Synergistic effects of metformin, resveratrol, and hydroxymethylbutyrate on insulin sensitivity

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1 Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy Open Access Full Text Article open ccess to scientific nd medicl reserch Originl Reserch Synergistic effects of metformin, resvertrol, nd hydroxymethylutyrte on insulin sensitivity Antje Bruckuer 1 Michel B Zemel 1,2 1 NuSirt Sciences Inc, 2 Deprtment of Nutrition, University of Tennessee, Knoxville, TN, USA Correspondence: Michel B Zemel 112 Solwy School Rd, Knoxville, TN 37931, USA Tel Emil mzemel@nusirt.com Bckground: The purpose of this study ws to determine whether mixture of the polyphenol, resvertrol, nd the leucine metolite, hydroxymethylutyrte (HMB), cts synergisticlly with low doses of metformin to impct insulin sensitivity nd AMP-ctivted protein kinsedependent outcomes in cell culture nd in dietic mice. Methods: C2C12 skeletl myotues nd 3T3-L1 dipocytes were treted with resvertrol.2 µm, HMB 5 µm, nd metformin.1 mm lone or in comintion. d/d mice were treted for 2 weeks with high (1.5 g/kg diet), low (.75 g/kg diet), or very low (.25 g/kg diet) doses of metformin lone or in comintion with diet contining resvertrol 12.5 mg nd CHMB 2 g/kg. Results: The comintion of metformin-resvertrol-hmb significntly incresed ft oxidtion, AMP-ctivted protein kinse, nd Sirt1 ctivity in muscle cells compred with metformin or resvertrol-hmb lone. A similr trend ws found in 3T3L1 dipocytes. In mice, the two lower doses of metformin exerted no independent effect ut, when comined with resvertrol- HMB, oth low-dose nd very low-dose metformin improved insulin sensitivity (HOMA IR ), plsm insulin levels, nd insulin tolernce test response to level comprle with tht found for high-dose metformin. In ddition, the metformin-resvertrol-hmb comintion decresed viscerl ft nd liver weight in mice. Conclusion: Resvertrol-HMB comined with metformin my ct synergisticlly on AMPctivted protein kinse-dependent pthwys, leding to incresed insulin sensitivity, which my reduce the therpeutic doses of metformin necessry in the tretment of dietes. Keywords: dietes, AMP-ctivted protein kinse, Sirt1, ft oxidtion Introduction The rpid growth in prevlence of type 2 dietes mellitus is minly ttriutle to chnges in lifestyle nd diet, nd hs een concomitnt with n increse in oesity. 1 It is lso recognized s one of the mjor cuses of other chronic moridities, such s crdiovsculr nd kidney disese, nd of erly mortlity, thus posing serious pulic helth prolem. 2 Therefore, identifiction of sfer pproches to improve insulin sensitivity is importnt s mens to control dietes nd to improve the efficcy of lifestyle pproches for the reduction of oesity nd risk of dietes. Metformin, igunide, either lone or s prt of comintion therpy, is the drug of choice for orl tretment of dietes, prticulrly in overweight nd oese people. 3 Its min ction is to lower lood glucose levels y inhiiting heptic gluconeogenesis s well s incresing insulin sensitivity. 3,7 Metformin cts, in prt, vi ctivtion of AMPctivted protein kinse (AMPK), therey stimulting oxidtion of ft in muscle. 4 6 sumit your mnuscript Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213: Bruckuer nd Zemel, pulisher nd licensee Dove Medicl Press Ltd. This is n Open Access rticle which permits unrestricted noncommercil use, provided the originl work is properly cited.

2 Bruckuer nd Zemel A dily orl dose of 2 25 mg/dy is typiclly required for optiml effect 8 nd orl iovilility is dose-dependent, with decresed iovilility t higher doses, suggesting n ctive, sturle sorption process. 9 Although severe dverse effects re rre, up to 3% of ptients report gstrointestinl symptoms, including dirrhe, crmps, nuse, nd vomiting, which cn cuse severe discomfort nd led to discontinution of the drug. 1 Therefore, finding strtegies to reduce the dose of metformin required without compromising its efficcy is useful pproch for mnging nd reducing dverse events. AMPK is key regultor of cell nd whole ody energy metolism. Increses in the AMP/ATP rtio ctivte AMPK, resulting in incresed glucose uptke nd shift from nolic to ctolic ATP-producing pthwys, 11 including ft oxidtion. AMP inding results in conformtionl chnge which promotes Thr 172 phosphoryltion y upstrem kinses, including tumor suppressor liver kinse B1 nd the C 2+ /clmodulin-dependent kinse (CmKKβ) nd prevents its dephosphoryltion y protein phosphtses. 12 When purified recominnt AMPK ctivity from cteril cell lystes ws mesured, AMP stimulted the ctivity of AMPK y up to 1-fold nd upstrem kinses y up to 1-fold, nd the comintion of AMP with the upstrem kinses y up to 1-fold. 13 In ddition, there is idirectionl interction etween the sirtuin, Sirt1, nd AMPK, leding to mutul ctivtion. 