Diurnal Variation in Vascular and Metabolic Function in Diet-induced Obesity

Divergence of Insulin Resistance and Loss of Clock Rhythm

Madhu J. Prasai; Romana S. Mughal; Stephen B. Wheatcroft; Mark T. Kearney; Peter J. Grant; Eleanor M. Scott

Disclosures

Diabetes. 2013;62(6):1981-1989. 

In This Article

Results

After 10 weeks of high-fat diet, animals were significantly obese in comparison with chow-fed controls (41.8 ± 0.3 vs. 29.8 ± 0.2 g, P < 0.0001) (Supplementary Fig. 2A). Abdominal obesity was pronounced (epididymal fat pad mass 1.30 ± 0.02 vs. 0.34 ± 0.01 g, P < 0.0001) (Supplementary Fig. 2B), and obese animals were markedly hyperinsulinemic over the 24-h day in comparison with controls (Supplementary Fig. 2C).

Figure 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. AD: 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. EG: 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.

Rhythmic Transcription of Core Clock Genes is not Impaired in Cardiovascular Tissues of Obese Mice But is Attenuated in Some Metabolic Tissues

There was no significant impairment of cycling of Bmal1 and Per2 in aorta (Fig. 1A). Adipose tissue showed marked and significant attenuation of cycling of Bmal1 (two-way ANOVA interaction F = 7.24, P < 0.01; obesity F = 4.86, P < 0.05; time F = 441.16, P < 0.001) and Per2 (two-way ANOVA interaction F = 6.64, P < 0.01; obesity F = 56.25, P < 0.001; time F = 44.58, P < 0.001) (Fig. 1C), but there was no significant disruption of rhythm in liver or muscle (Fig. 1B and D). There was no diurnal variation in the reference gene Gapdh and its expression did not differ between obese and lean animals (data not shown).

Diurnal Variation in Cardiovascular Physiological Measures is Preserved in Obesity

Lean aortas showed diurnal variation in constriction to stimulation with PE (maximal constriction at 8:00 A.M. 1.10 ± 0.08 vs. 0.94 ± 0.05 g at 8:00 P.M.) (Fig. 2A), and this variation was not attenuated by obesity (maximal constriction at 8:00 A.M. 0.54 ± 0.03 vs. 0.44 ± 0.04 g 8:00 P.M.; ANOVA P < 0.001) (Fig. 2A). Obese aortas showed marked impairment of constriction to PE, which was reversed by stimulation with the nonspecific NOS inhibitor L-NMMA (constriction at 8:00 A.M.: obese 1.03 ± 0.07 with and 0.49 ± 0.07 g without L-NMMA; lean 1.37 ± 0.08 with and 0.99 ± 0.11 g without L-NMMA; P < 0.001; data not shown). Expression of inducible NO synthase (iNOS) mRNA was increased in obese aortas over 24 h (data not shown). The daily pattern of BP did not differ significantly between obese and lean animals (Fig. 2B). Heart rate was significantly elevated in obese animals (two-way ANOVA, obesity F =18.27, P < 0.001) (Fig. 2C) with no impairment of diurnal variation. Diurnal variation in endothelial-dependent vasodilation to ACh was less evident (Fig. 2D), but there was statistically significant diurnal variation in the early phase of the curve, which was preserved in obese aortas; upon stimulation with 3 nmol/L ACh, there was a convincing vasodilator response at 8:00 P.M. (13.7 ± 3.3% in lean vs. 13.4 ± 2.4% in obese) but minimal vasodilation at 8:00 A.M. (0% in lean vs. 1.5 ± 0.9% in obese, P < 0.001) (Fig. 2E). There was no diurnal variation in endothelial-independent vasodilation to the NO donor sodium nitroprusside (data not shown). Both lean and obese aortas showed rhythmic phosphorylation of eNOS with an increase in the ratio of Ser1177 phospho-eNOS to total eNOS at 8:00 P.M.; however, this rhythm was not statistically significant in either group (Fig. 2F). Phospho-eNOS abundance was mildly reduced in obesity at both time points. Obese aortas were insulin resistant and showed no significant attenuation of constriction in response to insulin treatment at either time point (Fig. 2G). There was no evident diurnal variation in the magnitude of response to insulin in lean aortas.

Diurnal Variation in Systemic Glucose and Insulin Homeostasis is Impaired in Obesity

A distinct diurnal pattern of response to glucose challenge was seen both in lean and obese mice (ANOVA P < 0.001) (Fig. 3A); at 8:00 A.M., fasting blood glucose was lower but 30-min glucose peak was higher than at 8:00 P.M. Diurnal variation was further expressed as the percentage difference between the maximal 30-min glucose peaks attained at 8:00 A.M. vs. 8:00 P.M. (Fig. 3B). Obese animals showed blunting of this diurnal variation in peak glucose (Student t test P < 0.05). Insulin sensitivity also showed diurnal variation with increased sensitivity to insulin challenge at 8:00 P.M., both in lean and obese animals (ANOVA P < 0.001) (Fig. 3C). The percentage difference between the 60-min glucose nadirs attained at 8:00 A.M. vs. 8:00 P.M. revealed significant blunting of diurnal variation in insulin sensitivity in obesity (Student t test P < 0.01) (Fig. 3D).

