Effect of Chronic and Intermittent Calorie Restriction on Serum Adiponectin and Leptin and Mammary Tumorigenesis

Published OnlineFirst January 21, 2011; DOI: 10.1158/1940-6207.CAPR-10-0140

Cancer Prevention Research Article Research

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, 2 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. 2011 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 2011 American Association for Cancer Research. number of breast tumors was higher than that in control

568 Cancer Prev Res; 4(4) April 2011

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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. In humans, a long- tion of each mouse were determined daily. Each mouse term prospective study would be very difficult and expensive was weighed weekly and at that time mice were palpated to undertake. Thus, the use of a relevant preclinical model for mammary tumors. Once a tumor was detected, the would provide the opportunity to assess the role of serum mouse was closely monitored for signs of tumor-related adipokines in mammary tumorigenesis. We have utilized distress. Mammary tumors were measured weekly with MMTV-TGF-a mice as a model for human breast cancer as calipers. All mice were euthanized by CO2 overdose when tumors develop primarily in the second year of life and are they reached the predetermined terminal age of either considered to be hormone responsive (58, 59). 79 weeks (end of restriction) or 82 weeks (end of refeed- We have reported that calorie restriction prevents and ing) or when mammary tumor size exceeded 20 mm in delays tumor development in MMTV-TGF-a mice (54, 60, length or weight loss exceeded 25% from the previous 61). An interesting aspect of these calorie restriction studies week. When results are presented specific to the ICR mice

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during restriction periods they are further classified as immunoglobulin G were used as a secondary antibody. ICR-restricted and during refeeding periods they are clas- Antibody-bound proteins were detected by enhanced sified as ICR-refed. The Hormel Institute Animal Facility chemifluorescence (ECF substrate; Amersham Pharmacia is accredited by AAALAC. The University of Minnesota Biotech) and analyzed with the Storm 840 Machine Ima- IACUC approved the study and procedures. ging System (Molecular Dynamics). Standard molecular weight markers were run simultaneously for comparing Tissue sample collection and histopathologic analysis molecular weights of the visualized proteins. The intensity At sacrifice, fat pads (mammary, retroperitoneal, and of Western blot bands was quantified by densitometric parametrial), mammary tissues from the back of the neck analysis by using the ImageJ program. Results were and axillary areas, livers, mammary tumors, and any expressed as the ratio of intensity of the protein of interest abnormalities were removed and weighed. A sample of to that of b-actin (Delta Biolabs LLC) from the same each tissue was placed in 10% neutral buffered formalin. sample. The remaining tissues were stored at 70C. Left mammary fad pads, mammary tissues or mammary tumors Statistical Analysis and tissue samples that appeared abnormal were sent to Data are presented as mean SEM. Serum data were the Department of Pathology and Laboratory Medicine of analyzed by ANOVA followed by the Neuman–Keuls test or the Mayo Foundation (Rochester, MN) for histopatholo- t test. Mammary tumor incidence was analyzed by the "c2" gic analyses to determine malignancy and/or disease test and 2-group log-rank test. Graph Pad Prism version 4 status. was used for statistical analyses (GraphPad Software, http://www.graphpad.com/). Assessment of Serum Leptin and Adiponectin Concentrations Results Blood samples were collected from the orbital sinus from all mice at the terminal points and over the study (cycles 1, 3, Mammary tumor incidence, body and tissue weights, 5, 8, and 11 from 3 cohorts corresponding to the first, second, and terminal serum measurements and third weeks of restriction and refeeding of ICR mice). For The data presented here are part of an extensive study of each mouse 2 samples were obtained per cycle such that for serum factors potentially involved in mammary tumor cohort 1 samples were obtained corresponding to the first prevention resulting from different modes of calorie week of restriction and the first week of refeeding (week 1 and restriction. In our first paper (61), we focused on the 4 of the cycle), for cohort 2 after 2 weeks of restriction and insulin like growth factor I (IGF-I) axis and also fully 2 weeks of refeeding (week 2 and 4 of the cycle), and cohort 3 described tumor characteristics for the AL, CCR, and ICR after 3 weeks of restriction and 3 weeks of refeeding (week 3 groups. The mammary tumor incidence was 71.0%, and 6 of the cycle). Serum samples were stored in 20C 35.4%, and 9.1% for AL, CCR, and ICR mice (P < until used. Leptin was measured by the Mouse Leptin ELISA 0.0001), respectively, and all groups were significantly kit (EZML-82K; Linco Research). Serum Adiponectin levels different from each other (Fig. 1A). In addition, the age at were determined by a Mouse Adiponectin ELISA kit (EZML- which mammary tumors were detected (Fig. 1B) was 60K, Linco Research). Adiponectin/leptin ratio was calcu- extended from 61 (1.1) weeks of age in AL mice to lated for each mouse by dividing adiponectin serum con- 69 (1.6) and 67 (4.2) weeks of age in CCR and ICR centration by leptin concentration. mice, respectively, although only the CCR value was significantly different from AL mice (P < 0.005). Western blot analysis Although in our previous paper we also completely Tissue samples (mammary tissue from tumor-free mice, described body weight changes, here we present final mammary tumors, and mammary fat pad) were homo- body weights (Fig. 2Aa) as well as mammary genized in extraction buffer with protease inhibitors. Total (Fig. 2Ab) and internal fat pads weights (Fig. 2Ac). It protein was extracted by a Total Protein Extraction Kit can be seen that AL mice were the heaviest compared with (Chemicon International) and quantified by the Bradford all other groups, followed by CCR (P < 0.001) and ICR- assay. Extracted proteins were electrophoresed on 4% to refed (P < 0.001) with similar weights whereas ICR- 15% polyacrylamide gradient gels and then blocked in Tris- restricted mice weights were the lightest (P < 0.001). In Base solution containing 5% milk concentrate and 1% addition, ICR-restricted mice had significantly lower Tween-20 and then transferred to a Immobilon mem- body and fat pad weights than CCR (P < 0.01) and branes (Millipore) and probed by using primary antibodies ICR-refed (P < 0.05) mice. There were no significant for leptin (Ob; Santa Cruz Biotechnology), leptin long differences between ICR-refed and CCR mice for any of (ObRb), and total (ObR; Santa Cruz Biotechnology) forms these measurements. During each weight loss/regain of the leptin receptors and for adiponectin (Pro Sci Incor- cycle, food intake for both restricted groups was 22.9% porated), AdipoR1 (Santa Cruz Biotechnology), and Adi- to 28.7% lower than that of AL mice (not shown) and poR2 (Santa Cruz Biotechnology). Anti-rabbit (for Ob, foodintakesoftheCCRandICRgroupsasperthe ObR, ObRb, and adiponectin; Cell Signaling) or anti-goat experimental design were almost identical during each (for AdipoR1 and AdipoR2; Santa Cruz Biotechnology) food restriction/refeeding cycle (data not shown).

