RESUMEN
Alcohol use disorder (AUD) affects millions of people worldwide, causing extensive morbidity and mortality with limited pharmacological treatments. The liver is considered as the principal site for the detoxification of ethanol metabolite, acetaldehyde (AcH), by aldehyde dehydrogenase 2 (ALDH2) and as a target for AUD treatment, however, our recent data indicate that the liver only plays a partial role in clearing systemic AcH. Here we show that a liver-gut axis, rather than liver alone, synergistically drives systemic AcH clearance and voluntary alcohol drinking. Mechanistically, we find that after ethanol intake, a substantial proportion of AcH generated in the liver is excreted via the bile into the gastrointestinal tract where AcH is further metabolized by gut ALDH2. Modulating bile flow significantly affects serum AcH level and drinking behaviour. Thus, combined targeting of liver and gut ALDH2, and manipulation of bile flow and secretion are potential therapeutic strategies to treat AUD.
Asunto(s)
Consumo de Bebidas Alcohólicas , Aldehído Deshidrogenasa Mitocondrial , Etanol , Hígado , Hígado/metabolismo , Animales , Etanol/metabolismo , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Ratones , Consumo de Bebidas Alcohólicas/metabolismo , Acetaldehído/metabolismo , Inactivación Metabólica , Tracto Gastrointestinal/metabolismo , Alcoholismo/metabolismo , Humanos , Ratones Endogámicos C57BL , Masculino , Microbioma Gastrointestinal , Bilis/metabolismoRESUMEN
We have designed orally bioavailable, non-brain-penetrant antagonists of the cannabinoid-1 receptor (CB1R) with a built-in biguanide sensor to mimic 5'-adenosine monophosphate kinase (AMPK) activation for treating obesity-associated co-morbidities. A series of 3,4-diarylpyrazolines bearing rational pharmacophoric pendants designed to limit brain penetration were synthesized and evaluated in CB1R ligand binding assays and recombinant AMPK assays. The compounds displayed high CB1R binding affinity and potent CB1R antagonist activities and acted as AMPK activators. Select compounds showed good oral exposure, with compounds 36, 38-S, and 39-S showing <5% brain penetrance, attesting to peripheral restriction. In vivo studies of 38-S revealed decreased food intake and body weight reduction in diet-induced obese mice as well as oral in vivo efficacy of 38-S in ameliorating glucose tolerance and insulin resistance. The designed "cannabinoformin" four-arm CB1R antagonists could serve as potential leads for treatment of metabolic syndrome disorders with negligible neuropsychiatric side effects.
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Cannabinoides , Enfermedades Metabólicas , Síndrome Metabólico , Animales , Ratones , Síndrome Metabólico/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP , Biguanidas/farmacología , Biguanidas/uso terapéutico , Antagonistas de Receptores de Cannabinoides , Ratones ObesosRESUMEN
Molecular interactions between anorexigenic leptin and orexigenic endocannabinoids, although of great metabolic significance, are not well understood. We report here that hypothalamic STAT3 signaling in mice, initiated by physiological elevations of leptin, is diminished by agonists of the cannabinoid receptor 1 (CB1R). Measurement of STAT3 activation by semi-automated confocal microscopy in cultured neurons revealed that this CB1R-mediated inhibition requires both T cell protein tyrosine phosphatase (TC-PTP) and ß-arrestin1 but is independent of changes in cAMP. Moreover, ß-arrestin1 translocates to the nucleus upon CB1R activation and binds both STAT3 and TC-PTP. Consistently, CB1R activation failed to suppress leptin signaling in ß-arrestin1 knockout mice in vivo, and in neural cells deficient in CB1R, ß-arrestin1 or TC-PTP. Altogether, CB1R activation engages ß-arrestin1 to coordinate the TC-PTP-mediated inhibition of the leptin-evoked neuronal STAT3 response. This mechanism may restrict the anorexigenic effects of leptin when hypothalamic endocannabinoid levels rise, as during fasting or in diet-induced obesity.
