Diurnal variation in vascular and metabolic function in diet-induced obesity: divergence of insulin resistance and loss of clock rhythm

Abstract

Circadian rhythms are integral to the normal functioning of numerous physiological processes. Evidence from human and mouse studies suggests that loss of rhythm occurs in obesity and cardiovascular disease and may be a neglected contributor to pathophysiology. Obesity has been shown to impair the circadian clock mechanism in liver and adipose tissue but its effect on cardiovascular tissues is unknown. We investigated the effect of diet-induced obesity in C57BL6J mice upon rhythmic transcription of clock genes and diurnal variation in vascular and metabolic systems. In obesity, clock gene function and physiological rhythms were preserved in the vasculature but clock gene transcription in metabolic tissues and rhythms of glucose tolerance and insulin sensitivity were blunted. The most pronounced attenuation of clock rhythm occurred in adipose tissue, where there was also impairment of clock-controlled master metabolic genes and both AMPK mRNA and protein. Across tissues, clock gene disruption was associated with local inflammation but diverged from impairment of insulin signaling. We conclude that vascular tissues are less sensitive to pathological disruption of diurnal rhythms during obesity than metabolic tissues and suggest that cellular disruption of clock gene rhythmicity may occur by mechanisms shared with inflammation but distinct from those leading to insulin resistance.

FIG. 1.

The effect of obesity on rhythmic transcription of the core clock genes Bmal1 and Per2 in vascular and metabolic tissues. Aorta (A); liver (B); adipose tissue (C); skeletal muscle (D). Results from obese animals are denoted by circles and lean by triangles. Two-way ANOVA with Bonferroni post hoc correction: *P < 0.05 and #P < 0.001; n = 4 in each group.

FIG. 2.

The effect of obesity on diurnal variation in cardiovascular indices. A: Diurnal variation in constriction to PE. B: Diurnal variation in systolic BP. C: Diurnal variation in heart rate. D: Diurnal variation in endothelial-dependent vasodilation to ACh. E: Diurnal variation in endothelial vasodilation; vasodilator response to 3 nmol/L ACh. F: Diurnal variation in eNOS activation (ratio of phospho-eNOS to total eNOS protein). G: Diurnal variation in vasomotor response to insulin. A–D: Results from obese animals are denoted by circles and lean by triangles, and, where appropriate, values at 8:00 a.m. by open symbols and those at 8:00 p.m. by closed symbols. E–G: Obese animals are denoted by checkered bars and lean by solid bars, and 8:00 a.m. values are denoted by gray and 8:00 p.m. by black; in G, open bars denote insulin and filled bars vehicle treatment. Two-way ANOVA: *P < 0.05 and #P < 0.001. A, D, E, and G: n = 8–13 per group. B, C, and F: n = 4–6 per group.

FIG. 3.

The effect of obesity on diurnal variation in response to glucose and insulin challenge at 8:00 a.m. and 8:00 p.m. A: Glucose tolerance test; diurnal response to intraperitoneal glucose challenge in obese and lean animals. B: Glucose tolerance test; diurnal variation in glucose peak at 30 min is blunted in obesity. C: Insulin tolerance test; diurnal response to intraperitoneal insulin challenge in obese and lean animals. D: Insulin tolerance test; diurnal variation in glucose nadir at 60 min is blunted in obesity. A and C: Results from obese animals are denoted by circles and lean by triangles and values at 8:00 a.m. by open symbols and those at 8:00 p.m. by closed symbols. B and D: Obese animals are denoted by checkered bars and lean by solid bars. Student t test: *P < 0.05 and †P < 0.01; n = 16 in each group.

FIG. 4.

The effect of obesity on rhythmic transcription of clock-controlled genes regulating glucose and lipid homeostasis. A: Liver; Rev-erbα, Dbp, Ppar-α, and Pepck. B: Adipose tissue; Rev-erbα, Dbp, Pparα, and Pepck. Results from obese animals are represented by circles and lean by triangles. Two-way ANOVA with Bonferroni post hoc correction: *P < 0.05, †P < 0.01, and #P < 0.001; n = 4 in each group.

FIG. 5.

The effect of obesity on mRNA and protein rhythms of AMPK. A: Liver. B: Adipose tissue. Results from obese animals are represented by circles and lean by triangles. Two-way ANOVA with Bonferroni post hoc correction: *P < 0.05 and †P < 0.01; n = 4–6 in each group.

FIG. 6.

The effect of obesity on rhythmic transcription of adipokines in adipose tissue. A: Leptin. B: Adiponectin. Results from obese animals are represented by circles and lean by triangles. Two-way ANOVA with Bonferroni post hoc correction: *P < 0.05; n = 4 in each group.

FIG. 7.

Expression of inflammatory genes in obesity. A: Macrophage marker F4-80. B: Complement C3. C: VCAM-1 in aorta. D: ICAM-1 in aorta. E: TNF-α in adipose tissue. A–D: Results from obese animals are denoted by checkered bars and lean by solid bars. E: Results from obese animals are represented by circles and lean by triangles. Student t test (A–D); two-way ANOVA with Bonferroni post hoc correction (E): *P < 0.05, †P < 0.01, and #P < 0.001; n = 4 in each group.

FIG. 8.

The effect of obesity on Akt signaling and its diurnal variation at 8:00 a.m. and 8:00 p.m. A: Aorta. B: Liver. C: Adipose tissue. D: Skeletal muscle. Data are presented as expression of phospho-Akt normalized to β-actin or as the ratio of phospho-Akt to total Akt. Results from obese animals are denoted by checkered bars and lean by solid bars, and 8:00 a.m. values are denoted by gray and 8:00 p.m. by black. Student t test: *P < 0.05; n = 6 in each group.