Hibernation Proteins: Preparing for Life in the Freezer

Hibernation is an extreme response to a seasonal environment, yet we know almost nothing about how it is timed or how vital cellular functions are sustained in the face of plummeting body temperature. In this issue of Cell, Kondo et al. (2006) identify a liver-derived protein complex as an essential coordinator of this adaptation to the depredations of winter

Abnormal clock gene expression and locomotor activity rhythms in two month-old female APPSwe/PS1dE9 mice

In addition to cognitive decline, Alzheimer’s disease (AD) is also characterized by agitation and disruptions in activity and sleep. These symptoms typically occur in the evening or at night and have been referred to as ‘sundowning’. These symptoms are especially difficult for carers and there are no specific drug treatments. There is increasing evidence that these symptoms reflect an underlying pathology of circadian rhythm generation and transmission. We investigated whether a transgenic mouse model relevant to AD (APPswe/PS1dE9) exhibits circadian alterations in locomotor activity and expression of clock genes involved in the regulation of the circadian cycle. Female mice at 2 months of age were investigated in their home cage. Results show that the APPswe/PS1dE9 transgene alters levels and patterns in circadian rhythm of locomotor activity. Expression of the clock genes Per1, Per2, Cry1 and Cry2 was found to increase at night compared to day in wild-type control mice in the medulla/pons. This effect was blunted for Cry1 and Cry2 gene expression in APPswe/PS1dE9. In summary, this study suggests altered circadian regulation of locomotor activity is abnormal in female APPswe/PS1dE9 mice and that this alteration has biomolecular analogies in a widely available model of AD. Furthermore, the early age at which these effects are manifest suggests that these circadian effects may precede plaque development. The APPswe/PS1dE9 mouse genetic model may have potential to serve as a tool in understanding the neuropathology of circadian abnormalities in AD and as a model system to test novel therapeutic agents for these symptoms

Going Back to the Biology of FGF21: New Insights

© 2019 Elsevier Ltd Fibroblast growth factor 21 (FGF21) is a protein highly synthesized in the liver that exerts paracrine and endocrine control of many aspects of energy homeostasis in multiple tissues. In preclinical models of obesity and type 2 diabetes, treatment with FGF21 improves glucose homeostasis and promotes weight loss, and, as a result, FGF21 has attracted considerable attention as a therapeutic agent for the treatment of metabolic syndrome in humans. An improved understanding of the biological role of FGF21 may help to explain why its therapeutic potential in humans has not been fully realized. This review will cover the complexities in FGF21 biology in rodents and humans, with emphasis on its role in protection from central and peripheral facets of obesity

Contribution of serotonin and dopamine to changes in core body temperature and locomotor activity in rats following repeated administration of mephedrone

The psychoactive effects of mephedrone are commonly compared to those of 3,4-methylenedioxymethamphetamine, but because of a shorter duration of action users often employ repeated administration to maintain its psychoactive actions. This study examined the effects of repeated mephedrone administration on locomotor activity, body temperature and striatal dopamine and 5-hydroxytryptamine (5-HT) levels, and the role of dopaminergic and serotonergic neurons in these responses. Adult male Lister hooded rats received three injections of vehicle (1ml/kg, i.p.) or mephedrone HCl (10mg/kg) at 2h intervals for radiotelemetry (temperature and activity) or microdialysis (dopamine and 5-HT) measurements. Intracerebroventricular pre-treatment (21 to 28 days earlier) with 5,7-dihydroxytryptamine (5,7-DHT, 150μg) or 6-hydroxydopamine (6-OHDA, 300μg) was used to examine the impact of 5-HT or dopamine depletion on mephedrone-induced changes in temperature and activity. A final study examined the influence of i.p. pre-treatment (-30min) with the 5-HT1A receptor antagonist WAY-100635 (0.5mg/kg), 5-HT1B receptor antagonist GR 127935 (3mg/kg) or the 5-HT7 receptor antagonist SB-258719 (10mg/kg) on mephedrone-induced changes in locomotor activity and rectal temperature. Mephedrone caused rapid-onset hyperactivity, hypothermia (attenuated on repeat dosing), and increased striatal dopamine and 5-HT release following each injection. Mephedrone-induced hyperactivity was attenuated by 5-HT depletion and 5-HT1B receptor antagonism, whereas the hypothermia was completely abolished by 5-HT depletion and lessened by 5-HT1A receptor antagonism. These findings suggest that stimulation of central 5-HT release and/or inhibition of 5-HT reuptake play a pivotal role in both the hyperlocomotor and hypothermic effects of mephedrone, which are mediated in part via 5-HT1B and 5-HT1A receptors

Contribution of serotonin and dopamine to changes in core body temperature and locomotor activity in rats following repeated administration of mephedrone