RESUMEN
Growing evidence indicates that the crosstalk between the central nervous system and the periphery plays an important role in the pathophysiology of neuropsychiatric conditions, including addictive disorders. Fibroblast growth factor 21 (FGF21) is part of the liver-brain axis and regulates energy homeostasis, metabolism, and macronutrient intake. In addition, FGF21 signaling modulates alcohol intake and preference, and changes in FGF21 levels are observed following alcohol consumption. To further elucidate the relationship between alcohol use and FGF21, we assessed serum FGF21 concentrations in 16 non-treatment seeking individuals with alcohol use disorder (AUD) in a naturalistic outpatient setting, as well as a controlled laboratory experiment that included alcohol cue-reactivity, alcohol priming, and alcohol self-administration in a bar-like setting. FGF21 levels were stable during the outpatient phase when participants received placebo and had no significant lifestyle changes. During the bar-like laboratory experiment, a robust increase in serum FGF21 concentrations was found after the 2-hr alcohol self-administration session (F3, 49 = 23.39, p < 0.001). Percent change in FGF21 levels positively correlated with the amount of alcohol self-administered but did not reach statistical significance. No significant changes in FGF21 levels were found after exposure to alcohol cues or consuming the priming drink. Given the bidirectional link between FGF21 and alcohol, targeting the FGF21 system may be further examined as a potential pharmacotherapy for AUD.
Asunto(s)
Alcoholismo , Humanos , Consumo de Bebidas Alcohólicas , Factores de Crecimiento de Fibroblastos/metabolismo , EtanolRESUMEN
Alcohol consumption is associated with gut dysbiosis, increased intestinal permeability, endotoxemia, and a cascade that leads to persistent systemic inflammation, alcoholic liver disease, and other ailments. Craving for alcohol and its consequences depends, among other things, on the endocannabinoid system. We have analyzed the relative role of central vs. peripheral cannabinoid CB1 receptors (CB1R) using a "two-bottle" as well as a "drinking in the dark" paradigm in mice. The globally acting CB1R antagonist rimonabant and the non-brain penetrant CB1R antagonist JD5037 inhibited voluntary alcohol intake upon systemic but not upon intracerebroventricular administration in doses that elicited anxiogenic-like behavior and blocked CB1R-induced hypothermia and catalepsy. The peripherally restricted hybrid CB1R antagonist/iNOS inhibitor S-MRI-1867 was also effective in reducing alcohol consumption after oral gavage, while its R enantiomer (CB1R inactive/iNOS inhibitor) was not. The two MRI-1867 enantiomers were equally effective in inhibiting an alcohol-induced increase in portal blood endotoxin concentration that was caused by increased gut permeability. We conclude that (i) activation of peripheral CB1R plays a dominant role in promoting alcohol intake and (ii) the iNOS inhibitory function of MRI-1867 helps in mitigating the alcohol-induced increase in endotoxemia.
