The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts.

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Radicicol enhances the regeneration of skeletal muscle injured by crotoxin via decrease of NF-kB activation.

This study evaluated cellular and molecular effects of radicicol, a heat shock protein (HSP) inducer, on the regeneration of skeletal muscle injured by crotoxin, the main toxin isolated from Crotalus durissus terrificus venom. Regenerating muscles treated with radicicol had decreased NF-kB activation. Differentiating myoblasts treated with radicicol showed reduced number of NF-kB positive nuclei and increased fusion index. The results suggest that radicicol enhances regeneration of muscle by attenuating NF-kB activation and increasing myogenic differentiation.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Thyroid hormone upregulates MDM2 in rat type I fibre: Implications for skeletal muscle mass regulation.

AIM: Based upon a microarray assay, we have identified that triiodothyronine (T3) upregulates MDM2 gene expression in the rat skeletal muscle. As MDM2 protein is an E3 ligase, we hypothesized that this enzyme could play a role in T3 effects on skeletal muscle mass control. METHODS: To test our hypothesis, male rats (2 months old) were randomly assigned into the following groups: intact controls, treated with 20 physiological doses of T3 for 0.5, 1 and 7 days, or with 5, 20 and 50 physiological doses of T3 for 7 days. For in vitro experiments, myotubes and C2C12 cells were treated with T3 for 3 days. RESULTS: After validation of the microarray finding throughout RT-PCR and confirmation that T3 induces increases in MDM2 protein expression in a dose-dependent manner, we observed that MDM2 was upregulated by T3 exclusively in fibre type I. Moreover, detailed histological evaluation showed that MDM2 overexpression distributes punctiformily along the cross section of the fibre and also inside nuclei. MDM2 colocalizes with PAX7 in control muscle and T3 downregulates this myogenic factor. Pharmacological inhibition of MDM2 in cultured myotubes caused a severe decrease in their diameter (~35%, P < .001 vs Control), enhancing the effect of T3 (from ~12% to ~35%, P < .001) alone upon myotube diameter and mRNA levels of atrogenes. Finally, we observed that FOXO3 (MDM2 target) is kept outside the nucleus under T3 stimulation. CONCLUSION: Our results indicate that MDM2 might be involved in the pro-trophic effects of T3 in skeletal muscle.

Expression of genes related to muscle plasticity after strength and power training regimens.

The purpose of our study was to compare the effects of 8-week progressive strength and power training regimens on strength gains and muscle plasticity [muscle fiber hypertrophy and phenotype shift, mammalian target of rapamycin (mTOR), regulatory-associated protein of mTOR (RAPTOR), rapamycin-insensitive companion of m-TOR (RICTOR), calcineurin and calcipressin gene expression]. Twenty-nine physically active subjects were divided into three groups: strength training (ST), power training (PT) and control (C). Squat 1 RM and muscle biopsies were obtained before and after the training period. Strength increased similarly for both ST and PT groups (P<0.001). Fiber types I, IIa and IIb presented hypertrophy main time effect (P<0.05). Only type IIb percentage decreased from pre- to post-test (main time effect, P<0.05). mTOR and RICTOR mRNA expression increased similarly from pre- to post-test (P<0.01). RAPTOR increased after training for both groups (P<0.0001), but to a greater extent in the ST (P<0.001) than in the PT group. 4EBP-1 decreased after training when the ST and PT groups were pooled (P<0.05). Calcineurin levels did not change after training, while calcipressin increased similarly from pre- to post-test (P<0.01). In conclusion, our data indicate that these training regimens produce similar performance improvements; however, there was a trend toward greater hypertrophy-related gene expression and muscle fiber hypertrophy in the ST group.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice.

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom.

Early remodeling of rat cardiac muscle induced by swimming training.

The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35 degrees C. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35% in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-beta gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single approximately 60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.

Early remodeling of rat cardiac muscle induced by swimming training

The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35°C. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35 percent in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-ß gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single ~60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.

Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro.

Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A(2) (PLA(2)) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA(2) myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA(2)s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA(2) myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.

Cyclosporin-A does not affect skeletal muscle mass during disuse and recovery.

