Mouse Model Of Postmenopausal Obesity Research Articles

SAT-336 Time Restricted Feeding Delays Breast Cancer Initiation and Growth in a Mouse Model of Postmenopausal Obesity

Background: The prevalence of obesity and the metabolic syndrome (MetS) has increased dramatically in developed countries over the last three decades (Flegal et al., 2012). Numerous studies indicate that adiposity and the MetS are independent risk factors for multiple diseases including cancer, particularly postmenopausal breast cancer (Kim et al., 2018). Therefore improving the metabolic health of obese postmenopausal women may mitigate their risk for breast cancer. Accumulating evidence suggests that time-restricted feeding (TRF), a form of intermittent fasting in which food intake is limited to a defined period during the normal active phase, can have a positive influence on metabolic health. Importantly, interventional studies in obese mice and small clinical studies in humans have demonstrated that TRF can improve metabolic health even though obesity is maintained (Sutton et al., 2018). Time restriction rather than calorie restriction is thus a promising method to control the negative sequelae of obesity, due to the hunger and irritability that reduces compliance with long-term calorie restriction. The objective of this study was to investigate whether TRF attenuates breast cancer in a mouse model of postmenopausal obesity and whether this effect is mediated by reducing the hyperinsulinemia associated with obesity. Methods: Ovariectomized mice were used as postmenopausal mice model. The ovariectomized mice were initially made obese by feeding 60% high fat diet (HFD) for 10 weeks and then grouped into a continued ad libitum group (24 h access to food) or a TRF group (8 h access to food during active phase). For an orthotopic tumor model, mice were injected with E0771 breast cancer cells into four mammary fat pads per mouse three weeks following the start of TRF. As a tumor initiation model, transgenic PyMT mice were used to assess tumor onset and growth following the same TRF or AL access to the HFD. The insulin dependency of tumor growth was studied by increasing insulinemia using an implanted insulin pump, or by reducing insulin secretion using diazoxide. Insulin effects on tumor cell proliferation and migration was further validated in vitro. Results and Conclusion: TRF had a dramatic effect, reducing tumor growth in obese mice fed a high fat diet (HFD) to levels seen in lean mice. Tumor growth and initiation was also delayed in the transgenic PyMT model of mammary tumorigenesis. Our results further suggest that the antitumor effect of TRF is at least partially mediated by reducing hyperinsulinemia, suggesting that this intervention may be effective in breast cancer prevention and therapy.

Impact of central and peripheral estrogen treatment on anxiety and depression phenotypes in a mouse model of postmenopausal obesity.

Obesity and diabetes increase the risk of depression, and the incidence of these conditions increases rapidly after menopause, but few animal models of postmenopausal obesity have been available. We developed a mouse model of postmenopausal obesity that exhibited anxiety and depressive phenotypes in behavioral tests. To examine the effect of estradiol (E2) in the model, we prepared 4 experimental groups: 1) control, sham-operated female C57BL/6 mice fed a regular diet; 2) OVX-HF, ovariectomized (OVX) mice fed a high-fat diet (HF); 3) E2-SC, OVX-HF mice administered subcutaneous (SC) E2 (50 μg/kg/day); and 4) E2-ICV, OVX-HF mice administered intracerebroventricular (ICV) E2 (1 μg/kg/day). OVX-HF mice exhibited anxiety phenotypes in the open field test, but not in the light-dark box test, and E2 treatment via both routes effectively ameliorated it. OVX-HF mice demonstrated depressive phenotypes in the tail suspension test and forced swim test. Both E2 treatments achieved significant improvement in the tail suspension test, but not in the forced swim test. Serum corticosterone levels did not differ among the groups. Hippocampal expression of glucocorticoid receptor mRNA and serotonin 1A receptor mRNA was significantly increased in OVX-HF mice and was decreased in E2-treated mice. The hypothalamic level of pro-brain-derived neurotrophic factor (proBDNF) protein was tended to decrease in OVX-HF mice, but neither E2 treatment increased it. Since this mouse model exhibited anxiety and depressive phenotypes in relatively short experimental periods without genetic manipulations, it would be useful for further exploring psychiatric phenotypes or screening of therapeutic candidates in postmenopausal obesity.

