DOI 10.1515/hmbci-2013-0011      Horm Mol Biol Clin Invest 2013; 14(1): 15–24

Katherine C. Krueger and Brian J. Feldman*

Adipose circadian clocks: coordination of metabolic rhythms by clock genes, steroid hormones, and PPARs Abstract: A central clock consisting of interconnected positive and negative feedback gene loops operates in the brain, tying rhythmic activity to the 24-h day. The central clock entrains similar feedback loops present in most peripheral tissues to coordinate metabolic gene expression among organs and with feeding activity for more efficient utilization of resources. Recent studies are beginning to elucidate the intricate feedback mechanisms among central and peripheral clocks and their roles in activity and metabolic homeostasis. Adipose tissue serves as a major energy storage organ and releases paracrine and endocrine hormones to signal energy status to other organs. Within the adipose tissue, the transcriptional feedback regulation between clock genes and nuclear hormone receptors, together with direct protein associations among these molecules, ensures the expression of metabolic genes at the appropriate time. This review will summarize the important components and mechanisms of adipose clock entrainment, particularly highlighting instructive studies carried out in mice. This research not only illustrates the intricate connections between clocks and metabolism but also provides potential mechanisms to correct abnormalities induced by disrupted sleep or poor diet. Keywords: adipose tissue; circadian clock; circadian rhythm; cytoplasmic and nuclear; metabolism; receptors. *Corresponding author: Brian J. Feldman, Department of Pediatrics, Division of Endocrinology, School of Medicine, Stanford University, Lokey Stem Cell Research Building, MC5457, Stanford, CA 94305, USA, Phone: +1-650-723-5791, Fax: +1-650-725-8375, E-mail: [email protected] Katherine C. Krueger: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA

Introduction The circadian clock coordinates gene expression with daily activities and is necessary to ensure efficient utilization of

nutritional resources [1]. A central clock exists in the brain, and it communicates with and entrains numerous peripheral clocks [2]. The links between core clock function and metabolism are many: mice and humans have circadian cycles of glucose, insulin, fatty acids, and metabolites; metabolic disturbances are common in mice with mutations in core clock genes; mouse models of obesity, either genetic or diet-induced, show altered behaviors that are regulated by the central clock, such as overall activity and feeding patterns; and sleep disturbances, which also impact the clock, result in altered feeding patterns and metabolic abnormalities. This review will focus on recent studies that further elucidate the relationships between central clock abnormalities and metabolic derangements. In particular, mouse models of clock or metabolic disruption will be reviewed because the genetic tractability of this model allows insight into the role of tissue-specific clocks in this process, including adipose clocks. The mechanistic evidence of clock function in peripheral tissues to regulate gene expression in metabolically active tissues will be discussed, particularly those tasks specific to adipose tissue. Finally, the roles of steroid hormones and peroxisome proliferator-activated receptor proteins (PPARs), which act in concert with clock genes to direct tissue-specific gene expression patterns, will be reviewed.

Adipose tissue function Adipose tissue is a specialized connective tissue composed of adipocytes, which store lipid; adipocyte precursor cells called preadipocytes; endothelial cells, immune cells, and fibroblasts [3]. There are two types of adipose tissue, brown and white. Brown adipose tissue (BAT) has fundamentally different structure and function than white adipose tissue (WAT). BAT adipocytes have many lipid droplets, high concentration of mitochondria, and metabolize lipids to generate heat for thermoregulation [4]. WAT adipocytes have one large lipid droplet for storing energy when nutrients are in excess but release lipids when energy intake is low. Adipose tissue also acts as an endocrine organ, releasing

16      Krueger and Feldman: Adipose circadian clocks and metabolism endocrine and paracrine hormones (adipokines) to communicate with other organs [5]. Some of these, such as leptin and adiponectin (adipoQ) are adipose-specific, so their release and downstream effects are output indicators of adipose function. Adipose tissue can dramatically alter its size in response to energy intake. In pathologic states such as obesity, adipose tissue secretes a number of proinflammatory cytokines, such as tumor necrosis factor a and interleukin 6, resulting in increased macrophage recruitment to adipose tissue. Together, these impact insulin signaling and promote insulin resistance. Adipose tissue is widely distributed in the body and is associated with many internal organs. Most studies in mice focus on perigonadal WAT, which is attached to the epididymis in males or uterus and ovaries in females, and is the largest single depot. However, several other WAT depots are present in the peritoneal cavity and are associated with the kidney (perirenal and retroperitoneal), stomach and spleen (omental), and intestines (mesenteric). Subcutaneously, the largest WAT depot is the inguinal fat pad, but additional WAT is associated with the most prominent BAT depot, located between the scapulae (interscapular WAT and BAT, respectively).

