Alba Rebollo

Reduction of liver fructokinase expression and improved hepatic inflammation and metabolism in liquid fructose-fed rats after atorvastatin treatment

Consumption of beverages that contain fructose favors the increasing prevalence of metabolic syndrome alterations in humans, including non-alcoholic fatty liver disease (NAFLD). Although the only effective treatment for NAFLD is caloric restriction and weight loss, existing data show that atorvastatin, a hydroxymethyl-glutaryl-CoA reductase inhibitor, can be used safely in patients with NAFLD and improves hepatic histology. To gain further insight into the molecular mechanisms of atorvastatins therapeutic effect on NAFLD, we used an experimental model that mimics human consumption of fructose-sweetened beverages. Control, fructose (10% w/v solution) and fructose+atorvastatin (30 mg/kg/day) Sprague-Dawley rats were sacrificed after 14 days. Plasma and liver tissue samples were obtained to determine plasma analytes, liver histology, and the expression of liver proteins that are related to fatty acid synthesis and catabolism, and inflammatory processes. Fructose supplementation induced hypertriglyceridemia and hyperleptinemia, hepatic steatosis and necroinflammation, increased the expression of genes related to fatty acid synthesis and decreased fatty acid β-oxidation activity. Atorvastatin treatment completely abolished histological signs of necroinflammation, reducing the hepatic expression of metallothionein-1 and nuclear factor kappa B binding. Furthermore, atorvastatin reduced plasma (x 0.74) and liver triglyceride (x 0.62) concentrations, decreased the liver expression of carbohydrate response element binding protein transcription factor (x 0.45) and its target genes, and increased the hepatic activity of the fatty acid β-oxidation system (x 1.15). These effects may be related to the fact that atorvastatin decreased the expression of fructokinase (x 0.6) in livers of fructose-supplemented rats, reducing the metabolic burden on the liver that is imposed by continuous fructose ingestion.

Way back for fructose and liver metabolism: Bench side to molecular insights

The World Health Organization recommends that the daily intake of added sugars should make up no more than 10% of total energy. The consumption of sugar-sweetened beverages is the main source of added sugars. Fructose, together with glucose, as a component of high fructose corn syrups or as a component of the sucrose molecule, is one of the main sweeteners present in this kind of beverages. Data from prospective and intervention studies clearly point to high fructose consumption, mainly in the form of sweetened beverages, as a risk factor for several metabolic diseases in humans. The incidence of hypertension, nonalcoholic fatty liver disease (NAFLD), dyslipidemia (mainly hypertriglyceridemia), insulin resistance, type 2 diabetes mellitus, obesity, and the cluster of many of these pathologies in the form of metabolic syndrome is higher in human population segments that show high intake of fructose. Adolescent and young adults from low-income families are especially at risk. We recently reviewed evidence from experimental animals and human data that confirms the deleterious effect of fructose on lipid and glucose metabolism. In this present review we update the information generated in the past 2 years about high consumption of fructose-enriched beverages and the occurrence of metabolic disturbances, especially NAFLD, type 2 diabetes mellitus, and metabolic syndrome. We have explored recent data from observational and experimental human studies, as well as experimental data from animal and cell models. Finally, using information generated in our laboratory and others, we provide a view of the molecular mechanisms that may be specifically involved in the development of liver lipid and glucose metabolic alterations after fructose consumption in liquid form.

Liquid fructose downregulates Sirt1 expression and activity and impairs the oxidation of fatty acids in rat and human liver cells.

Fructose ingestion is associated with the production of hepatic steatosis and hypertriglyceridemia. For fructose to attain these effects in rats, simultaneous induction of fatty acid synthesis and inhibition of fatty acid oxidation is required. We aimed to determine the mechanism involved in the inhibition of fatty acid oxidation by fructose and whether this effect occurs also in human liver cells. Female rats were supplemented or not with liquid fructose (10% w/v) for 7 or 14 days; rat (FaO) and human (HepG2) hepatoma cells, and human hepatocytes were incubated with fructose 25mM for 24h. The expression and activity of the enzymes and transcription factors relating to fatty acid β-oxidation were evaluated. Fructose inhibited the activity of fatty acid β-oxidation only in livers of 14-day fructose-supplemented rats, as well as the expression and activity of peroxisome proliferator activated receptor α (PPARα). Similar results were observed in FaO and HepG2 cells and human hepatocytes. PPARα downregulation was not due to an osmotic effect or to an increase in protein-phosphatase 2A activity caused by fructose. Rather, it was related to increased content in liver of inactive and acetylated peroxisome proliferator activated receptor gamma coactivator 1α, due to a reduction in sirtuin 1 expression and activity. In conclusion, fructose inhibits liver fatty acid oxidation by reducing PPARα expression and activity, both in rat and human liver cells, by a mechanism involving sirtuin 1 down-regulation.

