Solute carrier 41A3 encodes for a mitochondrial Mg2+ efflux system

The important role of magnesium (Mg(2+)) in normal cellular physiology requires flexible, yet tightly regulated, intracellular Mg(2+) homeostasis (IMH). However, only little is known about Mg(2+) transporters of subcellular compartments such as mitochondria, despite their obvious importance for the deposition and reposition of intracellular Mg(2+) pools. In particular, knowledge about mechanisms responsible for extrusion of Mg(2+) from mitochondria is lacking. Based on circumstantial evidence, two possible mechanisms of Mg(2+) release from mitochondria were predicted: (1) Mg(2+) efflux coupled to ATP translocation via the ATP-Mg/Pi carrier, and (2) Mg(2+) efflux via a H(+)/Mg(2+) exchanger. Regardless, the identity of the H(+)-coupled Mg(2+) efflux system is unknown. We demonstrate here that member A3 of solute carrier (SLC) family 41 is a mitochondrial Mg(2+) efflux system. Mitochondria of HEK293 cells overexpressing SLC41A3 exhibit a 60% increase in the extrusion of Mg(2+) compared with control cells. This efflux mechanism is Na(+)-dependent and temperature sensitive. Our data identify SLC41A3 as the first mammalian mitochondrial Mg(2+) efflux system, which greatly enhances our understanding of intracellular Mg(2+) homeostasis

Overexpression of Na+/Mg2+ exchanger SLC41A1 attenuates pro-survival signaling

The Na+/Mg2+ exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg2+ efflux system, and its overexpression decreases [Mg2+]intracellular. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg2+]i impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan® RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg2+]extracellular in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr308 and/or Ser473 and of Erk1/2 on Thr202/Tyr204 in the presence of 0 or 1 mM (physiological) Mg2+ in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg2+]i in the presence of [Mg2+]e = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB – Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg2+]e (and consequently also [Mg2+]i) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells

Dietary methionine source alters the lipidome in the small intestinal epithelium of pigs

Methionine (Met) as an essential amino acid has key importance in a variety of metabolic pathways. This study investigated the influence of three dietary Met supplements (0.21% L-Met, 0.21% DL-Met and 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA)) on the metabolome and inflammatory status in the small intestine of pigs. Epithelia from duodenum, proximal jejunum, middle jejunum and ileum were subjected to metabolomics analysis and qRT-PCR of caspase 1, NLR family pyrin domain containing 3 (NLRP3), interleukins IL1β, IL8, IL18, and transforming growth factor TGFβ. Principal component analysis of the intraepithelial metabolome revealed strong clustering of samples by intestinal segment but not by dietary treatment. However, pathway enrichment analysis revealed that after L-Met supplementation polyunsaturated fatty acids (PUFA) and tocopherol metabolites were lower across small intestinal segments, whereas monohydroxy fatty acids were increased in distal small intestine. Pigs supplemented with DL-HMTBA showed a pronounced shift of secondary bile acids (BA) and sphingosine metabolites from middle jejunum to ileum. In the amino acid super pathway, only histidine metabolism tended to be altered in DL-Met-supplemented pigs. Diet did not affect the expression of inflammation-related genes. These findings suggest that dietary supplementation of young pigs with different Met sources selectively alters lipid metabolism without consequences for inflammatory status

Rhodolith Beds Heterogeneity along the Apulian Continental Shelf (Mediterranean Sea)

Rhodolith beds represent a key habitat worldwide, from tropical to polar ecosystems. Despite this habitat is considered a hotspot of biodiversity, providing a suite of ecosystem goods and services, still scarce quantitative information is available thus far about rhodolith beds occurrence and ecological role, especially in the Mediterranean Sea. This study reports the composition and patterns of distribution of rhodolith assemblages found in four study areas along ca. 860 km of coast in the Central Mediterranean Sea. These rhodolith beds were studied for the first time and significant differences at all spatial scales have been highlighted, documenting the high variability of this habitat. Rhodolith species composition, morphology and distribution have been discussed considering the potential role of environmental factors in driving these patterns. The need for improving their protection is discussed to complement present conservation and management initiatives, particularly in the frame of the EU Marine Strategy Framework Directive

