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Obesity

Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

GLUT4 Translocation is not Impaired After Acute Exercise in Skeletal Muscle of Women with Obesity and Polycystic Ovary Syndrome Wagner Silva Dantas1, Jos e Antonio Miguel Marcondes2, Samuel Katsuyuki Shinjo3, Luiz Augusto Perandini3, 4 Vanessa Olzon Zambelli , Willian Das Neves1, Cristiano Roberto Grimaldi Barcellos2, Michele Patrocınio Rocha2, a Pinto3, Viviane Dos Reis Vieira Yance2, Renato Tavares Dos Santos Pereira1, Igor Hisashi Murai1, Ana Lucia De S 1,3 1,3 Hamilton Roschel , and Bruno Gualano

Objective: The aim of this study was to examine the effects of acute exercise on insulin signaling in skeletal muscle of women with polycystic ovary syndrome (PCOS) and controls (CTRL). Methods: Fifteen women with obesity and PCOS and 12 body mass index-matched CTRL participated in this study. Subjects performed a 40-min single bout of exercise. Muscle biopsies were performed before and 60 min after exercise. Selected proteins were assessed by Western blotting. Results: CTRL, but not PCOS, showed a significant increase in PI3-k p85 and AS160 Thr 642 after a single bout of exercise (P 5 0.018 and P 5 0.018, respectively). Only PCOS showed an increase in Akt Thr 308 and AMPK phosphorylation after exercise (P 5 0.018 and P 5 0.018, respectively). Total GLUT4 expression was comparable between groups (P > 0.05). GLUT4 translocation tended to be significantly higher in both groups after exercise (PCOS: P 5 0.093; CTRL: P 5 0.091), with no significant difference between them (P > 0.05). Conclusions: A single bout of exercise elicited similar GLUT4 translocation in skeletal muscle of PCOS and CTRL, despite a slightly differential pattern of protein phosphorylation. The absence of impairment in GLUT4 translocation suggests that PCOS patients with obesity and insulin resistance may benefit from exercise training. Obesity (2015) 23, 2207-2215. doi:10.1002/oby.21217

Introduction Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by chronic anovulation and hyperandrogenism, affecting between 11% and 18% of women at reproductive age (1). A significant number of women also experience insulin resistance (IR; 2) and metabolic disturbances (3,4), increasing the risk of developing type 2 diabetes mellitus (T2DM; 3). IR in PCOS appears to result from a post-binding defect in insulin signaling, and skeletal muscle IR has been considered the leading cause of T2DM in these patients (4). In fact, impaired insulinstimulated phosphorylation of protein kinase B (Akt) and its 160 kDa substrate (AS160) was shown in skeletal muscle from PCOS patients (5). Furthermore, an increased phosphorylation of insulin receptor substrate type 1 (IRS-1) on Ser312 (i.e., a key inhibitory site) was demonstrated in PCOS skeletal muscle (6,7).

Physical exercise is one of the most important therapeutic strategies in the management of conditions characterized by IR (8). A number of experimental and clinical studies clearly support the role of muscle contraction in stimulating glucose uptake by enhancing the insulin-mediated molecular pathway and stimulating GLUT4 translocation via insulin-independent pathways (9-11). The effect of exercise training upon glucose uptake has been extensively examined in patients with obesity (12); however, studies involving PCOS patients are scarce. In fact, short-term exercise training has been shown to improve insulin sensitivity in PCOS patients (13,14). An intriguing finding is that the improvement experienced by PCOS patients was suboptimal when compared with that experienced by their control peers (14). In this respect, exercise training was shown to improve IR in overweight women with or without PCOS, but insulin sensitivity remained worse in PCOS compared with controls after the intervention. It has been speculated that persistent IR following exercise training in PCOS could be related to the aforementioned defects in

1 School of Physical Education and Sport, Department of Biodynamic of Human Movement, University of Sao Paulo, Brazil. Correspondence: Bruno Gualano ([email protected]) 2 Endocrinology Division, School of Medicine, University of Sao Paulo, Brazil 3 Rheumatology Division, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil 4 Laboratory of Pain and Signaling, Butantan Institute, S~ao Paulo, Brazil.

