Ablation of the Id2 Gene Results in Altered Circadian Feeding Behavior, and Sex-Specific Enhancement of Insulin Sensitivity and Elevated Glucose Uptake in Skeletal Muscle and Brown Adipose Tissue

Inhibitor of DNA binding 2 (ID2) is a dominant negative regulator of basic helix-loop-helix (bHLH) transcription factors, which acts by heterodimerizing with them and inhibiting their ability to bind to E-box elements within target gene promoters [1], [2]. A functional role of ID2 has been demonstrated in cell lineage determination, cell cycle progression, adaptive immunity and tumorigenesis [1], [2]. Id2 is expressed in a many adult tissues and shows circadian rhythmicity in its RNA and protein expression [3], [4].

Circadian rhythms are cyclical occurrences of physiological and/or behavioral events with a periodicity of ∼24 hrs, exhibited by all eukaryotic organisms [5]. This intrinsic time keeping mechanism is believed to have evolved as an adaptive response to the daily changes in the surrounding environment such as light and food availability. This enables organisms to anticipate such changes and coordinate, or temporally partition, different biological processes for optimal utilization of resources. In mammals the master circadian oscillator resides in the hypothalamic suprachiasmatic nuclei (SCN). The SCN synchronizes and phase resets the cell autonomous clock mechanisms in the peripheral tissues through humoral signals, autonomic innervation, body temperature and feeding related cues [6]. At the molecular level, the clock is encoded by transcriptional-translational autoregulatory feedback loops of core clock proteins. The positive loop is composed of bHLH-PAS transcriptional activators CLOCK (or NPAS2) and BMAL1. They heterodimerize and transactivate a proportion of the downstream clock controlled genes (CCGs) responsible for clock output, as well as transactivate the transcriptional repressor genes period and cryptochrome, that constitute the negative loop [5].

Recent evidence reveals that circadian rhythms and metabolism are tightly intertwined physiological processes. The circadian clock regulates glucose homeostasis at the levels of both SCN and peripheral clocks. Daily rhythms are observed in blood glucose levels, glucose tolerance, insulin sensitivity and glucose uptake by the brain, skeletal muscle and adipose tissue [7]–[9]. Moreover, disturbances in the circadian rhythmicity and sleep-wake cycle in humans, such as in shift-work personnel, are associated with diabetes, metabolic syndrome, hypoleptinemia, increased appetite and obesity [7]. Furthermore genetic disruption of several canonical clock genes in animal models results in diverse and profound metabolic disturbances, demonstrating the involvement of clock genes in glucose homeostasis [7].

Our previous studies have demonstrated that ID2 has roles in core clock function by interacting with CLOCK and BMAL1 through their HLH domain and inhibiting their transactivation potential [4], [10]. ID2 contributes to the input pathway of the circadian system as demonstrated by the abnormally rapid photoentrainment and increase in the magnitude of light-induced phase shifts exhibited by Id2 null (Id2−/−) mice [4]. In addition, a role for ID2 in the output pathways of the circadian clock is suggested by altered expression profiles of CCGs in the liver of Id2−/− mice, including those involved in lipid metabolism [11]. Moreover, absence of Id2 results in impaired adipogenesis in vitro[12] and reduced gonadal white adipose deposits and lipid content in liver in adult mice [11]. Due to these findings, the entwined nature of lipid and glucose metabolism and the interlocked relationship of the molecular circadian clock with metabolic processes [5], we hypothesized that ID2 contributes to the regulation of glucose homeostasis, energy utilization and the temporal regulation of feeding. It is well known that risk, development and manifestation of obesity, metabolic syndrome and insulin-resistance are sexually dimorphic, and animal models suggest contributing roles of both gonadal hormones and sex chromosomes [13]–[16]. Moreover, the effect of aging on the circadian clock and on glucose homeostasis has been reported in humans and animal models [17]–[20]. In the present study, our objective was to characterize key aspects of metabolic function and circadian regulation of activity and feeding in Id2−/− mice, and furthermore, to examine this in a sex- and age-specific context. We report here that Id2−/− mice have altered 24-hr patterns of locomotor and feeding behavior, and reduced weight gain despite elevated food intake, as well as sex-specific disturbances to adipocyte programing and to lipid accumulation in skeletal muscle and brown adipose tissue. We also report sex- and age-specific changes in glucose homeostasis, including fasting hypoglycemia, and increases in glucose tolerance, insulin sensitivity and glucose uptake that are most prominent in male animals.

