Mixed neuroendocrine non-neuroendocrine neoplasms in gastroenteropancreatic tract.

Mixed neuroendocrine non-neuroendocrine neoplasms (MiNENs) are a heterogeneous group of malignant neoplasms that can settle in the gastroenteropancreatic tract. They are composed of a neuroendocrine (NE) and a non-NE component in at least 30% of each tumour. The non-NE component can include different histological combinations of glandular, squamous, mucinous and sarcomatoid phenotypes, and one or both of the components can be low-or high grade malignant. Recent changes in the nomenclature of these neoplasms might lead to great deal of confusion, and the lack of specific clinical trials is the main reason why their management is difficult. The review aims to clarify the definition of MiNEN and analyze available evidence about their diagnosis and treatment options according to their location and extension through careful analysis of the available data. It would be important to reach a general consensus on their diagnosis in order to construct a classification that remains stable over time and facilitates the design of clinical trials that, due to their low incidence, will require long recruitment periods.

Observational Retrospective Study in Patients Treated with Galcanezumab as Preventive Treatment for Migraine: The ORYGAM Study.

INTRODUCTION: The efficacy of galcanezumab has been demonstrated in randomized controlled trials, but evidence about its use under clinical practice conditions is still limited. This study aimed to describe the characteristics of the patients treated with galcanezumab in routine clinical practice in Spain as well as treatment patterns, persistence, and effectiveness. METHODS: A retrospective chart review study was carried out in six hospitals. Information of adults with migraine, who started treatment with galcanezumab between November 2019 and September 2021, was analyzed until end or loss of follow-up. Continuous variables were described as mean (standard deviation, SD) and median (interquartile range, IQR), and categorical variables as frequency and percentages. Persistence to treatment was estimated using Kaplan-Meier analysis. RESULTS: A total of 314 patients were analyzed over median follow-up period of 17.5 months (13.8-20.7), with a mean age of 46.3 (12.6), 85% women, 80.6% chronic migraine, and reporting a mean of monthly migraine days of 16.7 (7.8). Overall, 72.9% had comorbid conditions, with anxiety and depression disorders being the most frequent. More than 60% had received ≥ 6 previous preventive drugs, the most common being antiepileptics, antidepressants, and botulinum toxin (95.2%, 89.8% and 84.1%, respectively). Overall, 60.3% of the patients with other preventive treatments maintained them after galcanezumab initiation. The median time on galcanezumab was 14.6 months (9.4-22.8); 95.7%, 82.0%, 76.2% and 59.8% of patients were persistent to treatment at 3, 6, 9 and 12 months, respectively. Of the patients who discontinued (151: 48.1%), 57.6% were due to lack of effectiveness and 31.1% were due to improvement in migraine. The average reduction of monthly migraine days at 3, 6, 9 and 12 months was 7.9 (7.2), 9.1 (7.5), 8.8 (6.6) and 9.0 (6.9) days, respectively. CONCLUSIONS: In real clinical practice, galcanezumab is an effective treatment and has a high persistence in patients with migraine, mostly chronic and with multiple use of previous preventive treatments.

Effectiveness, tolerability, and response predictors of preventive anti-CGRP mAbs for migraine in patients over 65 years old: a multicenter real-world case-control study.

OBJECTIVE: To evaluate clinical characteristics, effectiveness, and tolerability of preventive anti- calcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) in the elderly. Anti-CGRP mAbs have demonstrated efficacy and safety in patients with migraine although there is limited information regarding the elderly. DESIGN: We performed a multicenter case-control study of cases (patients over 65 years old) and controls (sex-matched patients under 55 years old) with migraine receiving anti-CGRP mAbs. METHODS: We included the demographic characteristics, effectiveness-reduction in the number of monthly headache days (MHD) and monthly migraine days (MMD), 30%, 50%, and 75% responder rates-and treatment emergent adverse events (TEAEs). The primary endpoint was the 50% response rate regarding MHD at weeks 20-24; exploratory 50% response predictors in the elderly were evaluated. RESULTS: In total, 228 patients were included: 114 cases , 114 controls-. Among cases 84.2% (96/114) were women, 79.8% (91/114) CM; mean age of cases 70.1 years old (range: 66-86); mean age of controls was 42.9 years old(range: 38-49). Cases had a higher percentage of vascular risk factors (P < .05),older age of onset (P < .001) and more reported prior preventive treatments (P < .001). Regarding effectiveness in cases, 50% response rate was achieved by 57.5% (42/73) at 20-24 weeks, with lower reduction in the MHD at 8-12 weeks (5 [7.2], 8 [9.1]; P = .001) and a higher reduction in MMD at 20-24 weeks (10.7 [9.1], 9.2 [7.7]; P = .04) compared to the control group. The percentage of TEAEs was similar in the 2 groups. Diagnosis of episodic migraine (EM) (P = .03) and lower number of MHD at baseline (P = .001) were associated with a 50% response in the elderly in univariate analysis. CONCLUSIONS: Our study provides real world evidence of effectiveness and safety of anti-CGRP mAbs for migraine in patients without upper age-limit and possible predictors of anti-CGRP response in the elderly.