~o de Amparo a Pesquisa do Estado de Sa ~o Paulo (FAPESP) 2012/02827-7 and 2012/14650-4. Funding agencies: This research was supported by Fundac¸a Disclosure: The authors declared no conflict of interest. Additional Supporting Information may be found in the online version of this article. Received: 11 March 2015; Accepted: 11 June 2015; Published online 16 September 2015. doi:10.1002/oby.21217

www.obesityjournal.org

Obesity | VOLUME 23 | NUMBER 11 | NOVEMBER 2015

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GLUT4 Translocation, Acute Exercise, and PCOS Dantas et al.

TABLE 1 Clinical, hormonal and metabolic profile in PCOS and controls (CTRL)

PCOS (n 5 15) Clinical profile Age (years) Body mass index (kg/m2) Waist circumference (cm) Fat mass (%) Lean mass (%) Ferriman-Gallwey score Ultrasonography Right ovary volume (cm3) Left ovary volume (cm3) Hormonal profile Total testosterone (ng/dL) Free testosterone (pg/dL) DHEAS (ng/mL) 17-OHP (ng/dL) Androstenedione (ng/mL) Estradiol (pg/mL) FSH (mUI/mL) LH (mUI/mL) SHBG (ng/mL) Prolactin (ng/mL) Metabolic profile Total cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL) VLDL (mg/dL) Oral glucose tolerance test Glucose 0’ (mg/dL) Glucose 30’ (mg/dL) Glucose 60’ (mg/dL) Glucose 90’ (mg/dL) Glucose 120’ (mg/dL) Insulin 0’ (mU/mL) Insulin 30’ (mU/mL) Insulin 60’ (mU/mL) Insulin 90’ (mU/mL) Insulin 120’ (mU/mL) AUC glucose (mg/dL/min) AUC insulin (mU/mL/min) HOMA-IR ISOGTT

CTRL (n 5 12)

P (PCOS vs. CTRL)

24.8 6 4.3 32.8 6 5.2 101.9 6 12.5 40.3 6 6.4 59.6 6 6.4 14 6 4

29.6 6 4.7 30.3 6 4.1 94.3 6 9.3 37.2 6 8.8 62.7 6 8.8 060

0.0114 0.2106 0.0970 0.3285 0.3274 0.9999 0.4965 >0.9999 >0.9999 >0.9999 >0.9999 0.3045 0.0020 5 ng/mL, normal circulating androgen levels, and the absence of hirsutism. The inclusion criteria for PCOS patients and CTRL were an age of between 18 and 35 years and a BMI between 25 and 40 kg/m2. Exclusion criteria included T2DM, smoking, and recent use of oral contraceptives or any other medications that could affect glucose metabolism within 6 months prior to study participation. The subjects were not engaged in any regular exercise training for at least 6 months before entering the study. Main subjects characteristics are shown in Table 1. All subjects provided written informed consent in accordance with the institutional review board guidelines for protection of human subjects.

Study design Prior to the intervention, PCOS and CTRL main characteristics were assessed, including body composition, laboratory and metabolic


parameters, physical activity level and physical capacity. To examine the effects of a single bout of exercise on insulin-signaling pathways, the subjects performed a single 40-min bout of aerobic exercise. Plasma glucose and insulin levels were assessed immediately before (PRE) and 0, 30, and 60 min after exercise. Muscle biopsies were taken at PRE and 60 min after the exercise (POST). Selected proteins involved in insulin signaling were assessed by Western blotting.