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AB Figure 1. Tumor incidence and 71.0% 70 †, latency of MMTV-TGF-a female 75 * * mice. A, tumor incidence of 60 † MMTV-TGF-a female mice. c2 ¼ P < 50 24.64, 2 degrees of freedom, 50 0.0001. AL, n ¼ 49/69; CCR, n ¼ 40 35.4% 23/65; (n ¼ 6/66). B, tumor latency 30 of TGF-a Female Mice. ANOVA P < n ¼ n ¼ 25

0.005. AL, 49; CCR, 23; Incidence (%) 20 ICR, n ¼ 6. *, †, z, columns with 10 9.1% different superscript symbol are significantly different. 0 Time of (week) detection 0 AL CR ICR C AL CCR ICR

Here we used serum and tissues from these mice with Adiponectin, AdipoR1, AdipoR2, leptin, ObR, and the focus on serum adiponectin and leptin levels in rela- ObRb protein expression in mammary tissue, tumors tionship to mammary tumor development as well as and mammary fat pads assessment of protein expression of these adipokines and A summary of the results of expression of proteins of their receptors in mammary tissues and tumors. At eutha- interest for mammary tissues, tumors, and fat pads is nasia, AL mice had serum leptin levels (Fig. 2Ba) signifi- presented in Table 2 and representative Western blots are cantly higher than CCR (P < 0.01), ICR-refed (P < 0.001), shown in Figure 3. Mammary tissue protein expression of and ICR-restricted (P < 0.001) mice. CCR mice had sig- adiponectin was not different among the groups. Adi- nificantly higher serum leptin values than ICR-refed (P < poR1 expression was somewhat higher in mammary 0.05) and ICR-restricted (P < 0.01) mice. There were no tissue obtained from ICR mice but only the ICR-restricted significant differences for serum leptin levels between ICR- mice value was significantly different (P < 0.05) compared refed and ICR-restricted mice. There was no statistical with that of AL mice. There was not a significant differ- difference (P > 0.05) for terminal adiponectin concentra- ence in AdipoR2 expression among any of the groups. tion among the groups (Fig. 2Bb). We also calculated Expression of leptin was lower in all calorie-restricted serum adiponectin/leptin ratios and found that at eutha- mice compared with AL mice but was only significant for nasia ICR-restricted mice had an adiponectin/leptin ratio CCR (P < 0.05) and ICR-restricted (P < 0.05) mice, and significantly higher than CCR (P < 0.01), ICR-refed (P < there was no significant difference in leptin expression 0.01), and AL (P < 0.01) mice. There were no significant amongICR-refed,CCR,andICR-restrictedmice.ObR differences in adiponectin/leptin ratio between ICR-refed, expression was significantly lower in ICR-refed mice (P CCR, and AL mice (Fig. 2Bc). Leptin (Fig. 2Ca) and adi- < 0.05) than AL mice but there was not a significant ponectin (Fig. 2Cb) serum concentrations were not sig- difference in ObR expression in mammary tissue among nificantly different for AL, CCR, ICR-restricted, and ICR- AL,CCR,andICR-restrictedmiceandalsoamongICR- refed mice that eventually developed mammary tumors restricted, CCR, ICR-refed groups. ObRb expression was compared with those that did not. Terminal adiponectin/ significantly lower in ICR-restricted mice (P < 0.05) than leptin ratio of ICR-restricted mice with mammary tumors AL mice, and values from all calorie-restricted mice were was significantly higher (P < 0.05) then ICR-restricted mice similar. without tumors; however, AL, CCR, and ICR-refed mice In mammary tumors expression levels of adiponectin, exhibited no differences in adiponectin/leptin ratio AdipoR1 and leptin were not significantly different among between tumor-bearing and tumor-free mice (Fig. 2Cc). the groups. AdipoR2 expression was significantly higher in Correlation between weight parameters and terminal mammary tumors from ICR-restricted mice (P < 0.05) than serum leptin, adiponectin, or adiponectin/leptin ratio tumors from AL, CCR, and ICR-refed mice. ObR and ObRb are presented in Table 1. At euthanasia, serum leptin proteins were not detected in mammary tumors from CCR concentration of all mice was positively correlated with and ICR-refed mice and were expressed in only 1 of 3 terminal body weights, total fat pad weights, and mam- tumors from ICR-restricted mice. For AL mice, all the mary fad pad weights. In contrast, terminal serum adipo- tumors expressed ObRb but only 2 of 5 tumors expressed nectin concentration was not correlated with terminal body ObR. weights, total fad pad weights, and mammary fad pad In mammary fat pads of mice without mammary tumor, weights. It was found, however, that the terminal adipo- adiponectin expression tended to be higher for both ICR- nectin/leptin ratio was negatively correlated with body restricted (P < 0.05) and ICR-refed mice than AL and CCR weights, total fat pad weights, and mammary fad pad mice although the ICR-refed value was not significantly weights. different from these 2 groups. In contrast, adiponectin

