Supporting Informtion Yun et l. 1.173/pns.1523236113 SI Mterils nd Methods Humn Sujects. All prticipnts in our study were recruited from the Center for Reproductive Medicine, Shndong Provincil Hospitl Affilited to Shndong University, during Septemer 214 to Mrch 215. Anthropometric vriles, such s ge, ody mss index (BMI), menstrul cycle, nd select endocrine nd iochemicl prmeters, were recorded nd re shown in Tle S3. The dignosis of PCOS ws sed on the following revised Rotterdm dignostic criteri for PCOS (37), which require the presence of t lest two of the following: (i) oligoovultion nd/or novultion; (ii) clinicl nd/or iochemicl signs of hyperndrogenism; nd (iii) polycystic ovries. Dignoses of PCOS were mde fter exclusion of other etiologies for hyperndrogenemi nd ovultory dysfunction (e.g., 21-hydroxylse deficiency, congenitl drenl hyperplsi, Cushing syndrome, ndrogen-secreting tumors, thyroid disese, nd hyperprolctinemi). All sujects in the control group hd regulr menstrul cycles nd norml ovrin morphology, nd totl testosterone ws evluted to exclude hyperndrogenism. Peripherl lood smples were collected from ll sujects during dys 2 4 of spontneous cycles fter n overnight fst. FSH, LH, nd testosterone were tested y chemiluminescence immuniztion (Beckmn Access Helth Compny). The hemtologicl iochemicl vriles were mesured t fsting. A 75-g orl glucose tolernce test (OGTT) ws crried out, nd the plsm glucose nd serum insulin t fsting nd 2-h postlod were mesured. The serum ws centrifuglly seprted from procogulnt peripherl lood. Written informed consent ws otined from ll prticipnts nd this study ws pproved y the Institutionl Review Bord of Reproductive Medicine of Shndong University. Animls. Femle nd mle Sprgue Dwley rts (3 wk old) were purchsed from Vitl River Lortory Animl Technology Co Ltd. Five rts per cge were housed under constnt environmentl conditions in the Office of Lortory Animl Welfre-certified niml fcility with 12-h light drk cycle. Food nd wter were provided d liitum. All niml studies were conducted with the pprovl of the Institutionl Animl Cre nd Use Committee of the Institute of Zoology, Chinese Acdemy of Sciences. Estlishment of the PCOS Model nd Assessment of the Estrous Cycle. The DHEA used for estlishing the PCOS model ws purchsed from Yngzhou Phrmceuticl Co., Ltd (ct. no. H19464). From 4 wk of ge, femle rts were injected dily (s.c.) with DHEA (6 mg/1 g ody weight) dissolved in.2 ml of PBS for 2 consecutive dys. rts were injected with.2 ml of PBS for n equivlent length of time. The successful PCOS rt model ws selected ccording to the previous criteri (38), which ws ssessment of estrous cycle y vginl cytology for eight consecutive dys, together with glucose tolernce test (GTT) for the following experiment. Tissue Trnsplnttion. The experiment ws conducted ccording to the methods descried previously (17, 18). The opertion ws crried out in sterile conditions. Age- nd sex-mtched donor nd recipient rts were used for the tissue trnsplnttion experiment. BAT nd prt of the qudriceps were hrvested from the donor rts, which were nesthetized with vertin (4 mg/kg ody weight i.p.), nd were plced in sterile sline. The recipient rts were nesthetized with vertin (4 mg/kg ody weight i.p.), nd the donor tissues (.5 g for ech recipient rt) were trnsplnted into the s.c. spce of the dorsl region djcent to the endogenous BAT s quickly s possile. The shm-operted rts underwent the sme procedure, except receiving donor tissues. Adiponectin Protein Tretment. Recominnt diponectin protein (5636-M8H-1) ws purchsed from Sino