Regulation of mitochondrial activity controls the duration of skeletal muscle regeneration in response to injury.

Thyroid hormone is a major regulator of skeletal muscle development and repair, and also a key regulator of mitochondrial activity. We have previously identified a 43 kDa truncated form of the nuclear T3 receptor TRα1 (p43) which stimulates mitochondrial activity and regulates skeletal muscle features. However, its role in skeletal muscle regeneration remains to be addressed. To this end, we performed acute muscle injury induced by cardiotoxin in mouse tibialis in two mouse models where p43 is overexpressed in or depleted from skeletal muscle. The measurement of muscle fiber size distribution at different time point (up to 70 days) upon injury lead us to unravel requirement of the p43 signaling pathway for satellite cells dependent muscle regeneration; strongly delayed in the absence of p43; whereas the overexpression of the receptor enhances of the regeneration process. In addition, we found that satellite cells derived from p43-Tg mice display higher proliferation rates when cultured in vitro when compared to control myoblasts, whereas p43-/- satellites shows reduced proliferation capacity. These finding strongly support that p43 plays an important role in vivo by controling the duration of skeletal muscle regeneration after acute injury, possibly through the regulation of mitochondrial activity and myoblasts proliferation.

Thyroid Hormone Action: The p43 Mitochondrial Pathway.

The possibility that several pathways are involved in the multiplicity of thyroid hormone physiological influences led to searches for the occurrence of T3 extra nuclear receptors. The existence of a direct T3 mitochondrial pathway is now well established. The demonstration that TRα1 mRNA encodes not only a nuclear thyroid hormone receptor but also two proteins imported into mitochondria with molecular masses of 43 and 28 kDa has provided new clues to understand the pleiotropic influence of iodinated hormones.The use of a T3 photo affinity label derivative (T3-PAL) allowed detecting two mitochondrial T3 binding proteins. In association with western blots using antibodies raised against the T3 nuclear receptor TRα1, mitochondrial T3 receptors were identified as truncated TRα1 forms. Import and in organello transcription experiments performed in isolated mitochondria led to the conclusion that p43 is a transcription factor of the mitochondrial genome, inducing changes in the mitochondrial/nuclear crosstalk. In vitro experiments indicated that this T3 mitochondrial pathway affects cell differentiation, apoptosis, and transformation. Generation of transgenic mice demonstrated the involvement of this mitochondrial pathway in the determination of muscle phenotype, glucose metabolism, and thermogenesis.

Mitochondrial T3 receptor and targets.

The demonstration that TRα1 mRNA encodes a nuclear thyroid hormone receptor and two proteins imported into mitochondria with molecular masses of 43 and 28 kDa has brought new clues to better understand the pleiotropic influence of iodinated hormones. If p28 activity remains unknown, p43 binds to T3 responsive elements occurring in the organelle genome, and, in the T3 presence, stimulates mitochondrial transcription and the subsequent synthesis of mitochondrial encoded proteins. This influence increases mitochondrial activity and through changes in the mitochondrial/nuclear cross talk affects important nuclear target genes regulating cell proliferation and differentiation, oncogenesis, or apoptosis. In addition, this pathway influences muscle metabolic and contractile phenotype, as well as glycaemia regulation. Interestingly, according to the process considered, p43 exerts opposite or cooperative effects with the well-known T3 pathway, thus allowing a fine tuning of the physiological influence of this hormone.

Protective Activity of Total Polyphenols from Genista quadriflora Munby and Teucrium polium geyrii Maire in Acetaminophen-Induced Hepatotoxicity in Rats.

