David Messenger,4 Mark Fowler,4 Gail Jenkins,4 Naila Rabbani,5 and Paul J. Thornalley1,5
Improved Glycemic Control andVascular Function in Overweight andObese Subjects by Glyoxalase 1Inducer FormulationDiabetes 2016;65:2282–2294 | DOI: 10.2337/db16-0153
Risk of insulin resistance, impaired glycemic control, andcardiovascular disease is excessive in overweight and obesepopulations. We hypothesized that increasing expressionof glyoxalase 1 (Glo1)—an enzyme that catalyzes themetabolism of reactive metabolite and glycating agentmethylglyoxal—may improve metabolic and vascularhealth. Dietary bioactive compounds were screened forGlo1 inducer activity in a functional reporter assay, hitswere confirmed in cell culture, and an optimized Glo1inducer formulation was evaluated in a randomized,placebo-controlled crossover clinical trial in 29 overweightand obese subjects. We found trans-resveratrol (tRES) andhesperetin (HESP), at concentrations achieved clinically,synergized to increase Glo1 expression. In highly overweightsubjects (BMI >27.5 kg/m2), tRES-HESP coformulation in-creased expression and activity of Glo1 (27%, P < 0.05)and decreased plasma methylglyoxal (237%, P < 0.05) andtotal body methylglyoxal-protein glycation (214%, P < 0.01).It decreased fasting and postprandial plasma glucose (25%,P < 0.01, and 28%, P < 0.03, respectively), increased oralglucose insulin sensitivity index (42 mL $ min21 $ m22, P <
0.02), and improved arterial dilatation Dbrachial artery flow-mediated dilatation/Ddilation response to glyceryl nitrate(95% CI 0.13–2.11). In all subjects, it decreased vascular in-flammationmarker soluble intercellular adhesionmolecule-1(210%, P < 0.01). In previous clinical evaluations, tRES andHESP individually were ineffective. tRES-HESP coformula-tion could be a suitable treatment for improved metabolicand vascular health in overweight and obese populations.
Increasing overweight and obese populations are driving aglobal epidemic of type 2 diabetes and cardiovascular diseasein Westernized countries. Glyoxalase 1 (Glo1) was linked toclinical obesity through association with measures of fatdeposition and Glo1 deficiency identified as a driver ofcardiovascular disease in a large integrative genomics study(1,2). It is currently unaddressed by therapeutic agents.Experimental studies found that overexpression of Glo1in mice suppressed inflammation and body weight gainin overfeeding models of obesity and prevented vasculardisease in diabetes (3).
Glo1 is part of the glyoxalase metabolic pathway, whichconsists of two enzymes, Glo1 and Glo2, and a catalyticamount of reduced glutathione (GSH) in the cytoplasm ofcells (Fig. 1A). The major function of the glyoxalase path-way is detoxification of the reactive dicarbonyl metabolite,methylglyoxal, converting it to D-lactate. Methylglyoxal isa highly potent glycating agent of protein that forms thequantitatively major advanced glycation end product,hydroimidazolone MG-H1, linked to protein inactiva-tion and cell dysfunction (4,5) (Fig. 1B). Degradation ofmethylglyoxal-modified proteins releases MG-H1 free ad-duct from tissues for urinary excretion (4). The steady-statelevel of protein MG-H1 is maintained at low tolerable levelsby Glo1 (3).
Abnormally high methylglyoxal concentration, dicarbonylstress, is a common characteristic of obesity and type 2diabetes. It is severe in diabetes, driven by increased flux of
1Clinical Sciences Research Laboratories, Warwick Medical School, University ofWarwick, University Hospital, Coventry, U.K.2Warwickshire Institute for the Study of Diabetes, Endocrinology and Metabolism,University Hospitals of Coventry and Warwickshire National Health Service Trust,Coventry, U.K.3Division of Health Sciences, Warwick Medical School, University of Warwick,Coventry, U.K.4Unilever Research & Development Colworth, Bedford, U.K.5Warwick Systems Biology Centre, University of Warwick, Coventry, U.K.
methylglyoxal formation and decreased Glo1 activity at sitesof vascular complications. Methylglyoxal is formed mainlyby the nonenzymatic degradation of triosephosphate inter-mediates of glycolysis—a minor “leak” of ;0.05% triose-phosphate flux. In obesity, dicarbonyl stress is mild andtriosephosphate flux is increased by glyceroneogenesis inadipose tissue–liver cycling of triglycerides and free fattyacids. Increased methylglyoxal protein modification in dicar-bonyl stress is implicated as a mediator of impaired meta-bolic and vascular health in obesity and diabetes (3).
An effective strategy to counter dicarbonyl stress is toincrease expression of Glo1 (6). We described a functionalregulatory antioxidant response element (ARE) in humanGLO1 with basal and inducible expression upregulatedby transcriptional factor nuclear factor E2-related factor2 (Nrf2). Recent advances in Nrf2 regulation suggestedthat potent induction of Glo1 expression could be achievedby a synergistic combination of Nrf2 activators addressingdifferent regulatory features (7,8). In this study, we soughtto screen dietary bioactive compounds for Glo1 induceractivity in a functional reporter assay, confirm hits in cellculture, and evaluate an optimized Glo1 inducer formula-tion in a randomized, placebo-controlled crossover clinicaltrial for improved metabolic and vascular health in over-weight and obese subjects.
RESEARCH DESIGN AND METHODS
Screening of Glo1 Inducers Using GLO1-AREand Related Stable Transfectant Reporter Cell LinesStable transfectant luciferase reporter cell lines with AREtranscriptional regulatory elements were developed fromhuman HepG2 cells, as described for quinone reductaseARE (7), incorporating regulatory elements: GLO1-ARE orfunctionally inactive mutant as negative control (ARE-1and ARE1m in previous work) (6). Stable transfectant celllines were incubated with and without bioactive com-pounds (0.625–20.0 mmol/L) for 6 h. Luciferase activitywas then determined in cell lysates, with correction for
blank response, and normalized to the highest effect(100%) achieved with 10 mmol/L trans-resveratrol (tRES)(6). Nrf2-dependent transcriptional response was verifiedby small interfering RNA silencing of Nrf2. Cytotoxicitywas assessed after 24 h exposure by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]method (9). Screening hit criteria were as follows: in-creased transcriptional response at #5 mmol/L withoutsignificant cytotoxicity to human aortal endothelial cells(HAECs) and BJ human foreskin fibroblasts in primaryculture. Dietary bioactive compound selection criteriawere ability to activate Nrf2 at concentrations achievedor likely achievable at tolerable doses clinically and/ordecrease glycation and/or toxicity by methylglyoxal orsimilar compounds.
