International multi-center evaluation of a novel chemiluminescence assay for the detection of anti-dsDNA antibodies.

OBJECTIVE: Anti-double stranded desoxyribonucleic acid (anti-dsDNA) antibodies are considered fairly specific for systemic lupus erythematosus (SLE) and their quantification is useful for the clinical management of SLE patients. We assessed the diagnostic performance of the QUANTA Flash dsDNA chemiluminescent immunoassay (CIA) in comparison to an ELISA, using patients from five participating countries. The main focus was to evaluate the correlation between anti-dsDNA antibody results from the CIA and global SLE disease activity, as measured by the SLE Disease Activity Index 2000 (SLEDAI-2K). PATIENTS AND METHODS: A total of 1431 samples (SLE, n = 843; disease controls, n = 588) from five countries (Canada, USA, Portugal, Sweden and Spain) were tested with QUANTA Flash dsDNA (Inova Diagnostics, San Diego, CA, USA). Data obtained with the QUANTA Lite dsDNA SC ELISA (Inova Diagnostics) were available for samples from three sites (Canada, USA and Sweden, n = 566). The SLEDAI-2K scores were available for 805 SLE patients and a cut-off of > 4 was used to define active disease. RESULTS: QUANTA Flash dsDNA had a sensitivity of 54.3% for the diagnosis of SLE, combined with 89.8% specificity. Anti-dsDNA antibody levels were significantly higher (p < 0.0001) in active SLE (SLEDAI-2K > 4; n = 232; median value 83.0 IU/mL) versus the inactive patients (n = 573; median value 22.3 IU/mL), and the SLEDAI-2K scoring correlated with their dsDNA antibody levels (Spearman's rho = 0.44, p < 0.0001). Similar but less pronounced findings were also found for the ELISA, in relation to disease activity. CONCLUSIONS: The QUANTA Flash dsDNA assay showed good clinical performance in a large international multi-center study. Additionally, the strong correlation between anti-dsDNA antibody results and SLEDAI-2K scores supported the potential utility of QUANTA Flash dsDNA for monitoring disease activity.

Does medicine still show an unresolved discrimination against women? Experience in two European university hospitals.

Have invisible barriers for women been broken in 2007, or do we still have to break through medicine's glass ceiling? Data from two of the most prestigious university hospitals in Barcelona with 700-800 beds, Hospital Clínic (HC) and Hospital de la Santa Creu i Sant Pau (HSCSP) address this issue. In the HSCSP, 87% of the department chairs are men and 85% of the department unit chiefs are also men. With respect to women, only 5 (13%) are in the top position (department chair) and 4 (15%) are department unit chiefs. Similar statistics are also found at the HC: 87% of the department chairs and 89% of the department unit chiefs are men. Currently, only 6 women (13%) are in the top position and 6 (11%) are department unit chiefs. Analysis of the 2002 data of internal promotions in HC showed that for the first level (senior specialist) sex distribution was similar. Nevertheless, for the second level (consultant) only 25% were women, and for the top level (senior consultant) only 8% were women. These proportions have not changed in 2007 in spite of a 10% increase in leadership positions during this period. Similar proportions were found in HSCSP where 68% of the top promotions were held by men. The data obtained from these two different medical institutions in Barcelona are probably representative of other hospitals in Spain. It would be ethically desirable to have males and females in leadership positions in the medical profession.

Prognostic markers of clinical remission in early rheumatoid arthritis after two years of DMARDs in a clinical setting.

