Abcc5 Knockout Mice Have Lower Fat Mass and Increased Levels of Circulating GLP-1.

OBJECTIVE: A previous genome-wide association study linked overexpression of an ATP-binding cassette transporter, ABCC5, in humans with a susceptibility to developing type 2 diabetes with age. Specifically, ABCC5 gene overexpression was shown to be strongly associated with increased visceral fat mass and reduced peripheral insulin sensitivity. Currently, the role of ABCC5 in diabetes and obesity is unknown. This study reports the metabolic phenotyping of a global Abcc5 knockout mouse. METHODS: A global Abcc5-/- mouse was generated by CRISPR/Cas9. Fat mass was determined by weekly EchoMRI and fat pads were dissected and weighed at week 18. Glucose homeostasis was ascertained by an oral glucose tolerance test, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test. Energy expenditure and locomotor activity were measured using PhenoMaster cages. Glucagon-like peptide 1 (GLP-1) levels in plasma, primary gut cell cultures, and GLUTag cells were determined by enzyme-linked immunosorbent assay. RESULTS: Abcc5-/- mice had decreased fat mass and increased plasma levels of GLP-1, and they were more insulin sensitive and more active. Recombinant overexpression of ABCC5 protein in GLUTag cells decreased GLP-1 release. CONCLUSIONS: ABCC5 protein expression levels are inversely related to fat mass and appear to play a role in the regulation of GLP-1 secretion from enteroendocrine cells.

Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly.

BACKGROUND: Acromegaly is produced by excess growth hormone secreted by a pituitary adenoma of somatotroph cells (ACRO). First-line therapy, surgery and adjuvant therapy with somatostatin analogs, fails in 25% of patients. There is no predictive factor of resistance to therapy. New therapies are investigated using few dispersed tumor cells in acute primary cultures in standard conditions where the cells do not grow, or using rat pituitary cell lines that do not maintain the full somatotroph phenotype. The RET/PIT1/p14ARF/p53 pathway regulates apoptosis in normal pituitary somatotrophs whereas the RET/GDNF pathway regulates survival, controlling PIT1 levels and blocking p14ARF (ARF) and p53 expression. METHODS: We investigated these two RET pathways in a prospective series of 32 ACRO and 63 non-functioning pituitary adenomas (NFPA), studying quantitative RNA and protein gene expression for molecular-clinical correlations and how the RET pathway might be implicated in therapeutic success. Clinical data was collected during post-surgical follow-up. We also established new'humanized' pituitary cultures, allowing 20 repeated passages and maintaining the pituitary secretory phenotype, and tested five multikinase inhibitors (TKI: Vandetanib, Lenvatinib, Sunitinib, Cabozantinib and Sorafenib) potentially able to act on the GDNF-induced RET dimerization/survival pathway. Antibody arrays investigated intracellular molecular pathways. FINDINGS: In ACRO, there was specific enrichment of all genes in both RET pathways, especially GDNF. ARF and GFRA4 gene expression were found to be opposing predictors of response to first-line therapy. ARF cut-off levels, calculated categorizing by GNAS mutation, were predictive of good response (above) or resistance (below) to therapy months later. Sorafenib, through AMPK, blocked the GDNF/AKT survival action without altering the RET apoptotic pathway. INTERPRETATION: Tumor ARF mRNA expression measured at the time of the surgery is a prognosis factor in acromegaly. The RET inhibitor, Sorafenib, is proposed as a potential treatment for resistant ACRO. FUND: This project was supported by national grants from Agencia Estatal de Investigación (AEI) and Instituto Investigación Carlos III, with participation of European FEDER funds, to IB (PI150056) and CVA (BFU2016-76973-R). It was also supported initially by a grant from the Investigator Initiated Research (IIR) Program (WI177773) and by a non-restricted Research Grant from Pfizer Foundation to IB. Some of the pituitary acromegaly samples were collected in the framework of the Spanish National Registry of Acromegaly (REMAH), partially supported by an unrestricted grant from Novartis to the Spanish Endocrine Association (SEEN). CVA is also supported from a grant of Medical Research Council UK MR/M018539/1.

