, in the presence of peroxidase, H2O2 reacts with 4-aminoantipyrine and phenol to give a red-colored quinoeimine that absorbs at 505 nm. NO was determined by quantification of NO metabolites nitrite and nitrate according to [30]. Tissue samples were also wet ashed in nitric acid (15.5 mol L-1),
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Dig Dis Sci Fig. 1 Protective effect of GSSE on HFD-induced liver weight (a), transaminases (b) and lipase activity (c) overload. Rats were fed a SD or HFD during 6 weeks and daily administered with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. # p \ 0.05 for SD ? GSSE versus SD. *p \ 0.05 for HFD vs. SD. §p \ 0.05 for HFD ? GSSE versus HFD
diluted, and filtered for zinc, copper and manganese determination by atomic absorption spectroscopy. Liver Histology A piece of liver was fixed in 10 % (v/v) formaldehyde, processed by successive dehydrations with ethanol baths and embedded in paraffin. Serial sections were cut 5 lm thick and stained with hematoxylin-eosin (HE) using standard procedures. Statistical Analysis Results are expressed as mean ± SEM. Statistical differences were evaluated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. All analyses were performed using Statistica. p value \ 0.05 was considered significant.
Results Effect of HFD on Body Weight Gain, Relative Liver Weight, and Toxicity At the end of the experiment, body weight gain reached 13.7 % for SD, 14 % for SD ? GSSE, 22 % for HFD and
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12 % for HFD ? GSSE animals. Moreover, HFD increased the relative liver weight by 11 % (Fig. 1a), transaminases as ASAT by 11 % and ALAT by 13 % (Fig. 1b) and lipase activity by 28 % (Fig. 1c). GSSE backed all HFD-induced disturbances to near control level. Noteworthy that GSSE also decreased lipase activity by 12 % in SD animals. Hepatic Lipid Content We reported in Fig. 2 the intra-hepatic level of various lipid species from SD- or HFD-treated animals. HFD increased triglyceride by 66 % (Fig. 2a), cholesterol by 41 % (Fig. 2b), phospholipid as assessed by LDL ? VLDL/HDL ratio by 15 % (Fig. 2c) and apo B/apo AI ratio by 20 % (Fig. 2d). All HFD-induced lipid accumulation was attenuated upon GSSE treatment. Non-enzymatic Antioxidant We further sought to determine whether lipid accumulation into the liver provoked an oxidative stress (Fig. 3). HFD increased liver MDA by 37 % (Fig. 3a), protein carbonylation by 36 % (Fig. 3b) but decreased the reducing power as assessed by GSH/GSSG ratio by 29 % (Fig. 3c). GSSE treatment brought all these parameters to near control level. Importantly, GSSE decreased MDA by 38 % and protein carbonylation by 25 % in SD group.
Dig Dis Sci Fig. 2 Protective effect of GSSE on HFD-induced liver triglyceride (a), cholesterol (b), phospholipid (c) and apolipoproteins (d) overload. Rats were fed a SD or HFD during 6 weeks and daily treated with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. *p \ 0.05 for HFD versus SD. §p \ 0.05 for HFD ? GSSE versus HFD
Fig. 3 Protective effect of GSSE on HFD-induced hepatic lipoperoxidation (a), carbonylation (b) and glutathione depletion (c). Rats were fed a SD or HFD during 6 weeks and daily administered with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. #Significant for SD ? GSSE versus SD. *Significant for HFD versus SD. § significant for HFD ? GSSE versus HFD
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Dig Dis Sci Fig. 4 Effect of GSSE on HFD-induced depletion of liver GPx (a), SOD (b) and CAT (c) activity. Rats were fed a SD or HFD during 6 weeks and daily treated with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. # Significant for SD ? GSSE versus SD. *Significant for HFD versus SD. §Significant for HFD ? GSSE versus HFD
Enzymatic Antioxidant
Histology
HFD depressed liver antioxidant enzyme activities as GPx by 17 % (Fig. 4a) and SOD by 40 % (Fig. 4b). In this latter case, the Cu/Zn and to a lesser extent the Mn isoform were depressed, whereas the Fe isoform was unchanged. No effect on CAT activity was detected (Fig. 4c). GSSE treatment corrected all the deleterious effects of HFD to near control level.
Histological examination of the liver sections revealed normal histology and architecture in SD- (Fig. 6a) and GSSE-treated SD animals (Fig. 6b). HFD provoked moderate to severe macrovesicular steatosis, dilatation and fat accumulation within hepatocytes and sinusoids (Fig. 6c). GSSE corrected almost all HFD-induced structural modifications to near control level (Fig. 6d).
Effect of HFD on Transition Metals HFD treatment strongly decreased liver zinc by 92 % (Fig. 5a), and GSSE brought all HFD-induced zinc disturbances to control level. Moreover, HFD depressed the activity of the zinc-dependant alcohol dehydrogenase by 38 % and GSSE counteracted this adverse effect. Remarkably, that GSSE increased significantly (8 %) the liver alcohol dehydrogenase activity in SD condition. HFD had no effect on hepatic manganese and only a slight (not significant) effect on liver copper (Table 3). Effect of HFD on Intracellular Mediators HFD treatment had no effect on intracellular mediators as H2O2, calcium or NO (Table 4).
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Discussion This study was undertaken in order to analyze the lipotoxic effect of a high-fat-enriched regime on rat liver as well as the protection offered by GSSE treatment. Overall, HFD increased body weight, caused abdominal fat deposition and induced dyslipidemia and C-reactive protein into plasma [31]. With regard to the liver which is a central organ for lipid metabolism, HFD induced hypertrophy and excessive deposition of various lipid species such as triglyceride, cholesterol and phospholipid. It also caused hepatic toxicity as illustrated by the increase in liver transaminases as well as in lipase activity [32]. Moreover, HFD provoked a clear oxidative stress characterized by high MDA and protein carbonylation, depressed GPx and Cu/Zn-SOD activities and a drastic depletion of Zn from
Dig Dis Sci Fig. 5 Effect of GSSE on HFD-induced depletion of liver zinc (a) and alcohol dehydrogenase activity (b). Rats were fed a SD or HFD during 6 weeks and daily treated with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. # Significant for SD ? GSSE versus SD. *Significant for HFD versus SD. §Significant for HFD ? GSSE versus HFD
Table 3 Effect of HFD on liver copper and manganese SD
SD ? GSSE
HFD
HFD ? GSSE
Copper (nmol/mg protein)
0.959 ± 0.019
1.060 ± 0.021
0.761 ± 0.017
0.885 ± 0.020
Manganese (nmol/mg protein)
0.891 ± 0.011
0.909 ± 0.006
0.907 ± 0.007
0.891 ± 0.011
Results are expressed as mean ± SEM HFD versus SD: * p \ 0.05; HFD ? GSSE versus HFD:
§
p \ 0.05
Table 4 Effect of HFD and GSSE on liver intracellular mediators
Hydrogen peroxide (mmol/mg protein)
SD
SD ? GSSE
HFD
HFD ? GSSE 39.92 ± 0.27
40.03 ± 0.17
39.81 ± 0.19
40.34 ± 0.28
Ionisable calcium (lmol/mg protein)
1.97 ± 0.04
1.97 ± 0.05
1.99 ± 0.04
1.97 ± 0.04
NO metabolites (lmol/mg protein)
8.07 ± 0.46
8.02 ± 0.34
8.15 ± 0.35
8.08 ± 0.56
Data are presented as mean ± SEM
the liver. Importantly, GSSE abrogated almost all the disturbances induced by HFD. Therefore, we accepted our original study hypothesis that GSSE efficiently counteracted the HFD-induced liver lipotoxicity. HFD increased liver MDA and protein carbonylation and depressed the antioxidant capacity as glutathione which constitutes the first line of defense against free radicals in the liver and also acts as a GPx substrate [33]. In addition, HFD which did not modify liver CAT, depressed GPx and SOD activity, more specifically the Cu/Zn isoform. This last result is supported by the strong depletion of zinc from the liver that did not occur in the other organs such as the brain, the heart and the kidney (data not shown). In fact, HFD treatment depleted Zn from the liver and pancreas (not shown), copper from the heart and kidney [34], and Mn from the brain [11]. As a consequence, HFD induced the accumulation of Zn, Mn and Cu into the plasma [35] and the depletion of iron from plasma that accumulates into the heart [31] and adipose tissue [35]. Such a relationship between obesity and transition metal distribution has been already mentioned several decades ago by Kennedy et al. [36] who demonstrated lower level of micronutrients into
tissues and higher level into plasma from genetically obese mice when compared to lean control. Thus, a role of copper deficiency in hypercholesterolemia has long been recognized [37] and high circulating level of copper was linked to obesity-induced left ventricular hypertrophy in elderly patients [38]. A growing number of studies suggest a potential link between obesity and altered iron metabolism. Iron chelation with deferoxamine improved obesity through decreased oxidative stress and adipocyte hypertrophy [39] and lipocalin 2 induced cardiomyocyte apoptosis by means of iron deposition [40]. Moreover, manganese overexposure of rats induced disturbances in brain lipid metabolism as assessed by high level of palmitate, oleate and cholesterol [41]. Our present data are consistent with several previous reports dealing with decreased zinc levels in both acute and chronic liver disease states [42, 43] and with the prominent role of zinc deficiency in oxidative stress [44]. It is our opinion that Zn ions constitute one of the missing links between HFDinduced obesity and liver steatosis, as this trace element plays a central role in liver function and homeostasis [45]. Zinc is an essential cofactor for many enzymes, including
