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Investigations into the mechanisms regulating obesity are frantic and novel translational approaches are needed. The raccoon dog (Nyctereutes procyonoides) is a canid species representing a promising model to study metabolic regulation in a species undergoing cycles of seasonal obesity and fasting. To understand the molecular mechanisms of metabolic regulation in seasonal adaptation, we analyzed key central nervous system and peripheral signals regulating food intake and metabolism from raccoon dogs after autumnal fattening and winter fasting. Expressions of neuropeptide Y (NPY), orexin-2 receptor (OX2R), pro-opiomelanocortin (POMC) and leptin receptor (ObRb) were analyzed as examples of orexigenic and anorexigenic signals using qRT-PCR from raccoon dog hypothalamus samples. Plasma metabolic profiles were measured with 1H NMR-spectroscopy and LC-MS. Circulating hormones and cytokines were determined with canine specific antibody assays. Surprisingly, NPY and POMC were not affected by the winter fasting nor autumn fattening and the metabolic profiles showed a remarkable equilibrium, indicating conserved homeostasis. However, OX2R and ObRb expression changes suggested seasonal regulation. Circulating cytokine levels were not increased, demonstrating that the autumn fattening did not induce subacute inflammation. Thus, the raccoon dog developed seasonal regulatory mechanisms to accommodate the autumnal fattening and prolonged fasting making the species unique in coping with the extreme environmental challenges.

Rhabdomyolysis (destruction of striated muscle) is a novel form of mushroom poisoning in Europe and Asia indicated by increased circulating creatine kinase levels. Particular wild fungi have also been reported to induce elevated creatine kinase activities in mice. Flammulina velutipes (enokitake or winter mushroom) is one of the most actively cultivated mushroom species globally. As it is marketed as a medicinal mushroom and functional food, it is important to examine whether it could induce potentially harmful health effects similar to some previously studied edible fungi. The present study examined the effects of F. velutipes consumption on the plasma clinical chemistry, hematology, and organ histology of laboratory mice. Wild F. velutipes were dried, pulverized, mixed with a regular laboratory rodent diet, and fed to the animals at 0, 3, 6, or 9 g/kg body mass/day for five days ( n = 6/group). F. velutipes consumption caused increased activities of plasma creatine kinase and the MB-fraction of creatine kinase at 6–9 g/kg/d, indicating potentially deleterious effects on both skeletal and cardiac muscle. The plasma total and high-density lipoprotein cholesterol concentrations (at 9 g/kg/d) and white blood cell and lymphocyte counts (at 6–9 g/kg/d) decreased. Although the cholesterol-lowering properties of F. velutipes can be beneficial, the previously unexamined, potentially hazardous side effects of mushroom consumption (myo- and cardiotoxicity) should be thoroughly investigated before recommending this mushroom species as a health-promoting food item. Impact statement This work is important to the field of functional foods, as it provides novel information about the potential myo- and cardiotoxic properties of an edible mushroom, Flammulina velutipes. The results are useful and of importance because F. velutipes is an actively cultivated mushroom and marketed as a health-promoting food item. The findings contribute to the understanding of the complexity of the balance between the beneficial and potentially harmful effects of mushroom consumption.

The wintering strategy of the Djungarian hamster (Phodopus sungorus) includes a naturally occurring decrease in food intake and body mass. Our aim was to investigate the conceivable role of the metabolic regulators, AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), in the seasonal adaptation of the Djungarian hamster. In addition, a rate-limiting enzyme in fatty acid synthesis and oxidation, acetyl CoA carboxylase (ACC), was studied. Relative protein expressions and phosphorylated forms (pAMPK and pACC) were determined by Western blot from subcutaneous white adipose tissues (sWAT), abdominal white adipose tissues (aWAT), interscapular brown adipose tissues (iBAT), skeletal muscle, and hypothalamus of winter- and summer-acclimatized hamsters. The winter group had higher AMPK expression in sWAT, aWAT, and iBAT, but the relative amount of phosphorylated protein (pAMPK/AMPK ratio) was lower in these tissues. Furthermore, ACC expression was higher in sWAT and iBAT of the winter animals. pACC (inactive form) levels were higher in all adipose tissues, yet a lower pACC/ACC ratio was detected in iBAT of the winter hamsters. Muscle AMPK expression was lower but pAMPK/AMPK ratio higher in the winter group. SIRT1 expression was higher in muscle and all adipose tissues of the winter hamsters. Hypothalamic protein expressions did not differ between the groups. Higher expressions of AMPK, ACC, and SIRT1 in WAT and iBAT of the winter hamsters suggest a role in the regulation of lipid reserves and increased thermogenic capacity characteristic to the winter-adapted Djungarian hamsters.

Until now, melanopsin (OPN4) - a specialized photopigment being responsive especially to blue light wavelengths - has not been found in the human brain at protein level outside the retina. More specifically, OPN4 has only been found in about 2% of retinal ganglion cells (i.e. in intrinsically photosensitive retinal ganglion cells), and in a subtype of retinal cone-cells. Given that Allen Institute for Brain Science has described a wide distribution of OPN4 mRNA in two human brains, we aimed to investigate whether OPN4 is present in the human brain also at protein level. Western blotting and immunohistochemistry, as well as immunoelectron microscopy, were used to analyse the existence and distribution of OPN4 protein in 18 investigated areas of the human brain in samples obtained in forensic autopsies from 10 male subjects (54 ± 3.5 years). OPN4 protein expression was found in all subjects, and, furthermore, in 5 out of 10 subjects in all investigated brain areas localized in membranous compartments and cytoplasmic vesicles of neurons. To our opinion, the wide distribution of OPN4 in central areas of the human brain evokes a question whether ambient light has important straight targets in the human brain outside the retinohypothalamic tract (RHT). Further studies are, however, needed to investigate the putative physiological phototransductive actions of inborn OPN4 protein outside the RHT in the human brain.

