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

Abstract and Introduction

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.

Introduction

Much research in the field of obesity is directed toward the role of obesity in the development of cardiovascular disease and insulin resistance. A critical early step in atherosclerotic cardiovascular disease is endothelial dysfunction, the hallmark of which is impaired nitric oxide (NO) production by endothelial NO synthase (eNOS) in vascular endothelial cells. We and others have shown that obesity is associated with endothelial dysfunction in human[1,2] and animal studies.[3] A further subject of investigation is the sequence of tissue-specific events in obesity that lead to insulin resistance in the canonical insulin-sensitive tissues: liver, adipose tissue, and skeletal muscle. Insulin also acts on the endothelium to promote NO release through a phosphoinositol-3-kinase (PI3K)-dependent pathway,[4] and a causal link is established between vascular insulin resistance and endothelial dysfunction.[5–7] Insulin resistance thus stands at the crossroads of cardiovascular and metabolic disease.

Circadian rhythms are pervasive in physiological processes. In the cell, timekeeping is maintained by an autoregulatory transcriptional-translational oscillator. The nucleus of this mechanism consists of a positive limb, composed of heterodimers of BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator [ARNT]-like) with either CLOCK (circadian locomotor output cycles kaput) or NPAS2 (neuronal PAS domain-containing protein), which promote transcription of PER (period) and CRY (cryptochrome) genes, which then close the negative feedback loop by inhibiting BMAL1 and CLOCK.[8] Rhythm is passed downstream through control of transcription of client clock-controlled genes, allowing the clock to influence a wide nexus of cellular physiology. In the endothelium, there is diurnal variation in NO production[9] and endothelial-dependent vascular tone.[10,11] Blood pressure (BP) and heart rate dip during the inactive or sleep phase. Responses to glucose and insulin challenge display a clear diurnal pattern[12] and many gate-keeping enzymes in metabolic pathways are under clock control.[13] There is some evidence that components of intracellular insulin signaling pathways, such as PI3K and its downstream kinase Akt, are similarly regulated by the clock,[14] but diurnal variation in these pathways is incompletely characterized.

Evidence is mounting that normal physiological rhythm may be lost in disease.[15] In transgenic mouse models, mutation of core clock genes leads to endothelial dysfunction[14,16] and obesity[17] with abnormal systemic glucose and insulin homeostasis.[18] Human genetic studies report associations between polymorphisms of CLOCK and obesity,[19] and BMAL1 and diabetes and hypertension.[20] Diet-induced obesity in wild-type mice leads to secondary blunting of rhythmic clock gene transcription in liver and adipose tissue[21] and in energy-sensing hypothalamic regions.[22] These studies complement old observations of a coexistence between rhythm loss and disease in humans. Obese humans show blunting of the normal diurnal variation in response to glucose challenge.[23] Nondipping, or loss of normal diurnal variation in BP, is associated with diabetes[24] and is linked to hypertensive complications.[25] There is a rhythm of onset of myocardial infarction, stroke, and other adverse cardiovascular events, which cluster in the early hours of the morning when endothelial reactivity falls and BP and hemostatic activity rise,[26] which too is lost in diabetes.[27]

Although knowledge of the association between rhythm loss and cardiovascular and metabolic disease is longstanding, it remains nonetheless poorly explored. As yet, no studies have examined the effect of obesity upon normal physiological variation in vascular function or upon rhythmic transcription of core clock genes in cardiovascular tissues. It is not known whether tissues vary in their sensitivity to clock disruption in disease. It is unknown whether the loss of clock rhythm occurs in conjunction with other pathological events in obesity, notably insulin resistance and inflammation. In a C57BL6J mouse model of diet-induced obesity, we examined the effect of obesity upon 1) rhythmic transcription of core clock genes; 2) diurnal variation in physiological measures of vascular and metabolic function; 3) rhythmic transcription of metabolic master regulatory genes; 4) rhythms of AMP-activated protein kinase (AMPK) mRNA and protein; 5) inflammation; and 6) insulin signaling and its diurnal variation in tissues.

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