Alexander W. Ross

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, ph...

Phosphorylation of CREB in Ovine Pars Tuberalis is Regulated both by cyclic AMP‐Dependent and cyclic AMP‐Independent Mechanisms

This study used a combination of Western blotting and immunocytochemistry to test whether signalling pathways independent of cyclic AMP have the potential to induce phospho‐CREB (pCREB)‐like immunoreactivity (‐ir) in the oPT. Western blot analysis of extracts of primary cultures of oPT using an antiserum against CREB, revealed a major band of CREB‐ir at 44 KDa. The intensity of this band did not vary systematically with treatment. In extracts from untreated cells, Western blot analysis revealed a major band of pCREB‐ir at 42 KDa which was not sensitive to agonist treatment. Treatment of cells with forskolin (10−6 M) increased the intensity of a number of other pCREB‐ir bands at between ca. 38 and 44 KDa. The band at 44 KDa probably represented native pCREB whilst the other bands induced by forskolin probably represented pCREB‐like proteins. Melatonin (10−6 M) alone had no effect on pCREB‐ir, but it did inhibit the effect of forskolin on the ca. 38 and 44 KDa pCREB‐ir bands. Treatment with lamb serum (1%) consistently increased the intensity of the ca. 38 and 44 KDa pCREB‐ir bands relative to control cells, as assessed by Western blot. However, Western blot analysis did not reveal a consistent effect of melatonin on the pCREB‐ir response to serum. The effect of serum on pCREB‐ir in oPTu2003cells was characterized further by immunocytochemical analysis. In contrast to experiments utilizing Western blotting, untreated cells did not possess detectable pCREB‐ir. In serum‐starved oPT and oPD cultures, treatment with serum induced exclusively nuclear pCREB‐ir. A large majority of oPTu2003cells (≥90%) were sensitive to serum (1%), and serum caused a time‐ and dose‐dependent increase of nuclear pCREB‐ir. Melatonin attenuated the response to serum in the oPT. This inhibition of the response to serum was not apparent in the oPD, demonstrating that the effect of melatonin was specific for a tissue known to express melatonin receptors. In oPT cultures, physiological concentrations of melatonin (10−9 M) partially reversed (ca. 70%) the inductive effect of 0.1% serum on nuclear pCREB‐ir. However, in contrast to studies applying forskolin, the induction of pCREB‐ir by serum occurred in the absence of measurable changes in the concentration of cyclic AMP, indicating that components of serum are able to stimulate the phosphorylation of CREB in the oPT through mechanisms independent of cyclic AMP. Both adenosine and prostaglandin E2 (PGE2) also induced nuclear pCREB‐ir in the absence of increased levels of cyclic AMP. These results demonstrate that transcriptional activities in the oPT which are under the control of CREB may be modulated by convergent cyclic AMP‐dependent and cyclic AMP‐independent pathways. Regulation of these pathways by melatonin and other factors present in serum may be an important control‐point in the generation of seasonal neuroendocrine cycles.

What can we learn from seasonal animals about the regulation of energy balance

Weight loss in humans requires, except during an illness, some form of imposed restriction on food intake or increase in energy expenditure. This necessitates overcoming powerful peripheral and central signals that serve to protect against negative energy balance. The identification of the systems and pathways involved has come from mouse models with genetic and targeted mutations, e.g., ob/ob and MC4 R(-/-) as well as rat models of obesity. Study of seasonal animals has shown that they undergo annual cycles of body fattening and adipose tissue loss as important adaptations to environmental change, yet these changes appear to involve mechanisms distinct from those known already. One animal model, the Siberian hamster, exhibits marked, but reversible, weight loss in response to shortening day length. The body weight is driven by a decrease in food intake with the magnitude of the loss of body weight being directly related to the length of time of exposure to short photoperiod. The most important facet of this response is that the point of energy balance is continuously re-adjusted during the transition in body weight reflecting an apparent sliding set point. Studies have focused on identifying the neural basis of this mechanism. Initial studies of known genes (e.g., NPY, POMC, and AgRP) both through the measurement of gene expression in the arcuate nucleus as well as following intracerebroventricular (i.c.v.) injection indicated that the systems involved are not those involved in restoring energy balance following energy deficits. Instead, a novel mechanism of regulation is implied. Recent studies have begun to explore the neural basis of the seasonal body weight response. A discrete and novel region of the posterior arcuate nucleus, the dorsal medial posterior arcuate nucleus (dmpARC) has been identified, where a battery of gene expression changes for signalling molecules (vgf and histamine H3 receptor) and transcription factors (RXRgamma and RAR) occur in association with seasonal changes in body weight. This work provides the basis of a potentially novel mechanism of energy balance regulation.