Central and peripheral clocks The core circadian clock is located in the superchiasmatic nucleus (SCN) of the hypothalamus in the brain and consists of interconnecting feedback loops that are regulated at the transcriptional and post-translational levels [2]. The positive arm includes Clock and Bmal1, which are E-box transcription factors. Together, these induce the transcription of components of the negative arm, Period (Per) 1–3 and Cryptochrome (Cry) 1–2 genes. Per and Cry proteins form a complex that translocates into the nucleus to repress the transcription of Clock and Bmal1 (and therefore Pers and Crys as well). An additional regulatory loop involves the nuclear hormone receptors (NRs) of the REV-ERB and ROR families, which also repress Clock and Bmal1. The phosphorylation status of clock proteins shows temporal variation and affects nuclear vs. cytoplasmic localization, the formation of protein complexes, their stability, and transcriptional activity [6]. Together, these processes help the central clock retain rhythmicity even in constant darkness, but the period is just under 24 h. Therefore, daily re-entrainment with light is necessary to coordinate with the 24-h day length. Strikingly, these same core clock genes are also expressed in peripheral tissues and oscillate at the RNA

and protein level with circadian periodicity. However, unlike the central clock, the ability of peripheral clocks to maintain their synchronous activity in culture is limited to a few days in the absence of further input. In vivo, the central clock communicates with peripheral clocks using a variety of signaling molecules, including glucocorticoids (GCs), but peripheral oscillators are also strongly entrained by feeding. A large percentage of the transcriptome in peripheral tissues, representing some 5000 genes, oscillates in expression with respect to circadian time [7]. Molecularly, this is carried out by the rhythmic binding of clock genes to specific genomic DNA sites. Interestingly, in the liver, core clock proteins bind mainly to genes involved in metabolic pathways and insulin signaling [8]. Circulating levels of triglycerides, free fatty acids, glucose, and insulin vary in a circadian fashion [9]. The core clock genes oscillate in adipose tissue, and the release of the leptin also follows a circadian pattern [7, 9].

Influences of central clock on metabolism Metabolic abnormalities in mouse circadian mutants Many circadian clock mutant mice display metabolic abnormalities, including changes to adiposity. For instance, mice with deletion of the Clock gene are obese and exhibit many symptoms of metabolic syndrome [10]. One targeted mutation of Bmal1 [11] generated mice that are obese, particularly after high-fat diet (HFD) [12–15], whereas a different Bmal1 mutation shows decreased adiposity even after HFD [16]. Neither Clock nor Bmal1 mutant mice show the expected circadian variation in glucose and insulin levels [14, 17]. Likewise, depending on the mutation, Per2 knockout (KO) mice are either obese [18] or have reduced body weight [19] relative to controls. Per3 KO mice have increased percent fat compared with wildtype (WT) controls on standard mouse diet [20]. Per1/2/3 KO, Per3 KO [21], and Cry1/2 KO [22] mice gain more weight on HFD. Cry1/2 KO mice are additionally hyperglycemic and hyperinsulinemic following HFD [22]. Mice with mutations in the orphan nuclear receptor REV-ERBα are hyperglycemic, and their insulin levels fail to follow a circadian pattern; metabolic profiling indicates abnormal utilization of lipids over glucose for energy [23].

