Potential Effects of Aerobic Exercise on the Expression of Perilipin 3 in the Adipose Tissue of Women with Polycystic Ovary Syndrome: a Pilot Study

J D Covington and others Exercise induces adipose PLIN3 172:1 47–58 Clinical Study expression in women with PCOS

Potential effects of aerobic exercise on the expression of perilipin 3 in the adipose tissue of women with polycystic ovary syndrome: a pilot study

Jeffrey D Covington, Sudip Bajpeyi1, Cedric Moro2, Yourka D Tchoukalova, Philip J Ebenezer, David H Burk, Eric Ravussin and Leanne M Redman Correspondence Laboratory of Reproductive Endocrinology and Woman’s Health, Pennington Biomedical Research Center, should be addressed 6400 Perkins Road, Baton Rouge, Louisiana 70808, USA, 1Department of Kinesiology, University of Texas in El Paso, to L M Redman 500 University Avenue, El Paso, Texas 79968, USA and 2Inserm UMR 1048, Institute of Metabolic and Cardiovascular Email Diseases and Paul Sabatier University, Toulouse, France [email protected]

Abstract

Objective: Polycystic ovary syndrome (PCOS) is associated with reduced adipose tissue lipolysis that can be rescued by aerobic exercise. We aimed to identify differences in the gene expression of perilipins and associated targets in adipose tissue in women with PCOS before and after exercise. Design and methods: We conducted a cross-sectional study in eight women with PCOS and eight women matched for BMI and age with normal cycles. Women with PCOS also completed a 16-week prospective aerobic exercise-training study. Abdominal subcutaneous adipose tissue biopsies were collected, and primary adipose-derived stromal/stem cell cultures were established from women with PCOS before 16 weeks of aerobic exercise training (nZ5) and controls (nZ5). Gene expression was measured using real-time PCR, in vitro lipolysis was measured using radiolabeled oleate, and perilipin 3 (PLIN3) protein content was measured by western blotting analysis. Results: The expression of PLIN1, PLIN3, and PLIN5, along with coatomers ARF1, ARFRP1, and bCOP was w80% lower in European Journal of Endocrinology women with PCOS (all P!0.05). Following exercise training, PLIN3 was the only perilipin to increase signiﬁcantly (P!0.05), along with coatomers ARF1, ARFRP1, bCOP, and SEC23A (all P!0.05). Furthermore, PLIN3 protein expression was undetectable in the cell cultures from women with PCOS vs controls. Following exercise training, in vitro adipose oleate oxidation, glycerol secretion, and PLIN3 protein expression were increased, along with reductions in triglyceride content and absence of large lipid droplet morphology. Conclusions: These ﬁndings suggest that PLIN3 and coatomer GTPases are important regulators of lipolysis and triglyceride storage in the adipose tissue of women with PCOS.

European Journal of Endocrinology (2015) 172, 47–58

Introduction

Polycystic ovary syndrome (PCOS) is a complex endocrine have increased adiposity (4), and between 20 and 43% have and reproductive disorder affecting w4–7% of women of insulin resistance and reduced glucose control (5, 6).One reproductive age (1, 2). As a principle cause of infertility possible culprit speculated to contribute to this irregular met- in reproductive aged women, PCOS is characterized by the abolic phenomena is defects within the adipose tissue (7). presence of menstrual disturbances, hyperandrogenemia, Adipose tissue functions as a storage reservoir for and ovarian cysts (3). Similarly, w70% of women with PCOS excess lipid, a reserve that must be readily lipolyzed upon

www.eje-online.org Ñ 2015 European Society of Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/EJE-14-0492 Printed in Great Britain

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increased energy demand. Inefﬁcient ability of the adipose GTPases from adipose tissue in a cross-sectional cohort of tissue to mobilize and secrete free fatty acids during eight women with PCOS compared with women with conditions of energy demand has been linked with the normal menses matched for age, BMI, and percent body conditions of impaired glucose tolerance and type 2 fat. Furthermore, in the women with PCOS, we investigated diabetes (8, 9, 10). It has been previously documented the effects of a 16-week aerobic exercise training program on that women with PCOS have dysfunctions in lipase acti- lipolysis both in adipose tissue and in stromal-derived vity and catecholamine-mediated lipolysis (11, 12, 13). adipose cultures. Our results indicate previously the unex- Furthermore it has been reported that testosterone can plored potential roles for PLIN3 and coatomers (ARF1, reduce: i) catecholamine-stimulated adipose tissue lipo- ARFRP1, b-COP1, and coatomer complex 2 subunit 23a lysis (14, 15); ii) expression of b-adrenergic receptors (Sec23a)) in the regulation of lipolysis in the adipose tissue. in adipose tissue (14, 15); and iii) adrenergic stimulated lipolysis in the brown adipose tissue (16). Possible molecular targets, such as the expression of lipases, have Materials and methods been implicated as factors regulated by testosterone Participants and study design (15, 17, 18), thus offering a possible relationship between impaired lipolysis and the hyperadrogenemic states seen Eight obese women with PCOS and eight age-, BMI-, and in women with PCOS. percent fat-matched healthy women without clinical signs Exercise training has been shown to improve several of abnormal menses or hyperandrogenemia were recruited metabolic impairments typical of PCOS (19, 20, 21), and in this study (anthropometric characteristics are provided has been shown to enhance adipose tissue lipolysis in Table 1). The results reported for this investigation were (22, 23). Previous studies from our group revealed an an ancillary project to a clinical study, which was designed increase in basal and pharmacologically stimulated and powered to determine the effect of an aerobic exercise lipolysis with isoproterenol from the adipose tissue program on body composition and whole-body insulin following exercise training (24). The molecular markers resistance in obese women diagnosed with PCOS. The that regulate lipolysis in the adipose tissue of women with original study was powered using the mean and S.D.of PCOS however have not been thoroughly studied. glucose disposal rate (GDR) to estimate sample sizes Prior investigations have shown that a single- required to measure a minimum 20% change in GDR nucleotide polymorphism within the perilipin gene from baseline. With a prospective study design (paired) (PLIN1) that exists in women with PCOS is associated with a target power of 80% and a signiﬁcance level set to

