
==== Front
PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1552355810.1371/journal.pbio.0020377Research ArticleAnimal BehaviorPhysiologyMus (Mouse)BMAL1 and CLOCK, Two Essential Components of the Circadian Clock, Are Involved in Glucose Homeostasis Metabolism and Peripheral ClocksRudic R. Daniel 
1
McNamara Peter 
2
Curtis Anne-Maria 
1
Boston Raymond C 
3
Panda Satchidananda 
4
Hogenesch John B 
4
FitzGerald Garret A garret@spirit.gcrc.upenn.edu
1
1Center for Experimental Therapeutics, University of PennsylvaniaPhiladelphia, PennsylvaniaUnited States of America2Phenomix Corporation, La Jolla, CaliforniaUnited States of America3School of Veterinary Medicine, University of PennsylvaniaKennett Square, PennsylvaniaUnited States of America4The Genomics Institute of the Novartis Research FoundationLa Jolla, CaliforniaUnited States of America11 2004 2 11 2004 2 11 2004 2 11 e37711 2 2004 31 8 2004 Copyright: © 2004 Rudic et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Sleeping, Waking, ... and Glucose Homeostasis 
Circadian timing is generated through a unique series of autoregulatory interactions termed the molecular clock. Behavioral rhythms subject to the molecular clock are well characterized. We demonstrate a role for Bmal1 and Clock in the regulation of glucose homeostasis. Inactivation of the known clock components Bmal1 (Mop3) and Clock suppress the diurnal variation in glucose and triglycerides. Gluconeogenesis is abolished by deletion of Bmal1 and is depressed in Clock mutants, but the counterregulatory response of corticosterone and glucagon to insulin-induced hypoglycaemia is retained. Furthermore, a high-fat diet modulates carbohydrate metabolism by amplifying circadian variation in glucose tolerance and insulin sensitivity, and mutation of Clock restores the chow-fed phenotype. Bmal1 and Clock, genes that function in the core molecular clock, exert profound control over recovery from insulin-induced hypoglycaemia. Furthermore, asynchronous dietary cues may modify glucose homeostasis via their interactions with peripheral molecular clocks.

Besides regulating various behavioral rhythms, the molecular clock plays a role in the control of glucose homeostasis
==== Body
Introduction
The master clock, which, in mammals, resides in the hypothalamic suprachiasmatic nucleus (SCN), is thought to synchronize multiple peripheral oscillators to ensure temporal coordination of behavior and metabolism. Peripheral clocks amplify or dampen central rhythms or exhibit autonomous behavior to facilitate local adaptive responses (Hastings et al. 2003). The central clock may communicate to modulate or entrain rhythms in the periphery via hormones (McNamara et al. 2001) or hemodynamic cues. Asynchronous environmental cues, such as eating, also influence the autonomous behavior of peripheral clocks (Damiola et al. 2000; Stokkan et al. 2001).

The variation in sleep and wakefulness (activity) is perhaps the most well-known circadian rhythm. Surgical ablation of the SCN in mice (Ibuka et al. 1980; Welsh et al. 1988) and rats (Ibuka et al. 1977; Mosko and Moore 1979) abolishes the nocturnal burst in locomotor activity. Similarly, disruption and/or mutation of Bmal1 (Bunger et al. 2000) or Clock (Vitaterna et al. 1994), transcription factors that compose the positive limb of an autoregulatory feedback loop in the core molecular clock (Young and Kay 2001; Reppert and Weaver 2002), also impairs circadian behavior. Bmal1 and Clock may influence behavioral rhythms by regulating the firing rate of SCN neurons (Herzog et al. 1998; Deboer et al. 2003).

