The effects of graded levels of calorie restriction: I. impact of short term calorie and protein restriction on body composition in the C57BL/6 mouse

Abstract

Faced with reduced levels of food, animals must adjust to the consequences of the shortfall in energy. We explored how C57BL/6 mice withdrew energy from different body tissues during three months of food restriction at graded levels up to 40% (calorie restriction: CR). We compared this to the response to equivalent levels of protein restriction (PR) without a shortfall in calories. Under CR there was a dynamic change in body mass over 30 days and thereafter it stabilized. The time to reach stability was independent of the level of restriction. At the end of three months whole body dissections revealed differential utilization of the different tissues. Adipose tissue depots were the most significantly utilized tissue, and provided 55.8 to 60.9% of the total released energy. In comparison, reductions in the sizes of structural tissues contributed between 29.8 and 38.7% of the energy. The balance was made up by relatively small changes in the vital organs. The components of the alimentary tract grew slightly under restriction, particularly the stomach, and this was associated with a parallel increase in assimilation efficiency of the food (averaging 1.73%). None of the changes under CR were recapitulated by equivalent levels of PR.

Keywords: Gerotarget; body composition; calorie restriction; dietary restriction; food intake; protein restriction.

Figure 1. Food intake and body mass changes recorded over 3 months graded calorie restriction

Daily records of a. food intake (FI) (kJ/day) and b. body mass (BM) (g) recorded over 2 weeks of baseline monitoring (days −14 to −1) and 12 weeks of treatment comprising 12 or 24h ad libitum (AL) feeding and graded levels of caloric restriction (CR) from 10 to 40% (10CR, 20CR, 30CR and 40CR respectively). Data are presented as daily mean ± SEM (g). c. Initial rates of weight loss for the four graded restriction groups. The value is the sum of the coefficients in x and x² of the second order fitted polynomial to the body weights over the first 30 days of restriction. d.Time taken (days) for weight loss to slow to zero in relation to the extent of restriction. The inflection points were calculated from the fitted polynomial curves to the weight loss data of days 1 to 30 inclusive. Error bars are SEM.

Figure 2. Body composition changes following 3 months of graded calorie restriction

a. body mass (BM), b. fat mass (FM) and c. fat free mass (FFM) measure by DXA at baseline (BL), 4, 8 and 12 weeks of ad libitum (AL) 12 & 24h or graded levels of caloric restriction (CR) (10CR, 20CR, 30CR and 40CR), Data represented as mean ± SEM (g).

Figure 3. Correlation matrix showing the magnitude of the correlation in the responses to calorie restriction (CR) of different organs across all individuals

The lower triangle represents animals under CR and the upper triangle animals under protein restriction (PR). EPI is epididymal white adipose tissue (WAT), Retro is retroperitoneal WAT, Sub Cut is subcutaneous WAT, rep orgs are the reproductive accessory organs, BAT is the interscapular brown adipose tissue. The scale for the correlations is shown at the top of the diagram increasing intensity of red was greater positive correlation and increasing intensity of blue was greater negative correlation (for actual correlation values see Supplementary Table 2).

Figure 4

Dendrograms showing the similarity in responses of the different organs to a. graded caloric restriction (CR) and b. graded protein restriction (PR). EPI is epididymal white adipose tissue (WAT), Retro is retroperitoneal WAT, Sub Cut is subcutaneous WAT, rep orgs are the reproductive accessory organs, BAT is the interscapular brown adipose tissue.

Figure 5. Changes in organ size following three months of graded calorie restriction (CR)

Histograms showing the extent of change in organ sizes relative to the sizes of the organs in the 12AL group (% difference) after three months of CR. Tissues are grouped in accord with the hierarchical clustering analysis in Figure 4a. panc is pancreas, EPI is epididymal white adipose tissue (WAT), Retro is retroperitoneal WAT, Sub Cut is subcutaneous WAT, Mes is mesenteric WAT, rep orgs are the reproductive accessory organs, BAT is the interscapular brown adipose tissue. Compare to Figure 10 for the same changes under PR.

Figure 6. Hierarchy of utilization of different organs following three months of caloric restriction (CR)

Hierarchy is reflected in the gradient of the relationship between the Log_e tissue weight and the Log_e final body mass (BM) (see Table 3 for actual regression coefficients). Tissues with values below 0 were invested in. Those with values between 0 and 1 were relatively protected. Those with values greater than 1 were preferentially utilized. Preferential utilization was greater at greater values.

Figure 7. Changes in assimilation efficiency (%) following calorie restriction (CR) or protein restriction (PR)

assimilation efficiency% of the different treatment groups measured during baseline (BL) and a. post-CR or b. post-PR. 24AL and 12AL refer to the two ad libitum groups and the 10CR, 20CR, 30CR and 40CR refer to 10, 20, 30 and 40%. 20PR, 30PR and 40PR refer to equivalent PR groups.

Figure 8. Food intake and body mass changes recorded over 3 months graded protein restriction

Daily records of a. food intake (kJ/day), b. body mass (BM) (g) recorded over 2 weeks of baseline monitoring (days −14 to −1) and 12 weeks of treatment: 12h ad libitum (12AL) feeding and graded levels of protein restriction (PR) from 20 to 40%. Data are presented as daily mean ± SEM (g). The plots use the same scale as the plots in Figure 1 to facilitate comparison to the changes under CR.

Figure 9. Body composition changes following 3 months of graded protein restriction

a. body mass (BM), b. fat mass (FM) and c. fat free mass (FFM) measured by DXA at baseline (BL), 4, 8 and 12 weeks of 12h ad libitum (12AL) or graded levels of protein restriction (PR) (20PR, 30PR and 40PR). Data represented as mean ± SEM (g).

Figure 10. Changes in organ size following three months of graded protein restriction

Histograms showing the extent of change in organ sizes after three months of protein restriction (PR) relative to the sizes of the organs in the ad libitum fed group (% difference). EPI is epididymal white adipose tissue (WAT), Retro is retroperitoneal WAT, Sub Cut is subcutaneous WAT, Mes is mesenteric, rep orgs are the reproductive accessory organs, BAT is the interscapular brown adipose tissue. Tissues are grouped in accord with the hierarchical clustering analysis in Figure 4b. The plots use the same scale as the plots in Figure 5 to facilitate comparison to the changes in the same tissues under CR.