Metabolic alterations derived from absence of Two-Pore Channel 1 at cardiac level.

Two-pore channels (TPCs or TPCNs) are novel voltage-gated ion channels that have been postulated to act as Ca2+ and/or Na+ channels expressed exclusively in acidic organelles such as endosomes and lysosomes. TPCNs participate in the regulation of diverse biological processes and recently have been proposed to be involved in the pathophysiology of metabolic disorders such as obesity, fatty liver disease and type 2 diabetes mellitus. Due to the importance of these pathologies in the development of cardiovascular diseases, we aimed to study the possible role of two-pore channel 1 (TPCN1) in the regulation of cardiac metabolism. To explore the cardiac function of TPCN1, we developed proteomic approaches as 2-DE-MALDI-MS and LC-MALDI-MS in the cardiac left ventricle of TPCN1 KO and WT mice, and found alterations in several proteins implicated in glucose and fatty acid metabolism in TPCN1 KO vs. WT mice. The results confirmed the altered expression of HFABP, a key fatty acid transport protein, and of enolase and PGK1, the key enzymes in the glycolytic process. Finally, in vitro experiments performed in neonatal rat cardiomyocytes, in which TPCN1 was silenced using siRNAs, confirmed that the downregulation of TPCN1 gene expression increased 2-deoxy-D-[3H]-glucose uptake and GLUT4 mobilization into cell peripherals in cardiac cells. Our results are the first to suggest a potential role for TPCNs in cardiac metabolism regulation.

Endolysosomal two-pore channels regulate autophagy in cardiomyocytes.

KEY POINTS: Two-pore channels (TPCs) were identified as a novel family of endolysosome-targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na(+) channels able to control endolysosomal fusion, a key process in autophagic flux. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure. We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy. We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts. ABSTRACT: Autophagy participates in physiological and pathological remodelling of the heart. The endolysosomal two-pore channels (TPCs), TPC1 and TPC2, have been implicated in the regulation of autophagy. The present study aimed to investigate the role of TPC1 and TPC2 in basal and induced cardiac autophagic activity. In cultured cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 transcripts and protein levels that paralleled the increase in autophagy identified by increased LC3-II and decreased p62 levels. Small interfering RNA depletion of TPC2 alone or together with TPC1 increased both LC3II and p62 levels under basal conditions and in response to serum starvation, suggesting that, under conditions of severe energy depletion (serum plus glucose starvation), changes in the autophagic flux (as assessed by use of bafilomycin A1) occurred either when TPC1 or TPC2 were downregulated. The knockdown of TPCs diminished cardiomyocyte viability under starvation and simulated ischaemia. Electron micrographs of hearts from TPC1/2 double knockout mice showed that cardiomyocytes contained large numbers of immature lysosomes with diameters significantly smaller than those of wild-type mice. In cardiac tissues from humans and rats, TPC1 and TPC2 transcripts and protein levels were higher in females than in males. Furthermore, transcript levels of TPCs correlated negatively with p62 levels in heart tissues. TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes (i.e. there is a negative effect on cell viability under stress conditions in their absence) and they are differentially expressed in male and female human and murine hearts, where they correlate with markers of autophagy.

Calcium signals regulated by NAADP and two-pore channels--their role in development, differentiation and cancer.

Ca(2+) signals regulate a wide range of physiological processes. Intracellular Ca(2+) stores can be mobilized in response to extracellular stimuli via a range of signal transduction mechanisms, often involving recruitment of diffusible second messenger molecules. The Ca(2+) mobilizing messengers InsP 3 and cADPR release Ca(2+) from the endoplasmic reticulum via InsP 3 and ryanodine receptors, respectively, while a third messenger, NAADP, releases Ca(2+) from acidic endosomes and lysosomes. Bidirectional communication between the ER and acidic organelles has functional relevance for endolysosomal function as well as for the generation of Ca(2+) signals. The two-pore channels (TPCs) are currently strong candidates for being key components of NAADP-regulated Ca(2+) channels. Ca(2+) signals have been shown to play important roles in embryonic development and cell differentiation; however, much remains to be established about the exact signalling mechanisms involved. Investigation of the role of NAADP and TPCs in development and differentiation is still at an early stage, but recent studies have suggested that they play important roles at key developmental stages in vivo and are important mediators of differentiation of neurons, skeletal muscle cells and osteoclasts in vitro. NAADP signals and TPCs have also been implicated in autophagy, an important process in differentiation. Moreover, potential links between TPC2 and cancer have been recently identified. Further studies will be required to identify the precise mechanisms of action of TPCs and their link with NAADP signalling, and to relate these to their roles in differentiation and other key developmental processes in the cell and organism.