ID2 is a HLH transcriptional repressor, rhythmically expressed in many mammalian tissues and previously reported to be involved in the input pathways, core clock function and output pathways of the circadian clock, and in adipogenesis [4], [10]–[12]. In the present study we have characterized an altered circadian rhythm and metabolic phenotype in Id2−/− mice, revealing a role for ID2 in the circadian regulation of feeding and locomotor behavior and in glucose metabolism.

Our studies show that Id2−/− mice exhibit lower levels of locomotor activity, and have extended activity patterns of feeding and locomotor activity. We also observed that WT females showed higher general activity and wheel running activity compared to WT males, a finding consistent with the reported sexual dimorphism in locomotor activity levels for various mouse strains, including C57BL/6J and 129x1/SvJ [23]. We also observed in Id2−/− mice a reduced level or absence of anticipatory activity 0–3 hr prior to the time lights off (ZT12). This finding is consistent with our earlier report in which Id2−/− mice were observed to have a significant ∼30 min delay in the phase angle of activity onset relative to WTs [4]. In WT mice the period of high activity is relatively short, peaking during the early dark/night phase of the LD cycle, in comparison with the longer and sustained profiles of Id2−/− mice, spanning early to late dark/night phase. That Id2−/− mice exhibit prolonged activity patterns of feeding and locomotor activity not only under LD but also DD conditions demonstrates the presence of an altered biological clock in Id2 mutants.

There are several possible mechanisms that could explain the altered locomotor and feeding rhythms in Id2−/− mice. Given that ID2 plays a role in regulating circadian clock output in the liver, including metabolic CCGs, and because Id2 is ubiquitously expressed and rhythmic [3], [4], [11], it is plausible that such a pivotal role is a feature of clocks throughout the body. This would include the hypothalamic brain and peripheral tissues involved in metabolic homeostasis.

Animals undergo vast changes in physiology to maintain metabolic homeostasis during the normal daily 24 hr feeding-fasting cycles and cycles of physical activity and arousal [5], [24]. Lack of precise time-of-day specific phase coordination between rhythmic processes within and between the metabolic organ systems (i.e. SCN, hypothalamic feeding-fasting centers, liver, WAT, BAT, skeletal muscle, pancreas, intestine), including impaired nutrient signaling (e.g. leptin, orexin, ghrelin), would predictably result in disruption of the normal balance of energy storage and utilization [5], [24]. Such disruption is thought to underlie the hyperphagic/obese phenotype of the clock mutant mouse [25]. It is noteworthy that ID2 can interact with CLOCK and its partner protein BMAL1 and modify both their localization signal and transactivation potential [10]. This general or perhaps tissue-specific disturbance in clock control may underlie both the behavioral and also physiological features (i.e. enhanced glucose uptake/reduced lipid storage) of the Id2−/− phenotype. We therefore suggest that the altered state of feeding and locomotor activity behaviors coupled with reduced physical activity, are a response to or the direct result of disruption in circadian metabolic homeostasis. These changes that we report in behavior would result in reduced energy expenditure and provide for a more sustained nutrient intake during the night. The most likely site for these changes in behavior is the SCN and hypothalamic feeding-fasting centers, known to regulate rhythmicity, wakefulness and feeding [5], [24]. Another possibility is that the behavioral changes reflect a disruption of normal SCN clock/output function [4], [10], [11], rather than a response to a system-wide metabolic alteration. Finally, reduced adipogenesis in the Id2−/− mouse [12] would likely compound any circadian disruption by reducing the lipid storage capacity of the animal. However, it is not sufficiently clear to what extent these processes can be considered distinct.

