Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress.

Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.

Steady-state regulation of COPII-dependent secretory cargo sorting by inositol trisphosphate receptors, calcium, and penta EF hand proteins.

Recently, we demonstrated that agonist-stimulated Ca2+ signaling involving IP3 receptors modulates ER export rates through activation of the penta-EF Hand proteins apoptosis-linked gene-2 (ALG-2) and peflin. It is unknown, however, whether IP3Rs and penta-EF proteins regulate ER export rates at steady state. Here we tested this idea in normal rat kidney epithelial cells by manipulation of IP3R isoform expression. Under standard growth conditions, spontaneous cytosolic Ca2+ oscillations occurred simultaneously in successive groups of contiguous cells, generating intercellular Ca2+ waves that moved across the monolayer periodically. Depletion of IP3R-3, typically the least promiscuous IP3R isoform, caused increased cell participation in intercellular Ca2+ waves in unstimulated cells. The increased spontaneous signaling was sufficient to cause increased ALG-2 and COPII coat subunit Sec31A and decreased peflin localization at ER exit sites, resulting in increased ER-to-Golgi transport of the COPII client cargo VSV-G. The elevated ER-to-Golgi transport caused greater concentration of VSV-G at ER exit sites and had reciprocal effects on transport of VSV-G and a bulk-flow cargo, though both cargos equally required Sec31A. Inactivation of client cargo sorting using 4-phenylbutyrate had opposing reciprocal effects on client and bulk-flow cargo and neutralized any effect of ALG-2 activation on transport. This work extends our knowledge of ALG-2 mechanisms and indicates that in normal rat kidney cells, IP3R isoforms regulate homeostatic Ca2+ signaling that helps determine the basal secretion rate and stringency of COPII-dependent cargo sorting.

Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.

Salivary potassium measured by genetically encoded potassium ion indicators as a surrogate for plasma potassium levels in hemodialysis patients-a proof-of-concept study.

BACKGROUND: Hyperkalemia is a common complication in cardiorenal patients treated with agents interfering with renal potassium (K+) excretion. It frequently leads to discontinuation of potentially life-saving medication, which has increased the importance of K+ monitoring. Non-invasive means to detect hyperkalemia are currently unavailable, but would be of potential use for therapy guidance. The aim of the present study was to assess the analytical performance of genetically encoded potassium-ion indicators (GEPIIs) in measuring salivary [K+] ([K+]Saliva) and to determine whether changes of [K+]Saliva depict those of [K+]Plasma. METHODS: We conducted this proof-of-concept study: saliva samples from 20 healthy volunteers as well as plasma and saliva from 29 patients on hemodialysis (HD) before and after three consecutive HD treatments were collected. We compared [K+]Saliva as assessed by the gold standard ion-selective electrode (ISE) with GEPII measurements. RESULTS: The Bland-Altmann analysis showed a strong agreement (bias 0.71; 95% limits of agreement from -2.79 to 4.40) between GEPII and ISE. Before treatment, patients on HD showed significantly higher [K+]Saliva compared with healthy controls [median 37.7 (30.85; 48.46) vs 23.8 (21.63; 25.23) mmol/L; P < .05]. [K+]Plasma in HD patients decreased significantly after dialysis. This was paralleled by a significant decrease in [K+]Saliva, and both parameters increased until the subsequent HD session. Despite similar kinetics, we found weak or no correlation between [K+]Plasma and [K+]Saliva. CONCLUSION: GEPIIs have shown an excellent performance in determining [K+]Saliva. [K+]Plasma and [K+]Saliva exhibited similar kinetics. To determine whether saliva could be a suitable sample type to monitor [K+]Plasma, further testing in future studies are required.

ALYREF, a novel factor involved in breast carcinogenesis, acts through transcriptional and post-transcriptional mechanisms selectively regulating the short NEAT1 isoform.

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1.

Nonclassical nuclear localization signals mediate nuclear import of CIRBP.

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice.

Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl-/-) and platelet-specific Mgl-deficient (platMgl-/-) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl-/- mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl-/- mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl-/- mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.

ALG-2 and peflin regulate COPII targeting and secretion in response to calcium signaling.

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

RNA editing of Filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure.

Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.

An unexpected effect of risperidone reveals a nonlinear relationship between cytosolic Ca2+ and mitochondrial Ca2+ uptake.

Mitochondria actively contribute to cellular Ca2+ homeostasis. The molecular mechanisms of mitochondrial Ca2+ uptake and release are well characterized and are attributed to the multi-protein assembly of the mitochondrial Ca2+ uniporter complex (MCUC) and the mitochondrial sodium-calcium exchanger (NCLX), respectively. Hence, Ca2+ transfer from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) into the mitochondrial matrix has been quantitatively visualized on the subcellular level using targeted fluorescent biosensors. However, a correlation between the amplitude of cytosolic Ca2+ elevation with that in the mitochondrial matrix has not been investigated in detail so far. In the present study, we combined the Ca2+-mobilizing agonist histamine with the H1-receptor antagonist risperidone to establish a well-tunable experimental approach allowing the correlation between low, slow, high, and fast cytosolic and mitochondrial Ca2+ signals in response to inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release. Our present data confirm a defined threshold in cytosolic Ca2+, which is necessary for the activation of mitochondrial Ca2+ uptake. Moreover, our data support the hypothesis of different modes of mitochondrial Ca2+ uptake depending on the source of the ion (i.e., ER vs SOCE).

