NaKtide Targeted to Adipocytes Ameliorates Western Diet Induced Obesity

Obesity has become a worldwide epidemic and is a major risk factor for metabolic syndrome. It is believed that cellular oxidant stress plays a key role in both the development and maintenance of obesity as well as its associated comorbidities such as diabetes, cardiovascular disease, and nonalcoholic steatohepatitis (NASH). We have previously reported that systemic administration of pNaKtide, which targets the Na/K-ATPase oxidant amplification loop was able to decrease oxidative stress and adiposity in mice fed a high fat and fructose supplemented western diet (WD). As adipocytes are believed to play an active role in the development of obesity and its related comorbidities, we examined whether lentiviral-mediated adipocyte-specific expression of NaKtide, the portion of pNaKtide derived from the α1 Na/K-ATPase N domain without the TAT leaders sequence used to make pNaKtide cell permeant, could have a similar ameliorative effect. To test this hypothesis, studies were first performed in isolated murine preadipocytes, 3T3-L1 cells; it was found that specific activation of Na/K-ATPase signaling (with the cardiotonic steroid ouabain which is specific to the Na/K-ATPase) significantly increased adipogenesis in 3T3-L1 cells in the presence of adipogenic medium, and pNaKtide administration attenuated oxidative stress and lipid accumulation. When administered through a lentiviral construct, NaKtide showed a similar decrease in oxidative stress. Complementary in vivo studies were performed as well. C57BL6 mice fed a WD activated adipocyte Na/K-ATPase signaling and increased adiposity, systemic oxidative stress and insulin resistance as well as induced development of NASH. Also of note, WD increased the plasma levels of leptin, IL-6 and TNFα along with decreased locomotor activity, expression of the D2 receptor and tyrosine hydroxylase in defined brain regions as well as markers of neurodegeneration including neuronal apoptosis. Selective adipocyte expression of NaKtide in these mice fed a WD attenuated all of these changes including the brain biochemical alterations and behavioral adaptations. These data suggest that adipocyte derived cytokines play an essential role in the development of obesity induced by a WD and that targeting the adipocyte Na/K-ATPase oxidant amplification loop (NKAL) may serve as an effective therapeutic strategy