Krueger and Feldman: Adipose circadian clocks and metabolism      17

Tissue-specific manipulations of clock gene expression To help tease apart the functions of circadian genes in the central vs. peripheral oscillators, several laboratories have either rescued circadian gene expression to selective tissues or removed genes from specific tissues. For instance, the greater importance of Clock in the central oscillator was revealed by the selective restoration of this gene to the central nervous system and adrenal gland [24]. Centrally mediated behaviors, such as overall activity, were rescued even under constant dark conditions. In the liver, where Clock remained deleted, the cycling and phase of gene expression were partially restored, particularly with respect to the core clock genes. However, the amplitude of highly oscillating genes was not fully restored, and the oscillations of ultradian rhythmic genes (genes that oscillate in 8- to 12-h cycles) switched to 24-h oscillations. Thus, the liver relies heavily on the central clock for 24-h oscillations but has evolved additional systems that rely upon its own clock for more specialized, ultradian gene expression [24]. Several tissue-specific KOs of Bmal1 have been generated. Liver-specific KOs have disruptions to many genes relevant for hepatic functions, including glucose release metabolism [12]. A pancreas-specific KO of Bmal1 resulted in similar phenotypes as the global Bmal1 KO, including persistently elevated glucose levels, impaired glucose tolerance, and reduced insulin function [17]. Interestingly, these phenotypes occurred at a younger age in conditional KO (CKO) mice as compared with the global KO, likely reflecting a lack of compensatory expression in CKO [17]. Deletion of Bmal1 in adipose tissue effectively abrogates clock gene expression in both white and brown fat [13]. These CKO mice have increased sensitivity to HFD as well as altered circadian profiles of glucose and triglycerides. Bmal1 CKOs have an attenuated feeding rhythm, which is consistent with altered expression of hypothalamic peptides that regulate feeding behavior in these mice. Gene microarray studies revealed decreased expression of two Bmal1 target genes, Elovl6 and Scd1, which are involved in the synthesis of polyunsaturated fatty acids. These fatty acids have been shown to cross the blood-brain barrier, signal within the hypothalamus, and alter feeding behavior [25]. Consistent with this hypothesis, Bmal1 CKO mice fed a diet with increased polyunsaturated fatty acids no longer showed differences in body weight, feeding behavior, and circadian expression of hypothalamic peptides [13]. Thus, the circadian clock in adipose tissue is integrated with mechanisms to relay the status of energy stores to the central clock, which may provide a mechanism to ensure stable energy homeostasis. The emergence of these

phenotypes in adipose-specific Bmal1 KOs, but not liveror pancreas-specific KOs, further underscores the specific importance of the adipose clock in energy homeostasis.

Disrupted sleep and metabolism Disruption of sleep, as part of shift work or jet lag, has negative health consequences. Mice that are prevented from sleeping for a portion of their inactive (light) phase have dampened clock gene rhythms in the liver [26]. Microarray analysis of the liver transcriptome revealed a large number of genes with reduced peak expression or diurnal rhythmicity, many of which are related to metabolic processes. Food intake during the inactive phase also increases and remains elevated even after the cessation of the sleep disruption protocol. In WT mice, this protocol results in major reprogramming of adipose gene expression, including both clock and metabolic gene expression changes [27]. The pattern of gene expression changes suggests a shift from glucose to lipid storage that persists after normal sleep was restored [27]. However, if mice are allowed to feed only during the active phase, sleep disruption during the inactive phase did not result in clock gene expression and metabolic disturbances [26]. Thus, the effects of sleep disruption are separable from peripheral entrainment cues such as food availability (see below). Interestingly, the same sleep-deprivation protocol carried out in Per1/2 double KO mice results in much milder effects on overall activity, food intake, and clock gene expression in adipose tissue [27].

Feedback between peripheral clock and central clock Entrainment mechanisms Peripheral oscillators are entrained by a number of stimuli, including GCs, food, and other circulating factors. Limiting food availability to the light phase also shifts circadian clock gene expression in adipose and liver tissues [7], and peak activity levels [28] are shifted to the light phase. Food consumption may alter clock gene expression through adenosine monophosphate kinase (AMPK) [29]. AMPK is a cellular energy sensor and activates the metabolism of glucose and fatty acids when levels of ATP are low. In the liver, AMPK oscillates between the nuclear and cytoplasmic compartments in a circadian fashion and antiphase to Cry1. AMPK can phosphorylate and destabilize Cry1, which may provide a mechanism by which information about

18      Krueger and Feldman: Adipose circadian clocks and metabolism nutritional status is coordinated with circadian clock function. Indeed, loss of AMPK leads to the stabilization of Cry1 and loss of circadian rhythmicity in liver [29].