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Aa Ba Ca with MT 35 * 25 30 * without MT 30 † † ‡ 20 25 † 20 20 15 15 ‡ 10 Weight (g) 10 ‡ 10 5 5 Leptin concentration (ng/mL) 0 Leptin concentration (ng/mL) 0 0 L R L R A C A C AL CR C -refed C -refed C refed R ICR-rest R ICR-rest R- CR-rest Ab IC Bb IC Cb IC I with MT 2 30 35 without MT

* g/mL) g/mL) µ µ 30 25 20 1 † † 20 15

Weight (g) ‡ 10 10 5 0 0 0 Adiponectin concentration ( Adiponectin concentration ( AL CR L R L R C refed A C A C - C refed C -refed CR ICR-rest R- CR-rest R CR-rest I IC I IC I

Ac Bc Cc with MT 20 50 2.5 * without MT 40 2.0 *

1.5 30 10 † † 1.0 20 Weight (g) † † 0.5 ‡ † Aiponectin/leptin ratio 10 * Aiponectin/leptin ratio

0.0 0 0 L R L R A C A C AL CR C -refed C -refed C refed R ICR-rest R ICR-rest R- CR-rest IC IC IC I

Figure 2. Terminal body, fat pad weights and leptin, adiponectin serum levels, and ratio of adiponectin/leptin of MMTV-TGF-a female mice. A, final body and fat pad weights of MMTV-TGF-a female mice. AL, n ¼ 69; CCR, n ¼ 66; ICR-refed, n ¼ 29; and ICR-restricted (ICR-rest), n ¼ 30 mice. a, terminal body weight; ANOVA P < 0.05. b, mammary fad pad weight (combined right and left mammary fat pads; ANOVA P < 0.05. c, internal fat pad weight (combined parametrial and retroperitoneal fat pads); ANOVA P < 0.05. B, terminal leptin and adiponectin serum level and ratio of adiponectin/leptin of TGF-a female mice. AL, n ¼ 26; CCR, n ¼ 23; ICR-refed, n ¼ 15–16; and ICR-restricted, n ¼ 15 mice. a, terminal leptin serum levels; ANOVA P < 0.05. b, terminal adiponectin serum levels; ANOVA P > 0.05. c, terminal adiponectin/leptin ratio; ANOVA P < 0.01. C, a, terminal leptin serum levels for mice with and without mammary tumors (MT); t test P > 0.05. b, terminal adiponectin serum levels for mice with and without mammary tumors; t test P > 0.05. c, terminal adiponectin/leptin ratio for mice with and without mammary tumors; t test P ¼ 0.03. AL without mammary tumors, n ¼ 13; AL with mammary tumors, n ¼ 13; CCR without mammary tumors, n ¼ 7; CCR with mammary tumors, n ¼ 16; ICR-refed without mammary tumors, n ¼ 13; ICR-refed with mammary tumors, n ¼ 2; ICR-restricted without mammary tumors, n ¼ 12; ICR-restricted with mammary tumors, n ¼ 3. In A, B, and C ICR-refed mice were euthanized during a refeeding period and ICR-restricted mice were euthanized during a restriction period. *, †, z, columns with different superscripts are significantly different from each other.

protein expression was only detected in mammary fat pad of Longitudinal adipokines serum levels 1 ICR-refed mouse with no expression in the other 3 groups One of the goals of this investigation was to prospectively of tumor-bearing mice. Leptin expression in mammary fat evaluate serum concentrations of the adipokines, leptin, pads for both tumor-free and tumor-bearing mice was not and adiponectin. Blood samples were obtained in cycles 1, statistically different either within or among the groups. 3, 5, 8, and 11. During each cycle, cohorts of mice had

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Table 1. Correlation (r) between terminal body weight, total fat pad weight, or mammary fat pad weight and leptin, adiponectin, or adiponectin/leptin ratio

Leptina Adiponectinb Adiponectin/leptin ratioc

rP r PrP Body weight 0.7687 <0.0001 0.0267 >0.05 0.2625 <0.0187 Total fat pad weight 0.7886 <0.0001 0.01260 >0.05 0.3440 <0.0018 Mammary fat pad weight 0.8005 <0.0001 0.0043 >0.05 0.3220 <0.0036