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Consumo de Bebidas Alcohólicas/patología , Antagonistas de Receptores de Cannabinoides/farmacología , Endotoxemia/patología , Etanol/efectos adversos , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Receptor Cannabinoide CB1/antagonistas & inhibidores , Consumo de Bebidas Alcohólicas/sangre , Animales , Ansiedad/sangre , Ansiedad/complicaciones , Conducta Animal/efectos de los fármacos , Catalepsia/inducido químicamente , Catalepsia/complicaciones , Ciclohexanoles/administración & dosificación , Prueba de Laberinto Elevado , Endotoxemia/sangre , Endotoxemia/complicaciones , Endotoxinas/sangre , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/metabolismo , Hipotermia Inducida , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Pirazoles/administración & dosificación , Receptor Cannabinoide CB1/metabolismo , Rimonabant/administración & dosificación , Rimonabant/farmacología , Estereoisomerismo , Sulfonamidas/administración & dosificaciónRESUMEN
Seven-transmembrane receptors signal via G-protein- and ß-arrestin-dependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced ß-arrestin-2 (ßArr2) recruitment over G-protein activation. In obese wild-type and ßArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB1R occupancy. By contrast, the unbiased global CB1R antagonist rimonabant elicits anxiety in both strains, indicating no ßArr2 involvement. Interestingly, obesity-induced muscle insulin resistance is improved by MRI-1891 in wild-type but not in ßArr2-KO mice. In C2C12 myoblasts, CB1R activation suppresses insulin-induced akt-2 phosphorylation, preventable by MRI-1891, ßArr2 knockdown or overexpression of CB1R-interacting protein. MRI-1891, but not rimonabant, interacts with nonpolar residues on the N-terminal loop, including F108, and on transmembrane helix-1, including S123, a combination that facilitates ßArr2 bias. Thus, CB1R promotes muscle insulin resistance via ßArr2 signaling, selectively mitigated by a biased CB1R antagonist at reduced risk of central nervous system (CNS) side effects.
RESUMEN
Cannabidiol (CBD) exhibits anti-inflammatory and neuroprotective properties and is suggested to be effective in the pre-clinical and clinical treatment of illnesses of the central nervous system (CNS). Two major types of CNS glial cells, astrocytes and microglia, play critical roles in the development and pathogenesis of CNS diseases. However, the mechanisms by which CBD plays an anti-inflammatory and neuroprotective role for these glial cells have not been fully elucidated. In this study, we examined the effects of CBD on the inflammatory response of mouse primary astrocytes and microglia. We also investigated whether the effect of CBD on cytokine release is mediated by the G protein coupled receptor 3 (GPR3), which was recently identified as a novel receptor for CBD. Our results showed that CBD inhibited inflammatory responses of astrocytes and microglia stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand in vitro and in vivo. In addition, CBD reduced the phosphorylation of STAT3 and NF-κB signaling pathways in LPS-stimulated astrocytes. However, the inhibitory effect of CBD on pro-inflammatory cytokine production was independent of GPR3 expression in both types of glial cells. Thus, although CBD is effective in ameliorating the activation of astrocytes and microglia, its mechanism of action still requires further study. Our data support the concept that CBD may have therapeutic potential for neurological disorders that involve neuroinflammation.
Asunto(s)
Antiinflamatorios/farmacología , Astrocitos/efectos de los fármacos , Cannabidiol/farmacología , Microglía/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Astrocitos/inmunología , Astrocitos/metabolismo , Cannabidiol/uso terapéutico , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Ratones , Microglía/inmunología , Microglía/metabolismo , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the key tissue for whole-body glucose disposal and utilization. New drugs aimed at improving insulin sensitivity of SKM would greatly expand available therapeutic options. ß-arrestin-1 and -2 (Barr1 and Barr2, respectively) are two intracellular proteins best known for their ability to mediate the desensitization and internalization of G protein-coupled receptors (GPCRs). Recent studies suggest that Barr1 and Barr2 regulate several important metabolic functions including insulin release and hepatic glucose production. Since SKM expresses many GPCRs, including the