European Journal of Endocrinology with impaired glucose tolerance and increased LDL (25), aZ0.05, we can conclude that only six subjects would perhaps implicating a potential role for the perilipin family be needed to detect a 20% change in GDR from baseline. of proteins, lipases, and related factors affecting adipose The main outcomes of this study were previously reported tissue lipolysis. Recent investigations have also identified in Moro et al. (24) and in Redman et al. (28). The diagnosis several coatomer GTPase proteins (ADP-ribosylation of PCOS was assessed by the Rotterdam criteria (3, 29). factor 1 (ARF1), SEC23A, coatomer complex 1 subunit Women with PCOS had to possess two of the following beta (b-COP1), GBF1, and ARF-related peptide 1 (ARFRP1)), criteria: confirmation by medical history of menstrual which are usually involved in endoplasmic reticulum irregularity (oligo- or amenorrhea), presence of more than (ER)-to-Golgi transport, and which are involved in the ten ovarian follicles of 2–9 mm in diameter as assessed delivery of adipose tissue triglyceride lipase (ATGL) to lipid by magnetic resonance imaging (MRI) or either clinical droplets, particularly those lipid droplets coated by (hirsutism score) or serum measures of androgen excess perilipin 3 (PLIN3) (26). We have recently shown that (elevated free androgen index (FAI)). Other causes of PLIN3 and coatomer GTPases in skeletal muscle tissue and oligomenorrhea (hyperprolactinemia, congenital adrenal in primary human skeletal muscle cultures were associated hyperplasia, Cushing’s syndrome, and hyperthyroidism) with increased fat oxidation following exercise and were excluded by medical history. Women in the control lipolytic stimulation (27). We, therefore, proposed group were excluded from participating in our study for a hypothesis that these novel mediators of lipolysis might exercise training, use of contraceptive medications, also be differentially expressed in the adipose tissue of and menstrual cycle irregularity together with androgen women with PCOS, where lipolysis is impaired and excess. All women in our control group had FAI values upregulated following aerobic exercise training. We investi- below 3.6 as defined as a cut-off value for FAI in the gated the expression of lipases, perilipins, and coatomer assessment of PCOS according to Hahn et al. (30).

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Table 1 Anthropometic, metabolic, and serum markers for both the cross-sectional study between controls and women with PCOS, and the exercise-training intervention in the women with PCOS only. This data reﬂect the ﬁve females with PCOS for whom we were able to obtain primary stromal-derived adipose cultures both before and after exercise intervention.

Women with PCOS P value

Control Pre-exercise Post-exercise Control vs PCOS Pre- vs post-exercise

Age (years) 29.7G12.4 27.0G2.9 – 0.65 – Weight (kg) 78.9G15.7 82.2G18.2 81.4G21.6 0.74 0.69 BMI (kg/m2) 26.5G5.4 30.8G4.2 30.4G5.8 0.17 0.66 Total body fat (%) 29.9G9.8 36.8G5.0 34.8G6.1 0.19 0.21 FM (kg) 23.3G9.3 30.8G11.1 29.3G13 0.23 0.39 FFM (kg) 55.6G15.9 51.4G7.9 52.1G9.0 0.60 0.24 Visceral AT (kg) 1.4G0.8 1.2G0.5 1.1G0.7 0.75 0.64 Subcutaneous AT (kg) 11.4G1.8 10.9G4.8 10.5G5.7 0.86 0.47 IHL (AU) 0.013G0.017 0.13G0.17 0.03G0.05 0.16 0.20 IMCL (soleus, AU) 0.006G0.003 0.006G0.004 0.007G0.005 0.89 0.27 Glucose, fasting (mg/dl) 103.5G28.6 82.2G6.3 89.6G5.5 0.14 0.08 Insulin, fasting (mg/dl) 11.0G10.2 9.8G3.9 12.4G5.6 0.81 0.51 HOMA-IR (AU) 3.4G4.2 2.0G0.8 2.7G1.2 0.50 0.42 GDR/EMBS (mg/kg FFMC17.7) 6.8G4.3 6.3G1.2 7.6G1.6 0.79 0.02 Resting metabolic rate/FFM 30.3G4.8 30.7G5.0 30.0G2.7 0.91 0.62 (kcal/day per kg) Resting RQ 0.80G0.03 0.81G0.02 0.80G0.01 0.37 0.21 Clamp RQ 0.93G0.07 0.90G0.04 0.90G0.05 0.50 0.83 DRQ (clamp vs resting) 0.13G0.07 0.09G0.02 0.10G0.03 0.26 0.72 VO2max (ml/min per kg) 27.7G10.0 29.5G3.0 33.6G4.3 0.71 0.04 Fat cell size (nl) 0.69G0.31 0.68G0.13 0.76G0.25 0.92 0.40 DHEA-S (mg/ml) 135.3G49.6 159.0G73.6 192.0G79.4 0.47 0.02 Testosterone (ng/dl) 35.4G11.7 92.4G41.2 64.8G25.3 0.002 0.61 SHBG (nmol/l) 24.1G4.6 30.9G31.7 34.2G19.3 0.56 0.75 FAI (AU) 1.2G0.6 18.8G14.2 13.9G17.5 0.02 0.67 Number of ovarian cysts 29.4G46.1 97G29.7 119.4G28.6 0.008 0.10 (2–9mm in diameter)*