ARE-Linked Gene and Other Cell Metabolism andFunctional Marker Gene Expression by Digital mRNAProfiling and ImmunoblottingHAECs, BJ fibroblasts, and HepG2 cells (5 3 105 cells/well) were seeded on sixwell plates in relevant mediumand cultured overnight at 37°C under 5% CO2/air. Cellswere treated with and without 5 mmol/L tRES, 5 mmol/Lhesperetin (HESP), and 5 mmol/L tRES plus HESP com-bined or vehicle (0.002% dimethylsulfoxide) and culturedfurther for up to 48 h. At the time points indicated,mRNA was extracted and analyzed by the NanoStringnCounter method (10). Immunoblotting was performedas previously described (6).
Clinical StudyA randomized, double-blind, placebo-controlled crossoverstudy of optimized Glo1 inducer was performed in 32overweight and obese healthy subjects (Healthy Ageingthrough functional food [HATFF]). The study was ap-proved by the National Research Ethics Service CommitteeWest Midlands—Coventry & Warwickshire (project num-ber 13/WM/0368) and registered at clinicaltrials.gov(NCT02095873). The procedures followed were in accordance
Figure 1—Glyoxalase pathway and protein glycation by methylglyoxal. A: Metabolism of methylglyoxal by the glyoxalase system.B: Formation of hydroimidazolone MG-H1 from arginine residues in protein.
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with institutional guidelines and the Declaration of Helsinki.Three participants failed to complete the study.
Main inclusion criteria were age 18–80 years, BMI 25–40kg/m2, and normal, impaired fasting, or impaired postpran-dial glucose. Main exclusion criteria were severe hypertri-glyceridemia, uncontrolled hypertension, cardiovasculardisease, relevant renal or hepatic disease, diabetes, andother relevant morbidity; severe excess alcohol consump-tion (.14/21 units [8g]/week for women/men), smoking,pharmacological treatment affecting glucose and lipid me-tabolism or blood coagulation, taking herbal remedies,known food allergies, pregnancy, and breast-feeding. Allsubjects were evenly randomized in tRES-HESP and pla-cebo arms (n = 16) by the Clinical Trials Unit, Universityof Warwick. Treatment was one capsule daily for 8 weeksand a washout period of 6 weeks—tRES-HESP (90 mgtRES and 120 mg HESP) and placebo—with starch inplace of bioactives in hard gelatin capsules. Previous stud-ies with dietary fiber supplementation indicated interven-tion for at least 8 weeks is required for improved glycemiccontrol (11). Participants were advised to maintain theirusual diet, confirmed by dietary questionnaires at the startand end of dosing periods, and physical activity, supportedby nurse and dietitian contact throughout the study.
Primary clinical end points were metabolic health (oralglucose insulin sensitivity [OGIS] index in an oral glucosetolerance test [OGTT] [75 g glucose; participants instructedto eat carbohydrate-rich diet, .150 g/day, for at least 3days before the test, followed by an overnight fast]) (12)and vascular health (brachial artery flow-mediated dilata-tion [FMD], including dilatation response to a subthera-peutic dose [25 mg] of glyceryl nitrate [GTND]) (13). OGTTand FMD/GTND assessments were performed at the startand end of each treatment period between 8:00 A.M. and10:00 A.M. in a quiet temperature-controlled room main-tained at 236 1°C. Markers of vascular inflammation werealso assessed by commercial ELISA.
Venous blood samples were also drawn in the fastingstate prior to the OGTT. Safety assessment of tRES-HESPcoformulation was assessed by electrocardiogram and anal-ysis of blood markers. Plasma methylglyoxal and glycationand oxidation adducts in plasma protein and urine (secondvoid after overnight fast) were assayed by stable isotopicdilution analysis liquid chromatography–tandem mass spec-trometry (LC-MS/MS) (14,15).
Total tRES and HESP Urinary MetabolitesTotal tRES and HESP urinary metabolites were deter-mined by stable isotopic LC-MS/MS after deconjugation ofglucuronides and sulfates. Urine (20 mL), from which cellshad been sedimented and removed prior to storage,with 42 mmol/L ammonium acetate buffer, pH 4.9(60 mL), internal standards (250 mmol/L [13C6]tRES and10 mmol/L d4-HESP; 20 mL), and b-glucuronidase (5 mL,85 units) and b-sulphatase (5 mL, 5 units), was incubatedfor 2 h at 37°C in the dark. Deconjugation was validatedwith authentic glucuronides and sulfates of tRES and
HESP before use. Thereafter, ice-cold methanol (100 mL)was added for deproteinization, centrifuged (10,000g, 10 min,4°C), and analyzed by LC-MS/MS. Calibration curves wereconstructed by analysis of 125–625 pmol tRES and HESP.
Cellular GSH and Oxidized Glutathione GSH–to–oxidized glutathioneCellular GSH and oxidized glutathione (GSSG) wereassayed by stable isotopic dilution analysis LC-MS/MS.Cells (;1 3 106 cells) were deproteinized with 10% tri-chloroacetic acid (40 mL) containing 0.15% NaCl and0.25% sodium azide in water and centrifuged (20,000g,30 min, 4°C). An aliquot of supernatant (10 mL) was mixedwith 10 mL isotopic standard cocktail (100 pmol [13C2,
15N]GSH and [13C4,
15N2]GSH–to–GSSG) and analyzed by LC-MS/MS. Calibration standards contained 100–2,000 pmol GSHand 5–100 pmol GSSG (Fig. 3).
Statistical AnalysisData are mean 6 SD or SEM for parametric data and me-dian (upper–lower quartile) for nonparametric data. Signifi-cance testing in paired data was assessed by paired Studentt test and Wilcoxon signed rank test (for two study groups),by ANOVA repeated measures and Friedman test (forfour study groups), and by correlation analysis by Pearsonand Spearman methods for parametric and nonparametricdata, respectively. For HATFF study power calculation, wejudged that decrease in area under the plasma glucose curve(AUCg) of the OGTT would be 10%, compared with 30%decrease with high cereal fiber intake (16). With a 30%dropout, 32 subjects were required for a = 0.05 and power(1 – b) = 0.80. Post hoc analysis of variables for highlyoverweight and obese subgroups was performed to exploreBMI as a factor influencing responsiveness to Glo1 inducers.