The psychoactive effects of mephedrone are commonly compared to those of 3,4-methylenedioxymethamphetamine, but because of a shorter duration of action users often employ repeated administration to maintain its psychoactive actions. This study examined the effects of repeated mephedrone administration on locomotor activity, body temperature and striatal dopamine and 5-hydroxytryptamine (5-HT) levels, and the role of dopaminergic and serotonergic neurons in these responses. Adult male Lister hooded rats received three injections of vehicle (1ml/kg, i.p.) or mephedrone HCl (10mg/kg) at 2h intervals for radiotelemetry (temperature and activity) or microdialysis (dopamine and 5-HT) measurements. Intracerebroventricular pre-treatment (21 to 28 days earlier) with 5,7-dihydroxytryptamine (5,7-DHT, 150μg) or 6-hydroxydopamine (6-OHDA, 300μg) was used to examine the impact of 5-HT or dopamine depletion on mephedrone-induced changes in temperature and activity. A final study examined the influence of i.p. pre-treatment (-30min) with the 5-HT1A receptor antagonist WAY-100635 (0.5mg/kg), 5-HT1B receptor antagonist GR 127935 (3mg/kg) or the 5-HT7 receptor antagonist SB-258719 (10mg/kg) on mephedrone-induced changes in locomotor activity and rectal temperature. Mephedrone caused rapid-onset hyperactivity, hypothermia (attenuated on repeat dosing), and increased striatal dopamine and 5-HT release following each injection. Mephedrone-induced hyperactivity was attenuated by 5-HT depletion and 5-HT1B receptor antagonism, whereas the hypothermia was completely abolished by 5-HT depletion and lessened by 5-HT1A receptor antagonism. These findings suggest that stimulation of central 5-HT release and/or inhibition of 5-HT reuptake play a pivotal role in both the hyperlocomotor and hypothermic effects of mephedrone, which are mediated in part via 5-HT1B and 5-HT1A receptors

Thyroid hormone and vitamin D regulate VGF expression and promoter activity

The Siberian hamster (Phodopus sungorus) survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. Hypothalamic tanycytes are pivotal for this process. In these cells, short-winter photoperiods upregulate deiodinase 3, an enzyme that regulates thyroid hormone availability, and downregulate genes encoding components of retinoic acid (RA) uptake and signaling. The aim of the current studies was to identify mechanisms by which seasonal changes in thyroid hormone and RA signaling from tanycytes might ultimately regulate appetite and energy expenditure. proVGF is one of the most abundant peptides in the mammalian brain, and studies have suggested a role for VGF-derived peptides in the photoperiodic regulation of body weight in the Siberian hamster. In silico studies identified possible thyroid and vitamin D response elements in the VGF promoter. Using the human neuroblastoma SH-SY5Y cell line, we demonstrate that RA increases endogenous VG expression (P!0.05) and VGF promoter activity (P!0.0001). Similarly, treatment with 1,25-ihydroxyvitamin D3 increased endogenous VGF mRNA expression (P!0.05) and VGF promoter activity (P!0.0001),whereas triiodothyronine (T3) decreased both (P!0.01 and P!0.0001). Finally, intrahypothalamic administration of T3 blocked the short day-induced increase in VGF expression in the dorsomedial posterior arcuate nucleus of Siberian hamsters. Thus, we conclude that VGF expression is a likely target of photoperiod-induced changes in tanycyte-derived signals and is potentially a regulator of seasonal changes in appetite and energy expenditure

Innervation of gonadotropin-releasing hormone neurons by peptidergic neurons conveying circadian or energy balance information in the mouse

Background: Secretion of gonadotropin-releasing hormone (GnRH) produced in neurons in the basal forebrain is the primary regulator of reproductive maturation and function in mammals. Peptidergic signals relating to circadian timing and energy balance are an important influence on the reproductive axis. The aim of this study was to investigate the innervation of GnRH neurons by peptidergic neurons. Methodology/Principal Findings: Immunohistochemistry and confocal microscopy were used to detect appositions of peptidergic fibers (NPY, β-endorphin, MCH) associated with energy balance and metabolic status in transgenic mice expressing a green fluorescent protein reporter construct in GnRH neurons. The frequency of these appositions was compared to those of vasoactive intestinal peptide (VIP), a hypothalamic neuropeptide likely to convey circadian timing information to the GnRH secretory system. The majority of GnRH neurons (73-87%) were closely apposed by fibers expressing NPY, β-endorphin, or MCH, and a significant proportion of GnRH neurons (28%) also had close contacts with VIP-ir fibers. Conclusions/Significance: It is concluded that GnRH neurons in the mouse receive a high frequency of direct modulatory inputs from multiple hypothalamic peptide systems known to be important in conveying circadian information and signalling energy balance. © 2009 Ward et al