EMBS, estimated mean body size; FM, fat mass; FFM, fat free mass; GDR, glucose disposal rate; HOMA-IR, Homeostatic model assessment of insulin resistance; RQ, respiratory quotient; SHBG, sex hormone-binding globulin; FAI, free androgen index; AT, adipose tissue; IHL, intrahepatic lipid; IMCL, intramyocellular lipid. European Journal of Endocrinology *Follicles measuring between 2-9mm in diameter were only detectable in 3 of the 8 control participants, but all women with PCOS possessed greater than 10 follicles measuring between 2-9mm in diameter.

In addition, all women in our control group reported stromal–vascular cells. For immunocytochemistry analysis regular menstruation, thus confirming that they did not of lipid droplet comparisons and protein immunoblotting, have PCOS based on the Rotterdam guidelines. The controls were taken from five women with normal potential subjects were excluded from participating in menstrual cycling unrelated to the original study that either group if they: smoked; were taking any medications; were matched for age (24.2G2.3 years, PZ0.37 compared had current or past history of cardiovascular disease; hyper- to women with PCOS) and BMI (26.1G2.5 kg/m2, PZ0.83 tension (O140/90 mmHg); diabetes (type 1 or type 2); compared with women with PCOS) – this was necessary kidney, liver, or heart disease; alcoholism or substance because the collection of stromal-derived adipose cultures abuse; were pregnant or trying to become pregnant; and was not part of the initial study protocol for the control were unable to comply with the exercise training program. subjects. The study design and protocol were approved by Adipose tissue measures are reported in all participants the Institutional Review Board of Pennington Biomedical included in this study. Fat cell size was determined Research Center, and all volunteers gave written informed using osmium fixation and counted using a multisizer consent. This study was registered at clinicaltrials.gov 3 Coulter counter (Beckman Coulter, Brea, CA, USA) as (NCT01150539). previously described (31). In vitro measures of stromal- All women were examined at baseline, and the women derived adipocytes are reported in five women with with PCOS were reexamined after 16 weeks of aerobic PCOS before and after exercise due to the availability of exercise training. At each time-point, study testing adipose tissue material for the isolation and culturing of occurred over 3 days. In order to reduce confounding

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factors associated with dietary differences and metabolic T1 inversion recovery (STIR), and T1-weighted testing, we provided participants with a standard diet of localizer), sagittal (T1-weighted localizer only), and axial 50% carbohydrate, 35% fat, and 15% protein 2 days before (T2-weighted FSE and STIR) sequences. Images were testing and throughout the duration of testing. Following acquired from the highest point of the uterus or ovaries an overnight fast, blood and subcutaneous abdominal through the bottom of the ovaries with 4-mm thick slices adipose tissue samples were collected, body composition and 1 mm intersection gap for a total of w40 images. (percent body fat surmised from all regions of the body) Images were analyzed using Analyze 8.1 (CNSoftware) by was assessed by dual-energy X-ray absorptiometry (DXA, a trained analyst. The ovaries, as well as ovarian follicles, QDR 4500A; Hologics, Bedford, MA, USA), and insulin were located and quantiﬁed on each coronal FSE image. sensitivity was determined by a hyperinsulinemic– The follicles were located on an image and followed down euglycemic clamp (120-min at 80 mU/min per m2)as through the remaining consecutive images to ensure that

