Miglitol increases energy expenditure by upregulating uncoupling protein 1 of brown adipose tissue and reduces obesity in dietary-induced obese mice

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1 Sugimoto et l. Nutrition & Metbolism 4, :4 RESERCH Open ccess Miglitol increses energy expenditure by upregulting uncoupling protein of brown dipose tissue nd reduces obesity in dietry-induced obese mice Storu Sugimoto, Hiskzu Nkjim *, Kzuki Kodo, Jun Mori, Kensuke Mtsuo, Kitro Kosk, Wtru oi, Knji Yoshimoto 3, Hiroshi Ikegy 3 nd Hjime Hosoi bstrct ckground: Miglitol is n orl nti-dibetic drug tht cts by inhibiting crbohydrte bsorption in the smll intestine. Recent studies hve shown tht miglitol reduces obesity in humns nd rodents. However, its mechnisms hve remined uncler. The purpose of this study ws to determine whether miglitol genertes het by ctivting uncoupling protein (UCP), n enzyme involved in thermogenesis, in brown dipose tissue (T) in mice. Methods: Four-week-old mle C57L/6 J mice were fed high-ft diet lone (HF) or high ft diet plus miglitol (HFM). Oxygen consumption (VO ) ws used to estimte metbolic rte. therml imging cmer ws used to quntify het genertion from interscpulr brown dipose tissue. We nlyzed the protein nd gene expressions of UCP nd the expressions of four proteins relted to β3-drenergic signling in the pthwy ctivting UCP (protein kinse (PK), hormone-sensitive lipse (HSL), p38 α mitogen-ctivted protein kinse (p38αmpk) nd peroxisome prolifertor-ctivted receptor gmm coctivtor α (PGCα)). Results: t 8 weeks, body weight, epididyml nd subcutneous white dipose tissue nd the HOM-R vlue of the HFM mice were significntly less thn those of the HF mice. Food intke ws not different between the HF nd HFM mice. VO nd T temperture were significntly higher in the HFM mice. Miglitol significntly enhnced the gene nd protein expressions of UCP nd the expressions of proteins relted to β3-drenergic signling. Conclusions: Miglitol s nti-obesity effect ws ttributed to incresed energy expenditure by upregulting UCP in T (i.e., by thermogenesis) nd to enhncement of β3-drenergic signling in T. Keywords: Miglitol, lph-glucosidse inhibitor, Obesity, Oxygen consumption, rown dipose tissue, Uncoupling protein, β3-drenergic signling ckground Obesity develops from n imblnce between energy intke nd energy expenditure []. t present, only medicines tht inhibit energy intke, such s ppetite suppressnts nd lipid bsorption inhibitors, re uthorized s nti-obesity drugs by the mericn Food nd Drug dministrtion * Correspondence: hiskzu@koto.kpu-m.c.jp Deprtment of Peditrics, Grdute School of Medicl Science, Kyoto Prefecturl University of Medicine, 465-Kjiicho, Hirokoji-Kwrmchi, Kmigyo-ku, Kyoto , Jpn Full list of uthor informtion is vilble t the end of the rticle (FD). Enhncement of energy expenditure hs emerged s potentil nd ttrctive strtegy for treting or preventing obesity. Wheres white dipose tissue cts to store surplus energy, brown dipose tissue (T) expends energy by het production through uncoupling protein (UCP) in its mitochondri []. In generl, it hs been believed tht humns lose T shortly fter infncy. However, recent studies using positron emission tomogrphy/ computed tomogrphy (PET/CT) hve shown tht dult humns still possess functionl T. T hs received much ttention s trget of obesity tretment [3-7]. 4 Sugimoto et l.; licensee iomed Centrl Ltd. This is n Open ccess rticle distributed under the terms of the Cretive Commons ttribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge of Miglitol is n lph-glucosidse inhibitor (αgi) nd is commonly used s n nti-dibetic drug [8]. In dibetic subjects, miglitol blunts postprndil hyperglycemi by inhibiting lph-glucosidse in the smll intestine nd prolongs crbohydrte bsorption. Recently, miglitol hs been reported to hve n nti-obesity effect [9-]. However, its mechnisms re not cler. Here we exmined the effect of miglitol on UCP, n enzyme involved in thermogenesis, in T. Our results show tht miglitol incresed energy expenditure, reduced obesity nd enhnced β3-drenergic signling nd upregultion of UCP in T. These dt provide further support for miglitol s n nti-obesity gent nd clrify its mechnism of ction. Methods nimls nd diets Four-week-old mle C57L/6 J mice were purchsed from CLE Jpn (Tokyo, Jpn). Four diets were prepred: norml chow (CLE Rodent diet CE-: % of clories from ft, 59.