Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

BACKGROUND: The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis RESULTS: Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA), relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 × PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0× PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 × PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 × PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucine CONCLUSION: In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway

Effect of early tranexamic acid administration on mortality, hysterectomy, and other morbidities in women with post-partum haemorrhage (WOMAN): an international, randomised, double-blind, placebo-controlled trial

Background Post-partum haemorrhage is the leading cause of maternal death worldwide. Early administration of tranexamic acid reduces deaths due to bleeding in trauma patients. We aimed to assess the effects of early administration of tranexamic acid on death, hysterectomy, and other relevant outcomes in women with post-partum haemorrhage. Methods In this randomised, double-blind, placebo-controlled trial, we recruited women aged 16 years and older with a clinical diagnosis of post-partum haemorrhage after a vaginal birth or caesarean section from 193 hospitals in 21 countries. We randomly assigned women to receive either 1 g intravenous tranexamic acid or matching placebo in addition to usual care. If bleeding continued after 30 min, or stopped and restarted within 24 h of the first dose, a second dose of 1 g of tranexamic acid or placebo could be given. Patients were assigned by selection of a numbered treatment pack from a box containing eight numbered packs that were identical apart from the pack number. Participants, care givers, and those assessing outcomes were masked to allocation. We originally planned to enrol 15 000 women with a composite primary endpoint of death from all-causes or hysterectomy within 42 days of giving birth. However, during the trial it became apparent that the decision to conduct a hysterectomy was often made at the same time as randomisation. Although tranexamic acid could influence the risk of death in these cases, it could not affect the risk of hysterectomy. We therefore increased the sample size from 15 000 to 20 000 women in order to estimate the effect of tranexamic acid on the risk of death from post-partum haemorrhage. All analyses were done on an intention-to-treat basis. This trial is registered with ISRCTN76912190 (Dec 8, 2008); ClinicalTrials.gov, number NCT00872469; and PACTR201007000192283. Findings Between March, 2010, and April, 2016, 20 060 women were enrolled and randomly assigned to receive tranexamic acid (n=10 051) or placebo (n=10 009), of whom 10 036 and 9985, respectively, were included in the analysis. Death due to bleeding was significantly reduced in women given tranexamic acid (155 [1·5%] of 10 036 patients vs 191 [1·9%] of 9985 in the placebo group, risk ratio [RR] 0·81, 95% CI 0·65–1·00; p=0·045), especially in women given treatment within 3 h of giving birth (89 [1·2%] in the tranexamic acid group vs 127 [1·7%] in the placebo group, RR 0·69, 95% CI 0·52–0·91; p=0·008). All other causes of death did not differ significantly by group. Hysterectomy was not reduced with tranexamic acid (358 [3·6%] patients in the tranexamic acid group vs 351 [3·5%] in the placebo group, RR 1·02, 95% CI 0·88–1·07; p=0·84). The composite primary endpoint of death from all causes or hysterectomy was not reduced with tranexamic acid (534 [5·3%] deaths or hysterectomies in the tranexamic acid group vs 546 [5·5%] in the placebo group, RR 0·97, 95% CI 0·87-1·09; p=0·65). Adverse events (including thromboembolic events) did not differ significantly in the tranexamic acid versus placebo group. Interpretation Tranexamic acid reduces death due to bleeding in women with post-partum haemorrhage with no adverse effects. When used as a treatment for postpartum haemorrhage, tranexamic acid should be given as soon as possible after bleeding onset. Funding London School of Hygiene & Tropical Medicine, Pfizer, UK Department of Health, Wellcome Trust, and Bill & Melinda Gates Foundation

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Nutrient and hormonal control of ubiquitin proteasome dependent proteolysis in skeletal muscle

