Effects of exposure temperature on brown adipose tissue uncoupling protein mRNA levels

The effect of temperature on the amount of uncoupling protein mRNA in rat brown adipose tissue was examined after 1 and 14 days of exposure to cold. The relative amounts after 1 day, compared with rats kept at a thermoneutral temperature of 28 degrees C, were 3.2 at 19 degrees C, 3.3 at 11 degrees C, and 2.1 at 3 degrees C. This suggests that in warm-acclimated rats, a maximal response to a cold stimulus in brown adipose tissue is reached by 19 degrees C. In contrast to these results, the relative amounts of uncoupling protein mRNA after 14 days of cold exposure, compared with rats left at 28 degrees C, were 1.2 at 19 degrees C, 1.9 at 11 degrees C, and 2.1 at 3 degrees C. Since it is known that the amount of uncoupling protein in cold-acclimated rats increases continuously with decrease in temperature, the amount of protein reflects the mRNA levels during later times but not the initial time of exposure to cold.     

Opposite regulation of uncoupling protein 1 and uncoupling protein 3 in vivo in brown adipose tissue of cold-exposed rats

Earlier we reported a 14-fold increase of glycogen in the brown adipose tissue (BAT) in rats when the animals were placed back from cold to neutral temperature. To elucidate the mechanism, here we compared the level of glucose transporter 4 (GLUT4) protein, uncoupling protein (UCP) 1 and UCP3 mRNA and protein expressions in the BAT under the same conditions. We found that the increased GLUT4 level in cold was maintained during the reacclimation. After 1 week cold exposure the mRNA and protein content of UCP1 increased parallel, while the protein level of UCP3 decreased, contrary to its own mRNA level.     

Selective loss of uncoupling protein mRNA in brown adipose tissue on deacclimation of cold-acclimated mice

The time course of changes in the level of uncoupling protein mRNA when cold-acclimated mice were returned to a thermoneutral environment (33 degrees C) was examined using a cDNA probe. Upon deacclimation, there was a marked loss of uncoupling protein mRNA within 24 h, which precedes the loss of uncoupling protein from mitochondria. This loss of uncoupling protein mRNA was selective, since there was no change in the relative proportion of cytochrome c oxidase subunit IV mRNA or poly(A)+ RNA in total RNA. The results suggest that the decrease in the mitochondrial content of uncoupling protein during deacclimation is likely the result of turnover of existing protein, with very little replacement due to a lower level of its mRNA.     

Increased nonshivering thermogenesis, brown fat cytochrome-c oxidase activity, GDP binding, and uncoupling protein mRNA levels after short daily cold exposure of Phodopus sungorus

In their natural environment, burrowing rodents experience rather fluctuating ambient temperatures and are acutely cold exposed only for short periods outside their burrows. The effect of short daily cold exposure on basal metabolic rate, nonshivering thermogenesis, brown fat thermogenesis, and uncoupling protein mRNA was studied in the Djungarian hamster, Phodopus sungorus. They were kept at 23 degrees C and exposed to 5 degrees C daily either for one 4-h period or twice for 2 h (in 12-h intervals). At the same time control hamsters were kept continuously either at thermoneutrality (23 degrees C) or at 5 degrees C. Two 2-h cold exposures daily were sufficient to increase basal metabolic rate and nonshivering thermogenesis to the same level as continuous cold exposure, whereas one 4-h cold period per day did not result in a significant increase of both parameters. Brown fat thermogenesis (as measured by cytochrome-c oxidase activity and GDP binding to the mitochondrial uncoupling protein) increased to the same extent by both treatments with short daily cold exposure. However, this increase was less than in the chronically cold-exposed hamsters. A similar result was found for uncoupling protein mRNA: both short-term cold-exposed hamsters increased uncoupling protein mRNA levels to a similar extent, but less than after chronic cold treatment. It is concluded that short daily cold exposures are sufficient to cause adaptive increases of the capacity of metabolic heat production as well as brown fat thermogenic properties.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

Uncoupling protein is expressed in liver mitochondria of cold-exposed and newborn rats

Uncoupling protein has been thought to be expressed only in the brown adipose tissue mitochondria of mammals. However, mRNA encoding mitochondrial uncoupling protein was detected in the liver of newborn rats and adult rats after cold exposure, although not in the liver of untreated adult rats.     

