Warisara Parichatikanond

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic β-Cells

Background: The molecular contributors of the mitochondrial Ca2+ uptake, which is essential for metabolism-secretion coupling in β-cells, are unknown. Results: Knockdown of MICU1 and MCU reduced agonist- and depolarization-induced mitochondrial Ca2+ sequestration, ATP production, and d-glucose-stimulated insulin secretion. Conclusion: MICU1 and MCU are integral to metabolism-secretion coupling in β-cells. Significance: The presented data identify MICU1 and MCU as important contributors to pancreatic β-cell function. In pancreatic β-cells, uptake of Ca2+ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca2+ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca2+ uptake 1 (MICU1), mitochondrial Ca2+ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca2+ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca2+ uptake upon both intracellular Ca2+ release and Ca2+ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca2+ uptake in response to either intracellular Ca2+ release or Ca2+ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca2+ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca2+ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca2+ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.

Study of anti-inflammatory activities of the pure compounds from Andrographis paniculata (burm.f.) Nees and their effects on gene expression.

In inflammation, the responses to noxious stimuli are controlled by the highly modulated interactions between various immune cells and chemical mediators. The purpose of this study is to evaluate and compare the anti-inflammatory effect of diterpenoids isolated from Andrographis paniculata, including dehydroandrographolide (AP1), andrographolide (AP2) and neoandrographolide (AP3), on the production of inflammatory cytokines and COX activities. Furthermore, the alteration of gene expression involved in this activity was investigated in the most potent compound to elucidate the other possible molecular mechanisms. AP1 (30.1 μM; 10 μg/ml) and AP2 (28.5 μM; 10 μg/ml) markedly inhibited COX-1 in ionophore A23187-induced human platelets. AP2 (28.5 μM) and AP3 (20.8 μM; 10 μg/ml) strongly suppressed the LPS-stimulated COX-2 activity in human blood. In addition, AP2 modulated the level of LPS-induced TNF-α, IL-6, IL-1β and IL-10 secretion in human blood in a concentration-dependent manner. The results revealed that AP2 exhibited the highest efficacy. Therefore, changes in the levels of mRNA transcripts by AP2 were further measured using human cDNA microarrays. The molecular response to AP2 was complex and mediated by various processes. Among the altered gene expressions, the genes involved in immune and inflammation processes were selectively down-regulated, such as cytokines and cytokine receptors (TNFSF14, TNF, TNFRSF6, and IL1A), chemokines (CCL8 and CXCL11), JAK/STAT signaling (JAK3 and STAT5A), TLRs family (TLR4 and TLR8) and NF-κB (NFKB1). Expression of some genes was validated using RT-PCR. The results demonstrated that AP1, AP2 and AP3 exhibited the anti-inflammatory effect by interfering COX and inflammatory cytokines and the underlying mechanisms of AP2 may be related to down-expression of genes involved in inflammatory cascade.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria.

Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.

Mitochondrial Ca 2+ uniporter (MCU)-dependent and MCU-independent Ca 2+ channels coexist in the inner mitochondrial membrane

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca2+ uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca2+ channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca2+ and Na+ currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca2+ current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca2+ current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca2+ channels.

Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.).

Mitochondrial Ca2+ uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca2+ uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca2+ channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca2+ signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca2+ rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca2+ uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca2+ rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca2+ concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.

PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake in immortalized cells.

Recent studies revealed that mitochondrial Ca2+ channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca2+ uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca2+ uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca2+ overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca2+ uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca2+ uptake. Our data demonstrate that mitochondrial Ca2+ uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca2+ sensitivity. UCP2/3 normalize Ca2+ sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca2+ uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca2+ uniporter by PRMT1 and UCP2/3.

Stimulation of Adenosine A2B Receptor Inhibits Endothelin-1-Induced Cardiac Fibroblast Proliferation and α-Smooth Muscle Actin Synthesis Through the cAMP/Epac/PI3K/Akt-Signaling Pathway

Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express α-smooth muscle actin (α-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and α-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A2 receptor inhibit ET-1-induced fibroblast proliferation and α-SMA expression in cardiac fibroblasts are not clearly identified. Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of α-SMA. Key results: Stimulation of adenosine subtype 2B (A2B) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced α-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A2B receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation. Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A2B receptor. Thus, A2B receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis.

Blockade of the Renin-Angiotensin System with Delphinidin, Cyanin, and Quercetin

Overactivation of the renin-angiotensin system is one of the most important risk factors for the development of hypertension. The use of the crude extracts and/or active compounds, such as anthocyanins and quercetin, of herbal plants that have antihypertensive effects is beneficial for decreasing of blood pressure level. However, the molecular mechanisms by which anthocyanins (delphinidin and cyanin) and quercetin regulate the renin-angiotensin system are not completely understood. In this study, we demonstrate that delphinidin, cyanin, and quercetin interrupt the renin-angiotensin system signaling pathway by inhibiting the angiotensin-converting enzyme activity and decreasing its mRNA production. Furthermore, treatment with either delphinidin or cyanin significantly inhibited renin mRNA production. However, delphinidin, cyanin, and quercetin did not act as the angiotensin II type 1 receptor antagonist and did not play roles in the regulation of its internalization. The direct inhibition of components of the renin-angiotensin system advances our understanding of the antihypertensive effects of these compounds.

Sustained βAR Stimulation Mediates Cardiac Insulin Resistance in a PKA-Dependent Manner

Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of β-adrenergic receptors (βARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained βAR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained βAR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT)4 expression that were mediated by the β2AR subtype in cardiomyocytes and heart tissue. Overstimulation of β2AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, βAR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with β-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in βAR-mediated insulin resistance.

β-Adrenergic Receptor and Insulin Resistance in the Heart

Insulin resistance is characterized by the reduced ability of insulin to stimulate tissue uptake and disposal of glucose including cardiac muscle. These conditions accelerate the progression of heart failure and increase cardiovascular morbidity and mortality in patients with cardiovascular diseases. It is noteworthy that some conditions of insulin resistance are characterized by up-regulation of the sympathetic nervous system, resulting in enhanced stimulation of β-adrenergic receptor (βAR). Over-stimulation of βARs leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, pathological consequences of the cross-talk between the βAR and the insulin sensitivity and the mechanism by which βAR over-stimulation promotes insulin resistance remain unclear. This review article examines the hypothesis that βARs over-stimulation leads to induction of insulin resistance in the heart.