Neuronal SIRT1 Regulates Metabolic and Reproductive Function and the Response to Caloric Restriction - PubMed (original) (raw)

Neuronal SIRT1 Regulates Metabolic and Reproductive Function and the Response to Caloric Restriction

Emily Rickert et al. J Endocr Soc. 2018.

Abstract

Sirt1 is an NAD-dependent, class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. In this study, we generated mice expressing an enzymatically inactive form (_N_-MUT) or wild-type (WT) SIRT1 (_N_-OX) in mature neurons. _N_-OX male and female mice had impaired glucose tolerance, and _N_-MUT female, but not male, mice had improved glucose tolerance compared with that of WT littermates. Furthermore, glucose tolerance was improved in all mice with caloric restriction (CR) but was greater in the _N_-OX mice, who had better glucose tolerance than their littermates. At the reproductive level, _N_-OX females had impaired estrous cycles, with increased cycle length and more time in estrus. LH and progesterone surges were absent on the evening of proestrus in the _N_-OX mice, suggesting a defect in spontaneous ovulation, which was confirmed by the ovarian histology revealing fewer corpora lutea. Despite this defect, the mice were still fertile when mated to WT mice on the day of proestrus, indicating that the mice could respond to normal pheromonal or environmental cues. When subjected to CR, the _N_-OX mice went into diestrus arrest earlier than their littermates. Together, these results suggested that the overexpression of SIRT1 rendered the mice more sensitive to the metabolic improvements and suppression of reproductive cycles by CR, which was independent of circadian rhythms.

Keywords: caloric restriction; fertility; glucose intolerance; neurons.

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Figures

Figure 1.

Figure 1.

Neuronal SIRT1 caused glucose intolerance in mice. Data for _N_-MUT mice are shown in red, in white for WT littermates, and green for _N_-OX mice in green. For (A–D), Two-way ANOVA indicated significant genotype and time effects (P < 0.0001) but no interaction. (A) Body weights for male mice from 3 to 13 wk of age (n = 13 for _N_-MUT; n = 48 for WT; n = 24 for _N_-OX). Inset shows final body weight at 8 to 9 mo. (B) Body weights for female mice from 3 to 13 wk of age (n = 10 for _N_-MUT; n = 44 for WT; n = 20 for _N_-OX). Inset shows final body weight at 8 to 9 mo. (C) Glucose tolerance testing in male mice at 3 mo of age (n = 8 for _N_-OX; n = 16 for WT; n = 4 for _N_-MUT). Two-way ANOVA indicated significant interaction effect (P = 0.005). Inset shows AUC data. (D) Glucose tolerance testing in female mice at 3 mo of age (n = 9 for _N_-OX; n = 16 for WT; n = 6 for _N_-MUT). Inset shows AUC data. (E) Insulin tolerance testing in male mice at 3 mo of age (n = 9 for _N_-OX; n = 16 for WT; n = 6 for _N_-MUT). (F) Insulin tolerance testing in female mice at 3 mo of age (n = 10 for _N_-OX; n = 16 for WT; n = 6 for _N_-MUT). (E, F) Two-way ANOVA indicated significant genotype and time effects (P < 0.001 and P < 0.0001, respectively) but no interaction. (G) Daily food intake over 10 wk for male mice (n = 3 for _N_-MUT; n = 12 for WT; n = 4 for _N_-OX). (H) Daily food intake over 10 wk for female mice (n = 6 for _N_-MUT; n = 11 for WT; n = 5 for _N_-OX). (I) Fasting blood glucose data of male mice (n = 17 for _N_-OX; n = 32 for WT; n = 8 for _N_-MUT). (J) Fasting blood glucose data of female mice (n = 20 for _N_-OX; n = 32 for WT; n = 4 for _N_-MUT). (K) Fasting insulin data of male mice (n = 6 for _N_-MUT; n = 14 for WT; n = 6 for _N_-OX). (L) Fasting insulin data of female mice (n = 6 for _N_-MUT; n = 12 for WT; n = 6 for _N_-OX). (M) Fasting leptin data of male mice (n = 6 for _N_-MUT; n = 14 for WT; n = 6 for _N_-OX). (N) Fasting leptin data of female mice (n = 6 for _N_-MUT; n = 12 for WT; n = 6 for _N_-OX). Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, *P < 0.001, and ****P < 0.0001 vs WT or as indicated. AUC, area under the curve.

