Obesity and Metabolic Syndrome in Circadian Clock Mutant Mice Fred W. Turek,1,3 Corinne Joshu,3,4* Akira Kohsaka,3,4* Emily Lin,3,4* Ganka Ivanova,2,4 Erin McDearmon,3,5 Aaron Laposky,3 Sue Losee-Olson,3 Amy Easton,3 Dalan R. Jensen,6 Robert H. Eckel,6 Joseph S. Takahashi,1,3,5 Joseph Bass2,3,4. 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. Major components of energy homeostasis, including the sleep-wake cycle, thermogenesis, feeding, and glucose and lipid metabolism, are subjected to circadian regulation that synchro- nizes energy intake and expenditure with changes in the external environment imposed by the rising and setting of the sun. The neural circadian clock located within the hypothalamic suprachiasmatic nucleus (SCN) orchestrates 24- hour cycles in these behavioral and physiolog- ical rhythms (1���3). However, the discovery that clock genes can regulate circadian rhythmicity in vitro in other central as well as peripheral tissues, including those involved in nutrient homeostasis (e.g., mediobasal hypothalamus, liver, muscle, and pancreas), indicates that cir- cadian and metabolic processes are linked at multiple levels (4���9). The recent finding that changes in the ratio of oxidized to reduced nicotinamide adenine dinucleotide phosphate control the transcriptional activity of the basic helix-loop-helix (bHLH) protein NPAS2���a homolog of a primary circadian gene, Clock��� suggests that cell redox may couple the expression of metabolic and circadian genes (10). Mice harboring a mutation in Clock show profound changes in circadian rhythmicity (11) and offer an experimental genetic model to analyze the link among circadian gene networks, behavior, and metabolism in vivo. Positional cloning and transgenic rescue of normal circadian phenotype identified Clock as a member of the bHLH Per-Arnt-Sim (PAS) transcription factor family (12, 13). Rel- ative to wild-type mice, the most pronounced alteration in circadian phenotype in Clock mutants is a 1-hour increase in the free- running rhythm of locomotor activity in het- erozygous mice in constant darkness (DD) and a 3- to 4-hour increase (i.e., period 0 27 to 28 hours in DD) in circadian period in homozygous mice, which is often followed by a total breakdown of circadian rhyth- micity (i.e., arrhythmicity) after a few weeks in DD. Although previous studies that used run- ning wheel behavior as a marker of locomotor activity did not reveal major differences between homozygous Clock mutant and wild-type mice maintained on a light-dark (LD) cycle, use of infrared beam crossing to monitor total activity revealed a signif- icant increase in activity during the light phase and a change in the temporal pattern of total activity during the dark phase (Fig. 1A) (14). In particular, wild-type mice showed two pronounced peaks of activity���one occurring after lights off, the other before lights on���whereas these peaks were atten- uated in Clock mutant mice. Surprisingly, despite there being a clear (but dampened) diurnal rhythm in locomotor activity in Clock mutant mice (Fig. 1B), the diurnal rhythm in food intake was severely altered in these 1 Department of Neurology and 2 Department of Medicine, Feinberg School of Medicine, 3Department of Neu- robiology and Physiology, Northwestern University, Evanston, IL 60208, USA. 4Evanston Northwestern Healthcare (ENH) Research Institute, Evanston, IL 60208, USA. 5Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. 6 Department of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA. *These authors contributed equally to this work. .To whom correspondence should be addressed. E-mail: j-bass@northwestern.edu Fig. 1. Altered diurnal rhythms in locomotor activity, feeding, and metabolic rate in Clock mutant mice. (A) Activity counts over the 24-hour cycle during light (unshaded) and dark (shaded) periods [wild-type, n 0 5, black line Clock, n 0 9, blue line]. Inset: Actograms showing locomotor activity over a 30-day period in representative adult wild-type (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-min intervals across 7 days of recording (selected days are indicated by red vertical lines to the left of the actograms). (B) Diurnal rhythm of locomotor activity for mice in (A). Activity counts were accumulated over the 12-hour light and 12-hour dark periods and are expressed in each period as a percentage of total 24-hour activity (*P G 0.05). Total activity over the 24-hour period was similar between wild-type (WT) and Clock mutant (CL) genotypes. (C) Diurnal rhythm of food intake. Different groups of adult WT (N 0 7) and Clock mutant (N 0 5) mice were maintained on a regular diet (10% kcal/fat), and food intake (in grams) 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 G 0.001). (D) Diurnal rhythm of metabolic rate. Metabolic rate was determined in additional groups of WT (N 0 7) and Clock mutant (N 0 9) mice by indirect calorimetry under 12:12 LD conditions over a 3-day continuous monitoring period (*P G 0.05). Results shown are average metabolic rates during the light and dark periods as a percentage of total metabolic rate. All results shown are expressed as group means T SEM. R E P O R T S www.sciencemag.org SCIENCE VOL 308 13 MAY 2005 1043

