Abstract Low-grade inflammation is seen with obesity and is suggested to be a mediator of insulin resistance. The eliciting factor of low-grade inflammation is unknown but increased permeability of gut bacteria-derived lipopolysaccharides (LPS) resulting in endotoxemia could be a candidate. Here we test the effect of LPS and the anti-inflammatory compound resveratrol on glucose homeostasis, insulin levels and inflammation. Mice were subcutaneously implanted with osmotic mini pumps infusing either low-dose LPS or saline for 28 days. Half of the mice were treated with resveratrol delivered through the diet. LPS caused increased inflammation of the liver and adipose tissue (epididymal and subcutaneous) together with enlarged spleens and increased number of leukocytes in the blood. Resveratrol specifically reduced the inflammatory status in epididymal fat (reduced expression of TNFa and Il1b, whereas the increased macrophage infiltration was unaltered) without affecting the other tissues investigated. By LC-MS, we were able to quantitate resveratrol metabolites in epididymal but not subcutaneous adipose tissue. LPS induced insulin resistance as the glucose-stimulated insulin secretion during an oral glucose tolerance test was increased despite similar plasma glucose level resulting in an increase in the insulinogenic index (IGI; delta 0-15 insulin / delta 0-15 glucose) from 13.73 to 22.40 pmol/mmol (P < 0.001). This aberration in insulin and glucose homeostasis was normalized by resveratrol. In conclusion: Low-dose LPS enhanced the glucose-stimulated insulin secretion without affecting the blood glucose suggesting increased insulin resistance. Resveratrol restored LPS-induced alteration of the insulin secretion and demonstrated anti-inflammatory effects specifically in epididymal adipose tissue possibly due to preferential accumulation of resveratrol metabolites pointing towards a possible important involvement of this tissue for the effects on insulin resistance and insulin secretion.

Citation: Nøhr MK, Dudele A, Poulsen MM, Ebbesen LH, Radko Y, Christensen LP, et al. (2016) LPS-Enhanced Glucose-Stimulated Insulin Secretion Is Normalized by Resveratrol. PLoS ONE 11(1): e0146840. https://doi.org/10.1371/journal.pone.0146840 Editor: Pratibha V. Nerurkar, College of Tropical Agriculture and Human Resources, University of Hawaii, UNITED STATES Received: August 27, 2015; Accepted: December 21, 2015; Published: January 11, 2016 Copyright: © 2016 Nøhr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: The project was supported by grants from The A.P. Møller Foundation for the Advancement of Medical Science (MKN). MKN was supported by a Ph.D. scholarship from the Graduate School of Health, Aarhus University. The study is part of the research program LIRMOI Research Center (www.LIRMOI.com), which is supported by the Danish Council for Strategic Research (Grant 10-093499). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Obesity and type 2 diabetes are interrelated and the current understanding is that as obesity develops, the body becomes increasingly insulin resistant which can progress into type 2 diabetes. The origin of the developing insulin resistance is not fully known but the concomitantly presence of a chronic low-grade inflammation seems to play an important role [1, 2]. For instance, it was shown decades ago that the proinflammatory cytokine tumor necrosis factor alpha (TNFa) induces insulin resistance [3, 4]. Later, other proinflammatory cytokines such as interleukin 1 beta (IL1b) [5] and interleukin 6 (IL6) [6, 7] have been found to induce insulin resistance in experimental settings. Metabolic endotoxemia, i.e., endotoxins or LPS in the blood derived from gram-negative bacteria due to increased epithelial permeability, has been suggested as a possible mechanism of low-grade inflammation [8]. Thus, it has been shown that chronic infusion of low-dose LPS commences obesity and insulin resistance in a CD14-dependent manner [8, 9], suggesting a causative link between LPS and development of insulin resistance. However, recent reports have not been able to replicate the effects of LPS on obesity and insulin resistance, and instead suggest that the glucose-stimulated insulin secretion (GSIS) is enhanced by LPS via the GLP-1 pathway [10, 11]. Resveratrol is a polyphenolic compound found in especially red wine, which has been heavily investigated the past decade for its potential anti-inflammatory and anti-diabetic effects [12–18]. Resveratrol suppresses the activation the transcriptional factors NFkB and AP-1, responsible for the induction of cytokines and stress-related stimuli [19, 20]. Previously, resveratrol has been investigated for its effects in acute phase (high dose) LPS stimulation as seen in sepsis [21–23]. However, the effect of resveratrol on chronic low-dose LPS as seen in metabolic endotoxemia, has not previously been studied. Furthermore, resveratrol has been reported as an ameliorating factor on the detrimental effects, such as glucose intolerance and insulin resistance, which is induced by high fat feeding [12, 13]. The molecular mechanism behind resveratrol has for long been debated. It has thus been suggested that resveratrol increases the activity of the intracellular deacetylase sirtuin-1 (SIRT1) either directly [24] or indirectly via AMP-activated protein kinase [25] or effects on phosphodiesterase activity [26], but the precise mechanism is yet to be found. SIRT1 activation is involved in multiple pathways such as PGC1a which is a regulator of mitochondrial biogenesis [13], NFkB involved in inflammatory pathways [27] and PPARg deacetylation and browning of white adipose tissue [28]. The overall aim of this study was to investigate the effect LPS and resveratrol on glucose, insulin and inflammatory status. As low-grade inflammation is seen with obesity, we speculated whether resveratrol could have an ameliorating effect on some of the morbidities.