Observing, Understanding, and Manipulating Biological Membranes

The phospholipid bilayer is one of the hallmarks of eukaryotic life. This complicated two dimensionally fluid surface is composed of a double layer of lipids which have a region that is hydrophobic and a region that is hydrophilic. The lipid bilayer membranes of a cell act as a barrier that distinguishes the cells and the organelles interiors from the outside environment. In order for the cell to be able to effectively communicate across these impermeable barriers they have evolved many intricate systems of lipid and protein interaction that serve to transmit information from one side of the membrane to the other. The abundance of functionality in the membrane has made them incredibly complicated and intricate structures. This complexity makes the study of any one membrane associated signaling pathway or system more often than not very challenging if not impossible. Because of this a simplified analogue of the biological membrane is necessary for controlled and quantitative studies. The amphiphilic nature of phospholipids allows researcher to create lipid bilayer that can be controlled for composition and placed on a surface that is accessible to many investigative systems. These supported lipid bilayer (SLB) systems have been used for decades to figure out the intricate series of actions and reactions that occur in a natural biomembrane. While there is still a lot to be learned from simple lipid bilayer systems there is also now the need to produce bilayers that incorporate more of the functions of a living cell. These will permit the study of signaling systems involving multiple molecules, the direct observation of cellular responses to surface stimuli, and even the control of cellular behavior. Along with providing greater versatility in the study of cells and membrane mediated systems these enhanced lipid bilayers will have major biomedical applications. Given that a lipid bilayer is the exterior presented by cells in nature there can be no more biocompatible surface than a lipid bilayer, for the surface treatment of medical devices this could have great implications. For instance, if sufficient knowledge is developed about these systems, then all types of medical implants could have a customized lipid bilayer based coating that would help it to integrate perfectly with the tissue to which it is embedded. Joint replacements could have a surface that would promote the growth of osteoblast cells, a cardiac stent could have a coating to prevent clot formation while promoting epithelial cell growth and discouraging smooth muscle cell growth, perhaps even neural implants capable of two way communication for the treatment of paralysis. It is with this long term vision that we set out to advance the field of lipid bilayer systems to increase the capacity for dynamic control, artificial enhancement, and tissue interface. A series of investigations were undertaken with the goal of promoting each one of these facets. In the first investigation we studied the dynamic behavior of the PIP2 phospholipid in varying physiological calcium concentrations. This anionic lipid has been hypothesized to have the capacity to organize itself spatially in response to fluctuations in calcium levels. No investigation has been so far carried out that look at PIP2s reaction to physiologically relevant changes in the calcium concentration. We used fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis to look at changes in the dynamics and the brightness of PIP2 in polymer supported lipid bilayers. We found that PIP2 appears to make electrostatic associations with zwitterionic lipids in the bilayer when there is no calcium present which are disrupted with the addition of calcium. In the second investigation we developed a genetically encoded protein/lipid anchoring system based on an aldehyde. This system allows proteins to be genetically modified to bear a 6 amino acid consensus sequence at, theoretically any position along its amino acid chain. This consensus sequence targets a cysteine within the sequence to a formylglycine generating enzyme (FGE) that converts the cysteine to a formylglycine. Formylglycine bears an aldehyde on one of its residues. Aldehydes are found very rarely in the mammals and are also highly reactive with certain elements such as aminooxy and hydrazides which are also very rare in mammalian biology. This makes an aldehyde an excellent anchoring method for medical applications. We produced aldehyde tagged enhanced green fluorescent proteins (EGFP) and successfully incorporated them with aminooxy modified lipids in a supported lipid bilayer. This showed that the system is a viable and in fact improved alternative to the lipid/protein anchoring systems currently in use. The third investigation centered on the capacity to culture cells on a SLB surface. Previous studies have found that SLBs have an anti-adhesion property for both proteins and cells. We investigated whether the inclusion of various quantities of both positively and negatively charged lipid into the bilayer would effect the capacity of fibroblast cells to adhere and proliferate. Our system prototyped a novel high throughput technique for bilayer/cell investigations. We found that cells initially had difficulty in adhering to all bilayer surfaces but the inclusion of greater quantities of negatively charged lipids produced a more favorable environment for cell growth. We also looked at how glycolipids, commonly held to be promoters of cell recognition and adhesion, effected fibroblast cells. Such bilayers with sugar elaborations were as suitable for fibroblast growth as the control subjects on collagenated glass. These findings are a significant advancement in the development of bilayer - cell interfaces. This body of work has provided exciting advances in the capacity to produce and understand biomimetic surfaces. While there is much work to be done yet before fully interfacial surfaces are possible, I have developed some novel tools and unearth some interesting findings that can provide some of the next steps towards this goa

Metabolic Plasticity in the Cellular Stress Response

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that 13C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained to a remarkable extent during an ischemic time course. These findings suggested an unexpected source of glucose in HIF-1α hearts during global ischemia. Accordingly, the presence of gluconeogenesis in hearts was evaluated. Indeed, gluconeogenic intermediates (i.e. m+3) including glucose-6-phosphate [m+3], fructose-6-phosphate [m+3], and fructose 1,6-bisphosphate [m+3] were observed at significantly elevated levels in the ischemic HIF-1α heart. Collectively, these data establish the surprising finding that HIF-1α supports active gluconeogenesis in the heart during ischemia. As less is known regarding the effects of CTRP3 we first tested whether CTRP3 overexpression would protect the ischemic heart. Our data indicate that CTRP3 failed to confer ischemic tolerance in heart ex vivo. However,we were able to show that CTRP3 protected the liver from lipid-induced stress and prevented hepatic lipid accumulation. To further investigate the mechanisms of hepatic protective effect mediated by CTRP3, we identified the receptor and established that CTRP3 increases oxygen consumption in response to lipid overloaded. Lysosomal-associated membrane protein 1 (LAMP-1), In summary, these data indicate that targeted metabolic rearrangements within cardiomyocyte/hepatocyte holds promise for the alleviation of common pathological conditions