% of clories from crbohydrte, 8.8% of clories from protein), high ft diet (HFD) (Cle High ft diet 3: 56.7% of clories from ft, 3.% of clories from crbohydrte, % of clories from protein), norml chow contining.8% miglitol nd HFD contining.8% miglitol. previous study of miglitol in mice [] used diet contining.8% miglitol. We chose to use lower dose becuse it ws closer to the dose used in clinicl medicine. Mice were divided into 4 groups: control group (NC), which ws fed norml chow, norml chow plus miglitol (NCM) group, which ws fed the norml chow plus miglitol, high ft (HF) group, which ws fed the HFD, nd high ft plus miglitol (HFM) group, which ws fed the HFD plus miglitol. The mice were kept in temperturecontrolled room (3 C) on h light/drk cycle (lights on 7: h; off 9: h) with free ccess to food nd wter. Individul food intke nd body weight gin were monitored once week. t 8 weeks, mice were fsted overnight nd nesthetized with sodium pentobrbitl (5 mg/kg, i.p.) nd blood ws obtined by crdiopuncture. Plsm ws seprted by centrifugtion t 4 C nd stored t 8 C until ssyed. The epididyml nd subcutneous white dipose tissues were dissected nd weighed. Interscpulr brown dipose tissue nd liver were immeditely dissected, frozen in liquid nitrogen nd stored t 8 C until further nlysis. ll niml experiments nd cre procedures were conducted in conformity with the Guidelines of the niml Cre nd Use Committee of Kyoto Prefecturl University of Medicine. Plsm prmeters lood glucose ws determined with compct glucose nlyzer ntsense II (Horib, Kyoto, Jpn). Plsm triglyceride (TG) nd totl cholesterol (T-Cho) levels were mesured with regents from Wko (Osk, Jpn). Plsm insulin level ws mesured by n ELIS kit (Moring Institute of iologicl Science, Kngw, Jpn). Plsm ctive glucgon-like peptide (GLP) levels were mesured with n ELIS kit (Shibygi, Gunm, Jpn). ll of the ssys were performed ccording to the mnufcturer s instructions. Serum concentrtion of miglitol ws mesured by liquid chromtogrphy - tndem mss spectrometry (LC/MS/MS). Oxygen consumption Oxygen consumption (VO ) ws mesured with n O / CO metbolism-mesuring system (model MK-5, Muromchi-Kiki, Tokyo, Jpn), which consists of two independent 56-ml chmbers (for mesuring two nimls simultneously), suction pump nd computer for dt nlysis []. The mice were plced in the chmbers t 3 C nd cclimted for more thn two hours. Every three minutes, the pump drws ir from one of the chmbers for one minute t rte of 65 ml/min to mesure O concentrtion. Oxygen consumption (VO ) ws clculted s [O-Oc]v m t, where O is the tmospheric oxygen concentrtion (%) tht flows into the chmber, Oc is the oxygen concentrtion in the chmber (%), v is the flow rte (65 milliliters/min), m is the mss of the mouse in kg nd t is the time in hours [3]. Interscpulr temperture Mice were fsted for 6 hours nd nesthetized (sodium pentobrbitl, 3 mg/kg, i.p.). Interscpulr temperture surrounding T ws recorded with therml imging cmer (FLIR i3, FLIR Systems, Tokyo, Jpn) nd nlyzed with FLIR QuickReport softwre. Histology T ws fixed in % buffered formlin. Sections (5 μm) were stined with hemtoxylin nd eosin. Slides were exmined nd photomicrogrphs tken under the sme exposure nd mgnifiction. Lipid droplets in cells of T were quntified s previously described [4]. One tissue section from ech mouse ws mesured under blinded conditions by one investigtor (S.S.) counting the number of nuclei surrounded by four or more lipid vcuoles/cell in two rndomly chosen res (6 μm ) of ech section, nd verging the results. Western blot nlysis T ws lysed with rdioimmunoprecipittion ssy (RIP) lysis buffer (Ncli Tesque, Kyoto, Jpn). Homogentes were centrifuged t, g for min t 4 C nd superntnts were collected. Protein concentrtions were determined with io-rd protein ssy kit (io-rd, Tokyo, Jpn). Tissue proteins were resolved on % polycrylmide gels in the presence of sodium dodecyl