Material and Methods Mice and diets Twelve-week old male C57BL/6N mice (Taconic, Ejby, Denmark) were used in the experiments. Mice were allowed free access to food and water and were housed on a twelve-hour light cycle. Mice had free access to a control diet (1324, Altromin, Lage, Germany) or a modified diet consisting of control diet mixed with resveratrol (4 g resveratrol/kg diet) (Evolva, Copenhagen, Denmark) and processed into pellets. Protocols were performed in accordance with the European Communities Directive of 24 November 1986 (86/609/ECC) and approved by the Danish Council for Animal Experiments and conducted under license no. 2013-15-2934-00899. Experimental design Mice, anesthetized with a mixture of Hypnorm/midazolam (0.079 mg/ml fentanyl citrate + 2.5 mg/ml fluanisone + 1.25 mg/ml midazolam), were subcutaneously implanted with osmotic mini-pumps (Model 2004, Alzet, Cupertino, CA) infusing either vehicle (saline) or low-dose LPS (Escherichia coli 055:B5, L2630, Sigma-Aldrich) for the duration of 28 days (Fig 1A). Initially, we used a dose of LPS at 300 ug/kg/day, which has been previously published [8], but did not observe any effect compared to saline infused mice in relation to insulin secretion. Thus, we doubled the dose to 600 ug LPS/kg/day and saw a similar degree of inflammation of the liver, as was reported by Cani et al. [8]. The mice were divided into four groups: 1) Ctr/saline–control diet with saline-filled pumps, 2) RSV/saline–resveratrol diet with saline-filled pumps, 3) Ctr/LPS–control diet with LPS-filled pumps and 4) RSV/LPS–resveratrol diet with LPS-filled pumps. Body weight was measured daily the first week after surgery and hereafter weekly. Food intake was measured weekly. Following 28 days of treatment, mice underwent oral glucose tolerance test (OGTT) [29]. Mice were euthanized under anesthesia (Hypnorm/midazolam) by cervical dislocation. Tissues were harvested after a 3–5 hour fast and snap frozen in liquid nitrogen for later quantitative polymerase chain reaction qPCR analyses. The following tissues were harvested: liver, epididymal and subcutaneous adipose tissue, muscle (gastrocnemius). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Body weight and food intake. (A) Schematic overview of the research design. (B) Body weight during the course of the experiment in mice treated with control diet and saline (Ctr/saline), resveratrol diet and saline (RSV/saline), control diet and LPS (Ctr/LPS) and resveratrol and LPS (RSV/LPS) (n = 28–29 per group). (C) Total weight gain expressed in g/mouse after 28 days of treatment (n = 28–29 per group). (D) The total food intake during the experimental period of 28 days in four independent experiments. (E) Average daily resveratrol consumption. Data are presented as means ± SEM. Means with different superscript letters are significantly different at P < 0.05 according to post-hoc ANOVA or unpaired t-test. https://doi.org/10.1371/journal.pone.0146840.g001 Oral glucose tolerance test Following a 5 hour fast in new cages, mice were administered an oral dose of 2 g/kg glucose from a 50% glucose solution. Blood glucose was measured from tail vein blood at 0, 15, 30, 60 and 120 min after glucose administration using a hand-held glucometer (Contour XT, Bayer, Leverkusen, Germany). Furthermore, 75 ul blood samples were drawn at time points 0 and 15 min in heparin-coated capillary tubes, centrifuged and snap frozen in liquid nitrogen for later insulin measurements. Gene expression analysis Total RNA was isolated from liver, muscle and adipose tissue using TRIzol® Reagent (Life Technologies, Carlsbad, CA) according to manufacturer’s protocol. The concentration and purity of the RNA was measured by absorbance at 260 and 280 nm. The integrity of the RNA was evaluated by gel electrophoresis. Reverse transcriptase PCR was performed using VersoTM cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). cDNA was run in duplicates against primer pairs (Table 1) on LightCycler480 (Roche, Basel, Switzerland) using KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA). Data are shown as relative copy number compared to housekeeping gene calculated by the Advanced Relative Quantification method in LightCycler480 software v. 1.5 and presented as fold change compared to control. Polr2a was used as housekeeping gene on liver and muscle samples whereas Gadph was used as housekeeping gene in adipose tissue. All housekeeping genes were tested and had a similar expression level between the four groups. Primer pairs were designed using QuantPrime [30]. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Primers used for qPCR analysis. https://doi.org/10.1371/journal.pone.0146840.t001 Western blot analysis Protein extraction and western blot analysis were performed as previously described [31]. Primary antibodies against GLUT4, AS160, SDHA, glycogen synthase, AKT (isoform 2), cytochrome c, HSP60 and pyruvate dehydrogenase were used. Primary and secondary antibodies, dilution and source can be found in S1 Table. Data were calibrated to an internal control and normalized to total protein. Biochemical analyses Insulin was measured in duplicates using ultra-sensitive mouse ELISA kit (90080, Crystal Chem, Downers Grove, IL) according to manufacturer’s instructions. Adiponectin was measured using a commercial available ELISA kit according to manufacturer’s protocol (ELM-Adiponectin, RayBiotech, Norcross, GA). For liver triglycerides measurements, 50 mg liver was weighted and added 125 ul ethanolic KOH in microfuge tubes. Samples were incubated overnight and added 175 ul H 2 O:EtOH (1:1), centrifuged 5 min at 5000 rpm and the supernantant was moved to new tubes. 100 ul EtOH was added, vortexed and 200 ul was moved to new tubes and added 215 ul 1M MgCl 2 . Samples were centrifuged, moved to new tubes and measured for triglycerides. Free fatty acids were measured by a commercial available kit according to the supplied instructions (NEFA-HA(2), Wako, Neuss, Germany). Leukocyte count analysis Non-fasted blood was collected from the tail vein in pre-chilled EDTA tubes and stored on ice. Samples were analyzed in duplicates for leukocyte count on a hematology analyzer (XP-300, Sysmex, Ballerup, Denmark). Resveratrol measurement by liquid chromatography-mass spectrometry 100 mg of frozen samples were homogenized in 1.5 ml microtubes with pestiles (VWRTM Pestle&Microtube, Argos Technologies, United Kingdom) with 200 ul of a solution of 1.5 M formic acid methanol (95:5, v/v), then 1 ml of the same solution was added to the microtube and processed in vortex (Vortex Mixer, Hounisen, Denmark) for 2 min prior centrifugation at 13 400 rpm at room temperature for 30 min. The procedure was repeated one time with 1 ml of a solution of 1.5 M formic acid methanol (95:5, v/v) and two times with 1 ml of a solution of 1.5 M formic acid methanol (20:80, v/v). Pooled supernatants were collected and evaporated to dryness under reduced pressure in ScanVac Speed Vacuum Concentrator (Thermo Scientific). The residue was reconstituted in mobile phase (acetonitrile-water (5:95) v/v) and filtered using syringeless filter device, 0.2 um pore size (Whatman). Liquid chromatography-mass spectrometry (LC-MS) analysis were performed using LTQ XL (Linear Quadrupole 2D Ion Trap Mass Spectrometer, Thermo Scientific, CA, USA) mass spectrometer operating in electrospray ionization (ESI) negative mode and attached to an Accela HPLC system. Settings for the mass spectrometer were 45, 3, and 0 (arbitrary units) for sheath, auxiliary, and sweep gas flow rates (N 2 ), respectively, a spray voltage of 1.10 kV, and a capillary temperature of 350°C. The settings for capillary voltage and tube lens voltage were 3 V and 90 V, respectively. Resveratrol metabolites (trans-resveratrol-3-O-sulfate, trans-resveratrol-sulfate-glucuronide, trans-resveratrol-3-O-glucuronide, trans-resveratrol-4´-O-glucuronide, trans-resveratrol-3,4´-O-disulfate) were separated by a solvent gradient with aqueous formic acid (0.1%, pH 2.5) as solvent A and 100% acetonitrile as solvent B on a Kinetex C18 reverse-phase column (100 mm length, 2.6 mm internal diameter, 1.7 μm particle size; Phenomenex) protected by a precolumn. Solvent gradient: 0 min 5% B, 2 min 5% B, 8 min 30% B, 11 min 95% B, 14 min 95% B and then equilibrating the column at 5% B for 5 min, the flow rate was 0.4 ml/min and the column temperature was 25°C. Glucuronides and sulfates were quantified by an external standard calibration curve of trans-resveratrol-3-O-β-D-glucuronide and trans-resveratrol-3-O-sulfate respectively, which were isolated from human urine according the procedure described by Radko et al. [32]. The structure of metabolites was identified based on their full scan MS and MS/MS spectra generated in negative