Our data revealed that Id2−/− males had a lower body weight while counter intuitively consuming greater relative quantities of food and simultaneously gaining less weight compared to WT males. This difference in body weight and weight gain was not observed in females. This observation could be explained in part by earlier findings of impaired adipogenesis and reduced adiposity in Id2−/− mice [11], [12]; and possibly by increased energy expenditure in BAT and skeletal muscle, as suggested by elevated FDG uptake specifically in Id2−/− males. This could also partly be attributed to the sex difference in the weight gain of WT mice, observed in C57Bl/6J mice, where males are more susceptible to obesity, and this advantage of females is abolished by ovariectomy [26]. Our results show that ablation of Id2 reduces body mass gain in male mice. In fact there was a reduction in body mass in male Id2−/− mice in contrast to an increase observed in WT males. This weight loss was only detected in the presence of a running wheel, even though Id2−/− mice do not run in the wheel as much as WTs. The wheel could act as an enriched environmental stimulus to which the Id2−/− mice might show a greater response, since an enriched environment can improve metabolic health through increasing adaptive thermogenesis and browning of white adipocytes and thus increasing energy dissipation [27].

Earlier studies from our laboratory and others have shown that Id2−/− mice have less gonadal white adipose deposits [11], [12]. The present histological data show no significant difference in the gonadal adipocyte cell size between male WT and Id2−/− mice, suggesting that the reduced adiposity observed in male Id2−/− mice is due to a lower number of adipocytes. However, we did identify a sex-specific difference in which cells were smaller in female Id2−/− mice relative to female WT controls. Thus, reduced adiposity observed in female Id2−/− mice might be the result of a different mechanism, in which adipocyte number does not change but lipid accumulation does. Interestingly, the difference observed between female Id2−/− and WTs is consistent with reports of smaller adipocytes in Id1−/− and Id4−/− mice [28], [29]. However, in these studies either no clear indication of animal sex was provided or no differentiation of sexes reported. Clearly this underlies yet another sex-specific feature of the Id2 null phenotype, that of differential adipocyte programing. These results could be attributed to the impaired adipocyte differentiation in the absence of Id2. It has also been observed that Id2 promotes PPARγ expression, morphological differentiation and lipid accumulation during adipogenesis [12]. Id2 gene expression is elevated in the initial phase of adipocyte differentiation and dramatically drops in the later phase [30], [31]. Here, ID2 potentially acts as a differentiation-inducing factor, as ID proteins are suggested to inhibit premature differentiation of progenitor cells and regulate cell fate determination [2]. A similar expression pattern has been observed for C/EBPβ, a key adipocyte differentiation inducer [32], which induces Id2 expression during this process [33]. Interestingly, C/EBPβ null mice also exhibit reduced epididymal WAT deposits, impaired adipocyte differentiation and reduced lipid accumulation in BAT [34]. Moreover, specific clock genes contribute to adipogenesis, including BMAL1, which acts as negative regulator of adipogenesis [35], [36]. Since previous studies in our laboratory have indicated that ID2 can interact with BMAL1 and reduce its transactivation potential [4], [10], it raises the possibility that ID2 might positivity regulate adipogenesis via inhibition of BMAL1 activity.

Male Id2−/− mice displayed enhanced glucose tolerance, greater insulin sensitivity, low basal insulin levels and lower levels of glucose-stimulated insulin secretion when compared to WTs. Furthermore, glucose tolerance and insulin sensitivity were even more pronounced in aged male Id2−/− mice. Our results revealed that the fasting blood glucose levels in the Id2−/− mice were significantly lower than age-matched WTs, and for both Id2−/− and WT mice, the fasting glucose levels decreased with age. It is conceivable that this difference between young and old animals, irrespective of genotype, is a result of habituation to handling occurring in older animals [37]. However, there still remains a large reduction in fasting glucose in Id2−/− animals at each age-matched group. Furthermore, these collective glucose homeostasis tests, including basal and glucose-stimulated insulin levels, demonstrate that the ability of male Id2−/− mice to improve glucose tolerance is not associated with enhanced circulating insulin levels. In fact, surprisingly, in the young male Id2−/− mice, significantly less insulin is produced, thereby revealing a dramatically enhanced insulin sensitivity despite reduced insulin concentrations.