Time‐Restricted Feeding Attenuates Breast Cancer Growth in a Mouse Model of Postmenopausal Obesity

BackgroundObesity continues to be a serious health issue worldwide and is mounting with time. Research indicates that obesity increases the risk for several cancers including breast cancer and the occurrence is further augmented by 40% in postmenopausal women. Therefore, effective and practical strategies that could have a far‐reaching impact are warranted to curb breast cancer risk in obese postmenopausal women. In this scenario, time‐restricted feeding (TRF), i.e.,consuming ad‐libitum energy during the normal active phase, has been shown to attenuate obesity‐driven metabolic dysregulations. However, implementation of TRF in a cancer setting has not been explored. Thus, the present study sought to understand the ability of TRF to enhance breast cancer remission in postmenopausal, obese female mice. Further, the work also explores the mechanistic link of TRF in reduced tumor growth.MethodsAs postmenopausal mice models, 4‐Vinylcyclohexene Diepoxide (VCD) treated or Ovariectomized mice were used. For the VCD model, mice were treated with VCD (160 mg/ml) for 15 days. The VCD and ovariectomized mice were fed a 60% high fat diet (HFD) to become obese and then grouped randomly into either an ad libitum group (24 hr access to food) or a TRF group (8 hr access to food during active phase) to evaluate the metabolic and tumor growth effect of TRF intervention. At 3 weeks following TRF, mice were injected with py230 breast cancer cells into the mammary fat pad for tumor development. Glucose tolerance test, pyruvate tolerance test, insulin tolerance test, lipid accumulation in liver by HE staining, body weight measurement, food intake, and measurement of the weight of different tissues assessed the metabolic effect of TRF. The change in tumor volume over time and tumor weight at time of harvest were measured to assess the effect of TRF on tumor growth. Also, insulin dependency of tumor growth was studied in mice fed with HFD with or without diazoxide in the ad libitum condition. Insulin dependent tumor growth was further confirmed in normal chow‐fed mice implanted with an insulin pump.ResultsThe studies in VCD treated or ovariectomized postmenopausal mice, revealed that confining access to western‐style HFD during active phase (night time) improves glucose tolerance, insulin resistance and hepatic steatosis without much change in quantity of food intake compared to the ad libitum group. Further, mice with TRF had reduced tumor growth in comparison to the ad libitum group in both models. More importantly, the results from the tumor growth study in mice fed with HFD with/without diazoxide or normal chow mice with/without insulin pump, suggest that the tumor growth is insulin‐dependent and TRF may act via attenuating insulin signaling.ConclusionThe results demonstrate that, TRF improves metabolic deregulation and reduces tumor growth in obese postmenopausal mice and imply that these effects are insulin‐dependent.Support or Funding InformationNIH/NCI RO1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Time-restricted feeding improves insulin resistance and hepatic steatosis in a mouse model of postmenopausal obesity

BackgroundMenopause is associated with significant hormonal changes that result in increased total body fat and abdominal fat, amplifying the risk for metabolic syndrome and diseases such as diabetes, cardiovascular disease and cancer in postmenopausal women. Intermittent fasting regimens hold significant health benefit promise for obese humans, however, regimens that include extreme daytime calorie restriction or daytime fasting are generally associated with hunger and irritability, hampering long-term compliance and adoption in the clinical setting. Time-restricted feeding (TRF), a regimen allowing eating only during a specific period in the normal circadian feeding cycle, without calorie restriction, may increase compliance and provide a more clinically viable method for reducing the detrimental metabolic consequences associated with obesity. MethodsWe tested TRF as an intervention in a mouse model of postmenopausal obesity. Metabolic parameters were measured using Clinical Laboratory Animal Monitoring System (CLAMS) and we carried out glucose tolerance tests. We also stained liver sections with oil red O to examine steatosis and measured gene expression related to gluconeogenesis. ResultsPreexisting metabolic disease was significantly attenuated during 7 weeks of TRF. Despite having access to the same high fat diet (HFD) as ad libitum fed (ALF) mice, TRF mice experienced rapid weight loss followed by a delayed improvement in insulin resistance and a reduced severity of hepatic steatosis by having access to the HFD for only 8h during their normal nocturnal feeding period. The lower respiratory exchange ratio in the TRF group compared with the ALF group early in the dark phase suggested that fat was the predominant fuel source in the TRF group and correlated with gene expression analyses that suggested a switch from gluconeogenesis to ketogenesis. In addition, TRF mice were more physically active than ALF fed mice. ConclusionsOur data support further analysis of TRF as a clinically viable form of intermittent fasting to improve metabolic health due to obesity.