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Fig. 6 Effect of GSSE on HFD-induced liver morphology alteration. SD (a), SD ? GSSE (b), HFD (c) and HFD ? GSSE (d). CV central vein, DH degenerated hepatocyte, LV lipid vacuole, S sinusoid and V vein. H&E 9 400
those implicated in the metabolism of vitamin A and ethanol [46]. Hypozincemia was shown to be linked to male obesity in humans [47], and Cu/Zn SOD deficiency induced hepatic lipid accumulation by impaired lipoprotein secretion [4], whereas zinc supplementation was used in the treatment of alcoholic liver disease [48]. Zinc is also known to regulate both enzyme activity and protein stability either as an activator or as an inhibitor; and zinc depletion is at the basis of alcoholic and non-alcoholic liver diseases leading to liver fibrosis [49]. Interestingly, we noted a drop in liver alcohol dehydrogenase activity, in accordance with previous [50] and more recent work [51] that linked zinc deficiency with reduced alcohol dehydrogenase activity. This latter result is liver specific as it was not observed in other organs as kidney where, as expected, HFD did not disturb zinc homeostasis (not shown). We did not yet know which zinc finger transcription factor, if any, is affected by this dramatic loss of zinc, nor did we know which zinc transporter as ZIP or ZnT could be involved in such depletion of zinc from the liver. Undoubtedly, the most relevant finding of the current work is the potential ability of our locally produced GSSE to prevent steatosis. GSSE prevented HFD-induced hypertrophy and lipid overload, as assessed by decreased lipid depletion as well as lipase activity and the prevention
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of liver histology from macrovesicular steatosis to normal architecture. Our results, which emphasized the antioxidative role of GSSE as well as its impact on hepatic lipid profile, are fully in line with recent works in the field [52, 53]. Moreover, our data showed that GSSE is also protective in SD conditions, which argued in favor of its safety and confirmed similar data found by other investigators [54]. Our contribution further extended the current knowledge on the putative treatment of fat-induced liver zinc depletion using natural grape polyphenols. GSSE prevented the HFD-induced zinc depletion from the liver as well as the drop in alcohol dehydrogenase activity, a zinc dependent enzyme. Our data are consistent with the clinical use of GSSE to improving liver function in patients with non-alcoholic fatty liver change [55]. GSSE was recently shown to reverse HFD-induced obesity in mice and hepatosteatosis through up- and down-regulation of mRNA for lipolytic and lipogenic enzymes, respectively [56]. To the best of our knowledge, our data are the first to link the preventive effect of GSSE with liver zinc repletion. They are consistent with in vitro studies using human Hep G2 cells in which dietary catechins and procyanidins were shown to modulate zinc metabolism and to increase the level of cytoplasmic labile zinc [57]. GSSE, which has been attributed the GRAS certification from US FDA, is
Dig Dis Sci
safe even when used at high doses. Moreover, the risk of zinc overloading constitutes a serious drawback in the treatment of many liver pathologies characterized by zinc depletion. A recent study using polaprezinc, a mixture of zinc and carnosine for the treatment of zinc deficiency in thioacetamide-induced liver fibrosis [58], proved the safety of such compound at a dose not exceeding 500 mg/kg/day. However, this dose of polaprezinc is far less from the optimal dose of 4,000 mg/kg/day GSSE, which is devoid of any toxicity even in long-term experiments. It is important to keep in mind that this high dose of GSSE, which corresponds to approximately 280 g/day for a 70 kg adult, has been daily administered to animals in 3-month-long experiment with no sign of toxicity (data not shown). GSSE should be viewed as a potential alternative to zinc supplementation in the treatment of liver pathologies characterized by zinc depletion [59]. Indeed, zinc supplementation, which inhibited the ethanol-increased liver weight, did not improve the weight gain [60], although in our current study, GSSE improved both the overall weight gain and the relative liver weight. Consequently, not only GSSE appeared more efficient than zinc supplementation but it also avoided several drawbacks linked to zinc supplementation. Future studies should analyze the relationship between HFD-induced liver steatosis, zinc depletion and the protection offered by GSSE. It would be particularly important to identify which lipid species or free fatty acid present in HFD provoked the drastic depletion of zinc from the liver, as well as the implication of zinc transporters in such a process [61]. In this respect, ZIP14 downregulation and zinc depletion were recently shown to be involved in the development and progression of hepatocellular cancer [62]. It would also be essential to identify which polyphenol present in GSSE could account for liver protection as described for resveratrol [63] or catechin and quercetin [64]. Importantly, SIRT1 activation by polyphenols as resveratrol is a successful strategy in the treatment of fatty liver disease and obesity [65]. Nevertheless, our work presents some limitations. First, GSSE was used at a suboptimal dose of 500 mg/kg which exhibited only a partial prevention of triglyceride accumulation. Indeed, in dose–response experiment, GSSE exerted antioxidative beneficial effects till 4,000 mg/kg (unpublished data) which is similar to the optimal dose claimed for the commercial IH636 californian grape extract [66]. Although HFD altered GPx activity and GSSE prevented it, selenium was not measured. Interestingly, grape seed extract was recently shown to act synergistically with selenium to decrease indomethacin-induced gastric ulcers in rats [67]. Future studies should analyze the relationship between GSSE-induced zinc repletion into the liver and selenium. Finally, we should have used the more specific
oil red lipid staining method as a more compelling evidence for HFD-induced hepato-lipotoxicity. In conclusion, HFD provoked zinc depletion from the liver and GSSE prevented HFD-induced hepato-steatosis. Clinical trials using GSSE for the prevention and treatment of alcoholic and non-alcoholic fatty liver diseases should be envisaged. Acknowledgments Financial support of the Tunisian Ministry of ‘‘Enseignement Supe´rieur, Recherche Scientifque et Technologie’’ is gratefully acknowledged. Conflict of interest
None.
References 1. Angulo P. Non alcoholic fatty liver disease. New Engl J Med. 2002;346:1221–1231. 2. Higdon JV, Frei B. Obesity and oxidative stress: a direct link to CVD? Arterioscl Throm Vas. 2003;23:365. 3. Adams LA, Angulo P, Lindor KD. Nonalcoholic fatty liver disease. Can Med Assoc J. 2005;172:899–905. 4. Uchiyama S, Shimizu T, Shirasawa T. CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice. J Biol Chem. 2006;281:31713–31719. 5. Bagchi D, Bagchi M, Stohs SJ, Ray SD, Sen CK, Preuss HG. Cellular protection with proanthocyanidins derived from grape seeds. Ann NY Acad Sci. 2002;957:260–270. 6. Bagchi D, Bagchi M, Stohs SJ, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology. 2000;7:187–197. 7. Curin Y, Ritz MF, Andriantsitohaina R. Cellular mechanisms of the protective effect of polyphenols on the neurovascular unit in strokes. Cardiovasc Hematol Agents Med Chem. 2006;4:277–288. 8. Moreno DA, Ilic N, Poulev A, Brasaemle DL, Fried SK, Raskin I. Inhibitory effects of grape seed extract on lipases. Nutrition. 2003;19:876–879. 9. Bomser JA, Singletary KW, Wallig MA, Smith MA. Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds. Cancer Lett. 1999;29:151–157. 10. Agarwal C, Sharma Y, Agarwal R. Anticarcinogenic effect of a polyphenolic fraction isolated from grape seeds in human prostate carcinoma DU145 cells: modulation of mitogenic signaling and cell-cycle regulators and induction of G1 arrest and apoptosis. Mol Carcinog. 2000;28:129–138. 11. Charradi K, Elkahoui S, Karkouch I, Limam F, Ben Hassine F, Aouani E. Grape seed and skin extract prevents high-fat dietinduced brain lipotoxicity in rat. Neurochem Res. 2012;37:2004–2013. 12. National Research Council. Guide for the care and the use of laboratory animals, vol. 20. Bethesda: National Institute of Health; 1985:85. 13. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509. 14. Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem. 1982;28:2077–2080.
