Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents.

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

Adrenodemedullation activates the Ca2+-dependent proteolysis in soleus muscles from rats exposed to cold.

Previous studies have shown that catecholamines in vivo and in vitro inhibit the activity of Ca2+-dependent proteolysis in skeletal muscles under basal conditions. In the present study we sought to investigate the role of catecholamines in regulating the Ca2+-dependent proteolysis in soleus and extensor digitorum longus (EDL) muscles from rats acutely exposed to cold. Overall proteolysis, the activity of proteolytic systems, protein levels and gene expression of different components of the calpain system were investigated in rats submitted to adrenodemedullation (ADMX) and exposed to cold for 24 h. ADMX drastically reduced plasma epinephrine and promoted an additional increase in the overall proteolysis, which was already increased by cold exposure. The rise in the rate of protein degradation in soleus muscles from adrenodemedullated cold-exposed rats was caused by the high activity of the Ca2+-dependent proteolysis, which was associated with the generation of a 145-kDa cleaved α-fodrin fragment, a typical calpain substrate, and lower protein levels and mRNA expression of calpastatin, the endogenous calpain inhibitor. Unlike that observed for soleus muscles, the cold-induced muscle proteolysis in EDL was not affected by ADMX. In isolated soleus muscle, clenbuterol, a selective ß2-adrenoceptor agonist, reduced the basal Ca2+-dependent proteolysis and completely abolished the activation of this pathway by the cholinergic agonist carbachol. These data suggest that catecholamines released from the adrenal medulla inhibit cold-induced protein breakdown in soleus, and this antiproteolytic effect on the Ca2+-dependent proteolytic system is apparently mediated through expression of calpastatin, which leads to suppression of calpain activation.NEW & NOTEWORTHY Although many effects of the sympathetic nervous system on muscle physiology are known, the role of catecholamines in skeletal muscle protein metabolism has been scarcely studied. We suggest that catecholamines released from adrenal medulla may be of particular importance for restraining the activation of the Ca2+-dependent proteolysis in soleus muscles during acute cold exposure. This finding helps us to understand the adaptive changes that occur in skeletal muscle protein metabolism during cold stress.

Activating cAMP/PKA signaling in skeletal muscle suppresses the ubiquitin-proteasome-dependent proteolysis: implications for sympathetic regulation.

Although we have recently demonstrated that plasma catecholamines induce antiproteolytic effects on skeletal muscle (Graça FA, Gonçalves DAP, Silveira WA, Lira EC, Chaves VE, Zanon NM, Garófalo MAR, Kettelhut IC, Navegantes LCC. Am J Physiol Endocrinol Metab. 305: E1483-E1494, 2013), the role of the muscle sympathetic innervation and, more specifically, norepinephrine (NE) in regulating the ubiquitin (Ub)-proteasome system (UPS) remains unknown. Based on previous findings that chemical sympathectomy acutely reduces UPS activity, we hypothesized that muscle NE depletion induces adrenergic supersensitivity in rat skeletal muscles. We report that surgical sympathetic denervation (SDEN), a condition in which only muscle NE from both hindlimbs is depleted, transiently reduced the overall proteolysis and the UPS activity (∼25%) in both soleus and extensor digitorum longus muscles. This antiproteolytic response was accompanied by increased activity of adenylyl cyclase (112%), levels of cyclic adenosine monophosphate (cAMP; 191%), and the serine phosphorylation of cAMP response element-binding protein (32%). In extensor digitorum longus from normal rats, NE (10(-4) M) in vitro increased the levels of cAMP (115%) and the serine phosphorylation of both cAMP response element-binding protein (2.7-fold) and forkhead box class O1 transcription factor. Similar effects were observed in C2C12 cells incubated with forskolin (10 µM). In parallel, NE significantly reduced the basal UPS (21%) activity and the mRNA levels of atrophy-related Ub-ligases. Similar responses were observed in isolated muscles exposed to 6-BNZ-cAMP (500 µM), a specific PKA activator. The phosphorylation levels of Akt were not altered by SDEN, NE, forskolin or 6-BNZ-cAMP. Our results demonstrate that SDEN induces muscle adrenergic supersensitivity for cAMP leading to the suppression of UPS, and that the suppressive effects of NE on UPS activity and expression of Ub-ligases can be mediated by the activation of cAMP/PKA signaling, with the inhibition of forkhead box class O1 transcription factor.

