Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance

Single-Cell Quantification of mRNA Expression in The Human Brain

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis

Insulin secretion in patients with latent autoimmune diabetes (LADA): half way between type 1 and type 2 diabetes: action LADA 9

Background: The study of endogenous insulin secretion may provide relevant insight into the comparison of the natural history of adult onset latent autoimmune diabetes (LADA) with types 1 and 2 diabetes mellitus. The aim of this study was to compare the results of the C-peptide response to mixed-meal stimulation in LADA patients with different disease durations and subjects with type 2 and adult-onset type 1 diabetes. Methods: Stimulated C-peptide secretion was assessed using the mixed-meal tolerance test in patients with LADA (n = 32), type 1 diabetes mellitus (n = 33) and type 2 diabetes mellitus (n = 30). All patients were 30 to 70 years old at disease onset. The duration of diabetes in all groups ranged from 6 months to 10 years. The recruitment strategy was predefined to include at least 10 subjects in the following 3 disease onset categories for each group: 6 to 18 months, 19 months to 5 years and 5 to 10 years. Results: At all time-points of the mixed-meal tolerance test, patients with LADA had a lower stimulated C-peptide response than the type 2 diabetes group and a higher response than the type 1 diabetes group. The same results were found when the peak or area under the C-peptide curve was measured. When the results were stratified by time since disease onset, a similar pattern of residual insulin secretory capacity was observed. Conclusions: The present study shows that the magnitude of stimulated insulin secretion in LADA is intermediate between that of type 1 and type 2 diabetes mellitus

Activation of Peroxisome Proliferator–Activated Receptor β/δ Inhibits Lipopolysaccharide-Induced Cytokine Production in Adipocytes by Lowering Nuclear Factor-κB Activity via Extracellular Signal–Related Kinase 1/2

OBJECTIVE—Chronic activation of the nuclear factor-κB (NF-κB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator–activated receptor (PPAR) β/δ activation prevents inflammation in adipocytes

Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus

The first measurable sign of arising autoimmunity in Type 1 Diabetes Mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of Flow Cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (Protocol A), anti-human IgG-biotin and streptavidindichlorotriazinyl aminofluorescein (DTAF) (Protocol B) or TrxGAD65-biotin and streptavidin- DTAF (Protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and it represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA; making it applicable to most medium-complexity laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Ureta, Daniela Beatriz. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentin

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

Background The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). Results The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

The BACE1 product sAPPβ induces ER stress and inflammation and impairs insulin signaling

Objective β-secretase/β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a key enzyme involved in Alzheimer's disease that has recently been implicated in insulin-independent glucose uptake in myotubes. However, it is presently unknown whether BACE1 and the product of its activity, soluble APPβ (sAPPβ), contribute to lipid-induced inflammation and insulin resistance in skeletal muscle cells. Materials/Methods Studies were conducted in mouse C2C12 myotubes, skeletal muscle from Bace1−/−mice and mice treated with sAPPβ and adipose tissue and plasma from obese and type 2 diabetic patients. Results We show that BACE1 inhibition or knockdown attenuates palmitate-induced endoplasmic reticulum (ER) stress, inflammation, and insulin resistance and prevents the reduction in Peroxisome Proliferator-Activated Receptor γ Co-activator 1α (PGC-1α) and fatty acid oxidation caused by palmitate in myotubes. The effects of palmitate on ER stress, inflammation, insulin resistance, PGC-1α down-regulation, and fatty acid oxidation were mimicked by soluble APPβ in vitro. BACE1 expression was increased in subcutaneous adipose tissue of obese and type 2 diabetic patients and this was accompanied by a decrease in PGC-1α mRNA levels and by an increase in sAPPβ plasma levels of obese type 2 diabetic patients compared to obese non-diabetic subjects. Acute sAPPβ administration to mice reduced PGC-1α levels and increased inflammation in skeletal muscle and decreased insulin sensitivity. Conclusions Collectively, these findings indicate that the BACE1 product sAPPβ is a key determinant in ER stress, inflammation and insulin resistance in skeletal muscle and gluconeogenesis in liver

Soluble epoxide hydrolase-targeting PROTAC activates AMPK and inhibits endoplasmic reticulum stress

Soluble epoxide hydrolase (sEH) is a drug target with the potential for therapeutic utility in the areas of inflammation, neurodegenerative disease, chronic pain, and diabetes, among others. Proteolysis-targeting chimeras (PROTACs) molecules offer new opportunities for targeting sEH, due to its capacity to induce its degradation. Here, we describe that the new ALT-PG2, a PROTAC that degrades sEH protein in the human hepatic Huh-7 cell line, in isolated mouse primary hepatocytes, and in the liver of mice. Remarkably, sEH degradation caused by ALT-PG2 was accompanied by an increase in the phosphorylated levels of AMP-activated protein kinase (AMPK), while phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) was reduced. Consistent with the key role of these kinases on endoplasmic reticulum (ER) stress, ALT-PG2 attenuated the levels of ER stress and inflammatory markers. Overall, the findings of this study indicate that targeting sEH with degraders is a promising pharmacological strategy to promote AMPK activation and to reduce ER stress and inflammation.This study was partly supported by grants RTI2018-093999-B-100 and PID2021-122116OB-I00 (M.V-C.), PID2020-118127RB-I00 (S.V.), PID2021-127693OB-I00 (C.G.) and PID2021-122766OB-I00 (A.M.V.) funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is a Carlos III Health Institute project. CERCA Programme/Generalitat de Catalunya. Partial support was provided by NIH – NIEHS (RIVER Award) R35 ES030443-01, NIH – NIEHS (Superfund Award) P42 ES004699 and NIH Counter Act Award U54 NS127758 (B.H.).Peer reviewe