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Dig Dis Sci 15. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470–475. 16. Tietz NW. Text book of clinical chemistry. 3rd ed. Philadelphia: W.B. Saunders; 1999:819–884. 17. Mainard F, Madec Y. Cholesterol, phospholipid and apoB composition of LDL: comparison of precipitation and ultracentrifugation methods. Ann Biol Clin (Paris). 1986;44:618–623. 18. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957;28:56–63. 19. Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990;186:421–431. 20. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:467–478. 21. Hartree EF. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972;48:422–427. 22. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502–522. 23. Nakamura K, Hosada S, Hayashi K. Purification and properties of rat liver glutathione peroxidase. Biochem Biophys Acta. 1974;358:251–261. 24. Aebi H. Methods of enzymatic analysis. 2e ed. New York: Chemia Weinheium; 1974. 25. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170–3175. 26. Tachiyashiki K, Imaizumi K. Lowering and delaying actions of bovine bile on plasma ethanol levels in rats. J Nutr Sci Vitaminol. 1992;38:69–82. 27. Humbert G, Guingamp MF, Linden G. Method for the measurement of lipase activity in milk. J Dairy Res. 1997;64:465–469. 28. Stern J, Lewis WH. The colorimetric estimation of calcium in serum with ocresolphthalein complexone. Clin Chim Acta. 1957;2:576–580. 29. Kakinuma K, Yamaguchi T, Kaneda M, Shimada K, Tomita Y, Chance B. A determination of H2O2 release by the treatment of human blood polymorphonuclear leucocytes with myristate. J Biochem. 1979;86:87–95. 30. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and [15 N] nitrate in biological fluids. Anal Biochem. 1982;126:131–138. 31. Charradi K, Sebai H, Elkahoui S, Ben Hassine F, Limam F, Aouani E. Grape seed extract alleviates high fat diet-Induced obesity and heart dysfunction by preventing cardiac siderosis. Cardiovasc Toxicol. 2011;11:28–37. 32. Chiu HK, Qian K, Ogimoto K, et al. Mice lacking hepatic lipase are lean and protected against diet-induced obesity and hepatic steatosis. Endocrinology. 2010;151:993–1001. 33. Hsu CL, Yen GC. Effect of gallic acid on high fat diet-induced dyslipidemia, hepatosteatosis, and oxidative stress in rats. Br J Nutr. 2007;98:727–735. 34. Charradi K, Elkahoui S, Karkouch I, et al. Grape seed and skin extract alleviates high-fat diet-induced renal lipotoxicity and prevents copper depletion in rat. Appl Physiol Nutr Metab. 2013;38:259–267. 35. Charradi K, Elkahoui S, Limam F, Aouani E. High-fat diet induced an oxidative stress in white adipose tissue and disturbed plasma transition metals in rat: prevention by grape seed and skin extract. J Physiol Sci. 2013. doi:10.1007/s12576-013-0283-6. 36. Kennedy ML, Failla ML, Smith JC Jr. Influence of genetic obesity on tissue concentrations of zinc, copper, manganese and iron in mice. J Nutr. 1986;116:1432–1441.
123
37. Kim S, Chao PY, Allen KG. Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia. FASEB J. 1992;6:2467–2471. 38. Lind PM, Olse´n L, Lind L. Elevated circulating levels of copper and nickel are found in elderly subjects with left ventricular hypertrophy. Ecotoxicol Environ Saf. 2012;86:66–72. 39. Tajima S, Ikeda Y, Sawada K, et al. Iron reduction by deferoxamine leads to amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes KKAy mice. Am J Physiol Endocrinol Metab. 2012;302:77–86. 40. Xu G, Ahn J, Chang S, et al. Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation. J Biol Chem. 2012;287:4808–4817. 41. Fordahl S, Cooney P, Qiu Y, Xie G, Jia W, Erikson KM. Waterborne manganese exposure alters plasma, brain, and liver metabolites accompanied by changes in stereotypic behaviors. Neurotoxicol Teratol. 2012;34:27–36. 42. Mohammad MK, Zhou Z, Cave M, Barve A, McClain CJ. Zinc and liver disease. Nutr Clin Pract. 2012;27:8–20. 43. Stamoulis I, Kouraklis G, Theocharis S. Zinc and the liver: an active interaction. Dig Dis Sci. 2007;52:1595–1612. 44. Eide DJ. The oxidative stress of zinc deficiency. Metallomics. 2011;3:1124–1129. 45. King JC, Shames DM, Woodhouse LR. Zinc homeostasis in humans. J Nutr. 2000;130:1360S–1366S. 46. Brandt EG, Hellgren M, Brinck T, Bergman T, Edholm O. Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site. Phys Chem Chem Phys. 2009;11:975–983. 47. Ozata M, Mergen M, Oktenli C, et al. Increased oxidative stress and hypozincemia in male obesity. Clin Biochem. 2002;35:627–631. 48. Kang YJ, Zhou Z. Zinc prevention and treatment of alcoholic liver disease. Mol Aspects Med. 2005;26:391–404. 49. Milman N, Laursen J, Pødenphant J, Asnaes S. Trace elements in normal and cirrhotic human liver tissue. I. Iron, copper, zinc, selenium, manganese, titanium and lead measured by X-ray fluorescence spectrometry. Liver. 1986;6:111–117. 50. Indo Y, Nagata N, Higashi A, Matsuda I, Kashiwabara N, Nakashima I. Effects of dietary zinc deficiency on hepatic ornithine carbamoyltransferase and alcohol dehydrogenase activities in rats. J Pediatr Gastroenterol Nutr. 1985;4:268–273. 51. Kawashima Y, Someya Y, Sato S, et al. Dietary zinc-deficiency and its recovery responses in rat liver cytosolic alcohol dehydrogenase activities. J Toxicol Sci. 2011;36:101–108. 52. Quesada H, del Bas JM, Pajuelo D, et al. Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver. Int J Obes (Lond). 2009;33:1007–1012. 53. Castrillejo VM, Romero MM, Esteve M, et al. Antioxidant effects of a grapeseed procyanidin extract and oleoyl-estrone in obese Zucker rats. Nutrition. 2011;27:1172–1176. 54. Goodrich KM, Fundaro G, Griffin LE, et al. Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occluding and reduces fecal calprotectin: a secondary analysis in healthy Wistar furth rats. Nutr Res. 2012;32:787–794. 55. Khoshbaten M, Aliasgarzadeh A, Masnadi K, et al. Grape seed extract to improve liver function in patients with nonalcoholic fatty liver change. Saudi J Gastroenterol. 2010;16:194–197. 56. Park SH, Park TS, Cha YS. Grape seed extract (Vitis vinifera) partially reverses high fat diet-induced obesity in C57BL/6 J mice. Nutr Res Pract. 2008;2:227–233. 57. Quesada IM, Bustos M, Blay M, et al. Dietary catechins and procyanidins modulate zinc homeostasis in human HepG2 cells. J Nutr Biochem. 2011;22:153–163.