NOTE: Number of XY pairs for each correlation is 80. AL, n ¼ 27; CCR, n ¼ 24; ICR-refed, n ¼ 15; and ICR-restricted, n ¼ 14 mice. ICR- refed mice were euthanized during a refeeding period and ICR-restricted mice were euthanized during a restriction period. aPositive correlation. bNo correlation. cNegative correlation. blood samples obtained at weeks 1 and 4, 2 and 5, and 3 well as for mice which eventually developed mammary and 6. For AL and CCR mice there were no differences in tumors compared with those that did not. values obtained over the 6-week periods and these results As shown in Figure 4Aa, serum adiponectin concentra- were combined. Data for AL and CCR mice are presented tions were not significantly different for AL mice that separately for mice which eventually developed mammary eventually developed mammary tumors compared with tumors from those that did not by the time the study was those that did not. Although serum adiponectin level of terminated. For ICR mice results were combined within tumor-free CCR mice appeared to increase to some degree every 3 weeks of restriction or 3 weeks of refeeding and between cycle 1 and cycle 3, tumor-bearing CCR mice presented separately for restriction and refeeding periods as continued to have a rise in adiponectin values until cycle

Table 2. Protein expression of adiponectin, AdipoR1, AdipoR2, leptin, ObR, and ObRb in mammary tissue, and aiponectin and leptin in mammary fat pad of MMTV-TGF-a mice with and without mammary tumor; densitometry units (optical density of protein/optical density of b-actin)

AL CCR ICR-restricted ICR-refed

Mammary tissue n ¼ 5 n ¼ 5 n ¼ 5 n ¼ 5 Adiponecin 1.00 0.03 0.99 0.07 0.88 0.13 0.95 0.01 AdipoR1 0.44 0.04b 0.47 0.05a,b 0.61 0.05a 0.56 0.01a,b AdipoR2 0.46 0.03 0.56 0.05 0.53 0.03 0.47 0.00 Leptin 0.90 0.05a 0.60 0.03b 0.63 0.08b 0.73 0.03a,b ObR 0.63 0.06a 0.58 0.13a,b 0.42 0.08a,b 0.26 0.04b ObRb 0.79 0.10a 0.68 0.04a,b 0.53 0.03b 0.61 0.06a,b Mammary tumor n ¼ 5 n ¼ 5 n ¼ 3 n ¼ 3 Adiponecin 0.75 0.12 0.53 0.06 0.55 0.15 0.63 0.01 AdipoR1 0.40 0.05 0.35 0.03 0.52 0.10 0.42 0.02 AdipoR2 0.53 0.07b 0.49 0.05b 0.76 0.03a 0.49 0.04b Leptin 0.77 0.03 0.79 0.02 0.75 0.06 0.76 0.02 ObR 0.62 0.01 nd 0.42 nd ObRb 0.55 0.05 nd 0.63 nd Mammary fat pad (tumor-free mice) n ¼ 5 n ¼ 5 n ¼ 5 n ¼ 5 Adiponectin 0.73 0.08b 0.70 0.06b 1.00 0.03a 0.95 0.05a,b Leptin 1.25 0.05 1.22 0.14 1.26 0.05 1.32 0.14 Mammary fat pad (tumor-bearing mice) n ¼ 5 n ¼ 5 n ¼ 3 n ¼ 3 Adiponectin nd nd nd 1.12 Leptin 1.42 0.23 1.48 0.20 1.49 0.03 1.59 0.06

NOTE: Values are mean SE; n is number of probed samples. a,bValues within a row with different superscript symbol are significantly different by ANOVA. Abbreviation: nd, not detected.

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ABMammary tissue Mammary tumor Adiponectin AdipoR1

AdipoR2 Leptin

ObR ObRb β-Actin AL ↑ CCR ICR-Rest ↑ ICR-Ref AL ↑ CCR ICR-Rest↑ICR-Ref

CDMammary fat pad Mammary fat pad tumor-free mice tumor-bearing mice Leptin Adiponectin

β-Actin AL ↑ CCR ICR-Rest ↑ ICR-Ref AL ↑ CCR ICR-Rest↑ICR-Ref

Figure 3. Protein expression of adiponectin, AdipoR1, AdipoR2, leptin, ObR, ObRb in mammary tissue and adiponectin, leptin in mammary fat pad of MMTV-TGF-a female mice. A, adiponectin, AdipoR1, AdipoR2, leptin, ObR, and ObRb protein expression in mammary tissue. B, adiponectin, AdipoR1, AdipoR2, leptin, ObR, and ObRb protein expression in mammary tumor. C, adiponectin and leptin protein expression in mammary fat pad of mice without mammary tumor. D, adiponectin and leptin protein expression in mammary fat pad of mice with mammary tumor. A–D, adiponectin, AdipoR1, AdipoR2, leptin, ObR, and ObRb protein expression levels were detected by Western blot analysis. b-Actin protein was probed as a loading control, ICR-refed (ICR-ref) were euthanized during a refeeding period, ICR-restricted (ICR-rest) mice were euthanized during a restriction period. The arrows at the bottom of the blots represent division between dietary groups.