The raccoon dog (Nyctereutes procyonoides) is a canid species with autumnal fattening and prolonged wintertime fasting. Nonpathological body weight cycling and the ability to tolerate food deficiency make this species a unique subject for studying physiological mechanisms in energy metabolism. AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating energy homeostasis. During acute fasting, AMPK promotes fatty acid oxidation and enhances glucose uptake. We evaluated the effects of prolonged fasting on muscle energy metabolism in farm-bred raccoon dogs. Total and phosphorylated AMPK and acetyl-CoA carboxylase (ACC), glucose transporter 4 (GLUT 4), insulin receptor and protein kinase B (Akt) protein expressions of hind limb muscles were determined by Western blot after 10 weeks of fasting. Plasma insulin, leptin, ghrelin, glucose and free fatty acid levels were measured, and muscle myosin heavy chain (MHC) isoform composition analyzed. Fasting had no effects on AMPK phosphorylation, but total AMPK expression decreased in m. rectus femoris, m. tibialis anterior and m. extensor digitorum longus resulting in a higher phosphorylation ratio. Decreased total expression was also observed for ACC. Fasting did not influence GLUT 4, insulin receptor or Akt expression, but Akt phosphorylation was lower in m. flexor digitorum superficialis and m. extensor digitorum longus. Three MHC isoforms (I, IIa and IIx) were detected without differences in composition between the fasted and control animals. The studied muscles were resistant to prolonged fasting indicating that raccoon dogs have an effective molecular regulatory system for preserving skeletal muscle function during wintertime immobility and fasting.

Energy deprivation during the wintertime poses a challenge to skeletal muscle function. The objective of the present study was to determine the expression of specific calcium (Ca 2+ ) regulating proteins in skeletal muscle of the reindeer during wintertime adaptation and moderate weight loss. To assess the possible changes in the dihydropyridine receptor (Cacna1), ryanodine receptor (RyR1), sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1) and calsequestrin 1 (CASQ1) proteins, six mature female reindeer were held under experimental outdoor conditions with limited energy intake and muscle biopsies were taken from m. semitendinosus . In addition, muscle biopsies from m. semitendinosus and m. gluteus medius taken from freely ranging reindeer after slaughter (October, n = 7; January, n = 7) were included in the study. The muscle samples were analysed by Western blot. The animals lost 10–21% of their body mass (BM) during the restricted feeding, with no change in the muscle total protein content. In general, the concentrations of nearly all Ca 2+ regulating components measured were maintained as a response to restricted feeding in experimental and freely ranging reindeer. Interestingly, the expression of Cacna1 was 2.3-fold up-regulated after the restricted feeding period in the m. semitendinosus of the experimental reindeer. On the contrary, in freely ranging animals, the expression of SERCA1 and RyR1 decreased 3.5- and 3.2-fold, respectively, in January compared to October. To conclude, the expression of Ca 2+ regulating proteins is not altered during moderate weight loss and winter adaptation in order to maintain effective muscle contraction capacity. Up-regulation of Cacna1, however, indicates the ability to allocate Ca 2+ cycling capacity to both oxidative and glycolytic metabolic pathways during times of energy deprivation.

Encephalopsin belongs to the family of extraretinal opsins having a putative role in CNS tissue photosensitivity. Encephalopsin mRNA has earlier been localized in rodent brains, but expression and localization of the protein has not yet been reported. In this study, we aimed to define encephalopsin protein abundance and localization in the rodent brain. The distribution and localization of encephalopsin protein in a mouse brain and selected peripheral tissues were analysed in ten mice, using Western blotting and immunohistochemistry. The specificity of immunoreaction was validated by primary antibody omitting and immunizing peptide blocking experiment. We found encephalopsin protein abundant in the mouse brain, but not in the periphery. Encephalopsin protein was present in neurons of the mouse cerebral cortex, paraventricular area, and cerebellar cells. Our results show that encephalopsin is expressed at the protein level in different brain areas of the mouse. Therefore, the suggested idea that encephalopsin plays a role in non-visual photic processes seems to be applicable. Evidently, further investigations are needed to find out the signalling mechanisms, and the potential physiological role of encephalopsin in phototransduction due to the changes in ambient light.

The deer ked (Lipoptena cervi) is a haematophagous parasitic fly of cervids that spread to Finland in the early 1960's. Presently its northern distribution limit lies at approximately 65°N and it is gradually spreading northwards. In Finland the principal host species has been the moose (Alces alces), but the deer ked is about to establish contact with another potential host, the semi-domesticated reindeer (Rangifer tarandus tarandus) causing possible threats to reindeer health and management. The aim of this study was to investigate if the deer ked would have an influence on the welfare of the reindeer. Eighteen adult reindeer were divided into three experimental groups: the control group and two infected groups with 300 deer keds per reindeer introduced in August-September. One of the infected groups was treated with subcutaneous ivermectin in November. To gather comprehensive data on potential health hazards caused by the deer ked a wide array of physiological variables was measured during and at the end of the experiment in December. The keds caused no clear changes in the complete blood count, plasma clinical chemistry, amino acids, endocrinology, energy stores, enzyme activities or tissue fatty acid profiles of the host. The haematological, clinical chemical and endocrinological values displayed changes that could be related to the seasonal physiological adaptations of the species. In conclusion, at the duration and intensity of infection that were employed, the effects of the deer ked on the measured physiological variables of the reindeer were insignificant.

The gastrointestinal tract of vertebrate species contains melatonin, which participates in several physiological functions. Some of these effects are mediated via specific membrane receptors (MT(1) and MT(2)). In the present study, the distribution of the MT(2) receptor protein in the gastrointestinal tract was localized, and changes in MT(2) receptor density were observed in relation to the expected circadian changes in melatonin concentrations. Immunohistochemistry was performed in the rat stomach, duodenum, colon and pancreas. The amount of MT(2) was determined by Western blot. Melatonin concentrations were measured by radioimmunoassay (RIA). In the stomach, duodenum and colon, the most intense immunoreactivity was observed in the Muscularis mucosae and in the Muscularis externa. In the pancreas, the immunolabelling was less intense. There was a clear daily rhythm of melatonin concentrations in the stomach, duodenum and pancreas, with higher levels during the dark period. The density of MT(2) receptors did not exhibit circadian variation. Moreover, circadian changes in melatonin concentrations were not found in the colon. The density of MT(2) was the highest in the colon. Our results provide evidence for the heterogeneous distribution of MT(2) receptors in different parts and layers of the gastrointestinal tract, which could indicate a physiological role of melatonin in the gastrointestinal tract.

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n=195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n=33), December 2006 (n=92), or January 2007 (n=70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.