Neoplastic appendiceal mucinous lesions: a narrative review of the literature from an oncologist's perspective.

Appendiceal mucinous lesions' classification and nomenclature has been modified several times along the last decades, reflecting their great heterogeneity and making difficult to compare results and draw conclusions. Despite its nearby origin, appendiceal mucinous lesions have a distinctive behaviour compared to colorectal cancer, including their molecular and genetic markers. Due to their low frequency, their management is not well standardised. However, surgery is considered the cornerstone of treatment. Their indolent behaviour has encouraged surgeons to apply more aggressive treatments, such as cytoreductive surgery (CRS) and heated intraperitoneal chemotherapy (HIPEC), that may extend overall survival. Chemotherapy is reserved for unresectable and/or disseminated disease and could play a role in the adjuvant and neoadjuvant setting. Pressurised intraperitoneal aerosol chemotherapy (PIPAC) is recently emerging as a possible alternative for treatment in advanced disease although its results in long-term survival are lacking Hereby, we review the available evidence in the management of appendiceal mucinous lesions, including localised and disseminated disease, with a special emphasis on the oncological perspective, focusing on the lights and shadows of the systemic treatments.

Brain Atrophy and Physical and Cognitive Disability in Multiple Sclerosis.

Introduction: Brain atrophy is associated with physical disability in multiple sclerosis (MS), but there is a great variability between different studies and methodologies, and its use is still limited to research projects. We aimed to analyze the relationship between several volumetric measurements and physical disability and cognitive functioning in MS patients in a clinical practice setting. Methods: This is a cross-sectional study. A total of 41 patients (31 relapsing-remitting MS, 6 secondary-progressive MS, and 4 primary-progressive MS) were included. Whole brain volume (WBV), gray matter volume (GMV), and T2 lesion load (T2L) were obtained using Icometrix® software. Physical disability was measured with the Expanded Disability Status Scale (EDSS), and cognitive status was evaluated with the brief repeatable battery of neuropsychological tests (BRB-N). The relationship between brain volumes and EDSS was analyzed through linear multivariate regression. The association between volumetry measurements and the number of affected cognitive domains was studied with negative binomial regression. Results: GMV was associated with age (b=-1.7, P=0.014) and with EDSS (b=-7.55, P=0.013). T2L was associated with EDSS (b=2.29, P=0.032). The number of affected cognitive domains was associated with clinical phenotype, worse in primary progressive MS (PPMS). There was not correlations between cognitive impairment and cerebral volumes. Conclusion: Brain atrophy measurement is feasible in clinical practice setting, and it is helpful in monitoring the EDSS progression. Primary progressive phenotype is associated with greater risk of cognitive dysfunction. Highlights: The T2 lesion load is associated with physical disability in patients with multiple sclerosis (MS).The gray matter volume is associated with age and physical disability in patients with MS.There is no significant correlation between cognitive impairment and cerebral volumes in patients with MS. Plain Language Summary: Conventional magnetic resonance imaging (MRI) is still used for diagnosing and monitoring multiple sclerosis (MS). Analysis of Brain volumes including Whole brain volume (WBV), gray matter volume (GMV), and T2 lesion load (T2L) allows the evaluation of its neurodegenerative mechanisms. Robust evidence links brain atrophy with disability in MS. This study aims to analyze the relationship between advanced MRI sequences and physical disability and cognitive functioning in MS patients. According to the results, T2L was associated with physical disability and GMV was associated with age and physical disability. There was no significant correlation between cognitive impairment and cerebral volumes in patients with MS.

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

Machine-learning-based approach for predicting response to anti-calcitonin gene-related peptide (CGRP) receptor or ligand antibody treatment in patients with migraine: A multicenter Spanish study.

