Annika Herwig

The circadian clock stops ticking during deep hibernation in the European hamster

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals.

Developmental changes in hypothalamic leptin receptor: relationship with the postnatal leptin surge and energy balance neuropeptides in the postnatal rat

In the adult brain, leptin regulates energy homeostasis primarily via hypothalamic circuitry that affects food intake and energy expenditure. Evidence from rodent models has demonstrated that during early postnatal life, leptin is relatively ineffective in modulating these pathways, despite the high circulating levels and the presence of leptin receptors within the central nervous system. Furthermore, in recent years, a neurotrophic role for leptin in the establishment of energy balance circuits has emerged. The precise way in which leptin exerts these effects, and the site of leptin action, is unclear. To provide a detailed description of the development of energy balance systems in the postnatal rat in relation to leptin concentrations during this time, endogenous leptin levels were measured, along with gene expression of leptin receptors and energy balance neuropeptides in the medial basal hypothalamus, using in situ hybridization. Expression of leptin receptors and both orexigenic and anorexigenic neuropeptides increased in the arcuate nucleus during the early postnatal period. At postnatal day 4 (P4), we detected dense leptin receptor expression in ependymal cells of the third ventricle (3V), which showed a dramatic reduction over the first postnatal weeks, coinciding with marked morphological changes in this region. An acute leptin challenge robustly induced suppressor of cytokine signaling-3 expression in the 3V of P4 but not P14 animals, revealing a clear change in the location of leptin action over this period. These findings suggest that the neurotrophic actions of leptin may involve signaling at the 3V during a restricted period of postnatal development.

Low body temperature governs the decline of circulating lymphocytes during hibernation through sphingosine-1-phosphate

Hibernation is an energy-conserving behavior consisting of periods of inhibited metabolism (‘torpor’) with lowered body temperature. Torpor bouts are interspersed by arousal periods, in which metabolism increases and body temperature returns to euthermia. In deep torpor, the body temperature typically decreases to 2–10 °C, and major physiological and immunological changes occur. One of these alterations constitutes an almost complete depletion of circulating lymphocytes that is reversed rapidly upon arousal. Here we show that torpor induces the storage of lymphocytes in secondary lymphoid organs in response to a temperature-dependent drop in plasma levels of sphingosine-1-phosphate (S1P). Regulation of lymphocyte numbers was mediated through the type 1 S1P receptor (S1P1), because administration of a specific antagonist (W146) during torpor (in a Syrian hamster at ∼8 °C) precluded restoration of lymphocyte numbers upon subsequent arousal. Furthermore, S1P release from erythrocytes via ATP-binding cassette (ABC)-transporters was significantly inhibited at low body temperature (4 °C) but was restored upon rewarming. Reversible lymphopenia also was observed during daily torpor (in a Djungarian hamster at ± 25 °C), during forced hypothermia in anesthetized (summer-active) hamsters (at ± 9 °C), and in a nonhibernator (rat at ∼19 °C). Our results demonstrate that lymphopenia during hibernation in small mammals is driven by body temperature, via altered plasma S1P levels. S1P is recognized as an important bioactive lipid involved in regulating several other physiological processes as well and may be an important factor regulating additional physiological processes in hibernation as well as in mediating the effects of therapeutic hypothermia in patients.

Differential Activity by Polymorphic Variants of a Remote Enhancer that Supports Galanin Expression in the Hypothalamus and Amygdala: Implications for Obesity, Depression and Alcoholism

