Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms

Abstract

Calorie restriction (CR) increases longevity in many species by unknown mechanisms. The circadian clock was proposed as a potential mediator of CR. Deficiency of the core component of the circadian clock-transcriptional factor BMAL1 (brain and muscle ARNT [aryl hydrocarbon receptor nuclear translocator]-like protein 1)-results in accelerated aging. Here we investigated the role of BMAL1 in mechanisms of CR. The 30% CR diet increased the life span of wild-type (WT) mice by 20% compared to mice on anad libitum(AL) diet but failed to increase life span ofBmal1(-/-)mice. BMAL1 deficiency impaired CR-mediated changes in the plasma levels of IGF-1 and insulin. We detected a statistically significantly reduction of IGF-1 in CRvs.AL by 50 to 70% in WT mice at several daily time points tested, while inBmal1(-/-)the reduction was not significant. Insulin levels in WT were reduced by 5 to 9%, whileBmal1(-/-)induced it by 10 to 35% at all time points tested. CR up-regulated the daily average expression ofBmal1(by 150%) and its downstream target genesPeriods(by 470% forPer1and by 130% forPer2). We propose that BMAL1 is an important mediator of CR, and activation of BMAL1 might link CR mechanisms with biologic clocks.-Patel, S. A., Chaudhari, A., Gupta, R., Velingkaar, N., Kondratov, R. V. Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms.

Keywords: aging; food anticipation; gene expression; glucose; insulin; transcription.

Figure 1.

BMAL1 deficiency did not affect CR-mediated increase in locomotion around the feeding time. In-cage locomotion of WT (A) and Bmal1^−/− (B) male mice. Mice were subjected to following diets: AL, gray circles, gray dotted lines; 30% CR, black squares, solid black lines. Each graph represents average normalized activity per hour and se (3 consecutive days for every animal, 5 mice per group). a.u., arbitrary units of normalized daily locomotor activity; total daily activity was set as 24 a.u. Zero and 24 h time points were double plotted. *Statistically significant increase in locomotor activity before feeding (food anticipation) for CR group compared to AL group. Light and dark bars represent light and dark phase of day. ZT0 is time when light is on and ZT12 is time when light is off. Food for CR group was provided at ZT14.

Figure 2.

Circadian clock protein BMAL1 is necessary for life span extension in response to CR. A) Kaplan-Meier survival curves of Bmal1^−/− mice on AL (n = 18, gray circles), 30% CR (n = 36, dark gray diamonds), and CR-adjusted (n = 21, black squares) feeding. Mice that died during first 3 wk of 30% CR were excluded from analysis. Mice of both genders were used. Difference between survival curves of AL and CR is statistically significant by log rank test; no statistically significant difference between AL and CR-adjusted groups was observed. B) Kaplan-Meier survival curves of female Bmal1^−/− mice on AL (n = 9, gray circles), 30% CR (n = 23, dark gray diamonds), and CR-adjusted (n = 14 black squares) feeding. Mice that died during first 3 wk of 30% CR were excluded from analysis. Difference between survival curves of AL and CR is statistically significant according to log rank test; no statistically significant difference between AL and CR-adjusted groups was observed. C) Kaplan-Meier survival curves of male Bmal1^−/− mice on AL (n = 9, gray circles), 30% CR (n = 13, dark gray diamonds), and CR-adjusted (n = 9 black squares) feeding. Mice that died during first 3 wk of 30% CR were excluded from analysis. Difference between survival curves of AL and CR is statistically significant according to log rank test; no statistically significant difference between AL and CR-adjusted groups was observed. D) Kaplan-Meier survival curves of WT mice on AL (n = 73, gray circles and gray dotted line) or CR (n = 31; black squares and solid black line) feeding. Mice of both genders were used. *Difference between survival curves of AL and CR statistically significant according to log rank test.

Figure 3.

Thirty percent CR affects body weight of WT and Bmal1^−/− mice. A) Changes in body weights of male WT mice (n = 16, black squares and solid black line) and Bmal1^−/− mice (n = 13, gray circles and gray dotted line). B) Changes in body weights for female WT (n = 14, black squares and solid black line) and Bmal1^−/− (n = 19, gray circles and gray dotted line) mice. Mouse weights were normalized to weight of animals at start of experiment. **Statistically significant difference between genotypes detected at indicated age range.

Figure 4.

Daily food and water consumptions in WT and Bmal1^−/− mice on 30% CR. A) Relative daily food intake of WT (black bars) and Bmal1^−/− (light gray bars) for male and female mice on 30% CR. Relative food intake was calculated by dividing daily food consumption by mouse body weight. *Statistically significant difference between genotypes (P < 0.05). B) Daily water consumption of WT and Bmal1^−/− mice on AL (light gray bars) or 30% CR (black bars) feeding. *Difference between genotypes statistically significant (P < 0.05).

Figure 5.

Circadian clock protein BMAL1 is necessary for reduction in plasma IGF-1 level in response to CR. A–C) Daily profiles of (A) glucose, (B) insulin, and (C) IGF-1 in plasma of WT and Bmal1^−/− mice on AL and CR. D) Daily profiles of liver IGF-1 protein level of WT and Bmal1^−/− mice on AL and CR. E) Daily profiles of liver IGF-1 mRNA level of WT and Bmal1^−/− mice on AL and CR. Three male mice of each genotype and on each feeding regimen were studied per time point. A–E) WT mice on AL feeding (black circles, black dashed lines); WT mice on CR feeding (black squares, black solid lines); Bmal1^−/− mice on AL feeding (gray triangles, gray dashed lines); Bmal1^−/− mice on CR feeding (gray diamonds, gray solid lines). ^aStatistically significant difference (P < 0.05) between WT mice on AL and CR feeding. ^bStatistically significant difference (P < 0.05) between WT and Bmal1^−/− mice on AL feeding. ^cStatistically significant difference (P < 0.05) between Bmal1^−/− mice on AL and CR feeding. ^dStatistically significant difference (P < 0.05) between WT and Bmal1^−/− mice on CR feeding.

Figure 6.

CR increases expression of Bmal1 and BMAL1 transcriptional target genes. mRNA expression of Bmal1 (A), 2 BMAL1 transcriptional target genes; Per1 (B) and Per2 (C), and Clock gene (D) have been studied in liver of mice subjected to following feeding regimens: AL, gray circles and gray dotted line; 30% CR, black squares and solid black lines. At least 3 male mice were used for every time point in both groups. Light and dark bars represent light and dark phase of day. ZT0 is time when light is on and ZT12 is time when light is off. Food for CR group was provided at ZT14. *Statistically significant (P < 0.05) with AL group at this time point.