0341: AMPK exerts an insulin-sensitizing effect on cardiac glucose uptake by multiple molecular mechanisms including cytoskeleton reorganization

BackgroundInsulin-resistant cardiomyocytes are characterized by a decreased ability of insulin to stimulate glucose uptake. We have previously shown that the activation of AMPK by metformin or phenformin restores insulin-sensitivity in insulin-resistant cardiomyocytes. The aim of our present work is to understand by which molecular mechanisms AMPK exerts its insulin sensitizing effect. In this study we focused on the mTOR/p70S6K pathway and on cytoskeleton reorganization. mTOR/p70S6K, which is known to be inhibited by AMPK, is able to reduce insulin signaling via a negative feedback loop involving serine phosphorylation of IRS-1. On the other hand, cytoskeleton reorganization, which is a known target of AMPK, is responsible for the translocation of the glucose transporter GLUT4 to the plasma membrane.MethodsAdult rat cardiomyocytes were primary cultured and treated with different agents including insulin, AMPK activator (phenformin), mTOR inhibitor rapamycin and/or actin cytoskeleton inhibitor latrunculin B. Glucose uptake was assessed by detritiation of 2-3H-glucose.ResultsFirst, we tested if rapamycin was able to mimic AMPK activators. Similarly to phenformin, rapamycin increased the insulin-dependent phosphorylation of Akt involved in the regulation of glucose uptake. Despite the ability of rapamycin to induce this Akt over-phosphorylation, rapamycin was not able to restore the insulin-dependent stimulation of glucose uptake like phenformin did. On the other hand, latrunculin B abolished the insulin-sensitizing action of phenformin on glucose uptake, in insulin-sensitive as well as in insulinresistant cells.Conclusionsactin cytoskeleton reorganization but not mTOR/p70S6K, is involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake. The role played by Small G proteins, known to be involved in the regulation of actin cytoskeleton is under investigation

Longitudinal changes in acylated versus unacylated ghrelin levels may be involved in the underlying mechanisms of the switch in nutritional phases in Prader-Willi syndrome

Introduction: Prader-Willi syndrome (PWS) is characterized by a switch from failure to thrive to excessive weight gain and hyperphagia in early childhood. An elevated, more unfavorable ratio between acylated and unacylated ghrelin (AG/UAG ratio) might play a role in the underlying mechanisms of this switch. We aimed to assess the evolution of the appetite-regulating hormones acylated ghrelin (AG) and unacylated ghrelin (UAG) and the AG/UAG ratio and their association with the change in eating behavior in children with PWS, compared to healthy age-matched controls. Methods: A longitudinal study was conducted in 134 children with PWS and 157 healthy controls, from the Netherlands, France, and Belgium. Levels of AG and UAG and the AG/UAG ratio were measured and nutritional phases as reported for PWS were scored. Results: The AG/UAG ratio was lower in the first years of life in PWS than in controls and started to increase from the age of 3 years, resulting in a high-normal AG/UAG ratio compared to controls. The AG levels remained stable during the different nutritional phases (p = 0.114), while the UAG levels decreased from 290 pg/mL in phase 1a to 137 pg/mL in phase 2b (p < 0.001). The AG/UAG ratio increased significantly from 0.81 in phase 2a to 1.24 in phase 2b (p = 0.012). Conclusions: The change from failure to thrive to excessive weight gain and hyperphagia in infants and children with PWS coincides with an increase in AG/UAG ratio. The increase in AG/UAG ratio occurred during phase 2a, thus before the onset of hyperphagia

Clinical Study Characteristics and Determinants of Partial Remission in Children with Type 1 Diabetes Using the Insulin-Dose-Adjusted A1C Definition

To evaluate the characteristics and determinants of partial remission (PR) in Belgian children with type 1 diabetes (T1D), we analyzed records of 242 children from our center. Clinical and biological features were collected at diagnosis and during follow-up. PR was defined using the insulin-dose-adjusted A1C definition. PR occurred in 56.2% of patients and lasted 9.2 months (0.5 to 56.6). 25.6% of patients entered T1D with DKA, which correlated with lower PR incidence (17.6% versus 82.3% when no DKA). In our population, lower A1C levels at diagnosis were associated with higher PR incidence and in young children (0-4 years) initial A1C levels negatively correlated with longer PR. Early A1C levels were predictive of PR duration since 34% of patients had long PRs (>1 year) when A1C levels were ≤6% after 3 months whereas incidence of long PR decreased with higher A1Cs. C-peptide levels were higher in patients entering PR and remained higher until 3 years after diagnosis. Initial antibody titers did not influence PR except for anti-IA2 titers that correlated with A1C levels after 2 years. Presence of 2 versus 1 anti-islet antibodies correlated with shorter PR. PR duration did not influence occurrence of severe hypoglycemia or diabetes-related complications but was associated with lower A1C levels after 18 months. We show that, at diagnosis of T1D, parameters associated with -cell mass reserve (A1C, C-peptide, and DKA) correlate with the occurrence of PR, which affects post-PR A1C levels. Further research is needed to determine the long-term significance of PR