The expression of the galanin gene (GAL) in the paraventricular nucleus (PVN) and in the amygdala of higher vertebrates suggests the requirement for highly conserved, but unidentified, regulatory sequences that are critical to allow the galanin gene to control alcohol and fat intake and modulate mood. We used comparative genomics to identify a highly conserved sequence that lay 42 kb 5′ of the human GAL transcriptional start site that we called GAL5.1. GAL5.1 activated promoter activity in neurones of the PVN, arcuate nucleus and amygdala that also expressed the galanin peptide. Analysis in neuroblastoma cells demonstrated that GAL5.1 acted as an enhancer of promoter activity after PKC activation. GAL5.1 contained two polymorphisms; rs2513280(C/G) and rs2513281(A/G), that occurred in two allelic combinations (GG or CA) where the dominant GG alelle occurred in 70-83 % of the human population. Intriguingly, both SNPs were found to be in LD (R2 of 0.687) with another SNP (rs2156464) previously associated with major depressive disorder (MDD). Recreation of these alleles in reporter constructs and subsequent magnetofection into primary rat hypothalamic neurones showed that the CA allele was 40 % less active than the GG allele. This is consistent with the hypothesis that the weaker allele may affect food and alcohol preference. The linkage of the SNPs analysed in this study with a SNP previously associated with MDD together with the functioning of GAL5.1 as a PVN and amygdala specific enhancer represent a significant advance in our ability to understand alcoholism, obesity and major depressive disorder.

Photoperiod and acute energy deficits interact on components of the thyroid hormone system in hypothalamic tanycytes of the Siberian hamster

In the Siberian hamster, seasonal weight loss occurs gradually over many weeks during autumn and winter. This is driven by a regulatory mechanism that is able to integrate duration of exposure to short days (SDs) with the size of body energy reserves. After food restriction in SDs, followed by ad libitum refeeding, body weight of the hamster does not return to its former level; rather, it increases to a level defined by the length of time spent in SDs. In this report, we show that components of the thyroid hormone system that are involved in seasonal weight loss change expression in response to 48 h of starvation. Eight weeks in an SD photoperiod induced weight loss in the Siberian hamster. In the hypothalamus of these hamsters, type II deiodinase expression was decreased and type III deiodinase expression was induced, but there was no change in hypothalamic neuropeptide Y or thyrotropin-releasing hormone gene expression. For the first time, we show that the thyroid hormone transporter monocarboxylate transporter 8 is expressed in tanycytes and is increased in response to an SD photoperiod. Food restriction (48 h of starvation) reversed the direction of gene expression change for type II and III deiodinase and monocarboxylate transporter 8 induced by SD photoperiods. Furthermore, fasting increased neuropeptide Y expression and decreased thyrotropin-releasing hormone expression. VGF, a gene upregulated in SDs in the dorsal region of the medial posterior area of the arcuate nucleus, was not changed by starvation. These data point to a mechanism whereby energy deprivation can interact with an SD photoperiod on hypothalamic tanycytes to regulate components of the thyroid hormone system involved in photoperiodic regulation of seasonal physiology.

Hypothalamic ventricular ependymal thyroid hormone deiodinases are an important element of circannual timing in the Siberian hamster (Phodopus sungorus).

Exposure to short days (SD) induces profound changes in the physiology and behaviour of Siberian hamsters, including gonadal regression and up to 30% loss in body weight. In a continuous SD environment after approximately 20 weeks, Siberian hamsters spontaneously revert to a long day (LD) phenotype, a phenomenon referred to as the photorefractory response. Previously we have identified a number of genes that are regulated by short photoperiod in the neuropil and ventricular ependymal (VE) cells of the hypothalamus, although their importance and contribution to photoperiod induced physiology is unclear. In this refractory model we hypothesised that the return to LD physiology involves reversal of SD expression levels of key hypothalamic genes to their LD values and thereby implicate genes required for LD physiology. Male Siberian hamsters were kept in either LD or SD for up to 39 weeks during which time SD hamster body weight decreased before increasing, after more than 20 weeks, back to LD values. Brain tissue was collected between 14 and 39 weeks for in situ hybridization to determine hypothalamic gene expression. In VE cells lining the third ventricle, expression of nestin, vimentin, Crbp1 and Gpr50 were down-regulated at 18 weeks in SD photoperiod, but expression was not restored to the LD level in photorefractory hamsters. Dio2, Mct8 and Tsh-r expression were altered by SD photoperiod and were fully restored, or even exceeded values found in LD hamsters in the refractory state. In hypothalamic nuclei, expression of Srif and Mc3r mRNAs was altered at 18 weeks in SD, but were similar to LD expression values in photorefractory hamsters. We conclude that in refractory hamsters not all VE cell functions are required to establish LD physiology. However, thyroid hormone signalling from ependymal cells and reversal of neuronal gene expression appear to be essential for the SD refractory response.