Survey of Molecular Mechanisms of Hyperbaric Oxygen in Tissue Repair.

For more than six decades, hyperbaric oxygen (HBO) has been used for a variety of indications involving tissue repair. These indications comprise a wide range of diseases ranging from intoxications to ischemia-reperfusion injury, crush syndrome, central nervous injury, radiation-induced tissue damage, burn injury and chronic wounds. In a systematic review, the molecular mechanisms triggered by HBO described within the last two decades were compiled. They cover a wide range of pathways, including transcription, cell-to-cell contacts, structure, adhesion and transmigration, vascular signaling and response to oxidative stress, apoptosis, autophagy and cell death, as well as inflammatory processes. By analyzing 71 predominantly experimental publications, we established an overview of the current concepts regarding the molecular mechanisms underlying the effects of HBO. We considered both the abovementioned pathways and their role in various applications and indications.

Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation.

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62- total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-ß-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.

Near-UV Light Induced ROS Production Initiates Spatial Ca2+ Spiking to Fire NFATc3 Translocation.

Ca2+-dependent gene regulation controls several functions to determine the fate of the cells. Proteins of the nuclear factor of activated T-cells (NFAT) family are Ca2+ sensitive transcription factors that control the cell growth, proliferation and insulin secretion in ß-cells. Translocation of NFAT proteins to the nucleus occurs in a sequence of events that starts with activating calmodulin-dependent phosphatase calcineurin in a Ca2+-dependent manner, which dephosphorylates the NFAT proteins and leads to their translocation to the nucleus. Here, we examined the role of IP3-generating agonists and near-UV light in the induction of NFATc3 migration to the nucleus in the pancreatic ß-cell line INS-1. Our results show that IP3 generation yields cytosolic Ca2+ rise and NFATc3 translocation. Moreover, near-UV light exposure generates reactive oxygen species (ROS), resulting in cytosolic Ca2+ spiking via the L-type Ca2+ channel and triggers NFATc3 translocation to the nucleus. Using the mitochondria as a Ca2+ buffering tool, we showed that ROS-induced cytosolic Ca2+ spiking, not the ROS themselves, was the triggering mechanism of nuclear import of NFATc3. Collectively, this study reveals the mechanism of near-UV light induced NFATc3 migration.

N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO-/- compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.

Glycogen Synthase Kinase 3 Beta Controls Presenilin-1-Mediated Endoplasmic Reticulum Ca²âº Leak Directed to Mitochondria in Pancreatic Islets and ß-Cells.

BACKGROUND/AIMS: In pancreatic ß-cells, the intracellular Ca²âº homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²âº store balances cellular Ca²âº. In this study basal ER Ca²âº homeostasis was evaluated in order to reveal potential ß-cell-specificity of ER Ca²âº handling and its consequences for mitochondrial Ca²âº, ATP and respiration. METHODS: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²âº and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3ß activity was measured with ELISA. RESULTS: An atypical ER Ca²âº leak was observed exclusively in pancreatic islets and ß-cells. This continuous ER Ca²âº efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3ß-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3ß. Expression of a presenlin-1 mutant that mimics GSK3ß-mediated phosphorylation established a ß-cell-like ER Ca²âº leak in HeLa and EA.hy926 cells. The ER Ca²âº loss in ß-cells was compensated at steady state by Ca²âº entry that is linked to the activity of TRPC3. CONCLUSION: Pancreatic ß-cells establish a cell-specific ER Ca²âº leak that is under the control of GSK3ß and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²âº handling for basal mitochondrial activity.

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and ß-Cells upon Elevated Glucose.

BACKGROUND/AIMS: In our recent work, the importance of GSK3ß-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in ß-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3ß-phosphorylated presenilin-1 for responsiveness of pancreatic islets and ß-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic ß-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in ß-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3ß in the pancreatic ß-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3ß in the pancreatic ß-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and ß-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3ß, a phenomenon that might be involved in the development of type 2 diabetes.

Calcium Signaling in ß-cell Physiology and Pathology: A Revisit.

Pancreatic beta (ß) cell dysfunction results in compromised insulin release and, thus, failed regulation of blood glucose levels. This forms the backbone of the development of diabetes mellitus (DM), a disease that affects a significant portion of the global adult population. Physiological calcium (Ca2+) signaling has been found to be vital for the proper insulin-releasing function of ß-cells. Calcium dysregulation events can have a dramatic effect on the proper functioning of the pancreatic ß-cells. The current review discusses the role of calcium signaling in health and disease in pancreatic ß-cells and provides an in-depth look into the potential role of alterations in ß-cell Ca2+ homeostasis and signaling in the development of diabetes and highlights recent work that introduced the current theories on the connection between calcium and the onset of diabetes.

Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.