The enhanced glucose uptake by skeletal muscle and BAT of the male Id2−/− mice could contribute to the elevation in insulin sensitivity. Consistent with the weight gain pattern, female mice did not show any significant differences in the glucose homeostasis parameters when compared to WTs, apart from the lower basal insulin levels in the older group. Moreover the positive effect of aging on insulin sensitivity was observed in females irrespective of genotype. Our findings suggest the role of sex and age in the development of enhanced insulin sensitivity in the absence of ID2. This is consistent with the report that females are less susceptible to develop insulin resistance associated with diet-induced obesity, and where ovarian hormones are implicated [16].

An evaluation of glucose uptake by FDG-MicoPET imaging revealed higher FDG levels in the skeletal muscle of Id2−/− males. Considering the total mass of skeletal muscle in the body, a small difference in glucose uptake in the forelimb muscle could be indicative of a highly elevated glucose uptake by total skeletal muscle in Id2−/− males. Furthermore, insulin resistance in skeletal muscle is associated with aging [20], [38], [39], and sexual dimorphism is observed in the development of insulin resistance [40], [41]. Since Id2 expression in the skeletal muscle is reported to increase during aging [42], it is possible that the difference between Id2−/− and WT males in skeletal muscle glucose uptake also increases with age. The fact that Id2 is up-regulated in the skeletal muscle of obese type II diabetic mice further suggests a role of Id2 in the development of skeletal muscle insulin resistance [43].

Accumulation of intramyocellular TG and especially DAG are associated with insulin resistance [22]. We found that the concentrations of these lipids were significantly reduced in the skeletal muscle of Id2−/− males as compared to Id2−/− females: 67% lower for TG, and 25% for total DAG. These data in part could explain both the enhanced insulin response and elevated skeletal muscle glucose uptake observed in the Id2−/− male but not female animals, through altered insulin signaling. Furthermore, the differences in the distribution of individual DAG species, observed between males and females (sex-specific differences), and especially between male Id2−/− versus male WT mice, reveal the complexity of the Id2−/− phenotype in terms of fatty acid composition. Interestingly, amongst several differences, the DAG species distribution data also indicate an elevated availability/metabolism of stearate (C18∶0) in the Id2−/− male compared to WT male controls. It has been suggested that alterations in specific DAG species may differentially contribute to insulin resistance [44], and so these disturbances in DAG distribution in the Id2−/− males could contribute to the increased glucose uptake by altering insulin signaling [11].

While the total DAG findings do not fully explain the genotypic differences we observed at the physiological level, they might in part constitute an explanation for the sex-specific element contributing to the glucose uptake and insulin sensitivity phenotype seen only in male Id2−/− mice. Likewise, these reductions in intramuscular TG/DAG may reflect reduced circulating lipids and/or increased myocyte mitochondrial fatty acid oxidation, as might be predicted from increased insulin sensitivity [44]. However, it still remains plausible that because of the intra-sex differences observed in DAG species composition between male Id2−/− and male WT mice, DAG might contribute to the genotypic differences. At the very least, low total DAG would be permissive to additional genotypic changes that are enhancing insulin sensitivity [42], [43].

We have observed that the average FDG uptake by iBAT is elevated in Id2−/− male mice. In addition, our data revealed a higher activated iBAT volume for Id2−/− mice of both sexes as well as the females of both genotypes. This is consistent with observations in adult humans, where females show increased mass of active iBAT [45]. However, since the actual mass of iBAT was lower in Id2−/− males and WT females than WT males, it is plausible that this elevated activated iBAT volume might also include recruitable brown adipocytes/beige adipocytes localized in the surrounding WAT mass [27], [46]. We also observed that the activated iBAT volume decreased with increase in body mass in WT mice, but not in Id2−/− mice. Similar observations were reported in humans, where an increase in body mass index (BMI) reduces the chance of detecting active BAT [45]; and BAT activity, measured as total FDG uptake, is negatively correlated with BMI and body fat percentage [47]. Our data from older mice revealed that WT male iBAT acquire greater weight and lipid deposits than Id2−/− males and WT females. We speculate that the presence of higher levels of lipid depositions in WT male iBAT might make it larger in size and less metabolically active per unit volume. In the Id2−/− male iBAT, higher glucose uptake, indicative of increased metabolic activity, suggests a higher thermogenic activity.