Effect of short-term cold exposure on skeletal muscle protein breakdown in rats.

Although it is well established that carbohydrate and lipid metabolism are profoundly altered by cold stress, the effects of short-term cold exposure on protein metabolism in skeletal muscle are still poorly understood. Because cold acclimation requires that an organism adjust its metabolic flux, and muscle amino acids may be an important energy source for heat production, we hypothesize that muscle proteolysis is increased and protein synthesis is decreased under such a stress condition. Herein, cold exposure for 24 h decreased rates of protein synthesis and increased overall proteolysis in both soleus and extensor digitorum longus (EDL) muscles, but it did not affect muscle weight. An increase in proteolysis was accompanied by hyperactivity of the ubiquitin-proteasome system (UPS) in both soleus and EDL, and Ca(2+)-dependent proteolysis in EDL. Furthermore, muscles of rats exposed to cold showed increased mRNA and protein levels of atrogin-1 and muscle RING finger enzyme-1 (MuRF1). Additionally, cold stress reduced phosphorylation of Akt and Forkhead box class O1 (FoxO1), a well-known effect that increases FoxO translocation to the nucleus and leads to activation of proteolysis. Plasma insulin levels were lower, whereas catecholamines, corticosterone, and thyroid hormones were higher in cold-exposed rats compared with control rats. The present data provide the first direct evidence that short-term cold exposure for 24 h decreases rates of protein synthesis and increases the UPS and Ca(2+)-dependent proteolytic processes, and increases expression of atrogin-1 and MuRF1 in skeletal muscles of young rats. The activation of atrophy induced by acute cold stress seems to be mediated at least in part through the inactivation of Akt/FoxO signaling and activation of AMP-activated protein kinase.

Insulin suppresses atrophy- and autophagy-related genes in heart tissue and cardiomyocytes through AKT/FOXO signaling.

Insulin is an important regulator of the ubiquitin-proteasome system (UPS) and of lysosomal proteolysis in cardiac muscle. However, the role of insulin in the regulation of the muscle atrophy-related Ub-ligases atrogin-1 and MuRF1 as well as in autophagy, a major adaptive response to nutritional stress, in the heart has not been characterized. We report here that acute insulin deficiency in the cardiac muscle of rats induced by streptozotocin increased the expression of atrogin-1 and MuRF1 as well as LC3 and Gabarapl1, 2 autophagy-related genes. These effects were associated with decreased phosphorylation levels of Akt and its downstream target Foxo3a; this phenomenon is a well-known effect that permits the maintenance of Foxo in the nucleus to activate protein degradation by proteasomal and autophagic processes. The administration of insulin increased Akt and Foxo3a phosphorylation and suppressed the diabetes-induced expression of Ub-ligases and autophagy-related genes. In cultured neonatal rat cardiomyocytes, nutritional stress induced by serum/glucose deprivation strongly increased the expression of Ub-ligases and autophagy-related genes; this effect was inhibited by insulin. Furthermore, the addition of insulin in vitro prevented the decrease in Akt/Foxo signaling induced by nutritional stress. These findings demonstrate that insulin suppresses atrophy- and autophagy-related genes in heart tissue and cardiomyocytes, most likely through the phosphorylation of Akt and the inactivation of Foxo3a.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle.

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of beta2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by beta2- and beta3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet.

We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.

Schistosoma mansoni: functional proteasomes are required for development in the vertebrate host.

Proteasomes are multi-subunit proteases involved in several mechanisms and thought to contribute to the regulation of cellular homeostasis. Here, we report for the first time biochemical evidence for the existence of a ubiquitin-proteasome proteolytic pathway in this parasite. Proteasomes from both cercariae and adult worms exhibited a high preference for hydrolysis of the substrate Suc-LLVY-AMC, although in the cercariae extract the rate of hydrolysis was 50% lower when compared to adult worms extracts. The same difference in proteasome activities was observed when endogenous proteins were broken down in the presence of ATP and ubiquitin. Additionally, accumulation of high molecular weight conjugates was observed when cercariae were pre-incubated with proteasome inhibitors. Finally, we present evidence that during experimental schistosomiasis, proteasome inhibitors were able to reduce the number of lung stage schistosomula, reduce the worm burden and consequently decrease the egg output in infected mice.