3 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 3 of sulfte, trnsferred electrophoreticlly to polyvinylidene difluoride membrnes, nd blocked by locking One (Ncli Tesque). The primry nd secondry ntibodies were diluted with Cn Get Signl (Toyobo, Osk, Jpn). The membrne ws incubted with primry ntibodies ginst prolifertor-ctivted receptor gmm coctivtor α (PGCα) (:,) (bcm, Tokyo, Jpn), UCP (:5,) (bcm), β3-drenergic receptor (β3r) (:,) (bcm), protein kinse (PK) (:5,) (Snt Cruz iotechnology, Snt Cruz, C), phosphoryltedprotein kinse (ppk) (:5,) (Snt Cruz iotechnology, Snt Cruz, C), hormone-sensitive lipse (HSL) (:,) (Cell Signling Technology, Tokyo, Jpn), crnitine plmitoyltrnsferse (CPT) (:5,) (Lifespn iosciences, Settle, W), p38α mitogen-ctivted protein kinse (p38αmpk) (:5,) (Cell Signling Technology), nd (:5,) (Cell Signling Technology). Secondry ntibody consisted of :5, dilution of HRP-conjugted donkey nti-rbbit IgG (for PGCα, UCP, β3r, PK, ppk, HSL, CPT, p38αmpk) (GE Helthcre, Tokyo, Jpn) or HRP-conjugted sheep ntimouse IgG (for ) (GE Helthcre). The immunocomplexes were detected using n enhnced HRP-luminol chemiluminescence system (ECL prime) (GE Helthcre) nd subjected to utordiogrphy (New mershm Hyperfilm) (GE Helthcre). Signls on the immunoblot were quntified using the NIH Imge computer progrm (NIH, ethesd, MD, version.45). To compre the results for protein expression, we ssigned vlue of to expression in T from control mice. Cyclic MP (cmp) ssy The selective β3-drenergic receptor gonist CL36,43 (Sigm, Tokyo, Jpn) ( mg/kg/body weight) nd sline ws given by intrperitonel injection 6 h before the end of the experiment. The mount of cmp in T ws mesured by cmp ssy kit (R&D Systems, Minnepolis, MN) ccording to the mnufcturer s instructions. Quntittive rel-time PCR Totl RN from T nd liver were isolted using NucleoSpin RN II kit (Mcherey-Ngel, Düren, Germny). Templte cdn synthesized from 5 ng totl RN with rndom hexmer primers ws used s the templte for ech rection with SuperScript First-Strnd Synthesis System (Invitrogen Life Technology, Osk, Jpn). Quntittive rel-time PCR (qrt-pcr) ws performed using SYR Green mster mix (Tkr, Shig, Jpn) with μm ofechprimerinn73rel- Time PCR System (pplied iosystems, Tokyo, Jpn). mplifiction ws performed with the following protocol: 4 cycles (5 sec t 95 C nd 3 sec t 6 C) fter n initil ctivtion step for 3 sec t 95 C. Primer sequences were shown s follows: (T), forwrd primer: 5 -GTCGTGCGTGCTCG-3, reverse primer: 5 -TGTGTTTCTGTGCCCG-3 ; (liver), forwrd primer: 5 -GGCTGTTTCCCCTCCTCG-3, reverse primer: 5 -CCGTTGGTCTGCCTGT-3 ; PGCα, forwrdprimer:5 -TGCGCCCTTGTGT GG-3, reverse primer: 5 - GGGTTTCTTGGTTGG CTTTTG-3 ; UCP, forwrd primer: 5 - GGCTTCC GTCCTTGGT -3, reverse primer: 5 -CTGGTG GGCGCTGTTT-3 ; CPT, forwrd primer: 5 -CC TCTCTGGGTGCTGG-3, reverse primer: 5 -G GCCCCGTGCCTC-3 ; glucokinse (GK), forwrd primer: 5 -CCTGGCCGGGCTTC-3, reverse primer: 5 -TGTGGCCCCGTGTCTTC-3. ws chosen s n internl stndrd. Sttisticl nlysis Dt re shown s mens ± SEM. Single-group dt were ssessed using Student s t-test. Repeted mesurements of nlysis of vrince (NOV) with Tukey-Krmer post hoc comprisons were performed for multiple comprisons. P vlues less thn.5 were considered sttisticlly significnt. Results Miglitol reduced body weight gin nd incresed energy expenditure in high ft diet-induced obese