Plasma free fatty acid levels during cold-induced and noradrenaline-induced nonshivering thermogenesis in the Djungarian hamster

Summary In Djungarian hamsters the cold-induced thermoregulatory heat production was preceeded and accompanied by an increase in the plasma level of free fatty acids. In warm-acclimated hamsters this increase was found more pronounced (0.85 to 1.48 mM) than in cold-acclimated hamsters (0.64 to 0.88 mM). Noradrenaline-induced thermogenesis at thermoneutrality provoked a similar increase in the free fatty acid level. Inhibition of nonshivering thermogenesis during cold exposure by propranolol abolished the increase in free fatty acids completely. The surgical removal of brown adipose tissue proportionately reduced the increase in free fatty acids. This indicates that the rise in plasma free fatty acids is functionally related to nonshivering thermogenesis and originates from brown adipose tissue.     

Effect of unilateral surgical denervation of brown adipose tissue on uncoupling protein mRNA level and cytochrom-c-oxidase activity in the Djungarian hamster

The bilateral lobe of interscapular brown adipose tissue of the Djungarian hamster was unilaterally denervated in order to study the role of the sympathetic innervation for maintenance and cold-induced increase of non-shivering thermogenesis. Denervation decreased the noradrenaline content of brown adipose tissue to less than 9% of the intact contralateral pad. This low noradrenaline level was maintained for 1–14 days after denervation. First, to study the role of the sympathetic innervation of brown adipose tissue in the maintenance of the high thermogenic capacity characteristic of the cold acclimated state, brown adipose tissue was denervated in hamsters either kept at thermoneutrality or acclimated to 5°C ambient temperature for 4 weeks. Cold-acclimated hamsters had elevated levels of uncoupling protein messenger ribonucleic acid (8.1-fold) and cytochrom-c oxidase-activity (3-fold). Denervation of brown adipose tissue decreased uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity as compared to the intact pad in thermoneutral and in cold-acclimated hamsters. However, in cold-acclimated hamsters uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity in denervated brown adipose tissue both were maintained on an elevated 6-fold higher levels as compared to thermoneutral controls. Second, to study the role of the sympathetic innervation of brown adipose tissue in the cold-induced increase in thermogenic capacity, hamsters were denervated prior to cold acclimation and responses were measured after 3 and 14 days of cold exposure. Uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity of intact brown adipose tissue increased after 14 days cold acclimation. Denervation did not completely prevent a cold-induced 1.5-fold increase of cytochrom-c-oxidase-activity and a 3.2-fold increase of the uncoupling protein-messenger ribonucleic acid level in denervated brown adipose tissue after 14 days of cold acclimation. In conclusion, high levels of uncoupling protein-messenger ribonucleic acid and cytochrom-c-oxidase activity in brown adipose tissue of cold-acclimated hamsters can partially be maintained without intact sympathetic innervation, suggesting a considerable contribution of trophic factors not requiring sympathetic innervation for maintenance. The cold-induced increase of cytochrom-c-oxidase activity and expression of uncoupling protein-messenger ribonucleic acid largely depends upon sympathetic innervation of brown adipose tissue.Abbreviations ANOVA analysis of variance - BAT brown adipose tissue - COX cytochrom-c-oxidase - HPLC high performance liquid chromatography - mRNA messenger ribonucleie acid - NA noradrenaline - T _a ambient temperature - UCP uncoupling protein     

Tissue-specific expression of the uncoupling protein family in streptozotocin-induced diabetic rats

The vulnerability of streptozotocin (STZ)-induced diabetic rats to cold stress has been established. One of the elements controlling body temperature is thermogenesis, in which uncoupling protein (UCP) is known to play an important role. We have examined UCP2 and UCP3 expressions in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle (MSL) during the acute and chronic phases of STZ-induced diabetes in rats. The long-term effect and the effect of insulin treatment thereafter were also unexplored previously and are examined in this study. In the acute phase of diabetes (2.5 days after STZ injection), UCP2 gene expression in BAT, WAT, and MSL, and UCP3 expression in the muscle were significantly increased. In the chronic phase of diabetes (21 days after STZ injection), UCP2 and UCP3 expression in the MSL were restored to the control levels without insulin supplementation. UCP2 in BAT and WAT remained high in the chronic phase, whereas UCP3 expression in BAT and WAT, which did not change in the acute phase, was significantly decreased. Insulin supplementation restored UCP2 expression in BAT and WAT, but over-corrected UCP3 in WAT above the control and did not affect UCP3 expression in BAT. Insulin supplementation depressed UCP3 expression in the MSL below control. These results indicate that the effects of STZ-induced diabetes on UCPs gene expression are tissue-specific as well as dependent on the duration of diabetes.     

