Voluntary physical activity counteracts the proliferative tumor growth microenvironment created by adipose tissue via high-fat diet feeding in female rats
2017; Wiley; Volume: 5; Issue: 13 Linguagem: Inglês
10.14814/phy2.13325
ISSN2051-817X
AutoresChristopher F. Theriau, Michael K. Connor,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoPhysiological ReportsVolume 5, Issue 13 e13325 Original ResearchOpen Access Voluntary physical activity counteracts the proliferative tumor growth microenvironment created by adipose tissue via high-fat diet feeding in female rats Christopher F. Theriau, Christopher F. Theriau School of Kinesiology and Health Science, York University, Toronto, ON, Canada Muscle Health Research Centre, York University, Toronto, ON, CanadaSearch for more papers by this authorMichael K. Connor, Corresponding Author Michael K. Connor mconnor@yorku.ca School of Kinesiology and Health Science, York University, Toronto, ON, Canada Muscle Health Research Centre, York University, Toronto, ON, Canada Correspondence Michael K. Connor, School of Kinesiology and Health Science, York University, 347 Bethune College, 4700 Keele Street, Toronto, ON M3J 1P3, Canada. Tel: 416-736-2100 ext 77206 Fax: 416-736-5774 E-mail: mconnor@yorku.caSearch for more papers by this author Christopher F. Theriau, Christopher F. Theriau School of Kinesiology and Health Science, York University, Toronto, ON, Canada Muscle Health Research Centre, York University, Toronto, ON, CanadaSearch for more papers by this authorMichael K. Connor, Corresponding Author Michael K. Connor mconnor@yorku.ca School of Kinesiology and Health Science, York University, Toronto, ON, Canada Muscle Health Research Centre, York University, Toronto, ON, Canada Correspondence Michael K. Connor, School of Kinesiology and Health Science, York University, 347 Bethune College, 4700 Keele Street, Toronto, ON M3J 1P3, Canada. Tel: 416-736-2100 ext 77206 Fax: 416-736-5774 E-mail: mconnor@yorku.caSearch for more papers by this author First published: 04 July 2017 https://doi.org/10.14814/phy2.13325Citations: 1 Funding Information: This work was supported by Natural Sciences and Engineering Research Council of Canada and Canadian Foundation for Innovation. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The adipokine secretion profile created from adipose tissue may represent the molecular mechanism behind the obesity-breast cancer association. Two adipokines, adiponectin (ADIPO), and leptin (LEP), are altered with obesity and exert antagonistic effects on breast cancer proliferation. We set out to determine whether the adipose-dependent tumor promoting growth environment created by a high-fat diet (HFD) in female Sprague-Dawley rats is altered compared to established responses in male rats and whether voluntary physical activity (PA) ameliorates any HFD-dependent effects. We found that conditioned media (CM) created from the adipose tissue of female HFD-fed rats increased the proliferation of MCF7 cells compared to those cells grown in CM prepared from lean adipose tissue. HFD-CM inhibited AMPK and activated Akt signaling, decreased p27 phosphorylation at T198, reduced total p27 and AdiporR1 protein levels and promoted cell-cycle entry. PA reversed the proliferative effects of HFD-CM on MCF7 cells by preventing the effects of HFD on AMPK, Akt, p27 and AdipoR1, ultimately resulting in cell-cycle withdrawal. Overexpressing AdipoR1 abolished the proliferative effects of the HFD-CM on MCF7 cells and enhanced the anti-proliferative effects PA on the HFD-CM. Thus, PA represents a means to prevent deleterious obesity-related alterations in tumor growth environment which are brought about by changes in adipokine secretion profile from adipose tissue in the presence of estrogen. Furthermore, although adipose produces hundreds of adipokines, the ADIPO:LEP ratio may serve to indicate the contribution of adipose in creating a tumor growth microenvironment. Introduction Breast cancer is the most commonly diagnosed malignancy among women in the world. For almost 50 years there has existed a statistical link between adiposity and an increased risk of breast cancer (Sneddon et al. 1968). Several clinical and preclinical