OBJECTIVE: To analyze the rate and baseline prognostic factors of clinical remission in a series of patients with early rheumatoid arthritis (RA) after 2 years of therapy based on a structured algorithm using disease-modifying anti-rheumatic drugs (DMARDs) in a clinical setting. To determine whether a good therapeutic response at 6 months of therapy is associated with remission at 2 years. METHODS: One hundred and five patients (81% female) with early RA (disease duration < 2 years) treated with the same therapeutic protocol using gold salts and methotrexate in a step-up strategy, together with methylprednisolone (4 mg/day), were followed up for 2 years. The outcome variable was clinical remission after 2 years of DMARD therapy using the 28-joint disease activity score (DAS28 < 2.6). Clinical, biological, immunogenetic and radiographic data (Larsen score) were analyzed at study entry and after 6, 12, 18 and 24 months of follow-up. Therapeutic response was analyzed using the ACR and EULAR criteria. RESULTS: Remission was observed in 34 patients (32.4%) after 2 years of follow-up. A baseline DAS28 score < 5.1 (p = 0.004), hemoglobin (p = 0.04) and male gender (p = 0.02) were associated with remission in the univariate analysis. In the multivariate logistic regression analysis, only a DAS28 < 5.1 was associated with remission at 2 years (OR 4.1, 95% CI: 1.56;10.77, p = 0.004). The percentage of ACR50 responses after 6 months was significantly higher in patients with remission at 2 years than in those without (66.7% vs 43.3%; p = 0.04). Similar results were obtained when analyzing the good EULAR response (50% vs 20.9%; p = 0.003). Furthermore, when the therapeutic response at 6 months was included in the logistic regression model, only an ACR50 response (OR 3.9, 95% CI 1.14;13.38, p = 0.03) and a good EULAR response (OR 6.23, 95% CI 1.61; 24.04, p = 0.008), but not an ACR20 response or a whole EULAR response were significantly associated with remission. CONCLUSION: In a series of early RA patients treated using a structured algorithm with DMARDs and very low doses of glucocorticoids, clinical remission was observed in one-third of patients after 2 years. Low or moderate disease activity (DAS28 < 5.1) at baseline and a good therapeutic response during the first months of therapy predicts clinical remission at 2 years.

Prognostic factors of radiographic progression in early rheumatoid arthritis: a two year prospective study after a structured therapeutic strategy using DMARDs and very low doses of glucocorticoids.

The objective of the study was to analyze the prognostic factors of radiographic progression in a series of patients with early rheumatoid arthritis (RA) after 2 years of therapy with a structured algorithm using disease-modifying antirheumatic drugs (DMARDs) and very low doses of oral glucocorticoids. One hundred and five patients (81% female) with early RA (disease duration <2 years) treated with the same therapeutic protocol using gold salts and methotrexate in a step-up strategy, together with methylprednisolone (4 mg/day), were followed up for 2 years. The outcome variable was radiographic progression after 2 years of DMARD therapy using the modified Larsen method. Clinical, biological, immunogenetic, and radiographic data were analyzed at study entry and after 1 and 2 years of follow-up. Radiographic progression (increase of four or more units in the Larsen score) was observed in 32% of patients after 2 years of follow-up. The percentage of erosive disease increased from 18.3% at baseline to 28.9% at 12 months and 44.6% at 24 months, in spite of a significant improvement in disease activity. New erosions appeared in 33% of patients after 2 years. Several baseline parameters were associated with radiographic progression in the univariate analysis: shared epitope (SE) homozygozity, HLA-DRB*04 alleles, female gender, hemoglobin, erythrocyte sedimentation rate, and anticyclic citrullinated peptide antibodies (anti-CCP). In the multivariate analysis, female gender [odds ratio (OR) 5.5, 95% confidence interval (CI): 1.1-28.2, p = 0.04], DRB1*04 alleles (OR 3.1, 95% CI 1.1-9, p = 0.03) and, marginally, anti-CCP antibodies (OR 3.6, 95% CI 0.9-14.5, p = 0.06), were associated with progression. Female patients with both DRB1*04 alleles and anti-CCP antibodies showed the highest scores in radiographic progression. The presence, but not the titer, of anti-CCP antibodies predicted progression. The positive predictive value of the multivariate model for progression was only 53.9% whereas the negative predictive value was 80.3%. In a series of early RA patients treated with a structured algorithm using DMARDs and very low doses of glucocorticoids, radiographic progression was observed in one third of patients after 2 years. Female gender, DRB1*04 alleles (rather than the SE), and the presence of anti-CCP antibodies at baseline (independently of the titer) were the most important predictors of progression. The utility of these parameters in clinical practice is limited by their relatively low positive predictive value.