Dig Dis Sci 58. Kono T, Asama T, Chisato N, et al. Polaprezinc prevents ongoing thiocetamide-induced liver fibrosis in rats. Life Sci. 2012;90: 122–130. 59. Oteiza PI, Mackenzie GG. Zinc, oxidant-triggered cell signaling, and human health. Mol Aspects Med. 2005;26:245–255. 60. Zhou Z, Liu J, Song Z, McClain CJ, Kang YJ. Zinc supplementation inhibits hepatic apoptosis in mice subjected to a long– term ethanol exposure. Exp Biol Med. 2008;233:540–548. 61. Cousins RJ, Liuzzi JP, Lichten LA. Mammalian zinc transport, trafficking, and signals. J Biol Chem. 2006;281:24085–24089. 62. Franklin RB, Levy BA, Zou J, et al. ZIP14 zinc transporter down regulation and zinc depletion in the development and progression of hepatocellular cancer. J Gastrointest Cancer. 2012;43: 249–257. 63. Gomez-Zorita S, Fernandez-Quintela A, Macarulla MT, et al. Resveratrol attenuates steatosis in obese Zucker rats by
64.
65.
66.
67.
decreasing fatty acid availability and reducing oxidative stress. Br J Nutr. 2012;107:202–210. Auger C, Teissedre PL, Ge´rain P, et al. Dietary wine phenolics catechin, quercetin and resveratrol efficiently protect hypercholesterolemic hamsters against aortic fatty streak accumulation. J Agric Food Chem. 2005;53:2015–2021. Li Y, Wong K, Giles A, et al. Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21. Gastroenterology. 2013;S0016–5085: 01577-1. doi:10.1053/j.gastro.2013.10.059. Ray S, Bagchi D, Lim PM, et al. Acute and long-term safety evaluation of a novel IH636 grape seed proanthocyanidin extract. Res Commun Mol Pathol Pharmacol. 2001;109:165–197. Abbas AM, Sakr HF. Effect of selenium and grape seed extract on indomethacin-induced gastric ulcers in rats. J Physiol Biochem. 2013. doi:10.1007/s13-0241-z.
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ORIGINAL ARTICLE
Protective Effect of Grape Seed and Skin Extract Against High-Fat Diet-Induced Liver Steatosis and Zinc Depletion in Rat Kamel Charradi • Salem Elkahoui • Ines Karkouch • Ferid Limam • Fethy Ben Hassine Miche`le Veronique El May • Ezzedine Aouani
•
Received: 30 May 2013 / Accepted: 20 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Obesity is a tremendous public health problem, characterized by ectopic deposition of fat into non-adipose tissues as liver generating an oxidative stress that could lead to steato-hepatitis. Grape seed and skin extract (GSSE) is a complex mixture of polyphenolics exhibiting robust antioxidative properties. Aim We hypothesize that GSSE could protect the liver from fat-induced lipotoxicity and have a beneficial effect on liver function. Methods Hepatoprotective effect of GSSE was measured by using an experimental model of fat-induced rat liver steatosis. Male rats were fed a standard diet or a high-fat diet (HFD) during 6 weeks and treated or not with 500 mg/ kg bw GSSE. Lipid deposition into the liver was assessed by triglyceride, cholesterol and phospholipid measurements. Fat-induced lipoperoxidation, carbonylation, depletion of glutathione and of antioxidant enzyme K. Charradi (&) S. Elkahoui I. Karkouch F. Limam E. Aouani Laboratoire des Substances Bioactives (LSBA), Centre de Biotechnologie de Borj Cedria, BP-901, 2050 Hammam-Lif, Tunisia e-mail: [email protected] K. Charradi E. Aouani Faculte´ des Sciences de Bizerte, Universite´ de Carthage, 7021 Jarzouna, Tunisia F. Ben Hassine Laboratoire de Biochimie, Polyclinique de la CNSS d’El Khadra, Avenue Zobeir Ibn El Awam, 1003 Cite´ El Khadra Tunis, Tunisia M. V. El May Unite´ de recherche no 01/UR/07-08, Faculte´ de Medicine de Tunis, Tunis, Tunisia
activities were used as oxidative stress markers with a special emphasis on transition metal distribution. Results HFD induced liver hypertrophy and inflammation as assessed by high liver transaminases. HFD also induced an oxidative stress characterized by increased lipid and protein oxidation, a drop in glutathione and antioxidant enzyme activities as glutathione peroxidase and superoxide dismutase and a drastic depletion in liver zinc. Importantly, GSSE prevented all the deleterious effects of HFD treatment. Conclusions Data suggest that GSSE could be used as a safe preventive agent against fat-induced liver lipotoxicity which could also have potential applications in other nonalcoholic liver diseases. Keywords Zinc
Polyphenol Liver Fat Oxidative stress
Introduction Obesity is a serious worldwide health problem, implicated in various pathologies including type 2 diabetes, hypertension, cancer, cardiovascular dysfunction and non-alcoholic fatty liver diseases (NAFLD) which could eventually lead to hepatic cirrhosis and, ultimately, to hepatocellular carcinoma [1]. Obesity is a state of low-grade and chronic oxidative stress and inflammation in relation to reduced antioxidant defense and strong reactive oxygen species (ROS) production [2]. Oxidative stress plays a pivotal role in the development of NAFLD, particularly in the progression from steatosis to steatohepatitis [3]. The emerging hypothesis for the pathogenesis of non-alcoholic steatohepatitis is a ‘‘two-hit theory’’ consisting of a hepatic fat accumulation as the first hit and an oxidative stress as the
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second hit [4]. GSSE is a complex mixture of polyphenolics as oligomeric proanthocyanidins and unsaturated fatty acids largely used as a nutritional supplement [5]. GSSE received much attention due to its pleiotropic biological activities and health-promoting properties, such as antioxidant [6], free radical scavenging [7], lipid lowering [8], anti-tumor [9, 10] or neuroprotective effects [11]. All these beneficial health properties led to consider GSSE as a potential health food ingredient which received the GRAS (generally recognized as safe) certification from US FDA. The present study aimed to evaluate the effect of a specific and self-elaborated HFD from animal origin on liver steatosis and oxidative stress, with a special emphasis on transition metal distribution. We hypothesize that a treatment with a high-dose GSSE will prevent the HFDinduced hepatotoxicity and improve liver function.
Methods and Materials
Table 1 LC-MS/MS data of some phenolic compounds found in carignan GSSE Compounds
Relative abundance (%)
Catechin
1.31
Epicatechin
1.61
Procyanidin dimer Procyanidin trimer
0.23 ND
Quercetin
0.55
Resveratrol
0.07
Rutin
1.00
Vanillin
9.21
Gallic acid
41.53
P-coumaric acid
0.19
Rosmarinic acid
0.37
2,5-Dihydroxybenzoı¨c acid
41.26
Caffeic acid
1.40
Chlorogenic acid
0.17
Ferulic acid
1.00
Reagents and Diets Grape seed and skin extract was processed from a grape cultivar (Carignan) of Vitis vinifera from northern Tunisia. Seeds were manually separated from skin, air-dried and grounded separately with a coffee grinder (FP 3121 Moulinex) until a fine powder was obtained. Both powders were then mixed at 50:50 ratio on a dry weight basis in 10 % ethanol (v/v) at dark. After vigorous stirring and centrifugation at 10,000g for 15 min at 4 °C, supernatant containing soluble polyphenols was administered to animals by intraperitoneal (i.p) route. Total phenolic content and GSSE composition (Table 1) were determined according to [13]. Standard diet (SD) for rodent in pelleted form was purchased from ALMAS Bizerta (Tunisia). Highfat diet (HFD) was prepared by soaking commercial food pellets into warmed (100 °C) and liquefied abdominal fat from animal origin (sheep) during 15 min and allowed to dry at room temperature. Sheep abdominal fat was used to feed the rats in order to mimic the local Tunisian feeding habits. Composition of SD and HFD is shown in Table 2. Animals and Experimental Design Twenty-four male Wistar rats (210–230 g) from Pasteur Institute (Tunis) were used in this study, in conformity with the local ethics committee of Carthage University and in agreement with the NIH guidelines [12]. They were maintained in animal facility at controlled temperature (22 ± 2 °C), a 12-h light/dark cycle, and divided into 4 groups of 6 animals each, fed with standard diet (SD) or HFD for 6 weeks. Rats received by daily i.p injection (8.00–9.00 am) either 10 % ethanol as vehicle (control SD and HFD) or
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500 mg/kg bw GSSE (SD ? GSSE and HFD ? GSSE). Parenteral administration of GSSE avoided the gastric barrier, the poor absorption of polyphenols through the intestine as well as hepatic and microflora transformation and inactivation of polyphenols. At the end of the treatment, rats were killed by decapitation, their blood collected using heparin as anticoagulant and plasma processed for lipids and oxidative stress assessment (not shown). Liver was isolated, weighed, homogenized in phosphate saline buffer pH 7.4 with an ultrathurax T25 homogenizator, centrifuged at 10,000g for 15 min at 4 °C and supernatant stored at -80 °C. After thawing, homogenates were used for the determination of transaminases, alcohol dehydrogenase activity, lipase activity, ectopic lipid content, oxidative stress status, metal ions distribution as well as intracellular mediators as calcium, nitric monoxide and hydrogen peroxide. The relative liver weight per 100 g body weight was expressed as [liver weight (g)/body weight on day 45 (g)] 9 100. Liver Analyses Lipids were extracted from whole liver according to [13]. Triglyceride and total cholesterol were determined using commercially available kits from Biomaghreb (Tunisia) according to [14] and [15], respectively. LDL-cholesterol was determined using a commercial kit from Biolabo, SA (France), according to [16]. Phospholipid (PL) and HDLPL were determined using assay kits purchased from BioMe´rieux, SA, Marcy-l’etoile, (France) according to [17]. Apo B and apo AI were determined using an assay kit from Konelab clinical chemistry analyzer (Thermo Clinical Labsystems, Espoo, Finland), according to [17].