studies have demonstrated that increased adiposity is associated with increased cancer incidence, morbidity, poorer response to therapy and higher disease mortality (Sneddon et al. 1968; Calle et al. 2003; Parekh et al. 2012). While this association appears to be strong in postmenopausal women (Xia et al. 2014), the relationship between obesity and breast cancer in premenopausal women is far less consistent. Studies have reported that obesity in premenopausal women is inversely associated with breast cancer (van den Brandt et al. 2000; Michels et al. 2006), shows no association (Kaaks et al. 1998; Lahmann et al. 2004), or shows a positive association with disease development (Cecchini et al. 2012). This increased incidence of breast cancer in obese postmenopausal women has been suggested to be due, in part, to the direct effects of estrogen on the peripheral fat depots via aromatization of androgens (Catalano et al. 2003, 2004). Adipose tissue has been identified as an active endocrine organ-producing adipocyte-derived factors, termed adipokines. These adipokines can act in an autocrine, endocrine and/or paracrine manner. While some adipokines are secreted from other tissues in the body, the vast majority are produced/secreted by white adipose tissue. Thus far, over 400 adipokines have been discovered and several have been shown to become dysregulated in obese individuals (Zhong et al. 2010). Adiponectin (ADIPO) and leptin (LEP) represent major potential contributors to the adipose-dependent microenvironment. Both are among the most abundant adipokines produced/secreted, are altered by obesity and have documented cell-cycle regulatory effects on breast cancer cells (Dieudonne et al. 2002, 2006; Jarde et al. 2009). LEP is predominately produced by white adipose tissue and its level in the peripheral circulation is directly proportional to BMI (Wauters et al. 2000). LEP activates several intracellular pathways implicated in breast carcinogenesis, including the phosphoinositide-3/Akt kinase signaling pathway (Garofalo and Surmacz 2006; Jarde et al. 2009). LEP activates Akt, which phosphorylates p27 at T157, preventing both its nuclear accumulation and inhibition of cyclin E/cdk2, thereby leading to cell-cycle entry (Dieudonne et al. 2002; Liang et al. 2002; Garofalo et al. 2006). Conversely, ADIPO production/secretion decreases with obesity and induces cell-cycle exit by activating AMPK, which directly phosphorylates p27 at T198, increasing p27 stability and inducing G1 arrest (Dieudonne et al. 2006; Liang et al. 2007; Grossmann et al. 2008). This is mediated by ADIPO binding to its receptor Adiponectin receptor 1 (AdpoR1) which is also implicated in breast cancer (Dieudonne et al. 2006; Theriau et al. 2016). ADIPO-dependent anti-proliferative effects are abolished by siRNA knockdown of AdipoR1 (Grossmann et al. 2008; Nakayama et al. 2008). Decreased ADIPO signaling through AdipoR1 has been shown to be associated with higher tumor grade and poorer patient outcomes in breast cancer patients (Pfeiler et al. 2010). We have previously demonstrated that increasing AdipoR1 levels in breast cancer cells increases the cell-cycle inhibitory effects of ADIPO, via AMPK signaling, and can counteract the antagonism of ADIPO by LEP (Theriau et al. 2016). Clinical studies in postmenopausal women also suggest that decreased ADIPO:LEP ratios, rather than the levels of each adipokine individually, are stronger predictors of breast cancer risk (Ollberding et al. 2013). In premenopausal women, this ADIPO:LEP breast cancer association is less clear. A sedentary lifestyle is widely accepted as a major contributor to the increase in obesity and its associated disorders (Schrauwen and Westerterp 2000), suggesting that physical activity (PA) can potentially serve as an intervention for obesity-associated effects on breast cancer (Enger et al. 2000; Bradley et al. 2008). Although there are some discrepancies for the role of obesity and breast cancer risk in pre- versus postmenopausal women, research shows that PA can lower the risk of breast cancer regardless of menopausal status (Bernstein et al. 1994; Carpenter et al. 1999). Obese and physically inactive breast cancer patients