Tissue-specific distribution of uncoupling proteins in normal rats and rats with high-fat-diet-induced obesity

Uncoupling proteins (UCPs) are a proton transporter family located in the mitochondrial inner membrane. Thus far, five molecules (UCP1–UCP5) have been identified as members of the UCP family. Recently, UCPs have attracted considerable interest in research on energy metabolism and obesity. However, to date, no study has focused on a comprehensive and systematic evaluation of the tissue-specific distribution of UCPs in obese individuals. Our study presents evidence of differential tissue expression profiles of five isoforms of UCPs in normal and diet-induced obese (DIO) rats using real-time polymerase chain reaction (PCR) analysis. The results clearly show that the tissue-specific expression patterns of individual isoforms between DIO and normal rats are quite distinct, which suggests a close relationship between the alterations in UCP expression and dietary obesity.     

Role of the sympathetic innervation in the cold-induced activation of 5'-deiodinase in brown adipose tissue of the Djungarian hamster.

The importance of the sympathetic innervation in the regulation of 5'-deiodinase activity in the interscapular brown adipose tissue (BAT) of the Djungarian hamster was studied. Interscapular BAT of Djungarian hamsters was either unilaterally or bilaterally denervated, and thereafter the animals were maintained at thermoneutral temperature or exposed to 0 degree C for 24 h. Denervation reduced the norepinephrine content to 2-10% of the level in the control groups. Unilateral denervation was as effective as bilateral denervation in depressing the norepinephrine content of the interscapular BAT. Cold exposure for 24 h resulted in a pronounced 5'-deiodinase activation. Denervation reduced, but did not completely prevent, the cold-induced increase in 5'-deiodinase activity. The basal level of 5'-deiodinase activity at thermoneutral temperature was not reduced by denervation. We conclude that cold-induced activation of BAT 5'-deiodinase primarily depends on the intact sympathetic innervation.     

Canine mitochondrial uncoupling proteins: structure and mRNA expression of three isoforms in adult beagles

Uncoupling proteins (UCPs) are members of the mitochondrial transporter family that dissipate the proton gradient as heat more than via ATP synthesis. In the present study, nucleotide and amino acid sequences of UCPs 1, 2 and 3 of a dog were determined, and their mRNA expression in various peripheral tissues was examined. The sequences were highly (76-97%) homologous to those of other species. Although lower homologies (60-74%) were found when compared among the three canine UCPs, their deduced amino acid sequences had some common domains, such as three mitochondrial carrier protein motifs, six transmembrane alpha-helix domains, and putative purine nucleotide binding domains. By Northern blot analyses, UCP1 mRNA was not detected in any tissues examined. UCP2 mRNA was expressed in most tissues, particularly abundantly in adipose tissue, spleen and lung. Two sizes of UCP3 mRNA were found exclusively in heart and skeletal muscle. These results suggest that canine UCPs have uncoupling activity, and are involved in the regulation of metabolic heat production and/or energy expenditure, as do those of other species.     

Tissue-specific modulation of insulin receptor mRNA levels in adrenaline-treated rats

Insulin receptor (IR) gene expression at the mRNA level was investigated in liver, hindlimb skeletal muscle, and epididymal adipose tissue of rats exposed to prolonged in vivo administration of adrenaline in relation to control rats. In the liver of adrenaline-treated rats, there were no differences in relation to controls when DNA and protein content were measured. In skeletal muscle, only a slight decrease in protein concentration was detected. By contrast, a clear increase in both protein and DNA content was observed in the adipose tissue of treated animals. Northern blot assays revealed two IR mRNA species of approximately 9.5 and 7.5 Kb in the three tissues from controls. Adrenaline treatment induced an increase of approximately 60% in the levels of both RNAs in adipose tissue but not in liver or skeletal muscle. These results provide evidence for an in vivo tissue-specific regulation of IR gene expression at the mRNA level in rats under an experimental condition of excess of catecholamines.     

Circadian regulation of pineal melatonin and reproduction in the Djungarian hamster

