👤 Iolanda Vendrell

Due to its proximity to cells of the central nervous system, cerebrospinal fluid (CSF) is an important source of novel biomarkers for neurological diseases. Membrane-bound extracellular vesicles (EVs) are enriched for proteins of intracellular and membrane origin, implicated in the pathogenesis of some neurological diseases, and secreted into CSF. Proteomic profiling of CSF-EVs, however, is limited by the large volumes required for typical EV isolation protocols. We appraised the performance of tetraspanin (CD81, CD63, CD9)-based immunocapture for EV isolation from 200 to 1000 µL CSF sample and compared to size-exclusion chromatography (SEC). EVs were profiled by library-free data independent-acquisition (DIA) mass spectrometry to assess protein depth and abundance of specific EV markers and known co-isolates. Abundance and precursor peptide locations for potential neuronal-specific immunocapture targets described in the literature were also assessed. Immunocapture was effective using CSF volumes as low as 200 µL, consistently detecting core EV markers and reducing relative levels of non-vesicular proteins such as Apolipoprotein B (APOB) and galectin 3 binding protein (LGALS3BP) compared with size-exclusion chromatography (SEC). Proteomic depth reached 811 ± 14 protein groups in EVs from 200 µL CSF, increasing to 1285 ± 224 when using feature alignment across runs with up to 1000 µL starting volume. These included eleven candidate biomarkers of neurological diseases that were detected in all preparation methods, with additional candidates detected by immunocapture only. Increased depth was observed for both transmembrane and secreted proteins using immunocapture compared with SEC, with proportional enrichment of transmembrane proteins. This work demonstrates the effectiveness of tetraspanin immunocapture for proteomic profiling of EVs in small volumes of CSF that can be adapted to use with cell-type-specific markers of choice. Show less

This case describes the individualised pharmacological management of a 2-month-old infant with genetically confirmed type I hypertriglyceridemia due to lipoprotein lipase (LPL) deficiency. After the failure of conventional treatment and contraindication to plasmapheresis, intravenous insulin therapy was initiated, followed by subcutaneous insulin and omega-3 fatty acid adjustment. The hospital pharmacist played a key role in selecting off-label treatments, adapting pharmaceutical forms for paediatric use and performing therapeutic reconciliation. The approach was effective and safe, achieving triglyceride levels below 1000 mg/dL and clinical stability. This report contributes practical evidence on alternative treatment strategies for a rare disease with limited therapeutic options in paediatrics, highlighting the importance of a multidisciplinary approach and pharmaceutical care. Show less

Dyslipidemia in gestational diabetes has been associated with worse perinatal outcomes. The ANGPTL3-4-8 axis regulates lipid metabolism, especially in the transition from fasting to feeding. In this study, we evaluated the response of ANGPTL3, 4, and 8 after the intake of a mixed meal in women with normal glucose tolerance and gestational diabetes, and we assessed their gene expressions in different placental locations. Regarding the circulating levels of ANGPTL3, 4, and 8, we observed an absence of ANGPTL4 response after the intake of the meal in the GDM group compared to its presence in the control group. At the placental level, we observed a glucose tolerance-dependent expression pattern of Show less

Glucose-dependent insulinotropic peptide (GIP) has a central role in glucose homeostasis through its amplification of insulin secretion; however, its physiological role in adipose tissue is unclear. Our objective was to define the function of GIP in human adipose tissue in relation to obesity and insulin resistance. GIP receptor (GIPR) expression was analyzed in human sc adipose tissue (SAT) and visceral adipose (VAT) from lean and obese subjects in 3 independent cohorts. GIPR expression was associated with anthropometric and biochemical variables. GIP responsiveness on insulin sensitivity was analyzed in human adipocyte cell lines in normoxic and hypoxic environments as well as in adipose-derived stem cells obtained from lean and obese patients. GIPR expression was downregulated in SAT from obese patients and correlated negatively with body mass index, waist circumference, systolic blood pressure, and glucose and triglyceride levels. Furthermore, homeostasis model assessment of insulin resistance, glucose, and G protein-coupled receptor kinase 2 (GRK2) emerged as variables strongly associated with GIPR expression in SAT. Glucose uptake studies and insulin signaling in human adipocytes revealed GIP as an insulin-sensitizer incretin. Immunoprecipitation experiments suggested that GIP promotes the interaction of GRK2 with GIPR and decreases the association of GRK2 to insulin receptor substrate 1. These effects of GIP observed under normoxia were lost in human fat cells cultured in hypoxia. In support of this, GIP increased insulin sensitivity in human adipose-derived stem cells from lean patients. GIP also induced GIPR expression, which was concomitant with a downregulation of the incretin-degrading enzyme dipeptidyl peptidase 4. None of the physiological effects of GIP were detected in human fat cells obtained from an obese environment with reduced levels of GIPR. GIP/GIPR signaling is disrupted in insulin-resistant states, such as obesity, and normalizing this function might represent a potential therapy in the treatment of obesity-associated metabolic disorders. Show less

Munc18c is associated with glucose metabolism and could play a relevant role in obesity. However, little is known about the regulation of Munc18c expression. We analyzed Munc18c gene expression in human visceral (VAT) and subcutaneous (SAT) adipose tissue and its relationship with obesity and insulin. We evaluated 70 subjects distributed in 12 non-obese lean subjects, 23 overweight subjects, 12 obese subjects and 23 nondiabetic morbidly obese patients (11 with low insulin resistance and 12 with high insulin resistance). The lean, overweight and obese persons had a greater Munc18c gene expression in adipose tissue than the morbidly obese patients (p<0.001). VAT Munc18c gene expression was predicted by the body mass index (B = -0.001, p = 0.009). In SAT, no associations were found by different multiple regression analysis models. SAT Munc18c gene expression was the main determinant of the improvement in the HOMA-IR index 15 days after bariatric surgery (B = -2148.4, p = 0.038). SAT explant cultures showed that insulin produced a significant down-regulation of Munc18c gene expression (p = 0.048). This decrease was also obtained when explants were incubated with liver X receptor alpha (LXRα) agonist, either without (p = 0.038) or with insulin (p = 0.050). However, Munc18c gene expression was not affected when explants were incubated with insulin plus a sterol regulatory element-binding protein-1c (SREBP-1c) inhibitor (p = 0.504). Munc18c gene expression in human adipose tissue is down-regulated in morbid obesity. Insulin may have an effect on the Munc18c expression, probably through LXRα and SREBP-1c. Show less