RESULTS
Screening of Small-Molecule Glo1 InducersAfter screening of ;100 dietary bioactive compounds withNrf2 activator activity, the highest maximum GLO1-AREtranscriptional activity response (Emax) was produced bytRES. The lowest half-maximal effective concentration(EC50) for GLO1-ARE transcriptional activity was foundwith HESP. For tRES, EC50 = 2.52 6 0.19 mmol/L andEmax 100 6 2%, and for HESP, EC50 = 0.59 6 0.01 mmol/Land Emax 24.4 6 0.1% (Fig. 2A and B). In previous clinicalstudies, dietary supplementation of 150 mg HESP achieveda peak plasma concentration of 6.7 mmol/L (17), suggestingthat HESP may be a competent Glo1 inducer for clinical usebut with low maximal effect, and dietary supplementation of500 mg tRES achieved a peak plasma concentration of ;0.3mmol/L (18), eightfold lower than the EC50 for GLO1-AREresponse. To enhance efficacy, we studied the pharmacolog-ical synergism of tRES and HESP together. Study of theGLO1-ARE transcriptional response of 5 mmol/L HESPwith 0.625–10 mmol/L tRES showed that HESP combinedsynergistically with tRES, decreasing the EC50 of tRES ap-proximately twofold to 1.466 0.10 mmol/L while maintain-ing the Emax (Fig. 2C). The predicted increase of GLO1-ARE
2284 Metabolic and Vascular Health With Glo1 Inducer Diabetes Volume 65, August 2016
Figure 2—Induction of Glo1 expression by tRES and HESP. GLO1-ARE transcriptional response reporter assay. Data of normalizedresponses for varied bioactive concentrations were fitted by nonlinear regression to the equation E = Emax 3 [bioactive]n/(EC50
n + [bio-active]n), solving for Emax, EC50, and n (Hill coefficient). A: Concentration-response curve for tRES. Data are mean 6 SD (n = 3) for5 concentrations. Nonlinear regression (red curve): E (%) = 100 3 [tRES]3.92/(2.523.92 + [tRES]3.92). B: Concentration-response curve forHESP. Data are mean 6 SD (n = 3–8) for 6 concentrations. Nonlinear regression (blue curve): E (%) = 24.4 3 [HESP]2.01/(0.592.01 +[HESP]2.01). C: Concentration-response curve for tRES in the presence of 5.0 mmol/L HESP. Data are mean 6 SD (n = 3–6) for5 concentrations. Nonlinear regression, green curve: E (%) = (83.4 3[tRES]1.36/(1.461.36 + [tRES]1.36)) + 11.6; green curve: tRES+5.0 mmol/L HESP; red dotted curve: tRES only (as for A). D: Expansion of C, also showing the response for 5.0 mmol/L HESP (blue dashed line).
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transcriptional response from concentration response curvesof 0.1–1.0 tRES in the presence of 5 mmol/L HESP was 3-to 79-fold, including up to 80% increase over additiveeffects (Fig. 2D). This suggests that marked benefits mayaccrue from use of tRES-HESP coformulation.
Safety assessments of tRES and HESP indicate that theyare highly tolerated (19,20). Studies of human BJ fibro-blasts in primary culture showed no toxicity of tRES andHESP individually or with 5 mmol/L combination with pri-mary bioactive compound at concentrations #20 mmol/L.tRES (40 mmol/L) with 5 mmol/L HESP and 40 mmol/LHESP with and without 5 mmol/L tRES showed minor de-crease in viability of BJ cells in vitro (Fig. 2E and F). tRES(5 mmol/L) with 0.625 mmol/L HESP gave a minor increasein cell number—possibly an effect of insulin-sensitizingactivity. (See below.) There was no toxicity of tRES,HESP, or both combined in HAECs in primary cultureunder similar conditions (Fig. 2G and H).
Validation of Glo1 Inducer Screening Resultsand Functional EffectsTo validate the Glo1 inducer studies, we measured thechange in Glo1 mRNA and protein and functional responsesin the human hepatocyte-like HepG2 cell line in vitro andHAECs and BJ fibroblasts in primary culture. There was a10–30% increase in Glo1 mRNA in cells incubated withtRES and HESP and combined, and similar increases inGlo1 protein (Fig. 2I–K). We then studied the effect onfunctional markers: inflammatory response proteins andmatrix metalloproteinase. In HAECs, treatments decreasedintercellular adhesion molecule-1 (ICAM-1), receptor foradvanced glycation end products (RAGE), and E-selectinprotein, with synergistic effects on ICAM1 and RAGE(Fig. 3A–C). In BJ fibroblasts, treatments decreased cellularvascular adhesion molecule-1 (VCAM-1), RAGE, and matrixmetalloproteinase-3 (MMP-3), with synergism for tRES andHESP in decrease of VCAM-1 and MMP-3 (Fig. 3D–F). Thissuggests that the tRES and HESP treatment lowers basalcell inflammation and extracellular matrix turnover.
Activation of Nrf2 is associated with increased cellularGSH and GSSG ratio through increased expression of genes ofGSH synthesis andmetabolism—particularly g-glutamylcysteineligase (modulatory and catalytic subunits [GCLM and GCLC])and glutathione reductase. Treatment of HAECs, BJ fibro-blasts, and HepG2 cells in vitro with 5 mmol/L tRES and5 mmol/L HESP individually, however, did not change cellular
levels of GSH and GSSG, whereas treatment with 5 mmol/LtRES and HESP combined increased cellular GSH content by43% in BJ fibroblasts and 32% in HepG2 cells (Fig. 3G–L).Increased cellular GSH concentration enhances in situ activityof Glo1 (3).
We also studied time-dependent changes in expressionof ARE-linked genes and other genes linked to metabolismand function in HAECs, BJ fibroblast, and HepG2 cellsby focused quantitative mRNA array (selected timecourse responses are given in Supplementary Fig. 1A–C). Overall, there were additive and synergistic changeson gene expression of tRES and HESP combined treat-ment. For example, in HAECs, mRNA of ARE-linkedgenes glutathione transferase A4 (GTSA4), heme oxygen-ase-1 (HMOX-1), GCLM, GCLC, and glutathione reduc-tase were increased. tRES and HESP together decreasedICAM1 mRNA. In BJ fibroblasts, tRES and HESP syner-gized to increase mRNA of GSTP1, HMOX1, NQO1, andaldoketo reductase 1C1 (AKR1C1) and to decrease ex-pression of inflammation markers CCL2 and ICAM1. InHepG2 cells, tRES and HESP combined synergistically toincrease mRNA of NQO1, GCLM, and GCLC, LDL receptor(LDLR), hexokinase-2 (HK2), and 6-phosphofructokinase/bisphosphatase-3 (PFKBP3).