mice (Fig. 1C): Only 53% of the food intake occurred during the dark phase in Clock mutant mice, versus 75% in wild-type mice. In a preliminary analysis, we found that the diurnal rhythm of food intake was already attenuated in 3-week-old mice before an increase in weight (fig. S1). Similarly, the rhythm in energy expenditure, as measured by respiratory gas analysis, was attenuated in the Clock mutant mice (Fig. 1D). Overall there was a net 10% decrease in energy expenditure in the mutants. Clock mutant mice fed either a regular or high-fat diet showed a significant increase in energy intake and body weight (Fig. 2, A and B). In Clock mutant and wild-type adult mice fed either a control or high-fat diet for a period of 10 weeks beginning at 6 weeks of age (Fig. 2C), the increase in body weight was 24% in wild-type and 29% in Clock mutant mice fed a regular diet, versus 38% in wild- type and 49% in Clock mutant mice fed a high-fat diet. Comparison of somatic growth and solid organ mass did not reveal genotype- specific differences. Instead, the marked weight gain in Clock mutants fed a regular diet was attributable to a 65% increase in lean mass and a 35% increase in fat mass, whereas in mutants fed a high-fat diet, the weight gain was due to a 25% increase in lean mass and a 75% increase in fat mass relative to wild-type control mice (fig. S2). Because the Clock mutation could affect early fetal growth and development, we analyzed the body weights of Clock and lit- termate pups throughout the first 8 weeks of life. Body weights were similar in Clock mutant and wild-type mice during the first 5 weeks, but by 6 weeks of age Clock mutant mice were consistently heavier (Fig. 2D), which suggests that the mutation did not affect fetal growth or nutrition. We next investigated whether the Clock mutation altered the adipose���central nervous system (CNS) axis that regulates feeding and energy expenditure. Histological analysis revealed adipocyte hypertrophy and lipid engorgement of hepatocytes with prominent glycogen accumulation (fig. S3A) in Clock mice fed a high-fat diet relative to wild-type controls these are hallmarks of diet-induced obesity in wild-type mice. At 6 to 7 months of age, Clock mutant mice also had hyper- cholesterolemia, hypertriglyceridemia, hyper- glycemia, and hypoinsulinemia (Table 1). In addition, serum leptin levels increased dur- ing the light phase in Clock mutant mice fed a regular diet this increase was enhanced in mice fed a high-fat diet (fig. S3B). These markers of metabolic dysregulation were not due to an increase in glucocorticoid pro- duction, because levels of corticosterone were lower in the Clock mutant mice across the 24-hour LD cycle (wild-type, 5.5 T 1.4 mg/dl Clock mutant, 2.6 T 0.4 mg/dl P G 0.05). Thus, the Clock mutant mice developed a spectrum of tissue and biochemical abnor- malities that are hallmarks of metabolic disease. To explore whether the Clock mutation affects expression of neuropeptides involved in appetite regulation and energy balance, we analyzed transcripts corresponding to selected orexigenic and anorexigenic neu- ropeptides expressed in the mediobasal hy- pothalamus (MBH). We studied the orexin transcript because the orexinergic system is involved in both feeding and sleep-wake regulation (15, 16) we also studied the tran- scripts for ghrelin and CART (cocaine- and amphetamine-regulated transcript) because the corresponding genes contain CLOCK- responsive E-box elements (17, 18). In ad- dition, we examined the expression of a second circadian clock gene, Per2, which has a diurnal rhythm of expression in the retro- chiasmatic area. The expression levels of Per2, orexin, and ghrelin mRNA were markedly reduced in Clock mutant mice at virtually all time points of the 12L:12D cycle (Fig. 3). A small but significant decrease in the expression level of CART in Clock mutant mice occurred at the beginning and end of the 12-hour light phase (Fig. 3). These broad effects of the Clock gene mutation on nutrient regulation reveal an unforeseen role for the circadian clock system in regulating more than just the timing of food intake and metabolic processes. The effect of Fig. 2. Obesity in Clock mutant mice. (A) 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 0 8 Clock, n 0 10) or high-fat chow (45% kcal/fat WT, n 0 7 Clock, n 0 11) for 10 weeks beginning at 6 weeks of age. Weekly food intake was analyzed in the two groups (*P G 0.01). (B) Body weights for the mice in (A) after the 10-week study (*P G 0.01). (C) Body weights of WT (open symbols) and Clock mutant (solid symbols) mice over the 10-week study for mice in (A) fed either regular (circles) or high-fat (squares) diets. (D) Body weight of mice after weaning, from 10 days to 8 weeks of age. Growth curves in WT (open circles) and Clock mutant (solid circles) mice on regular chow were obtained by weekly weighing. Significant differences did not appear until 6 weeks of age (*P G 0.05). All values represent group means T SEM. Table 1. Metabolic parameters in WT and Clock mutant mice. Serum triglyceride, cholesterol, glucose, insulin, and leptin concentrations were determined in 7- to 8-month-old WT and Clock mutant mice fed a regular diet ad libitum (n 0 4 to 8 mice per group). For measurement of glucose, insulin, and leptin, blood was collected at 4-hour intervals over a 24-hour time period via an indwelling catheter (40 ml per blood sample), and the data were pooled to provide an overall mean (TSEM) value. For triglyceride and cholesterol measurement, a single blood sample (160 ml) was collected at zeitgeber time 0. Metabolic parameter WT Clock P value Triglyceride (mg/dl) 136 T 8 164 T 8 G0.05 Cholesterol (mg/dl) 141 T 9 163 T 6 G0.05 Glucose (mg/dl) 130 T 5 161 T 7 G0.01 Insulin (ng/ml) 1.7 T 0.3 1.1 T 0.1 n.s. Leptin (ng/ml) 3.4 T 0.4 4.6 T 0.3 G0.05 R E P O R T S 13 MAY 2005 VOL 308 SCIENCE www.sciencemag.org 1044