previously described (28). Aerobic capacity (VO2max) was each individual follicle was identiﬁed as such. The total measured during a graded treadmill test (TrueMax 2400; number of follicles in each ovary was calculated using this ParvoMedics, Salt Lake City, UT, USA). Serum testosterone technique. The volume of each follicle was calculated and sex hormone-binding globulin (SHBG) were deter- based on the number of pixels measured in each image mined by an automated chemiluminescent immunoassay and the number of consecutive images in which the follicle on the Immulite 2000 (Siemens Healthcare Diagnostics, was located. Deerﬁeld, IL, USA). Serum glucose and serum insulin were measured by an enzymatic assay on a Beckman Coulter SAT biopsy DXC 600 (Beckman Coulter). Ovarian morphology, abdominal subcutaneous adipose tissue (SAT), and visceral SAT was obtained from the left upper quadrant of the adipose tissue (VAT) as well as intrahepatic lipid and abdomen with a 5-mm Bergstrom needle using the intramyocellular lipid (IMCL) of the soleus were examined Bergstrom technique. The skin was cleansed with povi- before and after 16 weeks of aerobic exercise training using done-iodine solution, a sterile drape was placed over a 3T MRI/MRS (GE 3.0T Signa EXCITE MRI, GE Healthcare, the incision site, and local anesthesia (5 ml 1:1 mixture Pittsburgh, PA, USA; results provided in Table 1). of 0.5% bupivacaine and 2% lidocaine) was administered. An w1 cm incision was made and adipose tissue was collected. MRI and spectroscopy

European Journal of Endocrinology Abdominal fat: an 8-channel torso-array coil was placed Exercise training program over the chest/abdomen area and 3.4 mm slices (1.7 mm intersection gap) were acquired from the highest point of Aerobic exercise was performed under supervision at the the liver to the inferior pole of the right kidney. A total of Pennington Health and Fitness Center, five times per w220 images were acquired on each participant. Total week. Aerobic exercise was prescribed on an individual adipose tissue, VAT, and subcutaneous abdominal adipose basis with the objective to achieve specified exercise tissue masses were calculated using the Analyze Software energy expenditure (ExEE) in each session. During the Package (CNSoftware, Rochester, MN, USA). Muscle and first 4 weeks, the target ExEE was 4% of the participants’ hepatic lipid content: for the muscle IMCL measures, the estimated energy requirement for weight maintenance, right leg was positioned inside a 1H knee coil with the knee and was incremented to 6% for weeks 5–8, to 8% for weeks in extension and the ankle in a neutral position. Separate 9–12, and to 10% for weeks 13–16. All exercises were

water-suppressed PRESS boxes (10!7.5!7.5 mm voxels) performed on a treadmill at 55% VO2max, a moderate were collected from the tibialis anterior (nZ1), soleus intensity. The necessary speed and gradient to achieve the

(nZ3), and peanut oil phantom (nZ1). For the liver lipid ExEE were estimated from a linear regression of O2 uptake 1 content, with the participant lying prone, a H body coil and workload during the VO2max test, and the ExEE was was placed over the torso and a single PRESS box (30!30! conﬁrmed once during each 4-week interval by indirect 30 mm) was collected in an area of the liver free calorimetry. Heart rate was monitored during all sessions from heavy vascularization. Data were analyzed using to verify ExEE. The exercise time necessary to complete the jMRUi Software Package (Lyon, France). Ovarian the energy expenditure target was 23G1min/session morphology: an 8-channel torso array coil was used to during weeks 1–4, 35G1 min/session during weeks 5–8, acquire coronal (T2-weighted fast spin echo (FSE), short 47G2 min/session during weeks 9–12, and 58G2 min/

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session during weeks 13–16. In order to prevent variation converted into cDNA using the High-Capacity cDNA RT in results due to dietary influences during the exercise Kit (Applied Biosystems) and stored at K20 8C until real- intervention, we asked participants to maintain their time PCR was performed. Gene expression was carried out dietary habits throughout the 16-week intervention as using real-time PCR with TaqMan gene expression assays they had before study enrollment. In addition, we on demand on the ABI 7900HT Fast Real-Time PCR System requested updates on food intake during site visits for (Applied Biosystems). Gene expression assays were per- exercise sessions. formed for the following genes: PLIN1 (Hs00160173_m1), PLIN2 (Hs00765634_m1), PLIN3 (Hs00998421_m1), PLIN4 (Hs00287411_m1), PLIN5 (Hs00965990_m1), GBF1 Stromal-derived adipocyte cultures, (Hs00188327_m1), ARF1 (Hs00796826_s1), ARFRP1 immunofluorescence staining, lipolysis, (Hs00182389_m1), COP (Hs00200674_m1), SEC23A triglyceride determination, and oleate oxidation (Hs00197232_m1), ATGL (Hs00386101_m1), CGI58/ABHD5 Adipose tissue samples from subcutaneous abdominal (Hs01104373_m1), MGL (Hs00200752_m1), DGAT2 depot were collected under aseptic conditions and isolated (Hs01045913_m1), mtGPAT (Hs00326039_m1), and PPIA stromovascular (SV) cells were cultured as previously (Hs99999904_m1). Relative gene expression was assessed described (32) and differentiated as described previously using a standard curve of known concentrations of mRNA (33) with modifications: DMEM–F12 (1:1) medium plus and normalized for cyclophilin A (PPIA) gene expression. 10 mg/ml transferrin, 33 mMbiotin,17mMcalcium Protein extracts from adipose culture were immuno- pantothenate, 0.5 mMinsulin,0.1mM dexamethasone, blotted and probed with an antibody against PLIN3 0.2 nM triiodothyronine, as well as 0.5 mM roziglitazone (Novus Biologicals, Littleton, CO, USA) and normalized and 540 mM 3-isobutyl-1-methylxanthine (IBMX) during against the loading control GAPDH (Abcam, Cambridge, the last 48 h of culture. All experiments were performed on MA, USA). cells following 9 days of differentiation. The cultures were stained for lipids (BODIPY, 10 mg/ml) and DNA Statistical analyses (4,6-diamidino-2-phenylindole (DAPI), 300 nM) (Invi- trogen). The images were obtained using the Leica TCS All analyses were performed using GraphPad Prism Soft- SP5 AOBS resonant scanning confocal microscope (Leica ware, version 5.0 (GraphPad Software, La Jolla, CA, USA). AG, Wetzlar, Germany). The lipolysis assay was performed The Mann–Whitney U test was used for cross-sectional over 3 h by adding 0.2 ml of HBSSCBSA 2%. At the end of comparisons between PCOS and obese control women