metabolically important ß2-adrenergic receptor, the goal of this study was to examine the potential roles of Barr1 and Barr2 in regulating SKM and whole-body glucose metabolism. Using SKM-specific knockout (KO) mouse lines, we showed that the loss of SKM Barr2, but not of SKM Barr1, resulted in mild improvements in glucose tolerance in diet-induced obese mice. SKM-specific Barr1- and Barr2-KO mice did not show any significant differences in exercise performance. However, lack of SKM Barr2 led to increased glycogen breakdown following a treadmill exercise challenge. Interestingly, mice that lacked both Barr1 and Barr2 in SKM showed no significant metabolic phenotypes. Thus, somewhat surprisingly, our data indicate that SKM ß-arrestins play only rather subtle roles (SKM Barr2) in regulating whole-body glucose homeostasis and SKM insulin sensitivity.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismo , Animales , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Glucosa/administración & dosificación , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Glucógeno/metabolismo , Humanos , Insulina/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Ratones Noqueados , Obesidad/etiología , Transducción de Señal/genética , beta-Arrestina 1/genética , Arrestina beta 2/genéticaRESUMEN
Endocannabinoids acting on the cannabinoid-1 receptor (CB1R) or ghrelin acting on its receptor (GHS-R1A) both promote alcohol-seeking behavior, but an interaction between the two signaling systems has not been explored. Here, we report that the peripheral CB1R inverse agonist JD5037 reduces ethanol drinking in wild-type mice but not in mice lacking CB1R, ghrelin peptide or GHS-R1A. JD5037 treatment of alcohol-drinking mice inhibits the formation of biologically active octanoyl-ghrelin without affecting its inactive precursor desacyl-ghrelin. In ghrelin-producing stomach cells, JD5037 reduced the level of the substrate octanoyl-carnitine generated from palmitoyl-carnitine by increasing fatty acid ß-oxidation. Blocking gastric vagal afferents abrogated the ability of either CB1R or GHS-R1A blockade to reduce ethanol drinking. We conclude that blocking CB1R in ghrelin-producing cells reduces alcohol drinking by inhibiting the formation of active ghrelin and its signaling via gastric vagal afferents. Thus, peripheral CB1R blockade may have therapeutic potential in the treatment of alcoholism.
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Consumo de Bebidas Alcohólicas/genética , Encéfalo/fisiología , Intestinos/fisiología , Receptor Cannabinoide CB1/genética , Aciltransferasas/genética , Aciltransferasas/fisiología , Consumo de Bebidas Alcohólicas/fisiopatología , Alcoholismo/genética , Alcoholismo/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Células Cultivadas , Eliminación de Gen , Ghrelina/metabolismo , Ghrelina/fisiología , Intestinos/efectos de los fármacos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Receptores de Ghrelina/genética , Receptores de Ghrelina/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas/farmacologíaRESUMEN
Adipocyte death occurs under various physiopathological conditions, including obesity and alcohol drinking, and can trigger organ damage particularly in the liver, but the underlying mechanisms remain obscure. To explore these mechanisms, we developed a mouse model of inducible adipocyte death by overexpressing the human CD59 (hCD59) on adipocytes (adipocyte-specific hCD59 transgenic mice). Injection of these mice with intermedilysin (ILY), which rapidly lyses hCD59 expressing cells exclusively by binding to the hCD59 but not mouse CD59, resulted in the acute selective death of adipocytes, adipose macrophage infiltration, and elevation of serum free fatty acid (FFA) levels. ILY injection also resulted in the secondary damage to multiple organs with the strongest injury observed in the liver, with inflammation and hepatic macrophage activation. Mechanistically, acute adipocyte death elevated epinephrine and norepinephrine levels and activated lipolysis pathways in adipose tissue in a chemokine (C-C motif) receptor 2-positive (CCR2+ ) macrophage-dependent manner, which was followed by FFA release and lipotoxicity in the liver. Additionally, acute adipocyte death caused hepatic CCR2+ macrophage activation and infiltration, further exacerbating liver injury. Conclusion: Adipocyte death predominantly induces liver injury and inflammation, which is probably due to the superior sensitivity of hepatocytes to lipotoxicity and the abundance of macrophages in the liver.