Figure 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.

Rhythmic Cellular Metabolism is Dysregulated in Obesity

Dysregulation of rhythmic clock gene transcription in metabolic tissues due to obesity was associated with effects upon downstream clock-controlled genes with major roles in control of glucose and lipid metabolism: Rev-erbα, Dbp (D-site albumin promoter binding protein), Pparα (peroxisome proliferator-activated receptor α), and Pepck (phosphoenolpyruvate carboxykinase). In adipose tissue, there was marked and statistically significant attenuation of rhythmic transcription of all genes in obese animals (two-way ANOVA: Rev-erbα interaction F = 8.15, P < 0.001; obesity F = 14.41, P < 0.001; time F = 11.61, P < 0.001; Dbp obesity F = 76.58, P < 0.001; time F = 8.52, P < 0.01; Pparα interaction F = 4.22, P < 0.05; obesity F = 13.69, P < 0.01; time F = 5.21, P < 0.01; Pepck obesity F = 33.76, P < 0.001; time F = 3.37, P < 0.05) (Fig. 4B). Rhythms of AMPK were significantly blunted both in mRNA (two-way ANOVA obesity F = 7.74, P < 0.05) and protein (two-way ANOVA interaction P = NS; obesity F = 5.34, P < 0.05; time F = 4.92, P < 0.01) (Fig. 5B), and peak expression of protein lagged 6 h behind that of mRNA. In liver, rhythmic gene transcription was largely unaffected by obesity, with the exception of Pepck, which was significantly blunted in obese animals (two-way ANOVA interaction P = NS; obesity F = 8.63, P < 0.01; time F = 3.20, P < 0.05) (Fig. 4A). There were significant differences between obese and lean in protein levels (obesity F = 12.89, P < 0.001) but not mRNA of AMPK (Fig. 5A).

Figure 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.

Figure 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.

Expression of Adipokines is Dysregulated in Obesity

We investigated the effect of obesity upon diurnal profiles of transcription of the adipokines leptin and adiponectin, which are known to be under clock control. Transcription of leptin was significantly upregulated in obese animals over the full 24-h day (two-way ANOVA obesity F = 18.76, P < 0.001) (Fig. 6A) and that of adiponectin was downregulated in comparison with lean (two-way ANOVA obesity F = 5.35, P < 0.05) (Fig. 6B). Diurnal variation was not impaired.

Figure 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.

Local Inflammation in Obesity is Most Pronounced in Adipose Tissue

F4-80, a marker of macrophage infiltration, was markedly upregulated in obese adipose tissue but not in other tissues (P < 0.001) (Fig. 7A). The complement protein C3 was expressed most strongly in liver, consistent with its predominant hepatic synthesis, but was upregulated in obesity only in adipose tissue (P < 0.01) (Fig. 7B). Neither gene showed diurnal variation in transcription in any tissue (data not shown). In aorta, the vascular inflammatory marker VCAM-1 (vascular cell adhesion molecule 1; P < 0.05) (Fig. 7C) was upregulated in obesity but ICAM-1 (intercellular adhesion molecule) (Fig. 7D) and E-selectin (data not shown) did not differ between obese and lean. Expression of TNF-α was markedly and significantly elevated in obese adipose tissue (two-way ANOVA interaction F = 1.66, P = NS; obesity F = 15.48, P < 0.001; time F = 0.93, P = NS) (Fig. 7E).

Figure 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. AD: 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 (AD); two-way ANOVA with Bonferroni post hoc correction (E): *P < 0.05, †P < 0.01, and #P < 0.001; n = 4 in each group.

Obesity is Associated With Impairment of Insulin Signaling in Liver and Aorta but not in Adipose Tissue or Skeletal Muscle

Diurnal variation in insulin signaling did not show statistically significant differences but aorta and adipose tissue showed a trend toward increased Thre308 phosphorylation of Akt at 8:00 P.M. (Fig. 8A and C). Abundance of the control protein β-actin did not show diurnal variation (data not shown). Impairment of insulin signaling was found both in vascular and metabolic systems, with significantly reduced phospho-Akt abundance in the aorta and liver of obese animals (P < 0.05) (Fig. 8A and B) but no statistically significant changes in adipose tissue or muscle (Fig. 8C and D). The ratio of phospho-Akt to total Akt was not significantly altered in obesity, since in liver and aorta, abundance of total Akt as well as of phospho-Akt was reduced in obesity (data not shown).

Figure 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.

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