Chronotherapeutic effect of orexin antagonists on glucose metabolism in diabetic mice

in Journal of Endocrinology

Correspondence should be addressed to T Sasaoka or H Tsuneki: tsasaoka@pha.u-toyama.ac.jp or htsuneki@pha.u-toyama.ac.jp

*(K Kon and H Tsuneki contributed equally to this work)

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Disrupted sleep is associated with increased risk of type 2 diabetes. Central actions of orexin, mediated by orexin-1 and orexin-2 receptors, play a crucial role in the maintenance of wakefulness; accordingly, excessive activation of the orexin system causes insomnia. Resting-phase administration of dual orexin receptor antagonist (DORA) has been shown to improve sleep abnormalities and glucose intolerance in type 2 diabetic db/db mice, although the mechanism remains unknown. In the present study, to investigate the presence of functional link between sleep and glucose metabolism, the influences of orexin antagonists with or without sleep-promoting effects were compared on glucose metabolism in diabetic mice. In db/db mice, 2-SORA-MK1064 (an orexin-2 receptor antagonist) and DORA-12 (a DORA) acutely improved non-rapid eye movement sleep, whereas 1-SORA-1 (an orexin-1 receptor antagonist) had no effect. Chronic resting-phase administration of these drugs improved glucose intolerance, without affecting body weight, food intake, locomotor activity and energy expenditure calculated from O2 consumption and CO2 production. The expression levels of proinflammatory factors in the liver were reduced by 2-SORA-MK1064 and DORA-12, but not 1-SORA-1, whereas those in the white adipose tissue were reduced by 1-SORA-1 and DORA-12 more efficiently than 2-SORA-MK1064. When administered chronically at awake phase, these drugs caused no effect. In streptozotocin-induced type 1-like diabetic mice, neither abnormality in sleep–wake behavior nor improvement of glucose intolerance by these drugs were observed. These results suggest that both 1-SORA-type (sleep-independent) and 2-SORA-type (possibly sleep-dependent) mechanisms can provide chronotherapeutic effects against type 2 diabetes associated with sleep disturbances in db/db mice.