Biologicl Inc. PCOS rts were treted with exogenous recominnt diponectin protein (1 μg/kg ody weight/dy i.p.) once dy for 2 consecutive dys. The control group ws treted with sterile PBS. Glucose Tolernce Test nd Insulin Tolernce Test. For glucose tolernce tests (GTTs), femle rts were fsted for 16 h (17 to 9), with free ccess to drinking wter, nd injected with D-glucose (2. g/kg ody weight) intrperitonelly. Blood glucose level ws mesured efore nd 15, 3, 6, 9, nd 12 min fter i.p. glucose injection y using n Accu-Chek glucose monitor (Roche Dignostics Corp.). For the insulin tolernce test (ITT), femle rts were fsted for 4 h (9 to 13), with free ccess to drinking wter, nd injected with insulin (1 U/kg ody weight) (Humulin; Eli Lilly) intrperitonelly. Blood glucose levels were mesured efore nd 15, 3, nd 6 min fter insulin injection. Resting Metolic Rte. The femle rts were housed with one rt per cge, with free ccess to food nd wter. Metolic rte ws determined y oxygen consumption mesurement performed for two consecutive dys using TSE lortory mster system s previously descried (39). Infrred Thermogrphy nd Core Temperture. Rts were exposed to cold chmer (4 C) with one rt per cge for up to 4 h, with free ccess to food nd wter. Imges were tken using n infrred digitl thermogrphic cmer (E6: Compct Infrred Therml Imging Cmer; FLIR) nd were nlyzed using FLIR Quick Report softwre (FLIR ReserchIR Mx 3.4; FLIR). Core ody temperture ws mesured using rectl proe connected to digitl thermometer (Yellow Spring Instruments). Micro PET/CT. PET/CT imging ws chieved with the Siemens Inveon Dedicted PET (dpet) System nd Inveon Multimodlity (MM) System (CT/SPECT) (Siemens Preclinicl Solutions) t the Institute of Lortory Animl Sciences, Chinese Acdemy of Medicl Sciences. Mice were llowed to fst overnight nd were lightly nesthetized with isoflurne, followed y til vein injection of 18 F-FDG (5 mci). Mice were sujected to PET/CT nlysis t 6 min fter rdiotrcer injection. Inveon Acquisition Workplce (IAW) softwre ws used for the scnning process. A 1-min CT X-ry for ttenution correction ws scnned with power of 8 kv nd 5 μa nd n exposure time of 1,1 ms efore PET scn. Ten-minute sttic PET scns were cquired, nd imges were reconstructed y n OSEM3D lgorithm followed y Mximiztion/ Mximum Posteriori (MAP) or FstMAP provided y IAW. The 3D regions of interest (ROIs) were drwn over the guided CT imges, nd the trcer uptke ws mesured using the softwre of Inveon Reserch Workplce (IRW) (Siemens). Individul quntifiction of the 18 F-FDG uptke in ech of the ROIs ws clculted. The dt for the ccumultion of 18 F-FDG on micro PET imges were expressed s the stndrd uptke vlues (SUVs), which were determined y dividing the relevnt ROI concentrtion y the rtio of the injected ctivity to the ody weight. The dt re presented s the men ± SEM. Fertility Assessment. To exmine fertility, femle rts mted with proven stud mles. Next dy, successful mting ws judged y 1of5
oservtion of vginl plug. After 1 d, the few femle rts were killed nd were exmined t implnttion sites to confirm pregnncy. The rest of the nimls were llowed to undergo nturl delivery to produce pups. Blood Anlysis. The lood smples were collected y crdic exsnguintion under Avertin nesthesi, nd the plsm smples were frozen nd stored t 8 C until further nlysis. Rt plsm levels of LH, FSH, nd insulin were nlyzed using ELISA kits (NnJing Jin Cheng Bioengineering Institute). The plsm level of diponectin in oth rt nd humn smples ws nlyzed using ELISA kits (R&D Systems). Gene Expression Anlysis. Totl RNA ws isolted using the RNesy Mini Kit. The cdna ws synthesized using rndom hexmers (Invitrogen) for susequent rel-time quntittive PCR nlysis (ABI Prism VIIA7; Applied Biosystems). PCR products were detected using SYBR Green nd normlized y cyclophilin expression. Primers were designed using Primer Quest (Integrted DNA Technologies). Western Blot Anlysis. Tissues were dissolved in RIPA uffer (15 mm sodium chloride, 1.