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Biomedical subjects

M Giralt

Publications and source records attributed to M Giralt.

At least 55 records · Page 3Linked to original sources
PubMed1999

Both afferent and efferent nerves are implicated in cholecytokinin motor actions in the small intestine of the rat.

Cholecystokinin (CCK) regulates intestinal motility after being released by several luminal nutrients. However the mechanism of action of CCK is still not well known. The aim of our study was to establish the mechanism of action of CCK in the rat intestine using an in vivo model and focusing on the nervous pathways involved in the response as well as type of receptors. Anesthetized rats were prepared with two strain-gauges, in duodenum and jejunum, to record circular muscle motor activity. A group of animals was also prepared with a catheter to infuse capsaicin inside the duodenum. Responses to CCK-octapeptide (CCK-8) as well as to CCK agonists were studied. CCK-8 was also infused after CCK antagonists, atropine, hexamethonium or L-nitroarginine. Results show that duodenal response to CCK-8 is excitatory although inhibitory responses can be induced by gastrin. In the jejunum, CCK-8 induces an inhibitory response that is mediated by both CCK-A and -B receptors. Excitatory responses to CCK-8 are due to stimulation of preganglionic receptors while inhibitory responses are NO mediated through stimulation of postganglionic CCK-B receptors. Capsaicin locally applied in duodenal mucosa significantly decreased CCK-8 response, whereas mucosal exposure to lidocaine completely blocked CCK-8 response. In conclusion our results show that CCK response varies along the intestine according to the predominance of excitatory or inhibitory efferent innervation. Moreover, CCK-8 actions are mediated through both extrinsic and intrinsic afferent fibres.

Anesthetics, Local↗
PubMed1999

CNS wound healing is severely depressed in metallothionein I- and II-deficient mice.

To characterize the physiological role of metallothioneins I and II (MT-I+II) in the brain, we have examined the chronological effects of a freeze injury to the cortex in normal and MT-I+II null mice. In normal mice, microglia/macrophage activation and astrocytosis were observed in the areas surrounding the lesion site, peaking at approximately 1 and 3 d postlesion (dpl), respectively. At 20 dpl, the parenchyma had regenerated. Both brain macrophages and astrocytes surrounding the lesion increased the MT-I+II immunoreactivity, peaking at approximately 3 dpl, and at 20 dpl it was similar to that of unlesioned mice. In situ hybridization analysis indicates that MT-I+II immunoreactivity reflects changes in the messenger levels. In MT-I+II null mice, microglia/macrophages infiltrated the lesion heavily, and at 20 dpl they were still present. Reactive astrocytosis was delayed and persisted at 20 dpl. In contrast to normal mice, at 20 dpl no wound healing had occurred. The rate of apoptosis, as determined by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, was drastically increased in neurons of ipsilateral cortex of the MT-I+II null mice. Our results demonstrate that MT-I+II are essential for a normal wound repair in the CNS, and that their deficiency impairs neuronal survival.

Animals↗
PubMed1999

Impaired inflammatory response to glial cell death in genetically metallothionein-I- and -II-deficient mice.

Metallothionein I+II (MT-I+II) are acute-phase proteins which are upregulated during pathological conditions in the brain. To elucidate the neuropathological importance of MT-I+II, we have examined MT-I+II-deficient mice following ip injection with 6-aminonicotinamide (6-AN). 6-AN is antimetabolic and toxic for bone marrow cells and grey matter astrocytes. In MT+/+ mice, injection with 6-AN resulted in breakdown of the blood-brain barrier (BBB) and absence of GFAP-positive astrocytes in specific grey matter areas of the brain stem. Reactive astrocytosis encircled the damaged grey matter areas, which were heavily infiltrated by microglia/macrophages. The recruitment of hematogenous macrophages was accompanied by leakage of the BBB. The immunoreactivity (ir) of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and the receptor for GM-CSF (GM-CSFrec) was significantly upregulated in astrocytes and microglia/macrophages, respectively. MT-I+IIir was also clearly increased in astrocytes surrounding the damaged areas, while that of the CNS-specific MT isoform, MT-III, was mildly increased in both astrocytes and microglia/macrophages. In MT-/- mice injected with 6-AN, the BBB remained almost intact. The damage to specific grey matter areas was similar to that observed in MT+/+ mice, but reactive astrocytosis, microglia/macrophages infiltration, and GM-CSFir and GM-CSFrecir were clearly reduced in MT-/- mice. In contrast, MT-IIIir was dramatically increased in MT-/- mice. Total zinc decreased and histochemically detectable zinc increased in the brain stem after 6-AN similarly in MT+/+ and MT-/- mice. Bone marrow myeloid monocytes and macrophages were increased as a reaction to 6-AN only in MT+/+ mice. The results demonstrate that the capability of MT-/- mice to mount a normal inflammatory response in the brain is severely attenuated, at least in part because of 6-AN-induced bone marrow affectation, involving MT-I+II for the first time as major factors during CNS tissue damage.