Absence of intracellular ion channels TPC1 and TPC2 leads to mature-onset obesity in male mice, due to impaired lipid availability for thermogenesis in brown adipose tissue.

Intracellular calcium-permeable channels have been implicated in thermogenic function of murine brown and brite/beige adipocytes, respectively transient receptor potential melastin-8 and transient receptor potential vanilloid-4. Because the endo-lysosomal two-pore channels (TPCs) have also been ascribed with metabolic functionality, we studied the effect of simultaneously knocking out TPC1 and TPC2 on body composition and energy balance in male mice fed a chow diet. Compared with wild-type mice, TPC1 and TPC2 double knockout (Tpcn1/2(-/-)) animals had a higher respiratory quotient and became obese between 6 and 9 months of age. Although food intake was unaltered, interscapular brown adipose tissue (BAT) maximal temperature and lean-mass adjusted oxygen consumption were lower in Tpcn1/2(-/-) than in wild type mice. Phosphorylated hormone-sensitive lipase expression, lipid density and expression of ß-adrenergic receptors were also lower in Tpcn1/2(-/-) BAT, whereas mitochondrial respiratory chain function and uncoupling protein-1 expression remained intact. We conclude that Tpcn1/2(-/-) mice show mature-onset obesity due to reduced lipid availability and use, and a defect in ß-adrenergic receptor signaling, leading to impaired thermogenic activity, in BAT.

Hypothalamic KLF4 mediates leptin's effects on food intake via AgRP.

Krüppel-like factor 4 (KLF4) is a zinc-finger-type transcription factor expressed in a range of tissues that plays multiple functions. We report that hypothalamic KLF4 represents a new transcription factor specifically modulating agouti-related protein (AgRP) expression in vivo. Hypothalamic KLF4 colocalizes with AgRP neurons and is modulated by nutritional status and leptin. Over-expression of KLF4 in the hypothalamic arcuate nucleus (ARC) induces food intake and increases body weight through the specific stimulation of AgRP, as well as blunting leptin sensitivity in lean rats independent of forkhead box protein 01 (FoxO1). Down-regulation of KLF4 in the ARC inhibits fasting-induced food intake in both lean and diet-induced obese (DIO) rats. Silencing KLF4, however, does not, on its own, enhance peripheral leptin sensitivity in DIO rats.

Review of novel aspects of the regulation of ghrelin secretion.

The role of ghrelin in regulating metabolism and energy balance has been a subject of intense focus ever since its discovery. Ghrelin regulates energy balance in the short term by induction of appetite and in the longer term by increasing body weight and adiposity. It is the only known peripheral orexigenic hormone and one of the most potent endogenous orexigenic factors discovered to date. However, whilst extensively studied, the mechanism of ghrelin secretion is not well understood. A better understanding of the pathways controlling ghrelin secretion could be useful in the development of new therapeutic approaches to appetite-related disorders. Here, we discuss current knowledge of the processes that control ghrelin secretion, focusing on neural, chemical and hormonal stimuli. In addition, we share our view on the potential of targeting ghrelin for the treatment of eating disorders such as obesity, anorexia nervosa and cachexia.

Pro B-type natriuretic peptide plasma value: a new criterion for the prediction of short- and long-term outcomes after transcatheter aortic valve implantation.

