Loss of brown adipose tissue uncoupling protein mRNA on deacclimation of cold-exposed rats

The effect of environmental temperature on the level of uncoupling protein mRNA from rat brown adipose tissue was examined using a cDNA probe. A 4.4 fold increase in the mRNA level was observed after 1 day exposure of rats to 6 degrees C, which was followed by a slow loss with longer times of exposure. When rats were returned to a thermoneutral environment, there was a dramatic loss of uncoupling protein mRNA within 1 day. Comparison wih poly(A)+ RNA levels suggest that the response to temperature is specific for uncoupling protein mRNA.     

Group 3 LEA Gene HVA1 Regulation by Cold Acclimation and Deacclimation in Two Barley Cultivars with Varying Freeze Resistance

The level of expression of the group 3 late embryogenesis abundant abscisic acid-regulated gene (HVA1) to cold treatment has been studied in winter barley (Hordeum vulgare) seedling tissue. The cDNA clone (pHVA1) encoding this late embryogenesis abundant protein was used as a hybridization probe to detect the corresponding mRNA. Expression of the HVA1 gene was determined after the tissue had been subjected to a regimen of 2°C exposure (cold acclimation), followed by a return to 25°C growth conditions (deacclimation). Accumulation of HVA1 mRNA occurred upon cold acclimation of the tissue and disappeared as early as 2 hours after exposure to deacclimation conditions. A comparison of the response to cold acclimation and deacclimation was made between seedling tissue of a freeze-resistant and less freeze-resistant cultivar. In both cultivars, the HVA1 gene was expressed and modulated by cold treatment. Within 2 hours of deacclimation HVA1 mRNA was no longer detectable in either cultivar independently of freeze resistance. The level of expression of HVA1 appeared to be greater in the less freeze-resistant cultivar (Winter Malt).     

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.     

Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine

Expression of the gene for the brown-fat specific uncoupling protein thermogenin was investigated in cell cultures by hybridization of isolated RNA with a cDNA clone corresponding to mouse thermogenin. The RNA was isolated 3-4 days after confluence from cells differentiated in culture from precursors isolated from the interscapular brown adipose tissue of 5-week-old mice. Very low thermogenin mRNA levels were found in cells derived from untreated mice, and there was only little effect of added norepinephrine on thermogenin gene expression in these cells. However, in cells derived from hypothyroid (methimazole-treated) mice there was a higher expression of thermogenin, and norepinephrine had a marked augmenting effect on the thermogenin mRNA level in these cells. These effects of thermogenin mRNA levels were specific, in that they contrasted with the effects of hypothyroidism and norepinephrine on the level of other mRNA species in these cells (coding for beta-actin, lipoprotein lipase, cytochrome-c oxidase, and glycerol-3-phosphate dehydrogenase). It was concluded that brown-fat cells in culture can reach a differentiated state, sufficiently advanced that the unique properties of these cells can be expressed, and that thermogenin gene expression (i.e., the level of thermogenin mRNA) is under direct control of norepinephrine.     

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1)

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.     

Effect of running training on uncoupling protein mRNA expression in rat brown adipose tissue

The effect was investigated of endurance training on the expression of uncoupling protein (UCP) mRNA in brown adipose tissue (BAT) of rats. The exercised rats were trained on a rodent treadmill for 5 days per week and a total of 9 weeks. After the training programme, a marked decrease in BAT mass was found in terms of weight or weight per unit body weight; there was a corresponding decrease in DNA content and a downward trend in RNA and glycogen levels. The UCP mRNA was present at a markedly decreased level in BAT of trained animals. In consideration of the reduced levels of mRNAs for hormone-sensitive lipase and acylCoA synthetase, the brown adipose tissue investigated appeared to be in a relatively atrophied and thermogenically quiescent state.     

Age-related changes in mitochondrial UCP, ANT and COX III gene expression in the breast muscle of quails (Coturnix coturnix japonica)

Efficient conversion of food into body mass has been associated with altered gene expression of some proteins of the electron transport chain. We evaluated the effect of age on mRNA expression of uncoupling protein, adenine nucleotide translocase and cytochrome c oxidase complex III in quails. One-day-old broiler quails were sacrificed after 7, 14, 21, or 28 days. Six quails of each age were slaughtered by cervical dislocation. Total RNA was extracted from the breast muscle and reverse transcribed into cDNA. Real-time PCR analysis was performed using specific primers for the genes. As the quails aged, there was reduced expression of all three genes. The greatest reduction when comparing 7- and 28-day-old birds was observed in the uncoupling protein mRNA levels (67%), followed by adenine nucleotide translocase (56%) and cytochrome c oxidase complex III (30%). We conclude that expression of some mitochondrial genes of the electron transport chain changes with age in quails.     