Relevance of MICA antibodies in acute humoral rejection in renal transplant patients.

The presence of MICA antibodies was examined in eleven patients diagnosed with AHR. MICA typing was performed in both recipients and donors. Sera were collected sequentially: pre-transplant, at the AHR episode and at follow-up. Sera from 30 patients with functioning graft were also analysed. A stable MICA()008 transfected cell line was used as target to identify MICA antibodies. MICA antibodies were not detected pre-transplant nor post-transplant in patients receiving a compatible graft. MICA antibodies were detected post-transplant AHR in patients receiving an incompatible graft. The persistence of MICA antibodies was associated with chronic graft dysfunction in 3 of 4 patients in this series; although it was not always associated with the graft loss in treated AHR. None of the 30 patients in the control group with long-term functioning grafts showed antibodies to MICA()008. This report provides some insights of the relevance of MICA antibodies in AHR.

Subtyping obsessive-compulsive disorder: clinical and immunological findings in child and adult onset.

It has been suggested that certain kinds of childhood OCD with specific clinical, biological and immunological characteristics may form a subgroup of OCD. We study the presence of these characteristics in child onset OCD and propose that the disorder be considered as a subtype of adult OCD. Forty adult patients with OCD were divided in two groups according to time of disease onset: 18 early onset and 21 late. Both sets were compared with a control group of 14 psychiatric patients. Child onset OCD was associated with higher mean ASLO titers, higher frequencies of history of tic disorders and tonsillitis in childhood and compulsive symptoms. No differences were found in D8/17 antibody titers or in other autoimmune parameters. The findings suggest that child onset OCD can be considered as a subgroup of adult OCD, although more specific biological markers are needed to identify it.

Antimitochondrial antibodies in patients with chronic hepatitis C virus infection: description of 18 cases and review of the literature.

To describe the clinical and immunologic patterns of disease expression of patients with chronic hepatitis C virus (HCV) infection and positive antimitochondrial antibodies (AMA). We investigated the presence of AMA in 237 consecutive HCV patients with extrahepatic manifestations from an International Registry. AMA were detected by indirect immunofluorescence in triple rat tissue (liver, stomach and kidney), aceton-fixed criosections and FITC-conjugated rabbit anti-human immunoglobulins. We found positive AMA in 18 (8%) out of 237 HCV patients. All patients were female with a mean age at protocol inclusion of 65.8 years (ranging from 37 to 87 years). Twelve (67%) patients fulfilled classification criteria for systemic autoimmune diseases (SAD), including Sjögren's syndrome (n = 7), systemic sclerosis (n = 3) and systemic lupus erythematosus (n = 2). Fourteen (78%) of the HCV-AMA patients presented at least one of the highly suggestive characteristics of primary biliary cirrhosis (PBC): 9 (50%) had a specific M2 pattern, 6 (33%) had more than twice normal levels of alkaline phosphatase, 5 (28%) had raised IgM levels and 4 (22%) a histological pattern compatible with PBC. Five (28%) patients developed neoplasia after detection of AMA. Seven (39%) patients died, due to neoplasia (n = 4), cirrhotic complications (n = 2) and hepatopulmonary syndrome (n = 1). We describe a subset of HCV patients with positive AMA who presented a broad spectrum of clinical features, including liver, autoimmune and neoplasic manifestations. Two-thirds of these patients presented an associated SAD, mainly Sjögren's syndrome or systemic sclerosis, together with a high frequency of multiple autoantibodies and an increased prevalence of cirrhosis and neoplasia.

Resumen del 4º Congreso Internacional sobre Autoinmunidad / Summary of the 4th International Congress on Autoimmunity

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Prevalence and clinical significance of anti-cyclic citrullinated peptide and antikeratin antibodies in palindromic rheumatism. An abortive form of rheumatoid arthritis?