14,15 The sirtuins, Sirt1 nd Sirt3, re lso well known regultors of glucose nd lipid metolism. Their ctivtion converges on the sme pthwys ctivted y AMPK 16 nd results in incresed mitochondril iogenesis nd ftty cid oxidtion. Becuse lipid nd glucose metolism is dysregulted in type 2 dietes mellitus, AMPK nd sirtuin modultors hve een suggested to e promising therpies. We hve recently demonstrted tht nutritionl mixture of resvertrol, nturlly occurring polyphenol found in the skin of red grpes, nd either leucine or HMB, nturlly occurring metolite of leucine, produces profound improvement in insulin sensitivity, muscle glucose, nd plmitte uptke, s well s reduction in inflmmtory nd oxidtive stress in mice with diet-induced oesity; notly, these effects occurred t concentrtions tht were without effect when the compounds were provided independently of ech other. 17 These effects re medited y ctivtion of AMPK, Sirt1, nd Sirt3. 17 Thus, they converge upon the sme metolic pthwys ctivted y metformin. Consequently, this study ws designed to determine whether lend of resvertrol-hmb would ct synergisticlly with metformin to control dietes in dietic mice. The intention ws to develop formultion tht uses very low levels of metformin in comintion with the resvertrol-hmb lend in order to lower the effective dose of metformin required for mngement of dietes. Mterils nd methods Cell culture 3T3-L1 predipocytes were incuted t density of 8 cells/cm 2 (1 cm 2 dish) nd grown in the sence of insulin in Dulecco s modified Egle s medium (25 mm glucose) contining 1% fetl ovine serum nd 1% penicillin-streptomycin (dipocyte medium) t 37 C in 5% CO 2 tmosphere. Confluent predipocytes were induced to differentite using stndrd differentition medium (DM2- L1, Zen-Bio Inc, Reserch Tringle Prk, NC, USA). The predipocytes were mintined in this differentition medium for 3 dys nd susequently cultured in dipocyte medium for further 8 1 dys to llow t lest 9% of cells to rech full differentition efore tretment. The medium ws chnged every 2 3 dys, nd differentition ws determined microscopiclly s inclusion of ft droplets. C2C12 muscle cells were incuted t density of 8 cells/cm 2 (1 cm 2 dish) nd grown in Dulecco s modified Egle s medium contining 1% fetl ovine serum nd ntiiotics t 37 C in 5% CO 2 tmosphere. Cells were grown to 1% confluence, trnsferred into differentition medium (Dulecco s modified Egle s medium with 2% horse serum nd 1% penicillin-streptomycin), nd fed with fresh differentition medium every dy until myotues were fully formed (6 dys). Tretment concentrtions for ll cell experiments were 2 nm resvertrol (Sigm-Aldrich, St Louis, MO, USA), 5 µm HMB (PureBulk Inc, Roseurg, OR, USA), nd.1 nm or 1 mm metformin (Sigm-Aldrich) s indicted. The incution time ws 4 48 hours, depending on the experiment. Ftty cid oxidtion Cellulr oxygen consumption ws mesured using n XF24 nlyzer (Sehorse Bioscience, Billeric, MA, USA) in 24-well pltes t 37 C, s descried y Feige et l, 18 with slight modifictions. Cells were seeded t 4, cells per well, differentited s descried ove, treted for 24 hours with the indicted tretments, wshed twice with nonuffered cronte-free ph 7.4 low glucose (2.5 mm) Dulecco s modified Egle s medium contining crnitine.5 mm, equilirted with 55 µl of the sme medium in non-co 2 incutor for 3 minutes, nd then inserted into the instrument for 15 minutes of further equilirtion. 94 sumit your mnuscript Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6

3 O 2 consumption ws mesured in three successive seline mesures t five-minute intervls prior to injection of plmitte (2 µm finl concentrtion). Post-plmitte injection mesurements were tken over 4-hour period with cycles consisting of 1-minute rek nd four successive mesurements of O 2 consumption. Sirt1 ctivity Sirt1 ctivity ws mesured using the Sirt1 fluorimetric drug discovery kit (BML-AK555, ENZO Life Sciences Interntionl Inc, Plymouth Meeting, PA, USA). The sensitivity nd specificity of this ssy kit hs een tested y Nin et l. 19 Using this ssy, Sirt1 ctivity is ssessed y the degree of decetyltion of stndrdized sustrte contining n cetylted lysine side chin. The sustrte utilized is peptide contining mino cids of humn p53 (Arg-His- Lys-Lys[Ac]), n estlished trget of Sirt1 ctivity; Sirt1 ctivity is directly proportionl to the degree of decetyltion of Lys-382. Smples were incuted with peptide sustrte (25 µm) nd NAD + (5 µm) in phosphte-uffered solution t 37 C on horizontl shker for 45 minutes. The rection ws stopped y ddition of 2 mm nicotinmide nd developing solution tht inds to the decetylted lysine to form fluorophore. Following 1 minutes of incution t 37 C, fluorescence ws red in plte-reding fluorometer with excittion nd emission wvelengths of 36 nm nd 45 nm, respectively. Resvertrol 1 mm served s Sirt1 ctivtor (positive control) nd surmin sodium 25 mm s Sirt1 inhiitor (negtive control). Endogenous Sirt1 ctivity in muscle cells