Mitochondrial respiration is decreased in visceral but not subcutaneous adipose tissue in obese individuals with fatty liver disease

Adipose tissue (commonly called body fat) can be found under the skin (subcutaneous) or around internal organs (visceral). Dysfunction of adipose tissue can cause insulin resistance and lead to excess delivery of fat to other organs such as the liver. Herein, we show that dysfunction specifically in visceral adipose tissue was associated with fatty liver disease

SLC41A1, SLC41A3 und CNNM2: Magnesium-responsive Genes mit potentieller Beteiligung an humanen Krankheiten

It is well known that magnesium deficiency or altered IMH can trigger many pathophysiological conditions, thus a correct functioning of Mg2+ transporters and channels is essential for normal cellular physiology. Mutations in many MRG induce hypomagnesemia which often represents one of the complications of many human ailments. Based on previous data which have characterized SLC41A1 as a plasma membrane Na+/Mg2+ exchanger (4, 5) and as being overexpressed in preeclamptic women (6), the present thesis aimed at a further characterization of SLC41A1 and Mg2+ efflux in some pathophysiological conditions. It further aimed at the molecular characterization of two others MRGs, CNNM2 and SLC41A3, in order to achieve a better understanding of Mg2+ homeostasis and to link the mechanisms of Mg2+ mobilization across the plasma membrane or between intracellular compartments with mitochondrial dysfunction and disease states (e.g.: Parkinson’s disease (PD), diabetes, etc). Proceeding from previous results of Kolisek et al (4, 5), describing SLC41A1 as the major Mg2+ efflux system of the cell, the first study examined the complex-forming ability of SLC41A1 in vivo and identified EBP and other members of the SLC superfamily as potential binding partners. Further experiments evaluated the transport activity of the PD-associated SLC41A1 variant pA350V and defined it as a “gain-of-function” mutation enhancing Mg2+ efflux compared with the wild-type protein. A next question was whether SLC41A1 transport activity could be influenced and regulated by insulin in order to explain the molecular basis of hypomagnesaemia often observed in diabetes patients. The present study shows that insulin reduces the SLC41A1-mediated Mg2+ efflux and, most importantly, it seems to have an effect on intracellular Mg2+ stores, since an earlier onset of Mg2+ release from intracellular stores was observed. In the second part of this thesis, experiments were conducted on stably transfected HEK293 cells overexpressing SLC41A3 in order to uncover the function of SLC41A3 with regard to its ability to transport Mg2+, its mode of Mg2+ transport and its role in cellular Mg2+ homeostasis. To assess the role of SLC41A3 for cellular Mg2+ homeostasis and gain insight into the regulation of transport activity and/or membrane insertion, knowledge about the precise cellular localization and the identification of the binding partners are essential. The present data reveal a specific mitochondrial localization for SLC41A3 and its function as mitochondrial Mg2+ efflux system. They further suggest that the effect of insulin on intracellular Mg2+ stores is most likely mediated via SLC41A3. Given that mitochondria serve as intracellular Mg2+ stores, a mitochondrial dysfunction might affect cellular Mg2+ homeostasis and this could be one reason for intracellular Mg2+ deficiency observed in diseases such as diabetes, PD and hypertension. The last part of the project focused on the physiological characterization of two isoforms of another Mg2+ responsive gene, CNNM2, because a mutation in this gene has been recently associated with severe familial hypomagnesaemia. A previous study showed that CNNM2 is overexpressed in diabetic patients (114) but its expression does