Teneligliptin improves metabolic abnormalities in a mouse model of postmenopausal obesity.

A decrease in serum estrogen levels in menopause is closely associated with the development of visceral obesity and the onset of type 2 diabetes in women. In the present study, we demonstrated the therapeutic effects of the novel DPP4 inhibitor, teneligliptin, on the features of postmenopausal obesity in mice. In the control group, female C57BL/6 mice were sham-operated and maintained on a standard diet. In the postmenopausal obese group, ovariectomized (OVX) mice were maintained on a high-fat diet, and were referred to as OVX-HF. In the treated group, teneligliptin at 60 mg/kg per day was administrated to OVX-HF, and were referred to as Tene. After a 12-week food challenge, the metabolic phenotypes of these mice were analyzed. Body weight, fat accumulation, and glucose intolerance were greater in OVX-HF than in control, while these abnormalities were markedly improved without alterations in calorie intake in Tene. Teneligliptin effectively ameliorated the characteristics of metabolic abnormalities associated with postmenopausal obesity. Regarding chronic inflammation in visceral adipose tissue, the numbers of F4/80(+)CD11c(+)CD206(-) M1-macrophages in flow cytometry, crown-like structure formation in immunohistochemistry, and proinflammatory cytokine expression were significantly attenuated in Tene. Hepatic steatosis was also markedly improved. Furthermore, decreased energy consumption in the dark and light phases, reduced locomotor activity in the dark phase, and lowered core body temperature in OVX-HF were ameliorated in Tene. Since obesity and reduced energy metabolism are a common physiology of menopause, teneligliptin appears to be beneficial as a treatment for type 2 diabetes in postmenopausal obesity.

Abstract 903: Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice

Abstract Metformin has been shown to decrease breast cancer incidence in diabetic women, suggesting that improvement of metabolic status may be a useful approach to decrease breast cancer risk. As the putative cellular target of metformin, AMP-activated protein kinase (AMPK) not only regulates energy metabolism in liver, muscle, and adipose tissue but also inhibits protein synthesis and induces cell cycle arrest and apoptosis. Naringenin is a flavonoid abundant in citrus fruits that may have beneficial effects on metabolic health and tumorigenesis. Our in vitro data showed that naringenin inhibited proliferation of E0771 murine breast cancer cells, increased AMPK activity, and reduced expression of cell cycle regulatory proteins Cyclin D1 and anti-apoptotic Bcl-2 protein. In this study, we evaluated whether dietary naringenin acts like metformin to suppress mammary tumor growth in a mouse model of postmenopausal obesity. Ovariectomized female C57BL/6J mice were fed a high-fat diet (60% energy as fat) for 3 weeks to induce obesity. Then mice were randomized to experimental diets of HF (continued the high-fat diet), LN (high fat + 1% wt/wt naringenin in diet), HN (high fat + 3% wt/wt naringenin in diet), and Met (high fat + 180 mg/kg metformin in water) for 5 weeks. After 2 weeks of treatment, 5 × 10(5) E0771 cells were injected into one thoracic mammary fat pad. Mice were maintained on study diets for another 3 weeks. For all mice tumor weight correlated with weight gain in the first 5 weeks prior to cell inoculation (r = 0.51, p < 0.01). There was no difference in fasting glucose levels among the groups on week 8. No difference was observed between the HF and LN groups regarding final body weight, food intake, tissue weights, and tumor mass. HN significantly decreased final body weight, food intake, and adipose depot mass. mRNA expression of two inflammatory cytokines, interleukin 6 and monocyte chemoattractant protein-1 were decreased in perigonadal fat of the HN group. Although tumor growth (cm2) decreased over time in HN mice, there was no significant difference in tumor mass between HF and HN groups at necropsy (p = 0.4). HN did not affect AMPK activity in tumor tissue, but decreased Bcl-2 protein expression. Additionally, dietary naringenin resulted in a dose ­dependent accumulation in plasma, tumor, and mammary fat. In comparison to naringenin enriched diets, metformin significantly reduced tumor growth and final tumor weight, without affecting body weight, food intake, and tissue weights. Metformin decreased protein expression of Cyclin D1 in mammary tumors. However, we did not observe AMPK activation and changes in Bcl-2 protein levels in tumors. Taken together, these data suggest that metformin may inhibit mammary tumor growth without affecting metabolic status as assessed by body weight, adiposity, and fasting glucose. There may be different mechanisms of action for naringenin and metformin to alter mammary tumorigenesis. Citation Format: Jia-Yu Ke, Yung-Hsuan Hsiao, Shana R. Straka, Lisa D. Yee, Martha A. Belury. Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 903. doi:10.1158/1538-7445.AM2015-903