Objective To investigate how winter acclimatization (WA), exogenous melatonin (MEL) or endogenous melatonin (SD) affect plasma leptin and ghrelin concentrations and how leptin and ghrelin contribute to the regulation of different adaptations to wintering in the Siberian hamster. Results The plasma leptin and insulin and testicular testosterone concentrations were lower in the WA, MEL and SD groups, whereas the plasma ghrelin concentration was higher due to the WA treatment. In the treated hamsters, body temperatures during photo- and scotophase were lower throughout the study and torpor bouts were observed. The absolute and relative body fat masses were lower in all treated groups. Conclusions Siberian hamsters reduce their feeding in winter, although just the opposite is suggested by the lower leptin concentrations in all treated groups and the higher ghrelin concentration in the WA group. The positive correlation between plasma leptin and testicular testosterone indicates a possible interaction between them. Torpor bouts were related to a lowered body mass and plasma leptin concentration. Exposure to short photoperiod did not induce elevated plasma ghrelin levels; the response required a low ambient temperature together with short day length.

We examined the effects of prolonged undernutrition on plasma leptin and insulin levels and some serum protein metabolites in reindeer (Rangifer tarandus tarandus L.) during winter and spring. The reindeer (male

Arvicolinae voles are small herbivores relying on constant food availability with weak adaptations to tolerate prolonged food deprivation. The present study performed a comparative analysis on the responses to 4-18 h of food deprivation in the common vole (Microtus arvalis) and the tundra vole (Microtus oeconomus). Both species exhibited rapid decreases in the plasma and liver carbohydrate concentrations during phase I of fasting and the decline in the liver glycogen level was more pronounced in the tundra vole. The plasma thyroxine concentrations of the common vole decreased after 4 h. Lipid mobilization (phase II of fasting) was indicated by the increased plasma free fatty acid levels after 8-18 (the common vole) or 4-18 h (the tundra vole) and by the elevated lipase activities. In the tundra vole, the plasma ghrelin concentrations increased after 12 h possibly to stimulate appetite. Both species showed increased liver lipid concentrations after 4 h and plasma aminotransferase and creatine kinase activities after 12-18 h of food deprivation implying liver dysfunction and skeletal muscle damage. No signs of stimulated protein catabolism characteristic to phase III of fasting were present during 18 h without food.

We examined the time course of changes in the synthesis and levels of endogenous melatonin and in the expression of MT(1) and MT(2) melatonin receptors 1 day, 2 and 4 wk after myocardial infarction (MI) in rats. MI was produced by ligation of the left anterior descending coronary artery. Transthoracic echocardiography was performed to characterize structural and functional changes after MI. mRNA levels were measured by real-time quantitative reverse transcription-polymerase chain reaction and proteins by Western blotting. One day after infarction, MI rats had 4.3 times (P < 0.001) higher pineal melatonin synthesis, than sham-operated animals, which was associated with the increased concentration of melatonin in plasma (P < 0.001) and left ventricle (LV) (P = 0.01). The amount of MT(1) receptor protein decreased significantly in MI LVs compared with control LVs 1 day after infarction (P < 0.01), followed by recovery during the next 2 wk. Furthermore, the expression of MT(1) receptor mRNA of the MI LVs was elevated 2 wk after infarction (P < 0.01) compared with control LVs. The amount of MT(2) receptor proteins in MI LVs was higher than in sham-operated LVs 1 day (P < 0.05) and 4 wk (P < 0.01) after MI. In conclusion, melatonin synthesis in the pineal gland increased rapidly in response to the MI, supporting an important role for endogenous melatonin in protecting the heart after MI. The observed changes in the expression of MT(1) and MT(2) receptors suggest that melatonin receptors may be involved in mediating, at least, in part, the protective effects of melatonin in the heart after infarction.

The raccoon dog (Nyctereutes procyonoides) is a canid with autumnal fattening and passive wintering strategy. We examined the effects of wintertime fasting and seasonality on AMP-activated protein kinase (AMPK), a regulator of metabolism, and its target, acetyl-CoA carboxylase (ACC) on the species. Twelve farmed raccoon dogs (eleven females/one male) were divided into two groups: half were fasted for ten weeks in December-March (winter fasted) and the others were fed ad libitum (winter fed). A third group (autumn fed, eight females) was fed ad libitum and sampled in December. Total AMPK, ACC and their phosphorylated forms (pAMPK, pACC) were measured from hypothalamus, liver, intra-abdominal (iWAT) and subcutaneous white adipose tissues (sWAT). The fasted animals lost 32% and the fed 20% of their body mass. Hypothalamic AMPK expression was lower and pACC levels higher in the winter groups compared to the autumn fed group. Liver pAMPK was lower in the winter fasted group, with consistently decreased ACC and pACC. AMPK and pAMPK were down-regulated in sWAT and iWAT of both winter groups, with a parallel decline in pACC in sWAT. The responses of AMPK and ACC to fasting were dissimilar to the effects observed previously in non-seasonal mammals and hibernators. Differences between the winter fed and autumn fed groups indicate that the functions of AMPK and ACC could be regulated in a season-dependent manner. Furthermore, the distinctive effects of prolonged fasting and seasonal adaptation on AMPK-ACC pathway could contribute to the wintering strategy of the raccoon dog.

The dynamics of animal populations are greatly influenced by interactions with their natural enemies and food resources. However, quantifying the relative effects of these factors on demographic rates remains a perpetual challenge for animal population ecology. Food scarcity is assumed to limit the growth and to initiate the decline of cyclic herbivore populations, but this has not been verified with physiological health indices. We hypothesized that individuals in declining populations would exhibit signs of malnutrition-induced deterioration of physiological condition. We evaluated the association of body condition with population cycle phase in bank voles (Myodes glareolus) during the increase and decline phases of a population cycle. The bank voles had lower body masses, condition indices and absolute masses of particular organs during the decline. Simultaneously, they had lower femoral masses, mineral contents and densities. Hemoglobin and hematocrit values and several parameters known to respond to food deprivation were unaffected by the population phase. There were no signs of lymphopenia, eosinophilia, granulocytosis or monocytosis. Erythrocyte counts were higher and plasma total protein levels and tissue proportions of essential polyunsaturated fatty acids lower in the population decline. Ectoparasite load was lower and adrenal gland masses or catecholamine concentrations did not suggest higher stress levels. Food availability seems to limit the size of voles during the decline but they can adapt to the prevailing conditions without clear deleterious health effects. This highlights the importance of quantifying individual health state when evaluating the effects of complex trophic interactions on the dynamics of wild animal populations.

