Food anticipatory activity on a calorie-restricted diet is independent of Sirt1

Abstract

A number of studies have demonstrated that the Sirtuin family member, Sirt1, is a key integrator of growth, metabolism, and lifespan. Sirt1 directly interacts with and deacetylates key regulators of the circadian clock, positioning it to be an important link between feeding and circadian rhythms. In fact, one study suggests that Sirt1 is necessary for behavioral anticipation of limited daily food availability, a circadian process termed food anticipatory activity (FAA). In their study, mice overexpressing Sirt1 had enhanced FAA, while mice lacking Sirt1 had little to no FAA. Based on the supposition that Sirt1 was indeed required for FAA, we sought to use Sirt1 deletion to map the neural circuitry responsible for FAA. We began by inactivating Sirt1 using the cell-type specific Cre-driver lines proopiomelanocortin, but after observing no effect on body weight loss or FAA we then moved on to more broadly neuronal Cre drivers Ca2+/calmodulin-dependent protein kinase II and nestin. As neither of these neuronal deletions of Sirt1 had impaired FAA, we then tested 1) a broad postnatal tamoxifen-inducible deletion, 2) a complete, developmental knockout of Sirt1, and 3) a gene replacement, catalytically inactive, form of Sirt1; but all of these mice had FAA similar to controls. Therefore, our findings suggest that FAA is completely independent of Sirt1.

Fig 1. Sirt1 expression, deletion, and testing FAA in mice lacking Sirt1 in Pomc neurons.

Immunostaining for Sirt1 in the adult mouse brain reveals a broad neuronal expression pattern: (A) CA1 region of the hippocampus (scale bar indicates 100 microns) and (B) low magnification image of the dorsal hypothalamus (scale bar indicates 500 microns), showing robust Sirt1 expression in the paraventricular nucleus. (C) Western blot showing the decreased molecular weight of Sirt1 in homogenates of cortex of CamKII-Cre; Sirt1^loxP/loxP mice (lane 2) indicated by an arrowhead. Lane 1 shows full length Sirt1 protein indicated by an arrow in Sirt1^loxP/loxP control cortex. Lanes 3 and 4 are from the cortex of a Sirt1 WT and Sirt1 KO mice. Note that there is a cross reacting protein of smaller molecular weight than the exon 4-deleted Sirt1 indicated by an asterisk. (D) Body weights of Pomc-Cre; Sirt1^loxP/loxP and Sirt1^loxP/loxP littermate controls. Mice were fed AL diets on Day -7. CR began on Day 0. Waveform of high activity behaviors at (E) day -7 (n = 6 WT, n = 8 KO), prior to initiating timed CR, (F) day 14 (n = 6 WT, n = 8 KO) of CR, (G) day 28 (n = 6 WT, n = 8 KO) of CR. (H) Total high activity (walking, hanging, jumping, and rearing) observed during the duration of the 23.5h to 24h video across the duration of the experiment. (I) High activity observed during the three hours preceding scheduled feeding divided by the total seconds of total high activity is depicted as fraction of high activity to represent FAA in mice. Data are shown as means +/- SEMS. Feeding time is indicated by an arrow. Lights on are indicated by yellow coloration while lights off time is indicated by grayed area. For body weight, statistical significance was determined using an unpaired t-test; for behavioral data, statistical significance was determined using the Mann-Whitney Test. * denotes p<0.05.

Fig 2. Testing FAA in forebrain and pan neuronal deletion mutants of Sirt1.

(A) Mean body weights for CamKII-Cre; Sirt1^loxP/loxP and Sirt1^loxP/loxP littermate controls. CR diets began on Day 0. Mean high activity data for home cage behaviors at (B) Day -7 (n = 8 WT and n = 9 for KO), (C) Day 14 of CR (n = 6 WT and n = 9 KO), and (D) Day 28 (n = 7 WT and n = 8 KO) of CR are shown. (E) Mean total high activity, measured in seconds, over the entire 23.5h-24h video recording for Sirt1^loxP/loxP controls and CamKII-Cre;Sirt1^loxP/loxP (F) Mean fraction of normalized high activity in the 3h prior to mealtime; there were no differences between groups on all days. (G) Mean body weight for Nestin-Cre; Sirt1^loxP/loxP and Sirt1^loxP/loxP littermate controls. Mean high activity data for home cage behaviors at (H) Day -7 (n = 5 WT, n = 7 KO), (I) Day 14 (n = 4 WT, n = 4 KO) of CR, and (J) Day 28 (n = 5 WT, n = 7 KO) of CR are shown. (K) The amount of high activity in seconds for Sirt1^loxP/loxP and Nestin-Cre; Sirt1^loxP/loxP mice. On Day 7, the amount of total activity was greater for than Nestin-Cre; Sirt1^loxP/loxP compared to Sirt1^loxP/loxP controls (P = 0.0333). (L) Fraction of high activity measures between Sirt1^loxP/loxP and Nestin-Cre; Sirt1^loxP/loxP groups exhibit a greater amount of FAA in controls on Day 7 (P = 0.0333). For body weight data, statistical significance was determined using an unpaired T test. For behavioral data, statistical significance was determined using the Mann-Whitney Test. * denotes p<0.05.

Fig 3. Testing FAA in post-natal knockout, complete null, and catalytic mutant Sirt1 mice.

(A) Mean body weight prior to starting CR (Day -7) through Day 42 of CR for tamoxifen-injected Sirt1^loxP/loxP and CAGG-Cre^Tm; Sirt1^loxP/loxP mice. CR diets began on Day 0. Mean high activity data for home cage behaviors at (B) Day -7 (n = 8 KO, n = 9 WT), (C) Day 14 (n = 9 KO and WT) of CR, and (D) Day 28 (n = 9 KO and WT) of CR are shown. Feeding time is indicated by an arrow. (E) The total high activity for Sirt1^loxP/loxP and CAGG-Cre^Tm; Sirt1^loxP/loxP groups were similar with the exception of Day 14, where the knockouts showed a significant decrease (n = 9 for both groups, P = 0.0315). (F) Mean normalized high activity in the 3-hours preceding scheduled mealtime were similar between tamoxifen-injected Sirt1^loxP/loxP and CAGG-Cre^Tm; Sirt1^loxP/loxP mice. (G) Mean body weight for control, Sirt1 KO, and Sirt1 Y/Y mutants starting Day -7, prior to starting CR, through Day 28 of CR. Mean home cage activity counts at (H) Day -7(n = 10 WT, N = 6 KO, and N = 9 YY), (I) Day 14 (n = 10 WT, n = 6 KO, and n = 8 YY) of CR, and (J) Day 28 (n = 10 WT, n = 6 KO, and n = 9 YY) of CR are shown. (K) Total activity counts over the course of the experiment. (I) When dividing the final 3h counts over total counts throughout the 23.5h-24h recording, all mice displayed relatively similar amount of FAA prior to scheduled feeding times on all days except for Day -7 (P = 0.0442). However, no significance was found when performing pairwise comparisons. All data is shown with mean +/- SEM and statistical significance was determined using the Kruskal-Wallis Test with Dunn’s post-test. * denotes p<0.05.