Liquid fructose down-regulates liver insulin receptor substrate 2 and gluconeogenic enzymes by modifying nutrient sensing factors in rats.

High consumption of fructose-sweetened beverages has been linked to a high prevalence of chronic metabolic diseases. We have previously shown that a short course of fructose supplementation as a liquid solution induces glucose intolerance in female rats. In the present work, we characterized the fructose-driven changes in the liver and the molecular pathways involved. To this end, female rats were supplemented or not with liquid fructose (10%, w/v) for 7 or 14 days. Glucose and pyruvate tolerance tests were performed, and the expression of genes related to insulin signaling, gluconeogenesis and nutrient sensing pathways was evaluated. Fructose-supplemented rats showed increased plasma glucose excursions in glucose and pyruvate tolerance tests and reduced hepatic expression of several genes related to insulin signaling, including insulin receptor substrate 2 (IRS-2). However, the expression of key gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was reduced. These effects were caused by an inactivation of hepatic forkhead box O1 (FoxO1) due to an increase in its acetylation state driven by a reduced expression and activity of sirtuin 1 (SIRT1). Further contributing to FoxO1 inactivation, fructose consumption elevated liver expression of the spliced form of X-box-binding-protein-1 as a consequence of an increase in the activity of the mammalian target of rapamycin 1 and protein 38-mitogen activated protein kinase (p38-MAPK). Liquid fructose affects both insulin signaling (IRS-2 and FoxO1) and nutrient sensing pathways (p38-MAPK, mTOR and SIRT1), thus disrupting hepatic insulin signaling without increasing the expression of key gluconeogenic enzymes.

Type II interleukin-1 receptor expression is reduced in monocytes/macrophages and atherosclerotic lesions

Type II interleukin-1 receptor (IL-1R2) is a non-signaling decoy receptor that negatively regulates the activity of interleukin-1 (IL-1), a pro-inflammatory cytokine involved in atherogenesis. In this article we assessed the relevance of IL-1R2 in atherosclerosis by studying its expression in monocytes from hyperlipidemic patients, in THP-1 macrophages exposed to lipoproteins and in human atherosclerotic lesions. Our results showed that the mRNA and protein expression of IL-1R2 was reduced in monocytes from patients with familial combined hyperlipidemia (-30%, p<0.05). THP-1 macrophages incubated with increasing concentrations of acetylated low density (ac-LDL) and very low density (VLDL) lipoproteins also exhibit a decrease in IL-1R2 mRNA and protein levels. Pre-incubation with agents that block intracellular accumulation of lipids prevents the decrease in IL-1R2 mRNA caused by lipoproteins. Lipoproteins also prevented the increase in IL-1R1 and IL-1R2 caused by a 4-h stimulation with LPS and reduced protein expression of total and phosphorylated IL-1 receptor-associated kinase-1. Finally, IL-1R2 expression in human atherosclerotic vessels was markedly lower than in non-atherosclerotic arteries (-80%, p<0.0005). Overall, our results suggest that under atherogenic conditions, there is a decrease in IL-1R2 expression in monocytes/macrophages and in the vascular wall that may facilitate IL-1 signaling.

Tissue factor pathway inhibitor 2 is induced by thrombin in human macrophages

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p<0.001) and human monocyte-derived macrophages (2.3-fold increase, p<0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.

Ritonavir increases CD36, ABCA1 and CYP27 expression in THP-1 macrophages.