Encephalopsin (OPN3) belongs to the light sensitive transmembrane receptor family mainly expressed in the brain and retina. It is believed that light affects mammalian circadian rhythmicity only through the retinohypothalamic tract, which transmits light information to the suprachiasmatic nucleus in the hypothalamus, although it has been shown that light penetrates the skull. Here we present the effect of transcranial light treatment on OPN3 expression and monoamine concentrations in mouse brain. Mice were randomly assigned to control group, morning-light group and evening-light group, and animals were illuminated transcranially five times a week eight minutes per mouse for four weeks. The concentrations of OPN3 and monoamines were analysed using Western blotting and HPLC, respectively. We report that transcranial light treatment affects OPN3 expression in different brain areas and plasma/adrenal gland monoamine concentrations. In addition, when light was administered at a different time of the day, the response varied in different tissues. These results provide new information on the effects of light on transmitters mediating mammalian rhythmicity.

Plasma cortisol concentrations were determined from the blood of eight mature female reindeer (Rangifer tarandus tarandus) after an intravenous injection of either saline (control) or 100, 250 or 500 μg of synthetic ACTH. Blood samples were collected at 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, 60, 75, 90, 120, 150, and 180 min after the injections. The aims were to determine the appropriate dose of ACTH for adrenal stimulation tests, to define the dose level of ACTH which elicited a maximal cortisol response and to describe the range of blood cortisol concentrations for reference when evaluating the stress responses of reindeer.The mean plasma cortisol concentrations (s.e.) at the zero sample times (t0) of the control and the ACTH treatments varied between 93·4 (11·8) and 132·5 (18·1) nmol/l. The total plasma cortisol response (area under curve, AUC, nmol/l × min) increased with increasing dose of ACTH (P < 0·001). The AUC of the control treatment was significantly smaller than of the ACTH treatments (P < 0·001). The highest dose of ACTH (500 μg) gave a significantly bigger AUC than the lowest dose (100 μg) (P = 0·008). The maximal plasma cortisol concentrations (CMAX) were achieved within 60 min of the ACTH injections. The ranges of individual CMAX were 59·0 to 136·8 nmol/l for the control treatment, and 110·0 to 252·0, 152·0 to 247·5 and 135·1 to 257·1 nmol/l for 100, 250 and 500 μg ACTH, respectively. The difference in CMAX was significant between the control treatment and the ACTH treatments (P < 0·001) but not between the different doses of ACTH. The plasma cortisol concentrations at the end of the observation period at t180 were not significantly affected by the ACTH treatment (P > 0·05).In conclusion, the 100-μg dose of ACTH was sufficient to produce a significant cortisol response compared with the control treatment. Increasing the dose did not increase the maximal response, but tended to elongate the response profile. The blood sampling frequency used in the study was found suitable for detection of the cortisol response in reindeer.

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT(1) and MT(2) melatonin receptors are expressed in certain tissues of the rat. The expression of MT(1) and MT(2) melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT(1) and MT(2) melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT(2) mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT(1) mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT(1) and MT(2) receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT(1) melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT(2) receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.

The raccoon dog (Nyctereutes procyonoides) is an omnivorous canid utilizing the passive wintering strategy in the boreal climate. Farmed raccoon dogs (n=12) were randomly assigned into two study groups on 26 November 2003. Between 3 December 2003 and 27 January 2004, half of the animals were fasted for 8 weeks and plasma weight-regulatory hormone concentrations determined on 26 November and 30 December 2003 and on 27 January 2004. The plasma peptide YY, ghrelin, and growth hormone (GH) concentrations increased due to food deprivation, while the T4 and Acrp30 concentrations decreased. Furthermore, the plasma GH concentrations were higher in the fasted raccoon dogs than in the fed animals, which had higher plasma insulin, glucagon, and T4 concentrations. However, fasting had no effect on the plasma leptin concentrations. The results confirm previous findings with unchanged leptin levels in fasting carnivores. Increased GH levels probably contribute to increased lipolysis and mobilization of fat stores. Ghrelin can also enhance lipolysis by increasing the GH levels. The decreased levels of T4 may reduce the metabolic rate. The plasma dopamine concentrations decreased due to fasting unlike observed previously in rats. Together with the unaffected adrenaline, noradrenaline, and cortisol concentrations, this suggests that food deprivation in winter does not cause stress to the raccoon dog but is an integral part of its natural life history. J. Exp. Zool. 301A:919–929, 2004. © 2004 Wiley-Liss, Inc.

Rodents inhabiting high latitudes, close to the Arctic Circle or above it, are exposed to near 24 h daylight during the summer season. An example to such rodent species is the Norwegian lemming Lemmus lemmus, which is distributed in northern Fennoscandia. We measured daily rhythms of heat production (VO2), body temperature (Tb), motor activity and melatonin secretion (measured from its metabolite 6-sulfatoximelatonin 6-SMT) in individuals exposed to natural day light, Oulu Finland, in August 1997 and at a controlled ambient temperature of 22 °C. Our results show a daily rhythm of VO2 with an acrophase at 04:00 h and minimal values measured at 16:00 h, with a significant difference (p

The objective was to study MgO and Mg alloy rumen boluses as Mg supplements for reindeer (Rangifer tarandus tarandus) by following the plasma and urinary Mg and Ca concentrations. In the first 116-day experiment, varying amounts of lichens or commercial reindeer feed were given to nine adult female reindeer allocated into three groups. Group 1 received lichens ad libitum for 76 days, followed by commercial reindeer feed for 40 days. Group 2a received lichens ad libitum for 6 days, followed by lichens at 80% of the ad libitum intake for 51 days with 10 g of MgO per day per animal and by lichens ad libitum for 59 days with MgO supplement. Group 2b was fed as group 2a, but without MgO supplementation. In the second experiment, six 10-month-old female reindeer calves with rumen cannulae were fed lichens for 18 days and then supplemented with two Mg alloy rumen boluses for 26 days of which they were fed commercial reindeer feed and some lichens for the last 11 days. The mean plasma Mg concentration of the animals supplemented with MgO increased from 0.73 to above 1 mmol/l when lichens were given as the only feed, whereas in the unsupplemented animals with otherwise the same feeding it declined below 0.70 mmol/l, which was considered hypomagnesemic, after 18 days. The differences in plasma Mg concentrations between the supplemented and unsupplemented groups were significant at all times when the animals fed on lichens (P Supplementation with Mg alloy rumen boluses increased plasma Mg (P In conclusion, both Mg supplements increased the plasma Mg concentration of reindeer, but the same plasma Mg level was also achieved by commercial reindeer feed. The considerable variation in the bolus weight loss suggests that their use as a Mg supplement may be limited to conditions where no other form of Mg supplementation or commercial reindeer feed can be provided.

