RESUMEN

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.

Asunto(s)

GTP Fosfohidrolasas , Fusión de Membrana , Animales , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Ratones , Fusión de Membrana/fisiología , Liposomas Unilamelares/metabolismo , Liposomas Unilamelares/química , Guanosina Trifosfato/metabolismo , Fosfatidiletanolaminas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mitocondrias/metabolismo

RESUMEN

BACKGROUND AND AIM: The excessive accumulation of lipid droplets (LDs) is a defining characteristic of nonalcoholic fatty liver disease (NAFLD). The interaction between LDs and mitochondria is functionally important for lipid metabolism homeostasis. Exercise improves NAFLD, but it is not known if it has an effect on hepatic LD-mitochondria interactions. Here, we investigated the influence of exercise on LD-mitochondria interactions and its significance in the context of NAFLD. APPROACH AND RESULTS: Mice were fed high-fat diet (HFD) or HFD-0.1 % methionine and choline-deficient diet (MCD) to emulate simple hepatic steatosis or non-alcoholic steatohepatitis, respectively. In both models, aerobic exercise decreased the size of LDs bound to mitochondria and the number of LD-mitochondria contacts. Analysis showed that the effects of exercise on HOMA-IR and liver triglyceride levels were independent of changes in body weight, and a positive correlation was observed between the number of LD-mitochondria contacts and NAFLD severity and with the lipid droplet size bound to mitochondria. Cellular fractionation studies revealed that ATP-coupled respiration and fatty acid oxidation (FAO) were greater in hepatic peridroplet mitochondria (PDM) from HFD-fed exercised mice than from equivalent sedentary mice. Finally, exercise increased FAO and mitofusin-2 abundance exclusively in PDM through a mechanism involving the curvature of mitochondrial membranes and the abundance of saturated lipids. Accordingly, hepatic mitofusin-2 ablation prevented exercise-induced FAO in PDM. CONCLUSIONS: This study demonstrates that aerobic exercise has beneficial effects in murine NAFLD models by lessening the interactions between hepatic LDs and mitochondria, and by decreasing LD size, correlating with a reduced severity of NAFLD. Additionally, aerobic exercise increases FAO in PDM and this process is reliant on Mfn-2 enrichment, which modifies LD-mitochondria communication.

Asunto(s)

Enfermedad del Hígado Graso no Alcohólico , Animales , Masculino , Ratones , Dieta Alta en Grasa , Ácidos Grasos/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo

RESUMEN

The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria. We found that endoplasmic reticulum stress-activated c-Abl interacts with and phosphorylates the mitochondrial fusion protein MFN2, resulting in mitochondrial fragmentation and apoptosis. Moreover, the pharmacological or genetic inhibition of c-Abl prevents MFN2 phosphorylation, mitochondrial fragmentation, and apoptosis in cells under endoplasmic reticulum stress. Finally, in the amyotrophic lateral sclerosis mouse model, where endoplasmic reticulum and oxidative stress has been linked to neuronal cell death, we demonstrated that the administration of c-Abl inhibitor neurotinib delays the onset of symptoms. Our results uncovered a function of c-Abl in the crosstalk between endoplasmic reticulum stress and mitochondrial dynamics via MFN2 phosphorylation.

RESUMEN

Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.

Asunto(s)

Dinámicas Mitocondriales , Hormonas Peptídicas , Humanos , Transducción de Señal , Eicosanoides , Ácido Araquidónico , Hormona Adrenocorticotrópica , Angiotensina II

RESUMEN

There is no effective treatment to halt peripheral nervous system damage in diabetic peripheral neuropathy. Mitochondria have been at the center of discussions as important factors in the development of neuropathy in diabetes. Photobiomodulation has been gaining clinical acceptance as it shows beneficial effects on a variety of nervous system disorders. In this study, the effects of photobiomodulation (904 nm, 45 mW, 6.23 J/cm2, 0.13 cm2, 60 ns pulsed time) on mitochondrial dynamics were evaluated in an adult male rat experimental model of streptozotocin-induced type 1 diabetes. Results presented here indicate that photobiomodulation could have an important role in preventing or reversing mitochondrial dynamics dysfunction in the course of peripheral nervous system damage in diabetic peripheral neuropathy. Photobiomodulation showed its effects on modulating the protein expression of mitofusin 2 and dynamin-related protein 1 in the sciatic nerve and in the dorsal root ganglia neurons of streptozotocin-induced type 1 diabetes in rats.

Asunto(s)

Ganglios Espinales/efectos de la radiación , Láseres de Semiconductores , Dinámicas Mitocondriales/efectos de la radiación , Nervio Ciático/efectos de la radiación , Animales , Glucemia/análisis , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Ganglios Espinales/metabolismo , Masculino , Ratas , Ratas Wistar , Nervio Ciático/metabolismo , Estreptozocina/toxicidad

RESUMEN

Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Proteínas Mitocondriales/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Preparación de Corazón Aislado , Masculino , Mitocondrias/efectos de los fármacos , Infarto del Miocardio/complicaciones , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Cultivo Primario de Células , Inhibidores de Proteasoma/uso terapéutico , Ratas , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