mice The body weight of mice fed high ft diet (HF mice) (7.3 ±.4 g t 8 weeks) ws significntly greter thn tht of mice fed norml chow (p <.5) (control mice) (.5 ±. g). The body weight of mice fed the high ft diet plus miglitol (HFM mice) (5.8 ±.4 g) ws significntly less thn tht of HF mice (p <.5) (Tble, Figure ) even though the two groups consumed the sme mount of food energy (Figure, C). On the other hnd, miglitol did not ffect the body weight under the condition of norml chow (Tble, Figure ). The miglitoltreted mice did not mnifest ny of the common dverse effects of miglitol, such s gstrointestinl bnormlities, dirrhe or norexi. Oxygen consumption (VO ), n indirect mesurement of metbolism, ws significntly incresed in HFM mice compred to HF mice in both the drk nd light phses (p <.5) (Figure ). y contrst, VO ws not different between NC nd NCM mice. Interscpulr T temperture in HFM mice (34.7 ±.7 C) ws significntly higher (p <.5) thn tht in HF mice (3.5 ±.3 C) nd ws significntly higher (p <.5) thn the tempertures in NC mice (3. ±.6 C) nd NCM mice (3.7 ±.5 C) (Figure 3). Miglitol meliorted insulin resistnce in high ft diet-induced obese mice lood glucose nd plsm T-Cho levels were significntly higher in HF mice thn in NC nd NCM mice. lood glucose levels were significntly lower in HFM mice

4 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 4 of Tble Metbolic prmeters t 8-weeks-old mice n NC NCM HF HFM ody weight (g) -.5 ±.. ±. 7.3 ±.4, c 5.8 ±.4 Glucose (mg/dl) 5 8 ± ± ± 4., c 4 ± 9. Totl cholesterol (mg/dl) ± ± ± 7. 4 ± 4. Triglyceride (mg/dl) 6 4 ± ± ± ± 5. Insulin (μu/ml) ±. 3.4 ±.5.3 ± ±. HOM-R 5.4 ±.3. ± ±.3, c 4. ±.7 Weight of epididyml white dipose tissue (g) ±..8 ±.. ±.8, c.85 ±.4 Weight of subcutneous white dipose tissue (g) 6.3 ±.3 Not mesured.5 ±.5.98 ±., c ctive glucgon-like peptide (pg/ml) ± ± ± ± 4.4 Concentrtion of miglitol (μmol/l) 3-4 Not mesured.6 ±. Not mesured.6 ±.3 Vlues re mens ± SE for 3 4 mice. lood smples were collected under fsting conditions. P <.5, vs mice fed norml chow diet (NC). b P <.5, vs mice fed norml chow diet plus miglitol (NCM). c P <.5, vs mice fed high-ft diet lone (HF). thn in HF mice. Plsm TG did not differ in the four groups. The HOM-R vlue, n index of insulin resistnce, ws significntly elevted in HF mice nd significntly lowered by miglitol (control.4 ±.3 vs. NM. ±.3 vs. HF 8.4 ±.3 vs. HFM 4. ±.7) (CC or NC vs. HF, p <.5; HF vs. HFM, p <.5). The msses of epididyml nd subcutneous white dipose were lower in the HFM mice thn in the HF mice (Tble ). Miglitol enhnces the secretion of ctive glucgonlike-peptide (GLP) in obese humns (see Discussion section). ecuse GLP decreses food intke, mny clinicins ttribute miglitol s nti-obesity effect to suppression of food intke. However, the ctive GLP level did not differ between HF nd HFM mice (Tble ). Miglitol decresed the number of lipid droplets in T cells of HF mice To evlute the degree of lipolysis, we investigted the microscopic ppernce of T. The HFD incresed the number of lipid droplets in cells of T, while miglitol significntly decresed the number of lipid droplets in cells of T (NC 9. ±.5 vs. HF 4.3 ±. vs. HFM 7. ±.3 cells/ re) (NC vs. HF or HFM, p <.5; HF vs. HFM, p <.5) (Figure 4). Miglitol enhnced the gene nd protein expressions of UCP in T of HFM mice The min function of T is thermogenesis, which is medited by upregultion of UCP. PGCα is trnscriptionl ody Weight (g) NC (n=) NCM (n=) HF (n=) HFM (n=), c, c, c Food intke (kcl/week) C NC (n=5) NCM (n=5) HF (n=5) HFM (n=5) Dy Dy Food intke (Kcl/month) 4 3 NC NCM HF HFM (n=5) (n=5) (n=5) (n=5) Figure Effect of miglitol on body weight gin. : ody weight chnge. : Weekly food intke. C: Totl food intke. Vlues re mens ± SE for 5 mice. P <.5, vs mice fed norml chow diet (NC). b P <.5, vs mice fed norml chow diet plus miglitol (NCM). c P <.5, vs mice fed high-ft diet lone (HF).