8 (Fig. 4Ab). Interestingly, tumor-bearing mice compared 0.02), cycle 5 (P < 0.05), and cycle 11 (P < 0.0001)]. There with those without mammary tumors from both CCR is one more important difference between AL and CCR [cycle 1 (P < 0.03), cycle 5 (P < 0.04), and cycle 8 (P < groups across the study (except mice with mammary 0.05)] and ICR [ICR-restricted: cycle 3 (P < 0.03); ICR- tumors in cycle 1), that is, leptin concentrations for AL refed: cycle 1 (P < 0.03), cycle 3 (P < 0.04)] groups (Fig. 4Ac mice were consistently significantly (P < 0.001) higher than and d) tended to have higher adiponectin levels prior to those for CCR mice. The mean value of leptin concentra- when tumors were eventually palpated in cycle 10. In cycle tion for AL group was 1.76 0.43 ng/mL in cycle 1 and 11, CCR mice with tumors had adiponectin levels similar to increased to 28.29 3.08 ng/mL in cycle 11, whereas CCR tumor-free CCR mice, whereas ICR-restricted tumor-bear- mice increased leptin concentration from 1.34 0.34 ng/ ing mice continued to have significantly higher adiponec- mL in cycle 1 to only 6.84 0.89 ng/mL in cycle 11. Higher tin values than mice without mammary tumors (P < 0.04). values of leptin for AL compared with CCR mice included Prospective serum leptin concentrations for AL (Fig. 4Ba) those with and without mammary tumors. and CCR (Fig. 4Bb) mice with or without mammary Serum leptin concentrations of ICR mice were reduced tumors increased from cycle 1 to cycle 8. In cycle 11, values by restriction compared with refeeding for most of the cycles were slightly decreased in both AL groups and in tumor-free regardless of whether mammary tumors developed (Fig. 4Bc CCR mice compared with earlier time points, whereas the and d) although only 4 ICR mice did so. Leptin values level of leptin for CCR mice with mammary tumors con- were significantly different between restriction and refeed- tinued to increase. Interestingly, at all time points AL mice ing for ICR mice in cycle 5 (P < 0.02), cycle 8 (P < 0.05), and without mammary tumors had higher serum leptin levels cycle 11 (P < 0.05) for tumor-free mice and in cycle 1 (P < than AL mice with mammary tumors with values signifi- 0.03), cycle 8 (P < 0.05), and cycle 11 (P < 0.05) for tumor- cantly different in cycle 1 (P < 0.001), cycle 5 (P < 0.05), bearing mice. With respect to the concentration of leptin cycle 8 (P < 0.05), and cycle 11 (P < 0.05). In contrast to the in relation to the development of mammary tumors, in AL group, CCR mice which developed mammary tumors the first half of study (cycles 1 and 3), ICR mice that had higher leptin values than mice which did not develop eventually developed mammary tumors had higher leptin mammary tumors [statistical significance in cycle 3 (P < concentrations in the corresponding restriction or refeeding

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aA aB aC - with MT LA - with MT LA - with MT LA 35 45 50 - without MT - without MT * * - without MT 30 * 40 40 25 35 30 30 20 25 15 20 20 * 10 15 * 10 10 5 5

Adiponectin/eptin ratio * * * 0 0 * 0 Leptin concentration (ng/mL) 1 3 5 8 11 1 3 5 8 11 1 3 5 8 11 Adiponectin concentration (µg/mL) Cycle Cycle Cycle bA bB bC - with MT RCC - with MT RCC RCC - with MT 9 45 - without MT 50 - without MT 8 - without MT 40 7 35 40 6 30 * * 30 5 * 25 * 4 20 20 3 * 15 * 2 * 10 10 1 5 Adiponectin/eptin ratio

Leptin concentration (ng/mL) 0 0 0 1 3 5 8 11 1 3 5 8 11 1 3 5 8 11 Adiponectin concentration (µg/mL) Cycle Cycle Cycle

cA cB cC - with MT - with MT ICR-restricted ICR-restricted - with MT ICR-restricted - without MT - without MT 60 6 - without MT 75 50 5 4 40 50 * * 30 3 * * * * * 20 * 2 25 1 10

0 Adiponectin/eptin ratio 0 0 Leptin concentration (ng/mL) 1 3 5 8 11 1 3 5 8 11

Adiponectin concentration (µg/mL) 1 3 5 8 11 Cycle Cycle Cycle

dA ICR-refed dB ICR-refed dC ICR-refed - with MT - with MT - with MT - without MT 60 - without MT - without MT 6 45 * 40 50 5 * 35 40 4 30 25 30 3 * 20 20 * 2 15 * 10 * 10 1 5 Adiponectin/eptin ratio 0 0 0 1 3 5 8 11 Leptin concentration (ng/mL) 1 3 5 8 11 1 3 5 8 11 Adiponectin concentration (µg/mL) Cycle Cycle Cycle

Figure 4. Serum adiponectin, leptin levels, and ratio of adiponectin/leptin levels for AL, CCR, and ICR MMTV-TGF-a mice with and without mammary tumors (MT) over the course of the study. A, serum adiponectin levels for AL, CCR, and ICR mice during cycles 1, 3, 5, 8, and 11 for mice that never developed mammary tumors compared with those that did. Bars represent mean of adiponectin concentrations; t test P > 0.05. a, AL without mammary tumors, n ¼ 10; AL with mammary tumors, n ¼ 9. b, CCR without mammary tumors, n ¼ 12; CCR with mammary tumors, n ¼ 11–12. c, ICR-restricted; d, ICR-refed. c and d, n ¼ 16 for mice without mammary tumors and n ¼ 4 for mice with mammary tumor. B, serum leptin levels for AL, CCR, and ICR mice during cycles 1, 3, 5, 8, and 11 for mice that never developed mammary tumors compared with those that did. Bars represent mean of adiponectin concentrations; t test P > 0.05. a, AL without mammary tumors, n ¼ 10; AL with mammary tumors, n ¼ 9. b, CCR without mammary tumors, n ¼ 12; CCR with mammary tumors, n ¼ 11–12. c, ICR-restricted; d, ICR-refed. c and d, n ¼ 15–16 for mice without mammary tumors and n ¼ 4 for mice with mammary tumor. C, serum adiponectin/leptin ratio for AL, CCR and ICR mice during cycles 1, 3, 5, 8, and 11 for mice that never developed mammary tumors compared with those that did. Bars represent mean of adiponectin concentrations; t test P > 0.05. a, AL without mammary tumors, n ¼ 10; AL with mammary tumors, n ¼ 9. b, CCR without mammary tumors, n ¼ 12; CCR with mammary tumors, n ¼ 11–12. c, ICR-restricted; d, ICR-refed. c and d, n ¼ 15–16 for mice without mammary tumors and n ¼ 4 for mice with mammary tumor. In A, B, and C ICR-refed mice were euthanized during a refeeding period and ICR-restricted mice were euthanized during a restriction period. *, †, z, columns with different superscripts are significantly different from each other. www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 575