Platelet dynamics during natural and pharmacologically induced torpor and forced hypothermia.

Hibernation is an energy-conserving behavior in winter characterized by two phases: torpor and arousal. During torpor, markedly reduced metabolic activity results in inactivity and decreased body temperature. Arousal periods intersperse the torpor bouts and feature increased metabolism and euthermic body temperature. Alterations in physiological parameters, such as suppression of hemostasis, are thought to allow hibernators to survive periods of torpor and arousal without organ injury. While the state of torpor is potentially procoagulant, due to low blood flow, increased viscosity, immobility, hypoxia, and low body temperature, organ injury due to thromboembolism is absent. To investigate platelet dynamics during hibernation, we measured platelet count and function during and after natural torpor, pharmacologically induced torpor and forced hypothermia. Splenectomies were performed to unravel potential storage sites of platelets during torpor. Here we show that decreasing body temperature drives thrombocytopenia during torpor in hamster with maintained functionality of circulating platelets. Interestingly, hamster platelets during torpor do not express P-selectin, but expression is induced by treatment with ADP. Platelet count rapidly restores during arousal and rewarming. Platelet dynamics in hibernation are not affected by splenectomy before or during torpor. Reversible thrombocytopenia was also induced by forced hypothermia in both hibernating (hamster) and non-hibernating (rat and mouse) species without changing platelet function. Pharmacological torpor induced by injection of 5′-AMP in mice did not induce thrombocytopenia, possibly because 5′-AMP inhibits platelet function. The rapidness of changes in the numbers of circulating platelets, as well as marginal changes in immature platelet fractions upon arousal, strongly suggest that storage-and-release underlies the reversible thrombocytopenia during natural torpor. Possibly, margination of platelets, dependent on intrinsic platelet functionality, governs clearance of circulating platelets during torpor.

Hypothalamic Gene Expression Rapidly Changes in Response to Photoperiod in Juvenile Siberian Hamsters (Phodopus sungorus)

Siberian hamsters are seasonal mammals that survive a winter climate by making adaptations in physiology and behaviour. This includes gonadal atrophy, reduced food intake and body weight. The underlying central mechanisms responsible for the physiological adaptations are not fully established but involve reducing hypothalamic tri‐iodthyronine (T3) levels. Juvenile Siberian hamsters born or raised in short days (SD) respond in a similar manner, although with an inhibition of gonadal development and growth instead of reversing an established long day (LD) phenotype. Using juvenile male hamsters, the present study aimed to investigate whether the central mechanisms are similar before the establishment of the mature LD phenotype. By in situ hybridisation, we examined the response of genes involved in thyroid hormone (Dio2 and Dio3, which determine hypothalamic T3 levels) and glucose/glutamate metabolism in the ependymal layer, histamine H3 receptor and VGF as representatives of the highly responsive dorsomedial posterior arcuate nucleus (dmpARC), and somatostatin, a hypothalamic neuropeptide involved in regulating the growth axis. Differential gene expression of type 2 and type 3 deiodinase in the ependymal layer, histamine H3 receptor in the dmpARC and somatostatin in the ARC was established by the eighth day in SD. These changes are followed by alterations in glucose metabolism related genes in the ependymal layer by day 16 and increased secretogranin expression in the dmpARC by day 32. In conclusion, our data demonstrate similar but rapid and highly responsive changes in gene expression in the brain of juvenile Siberian hamsters in response to a switch from LD to SD. The data also provide a temporal definition of gene expression changes relative to physiological adaptations of body weight and testicular development and highlight the likely importance of thyroid hormone availability as an early event in the adaptation of physiology to a winter climate in juvenile Siberian hamsters.

