A. Kalsbeek

SCN outputs and the hypothalamic balance of life

The circadian clock in the suprachiasmatic nucleus (SCN) is composed of thousands of oscillator neurons, each dependent on the cell-autonomous action of a defined set of circadian clock genes. Still, the major question remains how these individual oscillators are organized into a biological clock producing a coherent output able to time all the different daily changes in behavior and physiology. In the present review, the authors discuss the anatomical connections and neurotransmitters used by the SCN to control the daily rhythms in hormone release. The efferent SCN projections mainly target neurons in the medial hypothalamus surrounding the SCN. The activity of these preautonomic and neuroendocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together, the data on the SCN control of neuroendocrine rhythms provide clear evidence not only that the SCN consists of phenotypically (i.e., according to neurotransmitter content) different subpopulations of neurons but also that subpopulations should be distinguished (within phenotypically similar groups of neurons) based on the acrophase of their (electrical) activity. Moreover, the specialization of the SCN may go as far as a single body structure, that is, the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.

A Suprachiasmatic Nucleus Generated Rhythm In Basal Glucose Concentrations

The daily rhythm in feeding activity in mammals, as driven by the biological clock, largely determines the daily fluctuations in basal concentrations of glucose and insulin. To investigate a possible direct impact of the suprachiasmatic nucleus (SCN) on these parameters, we subjected intact rats and SCN‐lesioned rats to a fasting regimen of 36u2003h, or to a scheduled feeding regimen of six identical meals equally distributed over the light:dark‐cycle. Plasma profiles of glucose and insulin in rats during the final 24u2003h of the 36u2003h of fasting, and in rats subjected to the scheduled feeding regimen were compared to profiles in rats fed ad libitum. In rats fed ad libitum, in fasted rats and in rats subjected to a scheduled feeding regimen basal glucose concentrations showed a pronounced 24‐h rhythm that was not found in rats that had been SCN‐lesioned. Basal insulin levels showed a 24‐h rhythm in 50% of the rats fed ad libitum and in 50% of the rats subjected to a scheduled feeding regimen; neither rhythms were present in SCN‐lesioned rats. However, none of the fasted rats showed a 24‐h rhythm in basal insulin concentrations. These data provide clear evidence that the SCN directly controls basal glucose concentrations independent of its influence on feeding activity. At the same time, we found no consistent evidence for a strong impact of the SCN on basal insulin concentrations.

White adipose tissue: getting nervous.

Neuroendocrine research has altered the traditional perspective of white adipose tissue (WAT) as a passive store of triglycerides. In addition to fatty acids, WAT produces many hormones and can therefore be designated as a traditional endocrine gland actively participating in the integrative physiology of fuel and energy metabolism, eating behaviour and the regulation of hormone secretion and sensitivity. WAT is controlled by humoral factors, para‐ and intracrine factors and by neural regulation. Sympathetic nerve fibres innervate WAT and stimulate lipolysis, leading to the release of glycerol and free fatty acids. In addition, recent research in rats has clearly shown a functional parasympathetic innervation of WAT. There appears to be a distinct somatotopy within the parasympathetic nuclei: separate sets of autonomic neurones in the brain stem innervate either the visceral or the subcutaneous fat compartment. We therefore propose that the central nervous system (CNS) plays a major role in the hitherto unexplained regulation of body fat distribution. Parasympathectomy induces insulin resistance with respect to glucose and fatty acid uptake in the innervated fat depot and has selective effects on local hormone synthesis. Thus, the CNS is involved not only in the regulation of hormone production by WAT, but also in its hormone sensitivity. The developments in this research area are likely to increase our insights in the pathogenesis of metabolic disorders such as hypertriglyceridemia, diabetes mellitus type 2 and lipodystrophy syndromes.