6-Aminonicotinamide↗
PubMed1999

Glucagonlike peptide-1 (GLP-1) participation in ileal brake induced by intraluminal peptones in rat.

The aim of this study was to determine the mechanisms implicated in the gastrointestinal inhibition induced by ovoalbumin hydrolysate infused intraluminally. We studied the site of action, the possible implication of GLP-1, and the nervous mechanisms involved. We prepared anesthetized Sprague-Dawley rats with strain gauges in the antrum, duodenum, and proximal jejunum and a catheter in the duodenum or ileum for peptone infusion. Both intraduodenal (N = 6) and intraileal (N = 5) infusion of ovoalbumin hydrolysate induced inhibition of spontaneous motor activity in the antrum, duodenum, and proximal jejunum. Duodenal inhibition induced by intraduodenal (N = 6) or intraileal (N = 6) infusion of ovoalbumin hydrolysate was reversed by intraarterial infusion of GLP-1 receptor antagonist, exendin (9-39) (3 x 10(-8) mol/kg/40 min). Finally, a combination of the adrenergic blockers phentolamine and propranolol (1 mg/kg, each; N = 7) completely blocked inhibitory gastrointestinal motor actions caused by intraduodenal infusion of ovoalbumin hydrolysate. This study demonstrates that peptone, intraluminally infused, participates in the regulation of gastrointestinal motility through stimulation of adrenergic pathways in anaesthetized rats. Moreover, these effects are partly mediated by GLP-1 secretion. The ileum seems to be the site of action, indicating a role of GLP-1 on the ileal break mechanism.

Adrenergic alpha-Antagonists↗
PubMed1999

Retinoids and adipose tissues: metabolism, cell differentiation and gene expression.

Vitamin A derivatives (retinoids) play a key role in mammalian development and cell differentiation. Isomers of retinoic acid, the main active metabolite of vitamin A, activate retinoid receptors that act as ligand-dependent transcription factors and affect gene expression. White and brown adipose tissues are major sites of storage of vitamin A derivatives and they play an active role in whole body metabolism of retinoids. Moreover, adipose tissues are targets for the action of retinoic acid. In most cases retinoic acid impairs adipocyte differentiation although its final effects on adipose tissue development depend on retinoic acid concentration, isomers availability and expression of retinoid receptor subtypes in the white or brown adipocyte. Retinoic acid (RA) has a specific effect on brown adipose tissue, because it activates transcription of the gene for uncoupling protein-1, responsible for brown fat thermogenesis.

Adipose Tissue↗
PubMed1999

Metallothionein (MT)-III: generation of polyclonal antibodies, comparison with MT-I+II in the freeze lesioned rat brain and in a bioassay with astrocytes, and analysis of Alzheimer's disease brains.

Metallothionein-III is a low molecular weight, heavy-metal binding protein expressed mainly in the central nervous system. First identified as a growth inhibitory factor (GIF) of rat cortical neurons in vitro, it has subsequently been shown to be a member of the metallothionein (MT) gene family and renamed as MT-III. In this study we have raised polyclonal antibodies in rabbits against recombinant rat MT-III (rMT-III). The sera obtained reacted specifically against recombinant zinc-and cadmium-saturated rMT-III, and did not cross-react with native rat MT-I and MT-II purified from the liver of zinc injected rats. The specificity of the antibody was also demonstrated in immunocytochemical studies by the elimination of the immunostaining by preincubation of the antibody with brain (but not liver) extracts, and by the results obtained in MT-III null mice. The antibody was used to characterize the putative differences between the rat brain MT isoforms, namely MT-I+II and MT-III, in the freeze lesion model of brain damage, and for developing an ELISA for MT-III suitable for brain samples. In the normal rat brain, MT-III was mostly present primarily in astrocytes. However, lectin staining indicated that MT-III immunoreactivity was also present in microglia, monocytes and/or macrophages in the leptomeninges and lying adjacent to major vessels. In freeze lesioned rats, both MT-I+II and MT-III immunoreactivities increased in the ipsilateral cortex. The pattern of MT-III immunoreactivity significantly differed from that of MT-I+II, since the latter was evident in both the vicinity of the lesioned tissue and deeper cortical layers, whereas that of the former was located only in the deeper cortical layers. This suggests different roles for these MT isoforms, and indeed in a new bioassay measuring astrocyte migration in vitro, rMT-III promoted migration to a higher extent than MT-I+II. Thus, MT-III could not only affect neuronal sprouting as previously suggested, but also astrocyte function. Finally, MT-III protein levels of patients with Alzheimer's disease (AD) were, if anything, increased when compared with similarly aged control brains, which was in agreement with the significantly increased MT-III mRNA levels of AD brains.