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period than mice that did not develop tumors. In later women worldwide including the United States (64, 65). cycles ICR mice that did not eventually develop mammary Increased energy balance may be responsible for approxi- tumors had higher serum leptin than those that did not. mately one third of human mammary tumors (66). Thus, This occurred in both restriction (Fig. 4Bc) and refeeding modifying total energy intake and/or expenditure may be (Fig. 4Bd) periods. ICR mice that did not develop mammary one of the key elements of breast cancer prevention. In fact, tumors had significantly (P < 0.05) higher leptin levels chronic calorie restriction has been consistently reported to than those later confirmed to be tumor-bearing. Specifically prevent or delay mammary tumorigenesis in animal mod- in cycle 8 prior to tumor detection by palpation in cycle els (67–76). The consequence of multiple periods of calorie 10 and later when tumors were growing in cycle 11 in both restriction followed by refeeding, that is, intermittent cal- restriction (Fig. 4Bc) and refeeding (Fig. 4Bd) periods. orie restriction, has been less well studied. However, several Results for the adiponectin/leptin ratio over the course of previous studies indicated a protective effect on prevention the study are presented in Figure 4Ca–d. AL mice had the of spontaneous tumors (77, 78). Further, we have consis- highest adiponectin/leptin ratio in cycle 1 regardless of tently found that intermittent restriction prevents and eventual tumor status (Fig. 4Ca). Values of adiponectin/ delays mammary tumor development to a greater extent leptin ratio for AL mice declined with increasing age. At all compared with chronic calorie restriction matched for the ages, the adiponectin/leptin ratio was significantly higher same nutrient and calorie intake in two transgenic mouse in AL mice which developed mammary tumors than in AL strains (60–62, 79). Interestingly, it has been reported that mice which remained tumor-free (P < 0.03 in cycle 1, P < women with a history of anorexia nervosa and presumably 0.0001 in cycle 3, P < 0.0001 in cycle 5, P < 0.02 in cycle 5, having bouts of calorie restriction followed by refeeding and P < 0.0003 in cycle 11). In contrast, there was no had a reduced incidence of breast cancer (80). dramatic age-related change of the adiponectin/leptin ratio Our recent efforts have focused on identifying factors for CCR mice (Fig. 4Cb), and there was little consistent associated with mammary tumor development, which may difference between CCR mice which developed mammary be modified by the intermittent calorie restriction protocol. tumors and those that did not, except in cycle 8 when CCR We recently published detailed results for the impact of this tumor-bearing mice had significantly higher (P < 0.05) intervention protocol on the IGF-I axis (61). Here, we have adiponectin/leptin ratio than mice without mammary expanded our investigation to explore the effects of these tumor (Fig. 4Cb). The adiponectin/leptin ratio of ICR mice two calorie restriction protocols on adipokine serum levels depended on the feeding regimen, that is, regardless of and their signaling pathways within target tissues. Our whether they developed mammary tumors the adiponec- specific focus is on adiponectin and leptin. tin/leptin ratio was in almost all cases significantly higher We found at the termination of the study that in adult in restriction than in refeeding periods (Fig. 4Cc and female mice serum adiponectin levels were similar among Fig. 4Cd). As to differences of adiponectin/leptin ratio all groups with no impact of either long-term chronic or related to tumor status in ICR group, in the first half of intermittent calorie restriction (Fig. 2Bb). We also obtained study, ICR-restricted but not ICR-refed mice that later serum samples over the course of the study and found that developed mammary tumors had lower adiponectin/leptin adiponectin serum levels of AL and CCR mice were slightly ratio [cycle 1 (P < 0.01), cycle 3 (P < 0.05)] than mice that increased with aging. For ICR mice at some time points did not develop tumors. In contrast to the early time points, adiponectin concentration depended on the dietary regi- later in the study at cycles 8 (P < 0.05) and 11, both ICR- men and was somewhat higher in restriction than in restricted and ICR-refed mice with mammary tumors had refeeding. Reports in the literature for the effects of dietary significantly higher (P < 0.01) adiponectin/leptin ratio intervention on serum adiponectin vary. In humans, cal- than mice without mammary tumors. orie restriction and/or weight loss have been linked to When longitudinal data of adiponectin/leptin ratio were increased serum adiponectin concentration in some studies analyzed only for mice which developed mammary tumors, (81–84), but not in others (85, 86). There are only a few in cycle 1 AL mice had the highest ratio followed by CCR, reports of the influence of intermittent calorie restriction ICR-restricted, and ICR-refed mice; however, differences on serum adiponectin levels. Wan and colleagues (87) were not significant (P ¼ 0.92). In cycles 3, 5, and 8 prior reported that intermittent fasting of Wistar male rats to mammary tumor detection AL mice had the lowest resulted in increased levels of circulating adiponectin com- adiponectin/leptin ratio versus ICR-restricted values and pared with control rats. However, it was also reported that the difference reached significance in cycle 8 (P < 0.01). different levels of refeeding following alternate day calorie Interestingly, in cycle 11, when detected tumors were grow- restriction for 4 weeks did not affect adiponectin levels of ing, adiponectin/leptin ratio of ICR-restricted mice contin- C57BL/6J male mice (88), although it increased adiponec- ued to increase and was significantly higher than AL (P < tin concentration by 62% to 86% for female mice (89). We 0.01), CCR(P < 0.01), and ICR-refed mice (P < 0.01). have previously reported that neither chronic nor intermittent calorie restriction altered adiponectin in a cross- Discussion sectional study of female MMTV-TGF-a mice on the C57BL6 background (54). Similar results have been found There is no universal and effective preventive strategy for for C57BL6 TRAMP male mice in both a cross-sectional and breast cancer, which is the most common neoplasia among a long-term study (63, 90). We also found no effect of