BACKGROUND: To determine the prognostic value of pro B-type natriuretic peptide (pro-BNP) to predict mortality after transcatheter aortic valve implantation (TAVI). Logistic EuroSCORE (LES) overestimates observed mortality after TAVI. A new risk score specific to TAVI is needed to accurately assess mortality and outcome. METHODS: Eighty-five patients were included. Indications for TAVI were nonoperable or surgically high-risk patients (LES>20%). Pro-BNP was measured 24h before the procedure. Cox proportional hazards model was used to evaluate clinical factors. The predictive accuracy of these Cox models was determined by using time-dependent receiver operating characteristic (ROC) curves. RESULTS: Pro-BNP levels (log-transformed) were significantly higher in non-survivors than in survivors at 30 days (3.36 ± 0.43 vs. 3.81 ± 0.43, p<0.004) and at the end of follow-up (3.34 ± 0.42 vs. 3.63 ± 0.48, p<0.011). Multivariate analysis revealed that only increased log pro-BNP levels were associated with higher mortality rate at short [hazard ratio (HR) (95% confidence intervals (CI)]=5.35 (1.74-16.5), p=0.003] and long-term follow-ups [HR=11 (CI: 1.51-81.3), p=0.018]. LES was not associated with increased mortality at either time point [HR=1.03 (CI: 0.95-1.10), p=0.483 and HR=1.03 (CI: 0.98-1.07), p=0.230, respectively]. At 30, 90, 180, and 365 days, the c-index was 0.72 for log pro-BNP and 0.63 for LES (p=0.044). CONCLUSION: Pre-procedure log transform of plasma pro-BNP levels are an independent and strong predictor of short- and long-term outcomes after TAVI and are more discriminatory than LES.

Walking beyond the GRACE (Global Registry of Acute Coronary Events) model in the death risk stratification during hospitalization in patients with acute coronary syndrome: what do the AR-G (ACTION [Acute Coronary Treatment and Intervention Outcomes Network] Registry and GWTG [Get With the Guidelines] Database), NCDR (National Cardiovascular Data Registry), and EuroHeart Risk Scores Provide?

OBJECTIVES: This study sought to compare the in-hospital prognostic values of the original and updated GRACE (Global Registry of Acute Coronary Events) risk score (RS) and the AR-G (ACTION [Acute Coronary Treatment and Intervention Outcomes Network] Registry and the GWTG [Get With the Guidelines] Database) RS in acute coronary syndromes (ACS). To evaluate the utility of recalculating risk after percutaneous coronary intervention (PCI) with newer RS models (NCDR [National Cardiovascular Data Registry] and EHS [EuroHeart Score] RS). BACKGROUND: Defined in 2003, GRACE is among the most popular systems of risk stratification in ACS. An updated version of GRACE has since appeared and new RS have been developed, aiming to improve risk prediction. METHODS: From 2004 to 2010, 4,497 consecutive patients admitted to a single center in Spain with an ACS were included (32.1% ST-segment elevation myocardial infarction, 19.2% unstable angina). Discrimination (C-statistic) and calibration (Hosmer-Lemeshow [HL]) indexes were used to assess performance of each RS. A comparative analysis of RS designed to predict post-PCI mortality NCDR and EHS RS versus the GRACE and AR-G RS was performed in a subgroup of 1,113 consecutive patients included in the study. RESULTS: There were 265 in-hospital deaths (5.9%). Original and updated GRACE RS and the AR-G RS all demonstrated good discrimination for in-hospital death (C-statistics: 0.91, 0.90 and 0.90, respectively) with optimal calibration (HL p: 0.42, 0.50, and 0.47, respectively) in all spectra of ACS, according to different managements (PCI vs. conservative) and without significant differences between the 3 different RS. In patients undergoing PCI, EHS and NCDR RS (C-statistic = 0.80 and 0.84, respectively) were not superior to GRACE RS (C-statistic = 0.91), albeit in the subgroup of patients undergoing PCI who were categorized as high risk using the GRACE RS, both EHS and NCDR have contributed to decrease the false positive rate generated by using the GRACE RS. CONCLUSIONS: Despite having been developed over 8 years ago, the GRACE RS still maintains its excellent performance for predicting in-hospital risk of death among ACS patients.

Defining stem cell types: understanding the therapeutic potential of ESCs, ASCs, and iPS cells.