Oxidative stress is a major cause of drug-induced hepatic diseases and several studies have demonstrated that diet supplementation with plants rich in antioxidant compounds provides a variety of health benefits in these circumstances. Genista quadriflora Munby (Gq) and Teucrium polium geyrii Maire (Tp) are known to possess antioxidant and numerous biological properties and these endemic plants are often used for dietary or medicinal applications. Herein, we evaluated the beneficial effect of rich-polyphenol fractions of Gq and Tp to prevent Acetaminophen-induced liver injury and investigated the mechanisms involved in this protective action. Rats were orally administered polyphenolic extracts from Gq or Tp (300 mg/kg) or N-acetylcysteine (NAC: 200 mg/kg) once daily for ten days prior to the single oral administration of Acetaminophen (APAP: 1 g/kg). The results show that preventive administration of polyphenolic extracts from Gq or Tp exerts a hepatoprotective influence during APAP treatment by improving transaminases leakage and liver histology and stimulating antioxidant defenses. Besides, suppression of liver CYP2E1, GSTpi and TNF-α mRNA levels, with enhancement of mitochondrial bioenergetics may contribute to the observed hepatoprotection induced by Gq and Tp extracts. The effect of Tp extract is significantly higher (1.5-2 fold) than that of Gq extract and NAC regarding the enhancement of mitochondrial functionality. Overall, this study brings the first evidence that pretreatment with these natural extracts display in vivo protective activity against APAP hepatotoxicity through improving mitochondrial bioenergetics, oxidant status, phase I and II enzymes expression and inflammatory processes probably by virtue of their high total polyphenols content.

Temperature homeostasis in mice lacking the p43 mitochondrial T3 receptor.

Thyroid hormones and Thra gene play a key role in energy expenditure regulation, temperature homeostasis, and mitochondrial function. To decipher the function of the mitochondrial TRα receptor in these phenomena, we used mice lacking specifically the p43 mitochondrial T3 receptor. We found that these animals were hypermetabolic, hyperphagic, and displayed a down setting of the core body temperature. However, p43-/- animals do not present cold intolerance or defect of facultative thermogenesis. In addition, the mitochondrial function of BAT is slightly affected in the absence of p43. Our study, therefore, suggests a complementarity of action between the mitochondrial receptor and other proteins encoded by the Thra gene in the control of basal metabolism, facultative thermogenesis, and determination of the set point of temperature regulation.

p28, a truncated form of TRα1 regulates mitochondrial physiology.

We have previously identified in mitochondria two truncated forms of the T3 nuclear receptor TRα1, with molecular weights of 43kDa (p43) and 28kDa (p28) respectively located in the matrix and in the inner membrane. Previously, we have demonstrated that p43 stimulates mitochondrial transcription and protein synthesis in the presence of T3. Here we report that p28 is targeted into the organelle in a T3-dependent manner and displays an affinity for T3 higher than the nuclear receptor. We tried to generate mice overexpressing p28 using the human α-skeletal actin promoter, however we found an early embryonic lethality that was probably linked to a transient expression of p28 in trophoblast giant cells. This could be partly explained by the observation that overexpression of p28 in human fibroblasts induced alterations of mitochondrial physiology.

Chicoric acid is an antioxidant molecule that stimulates AMP kinase pathway in L6 myotubes and extends lifespan in Caenorhabditis elegans.

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 µM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

Mice lacking the p43 mitochondrial T3 receptor become glucose intolerant and insulin resistant during aging.

Thyroid hormones (TH) play an important regulatory role in energy expenditure regulation and are key regulators of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine (T3) receptor (p43) which acts as a mitochondrial transcription factor of the organelle genome, which leads in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Recently, we generated mice carrying a specific p43 invalidation. At 2 months of age, we reported that p43 depletion in mice induced a major defect in insulin secretion both in vivo and in isolated pancreatic islets, and a loss of glucose-stimulated insulin secretion. The present study was designed to determine whether p43 invalidation influences life expectancy and modulates blood glucose and insulin levels as well as glucose tolerance or insulin sensitivity during aging. We report that from 4 months old onwards, mice lacking p43 are leaner than wild-type mice. p43-/- mice also have a moderate reduction of life expectancy compared to wild type. We found no difference in blood glucose levels, excepted at 24 months old where p43-/- mice showed a strong hyperglycemia in fasting conditions compared to controls animals. However, the loss of glucose-stimulated insulin secretion was maintained whatever the age of mice lacking p43. If up to 12 months old, glucose tolerance remained unchanged, beyond this age p43-/- mice became increasingly glucose intolerant. In addition, if up to 12 months old p43 deficient animals were more sensitive to insulin, after this age we observed a loss of this capacity, culminating in 24 months old mice with a decreased sensitivity to the hormone. In conclusion, we demonstrated that during aging the depletion of the mitochondrial T3 receptor p43 in mice progressively induced an increased glycemia in the fasted state, glucose intolerance and an insulin-resistance several features of type-2 diabetes.