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goldthioglucose-induced obesity on adiponectin levels isoforms (103) with the isoform designated ObRb con- (57) and a recent online publication reported no effect sidered the active signaling protein. Previously, it was of high-fat feeding or sucrose ingestion in water on serum reported that ObR was overexpressed in breast tumor tissue adiponectin levels (91). Thus, it remains to be determined compared with noncancer breast epithelium and also the whether diet composition or other factors such as physical expression of leptin was found to be positively correlated activity may interact with calorie restriction to affect serum with expression of its receptor, suggesting that leptin acts adiponectin levels. on mammary tumor cells via an autocrine pathway (26). In In contrast to adiponectin, serum leptin levels were addition, the association between high intratumoral leptin significantly reduced by long-term chronic calorie restric- receptor mRNA level and poor breast cancer prognosis was tion. Further, serum leptin levels of the mice on the inter- shown in the presence of high, but not low serum and mittent calorie restriction protocol were reduced after intratumoral leptin levels (104). In this study, we deter- restriction as well as after refeeding compared with both mined expression of leptin in mammary tumors, mam- AL and CCR mice. Over the course of the study, serum mary tissue, and mammary fat pad, and also expression of leptin levels rose in AL mice with increasing age whereas total leptin receptor ObR and its long isoform ObRb in this response was muted by calorie restriction. These find- mammary tumors and mammary gland. Our data support ings are consistent with human studies (92–99). that caloric restriction affected leptin and leptin receptor Our interest in assessing these 2 adipokines in relation- expression in mammary tissue. CCR and ICR-restricted ship with mammary tumorigenesis was based on a number mice had significantly decreased leptin protein expression of previous studies. In vitro experiments by us and others in mammary tissue versus the AL group. In addition, ICR- have shown that leptin enhances proliferation of human restricted mice had significantly lower ObRb expression breast cancer cell lines (14, 15, 17, 18). In contrast, addition than AL mice. of adiponectin reduces human breast cancer cell prolifera- The mammary fat pad is a microenvironment for breast tion (20, 22, 23, 25). Human studies have not indicated a tissue and locally produced adipokines can play important consistent relationship of serum leptin levels with breast roles in mammary tumorigenesis. Although we did not find cancer (as recently summarized by Grossmann and collea- significant differences in leptin protein expression in mam- gues; ref. 100). However, serum adiponectin levels have mary fat pads among the dietary groups, earlier we showed been found to be reduced in women with breast cancer that chronic and intermittent calorie restriction signifi- compared with those without the disease (33, 34, 37, 45). In cantly decreased ObR and ObRb mRNA expression in most studies, the focus was on either leptin or adiponectin mammary fat pad in comparison with the AL diet (60). alone, but in one study, in which both proteins were In addition, Gallardo and colleagues (105) reported that measured, it was found that a low adiponectin/leptin ratio long-term 20% to 25% calorie restriction promoted a was associated with breast cancer (36). On the basis of these change in the distribution of ObRa between internal and findings, we conducted in vitro studies evaluating the impact plasma membranes in isolated rats’ adipocytes, increasing of different adiponectin/leptin ratios on human breast its presence at the cell surface. Interestingly, Sucajtys-Szulc cancer cell proliferation and the results support that this and colleagues (106) reported that leptin gene expression relationship may be more important in determining the in rat hypothalamus is downregulated by prolonged food overall consequence of how these two proteins affect tumor restriction as in white adipose tissue, but refeeding after development rather than their absolute amounts (101). long-term food restriction increased serum leptin concen- Here, although we did not show that individually either tration and leptin gene expression in white adipose tissue leptin or adiponectin was associated with mammary tumor but had no effect on hypothalamic leptin gene expression development either during the early stages of tumorigenesis indicating tissue specificity. or once mammary tumors were detected, we clearly show Interestingly, we determined that CCR and ICR-restricted that the intermittent calorie restriction protocol is asso- mice had significantly decreased leptin protein expression ciated with an elevated ratio of adiponectin/leptin and with in mammary tissue compared with the AL group; however, reduced mammary tumor incidence. This is consistent with mammary tumors as well as mammary fat pads showed no recent results from a cross-sectional study using this same difference in leptin protein expression and previously we mouse strain (54), as well as with our findings that inter- reported that neither the ICR nor CCR protocols changed mittent calorie restriction delays prostate tumor detection ObR and ObRb mRNA expression in mammary tumors and death in the TRAMP mouse model of prostate cancer (60). There are no published data about changes of leptin and this too was associated with an elevated adiponectin/ protein expression under long-term calorie restriction. In leptin ratio (63). adipose tissue, mRNA level for leptin has been reported to Circulating adipokines such as leptin and adiponectin decrease in response to short-term fasting or severe chronic exert their biological action on target cells not only by restriction (107). However, the effect of long-term inter- endocrine mechanism but also through paracrine and mittent or chronic calorie restriction on leptin protein autocrine pathways [reviewed by Vona-Davis and Rose; expression in fat tissue, in particular mammary fat, is ref. (102)]. Leptin acts by binding to its receptor, ObR, unknown. We assume that leptin may have a role in the which is structurally related to the cytokine receptor family. early phase of mammary tumor development. Analysis The ObR gene can be alternatively spliced into several of mammary tissues, tumors, and fat pads from our