Embryonic, adult, artificially reprogrammed, and cancer…- there are various types of cells associated with stemness. Do they have something fundamental in common? Are we applying a common name to very different entities? In this review, we will revisit the characteristics that define 'pluripotency', the main property of stem cells (SCs). For each main type of physiological (embryonic and adult) or synthetic (induced pluripotent) SCs, markers and functional behavior in vitro and in vivo will be described. We will review the pioneering work that has led to obtaining human SC lines, together with the problems that have arisen, both in a biological context (DNA alterations, heterogeneity, tumors, and immunogenicity) and with regard to ethical concerns. Such problems have led to proposals for new operative procedures for growing human SCs of sufficiently high quality for use as models of disease and in human therapy. Finally, we will review the data from the first clinical trials to use various types of SCs.

Increased expression of fatty-acid and calcium metabolism genes in failing human heart.

BACKGROUND: Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca(2+))-handling in the human heart. METHODS: RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6). RESULTS: Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca(2+)-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca(2+)-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM. CONCLUSION: DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca(2+)-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca(2+)-handling genes.

Comparing the predictive validity of three contemporary bleeding risk scores in acute coronary syndrome.

AIMS: Haemorrhagic complications are strongly linked with adverse outcomes in acute coronary syndrome (ACS) patients. Various risk scores (RS) are available to predict bleeding risk in these patients. We compared the performance of three contemporary bleeding RS in ACS. METHODS: We studied 4500 consecutive patients with ACS. We calculated the ACTION, CRUSADE, and Mehran et al. (2010) bleeding RS, and evaluated their performance for predicting their own major bleeding events and TIMI serious (major or minor) bleeding episodes, in patients with either non-ST-elevation ACS (NSTEACS) or ST-elevation myocardial infarction (STEMI). Calibration (Hosmer-Lemeshow test, HL) and discrimination (c-statistic) for the three RS were computed and compared. RESULTS: For RS-specific major bleeding, ACTION and CRUSADE showed the best prognostic discrimination in STEMI (c=0.734 and 0.791, respectively; p=0.04), and in NSTEACS (c=0.791 and 0.810; p=0.4); being CRUSADE significantly superior to Mehran et al. in both ACS types (p<0.05). All RS performed well in patients undergoing coronary arteriography using either a radial or femoral approach (all c≥0.718); however, their discriminative capacity was modest in patients not undergoing coronary arteriography and in those previously on oral anticoagulant (all c<0.70). For TIMI serious bleeding, ACTION and CRUSADE displayed the highest c-index values in both STEMI (0.724 and 0.703, respectively; p=0.3) and NSTEACS (c=0.733 and 0.744, respectively; p=0.6); however, calibration of ACTION was poor in both ACS types (HL p<0.05). CONCLUSIONS: Of contemporary bleeding RS, the CRUSADE score was found to be the most accurate quantitative tool for NSTEACS and STEMI patients undergoing coronary arteriography.

Aliskiren affects fatty-acid uptake and lipid-related genes in rodent and human cardiomyocytes.

PURPOSE: We investigated whether the direct renin inhibitor aliskiren can affect metabolism in cardiomyocytes from rat, mouse and human sources. METHODS AND RESULTS: At 10-50 µmol/L, aliskiren significantly increased medium-chain-fatty-acid uptake in primary-cultured neonatal-rat and HL-1 adult-mouse-derived cardiomyocytes (BODIPY-induced fluorescence intensity). The fatty-acid transporter CD-36 was correspondingly translocated to, but the glucose transporter Glut-4 away from, the sarcoplasmic reticulum/plasma membrane, in primary-cultured neonatal-rat (CD-36, Glut-4) and adult-human (CD-36) cardiomyocytes (confocal immunocytochemistry). Immunoblotting showed that aliskiren induced phosphorylation of ERK1/2 in cardiomyocytes from all three sources; responses were dose- and time-dependent, unaffected by renin treatment, and did not cause alterations in expression of (P)R or Igf2/M6P receptors. Microarray analysis of the complete genome of aliskiren-treated neonatal-rat cardiomyocytes, with RT-qPCR and immunoblot confirmation assays in rat and human primary cardiomyocytes, showed that aliskiren up-regulated mRNA and increased protein expression of several enzymes important in lipid and glucose metabolism and in cholesterol biosynthesis. Cardiomyocyte cell-cycle and viability were unaffected by aliskiren. CONCLUSIONS: Aliskiren can induce changes in fatty-acid and glucose uptake and expression of key enzymes of lipid and cholesterol metabolism, which are not associated with increased expression of (P)R or Igf2/M6P receptors, in cultured cardiomyocytes.