Genes relevant to the molecular clock are also expressed in peripheral tissues (Akhtar et al. 2002; Kita et al. 2002; Panda et al. 2002; Storch et al. 2002; Oishi et al. 2003) where approximately 5%–10% of the transcriptome is subject to circadian oscillation (Albrecht and Eichele 2003). Although the precise role of peripheral clocks and the mechanisms that link them to the SCN remain largely obscure, genetic mutation or deletion has implicated peripheral clocks in the regulation of some aspects of cellular function, including division (Matsuo et al. 2003), estrous cyclicity (Miller et al. 2004), and phospholipid metabolism (Marquez et al. 2004). Glucose and lipid homeostasis are also known to exhibit circadian variation (Seaman et al. 1965; Malherbe et al. 1969; Gagliardino and Hernandez 1971; Schlierf and Dorow 1973). Surgical ablation of the SCN impairs the control of glucose homeostasis (la Fleur et al. 2001). However, the proximity of satiety centres to the SCN has potentially confounded interpretation of these results. Indeed, there is no direct evidence implicating the molecular clock in the regulation of glycaemia or insulin sensitivity (Si).

Our studies revealed a profound role for core clock genes—Bmal1 and Clock—in regulating recovery from insulin-induced hypoglycaemia. Furthermore, the impact of a high-fat diet (HF) was to amplify the diurnal variation in glucose tolerance and Si in a manner dependent on the Clock gene. These studies suggest that the temporal distribution of a caloric load may influence the response to insulin and that circadian variability in glucose homeostasis may be subject to modulation by asynchronous dietary cues.

Results
We examined the role of the molecular clock in glucose homeostasis by using mice in which core clock genes are impaired (Clockmut) or deficient (Bmal1−/−). Both plasma glucose and triglycerides were subject to circadian variation in wild-type (WT) mice, peaking at approximately circadian time point 4 (CT4) and CT28 (where CT0 is subjective day beginning at 7 AM, and CT12 is subjective night beginning at 7 PM) (Figure 1A and 1B), as reported previously (Seaman et al. 1965; Schlierf and Dorow 1973). We also observed that corticosterone (Figure 1C), which stimulates gluconeogenesis during hypoglycaemia (Cryer 1993), and adiponectin (Figure 1D), which has been associated with insulin resistance (Yamauchi et al. 2001; Maeda et al. 2002), oscillated significantly, but out of phase with the glucose and triglyceride rhythms. Diurnal variation in glucose and triglycerides, but not in corticosterone, was disrupted in the mutant mice (Table 1).

Figure 1 Circadian Variation of Glucose, Triglyceride, and Hormone Levels in Circulating Blood
Plasma from whole blood isolated from unchallenged WT mice at different CTs was analyzed for glucose (A), triglyceride (B), corticosterone (C), and adiponectin levels (D) (n = 12 per time point). Results for Bmal1
−/− and Clockmut mice are shown in Table 1.

Table 1 Clock-Controlled Metabolic Rhythms
Utilizing peak and trough CTs described in Figure 2A, WT mice showed circadian variation in glucose and triglycerides at CT5 and CT17 that was absent in Bmal1−/− mice, though corticosterone rhythms were preserved in the mice with disrupted circadian rhythms. Values are presented as mean ± SEM; n = 7–12

*p < 0.05 versus corresponding time point (one-way ANOVA with the Kruskal-Wallis test)

ND, no data

Although there was no clear rhythm in the hypoglyacemic response to insulin, recovery of blood glucose exhibited a robust circadian variation (Figure 2A), with an excessive rebound from the effects of insulin evident at subjective dawn (CT19 and CT25) (Figure 2A). Insulin caused a profound hypoglyacemic response, independent of clock time, in both Bmal1
−/− and Clockmut mice (Figure 2B). This response was more pronounced in the former, consistent with the comparative severity of the molecular and behavioral phenotypes between the Bmal1−/− and Clockmut animals (King et al. 1997; Bunger et al. 2000). Despite exacerbation of the hypoglycaemic response to insulin in the mutants, the counterregulatory responses of both corticosterone and glucagon were retained (Figure 2C and 2D).

