The protective effect of intermittent calorie restriction on mammary tumorigenesis is not compromised by consumption of a high fat diet during refeeding

Abstract

Previously we reported that intermittent calorie restriction (ICR) provided greater prevention of mammary tumors (MTs) than chronic calorie restriction (CCR). Here the impact of increased fat intake during refeeding in an ICR protocol was evaluated. MMTV-TGF-α female mice were assigned to one of three groups: ad libitum (AL) fed (n = 45) with free access to a moderately high fat diet (22 % fat calories); ICR (n = 45) 50 % calorie restricted for 3-week intervals followed by 3 weeks of 100 % of AL intake; and CCR (n = 45) fed 75 % of AL mice, matching each 6-week cycle of ICR mice. ICR mice were further designated as ICR-Restricted or ICR-Refed for data obtained during these intervals. All mice consumed the same absolute amount of dietary fat. Mice were followed to assess MT incidence, body weight and serum IGF-1, IGFBP3, leptin and adiponectin levels until 79 (end of final 3-week restriction) or 82 (end of final 3-weeks refeeding) weeks of age. Age of MT detection was significantly extended for CCR (74 weeks) and ICR (82 weeks) mice, compared to 57.5 weeks for AL mice. MT incidence for AL, ICR and CCR mice was 66.7, 4.4, and 52.3 %, respectively. Mammary and fat pad weights were reduced significantly following 50 % calorie restriction in ICR-Restricted mice compared to AL, CCR and ICR-Refed mice. IGF-1 and leptin levels also tended to be reduced in ICR-Restricted mice over the course of the study while adiponectin was not compared to AL, CCR, and ICR-Refed mice. The adiponectin:leptin ratio was consistently higher following 50 % restriction in ICR-Restricted mice. There was no relationship of IGF-1, leptin, or adiponectin with the presence of MTs in any groups. Thus the manner in which calories are restricted impacts the protective effect of calorie restriction independently of high fat intake.

Fig 1

Body weight curves of TGF-α female mice over the course of the experiment. Ad libitum-Fed (AL) (■) n=12-45 depending on age; Intermittent Calorie Restricted (ICR) (▼) n=45; Chronic Calorie Restricted (CCR) (▲) n=19-45 depending on age. ANOVA P < 0.0001, All dietary groups are significantly different from each other (P<0.001).

Fig 2

Final Body and Fat Pad Weights of TGF-α Female Mice. A, B and C, AL (n = 42), CCR (n = 44), ICR-Refed (n = 22) and ICR-Restricted (n = 23) mice. A. Terminal Body Weight. Bars represent means of Terminal Body Weights. ANOVA P < 0.05. B. Mammary Fad Pad Weight (combined right and left mammary fat pads. Bars represent means of Mammary Fat Pad Weights. ANOVA P> 0.01. C. Internal Fat Pad Weight (combined parametrial and retroperitoneal fat pads). Bars represent means of Internal Fat Pad Weights. ANOVA P < 0.05. In A, B and C ICR-Refed mice were euthanized during a refeeding period and ICR-Restricted mice were euthanized during a restriction period. Columns with different superscripts are significantly different from each other.

Fig 3

Proportion of mice without palpable mammary tumor over the course of the experiment. Vertical plots represent number of new mice with palpable tumor in weekly basis. Kaplan-Meier analysis, P < 0.0001, χ²=40.9. Age of tumor detection is significantly different among all dietary groups.

Fig 4

Terminal IGF-1 and IGFBP-3 Serum Level and Ratio of IGF1:IGFBP-3 of TGF-α Female Mice. A, Terminal IGF-1 serum levels. Bars represent means of IGF-1 concentrations. ANOVA P < 0.05. AL (n = 37), CCR (n = 38), ICR-Refed (n = 22) and ICR-Restricted (n = 23) mice. B, Terminal IGFBP-3 serum levels. Bars represent means of IGFBP-3 concentrations. ANOVA P < 0.01. AL (n = 37), CCR (n = 38), ICR-Refed (n = 22) ICR-Restricted (n = 23) mice. C, Ratio of IGF-1 to IGFBP-3 levels. Bars represent means of IGF-1:IGFBP-3 Ratio. ANOVA P < 0.01. AL (n = 37), CCR (n = 38), ICR-Refed (n = 22) and ICR-Restricted (n = 23) mice. In A, B and C ICR-Refed mice were euthanized during a refeeding period and ICR-Restricted mice were euthanized during a restriction period. Columns with different superscripts are significantly different from each other.