Ritonavir, a protease inhibitor used in combination antiretroviral therapy for HIV-1 infection, is associated with an increased risk of premature atherosclerosis. The aim of the present study was to assess the effects of ritonavir, in the absence of added lipoproteins, on the expression of genes that control cholesterol trafficking in human monocytes/macrophages. Design: THP-1 cells were used to study the effects of ritonavir on the expression of CD36, ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1), scavenger receptor B class I (SR-BI), caveolin-1 and sterol 27-hydroxylase (CYP27). Exposure to ritonavir (2.5 μg/ml) increased CD36 protein (28%, P < 0.05) and mRNA (38%, P < 0.05) in differentiated THP-1 macrophages, but not in undifferentiated monocytes. This effect was not related to the increase in PPARγ expression (51%, P < 0.05) caused by ritonavir. Ritonavir also reduced SR-BI protein levels (46%, P < 0.05) and increased CYP27 (43%, P < 0.05) and ABCA1 (49%, P < 0.05) mRNA expression. Liver X receptor α (LXRα) mRNA, protein and binding activity were also increased by ritonavir treatment. Conclusions: We propose that ritonavir induces ABCA1 expression in THP-1 macrophages through LXRα. The increase in ABCA1 and other cholesterol efflux mediators, such as CYP27, may compensate CD36 induction. Therefore, we suggest that the net effect of ritonavir on macrophages in the absence of lipoproteins is not clearly proatherogenic.

El monocito/macrófago como diana terapéutica en la aterosclerosis

Monocitos y macrofagos son celulas que desempenan un papel clave en todas las etapas del desarrollo de la aterosclerosis. Uno de los fenomenos iniciales en este proceso es la union de monocitos circulantes al endotelio arterial mediante las moleculas de adhesion (MA), y la subsiguiente transmigracion hacia la capa intima bajo la influencia de quimiocinas como la proteina quimiotactica de monocitos (MCP-1). En fases posteriores, los monocitos reclutados se diferencian a macrofagos, proceso que comporta el incremento de la expresion de receptores toll - like (TLR), implicados en la respuesta inmune innata, y receptores scavenger (SR). Los TLR activan la respuesta inflamatoria en los macrofagos, induciendo la expresion de citocinas como IL-6, IL-1β y factor de necrosis tumoral (TNF). Por otra parte, los SR (principalmente SR-A y CD36) captan lipoproteinas modificadas de forma no regulada por los valores intracelulares de esteroles. El colesterol que el macrofago ha captado a traves de estos receptores es almacenado en forma de esteres de colesterol tras su esterificacion por la enzima acil-CoA:colesterol aciltransferasa (ACAT), conduciendo a la formacion de celulas espumosas. Para mantener la homeostasis lipidica, el macrofago depende de la existencia de mecanismos de exportacion de colesterol al espacio extracelular, incluyendo el transporte hacia las HDL maduras mediado por el receptor scavenger BI (SR-BI) y la transferencia de colesterol hacia la apolipoproteina AI a traves del transportador ATP- binding cassette A1 (ABCA1). La modulacion farmacologica de estas dianas (MA, quimiocinas, TLR, SR, ACAT y proteinas relacionadas con la exportacion de colesterol), directa o indirectamente a traves de factores de transcripcion que controlan la expresion genica (receptor hepatico X, factor nuclear κB), puede limitar el desarrollo de la aterosclerosis.

Ritonavir incrementa la expresión de CD36 y ABCA1 en macrófagos THP-1

El ritonavir es uno de los inhibidores de las proteasas que se utilizan como parte del tratamiento antirretroviral de gran actividad (TARGA) para la infeccion por el virus de la inmunodeficiencia humana, y su uso se ha asociado a un aumento en el riesgo de aterosclerosis prematura. Se ha propuesto que el ritonavir favorece la formacion de celulas espumosas, aunque los estudios previos realizados han mostrado efectos contradictorios de este farmaco sobre las concentraciones del receptor scavenger CD36 en monocitos y macrofagos. Nuestros resultados muestran que el tratamiento con ritonavir a concentraciones dentro del rango terapeutico produce un incremento en la expression de proteina y ARNm de CD36 en macrofagos THP-1 diferenciados, pero no en monocitos de la misma linea celular. Estas diferencias se podrian deber a la activacion de la proteincinasa C producida tras la diferenciacion con el ester de forbol PMA y la consiguiente activacion de proliferadores de peroxisomas γ (PPARγ). Este mecanismo tambien explicaria el incremento en la expresion de ABCA1 hallado en nuestro estudio, aunque no la reduccion de las concentraciones de proteina SR-BI, que no parece ser un efecto de tipo transcripcional. Finalmente, la induccion de PPARγ y CD36 causada por el ritonavir no se asocia a un incremento en las concentraciones de la forma madura de SREBP1.