Boosting antioxidants by lipophilization: a strategy to increase cell uptake and target mitochondria.

PURPOSE: To explore the possibility to boost phenolic antioxidants through their structural modification by lipophilization and check the influence of such covalent modification on cellular uptake and mitochondria targeting. METHODS: Rosmarinic acid was lipophilized by various aliphatic chain lengths (butyl, octyl, decyl, dodecyl, hexadecyl, and octadecyl) to give rosmarinate alkyl esters which were then evaluated for their ability (i) to reduce the level of reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria using MitoTracker® Red CMXRos. RESULTS: Increasing the chain length led to an improvement of the antioxidant activity until a threshold is reached for medium chain (10 carbon atoms) and beyond which lengthening resulted in a decrease of activity. This nonlinear phenomenon-also known as the cut-off effect-is discussed here in connection to the previously similar results observed in emulsified, liposomal, and cellular systems. Moreover, butyl, octyl, and decyl rosmarinates passed through the membranes in less than 15 min, whereas longer esters did not cross membranes and formed extracellular aggregates. Besides cell uptake, alkyl chain length also determined the subcellular localization of esters: mitochondria for medium chains esters, cytosol for short chains and extracellular media for longer chains. CONCLUSION: The localization of antioxidants within mitochondria, the major site and target of ROS, conferred an advantage to medium chain rosmarinates compared to both short and long chains. In conjunction with changes in cellular uptake, this result may explain the observed decrease of antioxidant activity when lengthening the lipid chain of esters. This brings a proof-of-concept that grafting medium chain allows the design of mitochondriotropic antioxidants.

How to boost antioxidants by lipophilization?

Covalent modification of antioxidants through lipophilization is an important field of research aiming at developing antioxidants with improved efficacy. However, due to insufficient knowledge on how hydrophobicity affects antioxidant activity, lipophilization strategies have been largely based on empirism. Often, the resulting lipophilized antioxidants were not optimal. Here we described how the body of knowledge regarding hydrophobicity has been dramatically redefined as unexpected results were recently published. Using a broad range of lipophilized antioxidants assessed in dispersed lipids models and cultured cells, it has been demonstrated that the antioxidant activity increases progressively with increasing chain length up to a critical point, beyond which the activity of the compounds dramatically decreases. Taking into account this nonlinear phenomenon, also known as cut-off effect, antioxidant drug designers now have to seek the critical chain length to synthesize the optimal drug in a rational manner. Here, we briefly presented three putative mechanisms of action to try to account for the cut-off effect.

Analysis of RXR/THR and RXR/PPARG2 heterodimerization by bioluminescence resonance energy transfer (BRET).

BACKGROUND: Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR in vitro or in living cells. The presence of a specific DNA target was required to induce in vitro a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl2 concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2. CONCLUSIONS/SIGNIFICANCE: The BRET approach described here allowed us to study the dynamic interactions which exist between NR in vitro or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.

Protein sequences involved in the mitochondrial import of the 3,5,3'-L-triiodothyronine receptor p43.