Dig Dis Sci Table 2 Composition of SD and HFD Parameter
SD
HFD
Lipid % (w/w)
3.00
28.00
Energetic contribution
5.00
39.00
% (w/w)
40.00
32.00
Energetic contribution
70.00
45.00
% (w/w)
14.00
12.00
Energetic contribution
25.00
16.00
Carbohydrate
Protein
Fiber % (w/w)
7.50
6.00
14.00 9.00
12.00 7.20
Methionine % (w/w)
0.46
0.37
Cystein % (w/w)
0.34
0.27
Threonine % (w/w)
0.78
0.62
Tryptophane % (w/w)
0.25
0.20
Moisture % (w/w) Ash % (w/w)
Copper (mg/kg)
10.00
8.00
Manganese (mg/kg)
50.00
40.00
Zinc (mg/kg)
50.00
40.00
Iron (mg/kg)
40.00
32.00
Calcium (mg/kg)
1.20
0.94
Magnesium (mg/kg)
0.05
0.04
Phosphorus (mg/kg)
0.50
0.40
Selenium (mg/kg)
0.10
0.08
Iodide (mg/kg)
0.70
0.56
Cobalt (mg/kg)
0.10
0.08
Liver alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) were determined using assay kits from Biomaghreb (Tunisia), according to [18]. Lipoperoxidation was evaluated by malondialdehyde (MDA) measurement [19]. Briefly, liver homogenates were precipitated with 20 % trichloroacetic acid (TCA) and MDA from supernatant was allowed to react with thiobarbituric acid (TBA). Spectrophotometric measurement was done at 532 nm and MDA concentration calculated using the absorbance coefficient of MDA–TBA complex 1.56 9 105 cm-1 M-1. Oxidative damage to proteins was evaluated by quantifying protein carbonylation in liver homogenates according to [20]. Briefly, after proteins precipitation with 20 % TCA and dissolution in 2,4 dinitrophenylhydrazine (DNPH)containing buffer, absorbance was measured at 366 nm and results expressed as nmol carbonyl/mg protein. Total protein was determined according to [21]. Reduced (GSH) and oxidized glutathione (GSSG) were determined according to [22]. Liver homogenates were also used for the determination of antioxidant enzyme activities. Glutathione peroxidase
(GPx; E.C.1.11.1.9.) was measured according to [23]. Briefly, liver homogenates (2 mg protein) were mixed with phosphate buffer 100 mmol pH 7,4 containing 4 mmol GSH and 5 mmol H2O2 in 1 mL final volume. The mixture was incubated at 37 °C for 1 min; then, 0.5 mL TCA (5 %, w/v) was added to stop the reaction. After centrifugation at 1,500g for 5 min, 0.2 mL supernatant was mixed with phosphate buffer 100 mmol pH 7.4 containing 10 mmol 2-nitrobenzoı¨c acid (DTNB). GPx activity was measured at 412 nm. Catalase (CAT; E.C.1.11.1.6.) was assayed by measuring the initial rate of H2O2 disappearance at 240 nm [24]. The reaction mixture contained 33 mmol H2O2 in 50 mmol phosphate buffer pH 7 and 5 lL extract in 1 mL final volume and CAT activity calculated using an extinction coefficient of 40 mmol-1 cm-1 for H2O2. Superoxide dismutase (SOD; E.C.1.15.1.1.) was determined using a modified epinephrine assay [25]. At alkaline pH, superoxide anion O2- causes the autoxidation of epinephrine to adenochrome. Liver extract was added to a 2 mL reaction mixture containing 4U bovine catalase, 100 lg epinephrine and 62.5 mmol sodium bicarbonate buffer pH 10.2, and changes in absorbance were recorded at 480 nm. Characterization of SOD isoforms was performed using KCN (3 mmol) as Cu–Zn inhibitor or H2O2 (3 mmol) which affects Cu/Zn and Fe-SOD, whereas MnSOD is insensitive to both inhibitors. Alcohol dehydrogenase activity (E.C.1.1.1.1) was evaluated by following NADH formation at 340 nm and 37 °C according to [26]. Lipase activity was determined according to [27] using p-nitrophenol dodecanoate in dimethylsulfoxide (DMSO) and ethanol as substrate. The reaction mixture containing 5 mmol p-nitrophenol dodecanoate, 50 mmol Tris–HCl buffer pH 8.5 and 50 lL of sample was incubated at 37 °C for 1 h, and the reaction stopped with 60 mmol EDTA. After centrifugation at 10,000g for 5 min, absorbance was measured at 412 nm. One unit is defined as the amount of enzyme catalyzing the release of 1 lmol p-nitrophenol (e = 18.3 mmol-1 cm-1). Ionizable calcium was determined according to [28] using an assay kit from Biomaghreb, Tunisia. At basic pH, calcium constitutes cresolphtalein a purple complex measurable at 570 nm. Briefly, 50 lL liver extract was added to 650 lL of reaction mixture containing 2-amino-2-methyl-1-propanolol buffer (500 mmol L-1), cresolphtalein (0.62 mmol L-1) and hydroxyl-8 quinoleine (69 mmol L-1). Incubation was carried out at room temperature for 5 min assuming the complex was stable for 1 h. H2O2 was determined according to [29] using an assay kit from Biomaghreb, Tunisia. Briefly, in the presence of peroxidase, H2O2 reacts with 4-aminoantipyrine and phenol to give a red-colored quinoeimine that absorbs at 505 nm. NO was determined by quantification of NO metabolites nitrite and nitrate according to [30]. Tissue samples were also wet ashed in nitric acid (15.5 mol L-1),
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Dig Dis Sci Fig. 1 Protective effect of GSSE on HFD-induced liver weight (a), transaminases (b) and lipase activity (c) overload. Rats were fed a SD or HFD during 6 weeks and daily administered with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. # p \ 0.05 for SD ? GSSE versus SD. *p \ 0.05 for HFD vs. SD. §p \ 0.05 for HFD ? GSSE versus HFD
diluted, and filtered for zinc, copper and manganese determination by atomic absorption spectroscopy. Liver Histology A piece of liver was fixed in 10 % (v/v) formaldehyde, processed by successive dehydrations with ethanol baths and embedded in paraffin. Serial sections were cut 5 lm thick and stained with hematoxylin-eosin (HE) using standard procedures. Statistical Analysis Results are expressed as mean ± SEM. Statistical differences were evaluated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. All analyses were performed using Statistica. p value \ 0.05 was considered significant.