nd mouse white dipose tissue ws mesured using modified ssy with 5 µl of cell lyste. The lystes were prepred y homogenizing cells in ice-cold RIPA uffer with protese inhiitor mix (MP Biomedicls LLC, Solon, OH, USA). After 1 minutes of incution on ice, the homogente ws centrifuged t 14, g nd the superntnt ws used for further experiments. Dt for endogenous Sirt1 ctivtion were normlized to cellulr protein concentrtion mesured using icinchoninic cid ssy. AMPK ctivity AMPK ctivity in cells ws mesured using n AMPK ssy kit (CycLex Co, Ltd, Ngno, Jpn) ccording to the mnufcturer s instructions. This ssy provides nonisotopic, sensitive, nd specific method in the form of n enzymelinked immunosorent ssy nd uses nti-phospho-mouse IRS-1 S789 monoclonl ntiody nd peroxidse-coupled nti-mouse IgG ntiody s reporter molecule. The mount of phosphorylted sustrte is determined y mesuring Metformin, resvertrol, HMB, nd insulin sensitivity sornce t 45 nm. Differentited cells were incuted with the tretments indicted for 24 hours. The cells were wshed three times with ice-cold phosphte-uffered solution, lysed in cell lysis uffer for 9 minutes on ice, nd centrifuged t 35 rpm for 15 minutes t 4 C. Next, 1 µl of cler superntnt ws used for ech ssy. Purified recominnt AMPK ctive enzyme ws included s positive control for phosphoryltion, nd.5 mm AICAR, potent AMPK ctivtor, ws included in some experiments s n dditionl positive control. To clculte the reltive AMPK ctivity of the smples, n inhiitor control with compound C for ech smple ws included once, nd inhiitor control sornce vlues were sutrcted from the test smple sornce vlues. Ech experiment ws ssyed in triplicte from four independent cell replictes. Western lotting The ntiodies P-AMPKα (Thr-172) nd AMPKα were otined from Cell Signling Technology Inc (Dnvers, MA, USA) nd EMD Millipore (Billeric, MA, USA), respectively. 3T3L1 dipocytes were treted s indicted, nd the cells were wshed with ice-cold phosphte-uffered solution, then lysed with ice-cold RIPA uffer nd protese nd phosphtse inhiitor cocktils (Sigm-Aldrich), centrifuged t 14, g t 4 C for 1 minutes, nd the resulting superntnt ws used for experiments. Protein ws mesured using icinchoninic cid kit (Thermo Scientific Inc, Wlthm, MA, USA). For Western lotting, equl mounts of protein per smple (1 µg) were resolved on 1% grdient polycrylmide gels (Criterion precst gel, Bio-Rd Lortories, Hercules, CA, USA), trnsferred to polyvinylidene difluoride memrnes, incuted in locking uffer (3% ovine serum lumin in Tris-uffered sline), then incuted with primry ntiody (1:1) overnight, wshed, nd incuted with secondry horserdish peroxidse-conjugted ntiody (1:1.) for one hour. Visuliztion nd chemiluminescent detection ws done using BioRd ChemiDoc instrumenttion nd softwre (Bio-Rd Lortories), nd nd intensity ws ssessed using Imge L 4. (Bio-Rd Lortories), with correction for ckground nd loding controls. Animls nd diets Dietic 8 1-week-old mle d/d mice (C57BLKS/Jlepr d /lepr d ) were purchsed from Hrln Lortories (Indinpolis, IA). After cclimtiztion to their environment for 5 7 dys, they were rndomized into six different dietry groups with 1 nimls per group nd kept on their diet for 2 weeks. The metformin concentrtions in the diets were Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6 sumit your mnuscript 95

4 Bruckuer nd Zemel sed on literture vlues of high (3 mg/kg ody weight), low (15 mg/kg ody weight), nd very low (5 mg/kg ody weight) metformin studies in mice, 2 23 sed on verge food consumption (8 g/dy) nd verge ody weight (4 g or.4 kg) for diet-induced mice; this ws clculted to e equivlent to 1.5 g,.75 g, nd.25 g metformin/kg diet, respectively. The dietry groups were either the stndrd diet (AIN 93G) only (control group), or the stndrd diet comined with one of the three different concentrtions of metformin (high [3 mg/kg ody weight], low [15 mg/kg ody weight], or very low metformin group [5 mg/kg ody weight]); the two lowest doses of metformin were provided with or without mixture of resvertrol (12.5 mg/kg diet) nd CHMB 2 g/kg diet (low-dose metformin + resvertrol- HMB nd very low-dose metformin + resvertrol-hmb groups, respectively). The nimls were housed in polypropylene cges t room temperture of 22 C ± 2 C nd on 12-hour light/drk cycle. The nimls hd free ccess to wter nd food throughout the experiment, nd food intke ws not mesured. Body weight ws mesured t dys, 7, nd 14 of the study period. At the end of the 14-dy tretment period, ll nimls were fsted overnight nd humnely euthnized with isoflurne overdose nd cervicl disloction. Blood nd tissues were collected for further experiments s descried elow. Liver nd viscerl dipose tissue mss ws weighed immeditely fter removl. The University of Tennessee is ccredited y the Americn Assocition for Accredittion of Lortory Animl Cre. This study nd ll niml procedures