IST Austria Thesis

Directed cell migration is a hallmark feature, present in almost all multi-cellular organisms. Despite its importance, basic questions regarding force transduction or directional sensing are still heavily investigated. Directed migration of cells guided by immobilized guidance cues - haptotaxis - occurs in key-processes, such as embryonic development and immunity (Middleton et al., 1997; Nguyen et al., 2000; Thiery, 1984; Weber et al., 2013). Immobilized guidance cues comprise adhesive ligands, such as collagen and fibronectin (Barczyk et al., 2009), or chemokines - the main guidance cues for migratory leukocytes (Middleton et al., 1997; Weber et al., 2013). While adhesive ligands serve as attachment sites guiding cell migration (Carter, 1965), chemokines instruct haptotactic migration by inducing adhesion to adhesive ligands and directional guidance (Rot and Andrian, 2004; Schumann et al., 2010). Quantitative analysis of the cellular response to immobilized guidance cues requires in vitro assays that foster cell migration, offer accurate control of the immobilized cues on a subcellular scale and in the ideal case closely reproduce in vivo conditions. The exploration of haptotactic cell migration through design and employment of such assays represents the main focus of this work. Dendritic cells (DCs) are leukocytes, which after encountering danger signals such as pathogens in peripheral organs instruct naïve T-cells and consequently the adaptive immune response in the lymph node (Mellman and Steinman, 2001). To reach the lymph node from the periphery, DCs follow haptotactic gradients of the chemokine CCL21 towards lymphatic vessels (Weber et al., 2013). Questions about how DCs interpret haptotactic CCL21 gradients have not yet been addressed. The main reason for this is the lack of an assay that offers diverse haptotactic environments, hence allowing the study of DC migration as a response to different signals of immobilized guidance cue. In this work, we developed an in vitro assay that enables us to quantitatively assess DC haptotaxis, by combining precisely controllable chemokine photo-patterning with physically confining migration conditions. With this tool at hand, we studied the influence of CCL21 gradient properties and concentration on DC haptotaxis. We found that haptotactic gradient sensing depends on the absolute CCL21 concentration in combination with the local steepness of the gradient. Our analysis suggests that the directionality of migrating DCs is governed by the signal-to-noise ratio of CCL21 binding to its receptor CCR7. Moreover, the haptotactic CCL21 gradient formed in vivo provides an optimal shape for DCs to recognize haptotactic guidance cue. By reconstitution of the CCL21 gradient in vitro we were also able to study the influence of CCR7 signal termination on DC haptotaxis. To this end, we used DCs lacking the G-protein coupled receptor kinase GRK6, which is responsible for CCL21 induced CCR7 receptor phosphorylation and desensitization (Zidar et al., 2009). We found that CCR7 desensitization by GRK6 is crucial for maintenance of haptotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. In the context of the organism, immobilized haptotactic guidance cues often coincide and compete with soluble chemotactic guidance cues. During wound healing, fibroblasts are exposed and influenced by adhesive cues and soluble factors at the same time (Wu et al., 2012; Wynn, 2008). Similarly, migrating DCs are exposed to both, soluble chemokines (CCL19 and truncated CCL21) inducing chemotactic behavior as well as the immobilized CCL21. To quantitatively assess these complex coinciding immobilized and soluble guidance cues, we implemented our chemokine photo-patterning technique in a microfluidic system allowing for chemotactic gradient generation. To validate the assay, we observed DC migration in competing CCL19/CCL21 environments. Adhesiveness guided haptotaxis has been studied intensively over the last century. However, quantitative studies leading to conceptual models are largely missing, again due to the lack of a precisely controllable in vitro assay. A requirement for such an in vitro assay is that it must prevent any uncontrolled cell adhesion. This can be accomplished by stable passivation of the surface. In addition, controlled adhesion must be sustainable, quantifiable and dose dependent in order to create homogenous gradients. Therefore, we developed a novel covalent photo-patterning technique satisfying all these needs. In combination with a sustainable poly-vinyl alcohol (PVA) surface coating we were able to generate gradients of adhesive cue to direct cell migration. This approach allowed us to characterize the haptotactic migratory behavior of zebrafish keratocytes in vitro. Furthermore, defined patterns of adhesive cue allowed us to control for cell shape and growth on a subcellular scale