Role for the Pineal and Melatonin in Glucose Homeostasis: Pinealectomy Increases Night-Time Glucose Concentrations

The effects of melatonin on glucose metabolism are far from understood. In rats, the biological clock generates a 24‐h rhythm in plasma glucose concentrations, with declining concentrations in the dark period. We hypothesized that, in the rat, melatonin enhances the dark signal of the biological clock, decreasing glucose concentrations in the dark period. We measured 24‐h rhythms of plasma concentrations of glucose and insulin in pinealectomized rats fed ad libitum and subjected to a scheduled feeding regimen with six meals equally distributed over the light/dark cycle and compared them with previous data of intact rats. Pinealectomy dampened the amplitude of the 24‐h rhythm in plasma glucose concentrations in rats fed ad libitum, and abolished it completely in rats subjected to the scheduled feeding regimen, while plasma insulin concentrations did not change under both conditions. Pinealectomy abolished the nocturnal decline in plasma glucose concentrations irrespective of whether rats were fed ad libitum or subjected to the scheduled feeding regimen. Melatonin replacement restored 24‐h mean plasma glucose concentrations in pinealectomized rats that were subjected to the scheduled feeding regimen but, interestingly, it did not restore the 24‐h rhythm. Melatonin treatment also resulted in higher meal‐induced insulin responses, probably mediated via an increased sensitivity of the β‐cells. Taken together, our data demonstrate that the pineal hormone, melatonin, influences both glucose metabolism and insulin secretion from the pancreatic β‐cell. The present study also demonstrates that removal of the pineal gland cannot be compensated by mimicking plasma melatonin concentrations only.

A network of (autonomic) clock outputs.

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each of which is dependent on the cell‐autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock producing a coherent output that is able to time all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre‐autonomic and neuro‐endocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together our data indicate that, with regard to the timing of their main release period within the light‐dark (LD) cycle, at least 4 subpopulations of SCN neurons should be discerned. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of 4 differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure; i.e., the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.

The diurnal modulation of hormonal responses in the rat varies with different stimuli

The circadian clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus not only controls the basal daily temporal organization of many neuroendocrine functions, but also its responsiveness. We studied the time‐of‐day influence on plasma changes in adrenocorticotropic hormone (ACTH), corticosterone, glucagon and leptin concentrations elicited by an insulin‐induced hypoglycaemic event. Male Wistar rats were exposed to an insulin challenge at six different times during the light/dark cycle. The time‐of‐day of exposure markedly affected the responses of all four hormones studied. Generally, the magnitude of the different hormone responses correlated with their basal daily release pattern (i.e. the responses of ACTH and corticosterone were largest around lights off, and glucagon and leptin responses were most pronounced during the dark period). With regard to the hormones of the hypothalamic‐pituitary‐adrenal axis, the presently reported time‐of‐day dependent modulation is completely opposite to that previously reported for novelty or restraint. Therefore, these findings provide further support for the existence of at least two different neural pathways that are able to activate the hypothalamic‐pituitary‐adrenal axis, and provide different substrates for modulation by the biological clock. This observation warrants a thorough examination of possible functional explanations for the observed differences.

Unaltered Instrumental Learning and Attenuated Body-Weight Gain in Rats During Non-rotating Simulated Shiftwork

Exposure to shiftwork has been associated with multiple health disorders and cognitive impairments in humans. We tested if we could replicate metabolic and cognitive consequences of shiftwork, as reported in humans, in a rat model comparable to 5 wks of non-rotating night shifts. The following hypotheses were addressed: (i) shiftwork enhances body-weight gain, which would indicate metabolic effects; and (ii) shiftwork negatively affects learning of a simple goal-directed behavior, i.e., the association of lever pressing with food reward (instrumental learning), which would indicate cognitive effects. We used a novel method of forced locomotion to model work during the animals normal resting period. We first show that Wistar rats, indeed, are active throughout a shiftwork protocol. In contrast with previous findings, the shiftwork protocol attenuated the normal weight gain to 76u2009±u20098u2009g in 5 wks as compared to 123u2009±u200915u2009g in the control group. The discrepancy with previous work may be explained by the concurrent observation that with our shiftwork protocol rats did not adjust their between-work circadian activity pattern. They maintained a normal level of activity during the “off-work” periods. In the control experiment, rats were kept active during the dark period, normally dominated by activity. This demonstrated that forced activity, per se, did not affect body-weight gain (mean±SEM: 85u2009±u200911u2009g over 5 wks as compared to 84u2009±u200911u2009g in the control group). Rats were trained on an instrumental learning paradigm during the fifth week of the protocol. All groups showed equivalent increases in lever pressing from the first (3.8u2009±u2009.7) to the sixth (21.3u2009±u20092.4) session, and needed a similar amount of sessions (5.1u2009±u2009.3) to reach a learning criterion (≥27 out of 30 lever presses). These results suggest that while on prolonged non-rotating shiftwork, not fully reversing the circadian rhythm might actually be beneficial to prevent body-weight gain and cognitive impairments. (Author correspondence: C.Leenaars@nin.knaw.nl)