Alzheimer Disease↗
PubMed1999

Activators of peroxisome proliferator-activated receptor-alpha induce the expression of the uncoupling protein-3 gene in skeletal muscle: a potential mechanism for the lipid intake-dependent activation of uncoupling protein-3 gene expression at birth.

The recently identified uncoupling protein-3 (UCP-3) gene, predicted to encode a new member of the family of uncoupling proteins, is preferentially expressed in skeletal muscle and has been related to phenotypes of obesity and type 2 diabetes. We have established that during mouse ontogeny, the expression of the UCP-3 gene is switched on in skeletal muscle just after birth. The induction of UCP-3 gene expression is dependent on the initiation of suckling and particularly on lipid intake. Treatment of newborn mice with activators of peroxisome proliferator-activated receptors (PPARs), such as clofibrate, bezafibrate, or (4-chloro-6-(2,3-xylidine)-pirimidinylthio)acetic acid (WY 14,643), mimics the action of food intake on UCP-3 gene expression. The specific ligand of PPAR-alpha WY 14,643 induces UCP-3 gene expression in a time- and dose-dependent manner, whereas the thiazolidinedione BRL 49653, specific for PPAR-gamma, has no effect. These treatments act without altering circulating free fatty acids. During development, skeletal muscle expresses constitutive levels of PPAR-delta mRNA, whereas expression of the PPAR-gamma gene is undetectable. PPAR-alpha gene expression is developmentally regulated in muscle as it is first expressed at birth, just before UCP-3 gene induction occurs. The induction of UCP-3 gene expression by WY 14,643 is impaired in skeletal muscle of premature neonates, which do not express PPAR-alpha. It is proposed that the UCP-3 gene is predominantly regulated in neonatal muscle by PPAR-alpha activation.

Animals↗
PubMed1999

Stage, percentage of basophils at diagnosis, hematologic response within six months, cytogenetic response in the first year: the main prognostic variables affecting outcome in patients with chronic myeloid leukemia in chronic phase treated with interferon-alpha. Results of the CML89 trial of the Spanish Collaborative Group on interferon-alpha2a and CML.

BACKGROUND AND OBJECTIVE: Interferon-a (IFN) is increasingly being used as the drug of choice in chronic myeloid leukemia patients. The main objectives of the study were to study the influence of the classic prognostic variables and response to IFN, and to assess the influence of this response on the course of the disease and survival. DESIGN AND METHODS: Single arm, prospective, multicenter study, without a control group. Only Ph1-positive CML patients were included. The treatment scheme was biphasic: the patients first received standard chemotherapy and thereafter IFN-a2a was used as monotherapy, with a target dose of 9 MU/d/s.c. RESULTS: Twenty-one centers in Spain enrolled 132 patients (72 men, 60 women). The median dose of IFN given was 5.8 MU/d, and the median treatment duration was 431 days (range: 18-2,597). Seventy-two percent of patients obtained a hematologic response in the first six months of IFN treatment. Genetic response was obtained in 47% of the patients, and the response was major or complete in 27% and 19%, respectively. The median time to obtain this response was 7, 9, and 18 months for minimal, partial and complete genetic response, respectively. Multivariant analysis showed that only a higher percentage of basophils at diagnosis was associated with a worse hematologic response at six months (p=0.001) (OR: 1.23) and with a worse cytogenetic response in the first year of IFN therapy (p=0.018) (OR: 1.4). Over an observation period of 8 years, 35.6% of the patients died, and 85 (64.4%) remained alive. With a median follow-up of 42 months (3.7-98), the 6-year projected probabilities of survival and transformation-free survival were 0.61+/-0.07 vs. 0.54+/-0.07, respectively. Patients with Kantarjian's stage 3 disease or in a high-risk Sokal group had lower probabilities of survival, but these systems did not adequately discriminate in our series. Obtaining a complete hematologic response in the first six months of IFN therapy was favorable in terms of overall survival (p=0.05; HR=0.33). Cox's analysis demonstrated that obtaining a cytogenetic response in the first year was independently associated with better overall survival (p=0.04; HR=0.19) and better transformation-free survival (p=0.0035; HR=0.11). INTERPRETATION AND CONCLUSIONS: Nearly half of the patients obtained some degree of Philadelphia suppression, which was major in 27%, and complete in 19%. A higher percentage of basophils at diagnosis was the only variable associated with a lower probability of cytogenetic response. Obtaining a cytogenetic response during the first year of IFN treatment was a favorable and independent variable in terms of survival and transformation-free survival. Obtaining a major cytogenetic response during this period decreased the risk of transformation twenty times. Our results suggest that the effect of IFN on survival is independent of the classic prognostic variables.