Figure 2 Disruption of Genes in the Core Molecular Clock Alters the Response to Insulin
(A) Insulin tolerance (IT) was examined in WT mice on CT7, CT13, CT19, and CT25 at 30 min, 60 min, and 90 min after insulin injection (n = 12 per time point, *p < 0.01).

(B) IT was examined in WT (black line), Bmal1
−/− (blue line), and Clockmut mice (green line) at CT1 (i) and CT13 (ii) (n = 6–10, *p < 0.05, †p < 0.01, ††p < 0.001).

(C and D) Plasma levels of the counterregulatory hormones corticosterone (C) and glucagon (D) were assessed 60 min after insulin injection in Bmal1
−/− and Clockmut mice (n = 7, corticosterone assay; samples were pooled for glucagon assay, *p < 0.05).

Gluconeogenesis also contributes to restoration of blood glucose after insulin-induced hypoglycaemia. Consistent with this observation, conversion of exogenously administered pyruvate to glucose, which reflects gluconeogenesis (Miyake et al. 2002), was impaired in the Clockmut animals. This impairment was most marked in Bmal1−/− mice, while Bmal1+/− and Clockmut mice exhibited an intermediate phenotype when compared with WT littermate controls (Figure 3A). Furthermore, activity of the key rate-limiting enzyme of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), exhibited diurnal variation in the liver and aorta that was blunted in Clockmut mice (Figure 3B). PEPCK activity in kidney was antiphasic to the rhythm in aorta and liver and was unimpaired in Clockmut mice (Figure 3B), suggesting tissue-specific regulation of enzyme activity.

Figure 3 Impaired Gluconeogenesis in Mice with a Disrupted Circadian Clock
(A) Pyruvate tolerance was compared among WT (black line), Bmal1+/− (blue line), and Bmal1−/− (purple line), and Clockmut (green line) mice at CT7 (n = 6–10).

(B) Relative PEPCK activity (units are expressed as luciferase activity × 103) was measured in liver (i), aorta (ii), and kidney (iii) from WT (white bars) and Clockmut mice (green bars).

The frequent sampling intravenous glucose tolerance test (FSIGT) was performed to assess more precisely the impact of the molecular clock on sensitivity to insulin. This test provides an estimate of Si, consistent with that obtained by the euglycaemic clamp (Pacini et al. 2001). Additionally, data modeling provides estimates of glucose-mediated glucose disposal (Sg), insulin secretion, and Si. Si and insulin secretion, but not Sg, exhibited a diurnal variation in WT mice fed a regular chow diet (RC) (Table 2). Circadian variation of glucose and lipid homeostasis might condition the metabolic response to asynchronous environmental cues, such as diet, that impinge on Si. Dyslipidemia coincides with insulin resistance in the metabolic syndrome (Brotman and Girod 2002), and a diet high in fat impairs Si (Grundleger and Thenen 1982; Coulston et al. 1983). Both HF-fed WT and HF-fed Clockmut mice increased body weight significantly and to a similar degree in comparison to their age-matched, RC-fed controls (Table 3). Body fat composition averaged 17.6% of lean body mass in RC-fed WT mice, rising to 27.7% (p < 0.002) on high-fat feeding. Again, fat composition was not significantly altered by the presence of the Clock mutation (Table 3).

Table 2 Indices of Noninsulin- and Insulin-Mediated Parameters of Glucose Disposal Derived from Modelling of FSIGT Data at CT1 and CT13
Values are presented as mean ± fractional standard deviation (n = 6–7 per group)


a 
p < 0.001 versus HF group (two-tailed, Student's t-test)


b 
p < 0.001 versus respective group at CT1

G0, blood glucose concentration at 1 minute post-glucose bolus; ND, valid Si value could not be determined

Table 3 Body Mass Index Was Assessed in Mice Feeding on RC and HF
Values are presented as mean ± SEM; n = 13 per group for WT; n = 7 per group for subsequent parameters

*p < 0.05 compared to RC (two-tailed Student's t-test); **p < 0.01 (two tailed Student's t-test)