not correlate with Mg2+ plasma levels. However, the present study shows an overexpression of CNNM2 in Jurkat and JVM-13 cells after Mg2+ starvation. The protein has an extensive localization in the cells, including the mitochondrial membrane, and its putative interactors include proteins involved in the regulation of mitochondrial homeostasis (mitophagy, clearance of ROS). A further question was whether the two isoforms (I1 and I2) of CNNM2 are able to transport Mg2+, but the data presented herein clearly indicate that CNNM2 transports Mg2+ neither in electrogenic nor in electroneutral mode in transgenic HEK293 cells overexpressing I1 or I2. This strongly suggests that CNNM2 might represent the first magnesium homeostatic factor without being a Mg2+ transporter per se. Instead CNNM2 can be postulated to sense the changes in extracellular and/or intracellular Mg2+ concentration and consequently activates other proteins responsible for Mg2+ mobilization in the cell. From these data a role of CNNM2 in intracellular Mg2+ homeostasis can be assessed and it can be speculated that the two SLC41 proteins act cooperatively with CNNM2-mediated Mg2+ sensing in controlling the cellular magnesium homeostasis.Es ist allgemein anerkannt, dass ein Magnesiummangel oder eine veränderte intrazelluläre Mg2+-Homöostase (IMH) viele pathophysiologische Zustände auslösen kann. Daher ist die einwandfreie Funktion von Mg2+-Transportern und Mg2+-Kanälen für physiologische Zellfunktionen essenziell. Mutationen in Mg2+-regulierten Genen (MRG) können eine Hypomagnesiämie induzieren, die bei vielen Erkrankungen zu Komplikationen führen kann. Basierend auf früheren Daten, die SLC41A1 als einen Na+/Mg2+-Austauscher in der Plasmamembran charakterisiert haben und als Gen, das bei Frauen mit Präeklampsie überexprimiert ist, war das Ziel dieser Arbeit eine weitere Charakterisierung von SLC41A1 und des durch dieses Protein vermittelten Mg2+-Effluxes unter pathophysiologischen Zuständen. Ein weiteres Ziel dieser Arbeit war die funktionelle Charakterisierung der MRGs CNNM2 und SLC41A3 im Hinblick auf ihre Bedeutung für die intrazelluläre Mg2+-Homöostase. Insbesondere sollten mögliche Beziehungen zwischen den Mechanismen des Mg2+-Transportes über die Plasmamembran bzw. zwischen intrazellulären Kompartimenten mit mitochondrialer Dysfunktion und bestimmten Krankheitszuständen (z.B.: Morbus Parkinson, Diabetes, etc.) aufgedeckt werden. Ausgehend von früheren Ergebnissen von Kolisek et al., die SLC41A1 als den Hauptmechanismus für den Mg2+-Efflux aus der Zelle beschrieben hatten, untersuchte der erste Teil der Studie die Fähigkeit von SLC41A1 Proteinkomplexe zu bilden. Emopamil binding protein (EBP) und andere Vertreter der SLC Superfamilie wurden als potentielle Bindungspartner identifiziert. In weiteren Experimenten wurde die Transportaktivität der mit Morbus Parkinson assoziierten SLC41A1 Variante pA350V evaluiert und gezeigt, dass es sich um eine „gain-offunction“ Mutation handelt, die im Vergleich zum Wildtyp-Protein einen erhöhten Mg2+-Efflux aus der Zelle bedingt. Anschließend wurde die Fragestellung untersucht, ob die Transportaktivität von SLC41A1 durch Insulin beeinflusst werden kann, da bei Diabetes-Patienten oft eine Hypomagnesiämie beobachtet wird. Die vorliegenden Ergebnisse zeigen, dass Insulin den SLC41A1-vermittelten Mg2+-Efflux reduziert und offensichtlich einen Effekt auf intrazelluläre Mg2+-Speicher hat, da ein früheres Einsetzen der Mg2+-Freisetzung aus intrazellulären Speichern beobachtet wurde. Im zweiten Teil der Arbeit wurde eine stabil transfizierte, SLC41A3-überexprimierende HEK293 Zelllinie verwendet, um die Fähigkeit dieses Proteins, Mg2+ zu transportieren, näher zu untersuchen und seine Rolle in der zelluläre Mg2+-Homöostase zu charakterisieren. Um die Regulation der Transportaktivität und die Rolle von