Adolescent↗
PubMed1998

9-cis retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes.

The expression of uncoupling protein-2 (UCP2) mRNA is up-regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down-regulated. 9-cis Retinoic acid causes a dose-dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all-trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9-cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

Adipocytes↗
PubMed1998

[Systemic mastocytosis. Study of 14 cases].

We have analyzed the clinical, analytical and evolutive data of 14 cases of systemic mastocytosis (SM) diagnosed in our hospital between 1991-1996 and we have compared these results with those of other published series. Clinical parameters, analytical profiles, peripheral hematologic data, radiologic data and histological study were collected. Following Metcalfe's criteria, the patients were classified in 4 groups: a) group 1, indolent mastocytosis; b) group 2, hematologic disorders; c) group 3, aggressive lymphadenopathic mastocytosis with eosinophilia, and d) group 4, mastocytic leukemia. The average age at diagnosis was 52.4 years, range 25-83, and 64% were females. The mean follow-up was 2 yr. In most of the cases (71.4%) the initial complaint was urticaria pigmentosa. The predominant clinical features were pruriginous-eritematous skin lesions (in 11 cases), and digestive symptoms (in 10 patients). The most usual biochemical disorder was the rise of serum alkaline phosphatase level (in 8 patients), while lactate-dehydrogenase (LDH) was normal in all the cases. The most striking roentgenologic features were oteopenia, observed in 50% of our patients. Pheripherical hematological disorders were discovered in 8 patients (64.3%) and in one of them circulating mast-cells were observed. The bone marrow was involved in all patients (100%) and in two of them mielodysplasic features were found. The diagnostic of SM is difficult in the absence of skin lesions. The skin lesions are very common in systemic mastocytosis. Bone marrow involvement is constant, so its study has a high diagnostic rentability.

Adult↗
PubMed1998

Beta-glucocerebrosidase gene locus as a link for Gaucher's disease and familial hypo-alpha-lipoproteinaemia.

BACKGROUND: Gaucher's disease is the most common lysosomal storage disorder, caused by deficiency of glucocerebrosidase resulting from homozygosity for any of several mutations of the glucocerebrosidase gene locus. Affected people have decreased concentrations of LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C). We assessed the association between mutations in the glucocerebrosidase locus and hypo-alpha-lipoproteinaemia. METHODS: We studied 258 people from 43 unrelated Spanish families. 57 participants were affected, 137 were non-affected carriers, and 64 were non-carriers. We determined glucocerebrosidase genotypes and measured plasmid lipids, apolipoproteins A-I, B, and E, and leucocyte glucocerebrosidase activity. FINDINGS: The most common glucocerebrosidase mutations were N370S (45%), L444P (23%), and G377S (5%). Deletions and recombinants accounted for another 5%, and point mutations in exons 5, 6, 9, and 10 were present in 12%. Affected participants had lower LDL-C and HDL-C concentrations than non-affected carriers (p<0.001) and non-carriers (p<0.001). HDL-C values were also significantly different between the non-affected carriers and non-carriers. Mutations at this locus may account for as much as 19.5% of the genetic variability in HDL-C in the population studied. INTERPRETATION: Heterozygosity for these mutations at the glucocerebrosidase locus does not result in clinical expression of Gaucher's disease but can decrease HDL-C concentrations. Given the high frequency of these mutations, the glucocerebrosidase locus might lead to familial low alpha-lipoproteinaemia in up to 2% of the general population and be one of the most common known genetic causes of HDL-C.