Melatonin suppresses the induction of AP-1 transcription factor components in the pars tuberalis of the pituitary.

In ovine pars tuberalis cells which express high affinity Mel 1a melatonin receptors, the ability of melatonin to directly stimulate or inhibit AP-1 transcription factor gene expression was studied. Effects of melatonin upon mRNA expression by forskolin, serum and IGF-1 were also investigated. Northern analysis showed melatonin had no direct stimulatory nor inhibitory effect upon transcription or translation. Melatonin was able to significantly inhibit forskolin-stimulated induction of c-fos and jun B mRNA whilst forskolin had no effect upon c-jun or jun D. Induction of c-Fos translation by forskolin was also inhibited by melatonin. Serum induced c-fos and c-jun, but melatonin was unable to affect these changes. Similarly IGF-1 stimulated c-fos and melatonin had no effect upon this induction. From these results it can be concluded that melatonin has no independent effects on expression of the AP-1 genes, rather its primary function is to inhibit cell activities through cyclic AMP-dependent routes of gene activation.

Melatonin Receptors and Signal Transduction Mechanisms

The ovine pars tuberalis (PT) still offers the best model for the study of signal transduction pathways regulated by the melatonin receptor. From the evidence accumulated so far, it seems likely that the cAMP signal transduction pathway will be a major effector of a stimulatory signal to the PT which can be regulated by melatonin. Thus a principal action of melatonin in the PT may be the repression of biochemical processes driven by cAMP. However, through the phenomenon of sensitization, melatonin may also act to amplify a stimulatory input to the cAMP signal transduction pathway in the PT. These events are mediated via the melatonin receptor, which is itself a target for regulation by the melatonin signal. Studies using the PT have identified several signalling pathways that may serve to positively or negatively regulate the expression of the melatonin receptor. These and other studies in the PT have alluded to cAMP-independent pathways regulated by the melatonin receptor.

RAPID COMMUNICATION oPer1 is an Early Response Gene Under Photoperiodic Regulation in the Ovine Pars Tuberalis

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2u2003h after lights‐on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night‐time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.

Hypothalamic thyroid hormone in energy balance regulation.

Thyroid hormone has been known for decades as a hormone with profound effects on energy expenditure and ability to control weight. The regulation of energy expenditure by thyroid hormone primarily occurs via regulation of the activity, or expression, of uncoupling proteins in peripheral tissues. However, mechanistically this requires a signal from the brain to change circulating levels of thyroxine and thyroid hormone or increased sympathetic drive to peripheral tissues to alter local thyroid hormone levels via increased expression of type 2 deiodinase. However, little consideration has been given to the potential role and involvement of thyroid hormones action in the brain in the regulation of energy balance. Recent evidence implicates thyroid hormone as a shortterm signal of energy deficit imposed by starvation. Furthermore, thyroid hormone action within the hypothalamus is involved in adjusting long-term energy expenditure in seasonal animals which endure food shortages in winter. Evidence from several studies suggests that regulation of type 2 and type 3 deiodinase enzymes in tanycytes of the third ventricle are gatekeepers of thyroid hormone levels in the hypothalamus. This paper reviews some of the evidence for the role of deiodinase enzymes and the actions of thyroid hormone in the hypothalamus in the regulation of energy balance.