SLC41A3 in der zelluläre Mg2+-Homöostase zu verstehen, war es notwendig die zelluläre Lokalisation und etwaige Bindungspartner zu identifizieren. Die vorliegenden Daten zeigen eine spezifische Lokalisation in den Mitochondrien und eine Funktion als mitochondriales Mg2+-Efflux System. Diese Ergebnisse legen nahe, dass der beobachtete Effekt von Insulin auf intrazelluläre Mg2+-Speicher wahrscheinlich durch SLC41A3 vermittelt wird. Vorausgesetzt, dass Mitochondrien als intrazelluläre Mg2+-Speicher fungieren, kann eine mitochondriale Dysfunktion auch die zelluläre Mg2+-Homöostase beeinflussen und ein Grund für die bei Krankheiten wie Diabetes, Morbus Parkinson oder Bluthochdruck beobachtete intrazelluläre Mg2+-Defizienz sein. Der letzte Teil der Arbeit konzentrierte sich auf die physiologische Charakterisierung zweier Isoformen des Mg2+-regulierten Gens CNNM2 (I1 und I2). Mutationen in diesem Gen sind mit schwerer, familiärer Hypomagnesiämie assoziiert. Eine vorangegangene Studie hatte gezeigt, dass CNNM2 in Diabetes-Patienten überexprimiert war, die Expression aber nicht mit den Mg2+- Spiegel im Plasma korrelierte. In der vorliegenden Arbeit konnte aber eine Überexpression von CNNM2 in Jurkat und JVM-13 Zellen nach Mg2+-Depletion gezeigt werden. Das Protein war in der Zelle weit verbreitet, auch in Mitochondrien, und seine potentiellen Interaktionspartner umfassen Proteine, die an der Regulation der Mitochondrien-Homöostase (Mitophagie, Beseitigung reaktiver Sauerstoffspezies) beteiligt sind. Es wurde auch untersucht, ob die beiden CNNM2-Isoformen in der Lage sind, Mg2+ zu transportieren. Die hier präsentierten Daten zeigen eindeutig, dass in HEK293 Zellen, die CNNM2 überexprimieren, das Protein weder in elektrogener noch in elektroneutraler Weise Mg2+ transportiert. Diese Ergebnisse legen nahe, dass CNNM2 der erste Mg2+-homöostatische Faktor ist, der selbst keine Transportaktivität besitzt. Das Protein scheint die extrazelluläre und/oder intrazelluläre Mg2+-Konzentration zu messen und darauffolgend andere Proteine, die für die Mg2+-Mobilisierung in der Zelle verantwortlich sind, zu aktivieren. Diese Daten charakterisieren CNNM2 als zentralen Faktor für die intrazelluläre Mg2+-Homöostase und es kann angenommen werden, dass die beiden SLC41 Proteine kooperativ mit dem Mg2+-Sensor CNNM2 die zelluläre Mg2+-Homöostase regulieren

Fixation, description and DNA barcode of a neotype for Botryllus schlosseri (Pallas, 1766) (Tunicata, Ascidiacea)

Botryllus schlosseri is a widespread colonial ascidian commonly considered cosmopolitan and amply used as model for researches ranging from developmental biology to immunobiology. Recently, molecular data lead to hypothesize that the species named B. Schlosseri may consist of more than a single taxon. Indeed, five highly divergent clades, named A-E, have been genetically identified and are referred as cryptic species. In this context, and lacking both a type and a detailed morphological description, we believe that it is necessary, as a taxonomic reference point, to designate a neotype and re-describe the species. Therefore, a sample from the Lagoon of Venice (Adriatic Sea, Italy) was deposited as neotype in the Natural History Museum of Venice (Italy), preserved both in formalin and in 90% ethanol. Here we provide a morpholo-gical description of the suggested neotype of B. Schlosseri that takes into account several developmental stages (oozooid, zooid of first blastogenic generations, and mature zooid) and is carefully compared with the previous descriptions of sam-ples coming from other European and non-European localities. Finally, we associate our morphological description to a "DNA barcode", consisting in a long fragment of the mitochondrial COI gene. Our description is associated to clade A, although at now we cannot guarantee that this association is univocal