Ablation of the <i>Id2</i> 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

<div><p>Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our earlier studies have demonstrated a role for ID2 in the input pathway, core clock function and output pathways of the mouse circadian system. We have also reported that <i>Id2</i> null (<i>Id2</i>−/−) mice are lean with low gonadal white adipose tissue deposits and lower lipid content in the liver. These results coincided with altered or disrupted circadian expression profiles of liver genes including those involved in lipid metabolism. In the present phenotypic study we intended to decipher, on a sex-specific basis, the role of ID2 in glucose metabolism and in the circadian regulation of activity, important components of energy balance. We find that <i>Id2</i>−/− mice exhibited altered daily and circadian rhythms of feeding and locomotor activity; activity profiles extended further into the late night/dark phase of the 24-hr cycle, despite mice showing reduced total locomotor activity. Also, male <i>Id2−/−</i> mice consumed a greater amount of food relative to body mass, and displayed less weight gain. <i>Id2−/−</i> females had smaller adipocytes, suggesting sexual-dimorphic programing of adipogenesis. We observed increased glucose tolerance and insulin sensitivity in male <i>Id2−/−</i> mice, which was exacerbated in older animals. FDG-PET analysis revealed increased glucose uptake by skeletal muscle and brown adipose tissue of male <i>Id2</i>−/− mice, suggesting increased glucose metabolism and thermogenesis in these tissues. Reductions in intramuscular triacylglycerol and diacylglycerol were detected in male <i>Id2</i>−/− mice, highlighting its possible mechanistic role in enhanced insulin sensitivity in these mice. Our findings indicate a role for ID2 as a regulator of glucose and lipid metabolism, and in the circadian control of feeding/locomotor behavior; and contribute to the understanding of the development of obesity and diabetes, particularly in shift work personnel among whom incidence of such metabolic disorders is elevated.</p></div

Glucose tolerance, insulin sensitivity and insulin release in <i>Id2−/−</i> females is unaltered.

<p>A) GTT of young female <i>Id2−/−</i> and WT mice (RM-ANOVA: time (T), P<0.001; genotype (G), n.s.; interaction (I), n.s.). B) GTT of old female <i>Id2−/−</i> and WT mice (T, P<0.001; G, n.s.; I, n.s.). C) ITT of young female <i>Id2−/−</i> and WT mice (T, P<0.001; genotype, n.s.; I, n.s.). D) ITT of old female <i>Id2−/−</i> and WT mice (T, P<0.001; G, P<0.01; I, n.s.). E) Glucose-stimulated insulin release in young female <i>Id2−/−</i> and WT mice (T, P<0.001; G, n.s.; I, n.s.). F) Glucose-stimulated insulin release in old female <i>Id2−/−</i> and WT mice (T, n.s.; G, P = 0.055; I, n.s.). No effect of aging was observed in the glucose tolerance of either WT or <i>Id2</i>−/− females (RM-ANOVAs, n.s.). An aging effect of insulin sensitivity was observed for WT and <i>Id2</i>−/− females (T, P<0.001; age, P<0.001; I P<0.001). Values shown represent mean ± SEM. **p<0.01.</p

Skeletal muscle triglyceride (TG) and diacylglycerol (DAG) profiles in <i>Id2−/−</i> mice.

<p>A) Total TG content of tibialis anterior muscle (ANOVA: genotype (G), n.s.; sex (S), P<0.05; interaction (I), n.s.). B) Total DAG content of tibialis anterior muscle (G, n.s.; S, P<0.01; I, n.s.). C) DAG species analysis. DAG species are abbreviated as two contributing fatty acyl groups: A, E, S, O, L and P denote arachidonoyl, eicosapentanoyl, stearoyl, oleoyl, linoleoyl and palmitoyl groups, respectively. Values represent mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001 are Turkey post-hoc tests following two way ANOVA for TG, DAG or each DAG species.</p

<i>Id2−/−</i> male mice display enhanced glucose tolerance and insulin sensitivity.