Fig 5

Serum IGF-1 Levels for Ad libitum-Fed (AL), Chronic Calorie Restricted (CCR) and Intermittent Restricted (ICR) TGF-α Mice (combined with and without mammary tumor) over the Course of the Study. Bars represent means of IGF-1 concentrations. Cycle 5: ANOVA P<0.001. AL n = 36, CCR n= 36, ICR-Refed n=18 and ICR-Restricted n=18. Cycle 8: ANOVA P<0.05. AL n = 36, CCR n= 36, ICR-Refed n=18 and ICR-Restricted n=18. Cycle 11: ANOVA P<0.001. AL n = 32, CCR n= 36, ICR-Refed n=18 and ICR-Restricted n=18.

Fig 6

Terminal Adiponectin, Leptin Serum Level and Ratio of Adiponectin:Leptin of TGF-α Female Mice. A. Terminal Adiponectin serum levels. Bars represent means of Adiponectin concentrations. ANOVA P > 0.05, AL (n = 23), CCR (n = 18), ICR-Refed (n = 22) and ICR-Restricted (n = 23) mice. B. Terminal Leptin serum levels. Bars represent means of Leptin concentrations. ANOVA P> 0.05, AL (n = 17), CCR (n = 18), ICR-Refed (n = 9) and ICR-Restricted (n = 7) mice. C. Terminal Adiponectin:Leptin Ratio. Bars represent means of Adiponectin:Leptin Ratio. ANOVA P < 0.001, AL (n = 17), CCR (n = 17), ICR-Refed (n = 9) and ICR-Restricted (n = 7) mice. In A, B and C ICR-Refed mice were euthanized during a refeeding period and ICR-Restricted mice were euthanized during a restriction period. Columns with different superscripts are significantly different from each other.

Fig 7

Serum Adiponectin, Leptin Levels and Ratio of Adiponectin:Leptin Levels for Ad libitum-Fed (AL), Chronic Calorie Restricted (CCR) and Intermittent Restricted (ICR) TGF-α Mice (combined with and without mammary tumor) over the Course of the Study. A, Serum Adiponectin Levels for AL, CCR, ICR-Refed and ICR-Restricted mice during cycles 5, 8, and 11. Bars represent means of Adiponectin concentrations. Cycle 5: ANOVA P<0.05. AL n = 36, CCR n= 36, ICR-Refed n=17 and ICR-Restricted n=17. Cycle 8: ANOVA P<0.05. AL n = 36, CCR n= 36, ICR-Refed n=17 and ICR-Restricted n=17. Cycle 11: ANOVA P<0.01. AL n = 32, CCR n= 36, ICR-Refed n=17 and ICR-Restricted n=16. B, Serum Leptin Levels for AL, CCR and ICR mice during cycles 5, 8, and 11. Bars represent means of Leptin concentrations. Cycle 5: ANOVA P<0.01. AL n = 36, CCR n= 34, ICR-Refed n=17 and ICR-Restricted n=15. Cycle 8: ANOVA P<0.01. AL n = 36, CCR n= 35, ICR-Refed n=17 and ICR-Restricted n=16. Cycle 11: ANOVA P<0.01. AL n = 32, CCR n= 35, ICR-Refed n=17 and ICR-Restricted n=17. C, Serum Adiponectin:Leptin Ratio for AL, CCR and ICR mice during cycles 5, 8, and 11.Bars represent means of Adiponectin:Leptin Ratio. Cycle 5: ANOVA P<0.01. AL n = 36, CCR n= 34, ICR-Refed n=17 and ICR-Restricted n=15. Cycle 8: ANOVA P<0.01. AL n = 36, CCR n= 35, ICR-Refed n=17 and ICR-Restricted n=16. Cycle 11: ANOVA P<0.01. AL n = 32, CCR n= 35, ICR-Refed n=17 and ICR-Restricted n=15. In A, B and C ICR-Refed mice were euthanized during a refeeding period and ICR-Restricted mice were euthanized during a restriction period. Columns with different superscripts are significantly different from each other.