BACKGROUND AND PURPOSE: Several variables have been reported to be associated with anti-calcitonin gene-related peptide (CGRP) receptor or ligand antibody response, but with differing results. Our objective was to determine whether machine-learning (ML)-based models can predict 6-, 9- and 12-month responses to anti-CGRP receptor or ligand therapies among migraine patients. METHODS: We performed a multicenter analysis of prospectively collected data from patients with migraine receiving anti-CGRP therapies. Demographic and clinical variables were collected. Response rates in the 30% to 50% range, or at least 30%, in the 50% to 75% range, or at least 50%, and response rate of at least 75% regarding the reduction in the number of headache days per month at 6, 9 and 12 months were calculated. A sequential forward feature selector was used for variable selection and ML-based predictive models for the response to anti-CGRP therapies at 6, 9 and 12 months, with model accuracy not less than 70%, were generated. RESULTS: A total of 712 patients were included, 93% were women, and the mean (SD) age was 48 (11.6) years. Eighty-four percent of patients had chronic migraine. ML-based models using headache days/month, migraine days/month and the Headache Impact Test (HIT-6) yielded predictions with an F1 score range of 0.70-0.97 and an area under the receiver-operating curve score range of 0.87-0.98. SHAP (SHapley Additive exPlanations) summary plots and dependence plots were generated to evaluate the relevance of the factors associated with the prediction of the above-mentioned response rates. CONCLUSIONS: Our results show that ML models can predict anti-CGRP response at 6, 9 and 12 months. This study provides a predictive tool that can be used in a real-world setting.

Risk and outcomes of COVID-19 in patients with multiple sclerosis.

BACKGROUND AND PURPOSE: Limited information is available on incidence and outcomes of COVID-19 in patients with multiple sclerosis (MS). This study investigated the risks of SARS-CoV-2 infection and COVID-19-related outcomes in patients with MS, and compared these with the general population. METHODS: A regional registry was created to collect data on incidence, hospitalization rates, intensive care unit admission, and death in patients with MS and COVID-19. National government outcomes and seroprevalence data were used for comparison. The study was conducted at 14 specialist MS treatment centers in Madrid, Spain, between February and May 2020. RESULTS: Two-hundred nineteen patients were included in the registry, 51 of whom were hospitalized with COVID-19. The mean age ± standard deviation was 45.3 ± 12.4 years, and the mean duration of MS was 11.9 ± 8.9 years. The infection incidence rate was lower in patients with MS than the general population (adjusted incidence rate ratio = 0.78, 95% confidence interval [CI] = 0.70-0.80), but hospitalization rates were higher (relative risk = 5.03, 95% CI = 3.76-6.62). Disease severity was generally low, with only one admission to an intensive care unit and five deaths. Males with MS had higher incidence rates and risk of hospitalization than females. No association was found between the use of any disease-modifying treatment and hospitalization risk. CONCLUSIONS: Patients with MS do not appear to have greater risks of SARS-CoV-2 infection or severe COVID-19 outcomes compared with the general population. The decision to start or continue disease-modifying treatment should be based on a careful risk-benefit assessment.

Endothelial Nox5 Expression Modulates Glucose Uptake and Lipid Accumulation in Mice Fed a High-Fat Diet and 3T3-L1 Adipocytes Treated with Glucose and Palmitic Acid.

Obesity is a global health issue associated with insulin resistance and altered lipid homeostasis. It has been described that reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity are involved in the development of these pathologies. The present study describes the role of endothelial NOX5 expression over adipose tissue by using two experimental systems: NOX5 conditional knock-in mice fed with a high-fat diet and 3T3-L1 adipocytes cultured with conditioned media of NOX5-expressing endothelial cells previously treated with glucose and palmitic acid. Animals expressing NOX5 presented lower body weight gain and less mesenteric and epididymal adipose mass compared to control mice fed with the same diet. NOX5-expressing mice also showed significantly lower glycaemia and improved insulin-induced glucose uptake. In addition, Glut4 and Caveolin 1 (Cav1) expression were significantly increased in the adipose tissue of these animals. Likewise, 3T3-L1 adipocytes treated with conditioned media from NOX5-expressing endothelial cells, incubated with high glucose and palmitic acid, presented a reduction in lipid accumulation and an increase in glucose uptake. Moreover, a significant increase in the expression of Glut4 and Cav1 was also detected in these cells. Taken together, all these data support that, in response to a highly caloric diet, NOX5 endothelial activity may regulate glucose sensitivity and lipid homeostasis in the adipose tissue.

NADPH Oxidase 5 Induces Changes in the Unfolded Protein Response in Human Aortic Endothelial Cells and in Endothelial-Specific Knock-in Mice.

