RESUMEN
Alcohol's impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol's inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD.
Asunto(s)
Ceramidas/metabolismo , Etanol/efectos adversos , Hígado Graso/metabolismo , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Animales , Composición Corporal , Homeostasis/efectos de los fármacos , Ratones , Especificidad de Órganos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTXs), potent neurotoxins that concentrate in fish causing ciguatera fish poisoning (CFP) in humans. While the structures and toxicities of ciguatoxins isolated from fish in the Pacific and Caribbean are known, there are few data on the variation in toxicity between and among species of Gambierdiscus and Fukuyoa. Quantifying the differences in species-specific toxicity is especially important to developing an effective cell-based risk assessment strategy for CFP. This study analyzed the ciguatoxicity of 33 strains representing seven Gambierdiscus and one Fukuyoa species using a cell based Neuro-2a cytotoxicity assay. All strains were isolated from either the Caribbean or Gulf of Mexico. The average toxicity of each species was inversely proportional to growth rate, suggesting an evolutionary trade-off between an investment in growth versus the production of defensive compounds. While there is 2- to 27-fold variation in toxicity within species, there was a 1740-fold difference between the least and most toxic species. Consequently, production of CTX or CTX-like compounds is more dependent on the species present than on the random occurrence of high or low toxicity strains. Seven of the eight species tested (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, Gambierdiscus ribotype 2, G. silvae and F. ruetzleri) exhibited low toxicities, ranging from 0 to 24.5 fg CTX3C equivalents cell-1, relative to G. excentricus, which had a toxicity of 469 fg CTX3C eq. cell-1. Isolates of G. excentricus from other regions have shown similarly high toxicities. If the hypothesis that G. excentricus is the primary source of ciguatoxins in the Atlantic is confirmed, it should be possible to identify areas where CFP risk is greatest by monitoring only G. excentricus abundance using species-specific molecular assays.
Asunto(s)
Ciguatoxinas/toxicidad , Dinoflagelados/química , Análisis de Varianza , Animales , Región del Caribe , Línea Celular , Golfo de México , Ratones , Especificidad de la Especie , Pruebas de ToxicidadRESUMEN
Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.