European Journal of Endocrinology incubation, the medium was collected for the measure- when data were not normally distributed, and an indepen- ment of glycerol, performed in duplicate using the free dent samples t-test was used when the data were normally glycerol reagent (Sigma–Aldrich), and adjusted for the distributed. The paired t-tests were used to compare triglyceride content. Triglyceride levels were then variables from baseline with the end of the exercise training measured using a glycerol phosphate oxidase triglyceride intervention, except when the data were not normally determination kit (Sigma–Aldrich) and normalized accord- distributed, Wilcoxon signed-ranked paired tests were ing to the protein content. The cultures were preincubated used. P!0.05 was considered statistically signiﬁcant. All 14 for 3 h with [1- C] oleic acid (1 mCi/ml; PerkinElmer, graphical data are presented as meanGS.E.M. Boston, MA, USA) and non-labeled (cold) oleic acid (100 mM). Oleic acid was coupled with fatty acid: free BSA 14 in a molar ratio of 5:1. Following incubation, CO2 was Results measured as described previously (34). The cells were Comparison of controls vs women with PCOS then lysed in 0.2 ml of 0.1% SDS for the determination of cell-associated label uptake and protein content for Compared with the control participants, women with normalization. All assays were performed in duplicate. PCOS had elevated serum testosterone and FAI, as expected, but they did not differ in terms of SHBG (Table 1). Otherwise, control participants were effectively Real-time qRT-PCR and western blotting matched with women with PCOS for body composition, Total RNA was extracted from w100 mg of adipose insulin sensitivity (fasting glucose, fasting insulin, GDR, tissue using the miRNEasy Kits (Qiagen) according to and HOMA-IR), and energy metabolism (resting meta- the manufacturer’s speciﬁcations. RNA extracts were bolic rate, fasting respiratory quotient (RQ), metabolic

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ﬂexibility (i.e. changes between the fasting RQ and RQ ARFRP1, and bCOP (Fig. 1B) were w80% lower in during the steady state of the hyperinsulinemic–euglyce- women with PCOS compared with controls, with GBF1

mic clamp), and VO2max)(Table 1). Importantly, women and SEC23A being expressed approximately nine- and with PCOS did not differ from control participants with sevenfold higher respectively. Finally, lipase mRNA regard to mean subcutaneous fat cell size (Table 1). expression of ATGL and monoglycerol lipase (MGL) was Women with PCOS did however differ signiﬁcantly reduced by 90 and 65%, respectively, with no difference in from controls in terms of SAT gene expression for the the expression of CGI58 (Fig. 1C). candidates responsible for lipolysis, lipid droplet perilipin proteins, and coatomer proteins. As shown in Fig. 1A, Effect of 16-week aerobic exercise training in PLIN1, PLIN3, and PLIN5 were w80–90% lower in women women with PCOS with PCOS when compared with controls, while PLIN2 and PLIN4 were approximately threefold higher. Aerobic exercise resulted in a w10% improvement in Z w Expression mRNA levels of coatomer GTPases ARF1, maximal aerobic capacity (VO2max, P 0.04) and a 20% improvement in GDR (PZ0.02) (Table 1). No other changes A 5 were noted in terms of body composition, abdominal Control adipose depot size (visceral or subcutaneous depots), 4 *** PCOS * ectopic lipid accumulation (in the liver or muscle), or in 3 fat cell size (Table 1). However, alterations were seen in the 2 gene expression in the adipose tissue following exercise. 1.0 Of all the measured perilipins, only expression of PLIN3 increased signiﬁcantly (P!0.05, Fig. 2A), whereas PLIN1,

mRNA expression 0.5 PLIN2, PLIN4, and PLIN5 did not change. On average, expression of ARF1, ARFRP1, bCOP,andSEC23A was *** *** *** increased by approximately ﬁve-, eight-, seven-, and 0.0 fourfold, respectively (P!0.05), with no changes in GBF1 PLIN1 PLIN2 PLIN3 PLIN4 PLIN5