Laboratory parameters Serum cortisol and androstenedione were assessed by a chemioimmunoassay (Immulite 2000, Siemens, Munich, Germany). Serum dehydroepiandrosterone sulfate (DHEAS) was measured by an electrochemiluminescence immunoassay (COBAS 6000, Roche Diagnostics, Mannheim, Germany). Serum prolactin, estradiol, total testosterone, and 17-hydroxyprogesterone (17-OHP) were measured by radioimmunoassays (RIA) (Roche Diagnostics, Mannheim, Germany and Imunotech, Prague, Czech Republic, respectively). Serum luteininzing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone, and sex-hormone binding globulin (SHBG) were assessed by chemiluminescent immunometric assays (Roche Diagnostics, Mannheim, Germany). Free testosterone was determined as previously described (18). Total cholesterol, triglycerides, and high-density lipoprotein (HDL) were assessed by colorimetric enzymatic methods (CELM, Sao Paulo, Brazil). Low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) were calculated following a previous description (19). In addition, a 75-g oral glucose tolerance test (OGTT) was performed. Samples were collected after a 12-hour overnight fast and 30, 60, 90, and 120 min following the glucose load. Plasma glucose was assessed by a colorimetric enzymatic assay (Bioclin, Belo Horizonte, Brazil) and insulin was assessed by a human-specific RIA method (Diagnostic Products Corp., Los Angeles, CA). Area under the curve of glucose (AUC glucose), insulin (AUC insulin), and homeostasis model assessment (HOMA-IR) was calculated from OGTT. An insulin sensitivity surrogate (ISOGTT) was derived from OGTT (20).


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The coefficient of variations (CVs) for total cholesterol, HDL, LDL, VLDL, fasting glucose, fasting insulin, androstenedione, total testosterone, SHBG, estradiol, FSH, LH, prolactine, 17-OHP, and DHEAS were 2.73%, 3.8%, 5.2%, 8.9%, 1.67%, 9.3%, 0.05). However, plasma insulin levels were significantly greater at the 60th, 90th, and 120th min of OGTT in PCOS patients when compared with CTRL (P < 0.05). Similarly, AUC insulin from OGTT was also greater in PCOS (P < 0.05). The aforementioned data are summarized in Table 1.

Body composition, physical activity level, and physical capacity Waist circumference tended to be greater (P 5 0.097) in PCOS when compared with CTRL. No between-group differences were found in body composition data.


Relative VO2peak and relative VO2 at the respiratory compensation point were significantly lower in PCOS patients when compared with CTRL. The remaining cardiopulmonary parameters were similar between groups. In addition, physical activity level parameters did not differ between groups. The aforementioned data are summarized in Tables 1 and (2).

Plasma glucose and insulin in response to a single bout of aerobic exercise PCOS patients showed significantly lower plasma glucose levels at the 30th minute following a single bout of aerobic exercise when compared with PRE values (25.39%, P < 0.05, within-group comparison, Figure 1, Panel A). No differences were observed for CTRL group at any time point (P > 0.05, Figure 1, Panel A). Similarly, neither the delta change in plasma glucose nor the glucose AUC showed any significant alterations after a single bout of aerobic exercise (P > 0.05, Figure 1, Panels C and E). Plasma insulin levels


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Figure 2 Protein phosphorylation of (A) IRS-1 Ser 307, (B) PI3-k p85, (C) Akt Ser 473, (D) Akt Thr 308, (E) AMPK Thr 172, and (F) AS160 Thr 642 before (PRE) and after a single bout of aerobic exercise (POST). # 5 significant difference vs. PRE (P 0.05). * 5 significant difference between PCOS vs. CTRL (P 0.05). & 5 tendency to significance between PCOS vs. CTRL (P 0.1).

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Figure 3 Expression of (A) total GLUT4, (B) membrane GLUT4, and (C) GLUT4 translocation (the ratio membrane GLUT4 to total GLUT4 before (PRE) and after a single bout of aerobic exercise (POST). & 5 tendency to significance vs. PRE (P 0.1).

were significantly higher in PCOS when compared with CTRL at PRE (236.36%, P < 0.05, Figure 1, Panel B). A significant reduction in plasma insulin levels were observed in PCOS patients 60 min after a single bout of aerobic exercise when compared with PRE values (72.91%, P < 0.05, within-group comparisons, Figure 1, Panel B). Delta change in plasma insulin levels after a single bout of aerobic exercise was significantly higher in PCOS (226.34%) than in CTRL (22.90%; P < 0.05, Figure 1, Panel D). AUC insulin in response to a single bout of aerobic exercise was significantly higher in PCOS than CTRL (143.94%, P < 0.05, Figure 1, Panel F).

Protein phosphorylation in response to a single bout of aerobic exercise Both groups showed a significant (PCOS: 245.61%, P 5 0.028, CTRL: 228.14%, P 5 0.018; within-group comparisons) reduction in IRS-1 phosphorylation after a single bout of aerobic exercise, although no significant differences were observed between the two groups (P > 0.05; Figure 2, Panel A). CTRL showed a significant increase in PI3-k p85 activation after acute exercise (151.66%, P 5 0.018, within-group comparison).