Improved Clinical Metabolic and Vascular HealthWith tRES-HESP Coformulation: HATFF StudyCoformulation of tRES-HESP was evaluated in healthy over-weight and obese subjects. Twenty-nine subjects completedthe study. Characteristics of subjects at study entry aregiven in Table 1. Subjects had mildly impaired glycemiccontrol, with only 9 subjects meeting criteria of prediabe-tes; all participants were overweight or obese, 20 werehighly overweight (BMI .27.5 kg/m2), and 11 were obese(BMI.30 kg/m2). tRES-HESP treatment increased urinaryexcretion of tRES and HESP metabolites by .2,000-foldand .100-fold, respectively, compared with placebo (Sup-plementary Fig. 2A and B). Dietary questionnaires, urinaryexcretion of pyrraline—an advanced glycation end productsourced only from food (21), and fasting plasma ketonebody concentrations in the normal range in all subjects(22) suggested that food consumption was similar through-out the study (Table 2). Clinical safety indicators werenormal at study entry and remained unchanged through-out the placebo and tRES-HESP treatment periods (Sup-plementary Table 1).
E–H: Evaluation of the effect of tRES and HESP individually and in combination on the growth and viability of BJ fibroblasts (E and F) andHAECs (G and H) in vitro. For E and G, cells were incubated with 0.625–40 mmol/L tRES and with (green bars) or without (red bars)5.0 mmol/L HESP. For F and H, cells were incubated with 0.625–40 mmol/L HESP and with (green bars) or without (blue bars) 5.0 mmol/LtRES. I–K: Validation of induction of Glo1 expression by 5.0 mmol/L tRES and HESP, individually and combined. Cell type: HAECs (I), BJfibroblasts (J), and HepG2 (K) cells. Panels (from left to right): GLO1 mRNA change with 5.0 mmol/L tRES (red line), 5.0 mmol/L HESP(blue line), and 5.0 mmol/L tRES and HESP (green line). Bar chart: Glo1 protein (16 h posttreatment) plus 5.0 mmol/L tRES (red bar) and5.0 mmol/L HESP (blue bar). Data are mean 6 SD (n = 3) for E–K. Significance: *P < 0.05, **P < 0.01, and ***P < 0.001. HAEC cells weregrown in proprietary large-vessel endothelial cell basal media supplemented with large-vessel endothelial cell growth supplement (con-taining hydrocortisone, human epidermal growth factor, and human fibroblast growth factor with heparin and in 2% [v/v] FBS), 25 mg/mLgentamicin, and 50 ng/mL amphotericin B. They were cultured in minimum essential medium with 10% FCS and 2 mmol/L glutamine underan atmosphere of 5% CO2 in air, 100% humidity, and 37°C.
2286 Metabolic and Vascular Health With Glo1 Inducer Diabetes Volume 65, August 2016
tRES-HESP produced a 22% increase in Glo1 activityof peripheral blood mononuclear cells (PBMCs) post-treatment compared with placebo in all subjects. PBMCGlo1 activity was increased 27% in highly overweight/obese and 30% in obese subgroups. Concomitant withthis there was a 37% decrease in plasma methylglyoxal
postsupplementation with tRES-HESP but not with pla-cebo in highly overweight/obese subjects. There was nochange in plasma D-lactate concentration with tRES-HESPtreatment—a surrogate indicator of flux of methylglyoxalformation (3) (Table 2). Therefore, increase of Glo1 activityby tRES-HESP in PBMCs, also expected in tissues, likely
Figure 3—Effect of tRES, HESP, and tRES-HESP coaddition on cell vitality markers and glutathione metabolism in human endothelial cells,fibroblasts, and HepG2 cells in vitro. A–F: Cell vitality markers. Control, 5.0 mmol/L tRES, 5.0 mmol/L HESP, and 5.0 mmol/L tRES-HESP.HAECs: ICAM-1 protein (A) and RAGE protein (B) at 18 h posttreatment and E-selectin protein (C ) and VCAM-1 protein (D) at 24 hposttreatment. BJ fibroblasts: RAGE protein (E) and MMP3 protein (F ) at 72 h posttreatment. G–L: Cellular GSH and GSSG at 24 hposttreatment. GSH (G–I) and GSSG (J–L) in HAECs, BJ fibroblasts, and HepG2 cells. Percentage change with respect to GSH of controlcultures is indicated. Data are mean 6 SD (n = 3). Significance: *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to control; oP < 0.05with respect to tRES control; and +P< 0.05 and ++P<0.01 with respect to HESP control. LC-MS/MS was performed using two HypercarbHPLC columns in series (50 3 2.1 mm and 250 3 2.1 mm, particle size 5 mm) at temperature of 30°C. Initial mobile phase was 0.1%trifluoroacetic acid in water and the elution profile was 1–15 min, a linear gradient of 0–30% acetonitrile, and 15–16 min, isocratic 30%acetonitrile; the flow rate was 0.2 mL/min and diverted into the mass spectrometer from 4 to 16 min. Mass spectrometric analysis wasperformed using electrospray positive ionization mode with the following detection settings: capillary voltage 3.4 kV, source and desolva-tion gas temperatures 120°C and 350°C, respectively; and cone and desolvation gas flows 146 and 550 L/h, respectively. For analytedetection, the retention time, mass transition molecular ion>fragment ion, cone voltage, and collision energy were as follows: GSH, 11.7min, 308.2 > 179.1 Da, 30 V, and 13 eV; [13C2,
15N]GSH, 11.7 min, 311.2 > 182.1 Da, 30 V, and 13 eV; GSSG, 14.4 min, 613.2 > 483.7 Da,52 V, and 18 eV; and [13C4,
15N2]GSSG, 14.4 min, 619.2> 489.7 Da, 52 V, and 18 eV. For GSH and GSSG, limits of detection were 0.92 pmoland 1.46 pmol, analytical recoveries 97 6 2% and 93 6 6%, and intrabatch coefficients of variation 8.8% and 10.9% (n = 6), respectively.
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decreased plasma methylglyoxal concentration withoutchange in flux of methylglyoxal formation.