B 15 ** Control 10 PCOS Figure 1

European Journal of Endocrinology ** Gene expression from abdominal subcutaneous adipose tissue 5 from women with PCOS vs age, BMI, and percent fat matched controls (cross-sectional study). (A) Perilipin family of protein 1.0 gene expression of all ﬁve perilipin proteins shows drastic differences in expression. Perilipin 1 (PLIN1), PLIN3, and PLIN5 mRNA expression 0.5 are all expressed w90% lower in females with PCOS compared *** *** *** to controls. PLIN2 and PLIN4 are expressed about three times 0.0 higher when compared with controls. (B) Coatomer proteins β GBF1 ARF1 ARFRP1 COP Sec23a involved in ER-to-Golgi transport were also differentially regulated on the gene expression level. ADP-ribosylation factor C 2.0 1 (ARF1), ARF-related protein 1 (ARFRP1), and beta-coatomer Control PCOS (bCOP, part of the COPI complex) were drastically reduced in 1.5 women with PCOS. GDP-exchange brefeldin A resistant factor 1 (GBF1, which operated in conjunction with ARF1) and Sec23a 1.0 (part of the COPII complex) were both expressed approximately eightfold higher in the women with PCOS. (C) Expression of adipose triglyceride lipase (ATGL) and monoglyceride lipase

mRNA expression 0.5 *** (MGL) were both reduced in women with PCOS on the gene *** expression level, while there was no statistical difference in the 0.0 expression of the ATGL co-activator CGI58. *P!0.05; **P!0.01; ATGL CGI58 MGL and ***P!0.001.

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A 8 (Fig. 2B). In addition, the lipases ATGL, MGL, and the ATGL Pre-exercise Post-exercise co-activator CGI58 increased signiﬁcantly after 16-week exercise training (P!0.05, Fig. 2C). 6

4 Primary adipocyte culture lipid droplet morphology and lipolysis *

mRNA expression 2 As shown in Fig. 3, primary adipocyte cultures revealed a larger lipid droplet morphology in women with PCOS 0 compared with controls, which decreased in size after PLIN1 PLIN2 PLIN3 PLIN4 PLIN5 exercise training, and thus more closely resembled the In vitro B 15 morphology of control females (Fig. 3). measures Pre-exercise of fat oxidation and lipolysis in primary cultured Post-exercise adipocytes showed that oleate oxidation and glycerol * 10 release were increased following exercise training * ! * (P 0.05, Fig. 4A and B). Likewise, total triglyceride content was reduced after exercise (P!0.05, Fig. 4C). 5 * mRNA expression Control

0 GBF1 ARF1 ARFRP1 βCOP Sec23a

C 15 Pre-exercise Post-exercise * 10

* 5

European Journal of Endocrinology * PCOS mRNA expression Pre-exercise Post-exercise

0 ATGL CGI58 MGL

Figure 2 Gene expression targets in adipose tissue known to be involved in lipid droplet regulation and lipolysis were altered at 16 weeks of exercise training in the women with PCOS (exercise training intervention, women with PCOS only). (A) Gene expression of the perilipin family of proteins from before to after exercise demonstrates that only PLIN3 is signiﬁcantly elevated following Figure 3 exercise training. (B) Coatomer gene expression reveals that Representative images of stromal-derived pre-adipocytes cul- ARF1, ARFRP1, and bCOP, which were grossly decreased when tured and differentiated into adipocytes. Women with PCOS compared with controls before exercise training, revealed an possessed large lipid droplets when compared with control increase in their expression. Sec23a was also increased donors matched by age, BMI, and % fat. Lipid droplet following exercise training. (C) Gene expression of ATGL and morphology appeared to resemble that of controls in our cross- MGL, which was blunted when compared with controls at sectional study following 16 weeks of aerobic exercise training baseline, increased with exercise training. Additionally, CGI58 in women with PCOS. Bodipy 494 (green) was used to identify increased following exercise training. *P!0.05. lipid and DAPI (blue) to identify nuclei.

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A BC

0.08 15 0.15 * * 0.06 0.10 10

release 0.04 * 2 0.05 5 CO 0.02 14 Glycerol release Triglyceride content

(nmol/h per mg protein) 0.00 0.00 0 Complete oleate oxidation Pre-exercise Post-exercise Pre-exercise Post-exercise Pre-exercise Post-exercise

D PCOS PCOS PCOS PCOS PCOS Ctr Pre Post Ctr Pre Post Ctr Pre Post Ctr Ctr Pre Post Pre Post PLIN3

GAPDH

Figure 4 Ex vivo oleate oxidation studies revealed that stromal-derived perilipin protein to increase in adipose tissue in the women with adipose cultures from women with PCOS had increased PCOS following exercise training on the gene expression level, 14 completed oleate oxidation as measured by C labeled CO2 were found to be virtually nonexistent in four out of the (A) after 16 weeks of exercise training. Furthermore, glycerol ﬁve females with PCOS donors when compared with controls release into the culture media was increased (B) and total in their primary adipocyte cultures. Likewise, expression levels triglyceride content was reduced (C) in the adipose cultures of increased in all ﬁve females with PCOS after exercise training women with PCOS after exercise training. (D) Both cross- in their primary adipose cultures. *P!0.05. sectional and exercise intervention expression of PLIN3, the only