Oxidative stress constitutes a key molecular mechanism in the development of cardiovascular diseases. A potential relationship between reactive oxygen species (ROS) driven by the NADPH oxidase family (NOX) and the unfolded protein response (UPR) has been postulated. Nevertheless, there is a lack of information about the crosstalk between NOX5 homologue and the UPR in a cardiovascular context. The main aim was to analyze NOX5-mediated ROS effects in the UPR and its importance in cardiovascular diseases. To this effect, we used an adenoviral NOX5-ß overexpression model in human aortic endothelial cells (HAEC) and a conditional endothelial NOX5 knock-in mouse. Using expression arrays, we investigated NOX5-induced genomic changes in HAEC. Compared with the control HAEC, 298 genes were differentially expressed. Gene ontology analysis revealed the activation of numerous cellular routes, the most relevant being the UPR pathway. Using real-time PCR and Western Blot experiments, we confirmed that NOX5 overexpression induced changes in the expression of the UPR components, which were associated with increased apoptosis. Moreover, in endothelial-specific NOX5 knock-in mice, we found changes in the expression of the UPR components genes. In these mice, myocardial infarction was performed by permanent coronary artery ligation; however, NOX5 expression was not associated with differences in the UPR components mRNA levels. In these animals, we found significant associations between the UPR components gene expression and echocardiographic parameters. Our data support the idea that NOX5-derived ROS may modulate the UPR pathway in endothelial cells, which might play a relevant role in cardiac physiology.

Endothelial NOX5 Expression Modulates Thermogenesis and Lipolysis in Mice Fed with a High-Fat Diet and 3T3-L1 Adipocytes through an Interleukin-6 Dependent Mechanism.

Obesity is a global health issue associated with the development of metabolic syndrome, which correlates with insulin resistance, altered lipid homeostasis, and other pathologies. One of the mechanisms involved in the development of these pathologies is the increased production of reactive oxygen species (ROS). One of the main producers of ROS is the family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, among which NOX5 is the most recently discovered member. The aim of the present work is to describe the effect of endothelial NOX5 expression on neighboring adipose tissue in obesity conditions by using two systems. An in vivo model based on NOX5 conditional knock-in mice fed with a high-fat diet and an in vitro model developed with 3T3-L1 adipocytes cultured with conditioned media of endothelial NOX5-expressing bEnd.3 cells, previously treated with glucose and palmitic acid. Endothelial NOX5 expression promoted the expression and activation of specific markers of thermogenesis and lipolysis in the mesenteric and epididymal fat of those mice fed with a high-fat diet. Additionally, the activation of these processes was derived from an increase in IL-6 production as a result of NOX5 activity. Accordingly, 3T3-L1 adipocytes treated with conditioned media of endothelial NOX5-expressing cells, presented higher expression of thermogenic and lipolytic genes. Moreover, endothelial NOX5-expressing bEnd.3 cells previously treated with glucose and palmitic acid also showed interleukin (IL-6) production. Finally, it seems that the increase in IL-6 stimulated the activation of markers of thermogenesis and lipolysis through phosphorylation of STAT3 and AMPK, respectively. In conclusion, in response to obesogenic conditions, endothelial NOX5 activity could promote thermogenesis and lipolysis in the adipose tissue by regulating IL-6 production.

Thrombopoietin Receptor Agonists for Severe Thrombocytopenia after Allogeneic Stem Cell Transplantation: Experience of the Spanish Group of Hematopoietic Stem Cell Transplant.

Persistent thrombocytopenia is a common complication after allogeneic hematopoietic stem cell transplantation (allo-SCT). Romiplostim and eltrombopag are the currently available thrombopoietin receptor agonists (TPO-RAs), and some studies with very small numbers of cases have reported their potential efficacy in the allo-SCT setting. The present retrospective study evaluated the safety and efficacy of TPO-RAs in 86 patients with persistent thrombocytopenia after allo-HSCT. Sixteen patients (19%) had isolated thrombocytopenia (PT), and 71 (82%) had secondary failure of platelet recovery (SFPR). TPO-RA therapy was started at a median of 127 days (range, 27 to 1177 days) after allo-SCT. The median initial and maximum administered doses were 50 mg/day (range, 25 to 150 mg/day) and 75 mg/day (range, 25 to 150 mg/day), respectively, for eltrombopag and 1 µg/kg (range, 1 to 7 µg/kg) and 5 µg/kg (range, 1 to 10 µg/kg), respectively, for romiplostin. The median platelet count before initiation of TPO-RA therapy was 14,000/µL (range, 1000 to 57,000/µL). Platelet recovery to ≥50,000/µL without transfusion support was achieved in 72% of patients at a median time of 66 days (range, 2 to 247 days). Eighty-one percent of the patients had a decreased number of megakaryocytes before treatment, showing a slower response to therapy (P = .011). The median duration of treatment was 62 days (range, 7 to 700 days). Grade 3-4 adverse events (hepatic and asthenia) were observed in only 2% of the patients. At last follow-up, 81% of patients had discontinued TPO-RAs and maintained response, and 71% were alive. To our knowledge, this is the largest series analyzing the use of TPO-RAs after allo-SCT reported to date. Our results support the efficacy and safety in this new setting. Further prospective trials are needed to increase the level of evidence and to identify predictors of response.