With tRES-HESP treatment, there was a positive corre-lation of change in OGIS from baseline (DOGIS) withBMI (r = 0.45, P , 0.05) (Fig. 4A). No similar correlationoccurred with placebo. In the subset of highly overweightsubjects, there was an increase in OGIS from baseline withtRES-HESP (DOGIS = 42 mL $ min21 $ m22) (Table 2 andFig. 4B) but not with placebo. This effect was further enhancedin obese subjects only (DOGIS = 58mL $min21 $m22) (Table2). The main contributory factors to this effect were: 1)decreased fasting plasma glucose (FPG) (P , 0.01) and 2)decreased AUCg (P , 0.03, ANOVA) (Table 2).
There was a negative correlation of change in FPG frombaseline (DFPG) to BMI with tRES-HESP treatment (r =20.41, P , 0.05) (Fig. 4C). No similar correlation occurredwith placebo. In highly overweight subjects, there was a 5%decrease in FPG postsupplementation with tRES-HESP(Fig. 4D). This effect was further enhanced in obese sub-jects only (29%) (Table 2). There was also decreased AUCgin the OGTT with tRES-HESP treatment in the highly over-weight study group to a similar extent (28%; P , 0.03,ANOVA) (Table 2). Therefore, tRES-HESP treatment wouldlikely decrease exposure to increase glucose concentration
in the fasting and postprandial states in the highly over-weight and obese populations.
Concomitant with increased metabolic health there weresmall decreases in BMI and body weight in the obesesubjects with tRES-HESP:20.5 kg/m2 and20.3 kg, respec-tively. Measurement at morning study visits excluded effectof diurnal variation. Other small changes were 3% increasein estimated glomerular filtration rate and 9% decrease inplasma urea with tRES-HESP. Further clinical variables un-changed by tRES-HESP treatment are given in Table 2.
In assessment of vascular function, we found nochange in FMD or GTND. For FMD-to-GTND ratio,normalizing from baseline, in the highly overweight/obesesubject group, the 95% CI for DFMD/DGTND with tRES-HESP was 0.13–2.11. Assessment of markers of vascularinflammation revealed a decrease in change of solubleintercellular adhesion molecule-1 (sICAM-1) from base-line with tRES-HESP in all subjects compared with increasewith placebo:23.66 6.9 vs. 25.86 6.9 ng/mL (P, 0.01), areversal of ;10% of postsupplementation placebo level.
To assess the effect on protein glycation in the HATFFstudy, we analyzed glycation and oxidative damage ofplasma protein. Plasma protein MG-H1 was unchangedwith tRES-HESP treatment (Table 1). We propose thatthis unexpected finding may be due to improved vascularfunction with tRES-HESP treatment, decreasing transca-pillary escape rate of albumin (23) and increasing vascularhalf-life of albumin and thereby maintaining methylglyoxalplasma protein glycation. The increase of transcapillary es-cape rate of albumin in obesity was 36% (24)—a magnitudesimilar to that of the decrease of plasma methylglyoxalherein with tRES-HESP. The oxidative cross-link dityr-osine was decreased 21% with tRES-HESP treatment butnot with placebo (Table 2 and Fig. 4E). To assess thewhole-body formation of MG-H1, we measured the uri-nary excretion of MG-H1 free adduct, corrected for MG-H1 absorbed from food by extrapolating regression ofurinary MG-H1 on urinary pyrraline in all subjects tozero pyrraline (and hence no contribution from food)(25,26) (Fig. 4F). The flux of endogenously generatedMG-H1 adducts was ;13 nmol/mg creatinine at baselineand decreased by 14% with tRES-HESP treatment but notwith placebo (Table 2 and Fig. 4G). The pentose-derivedcross-link, pentosidine, is a quantitatively minor and fluo-rescent glycation adduct. Urinary excretion of pentosidinefree adduct was decreased 32% by treatment with tRES-HESP but not by placebo (Fig. 4H).
We analyzed changes in gene expression of PBMCs in afocused quantitative mRNA array study. In all subjects,there was increased expression of GLO1 and decreasedinflammation-linked genes, IL8 and PTGS2. In obesesubjects, there was also decreased expression of CCL2and TNFA (Table 3).
DISCUSSION
Pharmaceutical doses of two dietary compounds—tRES,found in red grapes, and HESP, closely related to hesperidin,
Table 1—Characteristics of subjects in the HATFF studyat entry
Age (years) 45 6 13
Sex (male/female) 8/21
BMI (kg/m2) 30.0 6 3.8
Overweight/obese 18/11
FPG (mmol/L) 3.93 6 0.57
A1C (mmol/mol) 36.2 6 4.3
A1C (%) 5.5 6 0.7
Prediabetes (no/yes) 20/9
GFR (mL/min) 97 6 17
Systolic BP (mmHg) 133 6 12
Diastolic BP (mmHg) 83 6 10
Hypertension (no/yes) 18/11
Data are mean 6 SD or number of each classification (class1/class 2) unless otherwise indicated; n = 29. Hypertension wasdefined as systolic blood pressure (BP)$140 mmHg or diastolicblood pressure $90 mmHg on 4 occasions. Thirty-two subjectswere recruited at the University Hospitals of Coventry andWarwickshire National Health Service Trust in the period May–July 2014; the last participant left the study December 2014. Atprescreening, assessments made were 2-h plasma glucose inan OGTT and A1C, aspartate transaminase, alanine transami-nase, plasma triglycerides, and plasma creatinine (for estimatedglomerular filtration rate [GFR]). One participant was withdrawnfrom the study for diverging from the protocol, and 2 droppedout: 1 while on placebo and 1 on treatment. The 2 participantswho dropped out stated personal reasons related to interferingduties and therefore difficulties attending the scheduled follow-up appointments. None of the participants reported any relevantside effects (nausea, loss of appetite, gastrointestinal sideeffects, or other symptoms). Study data were analyzed perprotocol (n = 29).