ESI. Limit of detection of metabolites was 0.017 and 0.025 ug/g tissue; limit of quantification was 0.018 and 0.032 ug/g tissue for sulfates and glucuronides, respectively. Statistical analysis Data are presented as mean ± SEM. Differences of means were calculated by one-way ANOVA followed by Newman-Keuls post hoc test or unpaired t-test where appropriate. OGTT and insulin levels over time were evaluated by two-way ANOVA followed by Bonferroni post hoc test. Area under the curve (AUC) was calculated using the trapezoidal rule. The insulinogenic index (IGI) was calculated as the initial insulin secretion (delta 0-15 Insulin) divided by the initial glucose rise (delta 0-15 Glucose) following oral administration of glucose. Means were considered significantly different when P < 0.05. Data were analyzed using GraphPad Prism 5.01.

Discussion Low-grade inflammation is a key component of obesity and has previously been suggested to be induced by LPS-leakage through the gut epithelium [8]. In the present study, LPS treatment did not cause significant glucose intolerance during an OGTT (Fig 2A). However, the GSIS was elevated by LPS without affecting the blood glucose indicating an induction of insulin resistance. Furthermore, resveratrol restored the elevated LPS-induced GSIS (Fig 4D). LPS-treatment caused inflammation as the inflammatory markers Tnfa and Il1b were elevated in liver and subcutaneous and epididymal adipose tissues. Also, LPS-treated animals had increased leukocyte numbers in the blood and enlarged spleens, pointing towards an increased inflammatory state (Fig 3). Surprisingly, resveratrol showed a mixed picture of its conceivable anti-inflammatory function. Actually, resveratrol only reduced inflammation in epididymal adipose tissue whereas both liver, blood leukocytes, spleens and subcutaneous fat were unaffected (Fig 3). In agreement with recent publications [10, 11], LPS enhanced the GSIS. Nguyen and colleagues [10] showed that the enhancement of GSIS by LPS could be traced back to an increased GLP-1 secretion. GLP-1 is an incretin hormone released from the L-cells in the gut, which potentiates the insulin secretion from the beta-cells in the presence of glucose [37]. The physiological relevance for having increased GSIS during inflammation is of a complex nature and poorly understood. First, having a tight glucose control during endotoxemia and disease seems to be important for the body, which is also a predictor of the clinical outcome in critically ill patients [38, 39]. Second, insulin itself could have anti-inflammatory effects and is thus released in order to counteract the effect of LPS. Indeed, constant insulin infusion during normoglycemia decreases inflammation during endotoxemia in animals [40, 41] in a PI3K/Akt-dependent manner [42]. To further complicate the picture, a study by Ceasar et al. [43] demonstrated that monocolonisation with either E.coli or an isogenic strain with reduced LPS immunogenicity in mice resulted many of the same effects, e.g. increased adiposity, glucose intolerance and insulin resistance, even though LPS plasma concentration and inflammation was significantly reduced. Needless to say, much more data are required in order to elucidate the role of LPS in the context of metabolic disease. It was unexpected to find that resveratrol did not work uniformly as an anti-inflammatory agent, which has been showed previously in various cell cultures [44–46]. Actually, anti-inflammation was specifically seen in epididymal adipose tissue and not liver, skeletal muscle, leukocyte numbers or even subcutaneous adipose tissue (Fig 3). However, this is in agreement with a recent report stating that resveratrol only has an effect in visceral adipose tissue in high-fat fed monkeys, leaving the subcutaneous fat unaffected and inflamed [14]. This is very interesting, as especially visceral adipose tissue inflammation has long been correlated with the development of metabolic syndrome [47, 48]. To investigate whether the mixed anti-inflammatory properties is a result of altered tissue distribution of resveratrol, we quantified resveratrol metabolites in epididymal and subcutaneous adipose tissues by LC-MS. This analysis revealed that resveratrol metabolites are only measurable in visceral adipose tissue with no metabolites (except from small amounts of Trans-resveratrol-sulfate-glucuronide) found in