The raccoon dog (Nyctereutes procyonoides) is a canid omnivore with marked seasonal changes in its body adiposity. The aim of this study was to investigate the roles of melatonin, leptin, ghrelin and growth hormone (GH) in weight regulation and reproduction of the species. Sixteen raccoon dogs were treated with continuous-release melatonin implants in Aug 2000 and in Feb 2001 (the MEL group) and 16 animals were sham-operated (the SHAM group). Half of the raccoon dogs were fasted between Nov 27(th) 2000 and Jan 25(th) 2001. The autumnal results have been previously published and this paper reports the vernal data. The leptin concentrations of the SHAM females were high before the mating season, decreased before estrus, increased during gestation and reduced after parturition. The MEL females had higher leptin concentrations than the SHAM females in early March, whereas the MEL males had lower leptin concentrations than the SHAM males in late March. Also the ghrelin and GH concentrations of the SHAM females decreased before estrus. Continuous melatonin treatment advanced the vernal rise in the ghrelin concentrations and the vernal drop and the subsequent rise in the GH concentrations of the females. Melatonin also increased their body mass indices from July to Aug 2001, indicating that it triggers the autumnal accumulation of fat in the species.

Pigeons were allowed to feed for 1 h either 2 h after lights on (morning pulse) or 3 h before lights off (evening pulse). Body temperature was measured radiotelemetrically. Faecal excretion, as an index of rate of digestion, was measured using load cells. At 22 degrees C, faecal excretion peaked just after lights-on in morning-pulse condition, but not in evening-pulse condition. In the cold (+5 degrees C), the peak was absent. We conclude that at thermoneutrality, pigeons are able to postpone a major part of digestion until late in the dark phase when their body temperature is increasing to the diurnal level. Thus, the extra heat from digestion-related thermogenesis that otherwise would be dissipated into the environment can be used for rewarming. In the cold, such a delay is not necessarily advantageous as the extra heat can always be used to substitute for thermoregulatory thermogenesis. The occurrence of concentrated period of digestion only in the morning-pulse condition was correlated to a lower food intake and lower body temperature as compared with the evening-pulse condition. Restricted feeding may thus be needed to induce adaptive timing of digestion as a mechanism of energy sparing. In addition to storage of food, timing of digestion may be a significant factor in the evolution of the crop in birds.

Thermal and metabolic responses of pigeons maintained outdoors throughout the year in small cages (sedentary birds) or in large aviaries (active birds) were measured at three ambient temperatures (20, -10, -40 ° C) in midwinter and midsummer. In winter, all birds maintained a significantly lower heat production (measured as oxygen consumption and shivering), body temperature, and conductance than in summer at the two lower test temperatures. All birds maintained homeothermic body temperatures down to -40 ° C. Activity level had no effects on these thermoregulatory variables. Body mass was significantly higher in winter birds, and pectoral mass was correspondingly increased in winter. An additional specific increase in pectoral muscle mass was seen in sedentary birds. Muscle cytochrome oxidase levels were significantly higher in active birds, especially during winter. In winter, the birds had a higher plasma triiodothyronine level than in summer, and lowest values were found in active summer birds. We conc...

Encephalopsin (OPN3) belongs to the light-sensitive transmembrane receptor family mainly expressed in the brain and retina. It is believed that light affects mammalian circadian rhythmicity only through the retinohypothalamic tract, which transmits light information to the suprachiasmatic nucleus in the hypothalamus. However, it has been shown that light penetrates the skull. Here, we present the effect of transcranial light treatment on OPN3 expression and monoamine concentrations in mouse brain and other tissues. Mice were randomly assigned to control group, morning-light group and evening-light group, and animals were illuminated transcranially five times a week for 8 min for a total of 4 weeks. The concentrations of OPN3 and monoamines were analysed using western blotting and HPLC, respectively. We report that transcranial light treatment affects OPN3 expression in different brain areas and plasma/adrenal gland monoamine concentrations. In addition, when light was administered at a different time of the day, the response varied in different tissues. These results provide new information on the effects of light on transmitters mediating mammalian rhythmicity.

We investigated whether transcranial bright light (TBL) affects nocturnal melatonin and cortisol secretion in sham-controlled crossover trial. Young healthy adults were exposed in random order to 24 minutes of TBL or sham exposure via ear canals at 01:10 h. Saliva and urine samples were collected hourly between 21 h-03 h and 06 h-09 h. There were no significant differences in melatonin or cortisol concentrations between TBL and sham exposures at any sampling point indicating that TBL via ear canals does not suppress nocturnal melatonin secretion. Thus, non-visual effects of TBL are mediated via a pathway not involving melatonin suppression.

The raccoon dog (RD) is a Eurasian dog-like mammal with a peculiar wintering strategy. It is the only member of the family canidae that can spend cold winters in an inactive state. Our objective was to investigate the molecular signatures regulating energy homeostasis during the crucial phases of seasonal adaptation in RD (autumnal fattening and winter fasting). We analyzed several key regulators of metabolism /food intake on the transcription levels from RD hypothalamus (orexigenic: NPY, orexin-2 receptors (OX2R) and anorexigenic: POMC, leptin receptors (LepR)) and from the RD plasma (leptin, insulin, ghrelin, peptide YY, GIP, interleukin 8, MCP-1). The 10-week winter fasting period had no significant effect on the analyzed mRNA expression levels in the hypothalamus. Plasma peptides and cytokines were also not significantly different. In contrast, autumnal fattening decreased the expression of hypothalamic OX2R (p=0,002) and also LepR (p=0,006). Plasma leptin was increased in the autumnal fattening phase...