2288 Metabolic and Vascular Health With Glo1 Inducer Diabetes Volume 65, August 2016
Tab
le2—
Improve
men
tofmetab
olic
health
withtR
ES-H
ESPco
form
ulationin
theHATFF
stud
y
Variable
Study
grou
p
Place
bo
tRES-H
ESP
D+tRES-H
ESP
from
:bas
eline
[pos
tsup
plemen
t]
Significa
nceP
from
:bas
eline
[pos
tsup
plem
ent]
ANOVA/
Friedman
Bas
eline
Pos
tsup
plemen
tBas
eline
Pos
tsup
plemen
t
Urin
arypyrraline
(nmol/m
gcrea
tinine)
All
12.1
(6.4–15
.8)
10.6
(7.8–15
.0)
8.8(5.1–13
.4)
8.2(5.1–21
.3)
PBMC
Glo1ac
tivity
(mU/m
gprotein)
All
1,30
06
136
1,15
56
151
1,34
36
156
1,41
46
150
—[259
(22%
)]—
[,0.05
],0.02
Highly
overweigh
t1,34
66
154
1,15
66
185
1,34
56
189
1,46
36
177
—[307
(27%
)]—
[,0.05
],0.02
Obes
e1,45
16
242
1,08
56
253
1,14
06
246
1,41
36
264
—[328
(30%
)]—
[,0.05
]
Plasm
amethy
lglyox
al(nmol/L)
Highly
overweigh
t16
66
2822
86
3016
06
2913
36
18—
[284
(237
%)]
—[,
0.05
]
Plasm
aD-lac
tate
(mmol/L)
All
8.28
60.81
8.56
60.73
7.46
60.77
8.31
60.73
OGIS
(mL$min
21$m
22)
Highly
overweigh
t51
36
2354
16
2150
66
2254
86
2342
(8%
)[—]
,0.02
[—]
,0.05
Obes
e48
86
3252
26
3248
96
2954
76
3058
(12%
)[—]
,0.02
[—]
Fastingplasm
ainsu
lin(pmol/L)
All
39.6
66.3
38.2
65.2
43.1
66.9
36.0
63.2
OGTT
90-m
inplas
ma
insu
lin(pmol/L)
Highly
overweigh
t29
1(142
–48
7)32
6(223
–42
2)30
3(177
–50
8)24
0(110
–45
2)
FPG
(mmol/L)
Highly
overweigh
t3.92
60.12
3.80
60.13
4.02
60.15
3.82
60.16
20.20
(25%
)[—
],0.04
[—]
,0.01
Obes
e3.76
60.11
3.80
60.16
3.92
60.16
3.58
60.16
20.34
(29%
)[—
],0.02
[—]
,0.05
AUCg(m
mol/L
$h)
Highly
overweigh
t11
.06
0.7
10.5
60.6
10.8
60.7
9.96
0.6
20.9(2
8%)[—
]0.05
2[—
],0.03
BMI(kg
/m2)
Obes
e34
.26
0.7
34.3
60.6
34.0
60.7
33.8
60.6
—[2
0.5(2
1%)]
—[,
0.05
],0.02
Bod
yweigh
t(kg)
Obes
e93
.2(84.0–
109.4)
93.6
(84.1–
108.8)
93.8
(83.7–
106.6)
93.3
(83.2–10
7.7)
—[2
0.3(2
0.3%
)]—
[,0.05
]
eGFR
(mL/min)
All
996
210
16
297
62
1006
23(3%
)[—]
,0.03
[—]
,0.02
Plasm
aurea
(mmol/L)
All
4.60
60.19
4.35
60.18
4.67
60.17
4.23
60.15
20.44
(29%
)[—
],0.01
[—]
,0.01
Plasm
aac
etoa
cetate
(mmol/L)
All
61(42–
135)
74(49–
113)
62(48–
127)
77(65–
125)
Plasm
ab-hyd
roxy
butyrate
(mmol/L)
All
27(16–
47)
24(13–
46)
26(18–
49)
22(11–
46)
A1C
(mmol/m
ol)
All
36.7
60.8
36.5
60.8
36.8
60.8
36.5
60.9
A1C
(%)
5.56
0.1
5.56
0.1
5.56
0.1
5.56
0.1
OGTT
2-hgluc
ose(m
mol/L)
All
4.28
60.32
4.20
60.26
4.40
60.29
4.40
60.26
HOMA-IR
(mmol/L
3mU/L)
All
0.75
(0.46–
1.22
)0.68
(0.42–
1.25
)0.85
(0.38–
1.56
)0.69
(0.49–
1.34
)
Con
tinue
don
p.22
90
diabetes.diabetesjournals.org Xue and Associates 2289
Tab
le2—
Continue
d
Variable
Study
grou
p
Place
bo
tRES-H
ESP
D+tRES-H
ESP
from
:bas
eline
[pos
tsup
plemen
t]
Significa
nceP
from
:bas
eline
[pos
tsup
plem
ent]
ANOVA/
Friedman
Bas
eline
Pos
tsup
plemen
tBas
eline
Pos
tsup
plemen
t
Sys
tolic
BP(m
mHg)
All
1316
213
26
313
16
313
36
3
Diastolic
BP(m
mHg)
All
81.7
61.9
82.9
62.3
83.4
62.1
83.3
62.3
Totalc
holesterol
(mmol/L)
All
5.48
60.39
5.72
60.28
5.35
60.31
5.53
60.28
LDL/VLD
Lch
oles
terol(mmol/L)
All
3.93
60.26
4.16
60.23
4.01
60.23
4.06
60.23
HDLch
oles
terol(mmol/L)
All
1.36
60.10
1.39
60.11
1.36
60.10
1.37
60.11
Triglyce
rides
(mmol/L)
All
0.81
06
0.13
20.71
96
0.12
20.65
06
0.11
00.65
56
0.09
2
End
othe
lin-1
(pg/mL)
All
1.66
60.17
1.61
60.14
1.66
60.12
1.64
60.15
CRP(mg/mL)
All
2.32
(0.99–
5.46
)2.37
(0.63–
3.75
)2.23
(0.82–
3.97
)1.74
(0.76–
3.91
)
sE-selec
tin(m
g/mL)
All
39.0
62.9
38.8
63.2
39.2
63.0
38.3
63.0
sVCAM1(ng/mL)
All
4536
1644
96
1343
76
1244
56
14
Cys
tatin
-c(ng/mL)
All
6766
2867
86
2969
26
3269
16
31
Album
in-to-crea
tinineratio
(mgalbum
in/m
mol
crea
tinine)
All
0.25
7(0.171
–0.48
3)0.26
1(0.147
–0.79
0)0.23
1(0.150
–0.46
5)0.26
4(0.159
–0.58
1)
FMD
(mm)
All
0.18
(0.07–
0.49
)0.26
(0.07–
0.47
)0.17
(0.10–
0.35
)0.12
(0.06–
0.31
)
GTN
D(m
m)
All
0.41
(0.36–
0.77
)0.44
(0.28–
0.76
)0.45
(0.37–
0.75
)0.38
(0.29–
0.53
)
Plasm
asICAM-1
All
2576
1328
46
1528
06
1327
66
13,0.01
Plasm
aprotein
MG-H
1(m
mol/m
olArg)
All
0.37
0(0.348
–0.44
7)0.38
0(0.340
–0.42
0)0.35
7(0.330
–0.40
8)0.38
1(0.353
–0.41
6)
Plasm
aprotein
dity
rosine
(mmol/m
olTy
r)All
28.6
62.7
26.6
62.6
29.6
62.9
23.5
61.7
26.1(2
21%
)[—
],0.01
[—]
,0.02
Totalu
rinaryMG-H
1free
adduc
t(nmol/m
gcrea
tinine)
All
20.1
(16.3–
30.6)
22.7
(16.6–
29.3)
19.7
(12.5–
30.4)
19.8
(14.9–
27.4)
End
ogen
ousurinaryMG-H
1free
adduc
t(nmol/m
gcrea
tinine)
All
13.4
62.1
13.5
63.7
13.1
62.8
11.3
63.1
21.8(2
14%
)[—
],0.01
(,0.01
),0.01
Urin
arypen
tosidine
free
adduc
t(pmol/m
gcrea
tinine)
All
10.8
(6.8–20
.2)
12.7
(5.7–23
.6)
10.8
(7.4–21
.8)
7.4(4.3–16
.5)
23.4(2
32%
)[—
],0.05
[—]
Dataaremea
n6
SEM
ormed
ian(lo
wer–up
perq
uartile)u
nles
sothe
rwiseindica
ted.