Role of the Neutral Amino Acid Transporter SLC7A10 in Adipocyte Lipid Storage, Obesity and Insulin Resistance

Elucidation of mechanisms that govern lipid storage, oxidative stress, and insulin resistance may lead to improved therapeutic options for type 2 diabetes and other obesity-related diseases. Here, we find that adipose expression of the small neutral amino acid transporter SLC7A10, also known as alanine-serine-cysteine transporter-1 (ASC-1), shows strong inverse correlates with visceral adiposity, insulin resistance, and adipocyte hypertrophy across multiple cohorts. Concordantly, loss of Slc7a10 function in zebrafish in vivo accelerates diet-induced body weight gain and adipocyte enlargement. Mechanistically, SLC7A10 inhibition in human and murine adipocytes decreases adipocyte serine uptake and total glutathione levels and promotes reactive oxygen species (ROS) generation. Conversely, SLC7A10 overexpression decreases ROS generation and increases mitochondrial respiratory capacity. RNA sequencing revealed consistent changes in gene expression between human adipocytes and zebrafish visceral adipose tissue following loss of SLC7A10, e.g., upregulation of SCD (lipid storage) and downregulation of CPT1A (lipid oxidation). Interestingly, ROS scavenger reduced lipid accumulation and attenuated the lipid-storing effect of SLC7A10 inhibition. These data uncover adipocyte SLC7A10 as a novel important regulator of adipocyte resilience to nutrient and oxidative stress, in part by enhancing glutathione levels and mitochondrial respiration, conducive to decreased ROS generation, lipid accumulation, adipocyte hypertrophy, insulin resistance, and type 2 diabetes.acceptedVersio

Studies of the content and distribution of glutathione and glutathione S-transferases in cells

The Glutathione S-transferases (GSTs; EC 2.5.1.18) are a multigene family of dimeric isoenzymes which catalyse a variety of reactions utilizing the tripeptide glutathione (GSH; γ-Glu-Cys-Gly). Many of these, such as the conjugation of GSH to xenobiotic and endogenous electrophiles and the reduction of organic hydroperoxides, are involved in cellular detoxification and the GSTs are of particular interest for their anti-carcinogenic action. This thesis is concerned with the application of a variety of techniques to investigate the content and intracellular distribution of GSH and GSTs in rat liver, in cell lines derived from rat liver (IAR20 & IAR6.1) and mouse fibroblasts (Balb 3T3), and in two human tumour cell lines (HeLa & MCF7). Of particular interest was the controversial presence of the GSTs in the nucleus, which may be linked to their ability to protect against chemical carcinogenesis. The GSH and GST isoenzyme contents of these five cell lines were determined and the subcellular distribution of the GSTs was investigated using immunocytochemistry. Some evidence for a nuclear localization was obtained. However, analysis of the intracellular distribution of GSTs in rat liver using subcellular fractionation was inconclusive. Monochlorobimane (MCB), which undergoes GST-catalysed conjugation to GSH to yield a fluorescent product (MB-SG), was tested in conjunction with flow cytometry as a possible means of measuring cellular GSH. The GST enzyme kinetics were first analysed and considerable isoenzyme specificity towards MCB was observed. It was predicted that the kinetics of MCB conjugation in the above cell lines would vary according to their GSH and GST isoenzyme contents, and this was confirmed using flow cytometry. The implications of this for the use of MCB to measure cellular GSH are discussed. MCB-stained cell lines and primary rat hepatocytes were examined by fluorescence microscopy. Fluorescence was observed over the whole cell, but was often brighter over the nucleus. However, when the MB-SG conjugate was microinjected into the cytoplasm of primary rat hepatocytes, it diffused rapidly through the cell and into the nucleus, producing a very similar pattern of fluorescence to that observed using MCB. Therefore, MCB-staining may not indicate the true intracellular distribution of GSH and the GSTs