CTRL also showed higher PI3-k p85 values at POST when compared with PCOS (trend toward significance, P 5 0.073, Figure 2, Panel B). Akt Ser 473 phosphorylation was significantly higher in both groups after acute exercise (POST) when compared with PRE values (PCOS: 110.29%, P 5 0.008, CTRL: 145.09%, P 5 0.050; withingroup comparisons, Figure 2, Panel C). In terms of Akt Thr 308 phosphorylation, a significant within-group difference (pre to post change) was observed only in the PCOS patients (91.90%, P 5 0.018, Figure 2, Panel D). No between-group differences were observed for either protein (P > 0.05; Figure 2, Panels C and D). PCOS patients showed a significant PRE to POST change in AMPK phosphorylation (21.87%, P 5 0.008) whereas no changes were observed in the CTRL (P > 0.05). In addition, no between-group differences were observed (P > 0.05; Figure 2, Panel E). A significant increase in AS160 phosphorylation was observed following exercise (100.00%, P 5 0.017, within-group comparison) in the CTRL group. Significantly different phosphorylation values were also observed in CTRL when compared with PCOS at POST


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(P 5 0.017, between-group comparison, Figure 2, Panel F). Importantly, no changes in AS160 phosphorylation were observed in the PCOS group (P > 0.05). The aforementioned data are summarized in Figure 2.

Expression of total and membrane GLUT4 and GLUT4 translocation in response to a single bout of aerobic exercise No within- or between-group differences were observed in either total or membrane GLUT4 expression (P > 0.05; Figure 3, Panels A and B). GLUT4 translocation tended to be significantly higher in both groups after a single bout of aerobic exercise (PCOS: 84.19%, P 5 0.091; CTRL: 90.10%, P 5 0.093, Figure 3, Panel C). Importantly, no between-group differences were observed in this parameter (P > 0.05), suggesting a similar GLUT4 translocation response in both groups.

Discussion The novel finding of this study was that a single bout of aerobic exercise elicited similar GLUT4 translocation in PCOS patients and CTRL, despite a slightly different pattern of protein phosphorylation. In a previous study, exercise training was able to partially offset IR in PCOS patients (14,25). However, the exercise-induced improvements in IR appeared to be less pronounced in PCOS than CTRL (14). This partially blunted response has been attributed to possible defects in insulin signaling seen in PCOS skeletal muscle. To our knowledge, this was the first study to address this hypothesis by comparing the effects of exercise upon molecular pathways related to insulin signaling in skeletal muscle of PCOS patients and CTRL. Even though differences in protein phosphorylation were evident, GLUT4 translocation was equally modified in both groups, suggesting that the previous report of exercise-induced impairments in glucose uptake cannot be entirely attributed to defects in the molecular pathways leading to GLUT4 translocation. Several abnormalities in molecular pathways related to insulin signaling in skeletal muscle of PCOS patients have been reported (5,6). Based on these observations, one might expect a defective exerciseinduced GLUT4 translocation in PCOS patients, although this was not confirmed in the present study. Two main hypotheses may arise to explain this finding. First, it is possible that the sustained hyperinsulinemia experienced by PCOS patients may have been partially responsible for maintaining the activation of insulin-signaling pathways, resulting in a “normal” (i.e., at control levels) GLUT4 translocation. It is interesting to note that while type 2 diabetes and PCOS, both insulin-resistant conditions, share the feature of a normal total GLUT4 content in skeletal muscle, exercise-induced GLUT4 translocation is impaired only in the former (26). However, it is possible to speculate that IR in PCOS may progress to a point at which an effective exercise-induced GLUT4 translocation can no longer be sustained by hyperinsulinemia, thereby leading to progressive impairments in glucose homeostasis, similarly to that observed in type 2 diabetes. Secondly, it is worth noting that our sample showed high levels of physical activity (according to accelerometer), in spite