Results Effect of HFD on Body Weight Gain, Relative Liver Weight, and Toxicity At the end of the experiment, body weight gain reached 13.7 % for SD, 14 % for SD ? GSSE, 22 % for HFD and
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12 % for HFD ? GSSE animals. Moreover, HFD increased the relative liver weight by 11 % (Fig. 1a), transaminases as ASAT by 11 % and ALAT by 13 % (Fig. 1b) and lipase activity by 28 % (Fig. 1c). GSSE backed all HFD-induced disturbances to near control level. Noteworthy that GSSE also decreased lipase activity by 12 % in SD animals. Hepatic Lipid Content We reported in Fig. 2 the intra-hepatic level of various lipid species from SD- or HFD-treated animals. HFD increased triglyceride by 66 % (Fig. 2a), cholesterol by 41 % (Fig. 2b), phospholipid as assessed by LDL ? VLDL/HDL ratio by 15 % (Fig. 2c) and apo B/apo AI ratio by 20 % (Fig. 2d). All HFD-induced lipid accumulation was attenuated upon GSSE treatment. Non-enzymatic Antioxidant We further sought to determine whether lipid accumulation into the liver provoked an oxidative stress (Fig. 3). HFD increased liver MDA by 37 % (Fig. 3a), protein carbonylation by 36 % (Fig. 3b) but decreased the reducing power as assessed by GSH/GSSG ratio by 29 % (Fig. 3c). GSSE treatment brought all these parameters to near control level. Importantly, GSSE decreased MDA by 38 % and protein carbonylation by 25 % in SD group.
Dig Dis Sci Fig. 2 Protective effect of GSSE on HFD-induced liver triglyceride (a), cholesterol (b), phospholipid (c) and apolipoproteins (d) overload. Rats were fed a SD or HFD during 6 weeks and daily treated with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. *p \ 0.05 for HFD versus SD. §p \ 0.05 for HFD ? GSSE versus HFD
Fig. 3 Protective effect of GSSE on HFD-induced hepatic lipoperoxidation (a), carbonylation (b) and glutathione depletion (c). Rats were fed a SD or HFD during 6 weeks and daily administered with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. #Significant for SD ? GSSE versus SD. *Significant for HFD versus SD. § significant for HFD ? GSSE versus HFD
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Dig Dis Sci Fig. 4 Effect of GSSE on HFD-induced depletion of liver GPx (a), SOD (b) and CAT (c) activity. Rats were fed a SD or HFD during 6 weeks and daily treated with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. # Significant for SD ? GSSE versus SD. *Significant for HFD versus SD. §Significant for HFD ? GSSE versus HFD
Enzymatic Antioxidant
Histology
HFD depressed liver antioxidant enzyme activities as GPx by 17 % (Fig. 4a) and SOD by 40 % (Fig. 4b). In this latter case, the Cu/Zn and to a lesser extent the Mn isoform were depressed, whereas the Fe isoform was unchanged. No effect on CAT activity was detected (Fig. 4c). GSSE treatment corrected all the deleterious effects of HFD to near control level.
Histological examination of the liver sections revealed normal histology and architecture in SD- (Fig. 6a) and GSSE-treated SD animals (Fig. 6b). HFD provoked moderate to severe macrovesicular steatosis, dilatation and fat accumulation within hepatocytes and sinusoids (Fig. 6c). GSSE corrected almost all HFD-induced structural modifications to near control level (Fig. 6d).
Effect of HFD on Transition Metals HFD treatment strongly decreased liver zinc by 92 % (Fig. 5a), and GSSE brought all HFD-induced zinc disturbances to control level. Moreover, HFD depressed the activity of the zinc-dependant alcohol dehydrogenase by 38 % and GSSE counteracted this adverse effect. Remarkably, that GSSE increased significantly (8 %) the liver alcohol dehydrogenase activity in SD condition. HFD had no effect on hepatic manganese and only a slight (not significant) effect on liver copper (Table 3). Effect of HFD on Intracellular Mediators HFD treatment had no effect on intracellular mediators as H2O2, calcium or NO (Table 4).
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Discussion This study was undertaken in order to analyze the lipotoxic effect of a high-fat-enriched regime on rat liver as well as the protection offered by GSSE treatment. Overall, HFD increased body weight, caused abdominal fat deposition and induced dyslipidemia and C-reactive protein into plasma [31]. With regard to the liver which is a central organ for lipid metabolism, HFD induced hypertrophy and excessive deposition of various lipid species such as triglyceride, cholesterol and phospholipid. It also caused hepatic toxicity as illustrated by the increase in liver transaminases as well as in lipase activity [32]. Moreover, HFD provoked a clear oxidative stress characterized by high MDA and protein carbonylation, depressed GPx and Cu/Zn-SOD activities and a drastic depletion of Zn from
Dig Dis Sci Fig. 5 Effect of GSSE on HFD-induced depletion of liver zinc (a) and alcohol dehydrogenase activity (b). Rats were fed a SD or HFD during 6 weeks and daily treated with GSSE (500 mg/kg bw). Results are expressed as mean ± SEM. (n = 6). p \ 0.05 was considered significant. # Significant for SD ? GSSE versus SD. *Significant for HFD versus SD. §Significant for HFD ? GSSE versus HFD
Table 3 Effect of HFD on liver copper and manganese SD
SD ? GSSE
HFD
HFD ? GSSE
Copper (nmol/mg protein)
0.959 ± 0.019
1.060 ± 0.021
0.761 ± 0.017
0.885 ± 0.020
Manganese (nmol/mg protein)
0.891 ± 0.011
0.909 ± 0.006
0.907 ± 0.007
0.891 ± 0.011
Results are expressed as mean ± SEM HFD versus SD: * p \ 0.05; HFD ? GSSE versus HFD:
§
p \ 0.05
Table 4 Effect of HFD and GSSE on liver intracellular mediators
Hydrogen peroxide (mmol/mg protein)
SD
SD ? GSSE
HFD
HFD ? GSSE 39.92 ± 0.27
40.03 ± 0.17
39.81 ± 0.19
40.34 ± 0.28
Ionisable calcium (lmol/mg protein)
1.97 ± 0.04
1.97 ± 0.05
1.99 ± 0.04
1.97 ± 0.04
NO metabolites (lmol/mg protein)
8.07 ± 0.46
8.02 ± 0.34
8.15 ± 0.35
8.08 ± 0.56
Data are presented as mean ± SEM
the liver. Importantly, GSSE abrogated almost all the disturbances induced by HFD. Therefore, we accepted our original study hypothesis that GSSE efficiently counteracted the HFD-induced liver lipotoxicity. HFD increased liver MDA and protein carbonylation and depressed the antioxidant capacity as glutathione which constitutes the first line of defense against free radicals in the liver and also acts as a GPx substrate [33]. In addition, HFD which did not modify liver CAT, depressed GPx and SOD activity, more specifically the Cu/Zn isoform. This last result is supported by the strong depletion of zinc from the liver that did not occur in the other organs such as the brain, the heart and the kidney (data not shown). In fact, HFD treatment depleted Zn from the liver and pancreas (not shown), copper from the heart and kidney [34], and Mn from the brain [11]. As a consequence, HFD induced the accumulation of Zn, Mn and Cu into the plasma [35] and the depletion of iron from plasma that accumulates into the heart [31] and adipose tissue [35]. Such a relationship between obesity and transition metal distribution has been already mentioned several decades ago by Kennedy et al. [36] who demonstrated lower level of micronutrients into
tissues and higher level into plasma from genetically obese mice when compared to lean control. Thus, a role of copper deficiency in hypercholesterolemia has long been recognized [37] and high circulating level of copper was linked to obesity-induced left ventricular hypertrophy in elderly patients [38]. A growing number of studies suggest a potential link between obesity and altered iron metabolism. Iron chelation with deferoxamine improved obesity through decreased oxidative stress and adipocyte hypertrophy [39] and lipocalin 2 induced cardiomyocyte apoptosis by means of iron deposition [40]. Moreover, manganese overexposure of rats induced disturbances in brain lipid metabolism as assessed by high level of palmitate, oleate and cholesterol [41]. Our present data are consistent with several previous reports dealing with decreased zinc levels in both acute and chronic liver disease states [42, 43] and with the prominent role of zinc deficiency in oxidative stress [44]. It is our opinion that Zn ions constitute one of the missing links between HFDinduced obesity and liver steatosis, as this trace element plays a central role in liver function and homeostasis [45]. Zinc is an essential cofactor for many enzymes, including
