Obesity and metabolic syndrome in circadian Clock mutant mice

Abstract

The CLOCK transcription factor is a key component of the molecular circadian clock within pacemaker neurons of the hypothalamic suprachiasmatic nucleus. We found that homozygous Clock mutant mice have a greatly attenuated diurnal feeding rhythm, are hyperphagic and obese, and develop a metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis, hyperglycemia, and hypoinsulinemia. Expression of transcripts encoding selected hypothalamic peptides associated with energy balance was attenuated in the Clock mutant mice. These results suggest that the circadian clock gene network plays an important role in mammalian energy balance.

Fig. 1

Altered diurnal rhythms in locomotor activity, feeding and metabolic rate in Clock mutant mice. (A) Insert on left: Actograms showing locomotor activity over a 30 day period in representative adult wild-type (WT) (top) and Clock mutant (bottom) mice individually housed in 12:12 LD (at 23°C) and provided food and water ad libitum. Activity bouts were analyzed using ClockLab software in 6-minute intervals across 7 days of recording (selected days are indicated by red vertical lines to the left of the actograms). Shown over the 24 hr cycle are activity counts during light (unshaded) and dark (shaded) periods (WT, n=5, black line; Clock, n=9, blue line). (B) Diurnal rhythm of locomotor activity for mice shown in (A). Activity counts were accumulated over the 12-hour light and 12-hour dark periods and expressed in each period as a percent of total 24-hour activity (*, p<0.05). Total activity over the 24-hour period was similar between genotypes. (C) Diurnal rhythm of food intake. Different groups of adult WT (N=7) and Clock mutant (N=5) mice were maintained on a regular diet (10% kcal/fat) and food intake (g) was measured during light and dark periods. Results shown are average food intake during light and dark periods as a percentage of total food intake (*, p<0.001). (D) Diurnal rhythm of metabolic rate. Metabolic rate was determined in additional groups of WT (N=7) and Clock mutant (N=9) mice by indirect calorimetry under 12:12 LD conditions over a 3 day continuous monitoring period (*, p<0.05). Results shown are average metabolic rates during the light and dark periods as a percentage of total metabolic rate. Results shown (A–D) are expressed as group means ± SEM.

Fig. 2

Obesity in Clock mutant mice. (A) Energy intake. Average caloric intake over a 10 week period in male WT and Clock mutant mice. WT and Clock mutant mice were provided ad libitum access to regular (10% kcal/fat, WT, n=8, Clock, n=10) or high-fat chow (45% kcal/fat, WT, n=7, Clock, n=11) for 10 weeks beginning at 6 weeks of age. Weekly food intake was analyzed in the two groups (*, p<0.01). (B) Body weight. Body weights for the animals depicted in (A) after the 10 week study (*, p<0.01). (C) Longitudinal weight gain. Body weights WT (open) and Clock mutant (closed) mice over the 10 week study for animals depicted in (A) fed either regular (circle) or high fat (square) diets. (D) Post-weaning body weight of mice beginning at 10 days through 8 weeks of age. Growth curves in WT (open circle) and Clock mutant (closed circle) mice on regular chow were obtained by weighing animals weekly. Significant differences did not appear until 6 weeks of age (*, p<.05). All values (A–D) represent group means ± SEM

Fig. 3

Altered diurnal rhythms and abundance in Clock mutant mice of Per2 mRNA and mRNAs encoding selected hypothalamic peptides involved in energy balance. (A–D) mRNA relative abundance (RA) curves. Time-course variation of transcripts in the hypothalamus of WT (red line) and Clock mutant (black line) mice across a 12:12 LD cycle (indicated by white-black bar on bottom). Real-time PCR was used to determine transcript levels. Values are displayed as RA (mean ± SEM) after normalization to glyseraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels in the same sample. Note that for visual clarity the RA scales vary for the different transcripts and vary between genotypes for orexin and ghrelin. Brains were collected at 4-hour intervals across the 12:12 LD cycle using four WT and four Clock mutant mice at each time point. Genotype comparisons were made at each 4-hour time point using independent sample t-tests with a significance level of p<.05 (*). Per2 = Period-2; Orx = Orexin; Ghr = Ghrelin; CART = Cocaine- and amphetamine-regulated transcript.