As PLIN3 was the only perilipin to significantly increase with increases in oleate oxidation. We previously reported in adipose tissue on the mRNA level, we measured that exercise training in this cohort increased adipose the protein level of PLIN3 in primary adipocyte culture. tissue lipolysis under adrenergic stimulation (24). Our data We found virtually nonexistent levels of PLIN3 in four out suggest that PLIN3 may in part contribute to enhanced of five women with PCOS, while the expression of PLIN3 adipose tissue lipolytic stimulation. was present in controls (Fig. 4D). Exercise-induced protein The exercise benefits associated with improving the European Journal of Endocrinology expression of PLIN3 in those four women with PCOS, symptomatology of PCOS has been thoroughly investi- who had no protein expression of PLIN3 before exercise, gated. Studies have shown improvements for women with and was increased compared with pre-exercise expression PCOS in insulin resistance (19, 20, 35, 36), serum lipids in the one woman with PCOS, who had PLIN3 protein (35, 37), and cardiovascular disease risk (19, 20, 35, 38).In expression present before exercise training (Fig. 4D). addition, menstrual cycle improvements have been shown following exercise intervention in women with PCOS Discussion (19, 35, 39). Though some studies show reductions in body Our data highlight a previously unrecognized, potential weight with exercise (19, 20, 35), several studies have role of PLIN3 in adipose tissue lipolysis in women with shown that benefits occur without weight loss (28, 36, 40) PCOS. We show for the first time that the expression of and suggest that exercise for PCOS would be rec- PLIN3, along with PLIN1 and PLIN5, is greatly reduced in ommended even if weight loss were not achieved. Our the adipose tissue of women with PCOS when compared study showed improvements in insulin resistance as with females matched for age, body composition, and measured by euglycemic–hyperinsulinemic clamps metabolic phenotype. PLIN3 mRNA expression is (Table 1) and improvements in menstrual function (28). increased – the only perilipin protein to significantly However, despite all these benefits of exercise, few studies increase – following 16-week aerobic exercise training in have investigated molecular targets in the adipose tissue women with PCOS. In addition, stromal-derived primary from women with PCOS. This seems important given the adipose cultures from PCOS women revealed that virtually reported defects in adipose tissue function previously no PLIN3 protein is expressed before exercise training, but showninwomenwithPCOS(reviewedin(7)). We becomes expressed following exercise training coupled previously reported that 16 weeks of aerobic exercise can

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improve basal and catacholemine stimulated lipolysis of (48) found differential expression of coatomer GTPases adipose tissue (24). A prior investigation showed a single- with regards to differences in lipid droplet morphology. nucleotide polymorphism found in women with PCOS for Likewise, we show that independent of differences in fat the perilipin gene (25), indicating a potential for defects cell size, women with PCOS possess larger lipid droplet in the perilipin family of proteins, which are involved in morphology when compared with their age- and body lipolysis. In this study, we have investigated targets that composition-matched controls. Lipid droplet morphology regulate adipose tissue lipolysis, with an emphasis on more closely resembled control females following 16 weeks PLIN3 given the upregulation of gene expression seen in of aerobic exercise in the women with PCOS. Alterations the adipose tissue with exercise. in these coatomer GTPases may perhaps be involved in Studies centered on the perilipin family of proteins the regulation of lipolysis not only as a mediator of lipase have focused heavily on the involvement of PLIN1 in the delivery to lipid droplets, but also as a regulator of lipid regulation of lipolysis in adipose tissue (reviewed in (41)) droplet morphology. Further functional investigations via a coupling of PLIN1 phosphorylation and ATGL are warranted to determine these mechanisms. activation (42, 43). However, the possible role of PLIN3 We also observed reduced mRNA expression of lipases regulation in adipose lipolysis has been for the most part (both ATGL and MGL) in the adipose tissue of women with overlooked. Studies have shown PLIN3 to be highly PCOS compared with controls. The expression of lipases expressed in adipocytes and described to have a preference increased with exercise, which would be expected given for PLIN3 to coat smaller lipid droplets (44). In addition, the increase in both in vivo and in vitro lipolysis. One aspect studies in HeLa cells have revealed a colocalization of of lipase expression that remains elusive is that prior ATGL to with PLIN3-coated lipid droplets during lipolytic investigations have shown that testosterone mediates the stimulus (26, 45). Furthermore, a handful of studies expression of lipases (17, 18). Our data show that lipase investigating the role of PLIN3 in skeletal muscle lipolysis expression is increased following exercise training with no demonstrated that PLIN3 colocalizes to lipid droplets in decreases in circulating concentrations of total testoster- rats during epinephrine and muscle contractile stimu- one or FAI, perhaps indicating that aerobic exercise can lation (46, 47). Work from our group has recently shown increase adipose tissue lipolysis despite altering testo- that PLIN3 expression is upregulated following aerobic sterone expression. Furthermore, we speculate that the exercise in skeletal muscle, upregulated in response to increases in lipase expression we observed are not solely lipolytic stimulation in human primary skeletal myotube responsible for the increased lipolysis following exercise cultures, and is positively associated with both ex vivo training. In fact, previous reports show that lipases do