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae).

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.

Dietary sodium restriction exacerbates age-related changes in rat adipose tissue and liver lipogenesis.

To investigate the effects of prolonged dietary sodium restriction on lipid metabolism, male rats weighing 35 to 40 g (just weaned) were fed either a low-salt (LSD) or a normal salt diet (NSD) and used in metabolic experiments after 1, 2, or 3 months of diet consumption. After 2 and 3 months on the diet, LSD rats showed increased amounts of lipid in carcass and retroperitoneal tissue. In both LSD and NSD, extending the feeding period from 2 to 3 months resulted in a marked reduction in the in vivo rates of adipose tissue fatty acid synthesis that was accompanied by increases in liver lipogenesis and in the activity of adipose tissue lipoprotein lipase (LPL). However, these increases were more marked in LSD rats. Thus, in vivo rates of liver fatty synthesis and LPL activity in LSD rats, which were already higher (by about 35% and 20%, respectively) than in controls after 2 months, attained levels 50% higher than those in NSD animals after another month on the diet. Brown adipose tissue (BAT) thermogenic capacity, estimated after 2 and 3 months by the tissue temperature response to norepinephrine (NE) injection and by guanosine diphosphate (GDP) binding to BAT mitochondria, did not change in controls, but was significantly reduced in LSD rats. This raises the possibility that a decrease in overall energy expenditure, together with an LPL-induced increased uptake of preformed fatty acids from the circulation, may account for the excessive lipid accumulation in LSD rats. Taken together, the data indicate that prolonged dietary sodium restriction exacerbates normal, age-related changes in white and BAT metabolism.

Control of glyceroneogenic activity in rat brown adipose tissue.

Brown adipose tissue (BAT) glyceroneogenesis was evaluated in rats either fasted for 48 h or with streptozotocin-diabetes induced 3 days previously or adapted for 20 days to a high-protein, carbohydrate-free (HP) diet, conditions in which BAT glucose utilization is reduced. The three treatments induced an increase in BAT glyceroneogenic activity, evidenced by increased rates of incorporation of [1-14C]pyruvate into triacylglycerol (TAG)-glycerol in vitro and a marked, threefold increase in the activity of BAT phosphoenolpyruvate carboxykinase (PEPCK). BAT glycerokinase activity was not significantly affected by fasting or diabetes. After unilateral BAT denervation of rats fed either the HP or a balanced diet, glyceroneogenesis activity increased in denervated pads, evidenced by increased rates of nonglucose carbon incorporation into TAG-glycerol in vivo (difference between 3H2O and [14C]glucose incorporations) and of [1-14C]pyruvate in vitro. PEPCK activity was not significantly affected by denervation. The data suggest that BAT glyceroneogenesis is not under sympathetic control but is sensitive to hormonal/metabolic factors. In situations of reduced glucose use there is an increase in BAT glyceroneogenesis that may compensate the decreased generation of glycerol-3-phosphate from the hexose.

Beta2-agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle.

1. The role of beta2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective hbetaagonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10(-5)M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5)M ICI 118.551, a selective fbeta2antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4)M dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by fbeta2adrenoceptors, with the participation of a cAMP-dependent pathway.

Expression of glycerokinase in brown adipose tissue is stimulated by the sympathetic nervous system.

The effect of cold exposure (4 degrees C) or prolonged norepinephrine infusion on the activity and mRNA levels of glycerokinase (GyK) was investigated in rat interscapular brown adipose tissue (BAT). Cold exposure for 12 and 24 h induced increases of 30% and 100%, respectively, in the activity of BAT GyK, which was paralleled by twofold and fourfold increase in enzyme mRNA levels. BAT hemidenervation resulted in reductions of 50% and 30% in GyK activity and in mRNA levels, respectively, in denervated pads from rats kept at 25 degrees C, and suppressed in these pads the cold-induced increases in both GyK activity and mRNA levels. The increase in GyK activity induced by cold exposure was not affected by phenoxybenzamine, but was markedly inhibited by previous administration of propranolol or actinomycin D. BAT GyK activity did not change significantly after 6 h of continuous subcutaneous infusion of norepinephrine (20 microg/h), but increased twofold and fourfold after 12 and 24 h, with no further increase after 72 h of infusion. Norepinephrine infusion also activated mRNA production, but the effect was comparatively smaller than that on enzyme activity. beta-Adrenergic agonists also stimulated GyK activity with the following relative magnitude of response: CL316243 (beta(3)) > isoproterenol (non-selective) > dobutamine (beta(1)). In vitro rates of incorporation of glycerol into glyceride-glycerol were increased in BAT from rats exposed to cold. The data suggest that in conditions of a sustained increase in BAT sympathetic flow there is a stimulation of GyK gene expression at the pretranslational level, with increased enzyme activity, mediated by beta-adrenoreceptors, mainly beta(3).