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Adipocitos/fisiología , Tejido Adiposo/enzimología , Hepatopatías/etiología , Macrófagos/fisiología , Receptores CCR2/metabolismo , Animales , Bacteriocinas , Muerte Celular , Modelos Animales de Enfermedad , Epinefrina/sangre , Ácidos Grasos no Esterificados/sangre , Femenino , Inflamación/etiología , Isoproterenol , Lipólisis , Hepatopatías/sangre , Masculino , Ratones Transgénicos , Norepinefrina/sangre , Receptores CCR2/genéticaRESUMEN
Endocannabinoids promote energy conservation in obesity, whereas cannabinoid-1 receptor (CB1 R) blockade reverses body weight gain and insulin resistance and increases energy expenditure. Here we investigated the molecular mechanisms of the catabolic effects of CB1 R blockade in the liver. Exposure of primary mouse hepatocytes and HepG2 cells to the CB1 R agonist arachidonyl-2'-chloroethylamide inhibited the expression of Sirtuin-1 (Sirt1) and Rictor, a component of mechanistic target of rapamycin complex 2 (mTORC2) and suppressed insulin-induced Akt phosphorylation at serine 473. These effects were reversed by peripheral CB1 R antagonist JD5037 in control hepatocytes but not in hepatocytes deficient in Sirt1 and/or Rictor, indicating that these two proteins are required for the CB1 R-mediated inhibition of insulin signaling. Feeding C57BL/6J mice a high-fat diet (HFD) inhibited hepatic Sirt1/mTORC2/Akt signaling, and the inhibition was reversed by rimonabant or JD5037 in wild-type but not liver-specific Sirt1-/- (Sirt1-LKO) mice, to levels observed in hepatocyte-specific CB1 R-/- mice. A similar attenuation of hyperglycemia and hyperinsulinemia in wild-type mice with obesity but not in Sirt1-LKO mice could be attributed to insufficient reversal of HFD-induced mitochondrial reactive oxygen species generation in peripheral tissues in the latter. In contrast, JD5037 treatment was equally effective in HFD-fed wild-type and Sirt1-LKO mice in reducing hepatic steatosis, increasing fatty acid ß-oxidation, and activating 5'adenosine monophosphate-activated protein kinase (AMPK) through liver kinase B1 (LKB1), resulting in a similar increase in total energy expenditure in the two strains. Conclusion: Peripheral CB1 R blockade in mice with obesity improves glycemic control through the hepatic Sirt1/mTORC2/Akt pathway, whereas it increases fatty acid oxidation through LKB1/AMPK signaling.
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Resistencia a la Insulina , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Sirtuina 1/metabolismo , Sulfonamidas/farmacología , Adenilato Quinasa/metabolismo , Animales , Dieta Alta en Grasa , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Hepáticas/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
AIMS: To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB1 R) in the development of diabetic nephropathy (DN), relative to CB1 R in other renal cell types. MATERIAL AND METHODS: We developed a mouse model with a podocyte-specific deletion of CB1 R (pCB1Rko) and challenged this model with streptozotocin (STZ)-induced type-1 DN. We also assessed the podocyte response to high glucose in vitro and its effects on CB1 R activation. RESULTS: High glucose exposure for 48 hours led to an increase in CB1 R gene expression (CNR1) and endocannabinoid production in cultured human podocytes. This was associated with podocyte injury, reflected by decreased podocin and nephrin expression. These changes could be prevented by Cnr1-silencing, thus identifying CB1R as a key player in podocyte injury. After 12 weeks of chronic hyperglycaemia, STZ-treated pCB1Rko mice showed elevated blood glucose similar to that of their wild-type littermates. However, they displayed less albuminuria and less podocyte loss than STZ-treated wild-type mice. Unexpectedly, pCB1Rko mice also have milder tubular dysfunction, fibrosis and reduction of cortical microcirculation compared to wild-type controls, which is mediated, in part, by podocyte-derived endocannabinoids acting via CB1 R on proximal tubular cells. CONCLUSIONS: Activation of CB1 R in podocytes contributes to both glomerular and tubular dysfunction in type-1 DN, which highlights the therapeutic potential of peripheral CB1 R blockade.