Gonadal state, pineal melatonin rhythms, and locomotor activity rhythms were examined in juvenile male Djungarian hamsters exposed to non-24-hr light cycles ("T-cycles") or to full photoperiods. At the end of 1 month, hamsters exposed to a 1-hr pulse of light every 24.33 hr (T 24.33) exhibited small testes, whereas those receiving the same amount of light every 24.78 hr (T 24.78) displayed stimulated gonads, ten-fold larger in size. Accompanying the nonstimulatory effect of the T 24.33 cycle were nocturnal peaks in both pineal melatonin content and serum melatonin concentration which were longer by approximately 4 hr than those observed on the photostimulatory T 24.78 cycle. Exposure to an intermediate-length T-cycle (T 24.53) resulted in a mixed gonadal response and in pineal and serum melatonin peaks of intermediate duration. Wheel-running activity was entrained to the T-cycles such that light was present only near the beginning of the subjective night, its phase (relative to activity onset) differing only slightly among T-cycle groups. Hence the durational differences observed in the melatonin peaks were apparently not due to the acute suppressive or phase-advancing effects of morning light on melatonin biosynthesis, but were rather the result of differences in the endogenous control of pineal activity by the circadian pacemaker system. While no strong correlation was detected between gonadal state and the phase of locomotor activity onset relative to the light pulse, a significant correlation was observed between gonadal state and the duration of daily locomotor activity (alpha). These data were compared to similar measures obtained from hamsters exposed to long-versus short-day full photoperiods (LD 16:8 vs. LD 10:14). In summary, the results of this study indicate involvement of the circadian pacemaker system of Djungarian hamsters in the control of pineal melatonin synthesis and secretion, and in photoperiodic time measurement. Furthermore, these data strengthen the hypothesis that it is the duration of nocturnal pineal melatonin secretion that is the critical feature of this neuroendocrine gland's photoperiodic signal.     

Seasonal adaptation of brown adipose tissue in the Djungarian hamster

Summary The composition and oxidative capacity of brown adipose tissue (BAT) were investigated in Djungarian hamsters kept under natural photoperiod, either indoors at neutralT _a (23°C) or under outdoor conditions. BAT comprises up to 5% of the body weight in summer/indoor hamster, with lipid representing 86% of the total tissue mass. Tissue mass and thermogenic capacity are inversely related during seasonal adaptation: 30% decrease of total DNA, accompanied by extensive lipid depletion, reduces the amount of BAT by almost 60% during acclimatization from summer/indoor to winter/outdoor conditions. Mitochondrial protein in BAT is increased by a factor of 2.6 concomitantly, and by a factor of 4 when related to body weight (body weight reduction 36%).Cytochrome oxidase activity in different brown fat deposits varies by up to 150% in summer/indoor hamsters; depending on the fat pad, the enzyme activity is increased 200%–700% during adaptation to winter/outdoor conditions.Natural photoperiod is decisive in determining the seasonal adaptation of DNA content in BAT and of body weight. Short photoperiod alone may lead to depletion of lipid content of BAT and thus decrease the tissue mass practically to the lowest seasonal level, even though both parameters may be also influenced byT _a. One third of the maximum adaptive increase of tissue mitochondria may be attributed to seasonal changes in photoperiod and up to two thirds toT _a. Photoperiod establishes a fixed fundament of slow-reacting functional adaptation of BAT, whereas the effect of decreasedT _a depends on the rate and duration of cold influence.Abbreviations BAT brown adipose tissue - NST nonshivering thermogenesis - T _a ambient temperature     

Functional characterisation of UCP1 in the common carp: uncoupling activity in liver mitochondria and cold-induced expression in the brain

Mammalian uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis in brown adipose tissue. We previously reported on the presence of a UCP1 orthologue in ectothermic fish and observed downregulation of UCP1 gene expression in the liver of the common carp. Neither the function of UCP1, nor the mode of UCP1 activation is known in carp liver mitochondria. Here, we compared the proton conductance at 25°C of liver mitochondria isolated from carp either maintained at 20°C (warm-acclimated, WA) or exposed to 8°C (cold-acclimated, CA) water temperature for 7–10 days. Liver mitochondria from WA carp had higher state four rates of oxygen consumption and greater proton conductance at high membrane potential. Liver mitochondria from WA, but not from CA, carp showed a strong increase in proton conductance when palmitate (or 4-hydroxy-trans-2-nonenal, HNE) was added, and this inducible proton conductance was prevented by addition of GDP. This fatty acid sensitive proton leak is likely due to the expression of UCP1 in the liver of WA carp. The observed biochemical properties of proton leak strongly suggest that carp UCP1 is a functional uncoupling protein with broadly the same activatory and inhibitory characteristics as mammalian UCP1. Significant UCP1 expression was also detected in our previous study in whole brain of the carp. We here observed a twofold increase of UCP1 mRNA in carp brain following cold exposure, suggesting a role of UCP1 in the thermal adaptation of brain metabolism. In situ hybridization located the UCP1 gene expression to the optic tectum responsible for visual system control, the descending trigeminal tract and the solitary tract. Taken together, this study characterises uncoupling protein activity in an ectotherm for the first time. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acute central infusion of leptin modulates fatty acid mobilization by affecting lipolysis and mRNA expression for uncoupling proteins