The RECALCAR Project. Healthcare in the Cardiology Units of the Spanish National Health System, 2011 to 2014.

The RECALCAR project (Spanish acronym for Resources and Quality in Cardiology Units) uses 2 data sources: a survey of cardiology units and an analysis of the Minimum Basic Data set of all hospital discharges of the Spanish National Health System. From 2011 to 2014, there was marked stability in all indicators of the availability, utilization, and productivity of cardiology units. There was significant variability between units and between the health services of the autonomous communities. There was poor implementation of process management (only 14% of the units) and scarce development of health care networks (17%). Structured cardiology units tended to have better results, in terms of both quality and efficiency. No significant differences were found between the different types of unit in the mean length of stay (5.5±1.1 days) or the ratio between successive and first consultations (2:1). The mean discharge rate was 5/1000 inhabitants/y and the mean rate of initial consultations was 16±4/1000 inhabitants/y. No duty or on-call cardiologist was available in 30% of cardiology units with 24 or more beds; of these, no critical care beds were available in 45%. Our findings support the recommendation to regionalize cardiology care and to promote the development of cardiology unit networks.

Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes.

Adipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, insulin receptor (IR) and potein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure.

[New mutation in a young woman diagnosed with Niemann-Pick disease type C]. / Nueva mutación descrita en una mujer joven con esplenomegalia, diagnosticada de enfermedad de Niemann-Pick tipo C.

BACKGROUND AND OBJETIVE: To describe a new molecular variant of Niemann-Pick disease type C (NPC) in a 27 year-old patient with splenomegaly and abolition of osteotendinous reflexes. MATERIAL AND METHODS: NPC1 is the main gene with described mutation in NPC disease. Here we report a case with a new mutation, p.N916S, not described before in a patient diagnosed with NPC. RESULTS: p.N916S was described as a cause of NPC disease by predictive programmes Mutation Master, PolyPhen2 and SIFT. CONCLUSIONS: p.N916S is a new mutation detected as a cause of NPC disease in a patient without severe neurological symptoms.

Effect of TNF-Alpha on Caveolin-1 Expression and Insulin Signaling During Adipocyte Differentiation and in Mature Adipocytes.

BACKGROUND/AIMS: Tumor necrosis factor-α (TNF-α)-mediated chronic low-grade inflammation of adipose tissue is associated with obesity and insulin resistance. Caveolin-1 (Cav-1) is the central component of adipocyte caveolae and has an essential role in the regulation of insulin signaling. The effects of TNF-α on Cav-1 expression and insulin signaling during adipocyte differentiation and in mature adipocytes were studied. METHODS: 3T3-L1 cells were differentiated (21 days) in the presence TNF-α (10 ng/mL) and mature adipocytes were also treated with TNF-α for 48 hours. Cav-1 and insulin receptor (IR) gene methylation were determined as well as Cav-1, IR, PKB/AKT-2 and Glut-4 expression and activation by real time RT-PCR and western blot. Baseline and insulin-induced glucose uptake was measured by the 2-[C14]-deoxyglucose uptake assay. RESULTS: TNF-α slowed down the differentiation program, hindering the expression of some insulin signaling intermediates without fully eliminating insulin-mediated glucose uptake. In mature adipocytes, TNF-α did not compromise lipid-storage capacity, but downregulated the expression of the insulin signaling intermediates, totally blocking insulin-mediated glucose uptake. Insulin sensitivity correlated with the level of activated phospho-Cav-1 in both situations, strongly suggesting the direct contribution of Cav-1 to the maintenance of this physiological response. CONCLUSION: Cav-1 activation by phosphorylation seems to be essential for the maintenance of an active and insulin-sensitive glucose uptake.

Expression of Caveolin 1 is enhanced by DNA demethylation during adipocyte differentiation. status of insulin signaling.

Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.