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Nefropatías Diabéticas/fisiopatología , Glomérulos Renales/fisiología , Túbulos Renales Proximales/fisiología , Podocitos/metabolismo , Receptores de Cannabinoides/deficiencia , Animales , Arginasa/metabolismo , Hipoxia de la Célula/fisiología , Células Cultivadas , Diabetes Mellitus Experimental/fisiopatología , Glucosa/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón/irrigación sanguínea , Proteínas de la Membrana/metabolismo , Ratones , Microcirculación/fisiología , Estrés Oxidativo/fisiología , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/metabolismoRESUMEN
OBJECTIVE: Obesity-induced accumulation of ectopic fat in the liver is thought to contribute to the development of insulin resistance, and increased activity of hepatic CB1R has been shown to promote both processes. However, lipid accumulation in liver can be experimentally dissociated from insulin resistance under certain conditions, suggesting the involvement of additional mechanisms. Obesity is also associated with pro-inflammatory changes which, in turn, can promote insulin resistance. Kupffer cells (KCs), the liver's resident macrophages, are the major source of pro-inflammatory cytokines in the liver, such as TNF-α, which has been shown to inhibit insulin signaling in multiple cell types, including hepatocytes. Here, we sought to identify the role of CB1R in KCs in obesity-induced hepatic insulin resistance. METHODS: We used intravenously administered ß-D-glucan-encapsulated siRNA to knock-down CB1R gene expression selectively in KCs. RESULTS: We demonstrate that a robust knock-down of the expression of Cnr1, the gene encoding CB1R, results in improved glucose tolerance and insulin sensitivity in diet-induced obese mice, without affecting hepatic lipid content or body weight. Moreover, Cnr1 knock-down in KCs was associated with a shift from pro-inflammatory M1 to anti-inflammatory M2 cytokine profile and improved insulin signaling as reflected by increased insulin-induced Akt phosphorylation. CONCLUSION: These findings suggest that CB1R expressed in KCs plays a critical role in obesity-related hepatic insulin resistance via a pro-inflammatory mechanism.
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Resistencia a la Insulina , Macrófagos del Hígado/metabolismo , Obesidad/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Dieta Alta en Grasa , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Receptor Cannabinoide CB1/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein ß-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific ß-arrestin 2 deficiency did not affect hepatic insulin sensitivity or ß-adrenergic signaling. Adult mice lacking ß-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of ß-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic ß-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes.
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Glucemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Hígado/metabolismo , Receptores de Glucagón/metabolismo , Arrestina beta 2/metabolismo , Animales , Células COS , Chlorocebus aethiops , Dieta Alta en Grasa , Eliminación de Gen , Glucagón/metabolismo , Hepatocitos/metabolismo , Homeostasis/fisiología , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , beta-Arrestina 1/genéticaRESUMEN
Cytochrome P450-2E1 (CYP2E1) increases oxidative stress. High hepatic cholesterol causes non-alcoholic steatohepatitis (NASH) and fibrosis. Thus, we aimed to study the role of CYP2E1 in promoting liver fibrosis by high cholesterol-containing fast-food (FF). Male wild-type (WT) and Cyp2e1-null mice were fed standard chow or FF for 2, 12, and 24 weeks. Various parameters of liver fibrosis and potential mechanisms such as oxidative and endoplasmic reticulum (ER) stress, inflammation, and insulin resistance (IR) were studied. Indirect calorimetry was also used to determine metabolic parameters. Liver histology showed that only WT fed FF (WT-FF) developed NASH and fibrosis. Hepatic levels of fibrosis protein markers were significantly increased in WT-FF. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in FF-fed WT. Serum endotoxin, TLR-4 levels, and inflammatory markers were highest in WT-FF. FAS, PPAR-α, PPAR-γ, and CB1-R were markedly altered in WT-FF. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in FF-fed WT. Indirect calorimetry showed that Cyp2e1-null-mice fed FF exhibited consistently higher total energy expenditure (TEE) than their corresponding WT. These results demonstrate that CYP2E1 is important in fast food-mediated liver fibrosis by promoting nitroxidative and ER stress, endotoxemia, inflammation, IR, and low TEE.