Obesity-induced gene expression changes Mice fed a HFD have an increased period of free running as well as altered feeding rhythm, both of which precede significant weight gain [30]. Although total activity and caloric intake remained the same, the levels of both activities were abnormally increased during the light phase. Circadian gene expression in the hypothalamus was unaffected by HFD, suggesting central clock oscillations are unaffected. However, the amplitude of clock gene cycling was dampened modestly in the liver and more dramatically in WAT. In addition, leptin and glucose were chronically increased, whereas insulin and free fatty acids were specifically increased during the dark period. Within WAT, the levels of several NRs, including RORα, RXRα, and PPARγ, had decreased expression and reduced diurnal variation. Intriguingly, the expression of these same genes was abnormally increased in liver. Thus, HFD has opposite effects on certain genes in WAT and liver, which may underlie the metabolic abnormalities observed [30]. Clearly, although perturbation of the central clock has the ability to disrupt metabolism, these studies demonstrate that disrupted metabolism by HFD also feeds back and alters centrally mediated behaviors. Genetically obese mice (ob/ob), which lack functional leptin, have normal circadian gene expression in the SCN but reduced clock oscillations in the liver and WAT [31]. However, at 3 weeks of age, before the onset of obesity or metabolic changes, Per1, Per2, and Cry1 levels are already decreased in WAT. Injection of leptin for 7 days improved several metabolic parameters and body weight measures and restored the cycling of several circadian genes, particularly in the liver. Thus, circadian effects precede metabolic perturbations in ob/ob mice, but the correction of the leptin deficiency has the ability to restore circadian function and improve other metabolic measures [31].

Restricted feeding The timing of feeding may be more important than the fat content or even caloric content of food consumed. If food is restricted to the light phase, the phases of energy expenditure, clock gene expression, and metabolic gene expression are dramatically shifted, in comparison to mice in which food is restricted to the dark phase [32]. These

effects on clock and metabolic gene expression were relatively specific for the liver and WAT, with milder effects observed in muscles and the heart. Light phase-fed mice take in more calories but have lower energy expenditures and have a higher respiratory exchange ratio during the day, suggestive of decreased fatty acid oxidation. Thus, feeding during the phase in which mice are normally inactive leads to dyssynchrony of peripheral clocks and detrimental effects on metabolism [32]. Mice that were fed an isocaloric HFD but were only allowed to feed during the dark phase were protected against a large number metabolic and gene expression abnormalities that normally occur under ad libitum HFD conditions [33]. In particular, excessive weight gain, adipocyte hypertrophy, and inflammation of WAT were greatly attenuated with the restricted feeding regimen, despite equivalent caloric intake.

Circadian functions of adipokines Leptin The adipokine leptin shows circadian oscillations under normal feeding conditions, increasing after food intake and decreasing during fasting. Mice that lack this molecule are obese, hyperphagic, hyperglycemic, and hyperinsulinemic, but some phenotypes can be rescued with leptin treatment [31]. As mentioned above, restricted feeding to suboptimal times, such as the light phase in mice, results in shifted clock gene expression and metabolic dysregulation in WT mice. Interestingly, this phenotype is not observed in leptin-deficient mice, which fail to gain excess weight under this regimen [34]. Moreover, continuously applied exogenous leptin has no effect, but a timed pulse during the light phase does lead to weight gain [34]. Thus, oscillations in leptin levels are essential to its function and have critical implications for the regulation of metabolism. Leptin signals through its receptor, LepR, which is expressed in many tissues, including the brain, liver, and muscle. Exogenous leptin is able to phase advance the central clock, which is likely due to the expression of LepR on SCN neurons. LepR KO mice (also known as db/db) show altered expression of clock genes in WAT [35]. Consistent with leptin’s role in modulation of metabolism, the functions of AMPK and Sirt1 are also impaired. Together, these results suggest that leptin is responsive to circadian rhythms as well as metabolic status and also feeds back to influence the clock.