Cyclosporin-A (CsA) is an immunosuppressive drug that acts as an inhibitor of calcineurin, a calcium phosphatase that has been suggested to play a role in skeletal muscle hypertrophy. The aim of the present study was to determine the effect of CsA administration (25 mg kg(-1) day(-1)) on skeletal muscle mass and phenotype during disuse and recovery. Male Wistar rats received vehicle (N = 8) or CsA (N = 8) during hind limb immobilization (N = 8) and recovery (N = 8). Muscle weight (dry/wet) and cross-sectional area were evaluated to verify the effect of CsA treatment on muscle mass. Muscle phenotype was assessed by histochemistry of myosin ATPase. CsA administration during immobilization and recovery did not change muscle/body weight ratio in the soleus (SOL) or plantaris (PL). Regarding muscle phenotype, we observed a consistent slow-to-fast shift in all experimental groups (immobilized only, receiving CsA only, and immobilized receiving CsA) as compared to control in both SOL and PL (P < 0.05). During recovery, no difference was observed in SOL or PL fiber type composition between the experimental recovered group and recovered group receiving CsA compared to their respective controls. Considering the muscle/body weight ratio, CsA administration does not maximize muscle mass loss induced by immobilization. Our results also indicate that CsA fails to block skeletal muscle regrowth after disuse. The present data suggest that calcineurin inhibition by CsA modulates muscle phenotype rather than muscle mass.

Cyclosporin-A does not affect skeletal muscle mass during disuse and recovery

Cyclosporin-A (CsA) is an immunosuppressive drug that acts as an inhibitor of calcineurin, a calcium phosphatase that has been suggested to play a role in skeletal muscle hypertrophy. The aim of the present study was to determine the effect of CsA administration (25 mg kg-1 day-1) on skeletal muscle mass and phenotype during disuse and recovery. Male Wistar rats received vehicle (N = 8) or CsA (N = 8) during hind limb immobilization (N = 8) and recovery (N = 8). Muscle weight (dry/wet) and cross-sectional area were evaluated to verify the effect of CsA treatment on muscle mass. Muscle phenotype was assessed by histochemistry of myosin ATPase. CsA administration during immobilization and recovery did not change muscle/body weight ratio in the soleus (SOL) or plantaris (PL). Regarding muscle phenotype, we observed a consistent slow-to-fast shift in all experimental groups (immobilized only, receiving CsA only, and immobilized receiving CsA) as compared to control in both SOL and PL (P < 0.05). During recovery, no difference was observed in SOL or PL fiber type composition between the experimental recovered group and recovered group receiving CsA compared to their respective controls. Considering the muscle/body weight ratio, CsA administration does not maximize muscle mass loss induced by immobilization. Our results also indicate that CsA fails to block skeletal muscle regrowth after disuse. The present data suggest that calcineurin inhibition by CsA modulates muscle phenotype rather than muscle mass.

Cyclosporin A preferentially attenuates skeletal slow-twitch muscle regeneration.

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, is associated with muscle regeneration via NFATc1/GATA2-dependent pathways. However, it is not clear whether calcineurin preferentially affects the regeneration of slow- or fast-twitch muscles. We investigated the effect of a calcineurin inhibitor, cyclosporin A (CsA), on the morphology and fiber diameter of regenerating slow- and fast-twitch muscles. Adult Wistar rats (259.5 +/- 9 g) maintained under standard conditions were treated with CsA (20 mg/kg body weight, ip) for 5 days, submitted to cryolesion of soleus and tibialis anterior (TA) muscles on the 6th day, and then treated with CsA for an additional 21 days. The muscles were removed, weighed, frozen, and stored in liquid nitrogen. Cryolesion did not alter the body weight gain of the animals after 21 days of regeneration (P = 0.001) and CsA significantly reduced the body weight gain (15.5%; P = 0.01) during the same period. All treated TA and soleus muscles showed decreased weights (17 and 29%, respectively, P < 0.05). CsA treatment decreased the cross-sectional area of both soleus and TA muscles of cryoinjured animals (TA: 2108 +/- 930 vs 792 +/- 640 microm(2); soleus: 2209 +/- 322 vs 764 +/- 439 m(2); P < 0.001). Histological sections of both muscles stained with Toluidine blue revealed similar regenerative responses after cryolesion. In addition, CsA was able to minimize these responses, i.e., centralized nuclei and split fibers, more efficiently so in TA muscle. These results indicate that calcineurin preferentially plays a role in regeneration of slow-twitch muscle.

