Published OnlineFirst January 21, 2011; DOI: 10.1158/1940-6207.CAPR-10-0140 Cancer Prevention Research Article Research Effect of Chronic and Intermittent Calorie Restriction on Serum Adiponectin and Leptin and Mammary Tumorigenesis Olga P. Rogozina1, Melissa J.L. Bonorden1, Christine N. Seppanen1, Joseph P. Grande2, and Margot P. Cleary1 Abstract The effect of chronic (CCR) and intermittent (ICR) caloric restriction on serum adiponectin and leptin levels was investigated in relation to mammary tumorigenesis. 10-wks old MMTV-TGF-a female mice were assigned to ad libitum fed (AL; AIN-93M diet), ICR (3-week 50% caloric restriction, AIN-93M-mod diet, 2Â protein, fat, vitamins, and minerals followed by 3-wks 100% AL consumption of AIN-93M), and CCR (calorie and nutrient intake matched for each 6-wks ICR cycle, 75% of AL) groups. Mice were sacrificed at 79 (end of restriction) or 82 (end of refeeding) wks of age. Serum was obtained in cycles 1, 3, 5, 8, 11, and terminal. Mammary tumor incidence was 71.0%, 35.4%, and 9.1% for AL, CCR, and ICR mice, respectively. Serum adiponectin levels were similar among groups with no impact of either CCR or ICR. Serum leptin level rose in AL mice with increasing age but was significantly reduced by long-term CCR and ICR. The ICR protocol was also associated with an elevated adiponectin/leptin ratio. In addition, ICR- restricted mice had increased mammary tissue AdipoR1 expression and decreased leptin and ObRb expression compared with AL mice. Mammary fat pads from tumor-free ICR-mice had higher adiponectin expression than AL and CCR mice whereas all tumor-bearing mice had weak adiponectin signal in mammary fat pad. Although we did not show an association of either adiponectin or leptin with individual mice in relation to mammary tumorigenesis, we did find that reduced serum leptin and elevated adiponectin/leptin ratio were associated with the protective effect of intermittent calorie restriction. Cancer Prev Res; 4(4); 568–81. Ó2011 AACR. Introduction 23). Also, leptin receptors (ObRb and ObR; refs. 14, 15, 17, 24), and adiponectin receptors 1 (AdipoR1) and 2 (Adi- Epidemiologic and preclinical rodent studies suggest an poR2) as well as adiponectin (20, 22, 23, 25) itself have important, but still controversial, role of adipose tissue been found to be expressed in a number of human breast mass in mammary tumorigenesis (1–9). Factors produced cancer cell lines. directly in adipose tissue, adipokines, in particular leptin In addition to in vitro studies indicating that human and adiponectin, are now recognized for their influence on breast cancer cells respond to these adipokines, leptin, breast cancer risk and mammary tumor biology (10–13). adiponectin, and their receptors have been identified in Their physiologic and pathologic relationships are largely breast/mammary tumors and mammary tissues of humans in opposition to each other, as are their biological effects on and rodents (20, 25–29). Analyses of human tissue biop- mammary and breast cancer cells. For example, leptin sies revealed that leptin and its receptor are overexpressed stimulates the proliferation of estrogen receptor (ER)-posi- in breast tumors compared with noncancer breast epithe- tive breast cancer cell lines (14–17) whereas results for ER- lium and the expression of leptin is positively correlated negative breast cancer cell lines are less consistent (17–19). with expression of the leptin receptor in breast carcinoma In contrast, studies using human breast cancer cell lines cells (26, 27, 30, 31). With respect to adiponectin, Karadu- indicate that adiponectin inhibits proliferation of a num- man and colleagues (29) reported that adiponectin levels ber of ER-positive and -negative breast cancer cell lines (20– measured by ELISA were significantly higher in mammary tissue obtained from breast cancer patients than that obtained from healthy control subjects. In another study, adiponectin mRNA expression level was significantly Authors' Affiliations: 1The Hormel Institute, University of Minnesota, Austin and 2Mayo Clinic, Rochester, Minnesota higher in mammary tissue adjacent to breast tumors com- Corresponding Author: Margot P. Cleary, The Hormel Institute, University pared with either breast tumor tissue or control tissue from of Minnesota, 801 16th Ave NE, Austin, MN 55912. Phone: 507-437-9656; subjects without breast cancer, but AdipoR1 mRNA expres- Fax: 507-437-9606. E-mail: [email protected] sion level in mammary tissue adjacent to the breast tumor doi: 10.1158/1940-6207.CAPR-10-0140 was similar to the tumor itself. However, AdipoR1 level in a Ó2011 American Association for Cancer Research. number of breast tumors was higher than that in control 568 Cancer Prev Res; 4(4) April 2011 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst January 21, 2011; DOI: 10.1158/1940-6207.CAPR-10-0140 Adiponectin, Leptin and Mammary Tumors mammary tissue obtained from an individual without has been the