Adult↗
PubMed1998

Sympathetic pathways mediate GLP-1 actions in the gastrointestinal tract of the rat.

The aim of this study was to establish the actions of GLP-1 (7-37) on gastrointestinal motility in rats. We prepared anaesthetized Sprague-Dawley rats with strain-gauges in the antrum, duodenum and the proximal jejunum and a catheter in the aorta close to the coeliac artery for close infusion of substances. Intraarterial GLP-1 infusions (3 x 10(-10) and 3 x 10(-9) moles/kg per 10 min) (n = 8) induced inhibition of spontaneous motor activity in the antrum, duodenum and proximal jejunum. Inhibition induced by GLP-1 was reversed by i.v. infusion of GLP-1 receptor antagonist, Exendin (9-39) (3 x 10(-8) moles/kg per 10 min) (n = 6). Neither the presence of L-NNA (10(-5) moles/kg) (n = 9) nor the VIP receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP (3 x 10(-8) moles/kg per 10 min) (n = 8) modified responses to GLP-1. However, a combination of the adrenergic blockers phentolamine and propranolol (1 mg/kg each) (n = 8) completely blocked motor actions of GLP-1 in all the organs studied. Moreover, inhibition of gastrointestinal motor activity by GLP-1 was blocked by previous infusion of hexamethonium (10 mg/kg) (n = 4). This study demonstrates that GLP-1 inhibits gastrointestinal motor activity of the rat acting on specific GLP-1 receptors and via stimulation of adrenergic pathways.

Animals↗
PubMed1998

Regulation of mitochondrial biogenesis in brown adipose tissue: nuclear respiratory factor-2/GA-binding protein is responsible for the transcriptional regulation of the gene for the mitochondrial ATP synthase beta subunit.

The regulation of transcription of the gene for the beta subunit of the FoF1 ATP synthase (ATPsynbeta) in brown adipose tissue has been studied as a model to determine the molecular mechanisms for mitochondrial biogenesis associated with brown adipocyte differentiation. The expression of the ATPsynbeta mRNA is induced during the brown adipocyte differentiation that occurs during murine prenatal development or when brown adipocytes differentiate in culture. This induction occurs in parallel with enhanced gene expression for other nuclear and mitochondrially-encoded components of the respiratory chain/oxidative phosphorylation system (OXPHOS). Transient transfection assays indicated that the expression of the ATPsynbeta gene promoter is higher in differentiated HIB-1B brown adipocytes than in non-differentiated HIB-1B cells. A major transcriptional regulatory site was identified between nt -306 and -266 in the ATPsynbeta promoter. This element has a higher enhancer capacity in differentiated brown adipocyte HIB-1B cells than in non-differentiated cells. Electrophoretic shift analysis indicated that Sp1and nuclear respiratory factor-2/GA-binding protein (NRF2/GABP) were the main nuclear proteins present in brown adipose tissue that bind this site. Double-point mutant analysis indicated a major role for the NRF2/GABP site in the enhancer capacity of this element in brown fat cells. It is proposed that NRF2/GABP plays a pivotal role in the co-ordinated enhancement of OXPHOS gene expression associated with mitochondrial biogenesis in brown adipocyte differentiation.

Adipose Tissue, Brown↗
PubMed1998

Differential regulation of uncoupling protein-2 and uncoupling protein-3 gene expression in brown adipose tissue during development and cold exposure.

The expression of the two novel uncoupling proteins genes, UCP2 and UCP3, is differentially regulated during prenatal maturation of brown adipose tissue in the mouse. UCP2 gene is expressed early in prenatal development, when neither UCP1 nor UCP3 gene expression yet occurs. UCP3 mRNA is absent at any stage of fetal life; it appears suddenly at birth and reaches adult levels in a few hours. UCP2 mRNA increased after birth but more slowly than UCP3 and UCP1 mRNA. Short-time exposure of adult mice to cold caused a rise in UCP2 or UCP1 mRNA levels but not in that of UCP3. The postnatal rise in UCP2 gene expression appears to be a response to the thermic stress associated with birth, similarly to UCP1, whereas different biological signals may be responsible for the surge in UCP3 gene expression at birth.