were performed under the uspices of protocol pproved y the Institutionl Animl Cre nd Use Committee nd in ccordnce with Pulic Helth Service policy nd recommendtions of the guide. HOMA IR The homeostsis model ssessment of insulin resistnce (HOMA IR ) ws used s screening index for chnges in insulin sensitivity. HOMA IR is clculted vi stndrd formul from fsting plsm insulin nd glucose s follows: HOMA IR = [Insulin (µu/ml) glucose (mm)]/22.5. Plsm glucose nd insulin concentrtions were mesured using glucose ssy kit from Cymn (Ann Aror, MI, USA) nd n insulin kit from Millipore, respectively. Insulin tolernce test Insulin tolernce tests were performed t 2 pm on dy 7. The mice were injected intrperitonelly with insulin (.75 U/kg ody weight) in pproximtely.1 ml of.9% NCl. A 5 µl drop of lood ws tken from the cut til vein efore injection of insulin nd fter 15, 3, 45, nd 6 minutes for determintion of lood glucose. Chnge in lood glucose over the liner portion of the response curve ws then clculted. Sttisticl nlysis All dt re expressed s the men ± stndrd error of the men. Dt were nlyzed y one-wy nlysis of vrince, nd significntly different group mens (P,.5) were seprted y the lest significnt difference test using GrphPd Prism (GrphPd Softwre Inc, L Joll, CA, USA). Results Plmitte oxidtion, s mesured y plmitte-stimulted oxygen consumption rte, ws significntly incresed y oth metformin nd the resvertrol-hmb comintion in muscle cells (P,.2, Figure 1), while the comintion of metformin nd resvertrol-hmb showed 3% greter effect compred with either metformin or resvertrol-hmb lone (P,.4, Figure 1A). A similr pttern ws seen in dipocytes (Figure 1B), where the comintion of HMB with either resvertrol or metformin incresed the re under the concentrtion-time curve y out 1%. The comintion of metformin nd resvertrol-hmb enhnced this effect y further 16%. However, in contrst with muscle cells, metformin lone did not show ny effects in dipocytes. Consistent with these oservtions, AMPK ctivity in C2C12 ws significntly incresed y the comintion of metformin nd resvertrol-hmb (P,.1, Figure 2B), ut not y low-dose metformin or HMB lone or y metformin- HMB in the sence of resvertrol (Figure 2A). We lso ssessed AMPK ctivtion y mesuring phosphoryltion t Thr-172. Consistent with our ctivity mesurements, lowdose (.1 mm) metformin exerted no independent effect on P-AMPK, ut when low-dose metformin ws comined with either HMB or resvertrol-hmb comintion, it produced n increse of out 4%, similr to tht found with resvertrol-hmb (Figure 2D nd E, P,.1). Expressing the dt s n P-AMPK/AMPK rtio produced similr results (Figure 2F). In ddition, Sirt1 ctivity ws ugmented to significnt extent y the comintion of metformin nd HMB compred with either tretment lone (Figure 2C, P,.2). Bsed on these in vitro dt, we investigted the synergistic effects of low-dose metformin nd resvertrol-hmb on insulin sensitivity in dietic mice. Consistent with previous 96 sumit your mnuscript Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6

5 Metformin, resvertrol, HMB, nd insulin sensitivity A B OCR 188 A nd C C2C12 CTRL.1 mm MET RESV/HMB MET/HMB MET/R/HMB OCR 164 A nd C T3L OCR (%) OCR (%) AUC OCR (%) Time (min) c AUC OCR (%) Time (min) 5 5 CTRL.1MM MET RESV/HMB MET/R/HMB CTRL.1MM MET RESV/HMB MET/HMB MET/R/HMB Figure 1 Synergistic effects of metformin nd resvertrol-hmb on ftty cid oxidtion in C2C12 muscle cells nd 3T3L1 dipocytes. Differentited C2C12 muscle cells (A) nd 3T3L1 dipocytes (B) were treted with the tretments indicted for 24 hours. Notes: OCR ws mesured fter plmitte injection (point A nd C) nd represented s percentge of seline. The AUC is clculted y sutrcting the strting rte (third seline point) from ech verge rte point. Dt re expressed s the men ± stndrd devition (n = 4). Brs with the sme letter superscripts re not significntly different from ech other; rs with nonmtching letter superscripts indicte significnt differences etween groups (P,.4). Arevitions: AUC, re under curve; CTRL, control; HMB, hydroxymethylutyrte; MET, metformin; OCR, oxygen consumption rte; RESV, resvertrol. reports in the literture, 21,24,25 high-dose metformin reduced plsm insulin y 27% (P,.2, Figure 3A) nd HOMA IR y 35% (P,.25, Figure 3B), lthough no chnge ws found in plsm glucose levels in these highly insulin-resistnt mice (Tle 1). In contrst, low-dose metformin nd very low-dose metformin exerted no significnt independent effects, while comining either the low-dose or very low-dose of metformin with resvertrol nd HMB resulted in significnt decreses in plsm insulin from 62 µu/ml to 43 µu/ml (P,.2, Figure 3A), comprle with tht seen with high-dose metformin. Moreover, there ws no significnt difference etween the low-dose metformin-hmb comintion versus the very low-dose metformin-hmb comintion. Consistent with this oservtion, the HOMA IR index decresed from 29 