consistent finding that when calories are breast cancer, whereas there were no differences in Adi- restricted intermittently this protocol is more protective poR2 expression levels among control, adjacent, and tumor than the same degree of restriction implemented by chronic tissues (20). In the MMTV-TGF-a transgenic mouse, which restriction. The aim of this study was to determine whether develops hormone responsive mammary tumors, ObRb serum leptin and/or adiponectin play a role in the protec- was found to be expressed in both mammary fat pad and tive effect of calorie restriction in relationship to the man- mammary tumors (28). ner in which calories are reduced. Longitudinal blood A number of studies have evaluated serum leptin and samples were obtained at specific ages over the course of adiponectin levels in women with breast cancer. Results for the study as well as at the terminal end point. Thus, whether leptin have been inconsistent (32–44). However, serum serum adipokine levels were able to predict mammary adiponectin levels have been reported to be lower in tumors could be addressed. Additionally, the expression women with breast cancer compared with controls (20, of proteins associated with the signaling of leptin and 36, 38, 43, 45–47). In addition, it was reported that lower adiponectin was measured in mammary tissue, mammary serum adiponectin (47) and higher leptin (44) levels are fat pad, and mammary tumors. associated with higher mammary tumor grade. Under normal circumstances, higher serum leptin levels Material and Methods are associated with increased body weight and body fat in humans and rodents (8, 28, 48–54), whereas adiponectin Animals and study design levels are lower at higher body weights (49, 50, 55, 56). MMTV-TGF-a female mice overexpressing human TGF- Interventions which result in weight loss have been a were produced at the Hormel Institute (Austin, MN) reported to "normalize" these circulating factors such that and genotyped as previously described (62). At 8 weeks of leptin is reduced and adiponectin increased. With increas- age, mice were randomized and assigned to one of the ing body weight the relative relationship of these two following dietary groups: ad libitum fed (AL; n ¼ 75), adipokines becomes more divergent. Thus, dependent chronic calorie restricted (CCR; n ¼ 75), and intermittent on body weight status tissues would be exposed to very restricted (ICR; n ¼ 75). They were housed individually different relative amounts of these two proteins. For exam- and provided ad libitum access to water and powdered ple, we recently reported that mice with goldthioglucose- AIN-93M diet for 2 weeks to allow accommodation to the induced obesity had adiponectin/leptin ratio 10-fold less powdered diet. At 10 weeks of age mice began to consume than the lean mice (57). There are two studies related to their assigned diets. Those in the AL group continued to adiponectin/leptin ratio in women with breast cancer. have free access to AIN-93M diet. Mice in the CCR group Chen and coworkers (36) found that women with breast were given a diet formulated to be isocaloric with the cancer had increased ratio of leptin/adiponectin (conver- AIN-93M diet with 25% increases in protein, vitamin, sely a decreased adiponectin/leptin ratio). In another mineral, and fat content. This diet was given at 75% of report, the adiponectin/leptin ratio was calculated by using age-matched ad libitum consumption. Mice in the ICR average values and it was found to be 15% higher in age- group were provided a modified AIN-93M diet with 2- matched controls than in postmenopausal breast cancer fold increases in protein, vitamin, mineral and fat con- subjects, although for premenopausal women the average tent, and fed at 50% of the consumption level of AL mice adiponectin/leptin ratio was 30% lower in age-matched during every 3 weeks of restriction. Following each restric- controls than in women with breast cancer (43). These tion period, ICR mice were provided with AIN-93M diet results suggest that the balance of adiponectin to leptin at 100% of age-matched AL consumption for 3 weeks. The rather than either adiponectin or leptin levels alone may diet compositions have been previously described in play important roles in the development of cancer. detail (62, 63). This approach results in all mice receiving In general, these cited studies obtained blood samples at the same absolute intakes of all nutrients except carbo- the time of breast cancer diagnosis; thus, the findings are not hydrate during each 6-week cycle. Further, the only dif- definitive for adiponectin and leptin involvement in breast ference between CCR and ICR mice is the manner in tumor development, that is, the serum changes may reflect which the overall restriction of 25% is implemented. Over physiologic response to the presence of the tumor and/or the course of the study, food intakes and general condi- the tumor itself produces the adipokines.