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

Discussion

This study demonstrates novel findings with regard to the role of circadian clock dysfunction in the pathophysiology of vascular and metabolic disease in obesity. 1) Loss of diurnal rhythm associated with obesity is found in measured physiological indices in the metabolic system but not in the cardiovascular system, which corresponds to the preservation of core clock gene cycling in vascular tissues but disruption in some metabolic tissues. 2) Adipose tissue is most vulnerable to clock gene disruption secondary to obesity, which is associated with marked disruption of downstream clock-regulated genes in cellular metabolic homeostasis including AMPK and of AMPK protein. 3) There is divergence between rhythm loss and impairment of tissue insulin signaling, with adipose tissue most sensitive to rhythm loss and liver to insulin resistance, suggesting that insulin resistance and clock gene dysfunction arise by different mechanisms. 4) Tissue inflammation coincides with rhythm loss, suggesting possible common influences on inflammation and the cellular process of clock gene dysfunction.

Preservation of Diurnal Variation in Cardiovascular Measures in Obesity

The diurnal variation observed in this study is consistent with previous reports of increased endothelial responsiveness during the active period. Thus at 8:00 P.M., we found reduced vasoconstriction, increased NO-dependent vasodilation, and a trend toward greater phosphorylation of eNOS. The aortic vasomotor phenotype of our obese mice differs from other studies that have reported impaired endothelial-dependent vasodilation, marked eNOS protein dysfunction, hyperconstriction, and hypertension.[30,31] However, the findings of our study are consistent with our previous report of an obese vascular phenotype characterized by upregulation of iNOS in obese aortas, leading to increased NO production by iNOS rather than eNOS, hypoconstriction, and an exaggerated response to the NOS inhibitor L-NMMA.[3] Tumor necrosis factor-α (TNF-α) and leptin were found to be elevated and make a possible link between obese adipose tissue and the vasculature. Both are circulating mediators secreted by adipose tissue that are known to cause endothelial dysfunction, and furthermore, TNF-α has been shown to induce iNOS expression.[32,33]

Two alternatives may explain why diurnal rhythms were preserved in the cardiovascular system despite clear disruption of metabolic indices: vascular tissues are either resilient to disruption of the clock or they require longer exposure to develop the pathological effects of obesity upon diurnal variation. Hsieh et al.[34] found in mice that exposure to long-term high-fat diet for 11 months resulted in disruption of clock gene transcription in the liver and kidney, which was not evident with a shorter duration of obesity. Nondipping BP is associated with diabetes in humans but was not seen in these obese mice despite hyperglycemia and hyperinsulinemia consistent with type 2 diabetes, perhaps because a longer period of obesity is required. It is consistent with the narrative of cardiovascular disease caused by metabolic disease that loss of rhythm should occur first in metabolic tissues and subsequently appear in the vasculature.

Loss of Diurnal Variation in Metabolic Indices

Diurnal variation in insulin sensitivity was best seen in responses to metabolic challenge, consistent with previous reports of heightened insulin sensitivity during the active period, but diurnal variation was less clear in aortic vasomotion and Akt signaling. The primary mechanism by which obesity altered Akt signaling appears to be through suppression of the expression of Akt protein and not through impairment of its phosphorylation by upstream kinases in the insulin signaling pathway. Although there was a convincing loss of rhythm in glucose tolerance and insulin sensitivity in obesity, identification of the insulin-sensitive tissue primarily responsible for this defect is complex. Tissue insulin resistance, as measured by impairment of Akt signaling, was greatest in liver, but loss of rhythm was greatest in adipose tissue. Skeletal muscle demonstrated neither insulin resistance nor disruption of clock gene rhythms. The two major tissue events contributing to the loss of systemic insulin sensitivity are held to be failure to suppress hepatic glucose output and impairment of skeletal muscle glucose uptake in response to insulin stimulation, and adipose accounts for ~10% of insulin-induced glucose disposal and is thought to have a minor role in systemic glucose homeostasis.[35] Interestingly, rhythmic transcription of Pepck was disrupted in liver despite tight preservation of clock gene rhythms in this tissue.