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of not being engaged in regular exercise training (27). Such high levels of physical activity might have been partially responsible for “bypassing” IR and stimulating GLUT4 translocation, supporting the beneficial effects of a physically active lifestyle on glucose metabolism (28). Given the effective response to exercise concerning GLUT4 translocation experienced by PCOS patients, exercise training must be considered as a measure to improve IR in this population. Although GLUT4 translocation was similar in PCOS patients and CTRL, a slight different pattern of protein phosphorylation was apparent. Namely, PCOS patients did not show any increase in PI3k p85 activation after a single bout of aerobic exercise. PI3-k activation has been considered an important translational step towards GLUT4 translocation (29). Defects in PI3-k phosphorylation have been reported in T2DM and obesity (30). Despite the blunted response of PI3-k p85, PCOS patients had normal Akt phosphorylation after a single bout of aerobic exercise. In this respect, it is interesting to note that exercise led to increased Akt Thr 308 phosphorylation in PCOS patients, but not in CTRL, suggesting this as a compensatory mechanism to counteract the lack of PI3-k p85 activation in the former group. While Akt has been thought to be a downstream of PI3-k, a certain discrepancy between PI3-k p85 and Akt activation does exist, suggesting that Akt phosphorylation may effectively occur with only partial activation of PI3-k (31). Therefore, one may assume that the lower PI3-k p85 activation shown in PCOS has a minor (if any) role in Akt phosphorylation and, hence, in the impairment of insulin sensitivity in this syndrome. AMPK is known to play an important role in mediating exerciseinduced glucose uptake independent of insulin. This protein acts as an energy sensor, and is an important step in enabling GLUT4 translocation in this group. Interestingly, AMPK phosphorylation was paralleled by AS160 Thr 642 in CTRL, but not in PCOS patients. This latter protein is considered a target of AMPK, acting as an Akt substrate capable of mediating GLUT4 exocytosis at a step before fusion of GLUT4-containing vesicles with the plasma membrane (32). Exercise can phosphorylate AS160 Thr 642 through a mechanism involving AMPK (32). However, AMPK can directly regulate GLUT4 transcription, since it was identified a SLC2A4 region of approximately 895 bp that is responsive to AICAR (33). Therefore, the absence of AS160 phosphorylation in PCOS patients was possibly compensated by an effective AMPK phosphorylation. Although this study provided novel insights into the molecular mechanisms involved in exercise-induced glucose uptake in PCOS patients, some limitations must be acknowledged. First, the effects of exercise training upon insulin signaling have been classically interpreted as the summation of the effects produced by each acute session of exercise. However, acute and chronic molecular responses to exercise may differ, so that further studies must investigate the effects of exercise training on molecular pathways related to glucose uptake, along with clinical and metabolic outcomes. Second, to gather knowledge on the molecular mechanisms in PCOS, we selected some proteins due to their pivotal roles in glucose homeostasis. Third, this study comprised only two biopsies due to technical and ethical limitations. We chose to perform the second biopsy 60 min after exercise considering the pattern of exercise-induced protein expression related to insulin signaling in healthy subjects (34,35). Furthermore, there is also evidence that glucose uptake is increased 60 min following a single exercise session (36). Further


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studies involving PCOS patients undergoing a more comprehensive time-course of protein expression could be informative. Fourth, direct measures of glucose uptake (e.g., via stable isotopic techniques) were not possible in this study, precluding a direct link between the signaling events with the expected impairments in glucose uptake. Likewise, the clinical nature of this study hampered a proper distinction of the influence of physiological parameters underlying the molecular responses observed, as exercise can elicit remarkable changes in systemic factors (e.g., energetic substrates and neurohumoral parameters). The role of each exercise-induced systemic adjustments leading to the signaling events reported in this study remains to be explored by further mechanistic experimental studies. Further studies with a more comprehensive molecular approaches (e.g., metabolomics and proteomics) in a variety of tissues involved in PCOS pathophysiology are certainly warranted. In conclusion, a single bout of acute exercise elicited similar GLUT4 translocation in skeletal muscle of PCOS patients and controls, despite a slightly differential pattern of protein phosphorylation. The absence of impairment in GLUT4 translocation suggests that PCOS patients with obesity and IR may benefit from exercise training.O

Acknowledgments The authors are thankful to Vita Clınicas Medicina Especializada for the assistance with the body composition assessment. C 2015 The Obesity Society V

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