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Fig. 6 Effect of GSSE on HFD-induced liver morphology alteration. SD (a), SD ? GSSE (b), HFD (c) and HFD ? GSSE (d). CV central vein, DH degenerated hepatocyte, LV lipid vacuole, S sinusoid and V vein. H&E 9 400
those implicated in the metabolism of vitamin A and ethanol [46]. Hypozincemia was shown to be linked to male obesity in humans [47], and Cu/Zn SOD deficiency induced hepatic lipid accumulation by impaired lipoprotein secretion [4], whereas zinc supplementation was used in the treatment of alcoholic liver disease [48]. Zinc is also known to regulate both enzyme activity and protein stability either as an activator or as an inhibitor; and zinc depletion is at the basis of alcoholic and non-alcoholic liver diseases leading to liver fibrosis [49]. Interestingly, we noted a drop in liver alcohol dehydrogenase activity, in accordance with previous [50] and more recent work [51] that linked zinc deficiency with reduced alcohol dehydrogenase activity. This latter result is liver specific as it was not observed in other organs as kidney where, as expected, HFD did not disturb zinc homeostasis (not shown). We did not yet know which zinc finger transcription factor, if any, is affected by this dramatic loss of zinc, nor did we know which zinc transporter as ZIP or ZnT could be involved in such depletion of zinc from the liver. Undoubtedly, the most relevant finding of the current work is the potential ability of our locally produced GSSE to prevent steatosis. GSSE prevented HFD-induced hypertrophy and lipid overload, as assessed by decreased lipid depletion as well as lipase activity and the prevention
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of liver histology from macrovesicular steatosis to normal architecture. Our results, which emphasized the antioxidative role of GSSE as well as its impact on hepatic lipid profile, are fully in line with recent works in the field [52, 53]. Moreover, our data showed that GSSE is also protective in SD conditions, which argued in favor of its safety and confirmed similar data found by other investigators [54]. Our contribution further extended the current knowledge on the putative treatment of fat-induced liver zinc depletion using natural grape polyphenols. GSSE prevented the HFD-induced zinc depletion from the liver as well as the drop in alcohol dehydrogenase activity, a zinc dependent enzyme. Our data are consistent with the clinical use of GSSE to improving liver function in patients with non-alcoholic fatty liver change [55]. GSSE was recently shown to reverse HFD-induced obesity in mice and hepatosteatosis through up- and down-regulation of mRNA for lipolytic and lipogenic enzymes, respectively [56]. To the best of our knowledge, our data are the first to link the preventive effect of GSSE with liver zinc repletion. They are consistent with in vitro studies using human Hep G2 cells in which dietary catechins and procyanidins were shown to modulate zinc metabolism and to increase the level of cytoplasmic labile zinc [57]. GSSE, which has been attributed the GRAS certification from US FDA, is
Dig Dis Sci
safe even when used at high doses. Moreover, the risk of zinc overloading constitutes a serious drawback in the treatment of many liver pathologies characterized by zinc depletion. A recent study using polaprezinc, a mixture of zinc and carnosine for the treatment of zinc deficiency in thioacetamide-induced liver fibrosis [58], proved the safety of such compound at a dose not exceeding 500 mg/kg/day. However, this dose of polaprezinc is far less from the optimal dose of 4,000 mg/kg/day GSSE, which is devoid of any toxicity even in long-term experiments. It is important to keep in mind that this high dose of GSSE, which corresponds to approximately 280 g/day for a 70 kg adult, has been daily administered to animals in 3-month-long experiment with no sign of toxicity (data not shown). GSSE should be viewed as a potential alternative to zinc supplementation in the treatment of liver pathologies characterized by zinc depletion [59]. Indeed, zinc supplementation, which inhibited the ethanol-increased liver weight, did not improve the weight gain [60], although in our current study, GSSE improved both the overall weight gain and the relative liver weight. Consequently, not only GSSE appeared more efficient than zinc supplementation but it also avoided several drawbacks linked to zinc supplementation. Future studies should analyze the relationship between HFD-induced liver steatosis, zinc depletion and the protection offered by GSSE. It would be particularly important to identify which lipid species or free fatty acid present in HFD provoked the drastic depletion of zinc from the liver, as well as the implication of zinc transporters in such a process [61]. In this respect, ZIP14 downregulation and zinc depletion were recently shown to be involved in the development and progression of hepatocellular cancer [62]. It would also be essential to identify which polyphenol present in GSSE could account for liver protection as described for resveratrol [63] or catechin and quercetin [64]. Importantly, SIRT1 activation by polyphenols as resveratrol is a successful strategy in the treatment of fatty liver disease and obesity [65]. Nevertheless, our work presents some limitations. First, GSSE was used at a suboptimal dose of 500 mg/kg which exhibited only a partial prevention of triglyceride accumulation. Indeed, in dose–response experiment, GSSE exerted antioxidative beneficial effects till 4,000 mg/kg (unpublished data) which is similar to the optimal dose claimed for the commercial IH636 californian grape extract [66]. Although HFD altered GPx activity and GSSE prevented it, selenium was not measured. Interestingly, grape seed extract was recently shown to act synergistically with selenium to decrease indomethacin-induced gastric ulcers in rats [67]. Future studies should analyze the relationship between GSSE-induced zinc repletion into the liver and selenium. Finally, we should have used the more specific
oil red lipid staining method as a more compelling evidence for HFD-induced hepato-lipotoxicity. In conclusion, HFD provoked zinc depletion from the liver and GSSE prevented HFD-induced hepato-steatosis. Clinical trials using GSSE for the prevention and treatment of alcoholic and non-alcoholic fatty liver diseases should be envisaged. Acknowledgments Financial support of the Tunisian Ministry of ‘‘Enseignement Supe´rieur, Recherche Scientifque et Technologie’’ is gratefully acknowledged. Conflict of interest
None.
References 1. Angulo P. Non alcoholic fatty liver disease. New Engl J Med. 2002;346:1221–1231. 2. Higdon JV, Frei B. Obesity and oxidative stress: a direct link to CVD? Arterioscl Throm Vas. 2003;23:365. 3. Adams LA, Angulo P, Lindor KD. Nonalcoholic fatty liver disease. Can Med Assoc J. 2005;172:899–905. 4. Uchiyama S, Shimizu T, Shirasawa T. CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice. J Biol Chem. 2006;281:31713–31719. 5. Bagchi D, Bagchi M, Stohs SJ, Ray SD, Sen CK, Preuss HG. Cellular protection with proanthocyanidins derived from grape seeds. Ann NY Acad Sci. 2002;957:260–270. 6. Bagchi D, Bagchi M, Stohs SJ, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology. 2000;7:187–197. 7. Curin Y, Ritz MF, Andriantsitohaina R. Cellular mechanisms of the protective effect of polyphenols on the neurovascular unit in strokes. Cardiovasc Hematol Agents Med Chem. 2006;4:277–288. 8. Moreno DA, Ilic N, Poulev A, Brasaemle DL, Fried SK, Raskin I. Inhibitory effects of grape seed extract on lipases. Nutrition. 2003;19:876–879. 9. Bomser JA, Singletary KW, Wallig MA, Smith MA. Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds. Cancer Lett. 1999;29:151–157. 10. Agarwal C, Sharma Y, Agarwal R. Anticarcinogenic effect of a polyphenolic fraction isolated from grape seeds in human prostate carcinoma DU145 cells: modulation of mitogenic signaling and cell-cycle regulators and induction of G1 arrest and apoptosis. Mol Carcinog. 2000;28:129–138. 11. Charradi K, Elkahoui S, Karkouch I, Limam F, Ben Hassine F, Aouani E. Grape seed and skin extract prevents high-fat dietinduced brain lipotoxicity in rat. Neurochem Res. 2012;37:2004–2013. 12. National Research Council. Guide for the care and the use of laboratory animals, vol. 20. Bethesda: National Institute of Health; 1985:85. 13. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509. 14. Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem. 1982;28:2077–2080.