<p>A) Glucose tolerance test (GTT) of young male WT and <i>Id2−/−</i> mice (RM-ANOVA: time (T), P<0.001; Genotype (G), P<0.05; interaction (I), n.s). B) GTT of old male WT and <i>Id2−/−</i> mice (T, P<0.001; G, P<0.05; I, P = 0.001). C) Insulin tolerance test (ITT) of young male WT and <i>Id2−/−</i> mice (T, P<0.001; G, P<0.05; I, n.s.). D) ITT of old male WT and <i>Id2−/−</i> mice (T, P<0.001; G, P<0.01; I, P<0.001). E) Glucose-stimulated insulin release in young male WT and <i>Id2−/−</i> mice (T, P<0.01; G, P<0.01; I, n.s.). F) Glucose-stimulated insulin release in old male WT and <i>Id2−/−</i> mice (T, P = 0.103; G, P<0.01; I, n.s.). No effect of aging was observed in the glucose tolerance of either WT or <i>Id2</i>−/− males (RM-ANOVAs, n.s.). Comparison on young and old <i>Id2−/−</i> males reveal an increase in insulin sensitivity (T, P<0.001; A, P<0.001; I, P<0.01) in the older group. This large age effect was not observed in WTs (T, P<0.001; age (A), p = 0.06; I, P<0.05), although there was tendency for a slower recovery to baseline glucose levels at 90 and 120 mins (p<0.05). Values shown represent mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001.</p

<i>Id2−/−</i> mice exhibit less locomotor activity compared to WT mice.

<p>A) Daily feeding activity counts of <i>Id2−/−</i> and WT mice (ANOVA: genotype, n.s.; sex, n.s.; interaction, n.s). B) Daily general activity counts in <i>Id2−/−</i> and WT mice determined by passive infrared motion detectors (genotype, P<0.05; sex, P = 0.057; interaction, n.s). C) Daily wheel revolution counts of WT and <i>Id2−/−</i> mice (genotype, P<0.001; sex, P<0.05; interaction, P<0.01). Values represent mean ± SEM. ***p<0.001.</p

<i>Id2−/−</i> mice show altered daily and circadian patterns of feeding and locomotor activity.

<p>A) Daily feeding activity profile of WT and <i>Id2−/−</i> mice (ANOVA: time (T), P<0.001; genotype (G), n.s.; interaction (I), P<0.01). B) Circadian feeding activity profile of WT and <i>Id2−/−</i> mice (T, P<0.001; G, n.s.; I, P<0.05). C) Daily PIR motion detector general activity profile of WT and <i>Id2−/−</i> mice (T, P<0.001; G, P<0.05; I, P<0.001). D) Circadian general activity profile of WT and <i>Id2−/−</i> mice (T, P<0.001; G, P = 0.15; I, P<0.001). E) Daily wheel running activity profile of WT and <i>Id2−/−</i> mice (T, P<0.001; G, P<0.001; I, P<0.001). F) Circadian wheel running activity profile of WT and <i>Id2−/−</i> mice (T, P<0.001; G, P<0.01; I, P<0.001). The shaded area in the plots represents dark phase of the LD cycle or constant darkness. Values shown represent mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001.</p

Fasting glucose and Insulin levels while aging.

<p>A) Fasting blood glucose levels of young and old, WT and <i>Id2−/−</i> mice (ANOVA: genotype, P<0.01; age, P<0.001; interaction, n.s.). B). Fasting insulin levels of young and old, WT and <i>Id2−/−</i> mice (genotype, P<0.001; age, P<0.05; interaction, n.s.). Values shown represent mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001.</p

Cell size of the gonadal white adipose tissue is different between female <i>Id2−/−</i> and WT mice.

<p>A) Representative images of hematoxylin/eosin stained gonadal white adipose tissue sections of male and female WT and <i>Id2−/−</i> mice (scale bar = 50 µm). B) Cell area of gonadal WAT in WT and <i>Id2−/−</i> mice (ANOVA: genotype, P<0.05; sex, n.s.; interaction, P<0.001). No significant correlation was detected between cell size and age of animal (Spearman’s rank order correlation/linear regression, n.s.).</p