Abstract A005: MMP FRET ACPP imaging of mammary tumors in a mouse model of postmenopausal obesity

Abstract Obesity in postmenopausal women is a risk factor for breast cancer. Since 25-30% of women in the USA are obese, the public health implications of obesity and breast cancer are immense. Activation of inflammatory pathways is one plausible mechanism underlying the association between obesity and increased breast cancer risk. We have used a novel immunocompetent mouse model of luminal breast cancer to demonstrate that luminal tumor progression is enhanced in ovariectomized mice fed a high fat diet (HFD). The obese mammary fat pads are proinflammatory as measured by an increase in M1 polarized macrophages, increased TNF-α production and increased matrix metalloproteinase (MMP)-2 and -9 expression. To evaluate activatable cell penetrating peptide (ACPP) targeting of mammary tumors in the obese fat pad we utilized a version of the MMP cleavable ACPP that has a Cy5 fluorophore attached to the polycationic domain of the peptide and a Cy7 conjugated to the polyanionic domain. Upon cleavage of the peptide by MMPs, the fluorescence resonance energy transfer (FRET) between the two dyes is disrupted and the Cy5 emission increases while the Cy7 re-emission diminishes. This change in emission spectra is measured and the ratio of Cy5 to Cy7 fluorescence is used as a measure of peptide cleavage. The ACPP was administered at a dose of 0.4nmol/g and allowed to circulate for 2 hours prior to harvesting of the fat pads and imaging. The Cy5/Cy7 ratio was 24% higher in the obese fat pads, with a 45% increase in fluorescence when examining just the Cy5 emission. To determine if tumors could be identified in the obese fat pads with the higher background resulting from increased macrophage recruitment and MMP activity, normal chow (NC) and HFD fed C57Bl/6 mice were injected with syngeneic luminal Py230 and claudin low EMT Py8119 cell lines derived from spontaneous polyomavirus middle T (PyVmT) tumor homogenates. Mice harboring both Py230 and Py8119 tumors in their thoracic mammary fat pads were dosed with either the MMP cleavable FRET ACPP or an uncleavable control ACPP. Two hours after peptide injection, mice were sacrificed and the skin was removed to image the tumors in the context of the fat pad. In concordance with our previous results, tumor growth of the Py230 cells was significantly greater in the HFD mice. Interestingly, growth of the more aggressive Py8119 tumors was not enhanced in the HFD microenvironment. For both cell lines, the tumors were clearly delineated using the Cy5/Cy7 ratio. The Py8119 tumors had a ratio of 6.1±0.7 in NC mice and 5.4±0.3 in the HFD mice, and a similar trend was observed in the less aggressive luminal Py230 tumors, with ratios of 4.9±0.8 for NC and 3.8±0.4 for HFD fed mice. This was consistent with analysis showing increased macrophage recruitment in the Py8119 tumors compared with the Py230 tumors, but no difference in recruitment between tumors of either type in mice fed NC or HFD. Our results indicate that although inflammation associated with obesity results in higher background fluorescence in the mammary fat pad, this is not a barrier to visualizing the tumor tissue in the fat pad. Since a large amount of adipose tissue can be problematic during surgery, MMP FRET ACPPs may be useful for identifying and resecting breast cancers in obese patients leading to improved survival. Citation Format: Jessica L. Crisp, Elamprakash N. Savariar, Jonathan P. Hasselmann, Heekyung Chung, Roger Y. Tsien, Lesley G. Ellies. MMP FRET ACPP imaging of mammary tumors in a mouse model of postmenopausal obesity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A005.

