Development of diabetes does not alter behavioral and molecular circadian rhythms in a transgenic rat model of type 2 diabetes mellitus

Abstract

Metabolic state and circadian clock function exhibit a complex bidirectional relationship. Circadian disruption increases propensity for metabolic dysfunction, whereas common metabolic disorders such as obesity and type 2 diabetes (T2DM) are associated with impaired circadian rhythms. Specifically, alterations in glucose availability and glucose metabolism have been shown to modulate clock gene expression and function in vitro; however, to date, it is unknown whether development of diabetes imparts deleterious effects on the suprachiasmatic nucleus (SCN) circadian clock and SCN-driven outputs in vivo. To address this question, we undertook studies in aged diabetic rats transgenic for human islet amyloid polypeptide, an established nonobese model of T2DM (HIP rat), which develops metabolic defects closely recapitulating those present in patients with T2DM. HIP rats were also cross-bred with a clock gene reporter rat model (Per1:luciferase transgenic rat) to permit assessment of the SCN and the peripheral molecular clock function ex vivo. Utilizing these animal models, we examined effects of diabetes on 1) behavioral circadian rhythms, 2) photic entrainment of circadian activity, 3) SCN and peripheral tissue molecular clock function, and 4) melatonin secretion. We report that circadian activity, light-induced entrainment, molecular clockwork, as well as melatonin secretion are preserved in the HIP rat model of T2DM. These results suggest that despite the well-characterized ability of glucose to modulate circadian clock gene expression acutely in vitro, SCN clock function and key behavioral and physiological outputs appear to be preserved under chronic diabetic conditions characteristic of nonobese T2DM.

Keywords: HIP rat; SCN, hyperglycemia; circadian clocks; circadian rhythms; melatonin.

Fig. 1.

Diurnal plasma metabolic profiles and islet morphology in wild-type control and diabetic HIP (T2DM) rats. The mean diurnal plasma glucose (A), insulin (B), and glucagon (C) concentrations in diabetic HIP (T2DM) and corresponding wild-type (control) rats were sampled at 6-h intervals (n = 7 per group) throughout the 24-h day under standard LD cycle. The time of lights on is defined at ZT 0. Note development of overt diurnal hyperglycemia, hyperinsulinemia, and hyperglucagonemia in T2DM rats. D: representative examples of pancreatic islets imaged at ×20 magnification (scale bar = 50 μm) immunohistochemically stained to visualize β-cells with (top) and without (bottom) addition of the primary insulin antibody (ab) in diabetic HIP (T2DM) and corresponding wild-type (control) rats. Note distinct specificity of the primary insulin antibody and overt loss of insulin expression in T2DM rats. Data are expressed as means ± SE. *P < 0.05 vs. control.

Fig. 2.

Representative locomotor activity circadian rhythms in wild-type control and diabetic HIP (T2DM) rats. A–D: representative 24-h locomotor activity (double-plotted) actograms of wild-type (control; n = 7 total; A) and diabetic HIP (T2DM; n = 7 total; B–D) rats exposed to 70-day light regiment consisting of 1) 14 days of standard 12:12 LD cycle followed by 2) 14 days of 6-h phase advance and then 3) 14 days of 6-h phase delay and continuing to 4) 21 days in constant darkness (DD) followed by 5) 7-day reentrainment back to the standard 12:12 LD cycle. The gray shading represents periods of darkness. Note varying levels of hyperglycemia in control vs. T2DM rats. E–H: corresponding χ²-periodograms for control (E) and T2DM (F–H) rats exposed to 21 days of DD. Note robust circadian period amplitude in control and T2DM rats.

Fig. 3.

Quantification of behavioral circadian rhythms in wild-type control and diabetic HIP (T2DM) rats. Shown are mean χ²-periodogram-derived circadian period (A), amplitude (B), circadian rhythm fragmentation (C), average total daily (24-h) activity (D), as well as daily average activity during the active (E) and inactive (F) phase of the circadian cycle under either LD or DD conditions in wild-type (control) and diabetic HIP rats (T2DM). Bar graphs represent mean ± SE (n = 7 per group).

Fig. 4.

Assessment of molecular circadian rhythms using Per1:luciferase (Per1:LUC) bioluminescence in the SCN of wild-type control and diabetic HIP (T2DM) rats. A: schematic describing generation of HIP:Per1:LUC and WT:Per1:LUC reporter rats. RIP-II, rat insulin 2; h-IAPP, human islet amyloid polypeptide. B: representative example of Per1 bioluminescence waveforms in the SCN isolated from control and diabetic HIP rats (T2DM). Mean amplitude (C), period (D), and phase (E) of Per1:LUC bioluminescence circadian rhythms in the SCN isolated from control and diabetic HIP (T2DM) rats are shown. Data are expressed as means ± SE (n = 5 per group).

Fig. 5.

Assessment of molecular circadian rhythms using Per1:luciferase (Per1:LUC) bioluminescence in pancreatic islets and the aorta of wild-type control and diabetic HIP (T2DM) rats. Shown are representative examples of Per1 bioluminescence waveforms along with calculated mean period and the phase of Per1:LUC bioluminescence in pancreatic islets (A–C) and the aorta (D–F) isolated from control and diabetic HIP (T2DM) rats. Data are expressed as means ± SE (n = 5 per group).

Fig. 6.

Diurnal plasma melatonin levels for wild-type control and diabetic HIP (T2DM) rats. Shown are the mean diurnal plasma melatonin concentrations in diabetic HIP (T2DM) and corresponding wild-type (control) rats sampled at 6-h intervals (n = 7) across the 24-h day under standard LD cycle. Data are expressed as means ± SE.