Krueger and Feldman: Adipose circadian clocks and metabolism      19

Adiponectin AdipoQ is an anti-inflammatory adipokine involved in the metabolism of both glucose and insulin, and the circulating levels of adipoQ are inversely correlated with adipose tissue mass. When released, it binds to its receptors AdipoQR1 and R2, which are expressed in a variety of tissues, including WAT, to increase sensitivity to insulin via activation of AMPK and PPARα [36, 37]. Conversely, adipoQ KO mice are diabetic and have insulin resistance [38, 39]. AdipoQ is not released in a circadian rhythm in mice [9] but in a genetic model of diabetes and obesity [40], the restoration of adipoQ in the liver rescued the overall activity levels and liver circadian gene expression [41].

Role of steroid hormones and ­interactions with clock genes NRs play critical roles in directing metabolic gene expression programs in response to hormonal or lipid stimuli [42]. Among the metabolically active tissues, NRs are widely expressed, and at least 28 have circadian patterns of expression in liver, muscle, BAT, and WAT [43]. Moreover, some NRs oscillate in certain tissues but not others, which could imply different functions based on this cycling pattern in different tissues. For instance, PPARα, PPARγ, and PPARδ all oscillate in the liver. However, in fats, PPARα oscillates in both WAT and BAT, whereas PPARγ oscillates only in WAT and PPARδ only in BAT [43].

Glucocorticoids GCs are potent signaling molecules that regulate a variety of processes through binding to the GC receptor (GR). GCs are released from the adrenal gland in a circadian pattern, but they can also be induced by stress. These are separable mechanisms because Per2 KO mice have no circadian rhythm of corticosterone, the major GC in mice, but have normal responses to stress [18]. GCs are thought to be important entrainers of peripheral clocks, due to the wide expression of GR and the observation that synthetic GCs, such as dexamethasone or hydrocortisone, can synchronize clocks in vitro and in vivo [44, 45]. Moreover, adrenalectomy delays the phase of a clock reporter, Per1-luc, in several peripheral tissues, including the liver, whereas the SCN was unaffected [45]. GR directly binds to and

modulates Per2 regulatory elements in cultured cells, and Per2 KO mice are resistant to GC-induced metabolic abnormalities [46]. GR also interacts with Cry1 and Cry2, which serves to limit GR-regulated transcription in the context of gluconeogenesis and may affect negative feedback on GC release but does not appear to affect immunosuppression functions of GR [47].

PPARα The transcription factor PPARα plays a critical role in the liver for lipid metabolism under conditions of low energy. Several studies have demonstrated the importance of PPARα in circadian gene expression of metabolically active tissues. Clock [48] and Bmal1 [49] KO mice have reduced expression of PPARα in the liver. In clock KOs, this results in reduced or abrogated circadian oscillations in lipogenic gene expression in the liver [48]. Interestingly, although PPARα KO mice have normal clock gene expression in the SCN, oscillations of several clock genes are altered in the liver [49]. The altered expression of Per2, but not Bmal1, can be rescued by restricted feeding. A PPARα activator, fenofibrate, is able to generate synchronized Bmal1 oscillations in the liver of WT mice, but not in PPARα KO mice, perhaps through a PPAR response element upstream of Bmal1 [49]. Moreover, Per2 enhances PPARα-mediated transactivation of a Bmal1 reporter in a dose-dependent fashion [50]. In fact, Per2 protein associates with a number of NRs, including PPARα and REVERBα, and evidence suggests that Per2 directly modulates expression of NRs as well as their downstream genes [50]. The importance of these interactions deserves further study, but one hypothesis suggests input from clock genes helps amplify oscillations of NRs for more controlled regulation of target genes [50].

PPARγ PPARγ is a master regulator of adipogenesis [51, 52], and global KOs are embryonic lethal [53]. However, PPARγ conditional mice, in which PPARγ is deleted in all tissues in an acute fashion, have been generated [54]. These mice have altered metabolic rhythms but normal body weight. Gene expression studies reveal a preferential alteration to adipose and liver gene expression, with largely unaltered expression in muscle or the hypothalamus [54]. In a fibroblast cell line and primary preadipocytes, an endogenous ligand to PPARγ, 15d-PGJ2, can induce circadian gene cycling [54, 55]. This ligand occurs naturally and is

20      Krueger and Feldman: Adipose circadian clocks and metabolism excreted in urine with diurnal variation [55]. However, PPARγ-deficient preadipocytes have severely blunted circadian cycling. Together, these findings suggest a candidate mechanism by which PPARγ contributes to clock oscillations within adipose tissue.

