Abstract To determine the metabolic effects of grapefruit juice consumption we established a model in which C57Bl/6 mice drank 25–50% sweetened GFJ, clarified of larger insoluble particles by centrifugation (cGFJ), ad libitum as their sole source of liquid or isocaloric and sweetened water. cGFJ and control groups consumed similar amounts of liquids and calories. Mice fed a high-fat diet and cGFJ experienced a 18.4% decrease in weight, a 13–17% decrease in fasting blood glucose, a three-fold decrease in fasting serum insulin, and a 38% decrease in liver triacylglycerol values, compared to controls. Mice fed a low-fat diet that drank cGFJ experienced a two-fold decrease in fasting insulin, but not the other outcomes observed with the high-fat diet. cGFJ consumption decreased blood glucose to a similar extent as the commonly used anti-diabetic drug metformin. Introduction of cGFJ after onset of diet-induced obesity also reduced weight and blood glucose. A bioactive compound in cGFJ, naringin, reduced blood glucose and improved insulin tolerance, but did not ameliorate weight gain. These data from a well-controlled animal study indicate that GFJ contains more than one health-promoting neutraceutical, and warrant further studies of GFJ effects in the context of obesity and/or the western diet.

Citation: Chudnovskiy R, Thompson A, Tharp K, Hellerstein M, Napoli JL, Stahl A (2014) Consumption of Clarified Grapefruit Juice Ameliorates High-Fat Diet Induced Insulin Resistance and Weight Gain in Mice. PLoS ONE 9(10): e108408. https://doi.org/10.1371/journal.pone.0108408 Editor: Makoto Makishima, Nihon University School of Medicine, Japan Received: November 6, 2013; Accepted: August 20, 2014; Published: October 8, 2014 Copyright: © 2014 Chudnovskiy 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. Funding: The California Grapefruit Growers Cooperative provided financial support for this project. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. None of the authors have financial or non-financial competing interests to disclose. Competing interests: The California Grapefruit Growers Cooperative provided financial support for this project. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, and did not affect the outcome of these experiments.

Introduction The unabated increase in incidence of obesity and obesity-associated disorders, particularly type-2 diabetes, continues to present monumental challenges to health [1]. Dietary modification, including use of neutraceuticals, offer promising approaches to ameliorate obesity and its effects, and to increase health-span. Grapefruit juice (GFJ) is relatively rich in nutrients, including vitamins and minerals, and has fewer calories than other many juices [2], [3]. Putative health and weight-loss promoting effects of grapefruit or GFJ consumption have been popularized, but mostly in context of a hypocaloric diet, e.g. the “Hollywood diet”, which limits caloric intake to as low as 3349 kJ per day. Relatively few human studies have examined the effects of grapefruit or GFJ consumption per se on metabolism in well-controlled experiments, and these have produced intriguing, but contradictory results. Fujioka et al. reported that consumption of GFJ, whole grapefruit, or “grapefruit pills” led to weight loss and improved insulin sensitivity [4]. In contrast, Silver et al. reported that grapefruit or GFJ consumption had no significant effects on metabolic variables, except for a modest increase in HDL, in obese participants fed a restricted calorie diet [5]. Studies in animals have used GFJ administered ad libitum or have focused on one bioactive component, such as the flavonoid naringin, which contributes to GFJ's bitter taste, or on its aglycone, naringenin. These studies did not address differences in water consumption between treatment and control groups, and produced varied results. Mice are adverse to the bitter taste of GFJ and naringin, which could cause dehydration, reluctance to eat and weight loss independent of metabolic effects. For example, Jung et al. reported that naringin added to food decreases blood glucose in db/db mice, but has no effect on body weight [6]. Kannappan and Anuradha reported that naringin affects nutrient and energy metabolism, as well as insulin sensitivity [7]. Pu et al. reported that naringin added to the drinking water of mice fed a high-fat diet (HFD) leads to weight loss, decreased blood glucose, and improved insulin sensitivity [8]. Studies focusing solely on naringin overlook the complex phytochemical composition of GFJ with many potential nutraceutical compounds including bergamottin—a cytochrome P450 inhibitor with potential anti-tumor effects [9]. Other research has focused on GFJ and/or naringin-drug interactions [10], [11]. Naringin has been identified as an inhibitor of Cyp3A4 and organic anion transport protein, which mediate drug catabolism and enterocyte export, respectively. Combined effects of these two have been revealed as a mechanism whereby GFJ can alter intestinal first pass clearance of various drugs, such as statins [10], [12]. We report a model in which mice consumed centrifugation clarified GFJ (cGFJ) ad libitum at rates comparable to liquid consumption of control groups. cGFJ consumption did not modify food intake or absorption. In mice fed a HFD, cGFJ decreased the rate of weight gain, hepatic triacylglycerol accumulation, and fasting blood glucose, and improved insulin sensitivity. In mice fed a LFD, cGFJ consumption produced a two-fold decrease in fasting insulin. These data rely on a well-controlled animal model to reveal that GFJ consumption has health-promoting effects, and these effects are mediated by compounds in addition to naringin.