appear to be at an increased risk for both disease progression and cancer-related mortality, regardless of menopausal status (Pichard et al. 2008; Renehan et al. 2008; Doyle et al. 2012). Higher recreational PA has been shown to be associated with a 30–60% reduction in all estrogen receptor subtypes in premenopausal women compared to sedentary women (Enger et al. 2000). In mice, voluntary PA increases the circulating ADIPO:LEP ratio, decreases breast cancer incidence compared to sedentary controls by decreasing pAktT473 and increased pAMPKT172 and p27 within mammary carcinomas (Zhu et al. 2008, 2012; Thompson et al. 2010). In agreement, women who consumed a calorie restricted diet coupled with moderate PA experienced a 9.5% increase in ADIPO and a 40.1% decrease in LEP (Abbenhardt et al. 2013). Thus, there are clear positive effects of the volume of physical activity to breast cancer patient prognosis. However, the contribution of estrogen in these phenomena remains unclear. In male rats, which have very low circulating estrogen levels, visceral adipose of HFD-fed rats induced cell-cycle entry in MCF7 cells by activating Akt, inhibiting AMPK, decreasing ADIPO:LEP ratio and repressed AdipoR1 and p27 expression (Theriau et al. 2016). These HFD-dependent effects were abolished by both voluntary PA and increased expression of AdipoR1. Given some of the inconsistencies surrounding the role of estrogen in adipose-dependent increases in breast cancer development, we set out to determine whether the adipose-dependent tumor growth microenvironment created by an HFD in female Sprague-Dawley rats is altered compared to established responses in male rats and whether voluntary physical PA or increased expression of AdipoR1 ameliorates these effects. We show that the ADIPO:LEP ratio is decreased in the circulation of HFD-fed female rats. This altered adipokine secretion profile caused a decrease in pAMPKT172, p27T198, AdipoR1, and an increase in pAktT308 in MCF7 cells grown in conditioned media (CM) prepared from the adipose of HFD animals. This HFD-CM lead to cell-cycle entry by increasing the number of MCF7 cells in S-phase while decreasing the number in G0/G1. Voluntary PA increased the ADIPO:LEP ratio compared to HFD sedentary animals, abolishing the effects of HFD-CM on MCF7 proliferation. Overexpressing AdipoR1 in MCF7 cells counteracted the effects of the HFD-CM. The adipokine levels in the CM produced by adipose from females was very different than that shown in previous work in male animals (Theriau et al. 2016). This altered HFD-CM secretome blunted MCF7 proliferation compared to males (Theriau et al. 2016). These results highlight the importance of PA and stabilizing AdipoR1 signaling in order to overcome the positive tumor growth microenvironment created by obesity regardless of whether estrogen is present. Methods Animals All animal experiments were approved by the York University Animal Care Committee in accordance with Canadian Council for Animal Care guidelines. Thirty female Sprague-Dawley rats (7 weeks of age) were purchased from Charles River Laboratories (Montreal, QC, Canada) and were singly housed in standard clear, plastic cages. All animals had a 7-day habituation period to a 12 h light-dark cycle (lights on at 0600) in a temperature (22°C) and humidity (50–60%) controlled room. This age of animals was chosen as it is consistent with previously published data (Zhu et al. 2008, 2012; Thompson et al. 2010; Theriau et al. 2016) and ensured that the animals would maintain running distances throughout the entire protocol. After acclimatization, animals were randomly selected and given free access to a running wheel (wheel circumference, 106 cm; Harvard Apparatus, Holliston, MA) within the cage. A magnetic counter mounted to each wheel detected the revolutions and distance run was calculated every 24 h. Animals acclimated to the wheels for 7 days and were subsequently divided into two groups: chow diet (CD; n = 10) and high-fat diet (HFD; n = 20) with both groups given access to food and water (ad libitum). The CD (no. 5012 Lab Chows, Ralston Purina, St. Louis, MO) had caloric make-up of 14% fat, 54% carbohydrate, 32% protein (3.02 calories/g). The HFD (Harlan Laboratories, Madison, WI) had a caloric breakdown of 60% fat, 21% carbohydrate, 18% protein (5.1 calories/g). Daily caloric intake for all animals was calculated. HFD and CD-fed animals were further subdivided into sedentary and physical activity (PA) groups designated as chow diet-sedentary (CD; n = 6), chow diet-high physical activity (CD + HPA; >12.5 km/day; n = 4), HFD-sedentary (HFD; n = 8). Animals in the HFD-PA group were further subdivided depending on average wheel running distances into animals that run <12.5 km/day (n = 4) classified as “low physical activity” group (HFD + LPA) while animals that run more than 12.5 km/day (n = 8) were classed as “high physical activity” (HFD + HPA) to determine if volume effects of physical activity were evident. Our physical activity cutoff of 12.5 km/day has also been used previously with female Sprague-Dawley rats and wheel running to create a low and high PA groups (Tokuyama et al. 1982; Gollisch et al. 2009). Although these cutoffs have been previously established in the literature, they may also be attributed to an animal's predisposition to PA. Food intake and running distances were measured each day and body weight was measured three times per week. After a 6-week protocol, animals were anesthetized using isoflurane and tissues removed. Tissue collection and conditioned media Visceral adipose tissue (periovarian, perirenal, retroperitoneal) was quickly removed from all animals and cultured as previously described (Sutherland et al. 2009; Theriau et al. 2016). In order to ensure that our mass:volume preparation of CM was consistent among preparations across groups, we diluted 10–15 mg sections of adipose 30:1 in RIPA buffer for protein extraction. Equal volumes of lysate (25 μL) were subjected to SDS-PAGE, using 12% gels and membranes were probed for total Akt and β-actin to evaluate equivalency of specific protein content between preparations. Weights of all tissues collected were measured and normalized per 100 g of body weight. The sequence in which rats were killed was randomized across groups so as to minimize the likelihood that order effects would masquerade as treatment-associated effects. At the time of killing, the gastrocnemius, soleus and tibialis anterior were immediately excised, weighed, frozen, and stored at −84°C for future analyses. Also, 2–3 mL of blood was taken at the time of killing, left for 30 min on ice, and subsequently centrifuged. The serum was then extracted, frozen and stored at −84°C for future analyses. Cytochrome c oxidase activity assay In order to confirm that the physical activity protocol elicited a training effect, we measured cytochrome c oxidase (COX) activity in mixed gastrocnemius muscles. COX activity was determined as previously described (Theriau et al. 2016). Briefly, cross-sections of mixed gastrocnemius muscles (from the mid-section of the muscle belly) weighing roughly 20–30 mg were diluted 80-fold (sedentary) or 160-fold (PA) in extraction buffer (100 mmol/L Na-K-Phosphate, 2 mmol/L EDTA, pH 7.2). Muscle extracts were prepared by homogenization with metal beads in a magnetic homogenizer (Mixer Mill MM 400, Retsch, Haan, Germany). These homogenates were then used for the analyses of the maximum rate of oxidation of fully reduced cytochrome c at 30°C as indicated by changes in absorbance (550 nm). Conditioned media/serum adipokine and estradiol measurement The levels of ADIPO, LEP and estradiol (E2) produced and secreted into the co-culture media by the adipose tissue as well as in the serum at the time of killing was determined, using rat adiponectin sandwich ELISA kit (BioVision, Milpitas, CA), mouse/rat leptin quantikine sandwich ELISA kit (R&D Systems, Minneapolis, MN), and rat estradiol competitive-binding sandwich ELISA kit (Alpco Diagnostic, Salem, NH), as per manufacturer instructions. Aliquots of CM were diluted 50-fold (HFD) and 100-fold (PA and CD) for ADIPO ELISAs, fivefold (HFD) or undiluted (PA and CD) for LEP ELISAs and undiluted for E2 ELISAs. Aliquots of serum were diluted 1000-fold for all samples for ADIPO ELISAs, fivefold (HFD and HFD + PA) or undiluted (PA and PA + HPA) for LEP ELISAs and E2 ELISAs. The levels of each adipokine