The suppressor of cytokine signalling 3, SOCS3, may be one critical modulator of seasonal body weight changes in the Siberian hamster, Phodopus sungorus

The Siberian hamster, Phodopus sungorus, exhibits a remarkable cycle of body weight, reproduction and leptin sensitivity in response to a seasonal change in photoperiod. In the present study, we investigated the hypothesis that the suppressor of cytokine signalling 3 (SOCS3) plays a critical role in the regulation of the seasonal body weight cycle. We analysed arcuate nucleus SOCS3 gene expression in short day length (SD; 8u2003:u200316u2003h light/dark) acclimated Siberian hamsters that were transferred back to long day length (LD; 16u2003:u20038u2003h light/dark) and in hamsters that spontaneously became photorefractory to SD induced by prolonged exposure. SD acclimated hamsters that were transferred back to LD for 1, 2, 3, 4 or 6u2003weeks, increased arcuate nucleus SOCS3 gene expression to the LD level within 2u2003weeks, and maintained this higher level thereafter. The early increase of SOCS3 gene expression preceded the LD‐induced rise in body weight by approximately 3u2003weeks. Hamsters kept in SD for an extended period (25u2003weeks), began to become refractory to SD and to increase body weight. By this time, there was no difference in level of SOCS3 gene expression between LD and SD photoperiods, although body weight was still suppressed in SD hamsters. Finally, we addressed whether SOCS3 gene expression is related to SD‐induced gonadal regression or to body weight decrease by comparing Siberian hamsters with Syrian hamsters. The latter exhibited substantial SD‐induced gonadal regression but only limited seasonal changes in body weight. Acclimation to either LD or SD for 14u2003weeks had no effect on SOCS3 gene expression. This implies that arcuate nucleus SOCS3 gene expression is unlikely to be related to seasonal cycles in reproductive activity. Taken together, the findings further strengthen our hypothesis that SOCS3 may be one molecular trigger of seasonal cycles in body weight.

Altered Expression of SOCS3 in the Hypothalamic Arcuate Nucleus during Seasonal Body Mass Changes in the Field Vole, Microtus agrestis

We have previously shown that cold‐acclimated (8u2003°C) male field voles (Microtus agrestis) transferred from short day (SD, 8u2003h light) to long day (LD, 16u2003h light) photoperiod exhibit an increase in body mass lasting 4u2003weeks, after which they stabilise at a new plateau approximately 7.5u2003g (24.8%) higher than animals maintained in SD. By infusing voles with exogenous leptin, we have also demonstrated that SD voles respond to the hormone by reducing body mass and food intake, whereas LD animals increasing body mass are resistant to leptin treatment. In the present study, we investigated whether seasonal changes in body mass could be linked to modulation of the leptin signal by suppressor of cytokine signalling‐3 (SOCS3). We used in situ hybridisation to examine hypothalamic arcuate nucleus (ARC) expression of SOCS3, neuropeptide Y (NPY), agouti‐related peptide (AgRP), pro‐opiomelanocortin (POMC) and cocaine‐ and amphetamine‐regulated transcript (CART) genes in 90 voles exposed to either SD or LD for up to 11u2003weeks. LD voles increasing body mass had significantly higher levels of SOCS3 mRNA than SD or LD voles with a stable body mass. There were no associated changes in expression of NPY, AgRP, POMC and CART genes. These results suggest that voles that regulate body mass at either the lower (SD) or upper (LD) plateau remain sensitive to leptin action, whereas SOCS3‐mediated leptin resistance is a short‐term mechanism that enables animals to move between the stable body mass plateaus. Our data provide evidence that expression of SOCS3 in the ARC is involved in the modulation of the strength of the leptin signal to facilitate seasonal cycles in body mass and adiposity.

The pars tuberalis as a target of the central clock

Abstract. The pars tuberalis (PT) of the pituitary has emerged from being a gland of obscure and unknown function to a tissue of central importance to our understanding of how photoperiod regulates seasonal responses. The discovery of melatonin receptors on this gland first pointed to its involvement in seasonal physiology. However, the more recent demonstration of the expression of clock genes in the PT, such as Per1, has heightened interest in the gland. Recent work shows how photoperiod, through the hormone melatonin, affects the timing and amplitude of expression of the Per1 gene, as well as other genes such as Icer. The effect of photoperiod and melatonin on the expression of Per1 in the PT is distinct to its effects on the SCN, and this probably reflects distinct functions of the clock genes in the two tissues – acting as part of the biological clock in the SCN, but as an interval timing system within the PT. The changes in amplitude of Per1 gene expression in response to altered length of photoperiod have provided the first clues as to how the durational melatonin signal is decoded within the neuroendocrine system.