Acute cold exposure-induced down-regulation of CIDEA, cell death-inducing DNA fragmentation factor-α-like effector A, in rat interscapular brown adipose tissue by sympathetically activated β3-adrenoreceptors

The thermogenic activity of brown adipose tissue (BAT) largely depends on the mitochondrial uncoupling protein 1 (UCP1), which is up-regulated by environmental alterations such as cold. Recently, CIDEA (cell death-inducing DNA fragmentation factor-α-like effector A) has also been shown to be expressed at high levels in the mitochondria of BAT. Here we examined the effect of cold on the mRNA and protein levels of CIDEA in interscapular BAT of conscious rats with regard to the sympathetic nervous system. Cold exposure (4 °C for 3 h) elevated the plasma norepinephrine level and increased norepinephrine turnover in BAT. Cold exposure resulted in down-regulation of the mRNA and protein levels of CIDEA in BAT, accompanied by up-regulation of mRNA and protein levels of UCP1. The cold exposure-induced changes of CIDEA and UCP1 were attenuated by intraperitoneal pretreatment with propranolol (a non-selective β-adrenoreceptor antagonist) (2 mg/animal) or SR59230A (a selective β₃-adrenoreceptor antagonist) (2 mg/animal), respectively. These results suggest that acute cold exposure resulted in down-regulation of CIDEA in interscapular BAT by sympathetically activated β₃-adrenoreceptor-mediated mechanisms in rats.     

Muscle type difference in the regulation of UCP3 under cold conditions

We found opposite regulation of uncoupling protein 3 (UCP3) in slow-twitch soleus and fast-twitch gastrocnemius muscles of rats during cold exposure. Namely, the UCP3 mRNA level was downregulated in the soleus muscles, but upregulated in the gastrocnemius muscles after a 24-h cold exposure. In the analysis of UCP3 protein, we first succeeded in detecting UCP3 short-form as well as the long-form in vivo, which levels were decreased markedly in the cold-exposed soleus muscles. However, the levels of UCP3 and cytochrome oxidase subunit IV were well maintained in the cold-exposed gastrocnemius muscles with a rise in the total mitochondrial protein level, suggesting an increase of total oxidative ability. The fast-twitch muscle rather than the slow-twitch one may play an important role in adaptive responses, including thermogenesis under acute cold exposure.     

Nicotine infusion alters leptin and uncoupling protein 1 mRNA expression in adipose tissues of rats

We attempted to clarify whether leptin and uncoupling protein 1 (UCP1) are involved in the action of nicotine on the energy balance. Male Wistar rats were infused subcutaneously with nicotine (12 mg x kg(-1) x day(-1)) for 4 or 14 days. At the end of the 4-day period, the plasma concentrations of leptin of the nicotine-treated and pair-fed rats were lower than those of the freely fed rats, although the levels of leptin mRNA expression in various white adipose tissues did not differ among the three groups. At the end of the 14-day nicotine infusion period, plasma concentrations of leptin were higher, and leptin mRNA expression in the omentum and epididymal and retroperitoneal adipose tissues was stronger in the nicotine-treated rats than in the pair-fed and freely fed rats. UCP1 mRNA expression in the brown adipose tissue of nicotine-treated was stronger than that of the pair-fed rats. These results suggest that continuous nicotine infusion differentially affects the synthesis and secretion of leptin according to the duration of infusion and stimulates UCP1 mRNA expression, probably in a manner independent of leptin.     