AMP-activated protein kinase controls liposaccharide-induced hyperpermeability

Organ dysfunction determines the severity of sepsis and is correlated to mortality. Endothelial increased permeability contributes to the development of organ failure. AMP-activated protein kinase (AMPK) has been shown to modulate cytoskeleton and could mediate endothelial permeability. Our hypothesis is that AMPK controls sepsis-induced hyperpermeability in the heart and is involved in septic cardiomyopathy. Sepsis was induced by intraperitoneal injection of liposaccharide, 10 mg/kg (LPS). Alpha-1 AMPK knockout mice (α1KO) were compared with wild-type. Vascular permeability was characterized by Evans blue extravasation. Inflammatory cytokine mRNA expression was determined by qPCR analysis. Left ventricular mass was assessed by echocardiography. In addition, to emphasize the beneficial role of AMPK on heart vascular permeability, AMPK activator (acadesine) was administered to C57Bl6 mice before LPS injection. The ANOVA test with Bonferroni's post hoc test and the log-rank test were used. P < 0.05 was considered as significant. Increased cardiac vascular permeability was observed in the LPS group in comparison to untreated animals (2.5% vs. 16%; P < 0.05). The α1KO mice exhibited an increase vascular permeability after LPS injection in comparison to wild-type mice (41.5% vs. 16%; P < 0.05). α1KO animals had a significant mortality increase after LPS injection (70% vs. 10%; P < 0.05). LPS markedly induced the production of proinflammatory cytokines (TNFα, IL-1β, IL-6) that were significantly higher in the α1KO animals. More importantly, LPS treatment leads to an increased left ventricular mass in the α1KO mice within 24 hours, suggesting the onset of edema. Finally LPS-induced vascular hyperpermeability was greatly reduced after AMPK activation by acadesine (13.2% vs. 40%; P < 0.05). AMPK importantly regulates cardiac vascular permeability and could control the sepsis-induced cardiomyopathy. AMPK could represent a new pharmacological target of sepsis

Role of AMP-activated protein kinase in regulating hypoxic survival and proliferation of mesenchymal stem cells

Aims Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. Methods and results MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. Conclusion MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulatio

3D histopathology of stenotic aortic valve cusps using ex vivo microfocus computed tomography

BackgroundCalcific aortic stenosis (AS) is the most prevalent heart valve disease in developed countries. The aortic valve cusps progressively thicken and the valve does not open fully due to the presence of calcifications. In vivo imaging, usually used for diagnosis, does not allow the visualization of the microstructural changes associated with AS.MethodsEx vivo high-resolution microfocus computed tomography (microCT) was used to quantitatively describe the microstructure of calcified aortic valve cusps in full 3D. As case study in our work, this quantitative analysis was applied to normal-flow low-gradient severe AS (NF-LG-SAS), for which the medical prognostic is still highly debated in the current literature, and high-gradient severe AS (HG-SAS).ResultsThe volume proportion of calcification, the size and number of calcified particles and their density composition was quantified. A new size-based classification considering small-sized particles that are not detected with in vivo imaging was defined for macro-, meso- and microscale calcifications. Volume and thickness of aortic valve cusps, including the complete thickness distribution, were also determined. Moreover, changes in the cusp soft tissues were also visualized with microCT and confirmed by scanning electron microscopy images of the same sample. NF-LG-SAS cusps contained lower relative amount of calcifications than HG-SAS. Moreover, the number and size of calcified objects and the volume and thickness of the cusps were also lower in NF-LG-SAS cusps than in HG-SAS.ConclusionsThe application of high-resolution ex vivo microCT to stenotic aortic valve cusps provided a quantitative description of the general structure of the cusps and of the calcifications present in the cusp soft tissues. This detailed description could help in the future to better understand the mechanisms of AS

Congenital Hypothyroidism: A 2020-2021 Consensus Guidelines Update-An ENDO-European Reference Network Initiative Endorsed by the European Society for Pediatric Endocrinology and the European Society for Endocrinology

Background An ENDO-ERN initiative was launched which was endorsed by the European Society for Pediatric Endocrinology and the European Society for Endocrinology with 22 participants from the ENDO-ERN and the two societies. The aim was to update the practice guidelines for the diagnosis and management of congenital hypothyroidism (CH). A systematic literature search was conducted to identify key articles on neonatal screening, diagnosis and management of primary and central congenital hypothyroidism. The evidence-based guidelines were graded with the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system, describing both the strength of recommendations and the quality of evidence. In the absence of sufficient evidence, conclusions were based on expert opinion. Summary The recommendations include the various neonatal screening approaches for CH as well as the etiology (also genetics), diagnostics, treatment and prognosis of both primary and central CH. When CH is diagnosed, the expert panel recommends the immediate start of correctly dosed levothyroxine treatment and frequent follow-up including laboratory testing to keep thyroid hormone levels in their target ranges, timely assessment of the need to continue treatment, attention for neurodevelopment and neurosensory functions and, if necessary, consulting other health professionals, and education of the child and family about CH. Harmonisation of diagnostics, treatment and follow-up will optimise patient outcomes. Lastly, all individuals with CH are entitled to a well-planned transition of care from pediatrics to adult medicine. Conclusions This consensus guidelines update should be used to further optimize detection, diagnosis, treatment and follow-up of children with all forms of CH in the light of the most recent evidence. It should be helpful in convincing health authorities of the benefits of neonatal screening for CH. Further epidemiological and experimental studies are needed to understand the increased incidence of this conditio