Adipose Tissue, Brown↗
PubMed1998

Muscle/heart isoform of mitochondrial adenine nucleotide translocase (ANT1) is transiently expressed during perinatal development in rat liver.

A postnatal increase in the content of mitochondrial ANT in rat liver which is related to the maturation of mitochondrial function has previously been reported [Schönfeld et al., Biochim. Biophys Acta 1144 (1993) 353-358]. In order to define the contribution of the ANT isoforms to this postnatal increase we have studied the expression of ANT1 and ANT2 isoforms in the liver during this period. The results show that in contrast to adult liver, perinatal liver expressed the ANT1 isoform at the mRNA and protein level, and that during this period the expression of ANT1 increased to a similar extent as total ANT content. It is concluded that the postnatal increase in ANT is mainly due to the ANT1 isoform and therefore, a role for the ANT1 isoform in the postnatal maturation of mitochondrial respiration in rat liver is suggested.

Animals↗
PubMed1998

Mucosal mast cells are involved in CCK disruption of MMC in the rat intestine.

Our aim was to determine if mucosal mast cells could be activated by endogenous CCK and, as a consequence, mediate CCK actions in the small intestine. Rats were prepared for electromyography to record electrical activity in the small intestine. In another group of animals, the duodenum was perfused to measure rat mast cell protease II (RMCP II) as indicative of mast cell degranulation. Endogenous CCK release was induced by administration of soybean trypsin inhibitor (SBTI) in conscious rats or by intraduodenal perfusion of ovalbumin hydrolysate (OVH) in anesthetized rats. CCK concentration was measured by bioassay on pancreatic acini. SBTI in control rats disrupted migrating motor complexes (MMC) for >40 min. In rats treated with the mast cell stabilizer ketotifen, SBTI did not induce any change in the MMC pattern. RMCP II concentration in the duodenal perfusate significantly increased after OVH. Perfusate from ketotifen-treated animals did not show any significant increase in RMCP II values during OVH perfusion, although CCK plasma concentration was not different from the control group. Furthermore, infusion of the CCK-B receptor antagonist L-365,260 significantly blocked the increase of RMCP II concentration after OVH. Our results indicate that mucosal mast cells are degranulated by endogenous CCK release through stimulation of CCK-B receptors. Therefore mucosal mast cells participate in CCK intestinal actions.

Animals↗
PubMed1998

Dominant negative regulation by c-Jun of transcription of the uncoupling protein-1 gene through a proximal cAMP-regulatory element: a mechanism for repressing basal and norepinephrine-induced expression of the gene before brown adipocyte differentiation.

The brown fat uncoupling protein-1 (ucp-1) gene is regulated by the sympathetic nervous system, and its transcription is stimulated by norepinephrine, mainly through cAMP-mediated pathways. Overexpression of the catalytic subunit of protein kinase A stimulated a chloramphenicol acetyltransferase expression vector driven by the 4.5-kb 5'-region of the rat ucp-1 gene. Mutant deletion analysis indicated the presence of the main cAMP-regulatory element (CRE) in the proximal region between -141 and -54. This region contains an element at -139/-122 able to confer enhancer and protein kinase A (PKA)-dependent activity to the basal thymidine kinase promoter. The potency of this element was much higher in differentiated than in nondifferentiated brown adipocytes. Gel shift analyses indicated that a complex array of proteins from brown fat nuclei bind to the -139/-122 element, among which CRE-binding protein (CREB) and Jun proteins were identified. In transfected brown adipocytes, c-Jun was a negative regulator of basal and PKA-induced transcription from the ucp-1 promoter acting through this proximal CRE region. A double-point mutation in the -139/-122 element abolished both PKA- and c-Jun-dependent regulation through this site, and overexpression of CREB blocked c-Jun repression. Thus, an opposite action of these two transcription factors on the -139/-122 CRE is proposed. c-Jun content in brown adipocytes differentiating in culture correlated negatively with both ucp-1 gene expression and the acquisition of the brown adipocyte morphology. These findings indicate that c-Jun provides a molecular mechanism to repress the basal and cAMP-mediated expression of the ucp-1 gene before the differentiation of the brown adipocyte.

Adipocytes↗