Materials and Methods GFJ preparation GFJ was squeezed from fresh California Ruby Red grapefruit provided by the California Grapefruit Growers Cooperative, centrifuged at 10,400×g for 10 min at 4°C to remove pulp, amended with 0.15% saccharin (w/v), divided into 25 ml aliquots, and stored at -20°C [13], [14]. The pH of this clarified preparation (cGFJ) was 3.5, compared to 5.5 for the sweetened water used as control. We determined that the caloric content of the cGFJ was 1335 J/ml by bomb calorimetry of a lyophilized sample as previously described [15]. Control mice were given water with 4% glucose (w/v) and 0.15% saccharin (hereafter called control or control water), so that all groups consumed isocaloric liquids with the same amount of saccharin. Animals and diets Procedures were approved by the University of California-Berkeley Animal Care and Use Committee and were done according to AAALAC guidelines. Four-week-old male C57BL/6J mice were purchased from Jackson Laboratories (catalog # 000664). Mice were housed individually and were fed purified diets upon arrival (unless noted otherwise) with either 10% fat (LFD) (Research Diets Cat. # D12450B) or 60% fat (HFD) (Research Diets Cat. #D12492). Any stress induced by housing mice in isolation was normalized by equivalent and concurrent treatment of mice in each experiment. Mice were weighed three times per week. Food consumption was monitored twice per week. Mice were divided randomly into groups of six (unless noted otherwise): controls (water with 4% glucose and 0.15% saccharin); 50% cGFJ (50% cGFJ/water with 0.15% saccharin); 25% cGFJ (25% cGFJ/water with 4% glucose and 0.15% saccharin); naringin (0.72 mg/day in water with 4% glucose and 0.15% saccharin); metformin (7.5 mg/day metformin with 4% glucose and 0.15% saccharin); metformin + cGFJ (7.5 mg/day metformin with 0.15% saccharin in 50% cGFJ). Liquids were given in volumetric bottles (Med Associates, cat # PHM-127-15) to quantify consumption and were replaced daily. Blood glucose Glucose was measured Monday, Wednesday, and Friday between 9 and 11 AM with a NovaMax blood glucose monitor in blood from a tail prick (AmericanDiabetesWholesale). Glucometer values were corrected using a glucose enzymatic assay kit (Sigma, cat # GAHK20-1KT). Glucose (GTT), insulin (ITT), and pyruvate tolerance tests (PTT) For the GTT, mice were fasted overnight and injected i.p. with 0.2 ml of glucose in sterile water to deliver 2 g/kg glucose. For the ITT, mice were fasted 4 hr and injected i.p. with 0.75 units of insulin/kg. For the PTT, mice were fasted overnight and injected i.p. with 0.2 ml of pyruvate in sterile PBS to deliver 2 g/kg. Insulin ELISA Insulin concentrations were determined with a high-range insulin ELISA kit (ALPCO cat# 80-INSMSH-E01, E10) in blood taken retro-orbitally after an overnight fast. Mice were allowed access to food 4 hr and were re-sampled. Protein and triacylglycerol (TG) concentrations of organ lysates Protein concentrations were assayed with a BCA protein assay kit (Thermo Scientific cat# 23227). TG concentrations were assayed with the Infinity TG kit (Thermo Scientific cat# TR2241). Immunohistochemistry Livers were fixed 1 hr at 4°C with 4% paraformaldahyde, and were incubated overnight at 4°C with a cryopreservation medium of 30% sucrose, 20% Optimal Cutting Temperature medium (VWR cat# 25608-930), and 50% Superblock consisting of Block plus 2% normal donkey serum. Block consisted of 50 ml 10× Hanks balanced salt solution, 50 ml fetal calf serum, 5 g bovine serum albumin, and 0.25 g saponin in 500 ml. Blocks were sectioned into 8 µm strips at −23°C. Sections were stained 1 hr at room temperature with a nonpolar BODIPY probe (Molecular Probes cat# D-3922). Slides were mounted with DAPI/glycerol mounting medium (Life Technologies cat# S36938) and stored at −20°C until imaging. Real-time PCR Real-Time PCR was performed using the TaqMan Universal Master Mix II (Applied Biosystems). Primers were purchased from Integrated DNA Technologies (Table 1). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Sequences of primers and probes used for real-time PCR. https://doi.org/10.1371/journal.pone.0108408.t001 Western blotting Livers were homogenized with a Polytron PT2100 in radio immunoprecipitation lysis buffer containing protease and phosphatase inhibitors (Sigma cat# P8340 and cat# P5726) and centrifuged 5 min at 3220×g. Protein (50 µg) was loaded onto a 4–20% Tris-glycine gel. Antibodies were purchased from Cell Signaling. Signals were quantified with a LI-COR Odyssey gel analysis system and normalized to β-tubulin. Absorption assays At 4-weeks-old, mice (7 per group) were fed a HFD for 2 wk while drinking 50% cGFJ or control water ad libitum. Mice were fasted overnight and gavaged with 740 kBq [14C]oleate in 200 µL olive oil, or 740 kBq [3H]2-deoxy-D-glucose in 200 µl sterile PBS containing 2.5 g/kg glucose, or 740 kBq [14C]taurocholic acid in 500 µM taurocholic acid in sterile water. Blood was taken retro-orbitaly 15, 60, 120, 180, and 240 min after dosing. Radioactivity was measured in 10 µL serum. Indirect calorimetry Mice were assayed individually by indirect calorimetry (Columbus Instruments, Columbus Ohio, US) during a fast or after fasting 7 hr and re-feeding 1.1 g of the HFD, followed by fasting overnight. Experimental analyses were started between 3–4 PM and continued for ∼23 hr. Activity was monitored in 10 min intervals. Fatty acid concentrations and synthesis Total liver FA concentrations (C16:0, C16:1, C18:0, C18:1 and C18:2) were determined by gas chromatography-flame ionization detection [15]. Palmitate synthesis was measured by analysis of stable isotope incorporation. On day 0 mice were injected i.p. with 100% D 2 O (Sigma cat # 151890) containing 0.9% NaCl (0.35 ml/g body weight). Mice were given 8% D 2 O in their drinking solutions for 17 d. Deuterium incorporation into serum and liver was determined by GC/MS analysis [16]–[18]. Palmitate synthesis was calculated as the fraction of newly synthesized palmitate × total mg palmitate. Statistics Statistical analysis was performed as described in the figure legends. Data are means ± SE. Statistical significance was determined by two-tailed, unpaired t-tests.

