siRNA screening reveals that SNAP29 contributes to exosome release.

Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.

Extracellular vesicles and microRNAs are altered in response to exercise, insulin sensitivity and overweight.

Extracellular vesicles induced by exercise have emerged as potential mediators of tissue crosstalk. Extracellular vesicles and their cargo miRNAs have been linked to dysglycemia and obesity in animal models, but their role in humans is unclear. AIM: The aim of the study was to characterize the miRNA content in plasma extracellular vesicle isolates after acute and long-term exercise and to study associations between extracellular vesicle miRNAs, mRNA expression in skeletal muscle and adipose tissue, and cardiometabolic risk factors. METHODS: Sedentary men with or without dysglycemia and overweight underwent an acute bicycle test and a 12-week exercise intervention with extensive metabolic phenotyping. Gene expression in m. vastus lateralis and subcutaneous adipose tissue was measured with RNA sequencing. Extracellular vesicles were purified from plasma with membrane affinity columns or size exclusion chromatography. RESULTS: Extracellular vesicle miRNA profiling revealed a transient increase in the number of miRNAs after acute exercise. We identified miRNAs, such as miR-652-3p, that were associated to insulin sensitivity and adiposity. By performing explorative association analyses, we identified two miRNAs, miR-32-5p and miR-339-3p, that were strongly correlated to an adipose tissue macrophage signature. CONCLUSION: Numerous miRNAs in plasma extracellular vesicle isolates were increased by exercise, and several miRNAs correlated to insulin sensitivity and adiposity. Our findings warrant future studies to characterize exercise-induced extracellular vesicles and cargo miRNA to clarify where exercise-induced extracellular vesicles originate from, and to determine whether they influence metabolic health or exercise adaptation.

Type 2 diabetes: Prevalence of hypoglycemia in public versus private health care system

Social vulnerability has proved to be an independent risk factor for hypoglycemia in patients with diabetes. In some countries, patients who are in a vulnerable situation are assisted in the public health system which provides free medical care. This study compares the prevalence of hypoglycemia among patients with type 2 diabetes (T2D), in public versus private sector and its relationship with social vulnerability. This multicentric descriptive study included 600 patients with T2D from public and private care institutions of Argentina. Socioeconomic level (SEL) was evaluated through the Marketing Argentinean Association survey. Number of severe, documented symptomatic and asymptomatic hypoglycemias were registered. Among the patients included, 66% were assisted in the public sector. The 41% of patients (n = 246) registered at least 1 episode of any hypoglycemia event being more prevalent in the public sector compared to the private sector (50% vs. 22%). In the adjusted analysis it was observed a greater risk of hypoglycemia in public sector (OR 4, 95% CI 2.65-6.04) and in patients that did not have diabetological education (OR 2.28 95% CI 1.35-3.84). Similarly, unemployment (OR 5.04 95% CI 2.69-9.46), and marginal SEL (OR 60.79 95% CI 14.89-248.13) increased the risk of hypoglycemia. Several factors related to social vulnerability as unemployment, marginal SEL and poor sanitary education showed a significant increase in the hypoglycemia risk. Professionals working with people with diabetes must take into account these factors for a safe treatment of the disease.

