SIRT1 in the Ventromedial Hypothalamus: A Nutrient Sensor Input Into the Internal Timekeeper

There was a time when talking about clocks, rhythms, and timing of meals led to glazed eyes from colleagues in the field of diabetes and obesity (“diabesity”). The main focus at the turn of this century was on using newly developed molecular techniques to interrogate form and function of neural circuits linked to the control of appetite and metabolism. The problem of obesity and the metabolic conditions associated with nutrient excess facing most nations was, and likely still is by most, primarily viewed as a failure to coordinate energy consumption and energy expenditure (“energy homeostasis”). However, the publication in Science in 2005 of an obese phenotype in mice carrying the Δ19 mutation in the Circadian Locomotor Output Cycles Kaput (Clock) gene was important for bringing what is essentially an old idea back into the consciousness of those interested in how weight and metabolic health are maintained (1).

The ClockΔ19 mutation was produced during an N-ethyl-N-nitrosourea mutagenesis screening project; homozygous carriers of the mutation were identified by their inability to maintain a circadian rhythm in wheel-running behavior when housed in constant dark (2–4). These mice exhibited normal diurnal rhythms in a light-dark setting, suggesting that the protein produced by the locus is involved in maintaining an internal timing mechanism driving a circadian rhythm in the absence of photic cues. However, the use of infrared beams to measure ambulatory activity led to Turek et al identifying other roles related to energy homeostasis (1). They observed subtle increases in ambulatory behavior in the lights-on period and reduced activity at the onset of the dark period. More importantly, they observed far less subtle differences in energy balance. Diurnal rhythms in energy intake and energy expenditure were markedly dampened while energy expenditure was reduced by 10%. ClockΔ19 mutants maintained on rodent chow also exhibited fasting hyperglycemia, dyslipidemia, and enhanced diet-induced obesity, suggesting metabolic dysfunction.

The significance of this paper, at least for these authors, was in reintroducing circadian rhythms as a homeostatic concept. We say “reintroducing” because the concept of an anticipatory phase where foraging behavior and metabolic activity are optimized to coincide with nutrient consumption had been discussed for decades before the advent of molecular biology and the cloning of genes involved in obesity, metabolism, and circadian function (5, 6). Moreover, the work of other laboratories too numerous to cite in this short commentary had already revealed an intricate and innate connection between nuclear proteins that act as nutrient sensors with the core circadian oscillators responsible for the rhythms we all experience on a daily basis (7–9). In particular, the finding that circadian oscillators residing outside of the “master clock” in the suprachiasmatic nucleus (SCN) respond to nutrient sensors was important for suggesting clock functions in peripheral tissues that are independent of a central time-keeping mechanism (10). The core elements of the circadian oscillator system are now viewed as valid targets for developing pharmacotherapies against a range of ailments that includes diabetes and obesity (11, 12).

Much remains unclear about how circadian systems are organized, whereas the processes involved in how organisms coordinate a response to caloric cues to express rhythms anticipating mealtime are poorly understood (6, 7). In this issue of Endocrinology, Orozco-Solis et al explored the role of Sirtuin 1 (SIRT1) expressed in steroidogenic factor-1 (SF1)-positive neurons in the ventromedial hypothalamus (VMH) (13). SIRT1 is a nicotinamide adenine dinucleotide (NAD) (NAD+)-dependent deacytelase that counteracts the histone acetyltransferase activity of CLOCK (14, 15) and is considered a focal point in the interaction between metabolic condition and the core circadian oscillator complex.

Previous evaluation of the role of SIRT1 in the neurobehavioral adaptive response to dietary restriction using transgenic mice overexpressing SIRT1 in the brain and Sirt1 knockout mice suggested that SIRT1 in the dorsomedial and lateral hypothalamus may mediate increased physical activity during dietary restriction (16). In the current study, the authors used a strain of mice carrying a LoxP-flanked Sirt1 allele (Sirt1^loxP) to selectively target expression of the gene in the VMH. This strategy had already been used to target Sirt1 expression in the central nervous system. Deletion of Sirt1 in neurons expressing proopiomelanocortin results in an impaired ability to defend against weight gain (17). Sirt1 expressed agouti-related peptide neurons is required for the normal response to orexigenic inputs (18). Agouti-related peptide and proopiomelanocortin neurons are core components of the central nervous melanocortin system involved in regulating energy balance, acting to control both appetite and energy intake and the autonomic outputs that govern metabolic activity in the periphery (19).