Discussion We developed a well-controlled animal model, which showed that regardless of the amount of fat in the diet, consumption of cGFJ markedly lowered fasting serum insulin. In addition, consumption of 25% or 50% cGFJ reduced fasting glucose in mice fed a HFD, and 50% cGFJ reduced the rate of weight gain in mice fed a HFD. These outcomes did not depend on reduction of caloric uptake between cGFJ and control groups. The anti-glycemic effect of cGFJ occurred within five days, and was as pronounced as the effect of metformin, one of the most potent and widely-used anti-diabetic medications [23]. Although synergistic effects were not observed between GFJ and metformin, the two appear to act through different mechanisms, because metformin activated AMPK and canonical downstream signaling pathways in liver and muscle (p-ACC), whereas cGFJ decreased AMPK phosphorylation (liver) or had no significant effect (muscle)—a result reflected in unchanged p-ACC levels. Regardless, drinking centrifuged (pulp-free) GFJ corresponding to ∼3.5–4 cups (830–950 ml) per day for an average 70 kg human adult, had robust hypoglycemic effects in mice fed a HFD, warranting further study of its health-promoting effects, identification of bioactive components, and mechanisms of action. cGFJ behaved similarly, but not identically, to one of its bioactive compounds, naringin, which lowered blood glucose levels of HFD-fed animals without altering the activity of AMPK or ACC. This latter finding differs from results of Pu et al. [8], who reported robust activation of AMPK and inactivation of ACC in livers of HFD-fed C57Bl/6J mice in response to naringin, with comparable naringin doses, albeit presented in the diet, instead of in the drinking medium. We did not find that naringin caused weight loss or suppressed expression of the hepatic gluconeogenic enzymes PEPCK and G6Pase, as reported by Pu. The composition of the HFD used here differed from that of Pu (% J from fat/carbohydrate/protein, 60/20/20 vs. 37/43/20, respectively). Nevertheless, our results are consistent with reports that naringin has hypoglycemic, but not weight lowering effects [6], [24]. It should be noted for practical reasons we used cGFJ throughout the study to avoid clogging liquid intake monitors. Whether pulp-containing GFJ would have enhanced or reduced metabolic effects remains to be determined, but the fact remains that GFJ contains a compound or compounds other than naringin with health-promoting properties. We were unable to identify the proximate mechanism(s) of cGFJ effects. Possibly, subtle but cumulative differences in caloric absorption, respiration rates, or anti-inflammatory properties contribute to the phenotype. This possibility is supported by a need for 78 days of a HFD before weight differences induced by cGFJ became statistically significant [24]. Both the sweetened water control and cGFJ were acidic, but cGFJ had a lower pH at 3.5. All ingested liquids had to pass through a range of robust intraluminal pH gradients from the stomach (pH 1–3) to the small intestine (pH 6–7.4) [25], and it is unlikely that cGFJ consumption would alter duodenal pH to a degree that would impact pancreatic enzyme function or nutrient absorption. This conclusion is supported by the similarity in caloric value of feces collected over the final 24 hr of the 100-day-study from the cGFJ and control groups, and the lack of differences in absorption of glucose, oleic acid or taurocholic acid between cGFJ and the control mice. Interestingly, cGFJ decreased expression of the small heterodimer partner (SHP), which antagonizes function of multiple nuclear hormone receptors that regulate intermediary metabolism, such as LXRα, RARα, and PPARγ [26] [27]. Down regulation of Cyp7a1, FAS, SREBP1c, and PGC1α also are consistent with multiple alterations in lipid homeostasis [28], [29]. These data imply that cGFJ alters regulation of fat synthesis and storage. Potential benefits should be evaluated in context of reports that GF and GFJ components interact with several proteins that catalyze drug metabolism and absorption, and may cause health issues by modifying drug potency [30]. Many studies have shown that GF or GFJ, or their components alter drug pharmacokinetics, but altered pharmacokinetics doesn't necessarily alter pharmacodynamics [31]. In the ∼24 years since the potential for GFJ/GF consumption to alter drug potency was proposed, less than a dozen case reports have correlated GF or GFJ consumption with adverse clinical outcomes [10]. In most, if not all, the amount of GFJ consumed did not reflect normal consumption [32], [33], associations between GFJ and clinical manifestations were correlative [34], and patients had either severe pre-existing illnesses and/or confounding factors [35]. The possibility that excessive GFJ consumption could cause health issues in a select population taking specific drugs should not be dismissed, but nor is it appropriate to extrapolate these limited observations to the general population. A critical and evidence-based assessment of the potential beneficial vs. harmful effects of GF and GFJ consumption seems prudent. We have provided new evidence for potential health promoting properties of GFJ in murine HFD-driven obesity and non-obesity models. These results justify additional studies in animal models and humans to assess the mechanisms and scope of GFJ action.

Acknowledgments We are grateful to Greg Aponte for help with bomb calorimetry and to Mark Fitch for help with deuterated water analyses. The California Grapefruit Growers Cooperative provided financial support for this project. This does not alter our adherence to all the PlosOne policies on sharing data and materials, and did not affect the outcome of these experiments.

Author Contributions Conceived and designed the experiments: RC AT JLN AS. Performed the experiments: RC AT. Analyzed the data: RC AT JLN AS. Contributed reagents/materials/analysis tools: RC AT MH JLN AS. Wrote the paper: RC AT JLN AS. Analysis of grapefruit juice pH and caloric content: KT.