5 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 5 of Figure Effect of miglitol on energy expenditure. : -h Oxygen consumption. : Oxygen consumption in drk nd light phses. Vlues re mens ± SE for 3 mice. P <.5, vs mice fed norml chow diet (NC). b P <.5, vs mice fed norml chow diet plus miglitol (NCM). c P <.5, vs mice fed high-ft diet lone (HF). T T, c 35 T NC T NCM Interscpulrtemperture ( C) 3 5 NC NCM HF HFM (n=4) (n=4) (n=4) (n=5) HF HFM Figure 3 Effect of miglitol on T temperture. shows representtive infrred therml imge of norml chow diet mice (NC), miglitol-treted norml chow diet mice (NCM), high-ft diet mice (HF) nd miglitol-treted high-ft diet mice (HFM). : Interscpulr temperture ws mesured. Vlues re mens ± SE for 4 5 mice. P <.5, vs mice fed norml chow diet (NC). b P <.5, vs mice fed norml chow diet plus miglitol (NCM). c P<.5,vs mice fed high-ft diet lone (HF).

6 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 6 of C D Number of cells/re 8 6 4, c NC HF HFM (n =5) (n=6) (n=6) Figure 4 Effect of miglitol on the number of lipid droplets in T cells. Histology of T ws exmined by HE stining (Scle br = 4 μm)., nd C show the representtive histology of norml chow diet mice, high-ft diet mice nd miglitol-treted high-ft diet mice, respectively. D: The number of cells/re ws counted. Vlues re mens ± SE for 5 6 mice. P <.5, vs mice fed norml chow diet (NC). c P <.5, vs mice fed high-ft diet lone (HF). coctivtor tht is required for expression of the UCP gene. We evluted gene nd protein expressions of PGCα nd UCP. The mrn levels of PGCα showed no differences between the four groups. However, the level of PGCα protein of HFM mice ws.4-fold higher thn tht of HF mice (p <.5) (Figure 5, ). The expression of UCP mrn in HFM mice ws.5-fold higher thn tht of HF mice (p <.5). Miglitol did not enhnce the expression of UCP mrn in norml chow-fed mice. The level of UCP protein in HF mice ws.7-fold higher thn tht of control mice (p <.5), nd the level of UCP- protein of HFM mice ws.-fold higher thn tht of HF mice (p <.5) (Figure 5, ). We mesured CPT expression in T to evlute mitochondril β-oxidtion. The expressions of CPT mrn nd protein were significntly incresed in both HF mice nd HFM mice s compred with control mice. (Figure 6, ). Miglitol enhnced β3-drenergic signling in T of HFM mice β3-drenergic signling through the β3-drenergic receptor (β3r) ctivtes UCP nd thus hs role in reducing obesity. The protein expression of β3r ws not significntly different between HF nd HFM mice (Figure 7). However, the protein expressions of PK, HSL nd p38α MPK of HFM mice were significntly incresed s compred with HF mice (.7,. nd.5-fold, respectively) (p <.5) (Figure 7-D). To test whether miglitol s upregultion of UCP expression ws medited by β3-drenergic signling, we mesured the effect of selective β3r gonist (CL36,43). CL36,43 induced greter mounts of cmp nd ppk protein in HFM mice thn in HF mice (Figure 8, ). Heptic glucokinse expression did not ffect thermogenesis in T During the course of this study, it ws reported tht heptic glucokinse (GK) expression suppressed thermogenesis in T [5]. This rised the possibility tht miglitol cts by suppressing liver GK expression. However, miglitol did not suppress GK mrn expression (Figure 9). Discussion Our dt show tht miglitol reduced body weight gin nd insulin resistnce, consistent with previous study using spontneous-onset obese type dibetes mice []. Recent studies hve focused on T s trget of tretment for obesity [3,4,6]. We hypothesized tht the reson of suppressed body weight gin observed in miglitol-treted mice ws the upregultion of energy expenditure. T produces het by non-shivering thermogenesis to mintin body temperture [3]. The key component of this process is UCP. UCP, the rchetypl UCP, is uniquely expressed in mitochondri of brown dipocytes. UCP uncouples denosine-5 -triphosphte (TP) synthesis from substrte oxidtion in brown dipocytes [7]. When UCP is ctivted, chemicl energy is dissipted s het without TP synthesis. The upregultion of UCP expression indictes incresed thermogenesis nd energy expenditure, which helps to protect from ft ccumultion nd