Abstract A123: Omega-3 fatty acids reduce mammary tumor growth in a mouse model of postmenopausal breast cancer

Abstract Obesity is a risk factor for postmenopausal breast cancer. Inflammation, hyperinsulinemia, insulin resistance and altered levels of adipokines have been proposed as possible links between obesity and tumor progression. Furthermore, the composition of fats in the diet can influence inflammation and epidemiological studies have shown that an increase in fish oil (omega-3 fatty acids; ω-3 FAs) intake is associated with a reduction in breast cancer incidence in humans. Several mechanisms have been proposed for the anticancer effect of ω-3 FAs such as suppression of neoplastic transformation, cell proliferation or angiogenesis, or an increase in apoptosis. To study the effects of ω-3 FA on mammary tumor growth in a mouse model of postmenopausal obesity, ovariectomized C57BL/6 mice were fed either a high fat diet (HFD, 60% kcal from fat) or an isocaloric HFD containing 30% ω-3 FA replacement [wt/wt, 24% docosahexaenoic acid (DHA), 6% eicosapentaneoic acid (EPA)]. Fourteen weeks after starting HFDs, mice received orthotopic mammary fat pad injections of Py230 cells derived from a spontaneous polyomavirus middle T antigen (PyVmT) mammary tumor. Mammary tumor growth, inflammatory cytokine expression in perigonadal fat and the mammary fat pads and tumors, and apoptosis and cell proliferation in the tumors were determined at 22 weeks following initiation of the HFDs. There was no difference in body weights between mice fed a HFD with or without ω-3 FA, but ω-3 FA-fed mice had a significantly reduced mammary tumor burden (33%) compared to counterpart HFD-fed mice. The ω-3 FA-fed mice showed decreased expression of macrophages and inflammatory cytokines such as CD11c, TNF-α and iNOS in perigonadal fat. However, surprisingly the ω-3 FA-fed mice showed increased macrophage and inflammatory cytokine expression: CD11c, TNF-α, IL-1β and IL-6 in the mammary fat pads implanted with tumor cells, suggesting that the anti-inflammatory effect of ω-3 FAs may be modulated by implanted tumor cells. No difference was observed in the expression of macrophage marker and inflammatory cytokines in mammary tumors between mice fed a HFD with or without ω-3 FA. Immunohistochemical staining of mammary tumors revealed a high degree of variability in cell proliferation and apoptosis between tumors and no significant differences were observed. To examine whether ω-3 FAs may act directly on Py230 tumor cells to reduce mammary tumor growth, we tested the effects of ω-3 FA on cell growth, apoptosis and signaling in Py230 cells in vitro. DHA significantly inhibited Py230 cell growth in a dose-dependent manner. In addition, DHA inhibited TNF-α-induced cell growth and blocked NF-κB signaling activated by TNF-α in Py230 cells. Furthermore, DHA increased the activities of caspases 3 and 7 and induced apoptosis in Py230 cells. Our results suggest that ω-3 FAs may suppress mammary tumor growth by directly inhibiting the growth of mammary tumor cells via induction of apoptosis. Further analysis of the signaling pathways involved may identify new therapeutic targets for postmenopausal women with breast cancer. Citation Format: Heekyung Chung, Dorothy D. Sears, Nicholas J. Webster, Jerrold M. Olefsky, Lesley G. Ellies. Omega-3 fatty acids reduce mammary tumor growth in a mouse model of postmenopausal breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A123.