units on the control diet to 19 on the low-dose metforminresvertrol-hmb lend nd to 16 units on the very low-dose metformin-resvertrol-hmb lend (P,.25, Figure 3B), reflecting n improvement in insulin sensitivity comprle with tht found with high-dose metformin. Similr results were found for the insulin tolernce test (Figure 4). Animls on the control, low-dose, or very low-dose of metformin showed miniml chnges in lood glucose in response to insulin chllenge. In contrst, those on the stndrd metformin dose nd those on either low-dose or very low-dose metformin comined with resvertrol-hmb showed decreses in lood glucose of out 6 mg/dl over the 3-minute liner portion of the response curve (P,.2). Moreover, the metforminresvertrol-hmb comintion reduced viscerl diposity (Tle 1). Animls on the control diet hd men viscerl ft mss of 4.5 g, nd this ws not ffected y metformin t ny dose in the sence of resvertrol-hmb. Low-dose metformin comined with resvertrol-hmb nd very low-dose metformin comined with resvertrol-hmb reduced viscerl ft y out 2%, to 3.8 nd 3.6 g, respectively (P,.3). These tretments lso reduced liver mss, from 2.78 g (control) to 2.35 g nd 2.41 g, respectively (P,.5), while no effect on ody weight ws seen (Tle 1). Discussion Our results demonstrte potentition of the effect of the ntidietic drug, metformin, using mixture of resvertrol nd HMB, on ft oxidtion nd insulin sensitivity in vitro Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6 sumit your mnuscript 97

6 Bruckuer nd Zemel A AMPK ctivity in C2C12 D P-AMPK Asornce/mg protein MET HMB Met-HMB Bnd intensity MET (.1 mm) c c c c RESV/HMB MET/HMB MET/R/HMB AICAR (1 mm) B AMPK ctivity in C2C12 E C Asornce/mg protein MET MET/RESV/Leu MET/RESV/HMB Sirt1 ctivity in 3T3L1 F P-AMPK AMPK control Met R/HMB Met/HMB Met/R/HMB AICAR P-AMPK/AMPK AFU/ ug protein 2 c Rtio c c c c MET HMB MET-HMB MET (.1 mm) RESV/HMB MET/HMB MET/R/HMB AICAR (1 mm) Figure 2 Synergistic effects of metformin nd resvertrol-hmb on AMPK nd Sirt1 ctivity in C2C12 muscle cells nd 3T3L1 dipocytes. Differentited muscle cells nd dipocytes were treted with the tretments indicted. (A nd B) AMPK ctivity in C2C12 cell lystes. (C) Sirt1 ctivity in 3T3L1 cell lystes. (D) Quntittive nd nlysis of AMPK phosphoryltion in 3T3L1 dipocytes. (E) Western lots of AMPK in 3T3L1 cell lystes using nti-phospho-ampk-α (Thr172) nd nti-ampkα ntiodies. AICAR ws used s positive control. (F) P-AMPK/AMPK rtio in 3T3L1 dipocytes. Notes: Dt re expressed s the men ± stndrd error of the men (n = 4). Brs with the sme letter superscripts re not significntly different from ech other; rs with nonmtching letter superscripts indicte significnt differences etween groups (P,.5). Arevitions: AFU, ritrry fluorescent units; AMPK, AMP-ctivted protein kinse; HMB, hydroxymethylutyrte; MET, metformin; RESV, resvertrol. nd in vivo. These effects were found with concentrtions of ech compound tht were too low to exert significnt independent effects, demonstrting synergistic ction. We hve recently shown tht the mino cid leucine, s well s its metolite HMB, cts synergisticlly with the polyphenol, resvertrol, on insulin sensitivity nd ft metolism. 17 These effects were medited y ctivtion of AMPK, Sirt1, nd Sirt3 in muscle cells nd dipocytes. Therefore, we hypothesized tht the ddition of nother ctivtor of this pthwy my ct synergisticlly nd my e le to enhnce this effect. Indeed, consistent with our previous dt, the comintion of resvertrol with HMB enhnced ftty cid oxidtion y out 1% in dipocytes nd y out 16% in muscle when mesured over 2-hour period in this study. However, ddition of low-dose metformin, which hd little or no effect y itself, resulted in further significnt increse of 98 sumit your mnuscript Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6

7 Metformin, resvertrol, HMB, nd insulin sensitivity A B Std MET Insulin (µu/ml) Low MET HOMA this effect; these effects were ccompnied y corresponding increses in AMPK nd Sirt1 ctivity. Consistent with our previous oservtions, 17 leucine comined with resvertrol ws le to exert effects similr to those of the resvertrol-hmb comintion; however, in comintion with metformin, HMB ws superior (dt not shown). Although no significnt difference etween the metformin-hmb nd metformin-resvertrol-hmb comintions ws detected in dipocytes, there ws n enhnced effect of metformin-resvertrol-hmb in muscle cells. For this reson, we conducted our niml study using only the resvertrol-hmb comintion, nd found tht this Very low MET Low MET+RESV/HMB Very low MET+RESV/HMB Figure 3 Synergistic effects of metformin nd resvertrol-hmb on insulin sensitivity in mice. Effects of stndrd dose, low-dose, nd very low-dose of metformin compred with low-dose metformin-resvertrol-chmb nd with very low-dose metformin-resvertrol-chmb on (A) plsm insulin nd (B) HOMA IR in d/d mice. Notes: Dt re