BMC, bone mineral content

Glucose tolerance on an RC trended towards an intolerant phenotype at CT1 versus CT13, but this difference did not attain significance (Figure 4A). This is consistent with the temporal variation in insulin secretion observed in RC-fed WT mice in the FSIGT experiment (Table 2). However, when the mice were fed HF for 2 mo, this glycaemic excursion at CT1 evoked by the environmental challenge was amplified and significant (two way analysis of variance [ANOVA]; F = 63.2, p < 0.001) (Figure 4A). Similarly, although the hypoglycaemic response to insulin was not different in mice fed regular chow at CT1 and CT13 (Figure 4B), the HF induced a significant temporal variation (Figure 4B). Thus, the impact of a high fat intake on carbohydrate metabolism in WTs includes an amplification of the diurnal variation in the response to both glucose and insulin. This coincides with a modest impairment in the ability to restore euglycaemia after insulin. A similar impairment resulting from a defect in gluconeogenesis has been observed in rats (Oakes et al. 1997). The mutants failed to exhibit a significant time-dependent variation in their response to glucose or insulin, again reminiscent of the RC-fed, WT phenotype (Figure 4).

Figure 4 The Molecular Clock Conditions HF-Induced Circadian Variation in Glucose Homeostasis
(A) Glucose tolerance (GT) in RC-fed WT mice (i), HF-fed WT mice (ii), and HF-fed Clockmut mice (iii).

(B) IT in RC-fed WT mice (i), HF-fed WT mice (ii), and HF-fed Clockmut mice (iii) at respective times (n = 6–8; *p < 0.05, †p < 0.01).

Mice were also subjected to extended high-fat feeding (11 mo versus 2 mo). Long-term hyperlipidemia is known to induce frank diabetes with impaired release of insulin (Johnson et al. 1990), in contrast to short-term, high-fat feeding, which increases release of insulin, but impairs the response to it (Linn et al. 1999). The extended HF impaired insulin secretion, reflected by its marked reduction to negative values in HF-fed WT mice (Table 2). The lack of insulin secretion resulted in calculated values of Si that were imperceptibly low (see Material and Methods). However, Sg, insulin secretion, and Si were restored to a WT phenotype in Clockmut mice that were also HF-fed for 11 mo (Table 2). This is, again, consistent with a role for the molecular clock in conditioning the response of glucose metabolism to the intake of dietary fat. Remarkably, mutation of the molecular clock protected against the development of frank diabetes caused by chronic high-fat feeding.

Discussion
Maintenance of blood glucose levels within a narrow range is critical to mammalian survival, and environmental cues can trigger appropriate tissue disposition of glucose through adaptive behaviors such as in hibernation (Castex and Hoo-Paris 1987) or the “fight-or-flight” response (Surwit et al. 1992). In this sense, glucose regulation is a fundamental and ancestral defence mechanism. Our studies suggest that Bmal1 and Clock, core components of the molecular clock, contribute substantially to regulation of recovery from the hypoglycaemic response to insulin. However, other mechanisms also impinge on this ancient adaptive response. Thus, impaired recovery from insulin-induced hypoglycaemia is observed in mice lacking proopiomelanocortin. These animals lack adrenal glands and melanocortins and exhibit a defective glucagon response to insulin-induced hypoglycaemia (Hochgeschwender et al. 2003). They contrast with the Bmal1
−/− and Clockmut mice, where the counterregulatory hormone response is unimpaired. Thus, steroids, epinephrine, and glucagon appear to facilitate recovery from insulin-induced hypoglycaemia in a manner distinct from, but complementary to, the molecular clock.