Extracellular Ubiquitin: Role in Cardiac Myocyte Apoptosis and Myocardial Remodeling

Activation of sympathetic nervous system is a key component of myocardial remodeling that generally occurs following ischemia/reperfusion (I/R) injury and myocardial infarction. It induces cardiac myocyte apoptosis and myocardial fibrosis, leading to myocardial dysfunction. Intracellular ubiquitin (UB) regulates protein turnover by the UB-proteosome pathway. The biological functions of extracellular UB in the heart remain largely unexplored. Previously, our lab has shown that β-adrenergic receptor (β-AR) stimulation increases extracellular UB levels, and extracellular UB inhibits β-AR-stimulated apoptosis in adult rat ventricular myocytes (ARVMs). This study explores the role of extracellular UB in myocyte apoptosis, fibroblast phenotype and function, and myocardial remodeling following β-AR stimulation and I/R injury. First, left ventricular (LV) structural and functional remodeling was studied 7 days after chronic β-AR-stimulation in the presence or absence of UB infusion. Echocardiographic analyses showed UB infusion decreases β-AR-stimulated increases in percent fractional shortening and ejection fraction. It decreased cardiac myocyte apoptosis and myocardial fibrosis. UB activated Akt, and inhibition of Akt inhibited β-AR-stimulated increases in matrix metalloproteinase-2 expression. Second, using cardiac fibroblasts, we provide evidence that extracellular UB interacts with the cell surface and co-immunoprecipitates with CXCR4. UB treatment increased expression of α-smooth muscle actin (myofibroblast marker), and induced rearrangement of actin into stress fibers. It inhibited lamellopodia and filopodia formation, and cell migration into the wound. Third, using isolated mouse heart and I/R injury as a model, we provide evidence that UB treatment decreases I/R-mediated increases in infarct size. UB treatment improved functional recovery of the heart as measured by increased % LV developed pressure. Activation of proapoptotic proteins, p-STAT-1 and caspase-9, was significantly lower in UB I/R hearts versus I/R alone. In ARVMs, UB treatment decreased simulated I/R-induced apoptosis. It activated Akt (anti-apoptotic kinase) and inhibited activation of GSK-3β (pro-apoptotic kinase). It decreased I/R-induced oxidative stress and protected anoxia-induced mitochondrial polarization. In fibroblast and ARVMs, CXCR4 antagonism negated the effects of UB, while mutated UBs (unable to interact with CXCR4) had no effect. Thus, extracellular UB, most likely acting via CXCR4, modulates myocardial remodeling with effects on heart function, fibroblast phenotype and function and myocyte apoptosis

Investigation of the LRRC8 subunit composition and the activating signal transduction of the volume-regulated anion channel (VRAC)