The major effect of T3 on mitochondrial activity has been partly explained by the discovery of p43, a T3-dependent transcription factor of the mitochondrial genome. P43 is imported into mitochondria in an atypical manner which is not yet fully understood. Our aim was to characterize the p43 sequences inducing its mitochondrial import, using in organello import experiments with wild-type or mutated proteins and validation in CV1 cells. We find that several sequences define the mitochondrial addressing. Two alpha helices in the C-terminal part of p43 are actual mitochondrial import sequences as fusion to a cytosolic protein induces its mitochondrial translocation. Helix 5 drives the atypical mitochondrial import process, whereas helices 10/11 induce a classical import process. However, despite its inability to drive a mitochondrial import, the N-terminal region of p43 also plays a permissive role as in the presence of the C-terminal import sequences different N-terminal regions determine whether the protein is imported or not. These results can be extrapolated to other mitochondrial proteins related to the nuclear receptor superfamily, devoid of classical mitochondrial import sequences.

Mitochondrial T3 receptor p43 regulates insulin secretion and glucose homeostasis.

Thyroid hormone is a major determinant of energy expenditure and a key regulator of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine receptor (p43) that acts as a mitochondrial transcription factor of the organelle genome, which leads, in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Here we generated mice specifically lacking p43 to address its physiological influence. We found that p43 is required for normal glucose homeostasis. The p43(-/-) mice had a major defect in insulin secretion both in vivo and in isolated pancreatic islets and a loss of glucose-stimulated insulin secretion. Moreover, a high-fat/high-sucrose diet elicited more severe glucose intolerance than that recorded in normal animals. In addition, we observed in p43(-/-) mice both a decrease in pancreatic islet density and in the activity of complexes of the respiratory chain in isolated pancreatic islets. These dysfunctions were associated with a down-regulation of the expression of the glucose transporter Glut2 and of Kir6.2, a key component of the K(ATP) channel. Our findings establish that p43 is an important regulator of glucose homeostasis and pancreatic ß-cell function and provide evidence for the first time of a physiological role for a mitochondrial endocrine receptor.

Depletion of the p43 mitochondrial T3 receptor in mice affects skeletal muscle development and activity.

In vertebrates, skeletal muscle myofibers display different contractile and metabolic properties associated with different mitochondrial content and activity. We have previously identified a mitochondrial triiodothyronine receptor (p43) regulating mitochondrial transcription and mitochondrial biogenesis. When overexpressed in skeletal muscle, it increases mitochondrial DNA content, stimulates mitochondrial respiration, and induces a shift in the metabolic and contractile features of muscle fibers toward a slower and more oxidative phenotype. Here we show that a p43 depletion in mice decreases mitochondrial DNA replication and respiratory chain activity in skeletal muscle in association with the induction of a more glycolytic muscle phenotype and a decrease of capillary density. In addition, p43(-/-) mice displayed a significant increase in muscle mass relative to control animals and had an improved ability to use lipids. Our findings establish that the p43 mitochondrial receptor strongly affects muscle mass and the metabolic and contractile features of myofibers and provides evidence that this receptor mediates, in part, the influence of thyroid hormone in skeletal muscle.

P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression.

We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres.

Does hydrophobicity always enhance antioxidant drugs? A cut-off effect of the chain length of functionalized chlorogenate esters on ROS-overexpressing fibroblasts.