% Triton X-1,.5% sodium deoxycholte,.1% SDS, 5 mm Tris, protese nd phosphtse inhiitor mixture (Roche Dignostics). Protein concentrtions were determined using BCA ssy kit (Pierce Dignostics). Protein ws seprted y 1% (wt/vol) SDS/PAGE, trnsferred to PVDF memrne (Millipore), locked in 5% (wt/vol) skim milk in TBST (.2 M Tris se,.14 M NCl,.1% Tween 2, ph 7.4), nd incuted with primry ntiodies overnight t 4 C nd then incuted with secondry ntiodies conjugted with HRP. The following primry ntiodies were used: nti-ucp1 (1:1,; Acm), nti- OXPHOS (1:25; Acm), nd nti-gapdh (1:1,; Cell Signling Technology). Signls were detected with super signl west pico chemiluminescent sustrte (Pierce). Histology nd Immunohistochemistry Anlysis. Tissues were fixed in 4% prformldehyde overnight t room temperture nd then emedded in prffin. Sections of 5 μm thickness were stined with hemtoxylin nd eosin (H&E), nd then imges were tken y microscope (DS-RI1; Nikon). The numer of ntrl follicles (6 1, μm in dimeter) nd corpor lute were counted sed on morphology nd dimeter. The thickness of thec cell lyers ws mesured using ImgeJ softwre (version 1.48; NIH) (n = 6 per group, seril sections of ech ovry were used for mesurement). The stndrd streptvidin-iotin-peroxide immunostining procedure ws used for the detection of tyrosine hydroxylse (TH). Tissue specimens were locked with 1% norml got serum for 6 min nd then incuted with nti- UCP1 (1:4 dilution; Snt Cruz Biotechnologies) or nti-th (1:4 dilution; Pel FreeZ) ntiody overnight t 4 C, followed y 1-h incution with HRP-conjugted got nti-rit IgG t room temperture. Sttisticl Anlysis. Comprisons etween groups were mde y one-wy ANOVA with Tukey s post hoc test or Student s t tests. A difference etween groups of P <.5 ws considered significnt. Food intke (KJ/kg/h) 16 14 12 1 8 6 4 2 3 25 DHEA+shm DHEA+Mus DHEA+BAT Body weight(g) 2 15 1 5 DHEA+shm DHEA+Mus DHEA+BAT Fig. S1. Effect of BAT trnsplnttion on ody weight nd food intke. (A) Food intke nd (B) ody weight were not chnged mong groups. n = 8 1 per group. Error rs represent mens ± SEM. 2of5
2 Glucose mm) 15 1 5 15 3 6 9 12 Time fter glucose injection (min) DHEA+shm DHEA+Mus DHEA+BAT 8 Glucose mm) 6 4 2 c Insulin level(mu/l) 6. 4. 2.. 15 3 45 6 Time fter insulin injection (min) Fig. S2. BAT trnsplnttion reverses PCOS metolic normlity. BAT trnsplnttion could significntly reverse DHEA-induced glucose intolernce s evidenced y (A) glucose tolernce test (GTT) nd (B) insulin tolernce test (ITT), s well s insulin levels during GTT (C). Dt were nlyzed y one-wy ANOVA with Tukey s post hoc test. n = 8 1 per group. Different lowercse letters indicte significnt differences mong groups (One-wy ANOVA, with Tukey s post hoc test, P <.5). Humn PCOS diponectin (ng/ml) 9 6 3 Humn PCOS diponectin level ** PCOS Rt Adiponectin (ng/ml) 15 1 5 Rt PCOS diponectin level DHEA+shm DHEA+Mus DHEA+BAT PCOS c O2 consumption (ml/h/kg) 25 2 15 1 5 DHEA+Vehicle d 2 DHEA+AdipoQ 15 O2 consumption (ml/h/kg) 1 5 Fig. S3. Adiponectin tretment increses energy expenditure. Circulting diponectin levels were significntly decresed in oth the PCOS ptient nd rt model compred with their respective controls (A nd B). Dt were nlyzed y unpired t test in A. n = 4 per group. **P <.1 versus control or nlyzed y one-wy ANOVA with Tukey s post hoc test in B. n = 8 1 per group. Different chrcters indicte P <.5. Adiponectin tretment increses oxygen consumption (C nd D). Dt were nlyzed y one-wy ANOVA with Tukey s post hoc test. n = 6 per group. Different lowercse letters indicte significnt differences mong groups (One-wy ANOVA, with Tukey s post hoc test, P <.5). 3of5