Annual cycle of type I collagen formation and degradation and antler growth was studied in six adult female reindeer, Rangifer tarandus tarandus. Blood samples were collected twice a week during 1 year. Antler length was measured weekly during the antler growth period. An assay for human PICP, the carboxyterminal propeptide of type I procollagen, was used as an indicator of type I collagen formation and an assay for bovine ICTP, the carboxyterminal telopeptide of type I collagen, as an indicator of type I collagen degradation. PICP was clearly increased during the antler growth period. Also ICTP was slightly elevated during antler growth, but the highest values were found in autumn and winter. Our statistical analysis revealed that changes in lagged values (from 3 to 6 weeks) of PICP could be linked to the subsequent changes in the growth rate of the antler, although the highest values of PICP were found during the final third of antler growth. ICTP had significant predictive power as well, but the connection with the growth rate seemed more immediate than that of PICP. In conclusion, antler collagen synthesis can be predicted by PICP, but also ICTP was related to the antler growth.

The oxygen consumption of European finches, the siskin (Carduelis spinus), the brambling (Fringilla montifringilla), the bullfinch (Pyrhulla pyrhulla), the greenfinch (Carduelis chloris) and the hawfinch (Coccothraustes coccothraustes), was recorded continuously while ambient temperature was decreased stepwise from +30 down to-75°C. The oxygen consumption, body temperature (telemetrically), and shivering (integrated pectoral electromyography) of greenfinches were measured simultaneously at ambient temperatures between +30 and-75°C. Maximum heat production, cold limit, lower critical temperature, basal metabolic rate and thermal conductance (of the greenfinch) were determined. The diurnal variation of oxygen consumption of siskins and greenfinches was recorded at thermoneutrality and below the thermoneutral zone in winter- and summer-acclimatized birds. The diurnal variation of body temperature and thermal conductance of greenfinches were also determined. The diurnal variation of heat production was not seasonal or temperature dependent in the siskin and in the greenfinch. Nocturnal reduction of oxygen consumption saved 15–33% energy in the siskin and greenfinch. Body temperature of the greenfinch was lowered by 2.5–3.4°C. The nocturnal reduction of thermal conductance in the greenfinch was 39–48%. The basal metabolic rate was lowest in the largest bird (hawfinch) and highest in the smallest bird (siskin). The values were in the expected range. The heat production capacity of finches in winter was 4.7 times basal metabolic rate in the siskin, 4.2 times in the brambling, 3.5 times in the greenfinch and 2.9 times in the bullfinch and hawfinch. The heat production capacity of the siskin and greenfinch was not significantly lower in summer. The cold limit temperatures (°C) in winter were-61.2 in the siskin,-41.3 in the greenfinch,-37.0 in the bullfinch,-35.7 in the brambling and-28.9 in the hawfinch. The cold limit was 14.3°C higher in summer than in winter in the siskin and 8.7°C in the greenfinch. Thermal insulation of the greenfinch was significantly better in winter than in summer. The shivering of the greenfinch increased linearly when ambient temperature was decreased down to-40°C. Maintenance of shivering was coincident with season. In severe cold integrated pectoral electromyography did not correlate with oxygen consumption as expected. The possible existence of non-shivering thermogenesis in birds is discussed. It is concluded that the acclimatization of European finches is primarily metabolic and only secondly affected by insulation.

The physiological indicators such as body temperature, blood chemistry and hematology of seven European brown bears (Ursus arctos arctos) were used in the present study. They were kept in either the Zoological Garden of University of Oulu (65 degrees N, 25 degrees 24'E) or the Ranua Zoological Garden approx. 150 km NE of Oulu. Transmitters with a temperature-dependent pulse rate were implanted subcutaneously or into the abdominal cavity under anesthesia. Our data indicate that the body temperature of the bear decreases during the winter sleep to 4-5 degrees C below the normal level (37.0-37.5 degrees C). The lowest values, 33.1-33.3 degrees C, were measured several times in midwinter. Hematocrit, hemoglobin and erythrocyte counts seem to be higher, and the leucocyte count lower during the denning period than in the awake bear. Plasma N-wastes were lower during the winter sleep than before or after it. The analysed blood parameters showed that plasma catecholamines and thyroid hormones decreased in the fall.

Hepatic lipidosis is a common pathological finding in the American mink (Neovison vison) and can be caused by nutritional imbalance due to obesity or rapid body weight loss. The objectives of the present study were to investigate the timeline and characterize the development of hepatic lipidosis in mink in response to 0-7 days of food deprivation and liver recovery after 28 days of re-feeding. We report here the effects on hematological and endocrine variables, body fat mobilization, the development of hepatic lipidosis and the alterations in the liver lipid classes and tissue fatty acid (FA) sums. Food deprivation resulted in the rapid mobilization of body fat, most notably visceral, causing elevated hepatosomatic index and increased liver triacylglycerol content. The increased absolute amounts of liver total phospholipids and phosphatidylcholine suggested endoplasmic reticulum stress. The hepatic lipid infiltration and the altered liver lipid profiles were associated with a significantly reduced proportion of n-3 polyunsaturated FA (PUFA) in the livers and the decrease was more evident in the females. Likewise, re-feeding of the female mink resulted in a more pronounced recovery of the liver n-3 PUFA. The rapid decrease in the n-3/n-6 PUFA ratio in response to food deprivation could trigger an inflammatory response in the liver. This could be a key contributor to the pathophysiology of fatty liver disease in mink influencing disease progression.

We investigated the effect of 2 wk continuous postinfarction subcutaneous melatonin supply on the expression of the rat left ventricular (LV) dihydropyridine receptor (DHPR), ryanodine receptor (RyR(2)), and sarco-endoplasmic reticulum Ca(2+)-ATPase2 (SERCA2), as they are fundamental proteins in cardiac contractility. The levels of plasma and LV atrial (ANP) and brain natriuretic peptide and melatonin were also measured, as was the expression of LV MT(1) and MT(2) receptors and pineal arylalkylamine N-acetyltransferase. Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery and vehicle or melatonin (4.5 mg/kg per day) was administered by subcutaneous osmotic pumps. Echocardiography, real-time quantitative reverse transcription-polymerase chain reaction, and western blotting were used to analyze the samples. Echocardiography revealed that MI induced serious systolic LV dysfunction. The expression of DHPR, RyR(2), and SERCA2 mRNAs was significantly lower in the LVs of melatonin-treated MI rats compared with vehicle-treated rats (P < 0.01 for DHPR and P < 0.05 for RyR(2) and SERCA2). Melatonin also elevated the amount of LV MT(2) receptors to 1.9-fold (P < 0.05) and the concentration of LV ANP to over fivefold (P < 0.05) compared with vehicle rats after MI. Therefore, the results suggest that melatonin may influence the cardiac contractility after MI by regulating the expression of DHPR, RyR(2), and SERCA2, and melatonin receptors, particularly MT(2)s, might contribute to the postinfarction cardioprotective actions of melatonin. Furthermore, the finding of the relationship between melatonin and ANP suggests a novel mechanism for melatonin in protecting the heart after MI.

