Calcium signaling | EPFL Graph Search

Calcium signaling is the use of calcium ions (Ca2+) to communicate and drive intracellular processes often as a step in signal transduction. Ca2+ is important for cellular signalling, for once it enters the cytosol of the cytoplasm it exerts allosteric regulatory effects on many enzymes and proteins. Ca2+ can act in signal transduction resulting from activation of ion channels or as a second messenger caused by indirect signal transduction pathways such as G protein-coupled receptors. Concentration regulation The resting concentration of Ca2+ in the cytoplasm is normally maintained around 100 nM. This is 20,000- to 100,000-fold lower than typical extracellular concentration. To maintain this low concentration, Ca2+ is actively pumped from the cytosol to the extracellular space, the endoplasmic reticulum (ER), and sometimes into the mitochondria. Certain proteins of the cytoplasm and organelles act as buffers by bindi

Role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells

Isabelle Chareyron

Glucose sensing and regulation of insulin secretion are the two main functions performed by the pancreatic beta-cell. Together these processes contribute to the tight control of blood glucose in our body. Compromising the ability of the beta-cells to properly secrete insulin can lead to the development of Type 2 diabetes. Mitochondria play an important role in beta-cell function, by integrating nutrients signals and modulating insulin secretion. In this thesis, we aimed at better understanding the role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells. To that end, we designed three studies looking at different aspects of beta-cell mitochondrial function. In the first one, we developed an in vitro model of glucose overload in human islet. We report an over activation of the mitochondria when returned to basal conditions after a 4 day culture in elevated glucose. Additionally, these mitochondria only poorly responded to nutrient stimulation. Impaired metabolism secretion coupling and insulin secretion indicated a partial loss of beta-cell function, despite the increased mitochondrial activity. After 4 days of elevated glucose treatment, unusually high levels of glycerol-3-phosphate and pyruvate, even in sub-stimulatory extracellular glucose concentrations, were observed. We propose that the accumulation of these metabolites renders beta-cells blind to subsequent glucose stimulation. After removing the glucose stress for 24 hours the mitochondrial energy metabolism was fully restored and metabolism-secretion coupling was normalized. In the second approach, we measured for the first time the matrix pH in primary human pancreatic beta-cells. We showed that the mitochondrial pH in beta-cell is more acidic than in other cell types. Furthermore, in response to glucose, the beta-cell mitochondrial matrix slightly acidifies, possibly due to uptake and metabolism of pyruvate. Lastly, we observed that adenoviruses used to deliver the genetically encoded pH sensor can have an intrinsic impact on mitochondrial pH and calcium signaling. It must be used at low infection units per cell in the study of primary beta-cells. In the third project, we tried to modify the mitochondrial energy metabolism in beta-cells by inhibiting mitochondrial protein synthesis. We used different antibiotics to achieve this, in particular chloramphenicol, known to interfere with the activity of the mitochondrial ribosome. Surprisingly, we observe that when mitochondrial protein synthesis is inhibited, the respiration of insulin-secreting cells (INS-1E cells, human islets) is not affected. Expression of mtDNA encoded respiratory chain subunits is reduced and a mitochondrial unfolded protein response initiated without reducing respiratory rates. This raises the possibility that limited activation of unfolded protein response has a positive effect on the cells. This doctoral thesis provides novel insights into various aspects of mitochondrial behavior in pancreatic beta-cells, both during physiological and pathophysiological conditions. Additional work is needed to better understand the molecular mechanisms underlying the mitochondrial adaptation to nutrient overload and the associated beta-cell dysfunction leading to the accelerated impairment of glucose homeostasis during the development of Type 2 diabetes.