The situation in the Id2−/− male is indicative of the system-wide reduction in lipid accumulation, i.e. in addition to reduced quantities of WAT, this group has the lowest levels of skeletal muscle TG and DAG, and has low BAT lipids relative to WT males. Being that male Id2−/− mice on average have a smaller body mass, the changes in BAT physiology may be an adaptive response to an unfavorable surface area to mass ratio and compensating for increased loss of body heat [48]. It is plausible that to compensate for the increased energy expenditure, the mice increase food intake and reduce their locomotor activity. However, in the small cohort of body mass-matched male animals, male Id2−/− FDG uptake was still found to be elevated in respect to male WTs, suggesting that this tangible factor would not explain the entire extent of the BAT phenotype. Furthermore, in females, where there is no difference in body mass between mutant and WT animals, there still remains an elevation in activated BAT volume. In the Id2−/− females this increase in activated BAT volume also suggests a level increase in metabolic/thermogenic activity. Again, the observed reduction in activity might compensate for this increase energy expenditure.

Another mechanistic explanation for the observations in Id2−/− BAT and skeletal muscle and in glucose homeostasis would be associated with the circadian clock. As there are endogenous clock mechanisms within BAT, WAT and skeletal muscle [3], [8], [49], , the Id2 null phenotype might be as a result of disturbances to their normal intrinsic temporal coordination of CCGs and subsequent metabolic process [49], [50]. As ≥7% of genes are rhythmically regulated in these tissues, including Id2[3], [49], [51], [52], and as Id2 is implicated in regulating clock output [11], it is plausible that the absence of ID2 would result in a subset of CCGs being abnormally regulated, with potential consequences to local and systemic physiology.

Similar to the metabolic phenotype in Id2−/− mice, enhanced insulin sensitivity and increased glucose uptake by WAT and BAT were observed in a non-obese type 2 diabetic mice model in response to β3-adrenergic receptor activation [53]. Of note, β-adrenergic signaling induces PGC-1α, a thermogenic gene suggested to link the circadian clock and metabolism [54], and mice null for PGC-1α in WAT develop insulin resistance [55]. Interestingly, PGC-1α expression is elevated in Id2−/− liver [11].

In addition to PGC-1α, altered gene expression of other metabolic genes in Id2−/− liver could explain the present observations on Id2−/− mice. The rhythmically expressed genes Igfbp1 and Igfbp2, encoding insulin like growth factor (IGF) binding proteins, are up-regulated in Id2−/− liver [11]; in the case of Igfbp1, a change in the peak phase of its rhythmic profile is observed. IGFBPs are predominantly inhibitory to IGF action, and IGFBP1 can inhibit IGF1-mediated differentiation of preadipocytes [56]. IGFBP2 can inhibit adipocyte differentiation in vitro, and when overexpressed in vivo, mice are protected from developing age- and diet-induced obesity and insulin resistance [57]. Fibroblast growth factor (FGF) receptor 1 is also up-regulated in Id2−/− liver [11], interesting given that FGFs, including FGF21, are regulators of glucose and lipid metabolism [58].

In contrast to the metabolic phenotype observed in the absence of Id2, elevated expression of Id2 is associated with obesity and/or diabetes. Id2 expression in adipocytes is positively associated with obesity in mice and humans [12]. The Id2 gene promoter contains a conserved CREB binding site, and its expression is stimulated in adipocytes by cAMP or a high fat diet [31], [59]. Also CREB is activated in adipocytes of obese mice where it promotes insulin resistance [59]. Increased Id2 expression is observed in the muscle of obese type II diabetic mice and in the liver of type I diabetic mice [43], [60], and thiazolidinedione, a class of anti-diabetic drugs, inhibits Id2 expression in human aortic smooth muscle cells [61]. Moreover glucose is reported to induce Id2 expression in macrophages [62]. Taken together these findings clearly reveal the involvement of ID2 in the regulation of glucose/lipid metabolism and in the development of obesity and/or diabetes.