Increase of uncoupling protein and its mRNA in brown adipose tissue of rats fed on ''cafeteria diet''.

The effect of 'cafeteria diet' on mitochondrial uncoupling protein in brown adipose tissue of rats was examined. 'Cafeteria diet' induced an increase of the 32 kDa uncoupling protein in electrophoresed proteins of brown-fat mitochondria. Use of a cDNA probe corresponding to uncoupling-protein mRNA indicated that this mRNA was increased in rats fed on the 'cafeteria diet'. Nevertheless, this effect was weak compared with that observed in rats adapted to cold.     

Identification of serine phosphorylation in mitochondrial uncoupling protein 1

Native uncoupling protein 1 was purified from rat brown adipose tissue of cold-acclimated rats and rats kept at room temperature, in the presence of phosphatase inhibitors. The purified protein from cold-acclimated animals was digested with trypsin and immobilized metal affinity chromatography was used to select for phosphopeptides. Tandem mass spectroscopic analysis of the peptides derived from uncoupling protein 1, suggests phosphorylation of serine 3 or 4 and identified phosphorylation of serine 51. Furthermore, we were able to demonstrate that antibodies to phosphoserine detect full-length UCP 1 and that the proportion of phosphoserine on UCP1, purified from cold-acclimated rats, was significantly greater than that on UCP 1 from rats kept at room temperature (90+/-4% compared to 62+/-8%, p=0.013), respectively). We conclude that uncoupling protein 1 is a phosphoprotein and that cold-acclimation increases the proportion of UCP1 that is serine phosphorylated.     

Effects of chronic spinal cord injury on body weight and body composition in rats fed a standard chow diet

The inability to maintain body weight within prescribed ranges occurs in a significant portion of the human spinal cord injury (SCI) population. Using a rodent model of long-term high thoracic (spinal level T3) spinal cord transection (TX), we aimed to identify derangements in body weight, body composition, plasma insulin, glucose tolerance, and metabolic function, as measured by uncoupling protein 1 (UCP1) expression in interscapular brown adipose tissue (IBAT). Sixteen weeks after SCI, body weights of injured female rats stabilized and were significantly lower than surgical control animals. At the same time point, SCI rats had a significantly lower whole body fat:lean tissue mass ratio than controls, as measured indirectly by NMR. Despite lower body weight and fat mass, the cumulative consumption of standard laboratory chow (4.0 kcal/g) and mean energy intake (kcal.day(-1).100 g body wt(-1)) of chronic SCI rats was significantly more than controls. Glucose tolerance tests indicated a significant enhancement in glucose handling in 16-wk SCI rats, which were coupled with lower serum insulin levels. The post mortem weight of gonadal and retroperitoneal fat pads was significantly reduced after SCI and IBAT displayed significantly lower real-time PCR expression of UCP1 mRNA. The reduced fat mass and IBAT UCP1 mRNA expression are contraindicative of the cumulative caloric intake by the SCI rats. The prolonged postinjury loss of body weight, including fat mass, is not due to hypophagia but possibly to permanent changes in gastrointestinal transit and absorption, as well as whole body homeostatic mechanisms.     

Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats

The control of uncoupling protein-1, -2 and -3 (UCP-1, UCP-2, UCP-3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT-PCR assays. In rats reared at thermoneutrality (25 degrees C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP-1 mRNA level (-38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP-2 and -3 mRNA levels. Cold exposure (7 days, 4 degrees C) markedly increased UCP-1 (+180%), UCP-2 (+115%) and UCP-3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold-induced rise in BAT UCP-1 and UCP-2 mRNAs, but not that in BAT UCP-3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP-1, UCP-2 and UCP-3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure.     