Chronic administration of leptin has been shown to reduce adiposity through energy intake and expenditure. The present study aims to examine how acute central infusion of leptin regulates peripheral lipid metabolism, as assessed by markers indicative of their mobilization and utilization. A bolus infusion of 1 microg/rat leptin into the third cerebroventricle increased the expression of mRNA for hormone-sensitive lipase (HSL), an indicator of lipolysis, in white adipose tissue (WAT). This was accompanied by elevation of plasma levels of glycerol, but not of free fatty acids, as compared to the saline control (P < 0.03). The same treatment with leptin decreased plasma insulin levels but did not affect the plasma glucose level (P < 0.05 for insulin). Among the major regulators of the transportation or utilization of energy substrates, leptin treatment increased expression of mRNA for uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), UCP2 in WAT, and UCP3 in quadriceps skeletal muscle, but not those for fatty acid-binding protein in WAT, carnitine phosphate transferase-1, a marker for beta oxidation of fatty acids in muscle, nor glucose transporter 4 in WAT and muscle (P < 0.01 for HSL, P < 0.05 for UCP1, and P < 0.005 for UCP2 and UCP3). These results indicate that, even in a single bolus, leptin may regulate the mobilization and/or utilization of energy substrates such as fatty acids by affecting lipolytic activity in WAT and by increasing the expression of UCPs in BAT, WAT, and muscle.     

Ketamine-xylazine anaesthesia in the Djungarian hamster (Phodopus sungorus)

The combination of ketamine-xylazine was assessed as a surgical anaesthetic in Djungarian hamsters acclimatized to both long (16 h light: 8 h dark) and short (8 h light: 16 h dark) photoperiods. It was concluded that 50 mg/kg of ketamine with 10 mg/kg of xylazine or 100 mg/kg of ketamine with 5-10 mg/kg of xylazine when given together by intraperitoneal injection was a satisfactory general anaesthetic. Two hundred mg/kg of ketamine with 10 mg/kg xylazine caused death in 13 of 24 animals. There were no clinically significant effects on depth of anaesthesia due to photoperiod.     

Genetic analyses of photoresponsiveness in the Djungarian hamster,Phodopus sungorus

Summary Endotherms living at temperate and arctic latitudes must adjust their physiology and behavior in order to survive seasonal change. The Djungarian hamster uses photoperiod to cue annual cycles of reproduction and thermoregulation, and its responses to short photoperiod include loss of body weight and change in pelage color. Some individuals do not exhibit these responses when exposed to short days.In this study individual variation in photoresponsiveness is quantified, and four lines of evidence for a genetic component to that variation are provided. First, two separate breeding stocks differed in both the percent of animals responding to a short-day lighting regimen (SD) and in the degree and timing of their response. Second, analysis of variance within and between families of full sibs for a photoresponsive index, PI (body weightloss +2 (molt index –1)) following 12 weeks in SD demonstrated a significant family resemblance (intraclass correlation of 0.36±0.03). Third, heritability estimates from regression of offspring scores on parent scores for body weight loss, molt index and PI after 12 weeks in SD were 0.340.13, 0.36±0.10 and 0.37±0.12, respectively, indicating a strong additive genetic component for the three characters. Finally, a significant response occurred after one generation of artificial selection for and against photoresponsiveness.Abbreviations PI photoresponsive index (body weight loss+2 (molt index –1)) - SD short day light regimen     

Attenuated cold-induced increase in mRNA for uncoupling protein in brown adipose tissue of obese (ob/ob) mice

The level of mRNA for uncoupling protein was measured in brown adipose tissue of young (8-10 weeks) and old (11 months) lean and ob/ob mice using a cDNA clone constructed previously. The level of poly(A)+ RNA was also measured using an oligo(dT)18 probe. Mice were kept at 28 degrees C or exposed to 14 degrees C for 12 h. The level of mRNA for uncoupling protein was normal in brown adipose tissue of younger obese mice but reduced in brown adipose tissue of old obese mice. The cold-induced absolute increase in uncoupling protein mRNA was smaller in obese mice, regardless of age. It is concluded that the known attenuation of the acute thermogenic response of brown adipose tissue of the ob/ob mouse to cold is accompanied by a similar attenuation of the initiation of the trophic response. It is likely, however, that these defects are secondary to the chronic reduction in sympathetic nervous system activity in brown adipose tissue of the ob/ob mouse, which results in a functional atrophy of the tissue.     

Nonrandom karyotype changes in chemically induced tumors of the Djungarian hamster

In the cells of tumors induced with methylcholanthrene in wild type and mutant (pink-eyed dilution) Djungarian hamsters non-random involvement in structural changes of certain chromosomes (Xp, 3p and 3q, 7q, 8q) was revealed. In addition, characteristic feature of the majority of tumors was varied number of double-minutes chromosomes (DMs). In some tumors, the markers with long homogeneously or differentially stained regions (HSRs and DSRs) were also present. The DMs, HSRs and DSRs are known as the structures containing amplified genes.