The ubiquitin proteasome pathway is the predominant biological mechanism of myofibrillar protein (MF) degradation.  To test the hypothesis that amino acid and insulin act synergistically to regulate proteolysis, two experimental models were employed;  an in vivo study on growing calves and an in vitro C2C12 myotubes culture. Calves growing at 0.3kg/day, were constantly infused with glucose at a low (LDG) or high (HDG) dose (to stimulate insulin) with or without essential amino acids (EAA).  Glucose infusions increased plasma insulin and IGF-1 concentrations in a dose dependent manner (Pin vivo. In the in vitro experiments, amino acid deprivation (0.2 X physiological concentration amino acid;  PC AA) of myotubes for 8 h was associated with increased (P 3H-tyrosine release in the medium), compared to controls (1.0 X PC AA).  Addition of insulin inhibited this increase (P < 0.05).  Rapamycin significantly increased proteolysis in 1.0 X PC AA media suggesting amino acid might regulate proteolysis through mTOR signalling pathway.  Reduced amino acid supply also increased 14-kDa E2 and C2 mRNA expression compared to controls (P < 0.05).  Increasing leucine concentration in 0.2 X PC AA basal media showed a dose dependent decrease in protein degradation and expression of 14-kDa E2, in the presence of insulin.  In conclusion, the results suggested that decreased availability of amino acids was associated with increased total proteolysis and that anti-catabolic effect of amino acid in C2C12 muscle cell cultures, was additive to that of insulin.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterization of Mannose Binding Lectin (MBL) Levels in Type-2 Diabetes Mellitus Patients Amongst Pakistani Population

Introduction: Mannose Binding Lectin (MBL) is a pattern recognizing molecule in the Lectin complement pathway and acts by activating the complement cascade via binding with ligands. There is evidence of increased autoreactivity of Mannose Binding Lectin in various diseases especially diabetes. MBL deficiency can reduce pathogen clearance and impair atherogenic lipoprotein removal whereas higher levels are associated with exaggerated immune response due to complement activation in the presence of hyperglycemia. Aims & Objectives: This study aims to find out the association of MBL (Mannose Binding Lectin) with different clinical parameters in healthy controls and type 2 DM patients to predict disease outcome in type 2 diabetics. Mean level of MBL in type 2 diabetics and healthy population will be compared to characterize MBL levels amongst diabetics helping the clinicians to stratify patients according to disease severity. Place and duration of study: It was a cross sectional analytical study conducted at Chughtai Institute of Pathology from July 2019 to January 2020 on samples collected nationwide at Chughtai Lab collection centres. Material & Methods: We selected 300 adult male and females in this study after taking informed consent based on strict inclusion and exclusion criteria. Of these 200 were known cases of type 2 diabetes mellitus and 100 healthy controls. .Fasting blood samples were drawn from both groups were analyzed for hs-CRP, HbA1c, creatinine, Total Cholesterol, Alanine amino transfer as (ALT), HDL-Cholesterol, LDL-Cholesterol, Glucose, Triglyceride, and Mannose Binding lectin. eGFR (Estimated Glomerular Filtration rate) was calculated for each patient and control. Data was analyzed via Graph Pad Prism 5 and SPSS version 23.0, p value ? 0.05 was taken as significant. Results: Mean age of the participants was 47.9 years in diabetic group and 39.3 years in healthy controls. The healthy population had a mean MBL level of 110.1 (SD±3.94) pg/ml and mean MBL level of diabetic group was 197.9 (SD±12.84) pg/ml (p<0.05). No differences in MBL levels were detected based on gender distribution. There was a significant difference among HbA1c, LDL-C, HDL-C, Fasting Glucose, TG, creatinine and eGFR amongst the diabetic and the healthy group (p<0.05). There was a negative correlation between MBL levels and plasma glucose and a positive correlation between the former and HDL-C in the healthy controls. In diabetic patients having MBL above 178pg/ml, a positive correlation of HbA1C with MBL was found. CRP in the healthy population resembled levels in patients with elevated MBL and the ratio Trig/HDL was higher in this subgroup having a positive correlation with MBL. Conclusion: MBL plays a role in pathophysiology of diabetes mellitus and elevated MBL having positive correlation with HbA1c might show association of glycemic control with the biomarker levels. A direct relationship of MBL with development of cardiovascular complications in type 2 diabetics was suggested by a positive association of MBL with TG to HDL-C ratio