7 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 7 of UCP PGCα Reltive mrn level.5.5 NC (n=5-6) NCM (n=5-7) HF (n=5-6) HFM (n=5-6), c.5.5 NC (n=6) HF (n=6) HFM (n=5-7), c, c.5.5 PGCα UCP Figure 5 Effect of miglitol on the expressions of PGCα nd UCP in T. : Rel-time PCR experiments. : Western blot nlysis. Vlues re mens ± SE for 5 7 mice. P <.5, vs mice fed norml chow diet (NC). b P <.5, vs mice fed norml chow diet plus miglitol (NCM). c P <.5, vs mice fed high-ft diet lone (HF). PGCα UCP obesity [8]. The present study showed tht miglitol upregulted UCP in T of high ft diet-induced obese mice. Consistent with incresed UCP expression, oxygen consumption ws incresed in HFM mice (Figures nd 5). Miglitol induced n incresed interscpulr temperture, which cn be explined by its stimultion of UCP expression in T (Figure 3). rown dipocytes contin lrge number of mitochondri nd re highly innervted by the sympthetic nervous system (SNS). SNS nerve terminls of T relese nordrenline, which ctivtes β-drenergic receptors nd cscde of events leding to mitochondriogenesis nd incresed expression of UCP. rown dipocytes express vrious drenergic receptors tht include the α -ndβ 3 - receptors. β3-drenergic receptor (β3r) is, t lest in rodents, the min drenergic receptor in driving the cscde of events necessry for thermogenesis in T. CPT Reltive mrn level.5.5 NC (n=6) HF (n=6) HFM (n=5).5.5 NC (n=6) HF (n=6) HFM (n=5) CPT CPT Figure 6 Effect of miglitol on the expression of CPT. : Rel-time PCR experiments. : Western blot nlysis. Vlues re mens ± SE for 5 6 mice. P <.5, vs mice fed norml chow diet (NC). c P <.5, vs mice fed high-ft diet lone (HF).

8 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 8 of β3r PK β3r 3 PK, c NC HF HFM (n=6) (n=6) (n=5) NC HF HFM (n=6) (n=6) (n=5) HSL p38αmpk C.5 HSL, c D.5 p38αmpk.5.5, c.5 NC HF HFM (n=6) (n=6) (n=5) NC HF HFM (n=6) (n=6) (n=5) Figure 7 Effect of miglitol on β3-drenergic signling. Protein expressions of β3-drenergic signling in T were nlyzed. -D: Western blot nlysis. Vlues re mens ± SE for 5-6mice. P <.5, vs mice fed norml chow diet (NC). c P <.5, vs mice fed high-ft diet lone (HF). β3r intercts with Gα to stimulte denylyl cyclse ctivity, which promotes synthesis of cmp. Incresed sympthetic stimultion induces β3r ctivtion, incresed cmp genertion nd subsequent ctivtion of PK [9]. The protein levels of PK were higher in HFM mice thn in HF mice in our experiment (Figure 7), which suggests tht the upregultion of UCP observed in our study involved β3-drenergic signling. To confirm tht upregultion of UCP involves β3-drenergic signling, we evluted the downstrem signling of PK. PK induces lipolysis by ctivting hormone-sensitive lipse (HSL). HSL releses free ftty cids from intrcellulr lipid stores [9], which re then trnsformed into cyl-co. cyl-co is combined with crnitine by CPT nd trnsported into the mitochondril mtrix s cylcrnitine. cyl-crnitine is converted bck to cyl-co, which cn then enter the ftty cid β-oxidtion pthwy. Free ftty cids not only ct s substrtes for β-oxidtion but lso stimulte UCP ctivity [7,9]. The expressions of CPT mrn nd protein were not significntly different between HF mice nd HFM mice in our study (Figure 6, ), which suggests tht the β-oxidtion ctivity ws similr in the two groups. The HFM mice hd higher HSL protein levels thn the HF mice. The findings tht miglitol decresed the number of lipid droplets in T cells (Figure 4) nd incresed the protein expression of HSL (Figure 7C) suggest tht lipolysis ws ctivted by miglitol under the high-ft diet. The lipolysis induced by miglitol ctivted UCP. The chronic effects of PK ctivtion include mitochondril biogenesis nd incresed UCP gene expression [9]. p38αmpk is reported to induce UCP expression by stimulting the SNS [-]. In mouse dipocytes nd niml models, β-r stimultion triggers kinse cscde from PK to p38mpk, which phosphoryltes PGCα [,]. PGCα strongly coctivtes severl nucler