EPFL2019

Calcium dynamics and intercellular communication in arterial smooth muscle cells

Mathieu Lamboley

This thesis is a contribution to the field of cellular communication in arteries during vasomotion, i.e. rhythmic and spontaneous diameter oscillations of arteries. Investigating how individual smooth muscle cells (SMCs) and endothelial cells (ECs) calcium variations interact to induce an arterial response is a key to understanding the physical mechanisms leading to contraction and vasomotion. This study is composed of two main experimental parts using rat mesenteric arteries: a study on recruitment and synchronization of SMCs and an analysis on SMCs-ECs communication in arterial strips. The introduction is an overview of the mechanism leading to arterial vasomotion. We focused on the calcium signaling between SMCs, between ECs and between SMCs and ECs. In the chapter "Material and methods", we present the different experimental techniques we developed or improved in order to study calcium signaling in SMCs and/or ECs. For this purpose, we used arterial strips or cannulated arteries using a confocal microscope or a conventional microscope. Calcium concentration were measured using fluorescent dyes. These methods allow the correlation of calcium oscillations of individual SMCs, of individual ECs or both, together with mean calcium variations and arterial contraction. In chapter 3, we investigated the behavior of individual SMCs in order to determine if all cells presented the same variations of calcium concentration as the mean calcium variations or if vasomotion results of an unequal contribution of each SMC. Arterial strips were stimulated by increasing concentration of vasoconstrictors phenylephrine (PE) or potassium chloride (KCl). We revealed that the number of SMCs presenting calcium variations and their synchronization depend on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells present asynchronous calcium variations and no local contraction is detected. Recruitment of cells is complete and synchronous at medium concentration, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous oscillations and a fully contracted arterial strip, whereas high concentration of KCl leads to a sustained non-oscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling artery contraction and vasomotion. In chapter 4, we investigated the three main possible ways to synchronize the recruitment of SMCs when stimulating with PE and KCl. We tested the importance of calcium voltage-gated channels (VOCs) and thus the electrical communication, by using different inhibitors of the VOCs. We also tested the importance of stretch-activated channels (SACs) by inhibiting contraction of individual SMCs. Finally, we verified the importance of gap junctions and thus of chemical and/or electrical coupling. We applied a gap junction inhibitor and we also performed microinjection of fluorescent dyes in single SMCs. Our results provide evidence that under PE the synchronization of the SMCs recruitment occurred electrically through gap junctions. Under KCl, every SMC was simultaneously depolarized and calcium entered the cell simultaneously through VOCs. This leads to a global [Ca2+]i increase. When we studied the recruitment and synchronization of the SMCs under PE, we sometimes observed intercellular calcium waves propagating between SMCs in a direction corresponding to the arterial axis. In chapter 5, we used a tracking process to quantify these intercellular waves. We found that intercellular calcium waves propagating along the strip were linked to the local contraction variations. The velocity of both waves are similar (~60 µm/s). This method of tracking is particularly powerful for the study of cellular communication in arteries. It has the advantage of allowing the correlation of calcium with contraction dynamics. The role of the endothelium for vasomotion is matter of debate. SMCs and ECs are electrically and chemically coupled allowing exchange of intercellular signaling. In chapter 6, we investigated heterocellular communication at the cellular level in order to understand how SMCs can influence ECs calcium dynamics. SMCs of the arterial strip were stimulated by KCl and by PE. We verified that PE and KCl did not directly act on the calcium concentration of ECs. Depending on vasoconstrictor concentration, calcium concentration increased in some ECs 5 to 11 s after that a calcium concentration increase was observed in SMCs. The existence of this time interval suggests a chemical coupling between SMCs and ECs through gap junctions. Most probable chemical mediators are either calcium or inositol 1,4,5-trisphosphate (IP3). To discriminate between calcium and IP3 diffusion, first a phospholipase C inhibitor was applied to prevent IP3 production in response to the calcium concentration increase in SMCs. Under this inhibitor and KCl, all SMCs presented a global and synchronous calcium concentration increase, but no calcium concentration variations in ECs were detected. Secondly, 2-Aminoethoxydiphenylborate, an inhibitor of IP3-induced calcium release, reduced the number of flashing ECs by 75±3 %. The number of flashing ECs was also significantly reduced when palmitoleic acid, a gap junction uncoupler was added. Our results were compatible with a heterocellular communication based on IP3 diffusion through gap junctions from SMCs to ECs. The last chapter summarizes our findings and provides our conclusions with some perspectives.

EPFL2005

Augmented mitochondrial energy metabolism is an early response to chronic glucose stress in human pancreatic beta cells

Isabelle Chareyron, Loïc Dayon, Sofia Moco, Andreas Wiederkehr

Aims/hypothesis In islets from individuals with type 2 diabetes and in islets exposed to chronic elevated glucose, mitochondrial energy metabolism is impaired. Here, we studied early metabolic changes and mitochondrial adaptations in human beta cells during chronic glucose stress. Methods Respiration and cytosolic ATP changes were measured in human islet cell clusters after culture for 4 days in 11.1 mmol/l glucose. Metabolomics was applied to analyse intracellular metabolite changes as a result of glucose stress conditions. Alterations in beta cell function were followed using insulin secretion assays or cytosolic calcium signalling after expression of the calcium probe YC3.6 specifically in beta cells of islet clusters. Results At early stages of glucose stress, mitochondrial energy metabolism was augmented in contrast to the previously described mitochondrial dysfunction in beta cells from islets of diabetic donors. Following chronic glucose stress, mitochondrial respiration increased (by 52.4%,p < 0.001) and, as a consequence, the cytosolic ATP/ADP ratio in resting human pancreatic islet cells was elevated (by 27.8%,p < 0.05). Because of mitochondrial overactivation in the resting state, nutrient-induced beta cell activation was reduced. In addition, chronic glucose stress caused metabolic adaptations that resulted in the accumulation of intermediates of the glycolytic pathway, the pentose phosphate pathway and the TCA cycle; the most strongly augmented metabolite was glycerol 3-phosphate. The changes in metabolites observed are likely to be due to the inability of mitochondria to cope with continuous nutrient oversupply. To protect beta cells from chronic glucose stress, we inhibited mitochondrial pyruvate transport. Metabolite concentrations were partially normalised and the mitochondrial respiratory response to nutrients was markedly improved. Furthermore, stimulus-secretion coupling as assessed by cytosolic calcium signalling, was restored. Conclusion/interpretation We propose that metabolic changes and associated mitochondrial overactivation are early adaptations to glucose stress, and may reflect what happens as a result of poor blood glucose control. Inhibition of mitochondrial pyruvate transport reduces mitochondrial nutrient overload and allows beta cells to recover from chronic glucose stress.

2020