Figure 2.

Figure 2.

Neuronal SIRT1 regulated reproduction in female mice. Data for _N_-MUT mice are shown in red, in white for WT littermates, and green for _N_-OX mice in green. (A) Day of vaginal opening (n = 8 for _N_-MUT; n = 40 for WT; n = 18 for _N_-OX). (B) Day of first estrous (n = 8 for _N_-MUT; n = 41 for WT; n = 21 for _N_-OX). (C) Number of estrous cycles by vaginal lavage over 6 wk (n = 6 for _N_-MUT; n = 11 for WT; n = 5 for _N_-OX). (D) Cycle length during the 6 wk (n = 44 for _N_-MUT; n = 74 for WT; n = 19 for _N_-OX). (E) Relative-frequency histogram of cycle lengths. (F) Percentage of d spent in each stage of the estrous cycle during the 6-wk cycling (n = 6 for _N_-MUT; n = 11 for WT; n = 5 for _N_-OX). Two-way ANOVA indicated significant stage effect (P < 0.0001) and significant interaction of stage and genotype (P < 0.0001). (G) FSH levels during diestrus, the morning of proestrus (Pro-AM), the afternoon of proestrus (Pro-PM), and the morning of estrus (n = 8 for _N_-MUT; n = 16 for WT; n = 8 for _N_-OX). Two-way ANOVA indicated significant stage effect (P = 0.015) and significant interaction of stage and genotype (P = 0.013). (H) LH levels during diestrus, the morning of proestrus, and the morning of estrus (n = 8 for _N_-MUT; n = 16 for WT; n = 8 for _N_-OX). Two-way ANOVA indicated significant stage effect (P = 0.013) but no interaction of stage and genotype. (I) LH levels during the afternoon of proestrus (n = 8 for _N_-MUT; n = 16 for WT; n = 8 for _N_-OX). Proestrus LH values did not follow a normal distribution, so _N_-OX mice had significantly lower LH levels than did WT mice by Kruskal-Wallis test (P = 0.033). (J) Progesterone levels during the afternoon of proestrus (n = 7 for WT; n = 7 for _N_-OX). (K) Days to plug, days to first litter, and litter size during fertility test (n = 3 for _N_-MUT; n = 11 for WT; n = 6 for _N_-OX). Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, *P < 0.001, ****P < 0.0001 vs WT or as indicated.

Figure 3.

Figure 3.

Overexpression of SIRT1 impaired ovulation. Data for _N_-MUT mice are shown in red, in white for WT littermates, and green for _N_-OX mice in green.. (A) Ovarian morphology. Representative hematoxylin- and eosin-stained sections of ovaries obtained at euthanasia. (B) Quantification of ovarian cross-sectional area for _N_-MUT (n = 12), WT (n = 15), and _N_-OX (n = 8) ovaries. *P < 0.05 vs _N_-MUT. (C) Quantification of follicle stage. Percentage of follicles at each stage for _N_-MUT (n = 6), WT (n = 9), and _N_-OX (n = 4) mice. (D) Quantification of CL for _N_-MUT (n = 6), WT (n = 9), and _N_-OX (n = 4) mice. *P < 0.05 vs WT. Data are reported as mean ± SEM. *Significant difference by post hoc testing.

Figure 4.

Figure 4.