OBJECTIVE: To analyse the prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies (anti-CCP) and antikeratin antibodies (AKA) in patients with palindromic rheumatism (PR). METHOD: Sixty-three patients with PR were included: 33 were defined as pure or persistent PR at the time of serum test measurement, and 30 as associated PR, defined as patients with past history of PR who had developed persistent arthritis at the time of serum test: [21 with rheumatoid arthritis (RA)]. Sixty patients with early RA, and 80 with seronegative spondyloarthropathy were included as control groups. Anti-CCP were determined by a standardized ELISA test and AKA by indirect immunofluorescence in rat oesophagus. Clinical characteristics of these pure PR patients were compared according to the presence or absence of anti-CCP antibodies. A follow-up study was also performed. RESULTS: Anti-CCP were detected in 18 out of 32 (56.3%) patients with pure PR and 10 out of 30 (33.3%) with associated PR (38.1% in RA-associated PR patients). AKA were detected in 12 patients out of 33, with pure PR (36.4%), and in 9 out of 30 with associated PR (30%) (33.3% in RA-associated PR patients). The prevalence of anti-CCP and AKA in the RA control group was 55% (not significantly different from the pure PR group) and 61.7% (with respect to pure PR patients, P=0.02), respectively. In the spondyloarthropathy group, the prevalence of anti-CCP and AKA was 2.5 and 3.8%, respectively (P<0.001 compared with pure PR patients). No significant clinical differences were observed between pure PR patients with and without CCP antibodies. CONCLUSIONS: Anti-CCP and, to a lesser extent, AKA, were found in a high proportion of patients with PR, suggesting that this syndrome is an abortive form of RA. The predictive value of these antibodies in PR, as markers of progression to an established RA, remains uncertain.

Radiological progression in early rheumatoid arthritis after DMARDS: a one-year follow-up study in a clinical setting.

OBJECTIVE: To analyse the frequency and prognostic factors of radiographic progression in a series of Spanish patients with early rheumatoid arthritis (RA) after 1 yr of treatment with disease-modifying anti-rheumatic drugs (DMARDs). METHODS: Sixty patients (47 females, 13 males) with RA with a disease duration shorter than 2 yr [mean (s.d.) duration 9.5+/-6.6 months] were treated with the same therapeutic protocol using gold salts as the first DMARD and methotrexate as a second option, and were followed up for 1 yr. Radiographic progression in the hands and feet (total radiographic Larsen score and the erosion joint count) was used as the outcome variable. Clinical, laboratory, immunogenetic and radiographic data were obtained at study entry. Disease activity and response to therapy were measured at 6 and 12 months. RESULTS: Erosive disease was found in 21.7% of patients at baseline and in 38.3% after 1 yr. Although a substantial reduction in disease activity was observed during the 1 yr follow-up [disease activity score (DAS28) 5.8+/-0.8 at entry and 3.9+/-1.3 at 12 months, P < 0.001], the Larsen score rose from 1.9+/-3.3 to 5.6+/-9.8 after 1 yr. In 26.6% of patients, a raised erosion joint count was observed after 1 yr. Radiographic progression in the total joint radiographic damage (increase in Larsen score of >or=2) was observed in 36.6%. In the multivariate analysis, baseline pain [visual analogue scale (VAS)] and the presence of two copies of the shared epitope were associated with radiographic progression in the erosion joint count. Disease duration before study entry, VAS pain and Larsen score at baseline were significant predictors of radiographic progression in total damage (Larsen score). Baseline radiographic damage had the highest positive predictive value for progression. CONCLUSIONS: Radiographic progression was observed in up to 36.6% of patients with early RA after 1 yr of DMARD therapy in spite of a significant reduction in disease activity. Baseline factors, such as VAS pain, disease duration until DMARD therapy, damage score at baseline and the presence of two copies of the shared epitope, were associated with radiographic progression.

Anti-chromatin antibodies in systemic lupus erythematosus: a useful marker for lupus nephropathy.