The American marten (Martes americana) is a boreal forest marten with low body adiposity throughout the year. The aim of this study was to investigate the adaptations of this lean-bodied species to fasting for an ecologically relevant duration (48 h) by exposing eight farm-bred animals to total food deprivation with seven control animals. Selected morphological and hematological parameters, plasma and serum biochemistry, endocrinological variables and liver and white adipose tissue (WAT) enzyme activities were determined. After 48 h without food, the marten were within phase II of fasting with depleted liver and muscle glycogen stores, but with active lipid mobilization indicated by the high lipase activities in several WAT depots. The plasma ghrelin concentrations were higher due to food deprivation, possibly increasing appetite and enhancing foraging behavior. The lower plasma insulin and higher cortisol concentrations could mediate augmented lipolysis and the lower triiodothyronine levels could suppress the metabolic rate. Fasting did not affect the plasma levels of stress-associated catecholamines or variables indicating tissue damage. In general, the adaptations to short-term fasting exhibited some differences compared to the related farm-bred American mink (Mustela vison), an example of which was the better ability of the marten to hydrolyze lipids despite its significantly lower initial fat mass.

The aim of this study was to investigate whether the actively wintering Palearctic sable Martes zibellina has evolved physiological adaptations to tolerate nutritional scarcity. Sixteen farm-bred male sables were divided into a fed control group and an experimental group fasted for 4 days. The rate of weight loss in the sable was similar to other medium-sized mustelids. Fasting led to hypoglycaemia and to a decreased lymphocyte percentage. The sable derived metabolic energy from both subcutaneous and intraabdominal white adipose tissues and the relative decrease in fat mass was the largest for the retroperitoneal and subcutaneous depots. Metabolic energy derived partly from body proteins indicated by the increased plasma levels of urea, uric acid and total essential amino acids. Triacylglycerols accumulated in the livers of the fasted sables and the increased plasma aminotransferase activities suggested hepatic dysfunction. The decreased plasma insulin concentrations and the elevated cortisol levels probably contributed to stimulated lipolysis and protein catabolism. Moreover, fasting increased the plasma ghrelin concentrations of the sables and down-regulated the thyroid activity.

The aim of this study was to investigate the endocrine response to wintertime starvation in the male American mink (Mustela vison) fasted for 16 hrs, 2 days, 3 days, 5 days, or 7 days (n–10 per group). After 2 days of fasting, the plasma leptin concentrations decreased, along with the triiodothyronine, testosterone, and progesterone levels, and the blood monocyte counts. Leptin also seems to trigger the response to fasting in mustelids by inducing immunosuppression and downregulation of the reproductive and thyroid axes. The dramatic increase in the peptide YY concentrations after 3 days of fasting may be required to suppress gastrointestinal processes during food scarcity. The plasma insulin levels decreased, and those of glucagon increased after 5 days of fasting in association with efficient glucose sparing and lipid mobilization. Body energy stores cannot be wasted for growth during nutritional scarcity and, thus, the growth hormone levels of the minks decreased after 5 days of fasting. The plasma noradrenaline and cortisol concentrations also decreased after 3 and 7 days without food, respectively. The plasma ghrelin, adiponectin, resistin, thyroxine, adrenaline, or estradiol levels did not respond to fasting. The endocrine response to food deprivation is remarkably similar in divergent mammalian orders, indicating that the hormonal signals enhancing survival during nutritional scarcity must be evolutionarily old and well conserved.

Plasma total protein (TP), albumin (ALB) and globulin (GLOB) concentrations and albumin/globulin ratio (A/G) were analysed from blood samples collected from free-ranging reindeer (Rangifer tarandus tarandus) herds at varying times of year. The same parameters were followed in nine captive reindeer with varying protein and energy intake. Variation in the blood constituents of free-ranging reindeer was analysed in relation to different extrinsic and intrinsic factors and compared to findings from captive animals, allowing the analysis of effects of protein and energy intake. There was large overall variation in TP, ALB, GLOB and A/G ratio in the free-ranging animals, ranging between 36-110 g/L, 18-59 g/L, 17-59 g/L and 0.5-2.1. The variation between months and years was significant for all variables except the A/G ratio, where no year effect was noted. Increase in live body mass was associated with a small significant increase and pregnancy with a small significant decrease in all dependent variables, except for the A/G ratio. Age did not have a significant effect on any of the blood constituents when body mass was included in the same model. In captive animals, feeding lichens with low protein content was related to a significant decline in TP, ALB and GLOB, but not in the A/G ratio, whereas feeding commercial ration increased plasma TP, ALB and GLOB significantly. Extrinsic factors such as season and year explained majority of variation in the blood constituents of free-ranging reindeer, whereas body mass, pregnancy and age had only a minor influence. It is concluded that plasma TP and ALB, and to a lesser extent GLOB and A/G ratio may serve as nutritional biomarkers of reindeer.

The raccoon dog (Nyctereutes procyonoides) is an omnivorous canid utilizing the passive wintering strategy in the boreal climate. Farmed raccoon dogs (n=12) were randomly assigned into two study groups on 26 November 2003. Between 3 December 2003 and 27 January 2004, half of the animals were fasted for 8 weeks and plasma weight-regulatory hormone concentrations determined on 26 November and 30 December 2003 and on 27 January 2004. The plasma peptide YY, ghrelin, and growth hormone (GH) concentrations increased due to food deprivation, while the T4 and Acrp30 concentrations decreased. Furthermore, the plasma GH concentrations were higher in the fasted raccoon dogs than in the fed animals, which had higher plasma insulin, glucagon, and T4 concentrations. However, fasting had no effect on the plasma leptin concentrations. The results confirm previous findings with unchanged leptin levels in fasting carnivores. Increased GH levels probably contribute to increased lipolysis and mobilization of fat stores. Ghrelin can also enhance lipolysis by increasing the GH levels. The decreased levels of T4 may reduce the metabolic rate. The plasma dopamine concentrations decreased due to fasting unlike observed previously in rats. Together with the unaffected adrenaline, noradrenaline, and cortisol concentrations, this suggests that food deprivation in winter does not cause stress to the raccoon dog but is an integral part of its natural life history.