OBJECTIVES: Phenolic antioxidants are currently attracting a growing interest as potential therapeutic agents to counteract diseases associated with oxidative stress. However, their high hydrophilicity results in a poor bioavailability hindering the development of efficient antioxidant strategies. A promising way to overcome this is to increase their hydrophobicity by lipophilic moiety grafting to form the newly coined 'phenolipids'. Although hydrophobicity is generally considered as advantageous regarding antioxidant properties, it is nevertheless worth investigating whether increasing hydrophobicity necessarily leads to a more efficient antioxidant drug. METHODS: To answer this question, the antioxidant capacity of a homologous series of phenolics (chlorogenic acid and its methyl, butyl, octyl, dodecyl and hexadecyl esters) toward mitochondrial reactive oxygen species (ROS) generated in a ROS-overexpressing fibroblast cell line was investigated using 2',7'-dichlorodihydrofluorescein. KEY FINDINGS: Overall, the long chain esters (dodecyl and hexadecyl esters) were more active than the short ones (methyl, butyl, and octyl esters), with an optimal activity for dodecyl chlorogenate. Moreover, dodecyl chlorogenate exerted a strong antioxidant capacity, for concentration and incubation time below the cytotoxicity threshold, making it a promising candidate for further in-vivo studies. More importantly, we found that the elongation of the chain length from 12 to 16 carbons led unexpectedly to a 45% decrease of antioxidant capacity. CONCLUSION: The understanding of this sudden collapse of the antioxidant capacity through the cut-off theory will be discussed in this article, and may contribute towards development of a rational approach to design novel amphiphilic antioxidant drugs, especially phenolipids with medium fatty chain.

Overexpression of the mitochondrial T3 receptor induces skeletal muscle atrophy during aging.

In previous studies, we characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor. In in vitro and in vivo studies, we have shown that p43 increases mitochondrial transcription and mitochondrial biogenesis. In addition, p43 overexpression in skeletal muscle stimulates mitochondrial respiration and induces a shift in metabolic and contractile features of muscle fibers which became more oxidative.Here we have studied the influence of p43 overexpression in skeletal muscle of mice during aging. We report that p43 overexpression initially increased mitochondrial mass. However, after the early rise in mitochondrial DNA occurring at 2 months of age in transgenic mice, we observed a progressive decrease of mitochondrial DNA content which became 2-fold lower at 23 months of age relatively to control animals. Moreover, p43 overexpression induced an oxidative stress characterized by a strong increase of lipid peroxidation and protein oxidation in quadriceps muscle, although antioxidant enzyme activities (catalase and superoxide dismutase) were stimulated. In addition, muscle atrophy became detectable at 6 months of age, probably through a stimulation of the ubiquitin proteasome pathway via two muscle-specific ubiquitin ligases E3, Atrogin-1/MAFbx and MuRF1.Taken together, these results demonstrate that a prolonged stimulation of mitochondrial activity induces muscle atrophy. In addition, these data underline the importance of a tight control of p43 expression and suggest that a deregulation of the direct T3 mitochondrial pathway could be one of the parameters involved in the occurrence of sarcopenia.

[Triiodothyronine mitochondrial receptors: import and molecular mechanisms]. / Les récepteurs mitochondriaux de la triiodothyronine: import et mécanismes d'action.

Thyroid hormone exerts a diversity of physiological influences over developmental and metabolic processes. Searching for receptors able to mediate this extended regulation led to the identification of triiodothyronine (T3) nuclear receptors encoded by two different genes, c-erbA alpha (TR alpha) and c-erbA beta (TR beta). More recently, two N-terminally truncated forms of the triiodothyronine nuclear receptor TR alpha 1, with molecular weights of 43 and 28 kDa, have been discovered in mitochondria. Synthesized through the use of internal initiation sites of translation occurring in the TR alpha 1 transcript, they are addressed into mitochondria according to an atypical process. Two mitochondrial import sequences have been characterized in the C-terminal part of these proteins; in addition, their N-terminal part, devoid of negative charges, plays a permissive role in this import. Whereas the function of p28 remains unknown, p43 is a T3-dependent transcription factor of the mitochondrial genome, acting through dimeric complexes involving at least two other truncated forms of nuclear receptors, mtRXR and mtPPAR. P43 activation by T3 stimulates mitochondrial protein synthesis, respiratory chain activity and mitochondriogenesis. Through the mitochondrial/nuclear crosstalk, this direct T3 mitochondrial pathway influences the expression of nuclear genes involved in the regulation of cell proliferation and differentiation. In particular, in myoblasts, p43 overexpression stimulates terminal differentiation and induces a preferential expression of slow myosin, by down-regulating c-Myc expression and up-regulating calcineurin and myogenin expression. Comparison of the respective influences of the nuclear and mitochondrial T3 pathways demonstrates either both additivity (myoblast differentiation), complementarity (mitochondriogenesis, myoblast differentiation) or opposite influences (myosin expression), thus indicating that these two pathways introduce a fine-tuning of the hormone influence.

