Altered clock gene expression and vascular smooth muscle diurnal contractile variations in type 2 diabetic db/db mice

Abstract

This study was designed to determine whether the 24-h rhythms of clock gene expression and vascular smooth muscle (VSM) contractile responses are altered in type 2 diabetic db/db mice. Control and db/db mice were euthanized at 6-h intervals throughout the day. The aorta, mesenteric arteries, heart, kidney, and brain were isolated. Clock and target gene mRNA levels were determined by either real-time PCR or in situ hybridization. Isometric contractions were measured in isolated aortic helical strips, and pressor responses to an intravenous injection of vasoconstrictors were determined in vivo using radiotelemetry. We found that the 24-h mRNA rhythms of the following genes were suppressed in db/db mice compared with control mice: the clock genes period homolog 1/2 (Per1/2) and cryptochrome 1/2 (Cry1/2) and their target genes D site albumin promoter-binding protein (Dbp) and peroxisome proliferator-activated receptor-γ (Pparg) in the aorta and mesenteric arteries; Dbp in the heart; Per1, nuclear receptor subfamily 1, group D, member 1 (Rev-erba), and Dbp in the kidney; and Per1 in the suprachiasmatic nucleus. The 24-h contractile variations in response to phenylephrine (α(1)-agonist), ANG II, and high K(+) were significantly altered in the aortas from db/db mice compared with control mice. The diurnal variations of the in vivo pressor responses to phenylephrine and ANG II were lost in db/db mice. Moreover, the 24-h mRNA rhythms of the contraction-related proteins Rho kinase 1/2, PKC-potentiated phosphatase inhibitory protein of 17 kDa, calponin-3, tropomyosin-1/2, and smooth muscle protein 22-α were suppressed in db/db mice compared with control mice. Together, our data demonstrated that the 24-h rhythms of clock gene mRNA, mRNA levels of several contraction-related proteins, and VSM contraction were disrupted in db/db mice, which may contribute to the disruption of their blood pressure circadian rhythm.

Figures

Fig. 1.

Daily mRNA expression profiles of clock and target genes in aortas isolated from diabetic db/db mice and control mice. Male 9- to 10-wk-old db/db mice (● and dotted lines) and control mice (○ and solid lines) were euthanized at Zeitgeber time (ZT)5, ZT11, ZT17, and ZT23. Aortas were harvested and cleaned, and mRNAs were quantified using real-time PCR. A: circadian locomotor output cycles kaput (CLOCK). B: brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1). C: period homolog 1 (Per1). D: Per2. E: cryptochrome 1 (Cry1). F: Cry2. G: nuclear receptor subfamily 1, group D, member 1 (Rev-erb-α). H: D site albumin promoter-binding protein (DBP). I: peroxisome proliferator-activated receptor-γ (PPAR-γ). n = 5 for each mouse strain at each time point. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared between db/db mice and control mice at the same time point in the post hoc analysis.

Fig. 2.

Daily mRNA expression profiles of clock and target genes in mesentery arteries isolated from diabetic db/db mice and control mice. Male 9- to 10-wk-old db/db mice (● and dotted lines) and control mice (○ and solid lines) were euthanized at ZT5, ZT11, ZT17, and ZT23. Mesenteric artery beds were harvested and cleaned free of fat, and mRNAs were quantified using real-time PCR. A: CLOCK. B: BMAL1. C: Per1. D: Per2. E: Cry1. F: Cry2. G: Rev-erb-α. H: DBP. I: PPAR-γ. n = 5 for each mouse strain at each time point. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared between db/db mice and control mice at the same time point in the post hoc analysis.

Fig. 3.

Daily mRNA expression profiles of clock and target genes in hearts isolated from diabetic db/db mice and control mice. Hearts were isolated from 9- to 10-wk-old male db/db mice (● and dashed lines) and control mice (○ and solid lines), and the mRNAs of specific genes were quantified using real-time PCR. A: CLOCK. B: BMAL1. C: Per1. D: Per2. E: Cry1. F: Cry2. G: Rev-erb-α. H: DBP. I: PPAR-γ. ***P < 0.001 when compared between db/db mice and control mice at the same time point in the post hoc analysis.

