Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells

Skeletal muscle accounts for ~80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1−/− knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell

Established Tumour Biomarkers Predict Cardiovascular Events and Mortality in the General Population

Introduction: Several lines of evidence reveal that cardiovascular disease (CVD) and cancer share similar common pathological milieus. The prevalence of the two diseases is growing as the population ages and the burden of shared risk factors increases. In this respect, we hypothesise that tumour biomarkers can be potential predictors of CVD outcomes in the general population. Methods: We measured six tumour biomarkers (AFP, CA125, CA15-3, CA19-9, CEA and CYFRA 21-1) and determined their predictive value for CVD in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study. A total of 8,592 subjects were enrolled in the study. Results: The levels of CEA significantly predicted CV morbidity and mortality, with hazard ratios (HRs) of HR 1.28 (95% CI 1.08–1.53), respectively. Two biomarkers (CA15-3 and CEA) showed statistical significance in predicting all-cause mortality, with HRs 1.58 (95% CI 1.18–2.12) and HR 1.60 (95% CI 1.30–1.96), when adjusted for shared risk factors and prevalent CVD. Furthermore, biomarkers seem to be sex specific. CYFRA 21-1 presented as an independent predictor of CV morbidity and mortality in female, but not in male gender, with HR 1.82 (95% CI 1.40–2.35). When it comes to all-cause mortality, both CYFRA and CEA show statistical significance in male gender, with HR 1.64 (95% CI 1.28–3.12) and HR 1.55 (95% CI 1.18–2.02), while only CEA showed statistical significance in female gender, with HR 1.64 (95% CI 1.20–2.24). Lastly, CA15-3 and CEA strongly predicted CV mortality with HR 3.01 (95% CI 1.70–5.32) and HR 1.82 (95% CI 1.30–2.56). On another hand, CA 15-3 also presented as an independent predictor of heart failure (HF) with HR 1.67 (95% CI 1.15–2.42). Conclusion: Several tumour biomarkers demonstrated independent prognostic value for CV events and all-cause mortality in a large cohort from the general population. These findings support the notion that CVD and cancer are associated with similar pathological milieus

Diastolic function measurements and diagnostic consequences: a comparison of pulsed wave- and color-coded tissue Doppler imaging

Tissue Doppler imaging (TDI) plays an important role in assessing diastolic function using echocardiography. However, two different methods [pulsed wave (PW-TDI) and color-coded (CC-TDI)] are currently used. We aimed to compare both measurements. We included 114 patients that were referred to our echocardiography department for evaluation of diastolic left ventricular function. In these patients, we sequentially measured tissue velocities of basal lateral and septal myocardium of the left ventricle in an apical four-chamber view with both PW-TDI and CC-TDI. Our cohort consisted of a heterogeneous group of patients with and without a history of cardiac disease. Mean age of the patients was 52 +/- A 16.7 years, and 62% were males. We found a strong correlation between PW-TDI- and CC-TDI-derived myocardial velocities (r = 0.93; p = 0.001). However, E' (mean of lateral and septal) velocities measured with PW-TDI were consistently higher compared to CC-TDI values [PW-TDI E' 10.3 +/- A 3.9 (SD) cm/s vs. CC-TDI E' 7.7 +/- A 3.1 cm/s; p <0.001]. From these data, we calculated that the relation between E' measured with PW-TDI and CC-TDI can be described as: E' (PW-TDI) = 1.25 + 1.17 x E' (CC-TDI). Consequently, E/E' measured with PW-TDI was consistently lower compared with CC-TDI (9.1 +/- A 3.1 vs. 12.5 +/- A 5.7; p <0.001) From these data, we calculated that the relation between E/E' measured with PW-TDI and CC-TDI can be described as: E/E' (PW-TDI) = 2.13 + 0.56 x E/E' (CC-TDI). Despite a strong correlation, tissue velocities measured with PW-TDI will yield higher values as compared with CC-TDI. This should be taken into account when defining cut-off values for the evaluation of diastolic function

Insulin-like growth factor binding protein 7 (IGFBP7), a link between heart failure and senescence