An assumption intrinsic to our studies is that the phenotypes revealed in the mutant mice are attributable to their function as core elements of the molecular clock. However, as trans-activators, both Clock and Bmal1 may have pleiotropic effects independent of the circadian clock that could impinge on metabolism. Several lines of evidence argue against this hypothesis. First, genes relevant to these metabolic phenotypes display circadian oscillations in their steady-state mRNA levels (Young et al. 2001; Oishi et al. 2003). In addition, the mRNA levels of many of these key proteins are phase-aligned with Per1 (e.g., Enolase 3, Pgam, Transketolase, Lipase, Lpl, Dgat1, Ppar alpha) or Per2 (e.g., Mod1, Lpl, Pepck, lipin 1) (unpublished data). In addition, many of their mRNAs are at lower levels in Bmal1
−/− (e.g., Mod1, Pepck, Enolase3, Pgam) (unpublished data), consistent with a direct role of Bmal1 in their transcription. Thirdly, these metabolic parameters are disrupted in both circadian mutants with the same rank order of potency as the locomotor activity phenotypes (Bmal1−/− > Clockmut). Thus, the most parsimonious interpretation is that the observed metabolic deficiencies in the Bmal1
−/− and Clockmut mice are due to their roles in the circadian clock, rather than to “off-clock” effects.

We observed that the impact of HF on glucose homeostasis was apparently to emphasize the role of the molecular clock. Diet has previously been shown to interact with peripheral clocks. Changes in feeding shift the circadian pattern of gene expression in the liver, but not in the master clock in the SCN (Damiola et al. 2000), demonstrating the importance of food as a cue to circadian control. Individual constituents of food could also provide discrete stimuli. For example, glucose alone can induce rhythmic gene expression in isolated fibroblasts (Hirota et al. 2002). Thus, dietary composition, the size and timing of a feed might all be expected to interact differentially with an underlying circadian regulation of metabolic control.

Alterations in dietary content, the availability of “fast food,” inactivity, and sociocultural factors have all been implicated in the emergence of the metabolic syndrome as a major challenge to the public health (Zimmet et al. 2001). However, while mechanistic integration of the diverse elements of the syndrome has proven elusive, our studies suggest that timing may influence the functional consequences of ingesting a caloric load.

Materials and Methods

Animals
Mice were acclimatized for 2 wk in 12 h light–12 h dark cycles before being subjected to a 36-h period of constant darkness followed by experimentation in darkness. Experimental chronology is measured in CT, subjective day beginning at 7 AM (CT0), and subjective night beginning at 7 PM (CT12).

Diet
WT and Clockmut mice were placed on an HF (Teklad, TD02435) and compared to age-matched WT mice on a regular chow diet (RC). Mice were on RC for 8 wk except for those subjected to FSIGT where they received RC for 11 mo. Body mass composition was measured by dual energy X-ray absorptiometry at 10 mo.

Intraperitoneal tolerance
Tests were performed as described (Klaman et al. 2000) with a diminution in the glucose bolus (0.1 g/kg).

Intravenous glucose tolerance test and minimal modeling
The tolerance test was performed as described (Pacini et al. 2001) in unanesthetized mice, and the minimal model of Bergman et al. (1979) was applied to the data using MINMOD software (Boston et al. 2003). The derived values were Si, Sg, and acute insulin response to glucose, which measures insulin secretion. Si is the ratio of insulin delivery rate to the interstitium to insulin extraction rate from the interstitium. Long-term feeding of HF to WT mice resulted in imperceptibly small insulin sensitivity values. This could be the consequence of impaired delivery of insulin to the interstitium, exacerbated extraction rate, or a combination of both factors. Insulin secretion is derived from area under the insulin curve, above basal, from 0 to 10 min after glucose infusion; and disposition index, which equals the product of insulin sensitivity multiplied by insulin secretion and measures the degree to which insulin sensitivity can be compensated for by elevated insulin secretion (Pacini et al. 2001).

Assay methods
Insulin, leptin, corticosterone, and glucagon levels were measured by immunoassays from Crystalchem (Downers Grove, Illinois, United States), ICN Biochemicals (Costa Mesa, California, United States), and Linco Research (St. Charles, Missouri, United States). Plasma glucose was measured by the glucose oxidase method using a glucose analyzer machine for FSIGT and by glucometer for the intraperitoneal tolerance test. PEPCK activity was quantitated by a bioluminescent method (Wimmer 1988).