Supplementary Materials

    • Supplementary Figure 1 Effects of selective and dual orexin receptor antagonists on sleep and wakefulness in db/m+ mice. Mice were administered vehicle or DORA-12 (a DORA, 30 mg/kg) at ZT0, and subjected to sleep recordings for 24 h to analyze the awake (A), NREM (B) and REM sleep time (C). Left, total time spent in the awake (A), NREM sleep (B), and REM sleep (C) every 2 h. Middle, total time spent in the awake (A), NREM sleep (B), and REM sleep (C) during the early light phase (ZT2-6). Right, total time spent in the awake (A), NREM (B), and REM sleep (C) during the 12-h dark phase. In the middle and right panels, dashed lines indicate the mean levels of awake (A), NREM sleep (B), and REM sleep time (C) in vehicle-treated db/db (black dashed lines) and DORA-12-treated db/db mice (red dashed lines), calculated using the same dataset as Figure 1. Data are presented as the mean ± S.E.M. (n=3-4 per group). #p<0.05, ##p<0.01: vehicle-treated db/m+ vs. vehicle-treated db/db mice. *p<0.05: vehicle-treated db/db vs. DORA-12-treated db/db mice.
    • Supplementary Figure 2 Sleep-wake architecture in STZ-diabetic and non-diabetic mice. Mice were subjected to sleep recordings for 24 h to analyze the awake (A), NREM (B) and REM sleep time (C). Left, total time spent in the awake (A), NREM sleep (B), and REM sleep (C) every 2 h. Middle, total time spent in the awake (A), NREM sleep (B), and REM sleep (C) during the 12-h light phase. Right, total time spent in the awake (A), NREM (B), and REM sleep (C) during the 12-h dark phase. Data are presented as the mean ± S.E.M. (n=4 per group).
    • Supplementary Figure 3 Influences of resting-phase administration of DORA-P and diazepam on glucose metabolism in db/db mice. Vehicle, DORA-P (a BBB-impermeable DORA analog, 30 mg/kg), or diazepam (a GABAA receptor modulator, 10 mg/kg) was administered daily at ZT0. (A) Body weight changes. (B and C) Glucose tolerance test (B) conducted after 3 weeks of the treatment, and insulin tolerance test (C) conducted after 4 weeks of the treatment. Upper, time-course of changes in blood glucose. Lower, glucose AUC. Data are presented as the mean ± S.E.M. (n=4 per group).
    • Supplementary Figure 4 Influences of awake-phase administration of selective and dual orexin receptor antagonists on glucose metabolism in db/db mice. Vehicle, 1-SORA-1 (a 1-SORA, 30 mg/kg), 2-SORA-MK1064 (a 2-SORA, 30 mg/kg), or DORA-12 (a DORA, 30 mg/kg) was administered daily at ZT12. (A) Body weight changes. (B and C) Glucose tolerance test conducted at ZT6 after 3 (B) and 4 weeks (C) of the treatment. (D) Insulin tolerance test conducted at ZT6 after 5 weeks of the treatment. Upper in B, C and D, time-course of changes in blood glucose levels. Lower in B, C and D, area under the curve (AUC) of the changes in blood glucose. (E) Serum insulin levels in glucose-stimulated insulin secretion (GSIS) test conducted at week-6. Data are presented as the mean ± S.E.M. (n=5 per group). cp<0.05: DORA-12 vs. vehicle, ep<0.05, eep<0.01: 1-SORA-1 vs. DORA-12, fp<0.05: 2-SORA-MK1064 vs. DORA-12.
    • Supplementary Figure 5 Influences of awake-phase administration of selective and dual orexin receptor antagonists on energy balance in db/db mice. Vehicle, 1-SORA-1 (a 1-SORA, 30 mg/kg), 2-SORA-MK1064 (a 2-SORA, 30 mg/kg), or DORA-12 (a DORA, 30 mg/kg) was administered daily at ZT12, and the metabolic-cage analysis was conducted after 6 weeks of the treatment. (A-C) The drug effects on food intake (A), energy expenditure (B), and locomotor activity (C) were shown. Left, daily changes in the respective levels obtained every 2 h. Right, total levels obtained during the 12-h light and 12-h dark phase. (D) Respiratory quotient. The levels were averaged every 2 h (Left) or 12 h during the light and dark phase (Right). Data are presented as the mean ± S.E.M. (n=5 per group).
    • Supplementary Figure 6 Influences of resting-phase administration of selective and dual orexin receptor antagonists on glucose metabolism in STZ-diabetic mice. Vehicle, 1-SORA-1 (a 1-SORA, 30 mg/kg), 2-SORA-MK1064 (a 2-SORA, 30 mg/kg), or DORA-12 (a DORA, 30 mg/kg) was administered daily at ZT0. (A) Changes in body weight. (B and C) Glucose tolerance test (B) conducted after 3 weeks of the treatment, and insulin tolerance test (C) conducted after 4 weeks of the treatment. Upper, the time-course of changes in blood glucose levels. Lower, glucose AUC. (D-F) Total levels of food intake (FI, D), energy expenditure (EE, E), and locomotor activity (LA, F) obtained during the 12-h light and 12-h dark phase. (G) Respiratory quotient (RQ) in the light and dark phase. (H) Serum insulin levels at ZT6 under 6-h fasting condition after 7 weeks of treatment. Data are presented as the mean ± S.E.M. (n=3-5 per group).
    • Supplementary Figure 7 Influences of resting-phase administration of selective and dual orexin receptor antagonists on gene expression in the liver and eWAT of STZ-diabetic mice. Vehicle, 1-SORA-1 (a 1-SORA, 30 mg/kg), 2-SORA-MK1064 (a 2-SORA, 30 mg/kg), or DORA-12 (a DORA, 30 mg/kg) was administered daily at ZT0, and tissues were isolated after 7 weeks of the treatment. (A) The mRNA levels of gluconeogenic factors, phosphoenolpyruvate carboxykinase (Pepck) and peroxisome proliferator-activated receptor γ co-activator 1α (Pgc1α), and inflammation markers, Emr1 (encoding F4/80) and tumor necrosis factor α (Tnfα) in the liver. (B) The mRNA levels of inflammation markers, Emr1, Tnfα, monocyte chemotactic protein-1 (Mcp1), and IL-6 in the eWAT. Data are presented as the mean ± S.E.M. (n=3-5 per group).
    • Supplementary Table 1. Summary table for two-way repeated measures ANOVA.

 

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