Aims: Insulin like growth factor binding protein 7 (IGFBP7) is a marker of senescence secretome and a novel biomarker in patients with heart failure (HF). We evaluated the prognostic value of IGFBP7 in patients with heart failure and examined associations to uncover potential new pathophysiological pathways related to increased plasma IGFBP7 concentrations. Methods and results: We have measured plasma IGFBP7 concentrations in 2250 subjects with new‐onset or worsening heart failure (BIOSTAT‐CHF cohort). Higher IGFBP7 plasma concentrations were found in older subjects, those with worse kidney function, history of atrial fibrillation, and diabetes mellitus type 2, and in subjects with higher number of HF hospitalizations. Higher IGFBP7 levels also correlate with the levels of several circulating biomarkers, including higher NT‐proBNP, hsTnT, and urea levels. Cox regression analyses showed that higher plasma IGFBP7 concentrations were strongly associated with increased risk of all three main endpoints (hospitalization, all‐cause mortality, and combined hospitalization and mortality) (HR 1.75, 95% CI 1.25–2.46; HR 1.71, 95% CI 1.39–2.11; and HR 1.44, 95% CI 1.23–1.70, respectively). IGFBP7 remained a significant predictor of these endpoints in patients with both reduced and preserved ejection fraction. Likelihood ratio test showed significant improvement of all three risk prediction models, after adding IGFBP7 (P &lt; 0.001). A biomarker network analysis showed that IGFBP7 levels activate different pathways involved in the regulation of the immune system. Results were externally validated in BIOSTAT‐CHF validation cohort. Conclusions: IGFPB7 presents as an independent and robust prognostic biomarker in patients with HF, with both reduced and preserved ejection fraction. We validate the previously published data showing IGFBP7 has correlations with a number of echocardiographic markers. Lastly, IGFBP7 pathways are involved in different stages of immune system regulation, linking heart failure to senescence pathways

Cancer risk in DES daughters

We examined long-term risk of cancer in women exposed to diethylstilbestrol (DES) in utero. A total of 12,091 DES-exposed women in the Netherlands were followed prospectively from December 1992 till June 2008. Cancer incidence was assessed through linkage with the Dutch pathology database (PALGA) and the Netherlands Cancer Registry and compared with the Dutch female population. A total of 348 medically verified cancers occurred; median age at end of follow-up was 44.0 years. No overall increased risk of cancer was found (standardized incidence ratio [SIR] = 1.01; 95% confidence interval [CI] = 0.91, 1.13). The risk of clear cell adenocarcinoma of the vagina and cervix (CCA) was statistically significantly increased (SIR = 24.23; 95% CI = 8.89, 52.74); the elevated risk persisted above 40 years of age. The risk of melanoma diagnosed before age 40 was increased (SIR = 1.59; 95% CI = 1.08, 2.26). No excess risks were found for other sites, including breast cancer. Except for an elevated risk of CCA, persisting at older ages, and an increased risk of melanoma at young ages, we found no increased risk of cancer. Longer follow-up is warranted to examine cancer risk at ages when cancer occurs more frequently

Feasibility and reliability of PRISMA-Medical for specialty-based incident analysis

Aims and objectives: In this study, the feasibility and reliability of the Prevention Recovery Information System for Monitoring and Analysis (PRISMA)-Medical method for systematic, specialty-based analysis and classification of incidents in the neonatal intensive care unit (NICU) were determined. Methods: After the introduction of a Neonatology System for Analysis and Feedback on Medical Events (NEOSAFE) in eight tertiary care NICUs and one paediatric surgical ICU, PRISMA-Medical was started to be used to identify root causes of voluntary reported incidents by multidisciplinary unit patient safety committees. Committee members were PRISMA-trained and familiar with the department and its processes. In this study, the results of PRISMA-analysis of incidents reported during the first year are described. At t¿=¿3 months and t¿=¿12 months after introduction, test cases were performed to measure agreement at three levels of root cause classification using PRISMA-Medical. Inter-rater reliability was determined by calculating generalised ¿ values for each level of classification. Results: During the study period, 981 out of 1786 eligible incidents (55%) were analysed for underlying root causes. In total, 2313 root causes were identified and classified, giving an average of 2.4 root causes for every incident. Although substantial agreement (¿ 0.70–0.81) was reached at the main level of root cause classification of the test cases (discrimination between technical, organisational and human failure) and agreement among the committees at the second level (discrimination between skill-based, rule-based and knowledge-based errors) was acceptable (¿ 0.53–0.59), discrimination between rule-based errors (the third level of classification) was more difficult to assess (¿ 0.40–0.47). Conclusion: With some restraints, PRISMA-Medical proves to be both feasible and acceptably reliable to identify and classify multiple causes of medical events in the NICU

Specialty-based, voluntary incident reporting in neonatal intensive care: description of 4846 incident reports