Statistical analysis
The significance of differences amongst the tolerance test curves was assessed by distribution-free two-way ANOVA with a Bonferroni correction. FSIGT data were tested by one-way ANOVA with the Kruskal-Wallis test. Paired Student's t-tests were used to perform comparisons of corticosterone levels before and after insulin injection in Bmal−/− mice and between WT and Clockmut mice. Plasma samples for glucagon analysis were pooled and were thus not compared by a formal statistical analysis. Results are presented as mean ± standard error of the mean (SEM), except for the FSIGT data (Table 2), presented as mean ± fractional standard deviation. Differences were considered significant when p < 0.05.

We wish to thank Christopher A. Bradfield and Celeste Simon for graciously providing MOP3 knockout mice and Joseph S. Takahashi and Amita Sehgal for providing Clock mutant mice. We also thank the Penn Diabetes Center/Mouse Phenotyping, Physiology, and Metabolism Core (NIH P30DK19525). We are indebted to Daniel J. Rader, Karen Teff, and Mitch Lazar for their helpful suggestions. Finally, we wish to thank Darko Stefanovski, Margaret Lucitt, and Eileen Clark for technical assistance. Supported in part by grants from the National Institutes of Health (HL 62250 and HL 54500) and fellowship grants from the American Heart Association to RDR (0225478U) and to PM (0160504U). GAF is the Robinette Foundation Professor of Cardiovascular Medicine and the Elmer Bobst Professor of Pharmacology.


Conflicts of interest. The authors have declared that no conflicts of interest exist.


Author contributions. RDR, PM, and GAF conceived and designed the experiments. RDR, PM, AMC, and SP performed the experiments. RDR, RB, and JH analyzed the data. RB and JH contributed reagents/materials/analysis tools. RDR wrote the paper.

Academic Editor: Steve O'Rahilly, University of Cambridge

Citation: Rudic RD, McNamara P, Curtis AM, Boston RC, Panda S, et al. (2004) BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis. PLoS Biol 2(11): e377.

Abbreviations
Bmal1−/−
Bmal knockout mice

Clockmut
Clock mutant mice

CT[number]circadian time [designated time point]