9 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge 9 of sline CL36,43 ppk cmp (pmol/mg tissue) HF (n=5) HFM (n=4-5) d.5.5 ppk HF (n=5) HFM (n=4-5) c sline CL36,43 sline CL36,43 Figure 8 Effect of miglitol on cmp nd ppk in the presence of β3-drenergic receptor gonist. : cmp mount in T. : Western blot nlysis. Vlues re mens ± SE for 4 5 mice. c P <.5, vs mice fed high-ft diet lone (HF). d P <., vs mice fed high-ft diet lone (HF). receptors tht bind to the UCP enhncer nd upregultes UCP gene expression [3]. These events lso contribute totheorchestrtedresponsetoincresemitochondriogenesis nd the overll thermogenic cpcity of brown dipocytes. The finding tht protein levels of p38α MPK nd PGCα were higher in HFM mice thn in HF mice (Figure 5, Figure 7D) suggests tht the gene expression of UCP ws upregulted through the PK-p38 MPK-PGCα cscde by miglitol in high ft dietinduced obese mice. Figure 9 Effect of miglitol on the gene expression of liver glucokinse (GK). Rel-time PCR experiments. Vlues re mens ± SE for 5-6mice. P <.5, vs mice fed norml chow diet (NC). c P <.5, vs mice fed high-ft diet lone (HF). β3r gonist (CL36,43) ws found to increse PGCα mrn nd UCP mrn in 4 6 hours [4,5]. In our study, CL36,43 produced greter mounts of cmp nd ppk protein in HFM mice thn in HF mice (Figure 8, ), confirming tht miglitol enhnced β3- drenergic signling under the high ft diet. Glucgon-like peptide (GLP) is secreted from L cells in the intestine, nd promotes insulin secretion in glucose-dependent mnner following ingestion of crbohydrte [6]. GLP receptor gonists hve been used for the tretment of type dibetes ptients in recent yers. GLP hs the potentil to be used s n nti-obesity drug [7]. GLP not only stimultes insulin secretion but lso decreses ppetite nd reduces food intke when dministered either peripherlly or directly into the centrl nerve system [8]. Though miglitol enhnces GLP-secretion in obese humns [9-3], plsm ctive GLP levels in the HF nd HFM mice in this study were not significntly different (Tble ), which suggests tht GLP did not prticipte in the reduction of obesity in this study. It remins uncler how miglitol induces thermogenesis in T. One possibility is tht miglitol stimultes the SNS, which is known to enhnce β3-drenergic signling [9], which in turn induces thermogenesis in T. One wy in which miglitol could stimulte the SNS is by suppressing heptic glucokinse (GK) expression [5]. However, miglitol did not suppress GK mrn expression in our study (Figure 9). β3r gonists increse oxygen consumption nd led to weight loss in obese rodents [3,33]. However, β3r stimulnts hve not yet become vilble, prtly becuse

10 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge of the specificity of β3r gonists for humn β3r is low [34]. Miglitol hs few side effects in humns, nd is fesible for long-term use s n orl drug. The dverse effects of miglitol re minly minor gstrointestinl symptoms. If these side effects re cceptble, miglitol hs promise s n nti-obesity drug. If the side effects re not cceptble, nother pproch is to develop new therpeutics bsed on the mechnisms of miglitol. Conclusions Miglitol ) incresed energy expenditure, ) hd n ntiobesity effect in high ft diet-induced obese mice, 3) enhnced β3-drenergic signling nd 4) upregulted UCP in T under the condition of high ft diet. These findings suggest tht miglitol hs the potentil to be therpeutic for the tretment of obesity. bbrevitions αgi: lph-glucosidse inhibitor; T: rown dipose tissue; VO :Oxygen consumption; HOM-R: Homeostsis