OBJECTIVES: To examine the characteristics of incidents reported after introduction of a voluntary, non-punitive incident reporting system for neonatal intensive care units (NICUs) in the Netherlands; and to investigate which types of reported incident pose the highest risk to patients in the NICU. DESIGN: Prospective multicentre survey. METHODS: Voluntary, non-punitive incident reporting was introduced in eight level III NICUs and one paediatric surgical ICU. An incident was defined as any unintended event which (could have) reduced the safety margin for the patient. Multidisciplinary, unit-based patient safety committees systematically collected and analysed incident reports, and assigned risk scores to each reported incident. Data were centrally collected for specialty-based analysis. This paper describes the characteristics of incidents reported during the first year. Bivariate logistic regression analysis was conducted to identify high-risk incident categories. RESULTS: There were 5225 incident reports on 3859 admissions, of which 4846 were eligible for analysis. Incidents with medication were most frequently reported (27%), followed by laboratory (10%) and enteral nutrition (8%). Severe harm was described in seven incident reports, and moderate harm in 63 incident reports. Incidents involving mechanical ventilation and blood products were most likely to be assigned high-risk scores, followed by those involving parenteral nutrition, intravascular lines and medication dosing errors. CONCLUSIONS: Incidents occur much more frequently in Dutch NICUs than has been previously observed, and their impact on patient morbidity is considerable. Reported incidents concerning mechanical ventilation, blood products, intravascular lines, parenteral nutrition and medication dosing errors pose the highest risk to patients in the NIC

Shikonin Increases Glucose Uptake in Skeletal Muscle Cells and Improves Plasma Glucose Levels in Diabetic Goto-Kakizaki Rats

Glucose is the most common substrate for energy metabolism. Despite the varying demands for glucose, the body needs to regulate its internal environment and maintain a constant and stable condition. Glucose homeostasis requires harmonized interaction between several tissues, achieving equilibrium between glucose output and uptake. In this thesis we aimed to investigate factors modulating glucose homeostasis in a rat model of type 2 diabetes, the Goto-Kakizaki (GK) rat. In addition, we investigated sex differences in hepatic carbohydrate and lipid metabolism in healthy rats. In Paper I, three-week but not three-day treatment with a Southeast Asian herb, Gynostemma pentaphyllum (GP), significantly reduced plasma glucose (PG) levels in GK rats. An intra-peritoneal glucose tolerance test (IPGTT) was significantly improved in GP-treated compared to placebo-treated group. In the GP treated rats, the glucose response in an intra-peritoneal pyruvate tolerance test was significantly lower, indicating decreased gluconeogenesis, and hepatic glucose output (HGO) was reduced. GP-treatment significantly reduced hepatic glycogen content, but not glycogen synthase activity. The study provides evidence that the GP extract exerted anti-diabetic effect in GK rats, reducing PG levels and HGO, suggesting that GP improves the hepatic insulin sensitivity by suppressing gluconeogenesis. In Paper II, shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increased glucose uptake in L6 myotubes, but did not phosphorylate Akt. Furthermore we found no evidence for the involvement of AMP activated protein kinase (AMPK) in shikonin induced glucose uptake. Shikonin increased the intracellular levels of calcium in these cells and stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myotubes. In GK rats treated with shikonin once daily for 4 days, PG levels were significantly decreased. In an insulin sensitivity test, the absolute PG levels were significantly lower in the shikonin-treated rats. These findings suggest that shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. In Paper III, GK and control Wistar rats were injected daily for up to 4 weeks with either a non-hematopoietic erythropoietin analog ARA290 or with placebo. PG levels in GK but not Wistar rats were significantly lower in ARA290-treated compared to placebo. After 2 and 4 weeks, the IPGTT was significantly improved in ARA290 treated GK rats. In insulin and pyruvate tolerance tests, glucose responses were similar in ARA290 and placebo groups. In isolated GK rat islets, glucose-stimulated insulin release was two-fold higher and islet intracellular calcium concentrations in response to several secretagogues were significantly higher in ARA290-treated than in placebo-treated GK rats. These findings indicate that treatment with ARA290 significantly improved glucose tolerance in diabetic GK rats, most likely due to improvement of insulin release. In Paper IV, sex differences in hepatic carbohydrate and lipid metabolism were characterized in healthy rats. No sex-differences were observed regarding hepatic triglyceride content, fatty acid oxidation rates or insulin sensitivity. Male rats had higher ratios of insulin to glucagon levels, increased hepatic glycogen content, a lower degree of AMPK phosphorylation, a higher rate of glucose production and higher expression levels of gluconeogenic genes, as compared to female rats. A sex-dependent response to mild starvation was observed with males being more sensitive. In conclusion, sex-differences reflect a higher capacity of the healthy male rat liver to respond to increased energy demands. Key words: glucose homeostasis, type 2 diabetes, GK rats, L6 myotubes, hepatic glucose output, insulin sensitivity, sex differences