Foro
bese
,highlyov
erweigh
t/ob
ese,
andallstudy
grou
ps,n
=11
,20,
and29
,res
pectively.
Variables
failing
toac
hiev
eor
approa
chsign
ifica
ncearegive
nin
Sup
plem
entary
Table1.
tRES-H
ESPtrea
tmen
tcha
nges
,D+tRES-H
ESP,a
reab
solute
(perce
ntag
e)ch
ange
sfrom
base
line,
andch
ange
swith
resp
ecttopo
stsu
pplemen
tplace
boco
ntrolare
insq
uare
brac
kets.R
elated
sign
ifica
nceleve
lsarealso
give
nwhe
reP,
0.05
and,
inon
eca
se,b
orde
rline
failure
ofsign
ifica
nce,
P=0.05
2.Th
ere
wereno
tstatistic
ally
sign
ifica
ntdiffe
renc
esindu
cedby
plac
ebo.
BP,bloo
dpres
sure;eG
FP,es
timated
glom
erular
filtrationrate;sE
-selec
tin,so
lubleE-selec
tin.
2290 Metabolic and Vascular Health With Glo1 Inducer Diabetes Volume 65, August 2016
found in oranges—administered together acted synergis-tically to improve FPG, AUCg, OGIS, sICAM-1, arterialfunction, and renal function in highly overweight subjects.Most clinical effects were found in the highly overweightsubgroup (BMI .27.5 kg/m2), indicating that the tRES-HESP coformulation has decreasing potency as the leanrange of BMI is approached. This suggests a resetting togood metabolic and vascular health.
tRES and HESP administered individually in previousstudies were ineffective. From meta-analysis, it wasconcluded that tRES does not affect glycemic status inoverweight and obese human subjects (27). This is at oddswith evidence from rodent models (28) and is likely dueto interspecies differences in pharmacology, host interac-tions, and maximum tolerable dose. HESP absorbed from
clinical dosing with hesperidin did not improve plasmaglucose or insulin resistance (29).
We arrived at the tRES-HESP formulation throughmaximizing induction of Glo1 expression. While increasedGlo1 expression likely contributes to the observed beneficialhealth effects (3), changes in other gene expression occurred—such as induction of antioxidant enzymes and GSHsynthesis (Fig. 3H and I and Supplementary Fig. 1), and theirinterplay may also mediate the overall health benefit achieved.We exploited the regulatory ARE of GLO1 to increase expres-sion (6). We limited the small-molecule inducer screen to non-toxic dietary bioactive compounds of known or suspectedNrf2 activation activity to provide an option for use of Glo1inducers as functional food supplements as well as pharma-ceuticals. tRES and HESP also have a .50-fold safety margin
Figure 4—Outcomes from the HATFF clinical study. A: Correlation of change in OGIS from baseline (DOGIS) with BMI in the tRES-HESPtreatment arm. r = 0.45, P < 0.05 (Pearson); n = 29. B: OGIS in tRES-HESP treatment arm at baseline (BS) and postsupplementation (PS)study visits in highly overweight subjects; n = 20. OGIS was calculated from plasma glucose concentrations at 0, 90, and 120 min andplasma insulin concentrations at 0 and 90 min (12). C: Correlation of change in FPG from baseline with BMI in the tRES-HESP treatmentarm. r = 20.41, P < 0.05 Pearson; n = 29. D: FPG in tRES-HESP treatment arm at baseline and postsupplementation study visits in highlyoverweight; n = 20. E: Dityrosine residues in plasma protein at baseline and postsupplementation study visits; n = 29. F: Regression ofurinary excretion of MG-H1 free adduct on urinary excretion of pyrraline free adduct at baseline of the placebo arm. Regression line: urinaryMG-H1 (nmol/mg creatinine) = (0.592 6 0.180) 3 urinary pyrraline (nmol/mg creatinine) + (13.4 6 2.1); P = 0.003. Total urinary excretion ofMG-H1 free adduct correlated positively with urinary pyrraline for all 4 study visits, r = 0.43 – 0.63, P = 0.019 to<0.001. G: Endogenous fluxof MG-H1 formation at study visits. H: Urinary excretion of pentosidine free adduct at study visits. Significance: *P < 0.05 and **P < 0.01.B, D, E, and G: paired t test; H: Wilcoxon signed rank test.
Table 3—Summary of change in gene expression of PBMC with tRES-HESP coformulation in the HATFF study
Study group n
Genes
Increased Decreased
All 29 GLO1 (6%) HIF1A (26%), IL8 (239%), and PTGS2 (230%)
at doses used in the HATFF study (19,20). Activation of Nrf2by dietary bioactive compounds is mostly studied throughARE-linked induction of NQO1 or HMOX1 expression.Small-molecule activators of Nrf2 increase expression of dif-ferent ARE-linked gene subsets (6,7)—likely due to theability of Nrf2 activators to recruit the requisite accessoryproteins and increase nuclear concentration of functionallyactive Nrf2 to the level required for increased expression ofthe ARE-linked gene of interest (7,8). A specific functionalscreen for GLO1-ARE transcriptional activation was there-fore required.