The volume-regulated anion channel (VRAC) plays a key role in the regulation of osmotic cell volume as well as in various physiological processes such as apoptosis, insulin secretion, cell differentiation, and purinergic signaling. It is formed by hetero-hexamers of members of the leucine-rich repeat-containing protein family 8 (LRRC8), which consists of five members, LRRC8A-E. LRRC8A is the obligatory subunit, and its heteromerization with at least one other LRRC8 paralogue, LRRC8B-E, determines VRAC's biophysical properties. Subunit stoichiometry of VRAC is of physiological importance and largely influences its activation mechanism, as well as its response to regulatory inputs. However, the endogenous tissue-specific subunit composition of VRAC remains unknown. Furthermore, despite extensive research on VRAC's possible physiological functions, there is little consensus on its activation mechanism. In this thesis, I developed and applied a quantitative immunoblot method to quantify the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using glutathione-S-transferase (GST)-tagged recombinant fusion proteins for signal calibration. The subunits showed tissue-specific expression patterns, with relatively low expression of the obligatory LRRC8A subunit. Based on the co-immunoprecipitation of LRRC8B-E in excess with LRRC8A, I concluded that non-LRRC8A subunits predominate in native hetero-hexamers. In light of this information, I estimated ~10,000 VRACs per cell in the tested cell lines, which is consistent with an earlier calculation from the comparison of single-channel and whole-cell currents. Furthermore, I assessed VRAC activity by a Förster-resonance energy transfer (FRET)-based approach upon induction of apoptosis, sphingosine-1-phosphate (S1P)-induced signaling, and glucose feeding in pancreatic β-cells. I found that the pharmacological inhibition of protein kinase D (PKD) impaired the apoptotic-induced VRAC activation. Interestingly, signaling via S1P appeared to be mediated by an alliance between S1P receptors, specifically the Gq-coupled S1P receptors. I proposed that the Gq family of heterotrimeric G-proteins served as central mediators of the diacylglycerol (DAG)-PKD mediated VRAC activation induced by S1P in HeLa cells. PKD may phosphorylate VRAC, thereby activating it. This notion is supported by the observation that hypotonic activation of phospho-ablative LRRC8A mutant 8A-T169A is diminished due to the loss of the putative phosphorylation site. Lastly, I showed that an orphan G protein-coupled receptor (GPCR), GPCR5B, adversely modulated the VRAC activity in rat pancreatic β-cells, which may affect β-cell survival and insulin secretion. All in all, these results indicate a possible signaling pathway of VRAC activation, highlighting the importance of membrane-localized GPCRs and G-proteins in signal transduction.Die volumenregulierten Anionenkanäle (VRAC) spielen eine Schlüsselrolle bei der osmotischen Regulation des Zellvolumens sowie bei verschiedenen physiologischen Prozessen wie Apoptose, Insulinsekretion, Zelldifferenzierung und purinerger Signalübertragung. Es wird von Hetero-Hexameren der Leucine-Rich Repeat-Containing Protein Family 8 (LRRC8) gebildet, die aus fünf Mitgliedern, LRRC8A-E, besteht. LRRC8A ist die obligatorische Untereinheit, und ihre Heteromerisierung mit mindestens einem anderen LRRC8-Paralog, LRRC8B-E, bestimmt biophysikalische Eigenschaften von VRAC. Die Untereinheiten-Stöchiometrie von VRAC ist von physiologischer Bedeutung und beeinflusst weitgehend seinen Aktivierungsmechanismus sowie seine Reaktion auf regulatorische Signale. Die endogene gewebespezifische Untereinheitenzusammensetzung von VRAC ist jedoch weitestgehend unbekannt. Darüber hinaus gibt es trotz umfangreicher Forschung über die möglichen physiologischen Funktionen von VRAC wenig Konsens darüber, wie er aktiviert wird. In dieser Arbeit habe ich eine quantitative Immunoblot-Methode entwickelt und angewandt, um die fünf VRAC LRRC8-Untereinheiten in verschiedenen Maus-Zelllinien und -Geweben zu quantifizieren, wobei rekombinante Fusionsproteine mit Glutathion-S-Transferase (GST)-Markierung zur Signalkalibrierung verwendet wurden. Die Untereinheiten zeigten gewebespezifische Expressionsmuster, mit relativ geringer Expression der obligatorischen Untereinheit LRRC8A. Basierend auf der Co-Immunpräzipitation von LRRC8B-E im Überschuss mit LRRC8A schloss ich, dass Nicht-LRRC8A-Untereinheiten in nativen Hetero-Hexameren überwiegen. In Anbetracht dieser Informationen berechnete ich das Vorkommen auf ~10.000 VRACs pro Zelle in den getesteten Zelllinien, was mit einer früheren Berechnung aus dem Vergleich von Einzelkanal- und Ganzzellströmen übereinstimmt. Darüber hinaus habe ich die VRAC-Aktivität mittels Förster-Resonanz-Energie-Transfer (FRET)-basiertem Ansatz bei der Induktion von Apoptose, Sphingosin-1-Phosphat (S1P)-induzierter Signalgebung und Glukose-Stimulation in pankreatischen β-Zellen untersucht. Ich fand heraus, dass die pharmakologische Hemmung der Proteinkinase D (PKD) die apoptose-induzierte VRAC-Aktivierung beeinträchtigte. Interessanterweise wird die Signalisierung durch S1P über eine Allianz zwischen S1P-Rezeptoren, speziell den Gq-gekoppelten S1P-Rezeptoren, vermittelt. Dies deutet darauf hin, dass die Gq-Familie der heterotrimeren G Proteine als zentrale Vermittler der durch S1P induzierten Diacylglycerol (DAG)-PKD-vermittelten VRAC-Aktivierung in HeLa-Zellen dienen. PKD kann VRAC phosphorylieren und ihn dadurch aktivieren. Diese Vorstellung wird durch die Beobachtung unterstützt, dass die hypotone Aktivierung der phospho-ablativen XV LRRC8A-Mutante 8A-T169A aufgrund des Verlustes der möglichen Phosphorylierungsstelle vermindert ist. Schließlich habe ich gezeigt, dass einn orphan G Protein-gekoppelten Rezeptor (GPCR), GPCR5B, die VRAC-Aktivität in pankreatischen β-Zellen der Ratte negativ moduliert und damit das Überleben der β-Zellen und die Insulinsekretion beeinflusst. Alles in allem weisen diese Ergebnisse auf einen möglichen Signalweg der VRAC-Aktivierung hin und unterstreichen die Bedeutung von membranständigen GPCRs und G Proteinen in der Signaltransduktion