Relationships between serum adiponectin and soluble TNF-α receptors and glucose and lipid oxidation in lean and obese subjects

Insulin resistance might be associated with an impaired ability of insulin to stimulate glucose oxidation and inhibit lipid oxidation. Insulin action is also inversely associated with TNF-α system and positively related to adiponectin. The aim of the present study was to analyze the associations between serum adiponectin, soluble TNF-α receptors concentrations and the whole-body insulin sensitivity, lipid and glucose oxidation, non-oxidative glucose metabolism (NOGM) and metabolic flexibility in lean and obese subjects. We examined 53 subjects: 25 lean (BMI < 25 kg × m−2) and 28 with overweight or obesity (BMI > 25 kg × m−2) with normal glucose tolerance. Hyperinsulinemic euglycemic clamp and indirect calorimetry were performed. An increase in respiratory exchange ratio in response to insulin was used as a measure of metabolic flexibility. Obese subjects had lower insulin sensitivity, adiponectin and higher sTNFR1 (all P < 0.001) and sTNFR2 (P = 0.001). Insulin sensitivity was positively related to adiponectin (r = 0.49, P < 0.001) and negatively related to sTNFR1 (r = −0.40, P = 0.004) and sTNFR2 (r = −0.52, P < 0.001). Adiponectin was related to the rate of glucose (r = 0.47, P < 0.001) and lipid (r = −0.40, P = 0.003) oxidation during the clamp, NOGM (r = 0.41, P = 0.002) and metabolic flexibility (r = 0.36, P = 0.007). Serum sTNFR1 and sTNFR2 were associated with the rate of glucose (r = −0.45, P = 0.001; r = −0.51, P < 0.001, respectively) and lipid (r = 0.52, P < 0.001; r = 0.46, P = 0.001, respectively) oxidation during hyperinsulinemia, NOGM (r = −0.31, P = 0.02; r = −0.43, P = 0.002, respectively) and metabolic flexibility (r = −0.47 and r = −0.51, respectively, both P < 0.001) in an opposite manner than adiponectin. Our data suggest that soluble TNF-α receptors and adiponectin have multiple effects on glucose and lipid metabolism in obesity

Efficacy of vitamin D3-fortified-yogurt drink on anthropometric, metabolic, inflammatory and oxidative stress biomarkers according to vitamin D receptor gene polymorphisms in type 2 diabetic patients: a study protocol for a randomized controlled clinical trial

<p>Abstract</p> <p>Background</p> <p>Development of type 2 diabetes mellitus (T2DM) is determined by the interactions of genetic and environmental factors. This study was designed to evaluate the possible role of VDR single nucleotide polymorphisms (SNPs) on different aspects of diabetic host response (anthropometric, metabolic, oxidative stress and inflammatory) to daily intake of vitamin D through fortified yogurt drink for 12 weeks.</p> <p>Methods/Design</p> <p>This study comprises two parts: (i) a case-control study; and (ii) an intervention trial. In the first part, VDR polymorphisms <it>(Taq1</it>, <it>FokI</it>, <it>Apa1</it>, <it>Bsm1</it>, and <it>Cdx2) </it>are determined in 350 T2DM patients and 350 non-diabetic subjects. In the second part, the possible effects of daily intake of two servings of vitamin D3-fortified yogurt drink (FYD; 500 IU vitamin D/250 mL) on some selected metabolic (including insulin resistance), inflammatory and oxidative stress biomarkers in 135 T2DM patients are assessed. To relate the resulted changes in the biomarkers to vitamin D replenishment, another group of diabetic patients (n = 45) are also included in the study who receive 2 servings of plain yogurt drink (PYD) a day. The primary outcome is serum level of 25(OH) D, which it is expected to be elevated only in FYD group. Secondary outcomes include improvements in glycemic, metabolic, inflammatory and oxidative stress biomarkers in FYD group compared to PYD group. Three VDR <it>FokI </it>polymorphisms are determined only in FYD group followed by comparison of changes in the biomarkers among these genotypic variants.</p> <p>Discussion</p> <p>The present study, at least in part, elucidates the discrepancies in the results of different vitamin D-diabetes studies pertaining to the genetic variations of the population. If VDR polymorphisms are found to influence the response to our intervention, then knowing distribution of VDR polymorphisms in both diabetic and non-diabetic populations can give a picture of the proportion of the community in whom up to 1000 IU/d vitamin D may not be effective enough to improve insulin resistance and related morbidities. Therefore, they should ideally receive further nutritional support according to their genotype.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01236846">NCT01236846</a></p