were calculated in ng/mL values and ADIPO/LEP converted to nmol/L values for stoichiometric comparison. The levels of E2 were calculated in pg/mL values. We utilized the ADIPO:LEP ratio as a representation of the adipose-derived microenvironment as both have been shown to have cell-cycle regulatory effects on breast cancer cells and change predictably and reliably with obesity. This ratio allows for a prediction of the nature of the tumor growth microenvironment created by adipose tissue which may end up being of use in a clinical setting. Cell culture MCF7 cells were purchased from the American Tissue type Culture Collection (ATCC, Manassas, VA) and were maintained in AMEM, 10% FBS, 2% antimicotic/antibiotic, 1 mmol/L sodium pyruvate, nonessential amino acids, and 10 μg/mL insulin from human pancreas at 37°C and 5% CO2. MCF7 cells were transfected with an AdipoR1 overexpressing plasmid vector as previously described (Theriau et al. 2016). Mock transfected (MockT) MCF7 cells and stably transfected AdipoR1 (AdipoR1-T) cells were seeded in six well plates with AMEM for 24 h. At 70% confluence, cells were washed with PBS and incubated with CM produced from adipocytes for 24 h. MCF7 cells grown in AMEM supplemented with 10% FBS served as untreated controls (UT). Immunoblotting The effects of adipokines on specific cellular proteins were measured using standard SDS-PAGE protocols utilizing 12% polyacrylamide gels. Proteins (25 μg) were transferred to PVDF membranes (Bio-rad, Mississauga, ON, Canada), blocked for 2 h in 10% skim milk and subsequently incubated overnight with primary antibodies: p27Kip1 (BD Biosciences); p27T198 (R&D Systems); pAktT308, Akt, pAMPKT172 , and AMPK (Cell Signaling); AdipoR1 (Santa Cruz Biotech, Santa Cruz, CA) and β-actin (Abcam, Cambridge, MA). Anti-mouse, anti-rabbit (Promega, Madison, WI) and anti-goat (Santa Cruz) horseradish peroxidase secondary antibodies were used to visualize proteins using Immobilon-enhanced chemiluminescence substrate (Millipore, Whitby, ON, Canada) and detected/quantified on a Kodak In Vivo Pro imaging system (Marketlink Scientific, Burlington, ON, Canada). Cell-cycle analyses MCF7 cells isolated from 6-well plates were trypsinized, washed in cold PBS and fixed by drop wise addition of ice-cold 70% ethanol. Cells were washed twice in PBS, resuspended in a propidium iodide/RNAse solution and subjected to FACS analyses (Gallios Flow Cytometer, Beckman Coulter Mississauga, Canada). Cell-cycle profiles were determined, using Mod-fit software (Verity Software House, Topsham, ME), by fitting curves to profiles and measuring the areas under the curve to determine relative numbers of cells in G1, S, and G2/M phases. Statistical analyses All values are expressed as mean ± SEM of 5 to 8 separate experiments (as indicated) and statistical analyses were performed, using a one-way ANOVA with Tukey's post hoc tests conducted when significant main effects were found. Individual t-tests were used to identify differences in FACS analysis between groups. Group means were considered to be significantly different when P ≤ 0.05. Results HFD-dependent increases in adiposity are prevented by PA HFD-fed animals demonstrated a 26% higher total body mass compared to CD-fed sedentary animals (328.±21.3 vs. 260.4 ± 10.3, respectively; Fig. 1A). This increased total body weight was mirrored by a 4.5-fold increase total relative visceral fat mass (7.6 ± 0.97 vs. 1.68 ± 0.23 g/100 g body weight; Fig. 1B). The HFD sedentary animals also had an increased overall daily calorie intake compared to CD sedentary animals (76.6 ± 2.8 vs. 57.1 ± 3.2 cal/day; Fig. 1C), indicating that HFD animals were subjected to both increased fat and increased calorie ingestion. The difference in total body and total relative visceral weights between HFD and CD sedentary animals suggested that the HFD-induced obesity in the animals. Figure 1Open in figure viewerPowerPoint High-fat diet increases total visceral fat and is ameliorated with PA. Body mass changes over the 6-week protocol (A). Body weight adjusted total visceral fat mass in CD (open bar), HFD (black bar), HFD + LPA (light grey bar), HFD + HPA (dark gray bar) and CD + LPA (hatched bar) animals (B). Body weight adjusted muscle mass of the gastrocnemius (C), soleus (D) and tibialis anterior (E) muscles. Physical activity alters cytochrome C oxidase enzyme activity in the gastrocnemius muscles of CD, HFD, HFD + LPA, HFD + HPA and CD + LPA animals (F). *In A indicates different from HFD and HFD + LPA animals, **Indicates different from all other groups (P < 0.05). Different letters (B–F) indicate groups that are significantly different from each other (P < 0.05, n = 6/group). HFD, high-fat diet; CD, chow diet; HFD + LPA, high-fat diet + low physical activity; HFD + HPA, high-fat diet + high physical activity; CD + LPA, chow diet + low physical activity. HPA counteracted the HFD-dependent increase in visceral adiposity as the HFD + HPA animals were 13% lighter than their sedentary counterparts (287.9 ± 15.8 g vs. 328.1 ± 21.3 g; Fig. 1A). Consistent with these results we observed a 44% decrease in total visceral fat mass in HFD + HPA compared to HFD sedentary animals (Fig. 1B). In contrast, HFD + LPA animals showed no difference in total body weight or specific visceral fat mass compared to their sedentary counterparts (Fig 1A and B). This may be due to the HFD + LPA group having a 72% higher daily calorie intake compared to HFD sedentary animals (98.5 ± 7.5 vs. 57.1 ± 3.2 cal/day, respectively; Fig. 1C). CD + HPA animals were 22% lighter and had 85% less total visceral fat mass compared to HFD sedentary animals (Fig. 1A and B). The CD + HPA and HFD + HPA animals ran similar distances (16.3 ± 2.3 vs. 18.2 ± 2.3 km/day) and both groups were found to have run more than the HFD + LPA group (9.6 ± 1.1 km/day). In order to confirm that our PA protocol induced a specific aerobic training effect beyond that displayed by the decreases in body mass/adiposity, we looked for changes in the weights of the gastrocnemius, soleus and tibialis anterior muscles. PA increased gastrocnemius, soleus and tibialis anterior relative weights in both HFD + LPA and HFD + HPA animals compared to their sedentary counterparts (Fig. 1D–F). LPA and HPA increased muscle mass by an average of 29 ± 4% and 47 ± 3%, respectively, above HFD animals. This volume-dependent effect of PA was evidenced by changes in oxidative enzyme capacities of mixed gastrocnemius muscles (Fig. 1G). LPA increased mixed gastrocnemius COX activity by 2.0-fold while HPA further increased COX activity to levels that were 3.1-fold above those in sedentary HFD-fed animals. ADIPO:LEP ratio is decreased in the serum of HFD-fed animals and this is prevented by PA Serum ADIPO, LEP and E2 levels were determined at the time of sacrifice in order to determine if serum E2 levels correlated with any alterations in ADIPO and LEP due to HFD or PA. Serum E2 and LEP were both elevated by HFD and these increases were mitigated by diet (CD) and HPA but not LPA (Fig. 2A). The CD-fed animals were found to have 92% lower LEP (6.90 ± 1.61 vs. 0.55 ± 0.17 ng/mL), 95% lower E2 (57.2 ± 9.2 vs. 2.6 ± 2.0 pg/mL) compared to HFD-fed animals (Fig. 2A). Although the mean values for E2 and LEP were 29% and 47% lower in HFD + LPA than HFD sedentary animals, respectively, they were not different statistically. As a result of these ADIPO and LEP changes, there were corresponding changes in the ADIPO:LEP ratio (Fig. 2B). HFD caused a 97% decrease in the ADIPO:LEP ratio compared to CD animals (1854 ± 602 vs. 41,189 ± 15,901). LPA increased the ADIPO:LEP ratio 1.8-fold while HPA increased this ratio by 4.3-fold compared to that in HFD sedentary animals (Fig. 2B). Figure 2Open in figure viewerPowerPoint ADIPO is decreased, LEP and E2 increased in serum of HFD animals and reversed with HPA. Circulating serum ADIPO (open bar; μg/mL), LEP (closed bar; ng/mL) and E2 (gray bar; ng/mL) concentrations at time of killing (A). Circulating serum ADIPO:LEP ratio at time of killing in CD (open bar), HFD (black bar), HFD + LPA (light gray bar), HFD + HPA (dark gray bar) and CD + LPA (hatched bar) animals at time of killing (B). Plotting of ADIPO:LEP ratio in CM versus daily km run (C). Dotted line indicates divider between HPA and LPA groups. Western blots showing levels of Akt and β-actin in adipose tissues from the indicated groups (D). *In A indicates different