The anti-ageing potential of rooibos: preserving preadipocyte funtion

Treatments with natural products rich in anti-oxidants have attracted remarkable interest in the cosmetic and pharmaceutical industry to combat oxidative stress and reverse the effects of ageing. Rooibos (Aspalathus linearis) is a South African fynbos plant, well-known for its strong anti-oxidant capacity and use in many cosmetic products. However, little published research exists on its potential as an anti-ageing treatment. The anti-ageing properties of fermented and green rooibos were investigated using an in vitro cell culture model designed to evaluate the involvement of mitochondrial dysfunction in the age related decline in preadipocyte function. Mitochondrial DNA (mtDNA) deficient preadipocytes, ρ0 3T3-L1preadipocytes, were generated following continuous long-term exposure to sub lethal concentrations of ethidium bromide (EtBr). Depletion of the mtDNA resulted in a significantly reduced mitochondrial membrane potential, rate of proliferation in culture, as well as an increased glucose utilization and lactate production. Treatment with the green rooibos (100 μg/mL) stimulated cell growth rates for both the wildtype and mutant cell lines. MtDNA depleted cells showed arrest in the G1 phase (48.8 ± 3.34%) compared to wildtype cells (44.6 ± 1.38%), which was significantly attenuated after treatment with green rooibos for mutant (42.0 ± 0.83%) and wildtype (36.5 ± 5.80%) treated cells. The results obtained for glucose utilization and lactate production, indicated a significant increase in glucose utilization along with a concomitant increase in lactate production after treatment with both green and fermented rooibos for wildtype and mutant cell lines. A significant improvement in mitochondrial membrane potential was also later observed after treatment with green and fermented rooibos on both the wildtype and mutant cell lines. The results obtained indicate that rooibos extracts, particularly the green rooibos, exhibit effects which preserve the functional capacity of preadipocytes exposed to ageing related insults, and indicate that rooibos could cause a metabolic shift in cells redirecting carbon flow away from mitochondrial metabolism, and towards lactate production and consequently, cells become resistant to mitochondrial dysfunction