Expanding the adipokine network in cartilage: identification and regulation of novel factors in human and murine chondrocytes.

BACKGROUND: Obesity is a major risk factor for a plethora of diseases including joint disorders associated with cartilage destruction. Recently, it has been demonstrated that adipose tissue might contribute to degenerative joint diseases via the secretion of potent bioactive molecules termed adipokines. OBJECTIVE: To study expression of the novel adipokines chemerin, lipocalin 2 (LCN2) and serum amyloid A3 (SAA3) in murine and human chondrocytes, under basal conditions, in response to a range of biological and pharmacological treatments, and during chondrocyte differentiation. METHODS: Chemerin, LCN2 and SAA3 mRNA and protein expression were evaluated by quantitative real-time reverse transcription PCR and western blot analysis, respectively, in the ATDC-5 murine chondrocyte cell line, a human immortalised chondrocyte cell line (T/C-28a2) and primary cultured human chondrocytes. RESULTS: Human and murine chondrocytes expressed chemerin, LCN2 and SAA3 mRNA; interleukin (IL)-1ß was a potent inducer of these novel adipokines. Moreover, dexamethasone, lipopolysaccharides (LPS) and other relevant adipokines such as leptin and adiponectin were able to modulate chemerin, LCN2 and SAA3 mRNA expression alone and when coadministered. Intracellular signal transducers involved in the IL-1ß-mediated upregulation of LCN2 and SAA3 included Janus kinase (JAK) 2, phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein (MAP) kinases. Finally, expression of chemerin, LCN2 and SAA3 mRNA expression were modulated throughout chondrocyte differentiation. CONCLUSION: Chemerin, LCN2 and SAA3 are implicated in chondrocyte pathophysiology, and regulated by other relevant factors that drive inflammatory process such as IL-1ß, LPS and adipokines including leptin and adiponectin. It seems likely that JAK2, PI3K and MAP kinases are involved in mediating these responses.

Prognostic impact of atrial fibrillation progression in a community study: AFBAR Study (Atrial Fibrillation in the Barbanza Area Study).

INTRODUCTION: The aim of the study is to describe the natural history of an unselected population of patients with atrial fibrillation (AF) currently attending primary care services in a single health-service area in Galicia, north-western Spain. METHODS: AFBAR is a transverse prospective study in which 35 general practitioners within one health-service area have enrolled patients diagnosed with AF who presented at their clinics during a three-month recruiting period. Primary endpoints are mortality or hospital admission. Here we report the results of the first 7-month follow-up period. RESULTS: 798 patients (421 male) were recruited; mean age of cohort was 75 years old. Hypertension was the most prevalent risk factor (77%). 87% of the patients were both overweight and obese. Permanent AF was diagnosed in 549 patients (69%). In the follow-up period, 16.4% of the patients underwent a primary endpoint and the overall survival was 98%. The following independent determinants of primary endpoint were identified: change in AF status (Hazard Ratio (HR) 2.89 (95% confidence interval (CI) 1.28-6.55); p=0.011); ischemic heart disease (IHD) (HR 2.78 (95% CI 1.51-5.13); p=0.001); pre-recruitment hospital admission (HR 2.22 (95% CI 1.18-4.19); p=0.013); left ventricular systolic dysfunction (HR 2.19 (95% CI 1.11-4.32); p=0.023); or AF-related complications (HR 1.98 (95% CI 1.10-3.56); p=0.022). CONCLUSIONS: In the first 7-month follow-up period of patients with AF in a primary care setting the study identified several independent risk factors for mortality or hospital admission, i.e. change in AF status, ischemic heart disease, left ventricular systolic dysfunction, previous AF-related complications and hospital admission.