model ssessment of insulin resistnce; GLP: Glucgon-like peptide; UCP: Uncoupling protein ; PGCα: Peroxisome prolifertor-ctivted receptor gmm coctivtor α; β3r: β3-drenergic receptor; PK: Protein kinse ; ppk: Phosphorylted protein kinse ; HSL: Hormone -sensitive lipse; p38αmpk: p38 α mitogen-ctivted protein kinse; CPT: Crnitine plmitoyltrnsferse I; GK: Glucokinse; SNS: Sympthetic nervous system; NC group: Mice fed norml chow diet lone; NCM group: Mice fednormlchowdietplusmiglitol;hfgroup:micefedhighftdietlone; HFM group: Mice fed high ft diet plus miglitol. Competing interests The uthors hve no competing interests. uthors contributions SS, HN nd KK conceived the study nd designed the experimentl pln. SS performed ll of the experiments nd contributed to dt collection. SS nd HN contributed to dt nlysis, interprettion nd mnuscript writing. JM contributed to dt interprettion nd mnuscript writing. KK, KM nd W contributed to dt collection. KY, HI nd HH contributed to dt interprettion. ll uthors red nd pproved the finl mnuscript. cknowledgments prt of this work ws supported with JSPS KKENHI Grnt Number The bulk of miglitol ws provided by Snw Kgku Kenkyusho Co., Ltd. (Ngoy, Jpn). We thnk Dr Ikuyo Itoh for technicl ssistnce. We thnk Dr Tichiro Nishikw for guidnce nd technicl ssistnce. uthor detils Deprtment of Peditrics, Grdute School of Medicl Science, Kyoto Prefecturl University of Medicine, 465-Kjiicho, Hirokoji-Kwrmchi, Kmigyo-ku, Kyoto , Jpn. Lbortory of Helth Science, Grdute School of Life nd Environmentl Sciences, Kyoto Prefecturl University, Kyoto, Jpn. 3 Deprtment of Forensic Medicine, Grdute School of Medicl Science, Kyoto Prefecturl University of Medicine, Kyoto, Jpn. Received: 6 December 3 ccepted: 8 Mrch 4 Published: 6 Mrch 4 References. 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11 Sugimoto et l. Nutrition & Metbolism 4, :4 Pge of 6. hren : Glucgon-like peptide- (GLP-): gut hormone of potentil interest in the tretment of dibetes. ioessys 998, : Suzuki K, Jysen CN, loom SR: Obesity nd ppetite control. Exp Dibetes Res, : Smll CJ, loom SR: Gut hormones s peripherl nti obesity trgets. Curr Drug Trgets CNS Neurol Disord 4, 3: Lee, Ptrick P, Wishrt J, Horowitz M, Morley JE: The effects of miglitol on glucgon-like peptide- secretion nd ppetite senstions in obese type dibetics. Dibetes Obes Metb, 4: rkw M, Ebto C, Mit T, Fujitni Y, Shimizu T, Wtd H, Kwmori R, Hirose T: Miglitol suppresses the postprndil increse in interleukin 6 nd enhnces ctive glucgon-like peptide secretion in viscerlly obese subjects. Metbol 8, 57: Nrit T, Yokoym H, Ymshit R, Sto T, Hosob M, Morii T, Fujit H, TsukiymK,YmdY:Comprisonsoftheeffectsof -week dministrtion of miglitol nd voglibose on the responses of plsm incretins fter mixed mel in Jpnese type dibetic ptients. Dibetes Obes Metb, 4: rch JR, insworth T, Ellis RD, Piercy V, Thody VE, Thurlby PL, Wilson C, Wilson S, Young P: Tretment of obesity with thermogenic betdrenoceptor gonists: studies on RL 683 in rodents. Int J Obes 984, 8(Suppl ):. 33. Kto H, Ohue M, Kto K, Nomur, Toyosw K, Furutni Y, Kimur S, Kdowki T: Mechnism of meliortion of insulin resistnce by bet3- drenoceptor gonist J-9677 in the KK-y/T dibetic obese mouse model. Dibetes, 5: Perrone MG, Scilimti : β3-drenoceptor lignd development history through ptent review. Expert Opin Ther Pt, : doi:.86/ Cite this rticle s: Sugimoto et l.: Miglitol increses energy expenditure by upregulting uncoupling protein of brown dipose tissue nd reduces obesity in dietry-induced obese mice. Nutrition & Metbolism 4 :4. Submit your next mnuscript to iomed Centrl nd tke full dvntge of: Convenient online submission Thorough peer review No spce constrints or color figure chrges Immedite publiction on cceptnce Inclusion in PubMed, CS, Scopus nd Google Scholr Reserch which is freely vilble for redistribution Submit your mnuscript t