Overexpression of SIRT1 enhanced the metabolic response to CR. Data for _N_-MUT mice are shown in red, in white for WT littermates, and green for _N_-OX mice in green. (A) Glucose tolerance test conducted after CR (n = 8 for _N_-OX; n = 17 for WT; n = 6 for _N_-MUT; n = 20 males; n = 11 females). Two-way ANOVA indicated a significant time effect (P < 0.0001) and time-genotype interaction (P = 0.0025). (B) Fasting insulin levels during CR (n = 14 for _N_-MUT; n = 26 for WT; n = 12 for _N_-OX; n = 28 males; n = 24 females). (C) Fasting leptin levels during CR (n = 14 for _N_-MUT; n = 26 for WT; n = 12 for _N_-OX; n = 28 males; n = 24 females). Two-way ANOVA indicated a significant genotype effect (P < 0.0001). Data are reported as mean ± SEM. *P < 0.05, **P < 0.01 among _N_-OX and WT and _N_-MUT, or as indicated, from post hoc testing. AF, antral follicle; CL, corpus luteum.

Figure 5.

Figure 5.

Overexpression of SIRT1 enhanced the reproductive response to CR. Data for _N_-MUT mice are shown in red, in white for WT littermates, and green for _N_-OX mice in green. Estrous cycles were assessed by vaginal lavage in female mice during the 8 wk of CR. Data are reported as mean ± SEM. (A) Average number of d spent in met/diestrus, proestrus, or estrus during the four 2-wk stages of increasing CR for each genotype of the estrous cycle during the 6-wk cycling (n = 6 for _N_-MUT; n = 14 for WT; n = 5 for _N_-OX). Two-way ANOVA indicated a significant CR effect (P < 0.0001) and significant interaction of CR and genotype (P = 0.003) for met/diestrus, a CR effect (P < 0.0001) for proestrus; and a genotype effect (P = 0.017), a CR effect (P < 0.0001), and a significant interaction of CR and genotype (P = 0.011) for estrus. (B) LH levels during the four stages of CR (n = 6 for _N_-MUT; n = 12 for WT; n = 5 for _N_-OX). No significant differences were observed. (C) FSH levels during the four stages of CR (n = 6 for _N_-MUT; n = 12 for WT; n = 5 for _N_-OX). Two-way ANOVA indicated significant genotype (P = 0.022) and CR (P = 0.002) effects but no significant interaction. (D–F) Estradiol, progesterone, and testosterone levels before and after CR (n = 3 for _N_-MUT; n = 3 for WT; n = 3 for _N_-OX. Each sample was pooled from two animals). Two-way ANOVA indicated significant genotype (P = 0.0004) and CR (P = 0.0033) effects for estradiol, a CR effect (P = 0.0055) for progesterone, and genotype (P = 0.015) and CR (P = 0.019) effects for testosterone. No significant interactions of CR and genotype were observed. *P < 0.05, **P < 0.01, *P < 0.001, ****P < 0.0001 (post hoc testing). Met, metestrus.

Figure 6.

Figure 6.

Hypothalamic and pituitary gene expression in _N_-MUT and _N_-OX mice. Data for _N_-MUT mice are shown in red, in white for WT littermates, and green for _N_-OX mice in green. (A) Gene expression in the hypothalami from female mice, determined by qPCR. (B) Gene expression in the pituitaries from female mice, determined by qPCR. Data are reported as mean ± SEM; n = 5 for _N_-MUT; n = 10 for WT, and n = 5 for _N_-OX. *P < 0.05, **P < 0.01. Rel, relative.

Figure 7.

Figure 7.

Metabolic cage assessment of _N_-OX mice. Data of WT mice (n = 6) are shown in black and of _N_-OX mice (n = 6) in green. Graphs show diurnal patterns over 24 h measured per 13-min interval for the measures indicated by the _y_-axis labels. Horizontal black bar indicates period of lights off (6:00

pm

to 6:00

am

). (A) Ambulatory activity counts. (B) Rearing activity counts. (C) VO2 consumption. (D) Respiratory exchange ratio. (E) Food intake. (F) Water intake. (G) Heat produced. (H) Cage temperature. Data are reported as mean ± SEM. In all cases, repeated measures ANOVA indicated a significant time effect (P < 0.0001). Ambulatory activity, rearing activity, VO2, RER, and water intake were associated with significant genotype effects (P < 0.0001, P < 0.0001, P < 0.0001, P = 0.0004, and P = 0.04, respectively). *P < 0.05, **P < 0.01, by post hoc testing. RER, respiratory exchange ratio; Temp, temperature; VO2, oxygen consumption.

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