BACKGROUND: Anti-chromatin antibodies have recently been described in patients with systemic lupus erythematosus (SLE) and it has been suggested that their presence is associated with lupus nephritis. OBJECTIVE: To assess the prevalence and clinical associations of these antibodies in SLE. METHODS: The presence of anti-chromatin antibodies in 100 patients with SLE was investigated by an enzyme linked immunosorbent assay (ELISA). To determine the specificity of these antibodies, 100 patients with primary Sjögren's syndrome, 30 with primary antiphospholipid syndrome (APS), 10 with systemic sclerosis, and 100 normal controls were also tested. RESULTS: Positive levels were detected in 69/100 (69%) patients with SLE. In contrast, they were found in only 8/100 (8%) of those with primary Sjögren's syndrome, in 1/10 (10%) with systemic sclerosis, in 2/30 (7%) with primary APS, and in none of the 100 healthy controls. Patients with anti-chromatin antibodies had a twofold higher prevalence of lupus nephropathy than those without these antibodies (58% v 29%, p<0.01). A significant correlation was found between the levels of anti-chromatin antibodies and disease activity score as measured by the European Consensus Lupus Activity Measurement (ECLAM; p=0.011). CONCLUSIONS: The measurement of anti-chromatin antibodies appears to be a useful addition to the laboratory tests that can help in the diagnosis and treatment of SLE. These antibodies are both sensitive and specific for SLE, and are a useful marker for an increased risk of lupus nephritis.

Mitochondrial biogenesis in brown adipose tissue is associated with differential expression of transcription regulatory factors.

The differentiation of brown adipocytes during late fetal development or in cell culture is associated with enhanced mitochondrial biogenesis and increased gene expression for components of the respiratory chain/oxidative phosphorylation system. We have shown that this is due to a rise in mitochondrial DNA abundance and the corresponding increase in mitochondrial genome transcripts and gene products, as well as to the coordinate induction of nuclear-encoded genes for mitochondrial proteins. We studied how the expression of key components of the transcriptional regulation of mitochondrial biogenesis is regulated during this process. Changes in the expression of nuclear respiratory factor-2/GA-binding protein a and peroxisome proliferator-activated-receptor gamma coactivator-1 (increase) were opposite to those of nuclear respiratory factor-1 and Sp1 (decrease) during the developmental and differentiation-dependent induction of mitochondrial biogenesis in brown fat. These results indicate that the relative roles of transcription factors and coactivators in mediating mitochondrial biogenesis 'in vivo' are highly specific according to the cell type and stimulus that mediate the mitochondriogenic process.

Impaired expression of the uncoupling protein-3 gene in skeletal muscle during lactation: fibrates and troglitazone reverse lactation-induced downregulation of the uncoupling protein-3 gene.

The expression of uncoupling protein (UCP)-3 mRNA in skeletal muscle is dramatically reduced during lactation in mice. The reduction in UCP-3 mRNA levels lowers the amount of the UCP-3 protein in skeletal muscle mitochondria during lactation. Spontaneous or abrupt weaning reverses the downregulation of the UCP-3 mRNA but not the reduction in UCP-3 protein levels. In lactating and virgin mice, however, fasting increases UCP-3 mRNA levels. Changes in UCP-3 mRNA occur in parallel with modifications in the levels of free fatty acids, which are reduced in lactation and are upregulated due to weaning or fasting. Modifications in the energy nutritional stress of lactating dams achieved by manipulating litter sizes do not influence UCP-3 mRNA levels in skeletal muscle. Conversely, when mice are fed a high-fat diet after parturition, the downregulation of UCP-3 mRNA and UCP-3 protein levels due to lactation is partially reversed, as is the reduction in serum free fatty acid levels. Treatment of lactating mice with a single injection of bezafibrate, an activator of the peroxisome proliferator-activated receptor (PPAR), raises UCP-3 mRNA in skeletal muscle to levels similar to those in virgin mice. 4-chloro-6-[(2,3-xylidine)-pirimidinylthio] acetic acid (WY-14,643), a specific ligand of the PPAR-alpha subtype, causes the most dramatic increase in UCP-3 mRNA, whereas troglitazone, a specific activator of PPAR-gamma, also significantly increases UCP-3 mRNA abundance in skeletal muscle of lactating mice. However, in virgin mice, bezafibrate and WY-14,643 do not significantly affect UCP-3 mRNA expression, whereas troglitazone is at least as effective as it is in lactating dams. It is proposed that the UCP-3 gene is regulated in skeletal muscle during lactation in response to changes in circulating free fatty acids by mechanisms involving activation of PPARs. The impaired expression of the UCP-3 gene is consistent with the involvement of UCP-3 gene regulation in the reduction of the use of fatty acids as fuel by the skeletal muscle and in impaired adaptative thermogenesis, both of which are major metabolic adaptations that occur during lactation.