Differential effects of leptin administration on the abundance of UCP2 and glucocorticoid action during neonatal development

In the neonate, adipose tissue and the lung both undergo a rapid transition after birth, which results in dramatic changes in uncoupling protein abundance and glucocorticoid action. Leptin potentially mediates some of these adaptations and is known to promote the loss of uncoupling protein (UCP)1, but its effects on other mitochondrial proteins or glucocorticoid action are not known. We therefore determined the effects of acute and chronic administration of ovine recombinant leptin on brown adipose tissue (BAT) and/or lung in neonatal sheep. For the acute study, eight pairs of 1-day-old lambs received, sequentially, 10, 100, and 100 mug of leptin or vehicle before tissue sampling 4 h from the start of the study, whereas in the chronic study, nine pairs of 1-day-old lambs received 100 mug of leptin or vehicle daily for 6 days before tissue sampling on day 7. Acute leptin decreased the abundance of UCP2, glucocorticoid receptor, and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 mRNA and increased 11beta-HSD type 2 mRNA abundance in BAT, a pattern that was reversed with chronic leptin administration, which also diminished lung UCP2 protein abundance. In BAT, UCP2 mRNA abundance was positively correlated to plasma leptin and nonesterified fatty acids and negatively correlated to mean colonic temperature in the leptin group at 7 days. In conclusion, leptin administration to the neonatal lambs causes differential effects on UCP2 abundance in BAT and lung. These effects may be important in the development of these tissues, thereby optimizing lung function and fat growth.     

Expression of aquaporin 9 in rat liver and efferent ducts of the male reproductive system after neonatal diethylstilbestrol exposure.

Aquaporins (AQP) have important solute transport functions in many tissues including the epididymal efferent ducts (ED) and in the liver. We investigated the effect of neonatal exposure to diethylstilbestrol (DES) on AQP9 expressions in the ED and in the liver of rats. DES was administered from day 2 to day 20 postnatally at a dose of 4,8 microg/day, and AQP9 protein and mRNA were measured by immunoblotting and real-time PCR, respectively, along with immunohistochemistry. DES caused hepatic downregulation of AQP9 at both the protein and mRNA level; however, decreased AQP9 labeling was only observed in the periportal zone. In the ED, AQP9 protein expression was increased in the DES-treated animals by 300% that could be ascribed to a widening of the ED lumen, whereas no difference was observed in AQP9 mRNA expression. Immunohistochemical findings revealed that AQP9 expression was confined to the epithelial cells of the ED. In conclusion, neonatal DES exposure appears to upregulate AQP9 channels in the ED in male rats, whereas a downregulation in the hepatic expression was observed, particularly in the periacinous area.     

Molecular cloning of rat brain mitochondrial carrier protein-1 cDNA and its up-regulation during postnatal development

Brain mitochondrial carrier protein-1 (BMCP1), a new member of the mitochondrial uncoupling carrier, has been shown to be expressed predominantly in the brain of the mice and humans. We cloned rat BMCP1 cDNA and investigated its mRNA level during postnatal development and under various metabolic conditions. The nucleotide sequence of the cDNA revealed that rat BMCP1 protein was composed of 322 amino acid residues, and was 99 and 96% identical to the mouse and human proteins and 29, 33 and 35% identical to rat uncoupling protein (UCP) 1, UCP2 and UCP3, respectively. The molecular weight was predicted to be 36017 Da and the protein of this size was detectable when the cDNA was expressed in vitro. Using Northern blot analysis, the corresponding mRNA, approximately 1.8-kb in size, was found expressed predominantly in the cerebrum, cerebellum and hypothalamus. A unique developmental pattern was identified in the brain, where BMCP1 expression was low in their fetal life, but significantly elevated in the first postnatal week. Thereafter BMCP1 mRNA was maintained to be gradually increased. In 48-h fasted or insulin-induced hypoglycemic rats, BMCP1 mRNA expression in the hypothalamus slightly, but significantly, decreased compared with that in their appropriate controls. The present results indicate that BMCP1 may be involved in pathogenesis of mitochondrial dysfunction in neurons induced by aging or neurodegenerative disorders, and perhaps in energy balance in the brain.