Pgc1α

was not explored in this study, it seems plausible that a large number of genes dysregulated in ERRα KO mice are also regulated by components of the circadian clock.

Potential noncircadian functions of clock genes in metabolic regulation

The PPARγ coactivator, Pgc1α, is an important regulator of energy metabolism but can also induce several clock genes, including Bmal1, Clock, and REV-ERBα in several cell lines. The Bmal1 promoter contains a ROR response element that is bound by Pgc1α, which acts synergistically with RORα/γ to stimulate Bmal1 promoter activity. In Pgc1α KO mice, clock oscillations and expression of metabolic genes are altered [56]. Moreover, if Pgc1α is knocked down specifically in immortalized brown fat cells, the rhythmic expression of genes is abrogated. However, similar experiments in the SCN showed no disruption to circadian gene expression. Thus, Pgc1α appears to function mainly in peripheral tissues for clock gene oscillations. Interestingly, an adipose-specific KO of Pgc1α shows altered glucose homeostasis, insulin resistance, and lipid metabolism after HFD [57]. Whether clock gene expression was also altered in this model was not determined.

Per2

ERRα

Per3

The estrogen-related receptor α (ERRα) plays a critical role in energy metabolism, mainly through interactions with Pgc coativators (reviewed in [58]). ERRα is not expressed in the SCN, but has rhythmic expression in the liver that is lost in Clock mutants under constant darkness [59]. The oscillatory expression of ERRα and several core clock genes is attenuated in fasted mice, suggesting these genes are downstream of feeding. ERRα is induced by ClockBmal1 and is able to bind to response elements in many clock genes. Moreover, ERRα binds to a large number of target genes that are also bound by Bmal1, mostly genes involved in metabolic processes such as insulin receptor signaling and glycolysis/gluconeogenesis. Many of these targets are downregulated in ERRα KO mice, resulting in altered glucose homeostasis and levels of circulating metabolites [59]. Interestingly, ERRα KO mice have reduced adipose mass, adipocyte size, and are resistant to diet-induced obesity [60]. Consistent with these findings, gene expression profiling revealed imbalances in enzymes regulating lipid metabolism and lipogenesis. Although the expression of clock genes in relation to ERRα target genes

Per3 KO mice exhibit only subtle alterations to behavior, indicating a minor role in the central clock oscillator. Examination of peripheral clocks in Per3 KO mice revealed altered periods and phases in several tissues, with the most noticeable changes to the pituitary, liver, and lung [61]. However, although the phase was advanced in epididymal WAT, no effect was observed in perirenal WAT. Furthermore, clock oscillations were unaltered in cultured bone marrow mesenchymal stem cells of Per3 KO mice, yet these cells show increased adipogenic activity [20]. Per3 KO mice have increased adiposity, but they have similarsize adipocytes, suggesting an in vivo increase in adipogenesis as well. Notably, these mice also show decreased glucose tolerance. Similar to Per2, Per3 also interacts with PPARγ to repress PPARγ-dependent transcription and adipogenesis [20]. Thus, Per3 may have actions that are either independent or downstream of the circadian clock in modulating adipogenesis. Future studies should address whether these phenotypes are due to the lack of Per3 specifically in WAT or a feedback response due to misalignment of various peripheral clocks in Per3 KO mice [61].

Per2 KO mice have smaller fat pads than WT littermates, a higher metabolic rate, and lower levels of circulating lipids [19]. However, two circadian genes, Dbp and Per1, have normal circadian oscillations in WAT, suggesting that these metabolic disruptions may be separate from altered circadian cycling. In support of this hypothesis, PPARγ interacts with Per2 but not Clock, Bmal1, Per1, or Cry1, and Per2 decreases PPARγ-dependent transcription independently of Cry1 expression. Moreover, the expression of Clock-Bmal1 target genes remains circadian in Per2 KO mice, but the phase and amplitude of PPARγ targets are altered. These findings suggest a model in which Per2 acts outside the circadian clock to specifically regulate PPARγ transcriptional activity within WAT [19].