expressed s the men ± stndrd error of the men (n = 1). Brs with the sme letter superscripts re not significntly different from ech other; rs with nonmtching letter superscripts indicte significnt differences etween groups (P,.25). Arevitions: HOMA, homeosttic ssessment of insulin resistnce; HMB, hydroxymethylutyrte; MET, metformin; RESV, resvertrol minute glucose chnge (mg/dl) Std met Low MET comintion significntly potentited the effects of lowdose metformin on insulin sensitivity, resulting in efficcy comprle with tht of tretment with higher doses of metformin. Although men plsm glucose ws highest in the very low-dose metformin-resvertrol-hmb group, it ws not significntly different from tht in the other groups, while the insulin concentrtion nd clculted HOMA IR were significntly lower, indicting n improvement in insulin sensitivity. Further, consistent with our previous results in diet-induced oesity in mice, 17 comintion with resvertrol- HMB reduced viscerl ft mss nd liver weight, while no effect on these outcomes were found using metformin lone. Becuse the numer of mice used in the niml study ws limited, we did not include resvertrol-hmb group. Therefore, we cnnot rule out tht these effects were cused Very low MET Low MET+RESV/HMB Very low MET+RESV/HMB Figure 4 Synergistic effects of metformin nd resvertrol-hmb on insulin tolernce test in mice. Notes: Effects of stndrd dose, low-dose, nd very low-dose metformin compred with low-dose metformin-resvertrol-chmb nd with very low-dose metforminresvertrol-chmb on 3-minute plsm glucose response to insulin (.75 U/kg ody weight) in d/d mice. Dt re expressed s the men ± stndrd error of the men (n = 1). Brs with the sme letter superscripts re not significntly different from ech other; rs with nonmtching letter superscripts indicte significnt differences etween groups (P,.2). Arevitions: HMB, hydroxymethylutyrte; Met, metformin; RESV, resvertrol. Tle 1 Effects of indicted tretments on ody weight, viscerl ft mss, liver weight, plsm glucose, nd food intke in mice Stndrd metformin Low-dose metformin Very low-dose metformin Low-dose metformin + resvertrol-hmb Very low-dose metformin + resvertrol-hmb Body weight (g) (strt) 45. ± ± ± ± ± ±.6 Body weight (g) (end) 46.8 ± ± ± ± ± ± 1. Plsm glucose (mg/dl) ± ± ± ± ± ± 18.6 Viscerl ft (g) 4.5 ± ± ± ± ± ±.23 Liver (g) 2.78 ± ± ± ± ± ±.8 Notes: d/d mice were fed the indicted diets for 2 weeks. Dt re expressed s the men ± stndrd error of the men (n = 1). Vlues with the sme letter superscripts in ech row re not significntly different from ech other; vlues with nonmtching letter superscripts in ech row indicte significnt differences etween groups (P,.3). Arevition: HMB, hydroxymethylutyrte. Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6 sumit your mnuscript 99

8 Bruckuer nd Zemel simply y resvertrol-hmb tretment lone. However, the demonstrtion of synergy in the in vitro studies of myotues suggests tht low-dose metformin cts synergisticlly with resvertrol-hmb in vivo s well. The primry ction of metformin is to lower lood glucose concentrtion y inhiiting heptic glucose production nd stimulting glucose disposl in skeletl muscle. 3 Metformin lso ctivtes AMPK, lthough it is not cler whether this is y direct or indirect stimultion. 26 It hs een postulted tht inhiition of mitochondril respirtion t complex 1 resulting in decresed ATP production is responsile for AMPK ctivtion, 5,27 ut this model would e contrry to the finding of incresed ftty cid oxidtion in muscle with metformin. Metformin lso cts vi inhiition of AMP deminse, therey inhiiting AMP ctolism nd incresing the cellulr AMP/ATP rtio, resulting in phosphoryltion nd ctivtion of AMPK. 28 In ddition, metformin my interct directly with the γ-suunit of the AMPK complex, producing structurl chnge tht promotes phosphoryltion nd ctivtion y upstrem kinses. 6 Resvertrol hs lso een shown to ctivte AMPK y two mechnisms, ie, high doses of resvertrol inhiit campphosphodiesterse, thus incresing cellulr camp concentrtions nd ctivting the upstrem kinse CMKKβ, 29 nd moderte doses of resvertrol led to Sirt1-dependent ctivtion of AMPK. 3 We hve demonstrted direct effects of HMB on Sirt1 ctivtion in cell-free system 31 s well s the synergistic effects of resvertrol nd HMB on Sirt1 oth in vitro nd in vivo. 17 Considering the two different mechnisms of AMPK ctivtion of these compounds, it is possile tht metformin in the low concentrtions used in our study cts s n llosteric effector to promote AMPK phosphoryltion y resvertrol nd HMB. Although the pthogenesis of insulin resistnce in type 2 dietes mellitus is still not fully understood, there is evidence tht ccumultion of intrcellulr lipid metolites in skeletl muscle nd heptocytes, s oserved in oesity or lipodystrophy, leds to impirment in insulin signling pthwys. 