Photoperiodic regulation of FGF21 production in the Siberian hamster

FGF21 is an endocrine member of the fibroblast growth factor superfamily that has been shown to play an important role in the physiological response to nutrient deprivation. Food restriction enhances hepatic FGF21 production, which serves to engage an integrated response to energy deficit. Specifically, elevated FGF21 levels lead to reduced gluconeogenesis and increased hepatic ketogenesis. However, circulating FGF21 concentrations also paradoxically rise in states of metabolic dysfunction such as obesity. Furthermore, multiple peripheral tissues also produce FGF21 in addition to the liver, raising questions as to its endocrine and paracrine roles in the control of energy metabolism. The objectives of this study were to measure plasma FGF21 concentrations in the Siberian hamster, a rodent which undergoes a seasonal cycle of fattening and body weight gain in the long days (LD) of summer, followed by reduction of appetite and fat catabolism in the short days (SD) of winter. Groups of adult male hamsters were raised in long days, and then exposed to SD for up to 12 weeks. Chronic exposure of LD animals to SD led to a significant increase in circulating FGF21 concentrations. This elevation of circulating FGF21 was preceded by an increase in liver FGF21 protein production evident as early as 4 weeks of exposure to SD. FGF21 protein abundance was also increased significantly in interscapular brown adipose tissue, with a positive correlation between plasma levels of FGF21 and BAT protein abundance throughout the experimental period. Epididymal white adipose tissue and skeletal muscle (gastrocnemius) also produced FGF21, but levels did not change in response to a change in photoperiod. In summary, a natural programmed state of fat catabolism was associated with increased FGF21 production in the liver and BAT, consistent with the view that FGF21 has a role in adapting hamsters to the hypophagic winter state

The use of viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein eGFP

Introduction: The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro.New Method: To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo.Results: Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing VGF, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In-situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence.Comparison with old method: The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of vgf with fluorescent tracking without compromising viral capacity. Conclusion: The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects

VGF-peptides in the Siberian hamster

vgf is one of the few genes selectively induced in the hypothalamus of Siberian hamsters upon their typical change from an obese phenotype (long day adaptation, during summer) to a lean, catabolic phenotype (short day, or winter adaptation). In fact, the i.c.v. injection of the VGF-derived peptide TLQP-21 caused hypophagia and a decrease in body weight in long day hamster. Hence, we studied VGF multi-peptide profiles in brain cortex and hypothalamus of (male) Siberian hamsters, in the long day (LD) versus short day (SD) adapted state. Specific antisera were produced against short sequences at the C- or N-termini of VGF, and of several known/predicted VGF-derived products: TLQP, NERP-1, and PGH peptides, and used in immunohistochemistry (IHC) and ELISA. Hamsters were perfused with 4% paraformaldehyde (n= 4/group) for IHC or used for tissue sampling and extraction (n= 7/group). In IHC, VGF C- and N- terminus peptides were brightly labelled, as well as most abundant. They were found in both perikarya and axons, in different layers of brain cortex and in multiple hypothalamic areas, including the paraventricular (PVN), suprachiasmatic (SCN), supraoptic (SON) and arcuate nuclei, the lateral and anterior hypothalamic areas, and the median eminence (ME). TLQP peptides were largely restricted to SCN perikarya, and ME axons, while PGH and NERP-1 peptides were revealed in perikarya of the brain cortex, in ME axons, and certain perikarya of PVN and SON (NERP-1 only). Most VGF peptides studied were well represented in tissue extracts of hypothalamus and cortex, VGF C- and N- terminus peptides being again most abundant (hypothalamus: 1.8±0.3 and 10.9±0.6; cortex: 0.7±0.1 and 5±0.3 nmol/g, mean±SEM, C- and N-terminus, respectively, LD animals). A selective decrease in certain VGF peptides was revealed in SD animals, compared to LD ones, so that NERP-1 peptides were decreased in hypothalamus and cortex (61.3±12.7% and 45.8±11.1% of LD animals, respectively, mean±SEM, p<0.04), PGH peptides were reduced in hypothalamus (24.8±12.7% of LD group, mean±SEM, p<0.02), and both TLQP and N-terminus peptides in the brain cortex (31.8±10.9% and 41.5±10.8% of LD animals, respectively, mean±SEM, p<0.02). In conclusion, VGF peptides were well represented in the Siberian hamster brain, with a distinct, apparently selective modulation in the hypothalamus and brain cortex. A regionally specific, differential post-translational processing of the VGF precursor may be implicated. Supported by a RAS grant (Regione Autonoma Sardegna, PO FSE 2007-1013, L.R. 7/2007)