Should diabetic women with breast cancer have their own intervention studies?

This commentary on 'Calorie restriction and rapamycin inhibit MMTV-Wnt-1 mammary tumor growth in a mouse model of postmenopausal obesity' by Nogueira et al., published in this issue of Endocrine-Related Cancer, addresses the challenges of translating diet, exercise, and pharmacologic trials in diabetic mouse mammary tumor models to human studies. We propose that trials specifically designed to test such interventions in diabetic women with breast cancer would be valuable and informative.

Calorie restriction and rapamycin inhibit MMTV-Wnt-1 mammary tumor growth in a mouse model of postmenopausal obesity

Obesity is an established risk and progression factor for postmenopausal breast cancer. Interventions to decrease caloric intake and/or increase energy expenditure beneficially impact tumor progression in normoweight humans and animal models. However, despite the increasingly high global prevalence of obesity, the effects and underlying mechanisms of these energy balance modulating interventions are poorly characterized in obese individuals. The goal of this study was to better characterize the mechanism(s) responsible for the link between energy balance and breast cancer progression in the postmenopausal obesity context. We compared the effects of calorie restriction (CR), treadmill exercise (EX), and mammalian target of rapamycin (mTOR inhibitor) treatment on body composition, serum biomarkers, cellular signaling, and mammary tumor growth in obese mice. Ovariectomized C57BL/6 mice were administered a diet-induced obesity regimen for 8 weeks, then randomized into three treatment groups: control (semipurified diet fed ad libitum, maintained the obese state); 30% CR (isonutrient relative to control except 30% reduction in carbohydrate calories); and EX (control diet fed ad libitum plus treadmill exercise). Mice were implanted with syngeneic MMTV-Wnt-1 mammary tumor cells at week 12. Rapamycin treatment (5 mg/kg every 48 h) started at week 14. Tumors were excised at week 18. CR and rapamycin (but not EX) significantly reduced final tumor weight compared to control. In follow-up analysis, constitutive activation of mTOR ablated the inhibitory effects of CR on Wnt-1 mammary tumor growth. We conclude that mTOR inhibition may be a pharmacologic strategy to mimic the anticancer effects of CR and break the obesity-breast cancer progression link.

Abstract A75: Calorie restriction and rapamycin, but not exercise, inhibit mammary tumor growth in a mouse model of postmenopausal obesity

Abstract Obesity is a well-established risk factor for postmenopausal breast cancer and is associated with poor outcomes in both premenopausal and postmenopausal women. Given that approximately one third of the US population is obese, interventions to reverse the detrimental effects of obesity on breast cancer progression are urgently needed. The molecular target of Rapamycin (mTOR) modulates pathways regulating energy balance and cell growth. Therefore, agents that target the mTOR pathway are promising for breaking the obesity-breast cancer link. Here, we directly compared the two most recommended strategies for reversing obesity, calorie restriction (CR) and exercise (EX), and a well-established mTOR inhibitor (rapamycin) on mammary tumor growth and metabolic profiles in obese mice. Ovariectomized C57BL/6 mice were administered a diet-induced obesity regimen for 8 weeks, then randomized into 3 diet or exercise treatment groups: Control (AIN-76A diet fed ad libitum to maintain the obese state, n=30); 30% CR (70% of the carbohydrate calories but otherwise isonutrient relative to control, n=15); EX (control diet fed ad libitum plus treadmill exercise regimen, n=15). Mice were orthotopically implanted (4th mammary gland) with syngeneic MMTV-Wnt-1 mammary tumor cells at week 12. Of the 30 mice in the control group, 15 received rapamycin (5mg/kg, administered ip every 48hrs) starting at week 14. All other mice received vehicle treatment (saline, administered ip every 48 hrs) starting at week 14. Tumors were excised at week 18. Final tumor weights from mice in the CR (0.043 ± 0.007 g) and rapamycin (0.072 ± 0.010 g) groups, but not EX (0.380 ± 0.072 g) group, were significantly reduced compared to control (0.390 ± 0.066 g). The crucial role of mTOR in the link between obesity and breast cancer progression was confirmed in a follow up study where constitutive activation of mTOR (mTOR) ablated the inhibitory effects of CR on mammary tumor growth. CR failed to reduce tumor weight in mice injected with cells transfected with mTOR (0.38 ± 0.05 g), compared to control (0.55 ± 0.08 g). Transfection effect bias was discarded since CR significantly reduced weight of tumors (0.29 ± 0.03 g) in mice injected with cells transfected with WT mTOR, compared to control (0.62 ± 0.12 g). We conclude that mTOR inhibition may be a pharmacologic strategy to mimic the anticancer effects of CR and break the obesity-breast cancer progression link. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A75.