La vulnerabilidad social ha demostrado ser un factor de riesgo independiente de hipoglucemia en pacientes con diabetes. Los pacientes que se encuentran en situación de vulnerabilidad social reciben asistencia en el sistema de salud pública que brinda atención médica gratuita. Este estudio compara la prevalencia de hipoglucemia en pacientes con diabetes tipo 2 en el sector público frente al privado y su relación con la vulnerabilidad social. Se realizó un estudio multicéntrico descriptivo que incluyó 600 pacientes con diabetes tipo 2 de instituciones de atención pública y privada de Córdoba. El nivel socioeconómico se evaluó a través de la encuesta de la Asociación Argentina de Marketing que evalúa la dimensión social, educativa y económica para estratificar el nivel socioeconómico. Se registró el número de hipoglucemias graves, documentadas sintomáticas y asintomáticas. El 66% de los pacientes pertenecían al sector público. El 41% de los pacientes (n = 246) registró al menos 1 episodio de cualquier evento de hipoglucemia. En el análisis ajustado, se observó un mayor riesgo de hipoglucemia en el sector público (OR 4, 95% CI 2.65-6.04), en pacientes que no tenían educación diabetológica previa (OR 2.28, 95% CI 1.35-3.84), en desempleados (OR 5.04, 95% CI 2.69-9.46) y en aquellos con nivel socioeconómico marginal (OR 60.79 95% CI 14.89-248.13). Factores relacionados con la vulnerabilidad social como el desempleo, el nivel socioeconómico marginal y educación sanitaria deficiente mostraron un aumento en el riesgo de hipoglucemia. Los profesionales que trabajan con personas con diabetes deben tener en cuenta estos factores para un tratamiento seguro de la enfermedad.

Type 2 diabetes. Prevalence of hypoglycemia in public versus private health care system.

Social vulnerability has proved to be an independent risk factor for hypoglycemia in patients with diabetes. In some countries, patients who are in a vulnerable situation are assisted in the public health system which provides free medical care. This study compares the prevalence of hypoglycemia among patients with type 2 diabetes (T2D), in public versus private sector and its relationship with social vulnerability. This multicentric descriptive study included 600 patients with T2D from public and private care institutions of Argentina. Socioeconomic level (SEL) was evaluated through the Marketing Argentinean Association survey. Number of severe, documented symptomatic and asymptomatic hypoglycemias were registered. Among the patients included, 66% were assisted in the public sector. The 41% of patients (n = 246) registered at least 1 episode of any hypoglycemia event being more prevalent in the public sector compared to the private sector (50% vs. 22%). In the adjusted analysis it was observed a greater risk of hypoglycemia in public sector (OR 4, 95% CI 2.65-6.04) and in patients that did not have diabetological education (OR 2.28 95% CI 1.35-3.84). Similarly, unemployment (OR 5.04 95% CI 2.69-9.46), and marginal SEL (OR 60.79 95% CI 14.89-248.13) increased the risk of hypoglycemia. Several factors related to social vulnerability as unemployment, marginal SEL and poor sanitary education showed a significant increase in the hypoglycemia risk. Professionals working with people with diabetes must take into account these factors for a safe treatment of the disease.

La vulnerabilidad social ha demostrado ser un factor de riesgo independiente de hipoglucemia en pacientes con diabetes. Los pacientes que se encuentran en situación de vulnerabilidad social reciben asistencia en el sistema de salud pública que brinda atención médica gratuita. Este estudio compara la prevalencia de hipoglucemia en pacientes con diabetes tipo 2 en el sector público frente al privado y su relación con la vulnerabilidad social. Se realizó un estudio multicéntrico descriptivo que incluyó 600 pacientes con diabetes tipo 2 de instituciones de atención pública y privada de Córdoba. El nivel socioeconómico se evaluó a través de la encuesta de la Asociación Argentina de Marketing que evalúa la dimensión social, educativa y económica para estratificar el nivel socioeconómico. Se registró el número de hipoglucemias graves, documentadas sintomáticas y asintomáticas. El 66% de los pacientes pertenecían al sector público. El 41% de los pacientes (n = 246) registró al menos 1 episodio de cualquier evento de hipoglucemia. En el análisis ajustado, se observó un mayor riesgo de hipoglucemia en el sector público (OR 4, 95% CI 2.65-6.04), en pacientes que no tenían educación diabetológica previa (OR 2.28, 95% CI 1.35-3.84), en desempleados (OR 5.04, 95% CI 2.69-9.46) y en aquellos con nivel socioeconómico marginal (OR 60.79 95% CI 14.89-248.13). Factores relacionados con la vulnerabilidad social como el desempleo, el nivel socioeconómico marginal y educación sanitaria deficiente mostraron un aumento en el riesgo de hipoglucemia. Los profesionales que trabajan con personas con diabetes deben tener en cuenta estos factores para un tratamiento seguro de la enfermedad.