subcutaneous adipose tissue (Table 2). So our LC-MS measurement of resveratrol metabolites in the two adipose tissue depots might offer an explanation for the more pronounced effect of resveratrol in visceral adipose tissue. Cd14 expression, which is a marker of macrophage infiltration, was unaltered (Fig 3E) suggesting that resveratrol does not affect the actually number of residual macrophages, but instead shift their phenotype into a more anti-inflammatory state (M2 macrophage) in the epididymal adipose tissue. Despite the mixed anti-inflammatory properties in various tissues, resveratrol did reverse the LPS-induced increase in GSIS. Surprisingly, we saw that resveratrol caused a small decline in the food consumption during the treatment period without affecting the overall weight gain/loss (Fig 1C and 1D). One explanation could be due to the relative small reduction in food intake (≈ 7%, Ctr/saline vs RSV/saline) is not sufficient to detect alterations in body weight in the matter of a relative short treatment period (28 days). In a recent study, high-fat feeding (70% fat) for one month was not affecting the body weight in mice, though glucose intolerance and insulin resistance were commenced [49]. Only after three month of high-fat feeding, did also the body weight respond to the increased nutritional pressure. This suggests, in order to thoroughly investigate the effects of resveratrol treatment on body weight, longer treatment periods are needed, which were unfortunately not possible in our study due to limitations of the pumping capacity of the osmotic mini-pump. Finally, it was surprising to see that despite the GSIS was significantly increased by 29% compared to controls, the plasma glucose concentration was not lowered significantly 15 min after glucose administration (Fig 2A and 2C), which was also seen in the study by Nguyen et al. [10]. Thus, we speculate, despite lack of elevated fasting glucose and insulin levels, that LPS induces some subtle insulin resistance which only is revealed during a glucose challenge. A more sensitive method for assessing insulin sensitivity, like the euglycemic-hyperinsulinemic clamp, will probably be needed in order to study the degree of insulin resistance in more detail. Adiponectin expression has previously been described to be decreased by inflammation in murine adipocytes [50–52] and human subcutaneous adipose tissue [44, 53, 54]. We did see that the adiponectin expression was decreased (and partially rescued by resveratrol) in the subcutaneous but not the epididymal adipose tissue (Fig 4A). However, this effect of resveratrol was not translated into mature protein, where only a small non-significant decline by LPS of plasma adiponectin was seen (Fig 4A). This paper adds the growing field concerning the role of LPS and endotoxemia in the development of metabolic diseases. We here demonstrate that low-dose LPS enhance GSIS without affecting the glucose concentration suggesting increased insulin resistance. Resveratrol dampened the effect on the LPS-induced hyperinsulinemia and specifically reduced inflammation in epididymal adipose tissue pointing towards a possible important involvement of this tissue for the effects on insulin resistance and insulin secretion as a result of metabolic endotoxemia. Given the beneficial effect of resveratrol on specifically visceral adipose tissue, makes it an interesting candidate in ameliorating inflammation as seen in obesity and metabolic syndrome.

Supporting Information S1 Fig. Western blot analysis on skeletal muscle. AKT (isoform 2), AS160, glycogen synthase, cytochrome c, pyruvate dehydrogenase, SDHA and HSP60 were investigated by Western blot analysis. However, no significant alterations in protein expression were induced by resveratrol and/or LPS. Data are presented as means ± SEM. https://doi.org/10.1371/journal.pone.0146840.s001 (TIF) S1 Table. Primary and secondary antibodies used for Western blot analysis. https://doi.org/10.1371/journal.pone.0146840.s002 (DOCX)

Acknowledgments We wish to thank Lenette Pedersen, Pia Hornbæk, Helle Zibrandtsen, Sussi Kragh and Trine Kristensen for their much appreciated assistance in the laboratory and the animal facility.

Author Contributions Conceived and designed the experiments: MKN AD SL SBP. Performed the experiments: MKN YR LPC. Analyzed the data: MKN AD MMP BR SL NJ SBP LHE. Wrote the paper: MKN SBP.