A Thyroid Hormone Challenge in Hypothyroid Rats Identifies T3 Regulated Genes in the Hypothalamus and in Models with Altered Energy Balance and Glucose Homeostasis

BACKGROUND The thyroid hormone triiodothyronine (T3) is known to affect energy balance. Recent evidence points to an action of T3 in the hypothalamus, a key area of the brain involved in energy homeostasis, but the components and mechanisms are far from understood. The aim of this study was to identify components in the hypothalamus that may be involved in the action of T3 on energy balance regulatory mechanisms. METHODS Sprague Dawley rats were made hypothyroid by giving 0.025% methimazole (MMI) in their drinking water for 22 days. On day 21, half the MMI-treated rats received a saline injection, whereas the others were injected with T3. Food intake and body weight measurements were taken daily. Body composition was determined by magnetic resonance imaging, gene expression was analyzed by in situ hybridization, and T3-induced gene expression was determined by microarray analysis of MMI-treated compared to MMI-T3-injected hypothalamic RNA. RESULTS Post mortem serum thyroid hormone levels showed that MMI treatment decreased circulating thyroid hormones and increased thyrotropin (TSH). MMI treatment decreased food intake and body weight. Body composition analysis revealed reduced lean and fat mass in thyroidectomized rats from day 14 of the experiment. MMI treatment caused a decrease in circulating triglyceride concentrations, an increase in nonesterified fatty acids, and decreased insulin levels. A glucose tolerance test showed impaired glucose clearance in the thyroidectomized animals. In the brain, in situ hybridization revealed marked changes in gene expression, including genes such as Mct8, a thyroid hormone transporter, and Agrp, a key component in energy balance regulation. Microarray analysis revealed 110 genes to be up- or downregulated with T3 treatment (± 1.3-fold change, p<0.05). Three genes chosen from the differentially expressed genes were verified by in situ hybridization to be activated by T3 in cells located at or close to the hypothalamic ventricular ependymal layer and differentially expressed in animal models of long- and short-term body weight regulation. CONCLUSION This study identified genes regulated by T3 in the hypothalamus, a key area of the brain involved in homeostasis and neuroendocrine functions. These include genes hitherto not known to be regulated by thyroid status.

Photoperiodic regulation of glycogen metabolism, glycolysis, and glutamine synthesis in tanycytes of the Siberian hamster suggests novel roles of tanycytes in hypothalamic function.

The objective of this study is to investigate the impact of photoperiod on the temporal and spatial expression of genes involved in glucose metabolism in the brain of the seasonal mammal Phodopus sungorus (Siberian hamster). In situ hybridization was performed on brain sections obtained from male hamsters held in long photoperiod (high body weight and developed testes) or short photoperiod (reduced body weight with testicular regression). This analysis revealed upregulation in expression of genes involved in glycogen and glucose metabolism in short photoperiod and localized to the tanycyte layer of the third ventricle. On the basis of these data and a previously identified photoperiod‐dependent increase in activity of neighboring hypothalamic neurons, we hypothesized that the observed expression changes may reflect alteration in either metabolic fuel or precursor neurotransmitter supply to surrounding neurons. Gene expression analysis was performed for genes involved in lactate and glutamate transport. This analysis showed that the gene for the lactate transporter MCT2 and glutamate transporter GLAST was decreased in the tanycyte layer in short photoperiod. Expression of mRNA for glutamine synthetase, the final enzyme in the synthesis of the neuronal neurotransmitter precursor, glutamine, was also decreased in short photoperiod. These data suggest a role for tanycytes in modulating glutamate concentrations and neurotransmitter supply in the hypothalamic environment.