Immunological detection of cDNA clones encoding the uncoupling protein of brown adipose tissue: Evidence for an antigenic determinant within the C-terminal eleven amino acids

Poly(A)⁺ RNA was isolated from brown adipose tissue of cold acclimated rats and a fraction enriched for uncoupling protein mRNA was used to generate a cDNA library in pBR 322, Immunological screening of 1,500 colonies with an affinity-purified antiserum against the uncoupling protein yielded five positive clones, pUCP_ratl–5. Clone pUCP_rat2 encoded the C-terminal 54 amino acids of rat uncoupling protein and exhibited 90% amino acid homology with the hamster protein. Clones pUCP_rat3–5 encoded only the C-terminal 11 amino acids suggesting that an antigenic determinant lies within this sequence.     

Dietary regulation of ornithine transcarbamylase mRNA in liver and small intestine

In the rat, changes in dietary protein intake give rise to changes in the levels of ornithine transcarbamylase (OTC) in liver and small intestine--an increase in liver and decrease in small intestine. The changes in enzyme level are accompanied by similar changes in levels of specific mRNA. Thus in liver, there is an increase in the level of specific mRNA when protein intake is increased, whereas in small intestine there is a small decrease. Comparison of changes in specific mRNA with total poly-A-containing RNA showed that the change in OTC mRNA in liver paralleled the change in total RNA levels. In contrast, in small intestine the small decrease in OTC mRNA levels when protein intake was increased was in the face of an increase in the level of total mRNA. Whereas the level of OTC is 20-fold higher in liver than in small intestine, the mRNA level for the enzyme differs by only 2.5-fold.     

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.     

Molecular approach to thermogenesis in brown adipose tissue. Cell-free translation of mRNA and characterization of the mitochondrial uncoupling protein

In order to develop a molecular approach of the thermogenesis mechanism in brown adipose tissue, cell-free translation was performed with mRNA obtained from control or thermoactive brown adipose tissue. Alterations were observed on analysis of the newly synthesized proteins and in particular at the 32,000 dalton level. Experiments using antibodies against the purified characteristic 32,000-dalton uncoupling protein of brown fat mitochondria were carried out. They indicated that the uncoupling protein was synthesized in the reticulocyte lysate with the same apparent molecular weight as the mature form. It is suggested that the development of the thermogenic capacity of brown fat cells is accompanied by an increase in specific mRNA coding for the uncoupling mitochondrial protein and that such a system could be an interesting one for study of mitochondrial membrane biogenesis.     

Cloning, characterization, and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acid-induced protein families. The expression of this cDNA in Escherichia coli, using the T₇ RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.     

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34° C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 µl saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in inter-scapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.     

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.     

Atrophy responses to muscle inactivity. I. Cellular markers of protein deficits.

The goal of this study was to use the model of spinal cord isolation (SI), which blocks nearly all neuromuscular activity while leaving the motoneuron muscle-fiber connections intact, to characterize the cellular processes linked to marked muscle atrophy. Rats randomly assigned to normal control and SI groups were studied at 0, 2, 4, 8, and 15 days after SI surgery. The slow soleus muscle atrophied by approximately 50%, with the greatest degree of loss occurring during the first 8 days. Throughout the SI duration, muscle protein concentration was maintained at the control level, whereas myofibrillar protein concentration steadily decreased between 4 and 15 days of SI, and this was associated with a 50% decrease in myosin heavy chain (MHC) normalized to total protein. Actin relative to the total protein was maintained at the control level. Marked reductions occurred in total RNA and DNA content and in total MHC and actin mRNA expressed relative to 18S ribosomal RNA. These findings suggest that two key factors contributing to the muscle atrophy in the SI model are 1). a reduction in ribosomal RNA that is consistent with a reduction in protein translational capacity, and 2). insufficient mRNA substrate for translating key sarcomeric proteins comprising the myofibril fraction, such as MHC and actin. In addition, the marked selective depletion of MHC protein in the muscles of SI rats suggests that this protein is more vulnerable to inactivity than actin protein. This selective MHC loss could be a major contributor for the previously reported loss in the functional integrity of SI muscles. Collectively, these data are consistent with the involvement of pretranslational and translational processes in muscle atrophy due to SI.     

Androgenic control of polysomal poly(A)-containing RNA in the cultured rat ventral prostate

Testosterone stimulated, at the concentration of 10-7 M and independently of other hormones, the accumulation of polysomal poly(A)-containing RNA (mRNA) in cultured explants of rat ventral prostate and concomitantly also protein synthesis. The hormone-induced accumulation of polysomal mRNA, which reached its maximum at 24 h after testosterone addition, paralleled the preferential labeling of high molecular weight RNA demonstrable with the electrophoretic analysis of the double-isotope labeled RNA after a short pulse (30 min). These findings are consistent with the idea that testosterone activated the synthesis of precursor mRNA leading to an increased amount of polysomal mRNA and eventually an activated protein synthesis. The synthesis and maturation of rRNA appeared to proceed even in the absence of testosterone, which is in contrast to the vivo findings on castrated rats. This partial uncoupling of RNA synthesis from androgenic control may account for the slow and less marked hormonal responses found in protein synthesis and glucose metabolism in cultured explants from normal animals. Because of the lack of uniformity in the suture, routine light microscopic control to assess the viability of cultured explants was found to be a prerequisite for successful biochemical work on prostate culture.     

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.