Metabolic Master Genes and AMPK Make a Bidirectional Link Between the Clock and Metabolism

The link between the clock and cellular metabolism is an evolving area of research. Evidence of the intimate link between clock function and cellular energy balance comes from the discovery of NAMPT, NAD+,[36] and cAMP[37] as clock inputs and of heme as the ligand activating the accessory clock protein REV-ERBα.[38] Numerous master metabolic genes with wide-ranging effects in determining systemic glucose and lipid homeostasis are known to display rhythmic transcription,[39] and in this study, we confirm the findings of Kohsaka et al.[21] of the deleterious effect of obesity upon their diurnal profiles in metabolically active tissues. AMPK is of special interest because it embodies the two-way traffic between the clock and cellular redox status. As an energy-sensing kinase, it is activated by a rise in the intracellular AMP:ATP ratio indicative of falling energy stores; by direct binding to clock proteins, it enzymatically alters their stability,[40] and transcriptome studies suggest that AMPK itself may be clock controlled.[41] Its role in promoting systemic insulin sensitivity and impairment of its expression and activity in diet-induced obesity are recognized.[42,43] Although the effect of obesity upon AMPK from the perspective of diurnal variation has received some attention,[44,45] this study presents the first evidence of AMPK rhythms and loss of these rhythms in obesity. The 6-h lag observed between the peaks of mRNA and protein in this study is consistent with a delay required for translation.

Mechanism of Cellular Clock Disruption in Obesity

The finding of divergence of insulin resistance and loss of diurnal rhythm is novel and prompts discussion of how disruption of the clock fits into the context of what is already known about the cellular pathogenesis of obesity. Of the tissues studied, adipose was the most susceptible to cellular clock disruption, which suggests that the local adipose milieu in obesity may be especially pathogenic to the clock. Our finding of divergence between tissue-specific insulin resistance and inflammation is consistent with an elegant study that reported that in diet-induced obesity, insulin resistance developed early in liver but late in adipose tissue, and inflammation was concentrated in adipose tissue.[46] Broad convergence of inflammation and clock dysfunction raises the question of whether the two processes may share a common pathway and merits further investigation.

Implications of This Study

This animal study raises points directly applicable to human disease. Few studies have examined the effect of obesity on human clock gene function because of the requirement for repeated and invasive tissue sampling.[47,48] Otway et al.[47] did not find abnormalities of rhythmic transcription of clock genes in adipose tissue of obese humans, although this may be due to sampling of subcutaneous adipose tissue, which is less strongly linked to insulin resistance and dyslipidemia than visceral adipose tissue.[49] We found only mild impairment of core clock genes and metabolic genes in subcutaneous adipose tissue (data not shown). Indeed, differences in clock gene expression have been reported between visceral and subcutaneous depots in human adipose explants.[50] The prevalence of loss of diurnal rhythm in obesity and type 2 diabetes is not known and it remains to be seen whether it is an integral feature of acquired metabolic disease in human populations. Although loss of rhythm in the cardiovascular system was not found in these mice, it is possible that in humans with chronic vasculopathy, loss of endothelial rhythms may be found in conjunction with atherosclerosis. AMPK is further of interest because several pharmacological agents used in the treatment of diabetes, such as metformin and thiazolidinediones, exert their effects through its activation. Our findings prompt further investigation in human disease.

Conclusion

This study establishes important differences in the susceptibility of vascular and metabolic tissues to pathological loss of diurnal variation in diet-induced obesity. The novel finding that tissue-specific clock disruption occurs in conjunction with inflammation but not with insulin resistance demands further investigation of the underlying cellular mechanisms. We argue that loss of diurnal rhythm is an integral component of the pathophysiology of obesity and deserves closer attention.

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