Biology subjects

Vilas-Boas, E. A.

Publications and source records attributed to Vilas-Boas, E. A..

4 recordsLinked to original sources

bioRxiv2023

A practical and robust method to evaluate metabolic fluxes in pancreatic islets

Aims/hypothesisEfficient mitochondrial oxidative phosphorylation is essential for pancreatic beta cell responses to nutrient levels. Consequently, the evaluation of mitochondrial oxygen consumption and ATP production is important to investigate essential aspects of pancreatic islet pathophysiology. Currently, most studies use cell lines instead of primary islets due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables that are expensive, complicated to use, and poorly reproducible. The aim of this study is to establish a robust and practical method to assess primary islet metabolic fluxes using Extracellular Flux Technology and standard commercial consumables. MethodsPancreatic islets were isolated from 8 to 12-week-old mice and rats, and submitted to a dispersion protocol using trypsin. Dispersed islets were adhered overnight to pre-coated standard Seahorse microplates, and oxygen consumption rates were evaluated using a Seahorse Extracellular Flux Analyzer. We also validated the functionality of dispersed islets by analyzing glucose-stimulated insulin secretion (GSIS) and calcium (Ca2+) influx in response to different modulators by fluorescence microscopy. ResultsWe provide a detailed protocol with all steps necessary to optimize islet isolation and dispersion, in order to achieve a high yield of functional islets and perform metabolic flux analysis. With this method, which requires only a few islets per replicate, both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators (oligomycin, CCCP, antimycin and rotenone) and glucose addition. Both oligomycin and CCCP concentrations were titrated. Our method was also validated by other functional assays, which show these cells present conserved Ca2+ influx and insulin secretion in response to glucose. Conclusions/interpretationWe established a practical and robust method to assess ex vivo islet metabolic fluxes and oxidative phosphorylation. Our findings cover an important gap in primary islet physiology studies, providing a valuable tool we hope is useful to uncover basic beta cell metabolic mechanisms, as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols. Research in context What is already known about this subject?O_LIPancreatic beta cells efficiently couple oxidative phosphorylation and ATP production with insulin secretion; mitochondrial ATP production is crucial for proper insulin secretion. C_LIO_LIMost studies of beta cell respiration use cell lines instead of primary islets, which are a much more robust model to evaluate beta cell function. C_LIO_LIThe few works with primary islet respiration use specific equipment and consumables that are expensive, complicated, and poorly reproducible. C_LI What is the key question?O_LIIs it possible to develop a practical method to evaluate metabolic fluxes and ATP production in isolated islets, using the standard Seahorse Extracellular Flux Technology? C_LI What are the new findings?O_LIWe optimized rodent islet isolation and functional analysis protocols using standard extracellular flux analysis equipment and consumables. C_LIO_LIOur method allows for increased islet yield and robust islet respiration measurements. C_LI How might this impact on clinical practice in the foreseeable future?O_LIQuantitative measurements of metabolic fluxes and oxidative phosphorylation are the cornerstone of new discoveries in beta cells, and can contribute toward the establishment of new cellular protocols, such as for cell transplantation, as well as the development of new pharmacological agents targeted to these cells. C_LI

biochemistry↗

bioRxiv2023

Autophagy is Regulated by Mitochondrial Calcium Transporters NCLX and MCU

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporter, and efflux from by Na+/Ca2+ exchangers (NCLX). Here, we describe a novel relationship between NCLX, intracellular Ca2+, and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

cell biology↗

bioRxiv2022

Adiponectin Promotes Glucose-Sensitive Insulin Secretion and Prevents β-Cell Damage by Obesity

Obesity significantly decreases life expectancy and increases the incidence of age-related dysfunctions, including {beta}-cell dysregulation leading to inadequate insulin secretion. Here, we show that diluted plasma from obese human donors acutely impairs {beta}-cell integrity and insulin secretion relative to plasma from lean subjects. Similar results were observed with diluted sera from obese rats fed ad libitum, when compared to sera from lean, calorically-restricted, animals. The damaging effects of obese circulating factors on {beta}-cells occurs in the absence of nutrient overload, and mechanistically involves mitochondrial dysfunction, limiting glucose-supported oxidative phosphorylation and ATP production. We demonstrate that increased levels of adiponectin, as found in lean plasma, are the protective characteristic preserving {beta}-cell function; indeed, sera from adiponectin knockout mice limits {beta}-cell metabolic fluxes relative to controls. Furthermore, oxidative phosphorylation and glucose-sensitive insulin secretion, which are completely abrogated in the absence of this hormone, are restored by the presence of adiponectin alone, surprisingly even in the absence of other serological components, for both the insulin-secreting INS1 cell line and primary islets. The addition of adiponectin to cells treated with plasma from obese donors completely restored {beta}-cell functional integrity, indicating the lack of this hormone was causative of the dysfunction. Overall, our results demonstrate that low circulating adiponectin is a key damaging element for {beta}-cells, and suggest strong therapeutic potential for the modulation of the adiponectin signaling pathway in the prevention of age-related {beta}-cell dysfunction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/501128v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1df5041org.highwire.dtl.DTLVardef@e066borg.highwire.dtl.DTLVardef@c79132org.highwire.dtl.DTLVardef@d89d1_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: Incubation of -cells with sera or plasma from obese rats and humans hampers mitochondrial oxidative phosphorylation and glucose-stimulated insulin secretion (GSIS) relative to sera and plasma from lean rats and humans. Adiponectin, found at elevated levels in lean subjects, supports -cell function on its own, in the absence of sera, and also reverses the effects of obese plasma. Prepared using Biorender.com. C_FIG

biochemistry↗

bioRxiv2022

Goldilocks calcium and the mitochondrial respiratory chain: too much, too little, just right

Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways, and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca2+ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra or intramitochondrial Ca2+ modulate oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supra-physiological increases in cytosolic and mitochondrial Ca2+ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 {+/-} 0.6 M) and medium (22.0 {+/-} 2.4 M) Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and -ketoglutarate, respectively, but not complex II-linked succinate. In intact cells, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.

biochemistry↗