Glucose uptake, glucose transporter GLUT4, and glycolytic enzymes in brown adipose tissue from rats adapted to a high-protein diet.

In vivo rates of glucose uptake, insulin-responsive glucose transporter (GLUT4) content, and activities of glycolytic enzymes were determined in brown adipose tissue (BAT) from rats adapted to a high-protein, carbohydrate-free (HP) diet. Adaptation to the HP diet resulted in marked decreases in BAT glucose uptake and in GLUT4 content. Replacement of the HP diet by a balanced control diet for 24 hours restored BAT glucose uptake to levels above those in rats fed the control diet, with no changes in GLUT4 levels in 4 of 5 animals examined. BAT denervation of rats fed the control diet induced a 50% reduction in glucose uptake, but did not significantly affect the already markedly reduced BAT hexose uptake in HP diet-fed rats. It is suggested that the pronounced decrease in BAT glucose uptake in these animals is due to the combined effects of the HP diet-induced reductions in plasma insulin levels and in BAT sympathetic activity. Adaptation to the HP diet was accompanied by decreased activities of hexokinase, phosphofructo-1-kinase, and pyruvate kinase (PK). The activity of BAT PK in HP diet-fed rats was reduced to about 50% of controls, and approached normal levels 24 hours after diet reversion. BAT denervation induced a small (15%) decrease in BAT PK activity in control rats, but did not affect the activity of the enzyme in HP diet-adapted rats. Also, denervation did not interfere with the restoration of PK activity induced by diet substitution. Treatment with anti-insulin serum resulted in an almost 50% reduction in PK activity in both innervated and denervated BAT from rats fed the control diet, but caused a much smaller ( thick approximate 20%) decrease in BAT from HP diet-fed rats. Furthermore, anti-insulin serum administration completely suppressed the restoration of BAT PK activity induced by diet reversion. These data suggest that, differently from glucose uptake, BAT PK activity is predominantly controlled by hormonal/metabolic factors.

Anti-diabetic activity of Bauhinia forficata decoction in streptozotocin-diabetic rats.

The effects of using Bauhinia forficata leaf decoction (150 g leaf/l water; 35.2+/-7.8 ml/100 g body weight mean daily dose) as a drinking-water substitute for about 1 month on streptozotocin-diabetes (STZ-diabetes) in male Wistar rats were investigated. The physico-metabolic parameters measured were: body weight, food and liquid intake, urinary volume, hepatic glycogen, serum triglycerides and cholesterol, plasma glucose, urinary glucose and urea, and the weight of epididymal and retroperitoneal adipose tissue and soleus and extensor digitorum longus muscles. The STZ-diabetic rats treated with decoction showed a significant reduction in serum and urinary glucose and urinary urea as compared to the STZ-diabetic control, no difference being seen between decoction-treated and -untreated non-diabetic rats. The other physico-metabolic factors showed no changes in treated STZ-diabetic rats. The improvement in carbohydrate metabolism seen in the rats treated with Bauhinia forficata decoction does not appear to be linked to the inhibition of glycogenolysis or the stimulation of glycogenesis nor does it appear to act in a way similar to insulin or the sulfonylureas, although it may act by the inhibition of neoglycogenesis in a manner similar to that of the biguanides.

Relative importance of sympathetic outflow and insulin in the reactivation of brown adipose tissue lipogenesis in rats adapted to a high-protein diet.