123
Dig Dis Sci 15. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470–475. 16. Tietz NW. Text book of clinical chemistry. 3rd ed. Philadelphia: W.B. Saunders; 1999:819–884. 17. Mainard F, Madec Y. Cholesterol, phospholipid and apoB composition of LDL: comparison of precipitation and ultracentrifugation methods. Ann Biol Clin (Paris). 1986;44:618–623. 18. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957;28:56–63. 19. Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990;186:421–431. 20. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:467–478. 21. Hartree EF. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972;48:422–427. 22. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502–522. 23. Nakamura K, Hosada S, Hayashi K. Purification and properties of rat liver glutathione peroxidase. Biochem Biophys Acta. 1974;358:251–261. 24. Aebi H. Methods of enzymatic analysis. 2e ed. New York: Chemia Weinheium; 1974. 25. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170–3175. 26. Tachiyashiki K, Imaizumi K. Lowering and delaying actions of bovine bile on plasma ethanol levels in rats. J Nutr Sci Vitaminol. 1992;38:69–82. 27. Humbert G, Guingamp MF, Linden G. Method for the measurement of lipase activity in milk. J Dairy Res. 1997;64:465–469. 28. Stern J, Lewis WH. The colorimetric estimation of calcium in serum with ocresolphthalein complexone. Clin Chim Acta. 1957;2:576–580. 29. Kakinuma K, Yamaguchi T, Kaneda M, Shimada K, Tomita Y, Chance B. A determination of H2O2 release by the treatment of human blood polymorphonuclear leucocytes with myristate. J Biochem. 1979;86:87–95. 30. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and [15 N] nitrate in biological fluids. Anal Biochem. 1982;126:131–138. 31. Charradi K, Sebai H, Elkahoui S, Ben Hassine F, Limam F, Aouani E. Grape seed extract alleviates high fat diet-Induced obesity and heart dysfunction by preventing cardiac siderosis. Cardiovasc Toxicol. 2011;11:28–37. 32. Chiu HK, Qian K, Ogimoto K, et al. Mice lacking hepatic lipase are lean and protected against diet-induced obesity and hepatic steatosis. Endocrinology. 2010;151:993–1001. 33. Hsu CL, Yen GC. Effect of gallic acid on high fat diet-induced dyslipidemia, hepatosteatosis, and oxidative stress in rats. Br J Nutr. 2007;98:727–735. 34. Charradi K, Elkahoui S, Karkouch I, et al. Grape seed and skin extract alleviates high-fat diet-induced renal lipotoxicity and prevents copper depletion in rat. Appl Physiol Nutr Metab. 2013;38:259–267. 35. Charradi K, Elkahoui S, Limam F, Aouani E. High-fat diet induced an oxidative stress in white adipose tissue and disturbed plasma transition metals in rat: prevention by grape seed and skin extract. J Physiol Sci. 2013. doi:10.1007/s12576-013-0283-6. 36. Kennedy ML, Failla ML, Smith JC Jr. Influence of genetic obesity on tissue concentrations of zinc, copper, manganese and iron in mice. J Nutr. 1986;116:1432–1441.
123
37. Kim S, Chao PY, Allen KG. Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia. FASEB J. 1992;6:2467–2471. 38. Lind PM, Olse´n L, Lind L. Elevated circulating levels of copper and nickel are found in elderly subjects with left ventricular hypertrophy. Ecotoxicol Environ Saf. 2012;86:66–72. 39. Tajima S, Ikeda Y, Sawada K, et al. Iron reduction by deferoxamine leads to amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes KKAy mice. Am J Physiol Endocrinol Metab. 2012;302:77–86. 40. Xu G, Ahn J, Chang S, et al. Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation. J Biol Chem. 2012;287:4808–4817. 41. Fordahl S, Cooney P, Qiu Y, Xie G, Jia W, Erikson KM. Waterborne manganese exposure alters plasma, brain, and liver metabolites accompanied by changes in stereotypic behaviors. Neurotoxicol Teratol. 2012;34:27–36. 42. Mohammad MK, Zhou Z, Cave M, Barve A, McClain CJ. Zinc and liver disease. Nutr Clin Pract. 2012;27:8–20. 43. Stamoulis I, Kouraklis G, Theocharis S. Zinc and the liver: an active interaction. Dig Dis Sci. 2007;52:1595–1612. 44. Eide DJ. The oxidative stress of zinc deficiency. Metallomics. 2011;3:1124–1129. 45. King JC, Shames DM, Woodhouse LR. Zinc homeostasis in humans. J Nutr. 2000;130:1360S–1366S. 46. Brandt EG, Hellgren M, Brinck T, Bergman T, Edholm O. Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site. Phys Chem Chem Phys. 2009;11:975–983. 47. Ozata M, Mergen M, Oktenli C, et al. Increased oxidative stress and hypozincemia in male obesity. Clin Biochem. 2002;35:627–631. 48. Kang YJ, Zhou Z. Zinc prevention and treatment of alcoholic liver disease. Mol Aspects Med. 2005;26:391–404. 49. Milman N, Laursen J, Pødenphant J, Asnaes S. Trace elements in normal and cirrhotic human liver tissue. I. Iron, copper, zinc, selenium, manganese, titanium and lead measured by X-ray fluorescence spectrometry. Liver. 1986;6:111–117. 50. Indo Y, Nagata N, Higashi A, Matsuda I, Kashiwabara N, Nakashima I. Effects of dietary zinc deficiency on hepatic ornithine carbamoyltransferase and alcohol dehydrogenase activities in rats. J Pediatr Gastroenterol Nutr. 1985;4:268–273. 51. Kawashima Y, Someya Y, Sato S, et al. Dietary zinc-deficiency and its recovery responses in rat liver cytosolic alcohol dehydrogenase activities. J Toxicol Sci. 2011;36:101–108. 52. Quesada H, del Bas JM, Pajuelo D, et al. Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver. Int J Obes (Lond). 2009;33:1007–1012. 53. Castrillejo VM, Romero MM, Esteve M, et al. Antioxidant effects of a grapeseed procyanidin extract and oleoyl-estrone in obese Zucker rats. Nutrition. 2011;27:1172–1176. 54. Goodrich KM, Fundaro G, Griffin LE, et al. Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occluding and reduces fecal calprotectin: a secondary analysis in healthy Wistar furth rats. Nutr Res. 2012;32:787–794. 55. Khoshbaten M, Aliasgarzadeh A, Masnadi K, et al. Grape seed extract to improve liver function in patients with nonalcoholic fatty liver change. Saudi J Gastroenterol. 2010;16:194–197. 56. Park SH, Park TS, Cha YS. Grape seed extract (Vitis vinifera) partially reverses high fat diet-induced obesity in C57BL/6 J mice. Nutr Res Pract. 2008;2:227–233. 57. Quesada IM, Bustos M, Blay M, et al. Dietary catechins and procyanidins modulate zinc homeostasis in human HepG2 cells. J Nutr Biochem. 2011;22:153–163.
Dig Dis Sci 58. Kono T, Asama T, Chisato N, et al. Polaprezinc prevents ongoing thiocetamide-induced liver fibrosis in rats. Life Sci. 2012;90: 122–130. 59. Oteiza PI, Mackenzie GG. Zinc, oxidant-triggered cell signaling, and human health. Mol Aspects Med. 2005;26:245–255. 60. Zhou Z, Liu J, Song Z, McClain CJ, Kang YJ. Zinc supplementation inhibits hepatic apoptosis in mice subjected to a long– term ethanol exposure. Exp Biol Med. 2008;233:540–548. 61. Cousins RJ, Liuzzi JP, Lichten LA. Mammalian zinc transport, trafficking, and signals. J Biol Chem. 2006;281:24085–24089. 62. Franklin RB, Levy BA, Zou J, et al. ZIP14 zinc transporter down regulation and zinc depletion in the development and progression of hepatocellular cancer. J Gastrointest Cancer. 2012;43: 249–257. 63. Gomez-Zorita S, Fernandez-Quintela A, Macarulla MT, et al. Resveratrol attenuates steatosis in obese Zucker rats by
64.
65.
66.
67.
decreasing fatty acid availability and reducing oxidative stress. Br J Nutr. 2012;107:202–210. Auger C, Teissedre PL, Ge´rain P, et al. Dietary wine phenolics catechin, quercetin and resveratrol efficiently protect hypercholesterolemic hamsters against aortic fatty streak accumulation. J Agric Food Chem. 2005;53:2015–2021. Li Y, Wong K, Giles A, et al. Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21. Gastroenterology. 2013;S0016–5085: 01577-1. doi:10.1053/j.gastro.2013.10.059. Ray S, Bagchi D, Lim PM, et al. Acute and long-term safety evaluation of a novel IH636 grape seed proanthocyanidin extract. Res Commun Mol Pathol Pharmacol. 2001;109:165–197. Abbas AM, Sakr HF. Effect of selenium and grape seed extract on indomethacin-induced gastric ulcers in rats. J Physiol Biochem. 2013. doi:10.1007/s13-0241-z.
123