Tle S1. The effect of BAT trnsplnttion on estrous cycle Group Totl no. Norml estrous cycle Anorml estrous cycle 8 8 DHEA+shm 9 1 8 DHEA+Mus 9 2 7 DHEA+BAT 1 7 3 Tle S2. Group The effect of BAT trnsplnttion on ovrin phenotype Totl no. No. of corpor lute Thickness of thec cell lyer, μm 6 16 ±.58 19.34 ± 1.2 DHEA+shm 6 6.67 ±.88 36.4 ±.87 DHEA+Mus 6 5.67 ±.33 37.26 ± 2.11 DHEA+BAT 6 14.33 ± 1.2 19.99 ± 1.3 Dt were nlyzed y one-wy ANOVA with Tukey s post hoc test. Different lowercse letters indicte significnt differences mong groups (One-wy ANOVA, with Tukey s post hoc test, P <.5). Tle S3. Fertility ssessment in tissue trnsplnttion experiment Group Totl no. Pregnncy nd prturition 9 9 DHEA+shm 8 2 DHEA+Mus 7 2 DHEA+BAT 7 6 Tle S4. Clinicl chrcteristics of PCOS ptient compred with control suject Vrile PCOS P No. 4 4 Age, y 29.35 ± 4.4 29.38 ± 4.3.98 BMI, kg/m 2 27.36 ± 5.19 28.34 ± 4.63.379 FSH, miu/ml 5.61 ± 1.514 6.51 ± 1.42 <.1 LH, miu/ml 1.9 ± 5.94 4.37 ± 1.9.8 T, ng/dl 45.44 ± 16.4 28.9 ± 13.54 <.1 E2, pg/ml 44.47 ± 21.7 29.82 ± 15.8.1 min Glu, mmol/l 5.58 ±.65 5.83 ±.97.21 12 min Glu, mmol/l 6.95 ± 2.6 6.17 ± 2.5.11 min INS, mu/l 2.65 ± 19.67 16.43 ± 6.16.25 12 min INS, miu/l 16.48 ± 13.3 61.4 ± 63.81.25 Dt represent the men ± SD. BMI, ody mss index; E2, estrdiol; FSH, follicle-stimulting hormone; Glu, glucose; INS, insulin ; LH, luteinizing hormone; T, testosterone. P <.5 compred with the control group; P vlues were determined y Student s t test. Tle S5. Group The effect of diponectin tretment on estrous cycle nd ovrin phenotype Totl no. Norml estrous cycle Anorml estrous cycle No. of corpor lute Thickness of thec cell lyer, μm Pregnncy nd prturition 6 6 14.33 ±.67 19.25 ± 1.1 6 DHEA+Vehicle 6 1 5 6.67 ± 1.2 36.54 ± 1.23 1 DHEA+dipoQ 6 4 2 11.67 ± 1.2 19.6 ±.95 4 Dt were nlyzed y one-wy ANOVA with Tukey s post hoc test. Different lowercse letters indicte significnt differences mong groups (One-wy ANOVA, with Tukey s post hoc test, P <.5). 4of5
Tle S6. Hormonl profiles in tissue trnsplnttion experiment Vrile (n = 8) DHEA+Shm (n = 9) DHEA+Mus (n = 9) DHEA+BAT (n = 1) T3, pmol/l 4.48 ±.28 4.4 ±.331 4.24 ±.151 4.45 ±.251 T4, pmol/l 32.74 ± 1.454 29.84 ± 1.568 28.3 ± 1.916 29.35 ± 2.542 Estrdiol, pg/ml 31.13 ± 4.689 25.78 ± 1.597 28.14 ± 5.616 24.1 ± 5.177 Progesterone, ng/ml 4. ± 1.2 37.36 ± 1.621 38.61 ± 1.311 36.95 ± 1.61 Testosterone, ng/ml 2.41 ±.68 2.79 ±.136 2.79 ±.77 2.35 ±.133 CHO, mmol/l 1.34 ±.22 1.51 ±.116 1.33 ±.17 1.6 ±.114 TG, mmol/l.28 ±.32.4 ±.44.38 ±.43.28 ±.44 HDL, mmol/l 1.55 ±.174 1.34 ±.22 1.11 ±.79 1.15 ±.72 LDL, mmol/l.12 ±.16.14 ±.14.13 ±.17.12 ±.12 NGF, pg/ml 47.11 ± 5.658 48.42 ± 5.86 49.33 ± 8.42 55.35 ± 7.548 FGF21, pg/ml 45.5 ± 7.91 47.12 ± 8.1 5.17 ± 11.78 56.79 ± 1.726 Dt were nlyzed y one-wy ANOVA with Tukey s post hoc test. Different lowercse letters indicte significnt differences mong groups (One-wy ANOVA, with Tukey s post hoc test, P <.5). CHO, cholesterol; FGF21, firolst growth fctor 21; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NGF, nerve growth fctor; T3, triiodothyronine; T4, thyroxine; TG, triglyceride. 5of5