The Peptidic GHS-R antagonist [D-Lys3]GHRP-6 markedly improves adiposity and related metabolic abnormalities in a mouse model of postmenopausal obesity

It was demonstrated that estrogen deficiency and consuming high fat (HF) diet enhanced orexigenic activity of ghrelin. Therefore, we hypothesized that antagonizing of ghrelin action would attenuate food intake and body weight in mice obese both from ovariectomy (OVX) and feeding a HF diet.Ghrelin receptor antagonist [D-Lys3]GHRP-6 after seven days of subcutaneous treatment markedly decreased food intake in OVX mice fed both HF and standard diets; furthermore, it reduced body weight and blood glucose, insulin and leptin, and increased β-hydroxybutyrate level and uncoupling-protein-1 mRNA in brown adipose tissue. Pair-feeding revealed that effect of [D-Lys3]GHRP-6 was primary anorexigenic. Estrogen supplementation reduced anorexigenic effects of [D-Lys3]GHRP-6. OVX [D-Lys3]GHRP-6 treatment in mice on HF diet resulted in markedly increased circulating level and liver expression of a major metabolic regulator, fibroblast growth factor 21.Our data suggest that ghrelin antagonists could be especially beneficial in individuals with common obesity combined with estrogen deficiency.

Abstract 2930: Calorie restriction and rapamycin, but not exercise, inhibit MMTV-Wnt-1 mammary tumor growth in a mouse model of postmenopausal obesity

Abstract Obesity is an established breast cancer risk factor in postmenopausal women, and calorie restriction (CR) and exercise (EX) are the two most recommended ways to prevent or reverse obesity. The mammalian Target of Rapamycin (mTOR) pathway is a potential mechanistic target underlying the effects of these interventions, and rapamycin (mTOR inhibitor) has been shown to mimic many of the anticancer effects of CR. However, the effects of obesity reversal by CR, EX, or treatment with rapamycin, on mammary tumor development have not been well studied. Therefore, we administered a diet-induced obesity regimen to 60 ovariectomized C57BL/6 mice for 8 weeks to induce obesity. The mice were then randomized into 4 groups (n=15/group): Control (AIN-76A diet ad libitum); CR (30% calorie restriction relative to control); EX (control diet ad libitum + treadmill exercise at 20m/min, 45min/day, 5days/week); and Rapamycin (control diet ad libitum + 5mg/kg injected i.p. every 48 hrs). Body weight and total body fat were measured every two weeks (using MRI). All mice were injected (4th mammary fat pad) with 5 × 104 MMTV-Wnt-1 mammary tumor cells at week 12, and at week 18 tumors were extracted and analyzed by immunohistochemistry (IHC). At the end of the study, mice in the CR group weighed significantly less (27.6 ± 2.6 g) and displayed lower percent body fat (34.7 ± 5.2 %) than mice in the control group (41.7 ± 2.9 g and 47.9 ± 1.8 %). There were no differences in body weigh or percent body fat in the EX (38.3 ± 3.10 g and 45.9 ± 2.6 %) and Rapamycin (37.4 ± 2.7 g and 46.6 ± 5.6 %) groups compared to control. At the end of the study, the CR and Rapamycin groups had comparable tumor weights (0.04 ± 0.02 g and 0.07 ± 0.04 g) that were significantly smaller than those in the control and EX groups (both 0.3 ± 0.2 g and 0.3 ± 0.2 g). IHC analysis showed that Rapamycin significantly reduced p-mTOR, Cyclin D1 and p-S6 Ribosomal Protein (markers of mTOR activation) compared to control, Exercise and CR. Exercise displayed variable levels of p-mTOR, Cyclin D1 and p-S6 and CR displayed no differences compared to control. Rapamycin tumors were bimodal for both p-Akt (upstream of mTOR) and Ki67 (proliferation marker); those tumors displaying high p-Akt (∼50%) also displayed high Ki67 staining, but tumors with low pAkt displayed low Ki67 staining. CR and Exercise had no effect on p-Akt and Ki67 compared to control. There were no differences in Cleaved-Caspase 3 (marker of apoptosis) between the groups. We conclude that CR and Rapamycin reverse the effects of obesity on Wnt-1 mammary tumor growth, but apparently via different mechanisms, while EX does not appear to have anti-tumor growth effects in obese mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2930.