[Frequency and associated risk factors of cardiovascular autonomic neuropathy among patients with type 2 Diabetes.] / Frecuencia y factores de riesgo asociados de Neuropatía Autonómica Cardíaca en pacientes con Diabetes tipo 2.

Background: Cardiovascular autonomic neuropathy (CAN) is a common complication of diabetes and is associated with cardiovascular morbidity and mortality. Despite its prevalence, it is usually underdiagnosed. Objective to assess frequency of CAN in type 2 diabetes, treated at the Diabetology Service at Cordoba Hospital, Argentina. Method: Cross-sectional study was conducted from May 2015 to May 2016. Cardiovascular Autonomic Tests (Ewing´s battery) were performed: response to Valsalva maneuver, expiration-to-inspiration ratio (E/I ratio), standing to lying flat and postural hypotension. Results: 100 type 2 diabetes patients, 53% females. The mean age and diabetes duration 52 years old and 8.4 years, respectively. Hemoglobin A1c level of 8.7% and 47% were insulin-requiring type 2 diabetes patients. 29% had early CAN, 54% confirmed CAN (10% severe CAN) and 17% normal cardiovascular tests. Confirmed CAN was higher in those with longer duration of diabetes; longer duration of insulin therapy, older patients and severe hypoglycemia events was higher in those with confirmed CAN versus without CAN or early CAN (p 0.003). The risk factors associated were hypertension OR 2.55 (p 0.02), peripheral neuropathy OR 11.7 (p <0.0001), diabetic retinopathy OR 9.03 (p 0.001), diabetic nephropathy OR 3.12 (p 0.03) and hemoglobin A1c > 7% OR 2.57 (p 0, 03). Conclusions: frequency of CAN was high, was associated with hypertension, microvascular complications, older age, poor metabolic control, longer duration of disease and insulin therapy. Patients with a higher risk of developing CAN should be identified, in order to reduce the impact of this complication.

Introducción: La neuropatía autonómica cardíaca (NAC) es una complicación frecuente de la diabetes y aumenta la morbimortalidad cardiovascular. A pesar de su prevalencia e impacto está subdiagnosticada. Objetivo: Determinar la frecuencia de NAC y factores de riesgo asociados en pacientes con diabetes tipo 2(DM2), en el Servicio de Diabetología del Hospital Córdoba. Material y Métodos: estudio transversal, desde Mayo de 2015 a Mayo de 2016. Se realizó Test Autonómicos Cardiovasculares: Maniobra de Valsalva (MV), Respiración Profunda (RP), Razón 30/15 e Hipotensión ortostática. Resultados: Se reclutaron 100 pacientes con DM2. 53 % sexo femenino. Edad promedio 52 años, IMC 30,9; HbA1c 8,7 % y evolución de la enfermedad 8,4 años. El 17 % presentaron test normales. El 29 % tenía NAC temprana y 54% NAC confirmada (10 % NAC severa). Los pacientes con NAC confirmada tenían mayor tiempo de evolución (11 vs 5 años ), mayor duración de insulinoterapia (5 vs 1,5 años ), mayor edad ( 54 vs 49 años ) y mayor riesgo de hipoglucemias severas comparado con pacientes sin NAC o NAC temprana (p 0,003).Factores de riesgo asociados hipertensión arterial OR 2,55 (p 0,02), neuropatía periférica OR 11,7 (p<0,0001) , retinopatía OR 9,03 (p 0,001), nefropatía OR 3,12 (p 0,03) y HbA1c > 7% OR 2,57(p 0,03). Conclusiones: Hubo una alta frecuencia de NAC, se asoció con hipertensión, complicaciones microvasculares, mayor edad, duración de la enfermedad, de la insulinoterapia y mal control metabólico. Se debería identificar a pacientes con mayor riesgo, para reducir su aparición e impacto.