FSIGTfrequent sampling intravenous glucose tolerance test

GTglucose tolerance

HFhigh-fat diet

ITinsulin tolerance

PEPCKphosphoenolpyruvate carboxykinase

RCregular chow diet

SCNsuprachiasmatic nucleus

Sgglucose-mediated glucose disposal

Siinsulin sensitivity

WTwild-type
==== Refs
References
Akhtar RA  Reddy AB  Maywood ES  Clayton JD  King VM    Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus Curr Biol 2002 12 540 550 11937022 
Albrecht U  Eichele G   The mammalian circadian clock Curr Opin Genet Dev 2003 13 271 277 12787789 
Bergman RN  Ider YZ  Bowden CR  Cobelli C   Quantitative estimation of insulin sensitivity Am J Physiol 1979 236 E667 677 443421 
Boston RC  Stefanovski D  Moate PJ  Sumner AE  Watanabe RM    MINMOD millennium: A computer program to calculate glucose effectiveness and insulin sensitivity from the frequently sampled intravenous glucose tolerance test Diabetes Technol Ther 2003 5 1003 1015 14709204 
Brotman DJ  Girod JP   The metabolic syndrome: A tug-of-war with no winner Cleve Clin J Med 2002 69 990 994 12546272 
Bunger MK  Wilsbacher LD  Moran SM  Clendenin C  Radcliffe LA    Mop3 is an essential component of the master circadian pacemaker in mammals Cell 2000 103 1009 1017 11163178 
Castex C  Hoo-Paris R   Regulation of endocrine pancreas secretions (insulin and glucagon) during the periodic lethargy-waking cycle of the hibernating mammal Diabete Metab 1987 13 176 181 3301441 
Coulston AM  Liu GC  Reaven GM   Plasma glucose, insulin and lipid responses to high-carbohydrate low-fat diets in normal humans Metabolism 1983 32 52 56 6336816 
Cryer PE   Glucose counterregulation: Prevention and correction of hypoglycemia in humans Am J Physiol 1993 264 E149 155 8447379 
Damiola F  Le Minh N  Preitner N  Kornmann B  Fleury-Olela F    Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus Genes Dev 2000 14 2950 2961 11114885 
Deboer T  Vansteensel MJ  Detari L  Meijer JH   Sleep states alter activity of suprachiasmatic nucleus neurons Nat Neurosci 2003 6 1086 1090 12958601 
Gagliardino JJ  Hernandez RE   Circadian variation of the serum glucose and immunoreactive insulin levels Endocrinology 1971 88 1532 1534 5572981 
Grundleger ML  Thenen SW   Decreased insulin binding, glucose transport, and glucose metabolism in soleus muscle of rats fed a high fat diet Diabetes 1982 31 232 237 6759239 
Hastings MH  Reddy AB  Maywood ES   A clockwork web: Circadian timing in brain and periphery, in health and disease Nat Rev Neurosci 2003 4 649 661 12894240 
Herzog ED  Takahashi JS  Block GD   Clock controls circadian period in isolated suprachiasmatic nucleus neurons Nat Neurosci 1998 1 708 713 10196587 
Hirota T  Okano T  Kokame K  Shirotani-Ikejima H  Miyata T    Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts J Biol Chem 2002 277 44244 44251 12213820 
Hochgeschwender U  Costa JL  Reed P  Bui S  Brennan MB   Altered glucose homeostasis in POMC null mouse mutants lacking central and peripheral melanocortin Endocrinology 2003 144 5194 5202 12970157 
Ibuka N  Inouye SI  Kawamura H   Analysis of sleep-wakefulness rhythms in male rats after suprachiasmatic nucleus lesions and ocular enucleation Brain Res 1977 122 33 47 837222 
Ibuka N  Nihonmatsu I  Sekiguchi S   Sleep-wakefulness rhythms in mice after suprachiasmatic nucleus lesions Waking Sleeping 1980 4 167 173 7190752 
Johnson JH  Ogawa A  Chen L  Orci L  Newgard CB    Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes Science 1990 250 546 549 2237405 
King DP  Zhao Y  Sangoram AM  Wilsbacher LD  Tanaka M    Positional cloning of the mouse circadian clock gene Cell 1997 89 641 653 9160755 
Kita Y  Shiozawa M  Jin W  Majewski RR  Besharse JC    Implications of circadian gene expression in kidney, liver and the effects of fasting on pharmacogenomic studies Pharmacogenetics 2002 12 55 65 11773865 
Klaman LD  Boss O  Peroni OD  Kim JK  Martino JL    Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice Mol Cell Biol 2000 20 5479 5489 10891488 
la Fleur SE  Kalsbeek A  Wortel J  Fekkes ML  Buijs RM   A daily rhythm in glucose tolerance: A role for the suprachiasmatic nucleus Diabetes 2001 50 1237 1243 11375322 