Activation of Nrf2 by tRES has been studied previously byinduction of HMOX1 expression (30). Herein we found thattRES also induces expression of Glo1 with high Emax. Ourrecent studies (7,8) and those of others (31) suggest that thisis achieved by preventing nuclear acetylation and inactivationof Nrf2 via increasing in situ activity of sirtuin-1. At lowtRES concentrations, this occurs through inhibition ofcAMP phosphodiesterases, activation of AMPK, andincreased NAD+. HESP may also synergize for increasedactivity of sirtuin-1 through activation of AMPK by theprotein kinase A pathway (32,33).
Clinically achievable concentrations of tRES at highlytolerable doses are lower than the EC50 for induction ofGlo1 expression, however, so synergism with HESP is re-quired to achieve increased Glo1 expression in clinicaltranslation. HESP may activate Nrf2 through inductionand activation of protein kinase A, upstream of fyn ki-nase, which drives Nrf2 translocational oscillations andARE-linked gene expression (8,33). HESP is a partialagonist (Fig. 2B), which is likely due to inhibitory nuclearacetylation of Nrf2 blocking a high Emax. Combinationwith tRES and HESP provides for faster nuclear translo-cation and decreased inactivation of Nrf2 (7,8,31). Use ofHESP rather than related dietary glycoside hesperidinfound in citrus fruits (34) is likely crucial: HESP has;70-fold greater potency in Nrf2 activation and higherbioavailability than hesperidin (35).
tRES-HESP increased OGIS to levels typical of healthy, leansubjects. The magnitude ofDOGIS, 42–58 mL $min21 $m22,is comparable to that achieved with pharmaceutical treatmentof patients with type 2 diabetes (for example, 1.7 g metforminper day, DOGIS = increase of 54 mL $ min21 $ m22) (36) andextreme weight loss with gastric band surgery in morbid
Figure 5—Proposed mechanism of action of Glo1 inducer formulation. Yellow-filled arrows, mechanism of health improvement; red-filledarrows, damaging processes suppressed. See also 40,42–44. CBP, CREB-binding protein; FGFR1c, fibroblast growth factor receptor 1c;KLB, b-Klotho; Maf, small Maf protein, an accessory protein for Nrf2 activation.
2292 Metabolic and Vascular Health With Glo1 Inducer Diabetes Volume 65, August 2016
obesity (DOGIS = increase of 62 mL $ min21 $ m22) (37).These effects suggest the tRES-HESP can support thera-peutic improvement of insulin sensitivity in highly over-weight populations. OGIS was initially proposed as amarker of insulin resistance but is also improved withincreased b-cell sensitivity to glucose and decreasedglucose absorption (38). Regarding insulin resistance, de-creased activity of FGF21 due to downregulation of theFGF21 receptor cofactor b-Klotho may be involved (39).Methylglyoxal-driven protein glycation decreased expres-sion of b-Klotho (40). By inducing Glo1 expression anddecreasing methylglyoxal protein glycation, therefore, welikely corrected the functional deficit of b-Klotho andreengaged FGF21. This explains the resetting of insulinsensitivity to normal levels with the response greaterfor higher-BMI subject groups. Characteristics of in-creased b-Klotho was its blocking of inflammatory sig-naling to downregulate proinflammatory mediators IL8,MCP1, ICAM-1, and RAGE (40) and, via decreasingMCP-1, also suppressing of PTGS2 (41) (Fig. 5). Allfeatures of this transcriptional signature were foundin PBMCs of the HATFF study. Other effects increasingOGIS may be increased pancreatic b-cell sensitivity toglucose (42), decreased intestinal absorption of glucose(43), changes of gut microbiota, and decreased break-down and absorption of starch (44) (Fig. 5).
The 5% decrease in FPG herein exceeds and matcheseffects of metformin and Olristat, respectively, in similarintervention trials in overweight/obese subjects (45,46).Decrease in FPG in the normal range is associated withreduced risk of developing type 2 diabetes (47).
Decreased urinary excretion of pentosidine by tRES-HESP may be linked to decreased oxidative stress anddecreased pentose precursors expected from improvedinsulin resistance (48).
tRES-HESP treatment produced an increase in DFMD/DGTND. The effect is likely produced by improved nitricoxide responsiveness in both endothelium and smoothmuscle cells (13) related to induction of Glo1 and pre-vention of methylglyoxal glycation–driven impairment ofendothelial nitric oxide synthase (49). tRES-HESP also de-creased dityrosine content of plasma protein. Dityrosine isan oxidative cross-link of tyrosine residues and is a dom-inant cross-link of the extracellular matrix (50). Plasmaprotein dityrosine may be a surrogate marker of this andhence be reporting decreased dityrosine cross-linking,which may contribute to improved arterial function.
tRES-HESP decreased sICAM-1 in the HATFF study.The cell studies herein suggest that this is likely linked todecreased ICAM-1 expression. ICAM-1 expression wasdecreased in Glo1 transgenic rats (51). In clinical studies,tRES and HESP individually did not decrease ICAM1(52,53). sICAM-1 correlates with atherosclerosis burdenassessed by coronary artery calcification and is a risk pre-dictor of cardiovascular disease (54).
In summary, we present evidence that pharmaceuticaldoses of tRES and HESP coformulation produce improved
metabolic and vascular health in overweight and obesesubjects.
Acknowledgments. The authors thank Louise Goodbody, supportingresearch nurse, and Louise Halder, research dietician, in the HATFF study. A.S.thanks Taif University, the Ministry of Education, Government of Saudi Arabia, fora PhD studentship.Duality of Interest. This research was mainly funded by Unilever andInnovate UK (project no. 101129). D.M., M.F., and G.J. are employees of Unilever.No other potential conflicts of interest relevant to this article were reported.Author Contributions. M.X. performed screening, validation, and mostclinical chemistry analysis. M.O.W. was clinical lead and codesigned andanalyzed data of the HATFF study. S.Q. performed the clinical procedures. N.-B.K.was the HATFF study statistician. A.A. and A.S. performed some clinicalchemistry analysis. M.W. was the principal HATFF study research nurse. D.M.and M.F. participated in quarterly project steering meetings. G.J. participated inquarterly project steering meetings and raised funding for the study. N.R. wasstudy coordinator, codesigned the study, and performed some metaboliteanalysis. P.J.T. designed and led the study, raised funding for the study,performed some metabolite analysis, analyzed data, and wrote the manuscript.All authors read and approved the manuscript. P.J.T. is the guarantor of this workand, as such, had full access to all the data in the study and takes responsibilityfor the integrity of the data and the accuracy of the data analysis.
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2294 Metabolic and Vascular Health With Glo1 Inducer Diabetes Volume 65, August 2016
