Caloric restriction in C57BL/6J mice mimics therapeutic fasting in humans

Abstract

Background: Caloric restriction (CR) has long been recognized as a dietary therapy that improves health and increases longevity. Little is known about the persistent effects of CR on plasma biomarkers (glucose, ketone bodies, and lipids) following re-feeding in mice. It is also unclear how these biomarker changes in calorically restricted mice relate to those observed previously in calorically restricted humans.

Results: Three groups of individually housed adult female C57BL/6J (B6) mice (n = 4/group) were fed a standard rodent chow diet either: (1) unrestricted (UR); (2) restricted for three weeks to reduce body weight by approximately 15-20% (R); or (3) restricted for three weeks and then re-fed unrestricted (ad libitum) for an additional three weeks (R-RF). Body weight and food intake were measured throughout the study, while plasma lipids and levels of glucose and ketone bodies (beta-hydroxybutyrate) were measured at the termination of the study. Plasma glucose, phosphatidylcholine, cholesterol, and triglycerides were significantly lower in the R mice than in the UR mice. In contrast, plasma fatty acids and beta-hydroxybutyrate were significantly higher in the R mice than in the UR mice. CR had no effect on plasma phosphatidylinositol levels. While body weight and plasma lipids of the R-RF mice returned to unrestricted levels upon re-feeding, food intake and glucose levels remained significantly lower than those prior to the initiation of CR.

Conclusion: CR establishes a new homeostatic state in B6 mice that persists for at least three weeks following ad libitum re-feeding. Moreover, the plasma biomarker changes observed in B6 mice during CR mimic those reported in humans on very low calorie diets or during therapeutic fasting.

Figure 1

Flow chart of the study design. Body weight and food intake were measured every other day over the seven day pre-trial period. All mice received food ad libitum during the pre-trial period. After the pre-trial period, the mice were divided into three groups (n = 4 mice/group) where the average body weight of each group was similar. The mice in each group were then fed the same diet in different amounts: 1) the standard chow diet unrestricted (UR), 2) the standard chow diet restricted to achieve an approximate 15–20% body weight reduction from the pre-trial weight (R), or 3) the standard chow diet restricted to achieve an approximate 15–20% body weight reduction from the pre-trial weight for a period of three weeks, followed by unrestricted re-feeding for a period of three weeks (R-RF). Each mouse in the R and the R-RF groups served as its own control for body weight reduction as previously described [16]. Based on food intake and body weight during the pre-trial period, food in the R and the R-RF groups was reduced until each mouse achieved the target weight reduction of approximately 15–20%. The study was terminated and plasma was collected for two UR mice and four R mice on day 30, and for the remainder of the mice on day 51.

Figure 2

Influence of CR and re-feeding on food intake (A) and body weight (B). Values are expressed as means and 4–8 mice were analyzed in each group. The black arrow indicates the initiation of CR on day 8. The white arrow indicates the initiation of ad libitum re-feeding on day 30. The * indicates that the food intake average of the days 38 to 50 of re-feeding for the R-RF mice was significantly less than their food intake prior to initiation of CR, as determined by the paired t-test.

Figure 3

HPTLC of plasma neutral lipids in B6 mice. The amount of neutral lipids spotted per lane was equivalent to 2.5 μl of plasma. The plate was developed as described in the Methods. CE, cholesterol esters; TG, triglycerides; IS, internal standard; C, cholesterol; Cer, ceramide; CB, cerebrosides (doublet); PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomyelin; LPC, lysophosphatidylcholine; O, origin; and SF, solvent front of the first developing solvent system.

Figure 4

HPTLC of plasma acidic lipids in B6 mice. The amount of acidic lipids spotted per lane was equivalent to 15 μl of plasma. The plate was developed as described in the Methods. FA, fatty acids; IS, internal standard; CL, cardiolipin; PA, phosphatidic acid; Sulf, sulfatides (doublet); PS, phosphatidylserine; PI, phosphatidylinositol; O, origin; and SF, solvent front of the first developing solvent system.