from HFD (P < 0.05). Different letters (B) indicate groups that are significantly different from each other (P < 0.05, n = 6/group). HFD, high-fat diet; CD, chow diet; HFD + LPA, high-fat diet + low physical activity; HFD + HPA, high-fat diet + high physical activity; CD + LPA, chow diet + low physical activity; ADIPO, adiponectin; LEP, leptin. HFD decreases the ADIPO:LEP ratio in adipose-derived CM which is prevented by PA Visceral adipose tissue depots were excised from animals in each of the experimental groups and used to prepare CM. Similar to serum, HFD-CM showed a decreased ADIPO:LEP ratio compared to CD-CM (128:1 vs. 2051:1; Table 1). This decreased ratio was brought about by an increased ADIPO and a decreased LEP in HFD-CM. Compared to HFD-CM, HFD + LPA-CM had similar ADIPO but lower LEP levels, resulting in an overall increase in ADIPO:LEP ratio (128:1 vs. 341:1; Table 1). HFD + HPA-CM had higher levels of ADIPO (889.6 ± 82.4 ng/mL) and lower levels of LEP (0.43 ± 0.15 ng/mL) than the HFD-CM (ADIPO: 438.7 ± 71.5 ng/mL, LEP: 1.85 ± 0.21 ng/mL) with a resultant higher ADIPO:LEP ratio (128:1 vs. 1250:1). A linear relationship between the distance run and the ratio of ADIPO:LEP in the CM was found, suggesting that volume of PA is a major contributor regulating adipokine secretion from adipose tissue (m = 96.47 ± 25.47, R = 0.769, P = 0.0035; Fig. 2C). Individually, ADIPO was found to have a positive linear relationship with distance run (m = 36.06 ± 6.11, R = 0.882, P = 0.0001; Fig. 2D) while LEP has the opposite inverse relationship (m = −0.058 ± 0.018, R = 0.709, P = 0.0098). The levels of E2 were found to be no different in the CM among all groups (Table 1). Comparing the slopes of ADIPO:LEP and distance run between this study using female rats (m = 96.47 ± 25.47) and previously published data in males (m = 85.94 ± 21.83) (Theriau et al. 2016), we found no difference in the slopes of the linear regressions between the two studies despite a much greater distance run by the female animals in this study (F = 0.033, P = 0.86). Table 1. ADIPO:LEP ratio for adipose-derived conditioned media Group ADIPO (ng/mL) LEP (ng/mL) ADIPO:LEP Estradiol (pg/mL) CD 1166.0 ± 169.0a,b 0.31 ± 0.09a,b 2051.0 ± 336.3a,b 42.2 ± 3.0 HFD 438.7 ± 71.5 1.85 ± 0.21 128.8 ± 28.2 46.8 ± 4.0 HFD + LPA 569.4 ± 111.2 0.96 ± 0.44a 341.2 ± 91.1a 49.8 ± 7.2 HFD + HPA 889.6 ± 82.4a 0.43 ± 0.15a,b 1250.0 ± 467.5a,b 48.9 ± 4.7 CD + HPA 1692.0 ± 584.7a,b 0.39 ± 0.18a,b 2398.0 ± 459.0a,b 51.4 ± 12.2 HFD, high-fat diet; CD, chow diet; HFD + LPA, high-fat diet + low physical activity; HFD + HPA, high-fat diet + high physical activity; CD + LPA, chow diet + low physical activity; ADIPO, adiponectin; LEP, leptin. a Indicates significantly different from HFD. b Indicates significantly different from HFD + LPA (P < 0.05, n = 6/group). Changes in adipokine secretion bring about diet and PA-dependent effects on MockT MCF7 protein expression and signaling In order to verify that any effects observed between groups were not due to experimental design artifacts, we conducted western blot analyses using proteins isolated from the adipose tissue used in our CM preparations and measured the levels of Akt and β-actin proteins to ensure equal protein contents among groups (Fig. 2F). We found no consistent differences between groups, supporting the notion that the CM was not subjected to any preparation artefacts. HFD-CM decreased pAMPKT172, p27T198 and AdipoR1 while increasing pAktT308 levels compared to CD-CM-treated MockT MCF7 cells (Fig. 3A–F). No difference was seen between HFD-CM and CD-CM in p27 protein levels (Fig. 3D). Interestingly, HFD-CM appears to elicit the same effects on MCF7 cell protein expression as seen in UT cells (Fig. 3A). There were no changes evident in total AMPK and Akt. Figure 3Open in figure viewerPowerPoint Physical activity abolishes the effect of an HFD on adipose-dependent tumor growth microenvironment in MockT MCF7 cells. Representative western blots for selected proteins showing the effects of treatment with CM prepared from CD (open bar), HFD (black bar), HFD + LPA (light gray bar), HFD + HPA (dark gray bar) and CD
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