Uncoupling and oxidative stress in liver mitochondria isolated from rats with acute iron overload

One hypothesis for the etiology of cell damage arising from iron overload is that its excess selectively affects mitochondria. Here we tested the effects of acute iron overload on liver mitochondria isolated from rats subjected to a single dose of i.p. 500 mg/kg iron-dextran. The treatment increased the levels of iron in mitochondria (from 21 +/- A 4 to 130 +/- A 7 nmol/mg protein) and caused both lipid peroxidation and glutathione oxidation. The mitochondria of iron-treated rats showed lower respiratory control ratio in association with higher resting respiration. The mitochondrial uncoupling elicited by iron-treatment did not affect the phosphorylation efficiency or the ATP levels, suggesting that uncoupling is a mitochondrial protective mechanism against acute iron overload. Therefore, the reactive oxygen species (ROS)/H(+) leak couple, functioning as a mitochondrial redox homeostatic mechanism could play a protective role in the acutely iron-loaded mitochondria.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Background/Aims: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P , 0:01), as well as disrupted protein mobility(P , 0:001). Consistent with increased permeability, cytochrome c was released from the intermembrane space(P , 0:01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P , 0:01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Complex interplay between LPS and IL-10 in dendritic cells: uncoupling of inflammatory chemokine receptors and positive effects on B cell chemokines

Abstract: Protective immune responses against pathogen invasion and transformed cells requires the coordinated action of distinct leukocyte subsets and soluble factors, overall termed immunological network. Among antigen-presenting cells (APC), a crucial role is played by dendritic cells (DC), which initiate, amplify and determine the outcome of the immune response. Micro-environmental conditions profoundly influence DC in such ways that the resulting immune response ranges from successful immune stimulation to abortive response or immune suppression. For instance, the presence in the milieu of anti-inflammatory cytokine interleukin-10 (IL-10) reverts most of the effects mediated on DC by even strong pro-inflammatory agents such as bacterial Lipopolysaccharide (LPS), in terms of differentiation, activation and functions. In an environment containing both LPS and IL-10, uncoupling of receptors for inflammatory chemokines already occurs after a few hours and in a reversible manner on DC, allowing scavenging of chemokines and, consequently, attenuation of the inflammatory process which could be deleterious to the organism. By studying the effects on DC of concomitant stimulation by LPS and IL-10 from the gene expression point of view, we were able to define four distinct transcriptional programs: A. the inhibition of inflammation and immunity, B. the regulation of tissue remodeling, C. the tuning of cytokine/growth factor receptors and G protein-coupled receptors, D. the stimulation of B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrated that IL-10 synergizes with Toll-like receptor ligands for the production of functionally active B cell attracting chemokine CXCL13. Our data provide evidence that the combined exposure of APC to LPS and IL-10, via the production of CXCL13, involves humoral immunity by attracting antibody-producing cells. It is well known that the persistent release of CXCL13 leads to the development of ectopic lymphoid tissue aggregates and production of high levels of antibodies, thus favoring the induction of auto-immunity. Our findings suggest that the IL-10 produced in chronic inflammatory conditions may promote lymphoid tissue neogenesis through increased release of CXCL13. IL-10 is an anti-inflammatory cytokine inhibiting cellular-mediated TH 1-polarized immune responses. In this study we demonstrate that IL- 10 strongly supports the development of humoral immunity. IL-10 and CXCL13 can thus be targets for specific therapies in auto-immune diseases.

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.

Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity

Caloric restriction is the most effective non-genetic intervention to enhance lifespan known to date. A major research interest has been the development of therapeutic strategies capable of promoting the beneficial results of this dietary regimen. In this sense, we propose that compounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the protonophore 2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved serological glucose, triglyceride and insulin levels, decrease of reactive oxygen species levels and tissue DNA and protein oxidation, as well as reduced body weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and describe the first therapeutic intervention capable of effectively reproducing the physiological, metabolic and lifespan effects of caloric restriction in healthy mammals.

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: Uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca2+ at different conditions, a mitochondrial permeability transition (MPT) linked to protein,thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 mu M on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 mu M Ca2+; inhibition of these processes was assessed in non-energized organelles in the presence of 300 mu M t-butyl hydroperoxide plus 500 mu M Ca2+. Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca2+. Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin. (c) 2006 Published by Elsevier B.V..

Intrauterine food restriction alters the expression of uncoupling proteins in brown adipose tissue of rat newborns

A previous study from our laboratory showed that maternal food restriction (MFR) delays thermoregulation in newborn rats. In neonates brown adipose tissue (BAT) is essential for thermogenesis due to the presence of uncoupling proteins (UCPs). The aim of this study was to evaluate the influence of MFR on the UCPs mRNA and protein expression in BAT and skeletal muscle (SM) of the newborn rat. Female Wistar EPM-1 control rats (CON) received chow ad libitum during pregnancy, whereas food-restricted dams (RES) received 50% of the amount ingested by CON. Fifteen hours after birth, the litters were weighed and sacrificed. Blood was collected for hormonal analysis. BAT and SM were used for determination of UCPs mRNA and protein expression, and Ca2+-ATPase sarcoplasmic reticulum (SERCA1). RES pups showed a significant reduction in body weight and fat content at birth. MFR caused a significant increase in the expression of UCP1 and UCP2 in BAT, without changes in UCP3 and SERCA1 expression in BAT and SM. No differences between groups were found for leptin, T4 and glucose levels. RES pups showed increased insulin and decreased T3 levels. The delay in development of thermoregulation previously described in RES animals appears not to result from impairment in thermogenesis, but from an increase in heat loss, since MFR caused low birth weight in pups, leading to greater surface/volume ratio. The higher expression of UCP1 and UCP2 in BAT suggests a compensatory mechanism to increased thermogenesis. (C) 2011 Elsevier Ltd. All rights reserved.

Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling.

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Erythrocyte membrane vesiculation: Model for the molecular mechanism of protein sorting

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.

Kalata B8, a novel antiviral circular protein, exhibits conformational flexibility in the cystine knot motif

The cyclotides are a family of circular proteins with a range of biological activities and potential pharmaceutical and agricultural applications. The biosynthetic mechanism of cyclization is unknown and the discovery of novel sequences may assist in achieving this goal. In the present study, we have isolated a new cyclotide from Oldenlandia affinis, kalata B8, which appears to be a hybrid of the two major subfamilies (Mobius and bracelet) of currently known cyclotides. We have determined the three-dimensional structure of kalata B8 and observed broadening of resonances directly involved in the cystine knot motif, suggesting flexibility in this region despite it being the core structural element of the cyclotides. The cystine knot motif is widespread throughout Nature and inherently stable, making this apparent flexibility a surprising result. Further-more, there appears to be isomerization of the peptide backbone at an Asp-Gly sequence in the region involved in the cyclization process. Interestingly, such isomerization has been previously characterized in related cyclic knottins from Momordica cochinchinensis that have no sequence similarity to kalata B8 apart from the six conserved cysteine residues and may result from a common mechanism of cyclization. Kalata B8 also provides insight into the structure-activity relationships of cyclotides as it displays anti-HIV activity but lacks haemolytic activity. The 'uncoupling' of these two activities has not previously been observed for the cyclotides and may be related to the unusual hydrophilic nature of the peptide.