The SF1-Cre strain has been used extensively as a tool to dissect signaling pathways residing in the VMH. Indeed, the SF1-Cre strain had already been used to target Sirt1 expression in the VMH; these results suggested a role for SIRT1 expressed in SF1 neurons in the defense of body weight and maintaining insulin sensitivity in skeletal muscle (20). The VMH has long been considered an important hypothalamic center in the control of energy homeostasis. SF1 neurons in the VMH are primarily glutamatergic, and the excitatory properties of these neurons are required for the counter regulatory response to hypoglycemia induced by insulin and to maintain blood glucose levels during fasting (21). Neurons expressing Cre activity in the SF1-Cre strain define a population residing in predominantly in the dorsomedial part of the VMH that send projections throughout the brain, including areas in the brainstem governing autonomic outputs, which likely mediate metabolic actions (22). Within the hypothalamus, SF1 neurons project to sites known to influence appetite and energy expenditure, including POMC neurons in the arcuate nucleus and the medial portion of paraventricular nucleus that receives projections from POMC neurons residing the arcuate nucleus. Of particular relevance to the study by Orozco-Solis et al, SF1 neurons innervate the outer shell of the SCN providing a basis for communicating metabolic signals integrated by VMH neurons to the master clock.

The authors first determined whether suppression of SIRT1 in SF1 neurons alters activity of the master clock. It did not; SF1-Cre;Sirt^loxP/loxP mice exhibited a normal circadian rhythm when released into constant dark. They also observed no change when the mice were fed a high-fat diet (HFD) (60% kJ/fat), a challenge previously shown to suppress SIRT1 expression and to reprogram the endogenous clock. The authors then compared the response of SF1-Cre;Sirt^loxP/loxP and control mice with a restricted feeding paradigm, initially in a light-dark setting and then released into constant dark. For this procedure, food availability was limited between zeitgeber time 6 and 10. Again, the authors compared the response of standard diet (presumably rodent chow) and the HFD.

At this stage, it is worth reminding the reader about the consequences of this restricting feeding paradigm for the mouse. This feeding protocol forces mice to adapt behaviorally and metabolically to a cycle of binge eating and a protracted period of fasting that lasts for 20 hours. When provided food access, mice binge and consume 50%–60% of the caloric load in the first hour (23). The mice also rapidly switch from oxidizing fatty acids released from triacylglycerol stores that are required to bridge the gap in energy provided by exogenous source to using the calories provided by the meal. The cycle of prolonged fasting and rapid caloric loading also instigates metabolic adaptation that may also involve nutrient-responsive neurons in the hypothalamus (24, 25). Lipogenesis may be activated as a means to efficiently store energy for the long interval between meals (26). Mice adapt behaviorally by expressing foraging behaviors to coincide with the time of food presentation, exhibiting food anticipatory activity. Food anticipatory activity is thought to involve a distributed network of “food entrainable oscillators” that facilitate the synchronization of rhythms anticipating nutrient availability (6).

SF1-Cre;Sirt^loxP/loxP mice exhibited altered rhythms in locomotor activity compared with controls when subjected to the restricted feeding protocol, particularly when exposed to HFD. Rhythms in the expression of core components of the circadian oscillator were also altered in the SCN and VMH of SF1-Cre;Sirt^loxP/loxP mice subjected to restricted feeding. These are important results, implying that SIRT1 expressed in SF1 neurons is required for adapting rhythms to phase changes in nutrient availability. Cyclic changes in Sirt1 activity in the VMH could conceivably mediate some or all of these actions by sensing metabolic state locally, perhaps in response to changes in the ratio of nicotinamide adenine dinucleotide and its reduced form ratio associated with cycles of fasting and caloric loading. Although some may argue that a basic tenet of homeostasis is that energy sources derived from peripheral stores provide the brain with a constant influx of nutrients during situations of negative energy balance, there is evidence for changes in SIRT1 protein levels that has been observed in peripheral tissues, with an increase in protein levels during fasting (27). An alternative explanation is that Sirt1 could be required for synaptic plasticity of SF1 neurons in the VMH, with the absence of SF1 resulting in a loss of response of these neurons to signals of metabolic state (18).

The studies described by Orozco-Solis et al represent a significant advance in our understanding of the molecular basis for a nutrient entrainable oscillator within the hypothalamus. Regions of the hypothalamus such as the VMH and dorsomedial hypothalamus have been implicated as the source of the nutrient entrainable clocks, but until now, the identity of critical molecular nutrient/energy status sensors feeding into this clock was not known. SIRT1 is clearly a good candidate for such a role given its energy status specific activity as well as circadian regulation. With this study, SIRT1's role in such a nutrient entrainable clock within the VMH is solidified, but it is very likely that this clock is much more complex and will have many more “moving” parts.