The effect of denervation or acute insulin deficiency on brown adipose tissue lipogenesis was investigated in rats adapted to a high-protein diet before and after diet reversion to a balanced diet. Denervation of rats fed the balanced diet induced a 50% reduction in in vivo rates of brown adipose tissue fatty acid synthesis, with decreased activities of acetyl-CoA carboxylase and adenosine triphosphate (ATP)-citrate lyase. The markedly (80%) reduced fatty acid synthesis and enzyme activities in brown adipose tissue from rats adapted to the high-protein diet were not affected by denervation. Replacement of the high-protein diet by the balanced diet for 24 hours restored fatty acid synthesis to normal levels, but recovery of enzyme activities was only partial. Lipogenesis restoration and partial recovery of enzyme activities were impaired in denervated tissue from high-protein diet-fed rats. In all experimental conditions, the activities of acetyl-CoA carboxylase and ATP-citrate lyase showed a better correlation with brown adipose tissue lipogenesis than the generators of H(+), glucose-6-P dehydrogenase, and malic enzyme. Anti-insulin serum administration during the 12- to 24-hour period after diet reversion completely blocked lipogenesis recovery in innervated and denervated tissues and drastically reduced brown adipose tissue lipogenesis of concomitantly injected rats fed the balanced diet. The data suggest that efficient and rapid adjustments of brown adipose tissue lipogenesis require sympathetic activation, and that this tissue can maintain significant, albeit reduced, rates of lipogenesis in the absence of sympathetic activation, but not in the absence of insulin.

Glycerokinase activity in brown adipose tissue: a sympathetic regulation?

The effect of brown adipose tissue (BAT) sympathetic hemidenervation on the activity of glycerokinase (GyK) was investigated in different physiological conditions. In rats fed a balanced diet, the activity of the enzyme was approximately 50% lower in BAT-denervated pads than in intact, innervated pads. In rats adapted to a high-protein, carbohydrate-free diet, norepinephrine turnover rates and BAT GyK activity were already reduced, and BAT denervation resulted in a further decrease in the activity of the enzyme. Cold acclimation of normally fed rats at 4 degrees C for 10 days markedly increased the activity of the enzyme. Cold exposure (4 degrees C) for 6 h was insufficient to stimulate BAT GyK, but the activity of the enzyme was already increased after 12 h of cold exposure. The cold-induced BAT GyK stimulation was completely blocked in BAT-denervated pads. The data indicate that an adequate sympathetic flow to BAT is required for the maintenance of normal levels of GyK activity and for the enzyme response to situations, such as cold exposure, which markedly increase BAT sympathetic flow.

Effect of cold acclimation on brown adipose tissue fatty acid synthesis in rats adapted to a high-protein, carbohydrate-free diet.

The effect of cold acclimation on brown adipose tissue (BAT) fatty acid synthesis was investigated in rats adapted to a high-protein, carbohydrate-free diet. At an ambient temperature (25 degrees C), rates of fatty acid synthesis in BAT from rats adapted to the high-protein diet were reduced to 27% of rats fed the balanced diet and increased markedly after cold acclimation (10 days at 4 degrees C), although the increase was smaller than in control rats. BAT weight increase induced by cold acclimation was smaller in rats fed the high-protein diet (30%) than in controls (100%). When expressed per whole tissue, maximal activities of BAT glucose-6-phosphate dehydrogenase, malic enzyme, adenosine triphosphate (ATP)-citrate lyase, and acetyl-coenzyme A carboxylase were markedly reduced in high-protein diet-adapted rats at 25 degrees C and increased after cold acclimation in BAT from the 2 groups. However, when expressed per milligram protein, only acetyl-coenzyme A carboxylase showed an increase in both controls and in rats fed the high-protein diet. G6P-dehydrogenase, malic enzyme, and ATP-citrate lyase increased (per milligram protein) only in rats adapted to the high-protein diet and actually decreased in BAT from cold-acclimated control rats. Initial (before activation) pyruvate dehydrogenase (PDH) complex activity was lower in BAT from rats fed the high-protein diet at 25 degrees C and increased in cold-acclimated rats from the 2 groups. Circulating levels of insulin decreased in the 2 groups after cold acclimation. The data suggest that the cold acclimation-induced increase in BAT lipogenesis in rats adapted to the high-protein diet was due to a restoration of sympathetic activity, which induced both BAT hyperplasia and activation of adipocyte free fatty acid (FFA) synthesis, with an important participation of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase.

Role of ubiquitin-proteasome-dependent proteolytic process in degradation of muscle protein from diabetic rabbits.

The activity of ATP, ubiquitin (Ub)-dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ub-dependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ub-dependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ub-proteasome-dependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.