In steroid-producing cells, cholesterol transport from the outer to the inner mitochondrial membrane is the first and rate-limiting step for the synthesis of all steroid hormones. Cholesterol can be transported into mitochondria by specific mitochondrial protein carriers like the steroidogenic acute regulatory protein (StAR). StAR is phosphorylated by mitochondrial ERK in a cAMP-dependent transduction pathway to achieve maximal steroid production. Mitochondria are highly dynamic organelles that undergo replication, mitophagy and morphology changes, all processes allowed by mitochondrial fusion and fission, known as mitochondrial dynamics. Mitofusin (Mfn) 1 and 2 are GTPases involved in the regulation of fusion, while dynamin-related protein 1 (Drp1) is the major regulator of mitochondrial fission. Despite the role of mitochondrial dynamics in neurological and endocrine disorders, little is known about fusion/fission in steroidogenic tissues. In this context, the present work aimed to study the role of angiotensin II (Ang II) in protein subcellular compartmentalization, mitochondrial dynamics and the involvement of this process in the regulation of aldosterone synthesis. We demonstrate here that Ang II stimulation promoted the recruitment and activation of PKCε, ERK and its upstream kinase MEK to the mitochondria, all of them essential for steroid synthesis. Moreover, Ang II prompted a shift from punctate to tubular/elongated (fusion) mitochondrial shape, in line with the observation of hormone-dependent upregulation of Mfn2 levels. Concomitantly, mitochondrial Drp1 was diminished, driving mitochondria toward fusion. Moreover, Mfn2 expression is required for StAR, ERK and MEK mitochondrial localization and ultimately for aldosterone synthesis. Collectively, this study provides fresh insights into the importance of hormonal regulation in mitochondrial dynamics as a novel mechanism involved in aldosterone production.

Asunto(s)

Neoplasias de las Glándulas Suprarrenales/metabolismo , Carcinoma Corticosuprarrenal/metabolismo , Angiotensina II/farmacología , Colesterol/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas Quinasas/metabolismo , Vasoconstrictores/farmacología , Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Neoplasias de las Glándulas Suprarrenales/patología , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/patología , Transporte Biológico , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación , Células Tumorales Cultivadas

RESUMEN

Endoplasmic reticulum (ER) and mitochondria dysfunction contribute to insulin resistance generation during obesity and diabetes. ER and mitochondria interact through Mitofusin 2 (MTF2), which anchors in the outer mitochondrial and ER membranes regulating energy metabolism. Ablation of MTF2 leads to ER stress activation and insulin resistance. Here we determine whether lipotoxic insult induced by saturated lipids decreases MTF2 expression leading to ER stress response in hypothalamus and its effects on insulin sensitivity using in vitro and in vivo models. We found that lipotoxic stimulation induced by palmitic acid, but not the monounsaturated palmitoleic acid, decreases MTF2 protein levels in hypothalamic mHypoA-CLU192 cells. Also, palmitic acid incubation activates ER stress response evidenced by increase in the protein levels of GRP78/BIP marker at later stage than MTF2 downregulation. Additionally, we found that MTF2 alterations induced by palmitic, but not palmitoleic, stimulation exacerbate insulin resistance in hypothalamic cells. Insulin resistance induced by palmitic acid is prevented by pre-incubation of the anti-inflammatory and the ER stress release reagents, sodium salicylate and 4 phenylbutirate, respectively. Finally, we demonstrated that lipotoxic insult induced by high fat feeding to mice decreases MTF2 proteins levels in arcuate nucleus of hypothalamus. Our data indicate that saturated lipids modulate MTF2 expression in hypothalamus coordinating the ER stress response and the susceptibility to insulin resistance.

Asunto(s)

Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipotálamo/citología , Resistencia a la Insulina/fisiología , Neuronas/efectos de los fármacos , Ácido Palmítico/farmacología , Complejo Represivo Polycomb 2/metabolismo , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Dieta Alta en Grasa , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/ultraestructura , Proteína Oncogénica v-akt/metabolismo , Complejo Represivo Polycomb 2/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo

RESUMEN

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)-mitochondria communication, as it allows for a more efficient transfer of Ca(2+) into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER-mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3h of GLP-1 treatment, paralleled by increased Ca(2+) transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca(2+) increases in GLP-1 treated cells. Inhibiting both Ca(2+) release from the ER and Ca(2+) entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER-mitochondria communication in VSMC, resulting in higher mitochondrial activity.

Asunto(s)

Retículo Endoplásmico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Mitocondrias/metabolismo , Miocitos del Músculo Liso/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GTP Fosfohidrolasas , Receptor del Péptido 1 Similar al Glucagón , Glucosa/metabolismo , Glucógeno/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Ratas , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo

RESUMEN

Differentiation and dedifferentiation of vascular smooth muscle cells (VSMCs) are essential processes of vascular development. VSMC have biosynthetic, proliferative, and contractile roles in the vessel wall. Alterations in the differentiated state of the VSMC play a critical role in the pathogenesis of a variety of cardiovascular diseases, including atherosclerosis, hypertension, and vascular stenosis. This review provides an overview of the current state of knowledge of molecular mechanisms involved in the control of VSMC proliferation, with particular focus on mitochondrial metabolism. Mitochondrial activity can be controlled by regulating mitochondrial dynamics, i.e., mitochondrial fusion and fission, and by regulating mitochondrial calcium handling through the interaction with the endoplasmic reticulum (ER). Alterations in both VSMC proliferation and mitochondrial function can be triggered by dysregulation of mitofusin-2, a small GTPase associated with mitochondrial fusion and mitochondrial-ER interaction. Several lines of evidence highlight the relevance of mitochondrial metabolism in the control of VSMC proliferation, indicating a new area to be explored in the treatment of vascular diseases.