Both retinoic-acid-receptor- and retinoid-X-receptor-dependent signalling pathways mediate the induction of the brown-adipose-tissue-uncoupling-protein-1 gene by retinoids.

The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte.

Opposite regulation of PPAR-alpha and -gamma gene expression by both their ligands and retinoic acid in brown adipocytes.

The peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors involved in the regulation of lipid metabolism and adipocyte differentiation. Little is known, however, about the control of the expression of the genes encoding each of all three receptor subtypes: alpha, delta, and gamma. We have addressed this question in the brown adipocyte, the only cell type that co-expresses high levels of the three PPAR subtypes. Differentiation of brown adipocytes is associated with enhanced expression of PPAR genes. However, whereas PPARgamma and PPARdelta genes are already expressed in preadipocytes, the mRNA for PPARalpha appears suddenly in association with the acquisition of the terminally differentiated phenotype. Both retinoic acid isomers and PPAR agonists, specific for either PPARalpha or PPARgamma, regulate expression of each PPAR subtype gene in the opposite way: they up-regulate PPARalpha and down-regulate PPARgamma. The effects on PPARalpha mRNA are independent of protein synthesis, whereas inhibition of PPARgamma mRNA expression depends on protein synthesis, except when its specific ligand prostaglandin J2 is used. Our results indicate a strictly opposite autoregulation of PPAR subtypes, which supports specific physiological roles for them in controlling brown fat differentiation and thermogenic activity.

Contributions of p53 and PMA to gamma-irradiation induced apoptosis in Jurkat cells.

Several mutations prevent the expression of p53 in the human lymphoblastoid T cell line Jurkat. Restoration of p53 in Jurkat cells had no effect on the cell growth but markedly increased the amount of apoptosis induced by gamma-irradiation. Inhibition of RNA synthesis using 5,6-dichlorobenimidizole riboside had little effect on apoptosis induced by irradiation in the presence of p53 and did not affect the p53-independent apoptotic pathway. Expression of p53 also had no effect on the expression levels of proteins such as Fas, GADD45, Bax, Bcl-2, Bcl-x(L) or p53 induced proteins (PIGS) in resting cells or after irradiation. Activation of protein kinase C by phorbol 12-myristate 13-acetate produced an almost complete inhibition of p53-independent apoptosis following irradiation, whereas no significant effect was observed on the rate of p53-induced apoptosis. Although phorbol 12-myristate 13-acetate strongly induced p21 and stabilised p53 in the resting transfected Jurkat cells, neither apoptosis nor cell arrest was observed. In summary, this work shows that p53 enhances the radiosensitivity of Jurkat cells through an apoptotic process that is triggered by irradiation and is largely independent of RNA synthesis and protein kinase C activation. Apoptosis in p53- negative Jurkat cells is strongly inhibited by PMA indicating that the pathway triggered by p53 may be distinct from apoptotic pathways used in its absence.

Uncoupling protein-3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non-esterified fatty acids.

Uncoupling protein-3 gene expression in skeletal muscle is up-regulated during postnatal development of mice. A high-carbohydrate diet at weaning induces a decrease in uncoupling protein-3 mRNA levels that does not occur when mice were weaned onto a high-fat diet. Uncoupling protein-3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum non-esterified fatty acids, uncoupling protein-3 mRNA is up-regulated by starvation. Over-nutrition or under-nutrition during lactation increases or decreases, respectively, uncoupling protein-3 mRNA expression in skeletal muscle. Regulation of uncoupling protein-3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non-esterified fatty acids.

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.

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.