Cyclosporin A preferentially attenuates skeletal slow-twitch muscle regeneration

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, is associated with muscle regeneration via NFATc1/GATA2-dependent pathways. However, it is not clear whether calcineurin preferentially affects the regeneration of slow- or fast-twitch muscles. We investigated the effect of a calcineurin inhibitor, cyclosporin A (CsA), on the morphology and fiber diameter of regenerating slow- and fast-twitch muscles. Adult Wistar rats (259.5 ± 9 g) maintained under standard conditions were treated with CsA (20 mg/kg body weight, ip) for 5 days, submitted to cryolesion of soleus and tibialis anterior (TA) muscles on the 6th day, and then treated with CsA for an additional 21 days. The muscles were removed, weighed, frozen, and stored in liquid nitrogen. Cryolesion did not alter the body weight gain of the animals after 21 days of regeneration (P = 0.001) and CsA significantly reduced the body weight gain (15.5 percent; P = 0.01) during the same period. All treated TA and soleus muscles showed decreased weights (17 and 29 percent, respectively, P < 0.05). CsA treatment decreased the cross-sectional area of both soleus and TA muscles of cryoinjured animals (TA: 2108 ± 930 vs 792 ± 640 µm²; soleus: 2209 ± 322 vs 764 ± 439 m²; P < 0.001). Histological sections of both muscles stained with Toluidine blue revealed similar regenerative responses after cryolesion. In addition, CsA was able to minimize these responses, i.e., centralized nuclei and split fibers, more efficiently so in TA muscle. These results indicate that calcineurin preferentially plays a role in regeneration of slow-twitch muscle.

Deleteriuos effects of immobilization upon rat skeletal muscle: role of creatine supplementation.

AIM: The aim of the study was to investigate the impact of creatine feeding (5 g kg(-1) body weight day(-1)) upon the deleterious adaptations in skeletal muscle induced by immobilization. METHODS: Male Wistar rats were submitted to hind limb immobilization together with three dietary manipulations: control, supplemented with creatine for 7 days (along with immobilization) and supplemented with creatine for 14 days (7 days before immobilization and together with immobilization). Muscle weight (wet/dry) was determined in the soleus (SOL) and gastrocnemius (GAS). The analysis of lean mass was performed by DEXA and myosin heavy chain (MHC) distribution by SDS-PAGE. RESULTS: After 14 days of creatine loading, immobilized SOL and GAS total creatine content were increased by 25% and 18%, respectively. Regardless of dietary manipulation, the immobilization protocol induced a decrease in the weight of SOL and GAS (P < 0.001). However, creatine feeding for 14 days minimized mass loss in the SOL and GAS (P < 0.05). Our findings also indicate that creatine supplementation maximizes the expected slow-to-fast MHC shift driven by immobilization (P < 0.05). CONCLUSIONS: Previous creatine supplementation attenuates muscle wasting induced by immobilization. This effect is associated with the increment of intramuscular creatine content.

Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism.

The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 +/- 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05%; w/v) to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68% carbohydrates, 13% protein and 18% lipids) for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizol and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60% in hypothyroid rats and UCP3 mRNA expression was increased 70% in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals, confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40% lower as compared to euthyroid cafeteria-fed animals). The results show that a hypercaloric diet is a strong stimulator of UCP3 gene expression in skeletal muscle and requires T3 for an adequate action.

Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism

The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 ñ 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05 percent; w/v) to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68 percent carbohydrates, 13 percent protein and 18 percent lipids) for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizolå and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60 percent in hypothyroid rats and UCP3 mRNA expression was increased 70 percent in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals, confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40 percent lower as compared to euthyroid cafeteria-fed animals). The results show that a hypercaloric diet is a strong stimulator of UCP3 gene expression in skeletal muscle and requires T3 for an adequate action.

Cyclosporin A attenuates skeletal muscle damage induced by crotoxin in rats.

This work was undertaken to determine the role of the calcineurin pathway on the necrosis of skeletal muscle induced by crotoxin, the major component of the venom of Crotalus durissus terrificus. Rats were treated with cyclosporin A (CsA), a calcineurin inhibitor, for 5 days and, in the 6th day, received an intramuscular injection of crotoxin into the tibialis anterior muscle. Rats were also treated with diclofenac, a non-steroidal anti-inflammatory drug, for 5 days and, on the 6th day, injected with crotoxin. All treated groups were sacrificed 24 h after injection of crotoxin. Tibialis anterior and soleus muscles were removed, frozen and stored in liquid nitrogen. Histological sections were stained with Toluidine Blue and assayed for acid phosphatase. The results show that CsA, but not diclofenac, is able to significantly minimize myonecrosis promoted by crotoxin. In conclusion, CsA attenuates skeletal muscle necrosis induced by crotoxin, indicating that the calcineurin pathway is essential for crotoxin myotoxic activity. The myoprotective effect of CsA is not related to its anti-inflammatory effect since diclofenac, a cyclo-oxygenase inhibitor, was not able to produce myoprotection.