Des-acyl ghrelin has specific binding sites and different metabolic effects from ghrelin in cardiomyocytes.

The current study aimed to compare the effects of the peptide hormone ghrelin and des-G, its unacylated isoform, on glucose and fatty acid uptake and to identify des-G-specific binding sites in cardiomyocytes. In the murine HL-1 adult cardiomyocyte line, ghrelin and des-G had opposing metabolic effects: des-G increased medium-chain fatty acid uptake (BODIPY fluorescence intensity), whereas neither ghrelin alone nor in combination with des-G did so. Ghrelin inhibited the increase in glucose uptake normally induced by insulin (rate of 2-[(3)H]deoxy-d-glucose incorporation), but des-G did not; des-G was also able to partially reverse the inhibitory effect of ghrelin. In HL-1 cells and primary cultures of neonatal rat cardiomyocytes, des-G but not ghrelin increased insulin-induced translocation of glucose transporter-4 from nuclear to cytoplasmic compartments (immunohistochemistry and quantitative confocal analysis). AKT was phosphorylated by insulin but not affected by ghrelin or des-G, whereas neither AMP-activated protein kinase nor phosphatase and tensin homolog deleted from chromosome 10 was phosphorylated by any treatments. HL-1 and primary-cultured mouse and rat cardiomyocytes each possessed two independent specific binding sites for des-G not recognized by ghrelin (radioreceptor assays). Neither ghrelin nor des-G affected viability (dimethylthiazol diphenyltetrazolium bromide assays), whereas both isoforms were equally protective against apoptosis. Therefore, in cardiomyocytes, des-G binds to specific receptors and has effects on glucose and medium-chain fatty acid uptake that are distinct from those of ghrelin. Real-time PCR indicated that expression levels of ghrelin O-acyltransferase RNA were comparable between HL-1 cells, human myocardial tissue, and human and murine stomach tissue, indicating the possibility of des-G conversion to ghrelin within our model.

Foregut mesenchyme contributes cells to pancreatic acini during embryonic development in a chick-quail chimera model.

To understand causes of developmental abnormalities of the pancreas, it is essential to understand its normal embryonic development. Current understanding of the development of pancreatic exocrine tissue is that it develops solely from embryonic epithelium, while the role of the surrounding mesenchyme is to signal to this epithelium and form connective tissue. Recent work in our laboratory has shown that pancreatic bud mesenchyme can contribute cells to islets during embryonic development. However, no published studies have investigated in detail whether mesenchyme contributes cells to the exocrine structures of the pancreas. The aim of this study was to investigate whether cells from foregut mesenchyme can contribute to pancreatic acini during embryonic development. Chick-quail chimera recombinant organs were constructed using pancreatic epithelium and mesenchyme from either the pancreas (n=12) or stomach (n=25). These were cultured for 7 days in 3-D collagen gels. The resulting specimens were analysed using morphological criteria and fluorescent immunocytochemistry against pancreatic amylase, insulin, and the quail-specific nucleolar antigen QCPN. Two independent observers determined the origins of acini as either solely epithelial, solely mesenchymal, or of mixed origin. Results are expressed as percentages of total acini identified in each group. Statistical analysis was performed using chi(2) tests (P<0.01 was considered statistically significant). Recombinations of pancreatic epithelium and pancreatic mesenchyme yielded 11 acini, of which 45% were derived from epithelium only, 45% from mesenchyme only, and 10% of mixed origin. Recombinations of pancreatic epithelium and stomach mesenchyme yielded 78 acini, of which 40% were derived from epithelium only, 32% from mesenchyme only, and 28% of mixed origin. When acini with any mesenchymal cellular contribution were considered as a group, there was no significant difference between stomach and pancreatic mesenchymal contribution (P=0.72). This is the first study to demonstrate the cellular contribution of mesenchyme to pancreatic exocrine structures. Our data show that mesenchyme contributes cells to pancreatic acini during development in this model and that mesenchyme derived from stomach and pancreatic sources are both able to form acini.

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model.

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem-or progenitor-cells for islet transplantation.