33 Moreover, decresed mitochondril oxidtive cpcity my result in impired lipid oxidtion in skeletl muscle in oese nd dietic individuls, 34,35 potentilly excerting lipid overflow. Therefore, finding strtegies to improve mitochondril function nd to stimulte ctolic oxidtive pthwys my contriute to oth the prevention nd tretment of dietes. AMPK, s key regultor of energy metolism, medites the switch from nolic processes to ctolic pthwys. AMPK ctivity is decresed in oese insulin-resistnt ptients compred with oese insulin-sensitive ptients. 36 Further, lck of skeletl muscle AMPKα2 ctivity in trnsgenic mice excertes the development of diet-induced glucose intolernce nd insulin resistnce. 37 In contrst, ctivtion of AMPK improves symptoms of impired glucose homeostsis nd insulin resistnce Given tht lifestyle interventions such s cloric restriction or exercise, which re physiologicl ctivtors of AMPK, re difficult to incorporte or to mintin in the dily routine of most people, finding gents mimicking these effects re of gret interest. Unfortuntely, mny of these compounds require concentrtions which re ssocited with dverse effects. However, otining synergy from three unrelted compounds permits the use of very low concentrtions of ech individul component, reducing the likelihood of dverse effects while preserving the effects of higher-dose metformin on ft oxidtion nd insulin sensitivity. Conclusion In this study we hve demonstrted the synergistic effects of mixture of resvertrol nd HMB with low dose of metformin on ctivtion of AMPK nd Sirt1, with n ssocited increse in ft oxidtion in muscle nd dipose tissue in cell culture. Consistent with our in vitro dt, 2 weeks of tretment with comintion of these compounds incresed insulin sensitivity nd reduced diposity in mice, nd these effects were comprle with those found with high-dose metformin tretment. Therefore, this comintion my e useful pproch to lowering the therpeutic metformin concentrtion required nd my e eneficil for the mngement of type 2 dietes. Disclosure Finncil support for this study ws provided y NuSirt Sciences Inc, Knoxville TN. AB nd MBZ re employees nd stockholders of NuSirt Sciences Inc. References 1. Hrdoon SL, Morris RW, Thoms MC, Wnnmethee SG, Lennon LT, Whincup PH. Is the recent rise in type 2 dietes incidence from 1984 to 27 explined y the trend in incresing BMI?: evidence from prospective study of British men. Dietes Cre. 21;33(7): Srwr N, Go P, Seshsi SR, et l. Dietes mellitus, fsting lood glucose concentrtion, nd risk of vsculr disese: collortive met-nlysis of 12 prospective studies. Lncet. 21;375(9733): Viollet B, Guigs B, Snz Grci N, Leclerc J, Foretz M, Andreelli F. Cellulr nd moleculr mechnisms of metformin: n overview. Clin Sci (Lond). 212;122(6): Fryer LG, Pru-Ptel A, Crling D. The nti-dietic drugs rosiglitzone nd metformin stimulte AMP-ctivted protein kinse through distinct signling pthwys. J Biol Chem. 22;277(28): Hwley SA, Ross FA, Chevtzoff C, et l. Use of cells expressing gmm suunit vrints to identify diverse mechnisms of AMPK ctivtion. Cell Met. 21;11(6): sumit your mnuscript Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6

9 Metformin, resvertrol, HMB, nd insulin sensitivity 6. Zhng Y, Wng Y, Bo C, et l. Metformin intercts with AMPK through inding to gmm suunit. Mol Cell Biochem. 212;368(1 2): Grisourd J, Timper K, Rdimerski TM, et l. Mechnisms of metformin ction on glucose trnsport nd metolism in humn dipocytes. Biochem Phrmcol. 21;8(11): Grer AJ, Duncn TG, Goodmn AM, Mills DJ, Rohlf JL. Efficcy of metformin in type II dietes: results of doule-lind, plceocontrolled, dose-response tril. Am J Med. 1997;13(6): Proctor WR, Bourdet DL, Thkker DR. Mechnisms underlying sturle intestinl sorption of metformin. Drug Met Dispos. 28;36(8): Okysu S, Kitichi K, Hori A, et l. The evlution of risk fctors ssocited with dverse drug rections y metformin in type 2 dietes mellitus. Biol Phrm Bull. 212;35(6): Hrdie DG, Pn DA. Regultion of ftty cid synthesis nd oxidtion y the AMP-ctivted protein kinse. Biochem Soc Trns. 22;3(Pt 6): Srivstv RA, Pinkosky SL, Filippov S, Hnselmn JC, Crmer CT, Newton RS. AMP-ctivted protein kinse: n emerging drug trget to regulte imlnces in lipid nd crohydrte metolism to tret crdio-metolic diseses. J Lipid Res. 212;53(12): Suter M, Riek U, Tuerk R, Schlttner U, Wllimnn T, Neumnn D. Dissecting the role of 5 -AMP for llosteric stimultion, ctivtion, nd dectivtion of AMP-ctivted protein kinse. J Biol Chem. 26; 281(43): Slminen A, Hyttinen JM, Krnirnt K. AMP-ctivted protein kinse inhiits NF-kppB signling nd inflmmtion: impct on helthspn nd lifespn. J Mol Med (Berl). 211;89(7): Ln F, Ccicedo JM, Rudermn N, Ido Y. SIRT1 modultion of the cetyltion sttus, cytosolic locliztion, nd ctivity of LKB1. Possile role in AMP-ctivted protein kinse ctivtion. J Biol Chem. 28;283(41): Cnto C, Auwerx J. PGC-1lph, SIRT1 nd AMPK, n energy sensing network tht controls energy expenditure. Curr Opin Lipidol. 