Abstract B3: Exercise and calorie restriction differentially regulate energy balance-related cell signaling pathways in a model of post-menopausal obesity

Abstract B3 Epidemiological data suggest that induced negative energy balance through calorie restriction (CR) or increased exercise (EX) decreases postmenopausal breast cancer risk associated with obesity. Although the mechanism responsible for the beneficial effects of these anti-obesity strategies is not known, hormone alteration seems to play a critical role. Increased adiposity is associated with alterations in circulating adipokines, such as increased leptin and decreased adiponectin levels. In fact, reports show a positive correlation between leptin levels and breast cancer risk, and a negative association between adiponectin levels and breast-cancer patients. The aim of this study was to compare changes in adipokines and downstream signaling pathways proven to be altered in human breast cancers following either CR or EX in a mouse model of post-menopausal obesity. Ovariectomized female C57BL/6 mice (n=45) were administered a high-fat diet for 8 weeks to induce obesity. At week 9, these diet induced obesity (DIO) mice were randomized into 3 groups (n=15/group): control (AIN-76A diet ad libitum); CR (30% reduction in calories relative to control); EX (control diet + increased physical activity). The EX group was run on a variable speed treadmill for 45 minutes a day, five days a week at a maximum speed of 20 m/min At week 16, mice were euthanized and tissue was collected. At the end of the study, CR mice weighed significantly (p<.01) less than control mice (19.9 ± 0.5 vs. 28.8 ± 0.6 g, respectively) and the EX mice (26.0 ± 0.9). Although the CR mice weighed significantly less than the exercised mice, there was no difference in percent body fat between the groups (38.9 ± 1.7 vs. 33.7 ± 1.4 %, respectively), and both groups had significantly less percent body fat than the sedentary, ad libitum-fed controls (48.1 ± 1.9 %). CR, but not EX, demonstrated improved insulin sensitivity based on a glucose tolerance test compared to controls. Serum leptin levels were significantly reduced in CR and EX mice compared to controls and CR displayed increased serum adiponectin levels compared to exercise. Some of the cell signaling pathways regulated by these hormones, including AMPK, Akt and ERK, converge at mTOR; a molecule involved in coupling energy balance signals to protein translation and cell growth. Interestingly, mammary fat pads of CR, but not EX, displayed significantly higher AMPK phosphorylation and lower Akt phosphorylation relative to controls. Alternatively, mammary fat pads from both CR and EX displayed much lower levels of ERK phosphorylation compared to control. In addition, the key downstream effectors of mTOR activation such as S6 kinase and S6 ribosomal protein displayed decreased activation in both CR and EX compared to control. These data suggest that although EX can act on similar pathways as CR, CR possess a more global effect on cell signaling and therefore may produce a more potent anti-cancer effect. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B3.