European Journal of Endocrinology skeletal muscle and in vivo whole body fat oxidation (27). not act alone on lipid droplets to facilitate lipolysis, but Our data, using both in vivo and in vitro systems, show that require a chaperone such as the perilipin proteins the association between PLIN3 and lipolysis is evident (reviewed in (41)) or certain coatomer GTPases (26). in the adipose tissue of women with PCOS, indicating We recognize that our results and conclusions are PLIN3 as a potential novel mediator of lipolysis. based on observational data and associations between Our data also highlight previously uninvestigated increases in lipolysis, and expression of PLIN3 and coat- coatomer GTPases that might potentially be involved in omer GTPases following exercise, and does not establish the mediation of lipolysis. Our study shows a lower causative results. However, given the preponderance of our expression of ARF1, ARFRP1, and bCOP1 in women with ﬁndings and data presented in prior investigations from PCOS compared with controls; the expression of these our group and others, we believe that these associations are targets increases greatly following 16-week aerobic exer- novel and relevant to highlight potentially new, unrecog- cise training in the women with PCOS. Soni et al. (26) nized targets that may in part mediate adipose tissue showed that ATGL was delivered to PLIN3-coated lipid lipolysis. In addition, it is noted that we only conducted the droplets: a phenomenon that was inhibited when either exercise intervention in the women with PCOS, because treated with brefeldin A, a compound known to inhibit our original design was a prospective exercise intervention ARF1, or when several of the coatomer GTPases, such as study for the women with PCOS. Although we did not ARF1, SEC23A, bCOP, and GBF1 were knocked down. conduct an exercise intervention in our control group Studies from our group have shown that ARF1, SEC23A, because of later addition of the cross-section component, and ARFRP1 increase their expression with aerobic the goal of the study involving the control group was to exercise in skeletal muscle and with lipolytic stimulation perform the same set of state-of-the-art assessments (clamp, in primary skeletal muscle cultures (27). Finally, Guo et al. MRI, fat cell size, and DXA) and to obtain a control group

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matched on the basis of metabolic phenotype. The cross- Center (L M Redman) and a Nutrition Obesity Research Center (NORC) sectional comparison demonstrates novel and robust grant NIH 1P30 DK072476 (E Ravussin). This work utilized the Genomics Core Facility and Cell Biology and Bioimaging Core at Pennington differences between the women with PCOS and controls Biomedical Research Center, which are supported in part by COBRE (NIH and is able to further demonstrate that exercise in women P20 GM103528) and NORC (NIH 1P30-DK072476). L M Redman is supported with PCOS can rescue defects in lipolysis to levels observed by NIH grants R00HD060762 and U01DK094418. in control subjects. Owing to our rigorous study design, supervised and carefully monitored exercise interventions, and extensive phenotyping analyses of the women with Author contribution statement J D Covington conceived the experiment, performed experiments, analyzed PCOS before and after their exercise intervention, we are data, and wrote the manuscript; S Bajpeyi contributed to discussion confident that we were adequately able to identify the and reviewed/edited the manuscript; C Moro performed experiments upregulation of PLIN3 gene expression as well as the and reviewed/edited the manuscript; Y D Tchoukalova performed experiments and reviewed/edited the manuscript; P J Ebenezer performed coatomer GTPases, thus demonstrating their potential experiments and reviewed/edited the manuscript; D H Burk performed role in the improvements in adipose tissue lipolysis experiments and reviewed/edited the manuscript; E Ravussin contributed following exercise training. Further investigations would to discussion, conceived of the experiment, and reviewed/edited the manuscript; L M Redman provided helpful discussion, conceived the be necessary to understand specific mechanistic roles of experiment, and reviewed/edited the manuscript. All authors gave final PLIN3 in adipose tissue lipolysis. approval of the manuscript before submission. J D Covington is the In conclusion, based on prior evidence from single- guarantor of this work, and, as such, had full access to all the data in the nucleotide polymorphisms in the perilipin gene (25),we study and takes responsibility for the integrity of the data and the accuracy of the data analysis. investigated the expression of the perilipin family of proteins in the adipose tissue from women with PCOS, and have shown that several perilipin proteins are differentially Acknowledgements expressed in women with PCOS vs age- and body compo- The authors would like to thank the Nursing Staff of Pennington sition-matched control females. We have also shown that Biomedical Research Center. They also wish to thank Jamie LaGrange and coatomer GTPases (ARF1, SEC23a, bCOP, GBF1, and Blaine Masinter for their assistance with Adipose Tissue RNA isolation and thank Richard Carmouche and Susan Newman of the Genomics Core ARFRP1) are differentially expressed in women with PCOS. Facility of Pennington Biomedical Research Center. They also wish to thank A 16-week aerobic exercise training significantly increased the Cell Biology and Cell Imaging Core Facility of Pennington Biomedical PLIN3 expression as well as coatomer GTPases (ARF1, Research Center. Finally, they sincerely thank all the research volunteers for their participation in this study. SEC23A, bCOP, GBF1, and ARFRP1). Stromal-derived primary adipose cultures showed increases in in vitro

European Journal of Endocrinology lipolysis, oleate oxidation, and reductions in triglyceride content following exercise training. In addition, adipose References

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Received 17 June 2014 Revised version received 15 October 2014 Accepted 23 October 2014 European Journal of Endocrinology

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