Timing of fat and liquid sugar intake alters substrate oxidation and food efficiency in male Wistar rats

In addition to the amount of ingested calories, both timing of food intake and meal composition are determinants of body weight gain. However, at present, it is unknown if the inappropriate timing of diet components is responsible for body weight gain. In the present study, we therefore studied a time-dependent effect of the diet composition on energy homeostasis. Male Wistar rats were subjected to chow ad libitum (chow group) or a choice diet with saturated fat, a 30% sugar solution, chow and tap water. The choice diet was provided either with all components ad libitum (AL), with ad libitum access to chow, tap water and a 30% sugar solution, but with access to saturated fat only during the light period (LF), or with ad libitum access to chow, tap water and saturated fat, but access to a 30% sugar solution only during the light period (LS). Caloric intake and body weight gain were monitored during 31 days. Energy expenditure was measured in the third week in calorimetric cages. All rats on a choice diet showed hyperphagia and gained more body weight compared to the chow group. Within the choice diet groups, rats on the LS diet were most food efficient (i.e. gained most body weight per ingested calorie) and showed a lower respiratory exchange ratio (RER) with an anti-phasic pattern, whereas no differences in locomotor activity or heat production were found. Collectively these data indicate that the timing of the diet composition affects food efficiency, most likely due to a shifted oxidation pattern, which can predispose for obesity. Further studies are underway to assess putative mechanisms involved in this dysregulation.

Suprachiasmatic Nucleus Interaction with the Arcuate Nucleus; Essential for Organizing Physiological Rhythms

Abstract The suprachiasmatic nucleus (SCN) is generally considered the master clock, independently driving all circadian rhythms. We recently demonstrated the SCN receives metabolic and cardiovascular feedback adeptly altering its neuronal activity. In the present study, we show that microcuts effectively removing SCN-arcuate nucleus (ARC) interconnectivity in Wistar rats result in a loss of rhythmicity in locomotor activity, corticosterone levels, and body temperature in constant dark (DD) conditions. Elimination of these reciprocal connections did not affect SCN clock gene rhythmicity but did cause the ARC to desynchronize. Moreover, unilateral SCN lesions with contralateral retrochiasmatic microcuts resulted in identical arrhythmicity, proving that for the expression of physiological rhythms this reciprocal SCN-ARC interaction is essential. The unaltered SCN c-Fos expression following glucose administration in disconnected animals as compared to a significant decrease in controls demonstrates the importance of the ARC as metabolic modulator of SCN neuronal activity. Together, these results indicate that the SCN is more than an autonomous clock, and forms an essential component of a larger network controlling homeostasis. The present novel findings illustrate how an imbalance between SCN and ARC communication through circadian disruption could be involved in the etiology of metabolic disorders.

Administration of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) of male rats mimics the metabolic cold defence response

Background: Cold exposure increases thyrotropin-releasing hormone (TRH) expression primarily in the hypothalamic paraventricular nucleus (PVN). The PVN is a well-known hypothalamic hub in the control of energy metabolism. TRH terminals and receptors are found on PVN neurons. We hypothesized that TRH release in the PVN plays an important role in the control of thermogenesis and energy mobilization during cold exposure. Methods: Male Wistar rats were exposed to a cold environment (4°C) or TRH retrodialysis in the PVN for 2 h. We compared the effects of cold exposure and TRH administration in the PVN on plasma glucose, corticosterone, and thyroid hormone concentrations, body temperature, locomotor activity, as well as metabolic gene expression in the liver and brown adipose tissue. Results: Cold exposure increased body temperature, locomotor activity, and plasma corticosterone concentrations, but blood glucose concentrations were similar to that of room temperature control animals. TRH administration in the PVN also promptly increased body temperature, locomotor activity and plasma corticosterone concentrations. However, TRH administration in the PVN markedly increased blood glucose concentrations and endogenous glucose production (EGP) compared to saline controls. Selective hepatic sympathetic or parasympathetic denervation reduced the TRH-induced increase in glucose concentrations and EGP. Gene expression data indicated increased gluconeogenesis in liver and lipolysis in brown adipose tissue, both after cold exposure and TRH administration. Conclusions: We conclude that TRH administration in the rat PVN largely mimics the metabolic and behavioral changes induced by cold exposure indicating a potential link between TRH release in the PVN and cold defense.