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cross-sectional study (54) will provide details about leptin of young but not senescent rats (110, 111). In senescent protein expression in target tissues at earlier stages of rats, a long-term fairly severe calorie reduction (40%) was mammary tumor development. needed for mild induction of adiponectin expression in We also determined expression levels of adiponectin and visceral fat. In our study, 25% chronic calorie reduction did its two receptors, AdipoR1 and AdipoR2 (108), in target not change adiponectin protein expression in mammary fat tissues. Adiponectin and its receptors have been shown to pad of tumor-free mice, but 50% restriction significantly be expressed in healthy breast tissue and also in mammary increased adiponectin expression in mammary fat of ICR- malignancy (20, 25, 29, 109). Previously, it was reported restricted mice compared with AL and CCR mice. It is that breast tissue adiponectin levels measured by ELISA in important to note that tumor-bearing mice had a very patients with breast cancer were significantly increased in low signal of adiponectin expression in mammary fat comparison with controls although there was no associa- pad with strong leptin protein expression. Unbalance of tion between tumor stage and tumor size (29). Korner and these two adipokines in mammary tumor environment colleagues (20) showed that adiponectin mRNA expression could be critical in the process of tumorigenesis. level was significantly higher in mammary tissue adjacent In summary, we showed that two modes of calorie to breast tumors compared with either breast tumor tissue restriction had different influences on serum leptin levels or control tissue from subjects without breast cancer. In the and on the adiponectin/leptin ratio. Mice subjected to same study, AdipoR1 mRNA expression, but not AdipoR2, chronic calorie restriction had reduced serum leptin level was higher in breast tumors compared with control mam- and increased adiponectin/leptin ratio compared with AL mary tissue from cancer-free subjects. Jarde and colleagues mice whereas ICR mice had fluctuations of leptin concen- (109) used immunohistochemical staining to assess adi- tration and adiponectin/leptin ratio. However, it should be ponectin receptor expression in breast tissues. In contrast to noted that 3 weeks of the same calorie and nutrient intake as Korner’s study, they showed that difference in AdipoR1 that of AL mice during refeeding did not restore values of expression in breast cancer tumors and normal adjacent ICR mice to levels of either AL and CCR mice. Serum leptin tissues was not statistically significant; however, AdipoR2 levels were influenced by age whereas there was very little expression was more pronounced in malignant cells than effect on adiponectin. We also provided evidence for a in patients’ normal tissues. In our cross-sectional study strong association of serum leptin concentration as well (Dogan and Cleary, unpublished data) expression level of as adiponectin/leptin ratio with fat mass and body weight. AdipoR2 was similar in mammary tumor and control Some effects of the calorie restriction protocols were found tissues, but protein expression levels of AdipoR1 were on tissue expression levels of leptin and adiponectin recep- significantly lower in mammary tumors than in control tors, although no consistent effects were noted. However, in mammary tissue obtained from 74-week-old MMTV-TGF-a this and other studies we have found that a high adiponec- mice. Here we found that in ICR-restricted mice there was tin/leptin ratio is associated with reduced tumorigenesis in increased AdipoR1 expression in mammary tissues and ICR mice. Thus, although it seems that adiponectin and increased AdipoR2 expression in mammary tumors. We leptin are involved in mammary tumorigenesis there are still also showed that caloric restriction did not affect adipo- many aspects of the relationship to be determined. nectin protein expression in mammary tissues, whereas in mammary tumors it was decreased in both CCR and ICR- Disclosure of Potential Conflicts of Interest restricted mice in comparison with the AL group; however, the difference was not significant. Integration of our data No potential conflicts of interest were disclosed. with previously published studies indicates an involvement The costs of publication of this article were defrayed in part by the payment of adiponectin receptors in breast tumorigenesis, but the of page charges. This article must therefore be hereby marked advertisement in relevance of these observations is still unclear. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Previously, it was reported that 2 months of 16% calorie Received June 22, 2010; revised December 21, 2010; accepted January 13, restriction enhanced adiponectin expression in visceral fat 2011; published OnlineFirst January 21, 2011.

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Effect of Chronic and Intermittent Calorie Restriction on Serum Adiponectin and Leptin and Mammary Tumorigenesis

Olga P. Rogozina, Melissa J.L. Bonorden, Christine N. Seppanen, et al.

Cancer Prev Res 2011;4:568-581. Published OnlineFirst January 21, 2011.

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