Krueger and Feldman: Adipose circadian clocks and metabolism      21

Gaps in current knowledge and future studies Most metabolic effects on adipose tissue are studied in the context of global gene KOs. Only one study has addressed the role of Bmal1 specifically in adipose tissue [13], and other studies of tissue-specific Bmal1 KOs have not evaluated adipose tissue function. Thus, the relationship of the adipose clock to other peripheral clocks, including potential feedback mechanisms, is largely unexplored. The required clock components have not been definitively identified nor the key metabolic processes that are specifically controlled by adipose-specific clock gene functions. Given the general hypothesis that most homeostatic processes are under circadian control, it is interesting to speculate how circadian genes might control adipose-specific functions. For instance, adipocytes are replaced on a regular basis [62], which requires the continuous generation and differentiation of preadipocytes, but the mechanics and kinetics of these processes are only beginning to be addressed [63]. Also, it would be interesting to compare adipose clock functions among adipose depots, which have different gene expression profiles, functions, metabolism, adipokine release [64, 65], and cellular response to HFD [66]. Circadian gene cycling has been observed in inguinal, epididymal, and perirenal fat depots [7, 61], but comparative effects of circadian perturbations in these depots are largely unexplored. Finally, a major unresolved question is the relevance of mouse studies to human pathophysiology. Although several studies have examined circadian gene expression [67] and adipokine release [68], these analyses are mainly carried out on tissues from obese individuals at a single time point. Thus, it remains a considerable challenge to assess circadian rhythms in human tissues [69], which will be important to determine if pathways relating to metabolism and adipose tissue are conserved between humans and mice.

Expert opinion Clock and nuclear hormone expression is tightly linked with metabolism, as strongly illustrated by metabolic derangements associated with clock disruption. In adipose and many peripheral tissues, clock genes and NRs mutually regulate each other’s expression, and they interact at the protein level as well as regulate metabolic genes (Figure 1). These associations are essential in adipose tissue, as elegantly demonstrated with adipose-specific KOs of Bmal1 or Pgc1α. Future studies should address

Central clock

Clock/Bmal

Pers/Crys

Circulating factors Food Adipose clock

Clock/Bmal

Pparα Pparγ Pgc1α

Pers/Crys

ERRα

Metabolic gene expression

Figure 1 Simplified schematic of the central and peripheral clocks. The central oscillator is located in the hypothalamus of the brain and entrains peripheral clocks by cueing the release of circulating factors, such as GCs. Peripheral clocks are also influenced by food intake. The adipose tissue expresses core clock genes as well as a number of NRs, and these mutually regulate each other’s expression and coordinate the expression of metabolic genes.

the function of other clock components or other nuclear receptors specifically in adipose tissue. Additional refinements of these studies are required to examine multiple adipose depots, which differ in their metabolic rates and inflammatory signaling. More­over, the relevance of these studies to pathways operating in humans is unclear because studies of circadian signaling in human adipose tissues present significant challenges.

Outlook Studies to more precisely target and manipulate gene expression in specific adipose depots will further elucidate the contributions of clock-regulated genes in this important endocrine organ. In addition, greater

22      Krueger and Feldman: Adipose circadian clocks and metabolism understanding of the relationships among peripheral and circadian clocks will lead to more targeted therapies for metabolic abnormalities that also take into account the timing of treatments for optimal outcomes.

Highlights –– The central clock regulates activity and feeding and entrains the peripheral clock. –– Feeding also entrains the peripheral clock, altering metabolic gene expression in tissues. –– Dysregulation among central and peripheral clocks is pathogenic. –– Clock genes and many NRs coordinate metabolic gene expression in peripheral tissues, including adipose.

–– Disruption of the clock specifically in adipose alters adiposity, glucose homeostasis, and metabolism. –– Restriction of food intake to specific circadian times can overcome dysregulation caused by disrupted sleep. –– Clock functions among various adipose depots have not been explored, despite important differences in several properties, including gene expression and adipokine release. –– The relevance of studies in mice to pathways operating in humans is unclear. Acknowledgments: This work is supported in part by NIH F32 DK093191 to K.C.K. and DP2 OD006740 to B.J.F. B.J.F. is a Bechtel Endowed Faculty Scholar. Received April 15, 2013; accepted April 30, 2013; previously published online May 23, 2013

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