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Citocromo P-450 CYP2E1/metabolismo , Inflamación/metabolismo , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Animales , Colesterol/metabolismo , Citocromo P-450 CYP2E1/genética , Estrés del Retículo Endoplásmico , Metabolismo Energético , Comida Rápida , Fibrosis , Humanos , Mediadores de Inflamación/sangre , Resistencia a la Insulina , Hígado/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Estrés OxidativoRESUMEN
Islet inflammation promotes ß-cell loss and type 2 diabetes (T2D), a process replicated in Zucker Diabetic Fatty (ZDF) rats in which ß-cell loss has been linked to cannabinoid-1 receptor (CB1R)-induced proinflammatory signaling in macrophages infiltrating pancreatic islets. Here, we analyzed CB1R signaling in macrophages and its developmental role in T2D. ZDF rats with global deletion of CB1R are protected from ß-cell loss, hyperglycemia, and nephropathy that are present in ZDF littermates. Adoptive transfer of CB1R-/- bone marrow to ZDF rats also prevents ß-cell loss and hyperglycemia but not nephropathy. ZDF islets contain elevated levels of CB1R, interleukin-1ß, tumor necrosis factor-α, the chemokine CCL2, and interferon regulatory factor-5 (IRF5), a marker of inflammatory macrophage polarization. In primary cultured rodent and human macrophages, CB1R activation increased Irf5 expression, whereas knockdown of Irf5 blunted CB1R-induced secretion of inflammatory cytokines without affecting CCL2 expression, which was p38MAPKα dependent. Macrophage-specific in vivo knockdown of Irf5 protected ZDF rats from ß-cell loss and hyperglycemia. Thus, IRF5 is a crucial downstream mediator of diabetogenic CB1R signaling in macrophages and a potential therapeutic target.
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Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/genética , Hiperglucemia/genética , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Macrófagos/metabolismo , Receptor Cannabinoide CB1/genética , Animales , Quimiocina CCL2/metabolismo , Nefropatías Diabéticas/metabolismo , Técnicas de Inactivación de Genes , Hiperglucemia/metabolismo , Factores Reguladores del Interferón/metabolismo , Interleucina-1beta , Masculino , Ratas , Ratas Zucker , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.
Asunto(s)
Hígado Graso Alcohólico/prevención & control , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Hígado Graso Alcohólico/genética , Hígado Graso Alcohólico/metabolismo , Humanos , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NAD/metabolismo , Estrés Nitrosativo/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fenantrenos/farmacología , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Poli(ADP-Ribosa) Polimerasa-1/genética , Quinolinas/farmacología , Sirtuina 1/metabolismoRESUMEN
Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvß6 signaling, as judged by its ability to inhibit these pathways in cnr1-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.
RESUMEN
We report an unexpected link between aging, thermogenesis and weight gain via the orphan G protein-coupled receptor GPR3. Mice lacking GPR3 and maintained on normal chow had similar body weights during their first 5 months of life, but gained considerably more weight thereafter and displayed reduced total energy expenditure and lower core body temperature. By the age of 5 months GPR3 KO mice already had lower thermogenic gene expression and uncoupling protein 1 protein level and showed impaired glucose uptake into interscapular brown adipose tissue (iBAT) relative to WT littermates. These molecular deviations in iBAT of GPR3 KO mice preceded measurable differences in body weight and core body temperature at ambient conditions, but were coupled to a failure to maintain thermal homeostasis during acute cold challenge. At the same time, the same cold challenge caused a 17-fold increase in Gpr3 expression in iBAT of WT mice. Thus, GPR3 appears to have a key role in the thermogenic response of iBAT and may represent a new therapeutic target in age-related obesity.