Fig. 4.

Daily mRNA expression profiles of clock and target genes in kidneys isolated from diabetic db/db mice and control mice. Kidneys were isolated from 9- to 10-wk-old male db/db mice (● and dotted lines) and control mice (○ and solid lines), and the mRNAs of specific genes were quantified using real-time PCR. A: CLOCK. B: BMAL1. C: Per1. D: Per2. E: Cry1. F: Cry2. G: Rev-erb-α. H: DBP. I: PPAR-γ. n = 5 for each mouse strain at each time point. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared between db/db mice and control mice at the same time point in the post hoc analysis.

Fig. 5.

Time of day variation in suprachiasmatic nucleus (SCN) Per1 mRNA expression is altered in diabetic db/db mice compared with control mice. Brains were isolated from 9- to 10-wk-old male db/db mice and control mice and cut into slides. Per1 mRNA expression was quantified by in situ hybridization. A: representative autoradiogram. B: quantification of the data. n = 6 for each mouse strain at each time point. **P < 0.01 when compared between the two time points of the same strain of mice.

Fig. 6.

The diurnal variation of the contractile response to phenylephrine (PE) is altered in db/db compared with control mouse abdominal aorta strips. Male 9- to 10-wk-old control mice (A–C) and db/db mice (D–F) were euthanized at ZT5, ZT11, ZT17, and ZT23. Isometric contractions in response to cumulative does of PE were determined in endothelium-denuded abdominal aortic helical strips. **P < 0.01 compared with the corresponding ZT5 point in the post hoc analysis.

Fig. 7.

Diurnal variations of maximal contractile responses to high K+ and ANG II are altered in db/db compared with control mouse abdominal aorta strips. Male 9- to 10-wk-old control mice (A and B) and db/db mice (C and D) were euthanized at ZT5, ZT11, ZT17, and ZT23. Isometric contractions in response to maximal does of high K+ depolarization (143 mM; A and C) or ANG II (100 nM; B and D) were determined in endothelium-denuded abdominal aortic helical strips. n = 8–9 for each time point. *P < 0.05 compared with the corresponding ZT5 point in the post hoc analysis.

Fig. 8.

Diurnal variations of in vivo pressor responses to intravenous PE and ANG II injection were lost in db/db mice. Male 10-wk-old control mice and db/db mice were instrumented with radiotelemetry, recovered for 10 days, and underwent basal blood pressure collection for 3 days. Mice were then anesthetized with isoflurane, and various doses of PE or ANG II were injected via a femoral vein. A and B: representative original blood pressure recordings. C–F: quantification of maximal net blood pressure increases in response to PE and ANG II injections. MAP, mean arterial pressure; ns, not significant. n = 3–6. *P < 0.05. **P < 0.01.

Fig. 9.

Daily mRNA expression profiles of contraction-related proteins in aortas (A–K) and mesenteric arteries (L–U) isolated from diabetic db/db mice and control mice. Male 9- to 10-wk-old db/db mice (● and dotted lines) and control mice (○ and solid lines) were euthanized at ZT5, ZT11, ZT17, and ZT23. Aortas and mesenteric arteries were harvested and cleaned, and mRNAs were quantified using real-time PCR. A: RhoA. B and L: Rho kinase 1 (ROCK-1). C and M: ROCK-2. D and N: PKC-potentiated phosphatase inhibitory protein of 17 kDa (CPI-17). E and O: Ca2+-independent phospholipase A2-β (iPLA2β). F and P: calponin-1. G and Q: calponin-2. H and R: calponin-3. I and S: tropomyosin-1. J and T: tropomyosin-2. K and U: smooth muscle protein 22-α (SM22α). n = 5 for each mouse strain at each time point. *P < 0.05; **P < 0.01, and ***P < 0.001 when compared between db/db mice and control mice at the same time point in the post hoc analysis.