Linn T  Strate C  Schneider K   Diet promotes beta-cell loss by apoptosis in prediabetic nonobese diabetic mice Endocrinology 1999 140 3767 3773 10433238 
Maeda N  Shimomura I  Kishida K  Nishizawa H  Matsuda M    Diet-induced insulin resistance in mice lacking adiponectin/ACRP30 Nat Med 2002 8 731 737 12068289 
Malherbe C  De Gasparo M  De Hertogh R  Hoet JJ   Circadian variations of blood sugar and plasma insulin levels in man Diabetologia 1969 5 397 404 5372892 
Marquez S  Crespo P  Carlini V  Garbarino-Pico E  Baler R    The metabolism of phospholipids oscillates rhythmically in cultures of fibroblasts and is regulated by the clock protein PERIOD 1 FASEB J 2004 18 519 521 14715703 
Matsuo T  Yamaguchi S  Mitsui S  Emi A  Shimoda F    Control mechanism of the circadian clock for timing of cell division in vivo Science 2003 302 255 259 12934012 
McNamara P  Seo SP  Rudic RD  Sehgal A  Chakravarti D    Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: A humoral mechanism to reset a peripheral clock Cell 2001 105 877 889 11439184 
Miller BH  Olson SL  Turek FW  Levine JE  Horton TH    Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy Curr Biol 2004 14 1367 1373 15296754 
Miyake K  Ogawa W  Matsumoto M  Nakamura T  Sakaue H    Hyperinsulinemia, glucose intolerance, and dyslipidemia induced by acute inhibition of phosphoinositide 3-kinase signaling in the liver J Clin Invest 2002 110 1483 1491 12438446 
Mosko S  Moore RY   Retinohypothalamic tract development: Alteration by suprachiasmatic lesions in the neonatal rat Brain Res 1979 164 1 15 427554 
Oakes ND  Cooney GJ  Camilleri S  Chisholm DJ  Kraegen EW   Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding Diabetes 1997 46 1768 1774 9356024 
Oishi K  Miyazaki K  Kadota K  Kikuno R  Nagase T    Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes J Biol Chem 2003 278 41519 41527 12865428 
Pacini G  Thomaseth K  Ahren B   Contribution to glucose tolerance of insulin-independent vs. insulin-dependent mechanisms in mice Am J Physiol Endocrinol Metab 2001 281 E693 703 11551845 
Panda S  Antoch MP  Miller BH  Su AI  Schook AB    Coordinated transcription of key pathways in the mouse by the circadian clock Cell 2002 109 307 320 12015981 
Reppert SM  Weaver DR   Coordination of circadian timing in mammals Nature 2002 418 935 941 12198538 
Schlierf G  Dorow E   Diurnal patterns of triglycerides, free fatty acids, blood sugar, and insulin during carbohydrate-induction in man and their modification by nocturnal suppression of lipolysis J Clin Invest 1973 52 732 740 4685092 
Seaman GV  Engel R  Swank RL  Hissen W   Circadian periodicity in some physicochemical parameters of circulating blood Nature 1965 207 833 835 5885941 
Stokkan KA  Yamazaki S  Tei H  Sakaki Y  Menaker M   Entrainment of the circadian clock in the liver by feeding Science 2001 291 490 493 11161204 
Storch KF  Lipan O  Leykin I  Viswanathan N  Davis FC    Extensive and divergent circadian gene expression in liver and heart Nature 2002 417 78 83 11967526 
Surwit RS  Schneider MS  Feinglos MN   Stress and diabetes mellitus Diabetes Care 1992 15 1413 1422 1425110 
Vitaterna MH  King DP  Chang AM  Kornhauser JM  Lowrey PL    Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior Science 1994 264 719 725 8171325 
Welsh D  Richardson GS  Dement WC   Effect of running wheel availability on circadian patterns of sleep and wakefulness in mice Physiol Behav 1988 43 771 777 3237790 
Wimmer M   A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples Anal Biochem 1988 170 376 381 3394934 
Yamauchi T  Kamon J  Waki H  Terauchi Y  Kubota N    The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity Nat Med 2001 7 941 946 11479627 
Young ME  Razeghi P  Cedars AM  Guthrie PH  Taegtmeyer H   Intrinsic diurnal variations in cardiac metabolism and contractile function Circ Res 2001 89 1199 1208 11739286 
Young MW  Kay SA   Time zones: A comparative genetics of circadian clocks Nat Rev Genet 2001 2 702 715 11533719 
Zimmet P  Alberti KG  Shaw J   Global and societal implications of the diabetes epidemic Nature 2001 414 782 787 11742409