Similar to the Id2−/− mouse, a metabolic phenotype of reduced fat mass, increased insulin sensitivity and enhanced energy expenditure has been observed in Id1 null mice. In contrast to ID2, ID1 is not required for adipocyte differentiation, and adipocyte differentiation is in fact accelerated in Id1−/− cells in vitro[29]. Defective adipogenesis is not implicated in the lower adiposity of Id1−/− mice [29]. Even though basal insulin levels are lower in Id1−/− mice, their glucose-stimulated insulin secretion is highly elevated, enhancing their glucose tolerance [63]. This is in contrast to the reduced levels of glucose-stimulated insulin we observed in Id2−/− mice. However enhanced gene expression of thermogenic proteins is also reported in Id1−/− mice [29], suggesting their BAT metabolic activity may be elevated as observed in Id2−/− mice. Similar to Id2, Id4 is also reported to be necessary for adipogenesis, and Id4−/− mice exhibit reduced body weight and adipose deposits, including smaller adipocytes [28]. Loss of Id3 does not affect weight gain or adipose deposit size in regular chow fed mice, but does result in reduced high fat diet induced visceral fat pad expansion [64]. Therefore the metabolic phenotypes observed in the absence of different ID proteins may not be the result of similar mechanisms, in spite of the possible functional redundancies amongst them.

Considering the involvement of ID2 in input pathways, core clock function and output pathway of circadian clock and also in adipogenesis [4], [10]–[12], we propose possible mechanisms for the development of the observed circadian and metabolic phenotypes in Id2−/− mice. Ablation of Id2 could possibly alter the master clock in the SCN, thereby altering feeding and locomotor activity rhythms. Another possibility is an altered feeding pattern, either because of the altered CCG expression in periphery, as seen in liver [11], or centrally within the hypothalamic feeding centers [6], [24]; or because of a more persistent drive for feeding owing to lower energy storage capabilities (i.e. lipids), at least in part, due to reduced adipogenesis [48]. This would generate a drive for the animal to be active for a longer duration and thus alter locomotor activity rhythms. The lean phenotype in Id2−/− males could be a result of increased glucose uptake and utilization in both skeletal muscle and BAT, as well an increase in BAT thermogenesis. Likewise, there may be a generalized increase in metabolic activity, i.e. both glucose and fatty acid oxidation, as might be predicted from the increased insulin sensitivity and elevated FDG uptake. These changes in energy metabolism may reflect a disturbance in circadian clock function, occurring specifically within skeletal muscle and BAT [3], [8], [49], [50] and beyond these tissues, and thereby disrupting normal temporal coordination of metabolic processes; or the changes in energy metabolism may be a poor adaptation to the lower energy storage capabilities. The changes in glucose uptake coincide with enhanced insulin sensitivity and glucose tolerance in Id2−/− males. The sex-specific lower intramuscular total DAG, as well as differential profile of DAG species in male Id2−/− mice, might also contribute to the increased insulin sensitivity and glucose uptake; although a low concentration of intramuscular lipid in itself is a reflection of the lean state, most pronounced in the male Id2−/− mouse. The lean phenotype in Id2−/− males might also necessitate increased thermogenesis to compensate for a loss of body heat. It is plausible that to compensate for the increased energy expenditure, Id2−/− males increase food intake and reduce their locomotor activity.

In conclusion, we find that Id2−/− mice exhibit altered feeding and locomotor rhythms, fasting hypoglycemia, sex- and age-dependent enhanced glucose tolerance and insulin sensitivity, and sex-dependent elevated glucose uptake in skeletal muscle and BAT. Our observations clearly suggest the role of the ubiquitously expressed and rhythmic Id2 gene in regulating pathways involved in glucose and lipid metabolism, as well as in the circadian control of feeding and locomotor activity behavior. BAT has received considerable attention recently as a potential therapeutic system to combat obesity, especially after it was discovered to occur in adult humans [65]. Our current data and earlier reports of Id2 up-regulation during aging, suggests ID2 to be a potential therapeutic target for treating metabolic and circadian disorders in adults. Further, these data reinforce the relevance of sex and age-specific analyses in studying models of metabolic and circadian function as they pertain to human health and disease. In particular they also help to improve understanding of the development of obesity and diabetes, diseases prevalent in shift work personnel.