Overexpression of the mitochondrial T3 receptor p43 induces a shift in skeletal muscle fiber types.

In previous studies, we have characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor and consequently stimulating mitochondrial activity and mitochondrial biogenesis. We have established the involvement of this T3 pathway in the regulation of in vitro myoblast differentiation. We have generated mice overexpressing p43 under control of the human alpha-skeletal actin promoter. In agreement with the previous characterization of this promoter, northern-blot and western-blot experiments confirmed that after birth p43 was specifically overexpressed in skeletal muscle. As expected from in vitro studies, in 2-month old mice, p43 overexpression increased mitochondrial genes expression and mitochondrial biogenesis as attested by the increase of mitochondrial mass and mt-DNA copy number. In addition, transgenic mice had a body temperature 0.8 degrees C higher than control ones and displayed lower plasma triiodothyronine levels. Skeletal muscles of transgenic mice were redder than wild-type animals suggesting an increased oxidative metabolism. In line with this observation, in gastrocnemius, we recorded a strong increase in cytochrome oxidase activity and in mitochondrial respiration. Moreover, we observed that p43 drives the formation of oxidative fibers: in soleus muscle, where MyHC IIa fibers were partly replaced by type I fibers; in gastrocnemius muscle, we found an increase in MyHC IIa and IIx expression associated with a reduction in the number of glycolytic fibers type IIb. In addition, we found that PGC-1alpha and PPARdelta, two major regulators of muscle phenotype were up regulated in p43 transgenic mice suggesting that these proteins could be downstream targets of mitochondrial activity. These data indicate that the direct mitochondrial T3 pathway is deeply involved in the acquisition of contractile and metabolic features of muscle fibers in particular by regulating PGC-1alpha and PPARdelta.

Leptin downregulates heat shock protein-70 (HSP-70) gene expression in chicken liver and hypothalamus.

Heat shock protein (HSP)-70 is expressed in normal and stressed cells but is highly stress-inducible. Although leptin has long been suggested to be involved in the regulation of stress response, its interaction with the HSP-70 gene is still unknown, under both unstressed and stressed conditions. The present study has aimed to investigate the effect of leptin on HSP-70 gene expression in normal chicken liver, hypothalamus, and muscle. Continuous infusion of recombinant chicken leptin (8 mug/kg per hour) at a constant rate of 3 ml/h for 6 h in 3-week-old broiler chickens significantly (P < 0.05) decreased food intake and HSP-70 mRNA levels in liver and hypothalamus, but not in muscle. In an attempt to discriminate between the effect of leptin and of leptin-reduced food intake on HSP-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses in two broiler chicken lines genetically selected for low (LL) or high (FL) abdominal fat pad size. Food deprivation for 16 h did not affect HSP-70 gene expression in any of the studied tissues indicating that the effect of leptin was independent of the inhibition of food intake. Regardless of the nutritional status, HSP-70 mRNA levels were significantly (P < 0.05) higher in the hypothalamus of FL compared with LL chickens consistent with higher mRNA levels for hypothalamic corticotropin-releasing factor. To assess, whether the effects of leptin were direct or indirect, we carried out in vitro studies. Leptin treatments did not affect HSP-70 mRNA levels in a leghorn male hepatoma cell line or quail myoblast cell line suggesting that the effect of leptin on HSP-70 gene expression is mediated through the central nervous system. Furthermore, HSP-70 gene expression was gender-dependent with significantly (P < 0.05) higher levels in male than in female chickens.