Role of nitric oxide in myotoxic activity induced by crotoxin in vivo.

This study was aimed to determine the role of nitric oxide on the skeletal myotoxic activity induced by crotoxin, the major component of the venom of Crotalus durissus terrificus. Rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of nitric oxide synthase or vehicle for 4 days, and on the 5th day received an intramuscular injection of crotoxin into the tibialis anterior muscle. Rats were also treated with aminoguanidine bicarbonate salt or 7-nitroindazole, inhibitors of the inducible and neuronal isoforms of nitric oxide synthase, respectively, for 4 days and on the 5th day injected with crotoxin. All treated groups were sacrificed 24 h after injection of crotoxin. Tibialis anterior and soleus muscles were removed, frozen and stored in liquid nitrogen. Histological sections were stained with toluidine blue and assayed for acid phosphatase. The results show that L-NAME significantly minimizes myonecrosis induced by crotoxin and both aminoguanidine and 7-nitroindazole partially prevented myonecrosis induced by crotoxin. Based on the present results we conclude that nitric oxide is a very important intracellular signaling molecule that mediates crotoxin myotoxic activity.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds.

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5' and 3' ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being approximately 72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3',3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room's temperature to 12-14 degrees C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

Thyroxine 5'-deiodination mediates norepinephrine-induced lipogenesis in dispersed brown adipocytes.

In euthyroid rats, maximal sympathetic nervous system stimulation (e.g. during cold exposure) results in a 3- to 4-fold increase in brown adipose tissue lipogenesis, a response that is blunted in hypothyroid rats. To further investigate this phenomenon, the role of local type II 5'-deiodinase (5'-DII) was studied in freshly isolated brown adipocytes. In a typical experiment, 1.5 x 10(6) cells were incubated for up to 48 h in a water-saturated 5% CO2-95% O2 atmosphere. After incubation with medium alone or with different concentrations of T4, T3, and/or norepinephrine (NE), lipogenesis was studied by measuring 1) the rate of fatty acid synthesis as reflected by 3H2O incorporation into lipids and 2) the activity of key rate-limiting enzymes, i.e. acetyl coenzyme A carboxylase and malic enzyme, and the results are reported in terms of DNA content per tube. Lipogenesis decreased progressively over time (approximately 40%) when no additions were made to the incubation medium. T4 or T3 partially prevented that inhibition at physiological concentrations (65 x 10[-9] and 0.77 x 10[-9] M, respectively), whereas a receptor-saturating concentration of T3, (154 x 10[-9] M) doubled the lipogenesis rate. The addition of 10(-6) M NE inhibited lipogenesis acutely (approximately 50% by 12 h) and was followed by a progressive stimulation that reached approximately 2-fold by 48 h, but only in the presence of T4. Furthermore, NE did not attenuate T3 (154 x 10[-9] M)-induced lipogenesis. Both the inhibition and the stimulation of lipogenesis caused by NE showed a strong dose-response relationship within the range of 10(-11)-10(-5) M. The role of local 5'-DII was further tested by incubating brown adipocytes with 10(-6) M NE and T4 (65 x 10[-9] M) in the presence of 100 microM iopanoic acid, a potent inhibitor of 5'-DII. Although iopanoic acid did not affect the T3 stimulation of lipogenesis, it did block the approximately 2-fold stimulation of lipogenesis triggered by NE in the presence of T4, confirming the mediation of 5'-DII in this process. In conclusion, lipogenesis in brown adipose tissue is under complex hormonal control, with key roles played by NE, thyroid hormones, and local 5'-DII. As in other tissues, NE-generated signals acutely (12 h) inhibited lipogenesis. However, the presence of the 5'-DII generated enough T3 to stimulate lipogenesis and gradually reverse the short-lived NE-induced inhibition, leading to the 2- to 3-fold response observed at later time points.