The raccoon dog (Nyctereutes procyonoides) is a middle-sized canid with profound autumnal fattening followed by winter sleep. This study investigated the effects of prolonged fasting-induced winter sleep on the fat and nitrogen metabolism of the species. Half of the animals were treated with continuous-release melatonin implants to induce artificial short photoperiod. Autumnal accumulation of fat was characterized by low plasma free fatty acid (FFA), diacylglycerol (DG), and triacylglycerol (TG) levels. After transition to winter catabolism, the circulating lipid levels increased due to enhanced lipolysis. Two months of fasting resulted in a steady 3.1 kg weight loss (28% of body mass, 0.47% day(-1)). Storage fat was mobilized during the winter sleep reflected by the elevated FFA and DG concentrations. The lowered insulin levels could be a stimulator for TG hydrolysis. The plasma total amino acid concentrations, urea levels, and urea-creatinine ratios decreased due to fasting, whereas ammonia and total protein concentrations remained stable. The effects of melatonin on energy metabolism were modest. The results indicate that the raccoon dog is well adapted to long-term wintertime fasting utilizing fat as the principal metabolic fuel. The species can maintain its protein catabolism constant for at least 60 days. Decreased cortisol and thyroid hormone concentrations may contribute to protein sparing.

The pigeon's main source of regulated heat production, shivering, is especially likely to be used for thermoregulation during the dark phase of the day when there is little heat from locomotor activity. However, food stored in the pigeon's crop is digested during the night, and digestion-related thermogenesis (DRT) will provide heat that should decrease the need for shivering to maintain body temperature (Tb). We investigated the conditions under which DRT alters the occurrence of nocturnal shivering thermogenesis in pigeons. In fasting experiments, in which DRT was minimal, variations in pectoral shivering were closely related to the kinetics of nocturnal Tb when the ambient temperature (Ta) was moderate (21 degrees C). In that case, shivering was low while Tb fell at the beginning of the night, moderate during the nocturnal plateau in Tb, and strong during the prelight increase in Tb. Similar kinetics of nocturnal Tb occurred when Ta = 28 degrees C, but shivering was negligible throughout the dark phase. In restricted feeding experiments, nocturnal DRT was varied by providing different amounts of food late in the light phase. When Ta = 21 degrees C, 11 degrees C, and 1 degrees C, nocturnal Tb and O2 consumption were directly related to the amount of food ingested. However, nocturnal shivering tended to decrease as the food load increased and was significantly reduced at the higher loads. Because nocturnal shivering did not become more efficient in producing heat as the size of the food load increased, we conclude that nocturnal DRT decreased the need for shivering thermogenesis.

Introduction OPN4 (Melanopsin) is extravisual opsinreceptor sensing light outside of visual system. Earlier literature has described OPN4 in entraining circadian rhythms via retinohypothalamic track in human brain. Objectives To investigate whether OPN4 protein is present also outside the retinohypothalamic track. Aims To study the distribution on OPN4 protein in functionally central areas of human brain. Methods The localization of OPN4 protein in human brain and peripheral tissues was assessed by immunohistochemical staining using polyclonal antibody against OPN4. OPN4 protein content was measured using Western blotting and SDS-PAGE. The samples from altogether nine cadavers were assessed during forensic examination and their tissues prepared. Samples were cut into sections and stained with fluorescent dye labeled antibody before confocal laser scanning microscopy. The specificity of labeling and immununoreaction was verified by primary antibody omitting and immunizing peptide blocking. Results We found OPN4 protein abundant in Human brain, but not in periphery or in negative controls. Neuronal OPN4 was present in granular pattern intracellularly altogether in all eighteen examined sites, including numerous cerebral cortical areas, cerebellar cortex and several nuclei in phylogenetically old regions. Immunoreaction expressed mostly in neuronal soma, but not in nuclei. Conclusions Previously OPN4 has been known to be present only in non-image forming visual system mediating circadian timing properties of light to the pacemaker (Zeitgeber). However, our findings of abundant presense of OPN4 protein in neurons of human brain call further studies of effects on direct photic stimuli to brain.

Accumulated information shows that, besides its role in the timing of seasonal reproduction, melatonin also plays an important role in seasonal thermoregulatory adjustments of animals including torpor and hibernation. Furthermore, melatonin has a crucial role in circadian thermoregulatory adjustments of body temperature (Tb). Melatonin appears to send signals to the preoptic area of anterior hypothalamus (PoAH) where it adjusts the set point of Tb consistent with the metabolic rate of the animal. This new function for melatonin as a transducer mediating information about energy balance has been suggested in this review. Melatonin also adjusts the activity of the biological clock in vertebrates.

Captive reindeer were fed four different rations which supplied equal energy but varying amount of crude protein: lichens (3%), mixed hay plus lichens (10%) and two feed concentrates with medium (12%) or high (18%) protein content. Kinetics of total body water were measured with tritiated water from February to March 1985, and thermal energy cost of daily water intake was estimated. Biological half-time of water was shorter, and daily water intake significantly higher on the hay plus lichens diet and on the medium- or high-protein concentrates diet than on the lichens diet during March 1985. Similar differences were found between reindeer on medium-protein concentrate and on lichens at the end of the corresponding feeding period during April 1986. Daily water inflow was positively correlated with a dietary supply of digestible crude protein (r = 0.916). Thermal energy costs of daily water intake were highest 1.9 MJ (3.7 l-1) in reindeer on high-protein concentrate during March 1985, and 2.0 MJ (3.9 l-1) in reindeer on medium-protein concentrate during April 1986. The reindeer fed on lichens had minimal and nearly twice as small thermal energy cost of daily water intake (mean 1.1 MJ 2.1 l-1) as on either of the concentrates. Our results show that even a moderate feed protein ration can significantly increase free water intake and its thermal energy cost in reindeer as compared to dominant natural feed (lichens) during winter. Careful protein supplementation is recommended to support body condition with a concomitant addition of easily soluble carbohydrates to compensate for increased thermal costs of water intake.