29;2(2): Bruckuer A, Zemel MB, Thorpe T, et l. Synergistic effects of leucine nd resvertrol on insulin sensitivity nd ft metolism in dipocytes nd mice. Nutr Met (Lond). 212;9(1): Feige JN, Lgouge M, Cnto C, et l. Specific SIRT1 ctivtion mimics low energy levels nd protects ginst diet-induced metolic disorders y enhncing ft oxidtion. Cell Met. 28;8(5): Nin V, Escnde C, Chini CC, et l. Role of deleted in rest cncer 1 (DBC1) protein in SIRT1 decetylse ctivtion induced y protein kinse A nd AMP-ctivted protein kinse. J Biol Chem. 212; 287(28): Souz-Mello V, Gregorio BM, Crdoso-de-Lemos FS, de Crvlho L, Aguil MB, Mndrim-de-Lcerd CA. Comprtive effects of telmisrtn, sitgliptin nd metformin lone or in comintion on oesity, insulin resistnce, nd liver nd pncres remodelling in C57BL/6 mice fed on very high-ft diet. Clin Sci (Lond). 21;119(6): Okmoto T, Knemoto N, Ohuchi Y, Okno M, Fukui H, Sudo T. Chrcteriztion of STZ-induced type 2 dietes in Zucker ftty rts. Exp Anim. 28;57(4): Grce MH, Rinicky DM, Kuhn P, et l. Hypoglycemic ctivity of novel nthocynin-rich formultion from lowush lueerry, Vccinium ngustifolium Aiton. Phytomedicine. 29;16(5): Hou M, Venier N, Sugr L, et l. Protective effect of metformin in CD1 mice plced on high crohydrte-high ft diet. Biochem Biophys Res Commun. 21;397(3): Cton PW, Nyuni NK, Kieswich J, Khn NQ, Yqoo MM, Corder R. Metformin suppresses heptic gluconeogenesis through induction of SIRT1 nd GCN5. J Endocrinol. 21;25(1): Cton PW, Kieswich J, Yqoo MM, Holness MJ, Sugden MC. Metformin opposes impired AMPK nd SIRT1 function nd deleterious chnges in core clock protein expression in white dipose tissue of geneticlly-oese d/d mice. Dietes Oes Met. 211;13(12): Hwley SA, Gdll AE, Olsen GS, Hrdie DG. The ntidietic drug metformin ctivtes the AMP-ctivted protein kinse cscde vi n denine nucleotide-independent mechnism. Dietes. 22;51(8): Owen MR, Dorn E, Hlestrp AP. Evidence tht metformin exerts its nti-dietic effects through inhiition of complex 1 of the mitochondril respirtory chin. Biochem J. 2;348 Pt 3: Ouyng J, Prkhi RA, Ochs RS. Metformin ctivtes AMP kinse through inhiition of AMP deminse. J Biol Chem. 211; 286(1): Prk SJ, Ahmd F, Philp A, et l. Resvertrol meliortes ging-relted metolic phenotypes y inhiiting camp phosphodiesterses. Cell. 212;148(3): Price NL, Gomes AP, Ling AJ, et l. SIRT1 is required for AMPK ctivtion nd the eneficil effects of resvertrol on mitochondril function. Cell Met. 212;15(5): Bruckuer A, Zemel MB. Effects of diry consumption on SIRT1 nd mitochondril iogenesis in dipocytes nd muscle cells. Nutr Met (Lond). 211;8: Ismil-Beigi F. Pthogenesis nd glycemic mngement of type 2 dietes mellitus: physiologicl pproch. Arch Irn Med. 212; 15(4): Petersen KF, Shulmn GI. Etiology of insulin resistnce. Am J Med. 26;119(5 Suppl 1):S1 S Morino K, Petersen KF, Dufour S, et l. Reduced mitochondril density nd incresed IRS-1 serine phosphoryltion in muscle of insulinresistnt offspring of type 2 dietic prents. J Clin Invest. 25; 115(12): Petersen KF, Befroy D, Dufour S, et l. Mitochondril dysfunction in the elderly: possile role in insulin resistnce. Science. 23; 3(5622): Guthier MS, O Brien EL, Bigorni S, et l. Decresed AMPctivted protein kinse ctivity is ssocited with incresed inflmmtion in viscerl dipose tissue nd with whole-ody insulin resistnce in moridly oese humns. Biochem Biophys Res Commun. 211;44(1): Fujii N, Ho RC, Mne Y, et l. Altion of AMP-ctivted protein kinse lph2 ctivity excertes insulin resistnce induced y high-ft feeding of mice. Dietes. 28;57(11): Bergeron R, Previs SF, Cline GW, et l. Effect of 5-minoimidzole-4- croxmide-1-et-d-riofurnoside infusion on in vivo glucose nd lipid metolism in len nd oese Zucker rts. Dietes. 21;5(5): Pold R, Jensen LS, Jessen N, et l. Long-term AICAR dministrtion nd exercise prevents dietes in ZDF rts. Dietes. 25;54(4): Cool B, Zinker B, Chiou W, et l. Identifiction nd chrcteriztion of smll molecule AMPK ctivtor tht trets key components of type 2 dietes nd the metolic syndrome. 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10 Bruckuer nd Zemel Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy Pulish your work in this journl Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy is n interntionl, peer-reviewed open-ccess journl committed to the rpid puliction of the ltest lortory nd clinicl findings in the fields of dietes, metolic syndrome nd oesity reserch. Originl reserch, review, cse reports, hypothesis formtion, expert Sumit your mnuscript here: opinion nd commentries re ll considered for puliction. The mnuscript mngement system is completely online nd includes very quick nd fir peer-review system, which is ll esy to use. Visit to red rel quotes from pulished uthors. 12 sumit your mnuscript Dietes, Metolic Syndrome nd Oesity: Trgets nd Therpy 213:6