Role of monounsaturated fatty acids in the improvement of non-alcoholic fatty liver disease and type 2 diabetes mellitus

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biotecnología, Biomedicina y Ciencias de la SaludClave Programa: DBICódigo Línea: 110En los últimos años, debido a la creciente incidencia de obesidad, estilos de vida sedentarios y dietas poco saludables en todo el mundo, ha aumentado la prevalencia del síndrome metabólico y sus comorbilidades, como la diabetes mellitus tipo dos (T2D) y la enfermedad del hígado graso no alcohólico (EGHNA). Europa ha sido testigo de entre el 20 % y el 30 % de los casos. Por lo tanto, el síndrome metabólico se considera como un importante problema de salud pública. En un intento por comprender estas enfermedades y descubrir los mecanismos moleculares implicados para encontrar posibles dianas terapéuticas, en esta Tesis Doctoral se han estudiado varios modelos animales que recapitulan todas las características de estas enfermedades de la forma más parecida posible a los humanos ya que a pesar de la alta incidencia tanto del síndrome metabólico como de sus comorbilidades, no existe un tratamiento farmacológico efectivo. En general, una dieta hipercalórica, y en particular una rica en grasas trans, grasas saturadas, colesterol y bebidas endulzadas con fructosa, parece aumentar la adiposidad visceral y estimular la acumulación de lípidos en el hígado, así como la progresión de EGHNA y resistencia a la insulina. Sin embargo, la reducción de la ingesta calórica y la suplementación con ácidos grasos monoinsaturados omega-3 parece tener efectos preventivos y terapéuticos. Hay muchos estudios que demuestran que las dietas con grasas que representan al menos el 40% de la ingesta calórica conducen a la aparición de síndrome metabólico, T2D y EGHNA. Por el contrario, varios estudios indican que las grasas monoinsaturadas y poliinsaturadas tienen efectos beneficiosos sobre las enfermedades metabólicas. Por lo tanto, estos hallazgos contradictorios nos llevaron a considerar si una dieta alta en grasas con un alto consumo de ácidos grasos monoinsaturados y compuestos polifenólicos presentes en el aceite de oliva virgen extra puede tener efectos beneficiosos sobre estas enfermedades. Es por ello que la siguiente Tesis Doctoral que se presenta mediante la modalidad de compendio de publicaciones y en la que se aportan distintas contribuciones científicas en revistas con alto índice de impacto en el Journal of Citation Reports (JCR), se han evaluado los siguientes aspectos: (i) El papel de la ingesta de una dieta alta en grasas enriquecida en grasas monoinsaturadas en la etiopatogenia de la EGHNA y T2D. (ii) La optimización de diferentes modelos de ratones para el estudio de trastornos hepáticos relacionados con EGHNA. (iii) El estudio de los mecanismos responsables de los efectos beneficiosos del aceite de oliva virgen extra en tejidos metabólicos activos (hígado y páncreas). (iv) La comprensión de las rutas y redes de señalización molecular, para obtener una visión integradora necesaria para explicar las mejoras producidas a nivel fisiopatológico, en animales alimentados con dietas altas en grasas enriquecidas con ácidos grasos monoinsaturados provenientes del aceite de oliva virgen extra.Universidad Pablo de Olavide de Sevilla. Departamento de Biología Molecular e Ingeniería Bioquímic

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr −/−). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m), diabetes mellitus and ALT werefound to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.The research leading to these results has received funding from the Consejería de Salud de la Junta de Andalucía under grant agreement PC-0148-2016-0148 and PE-0451-2018 and Instituto de Salud Carlos III under grant agreements CD21/00095, PI16/01842, PI19/01404, PI19/00589, IFI18/00041, FI20/00201, CD18/00126 and EHD18PI04/2021. Rocío Gallego-Durán has received the Andrew K Burroughs Fellowship from European Association for the Study of the Liver (EASL), Aprendizaje de Nuevas Tecnologías fellowship from Asociación Española para el Estudio del Hígado (AEEH) and CIBERehd Grant to support researcher’s mobility

Extra‐virgin olive oil with Natural phenolic content exerts an anti‐inflammatory effect in adipose tissue and attenuates the severity of atherosclerotic lesions in Ldlr−/−.Leiden Mice

[Scope] The present study investigates the effect of olive oils with different phenolic content in high‐fat diets (HFDs) on hypertrophy and inflammation in adipose tissue and associated atherosclerosis, in the context of obesity.[Methods and results] Ldlr−/−.Leiden mice were fed three different HFDs for 32 weeks and were compared with mice fed the standard low‐fat diet (LFD). The different fats provided in the HFDs were lard (HFD‐L), extra‐virgin olive oil (EVOO; 79 mg kg–1 of phenolic compounds, HFD‐EVOO), or EVOO rich in phenolic compounds (OL, 444 mg kg–1 of phenolic compounds, HFD‐OL). All HFD‐fed mice became obese, but only HFD‐L–induced adipocyte hypertrophy. HFD‐EVOO mice exhibited the greatest levels of Adiponectin in adipose tissue and presented atherosclerotic lesions similar to the LFD group, with a very low count of monocyte/macrophage compared with HFD‐L and HFD‐OL mice. Enrichment of the phenolic content of olive oil reduced the secretion of nitrites/nitrates in the aorta, but atherosclerosis was not attenuated in HFD‐OL mice compared to other HFD mice.[Conclusion] Consumption of olive oil with a natural content of phenolic compounds attenuates adipose tissue hypertrophy and inflammation and exerts antiatherosclerotic effects in mice. A higher phenolic content of olive oil did not provide further benefits in the prevention of atherosclerosis.This work was supported by the Ministerio de Economía ́ıa y Competitividad (grant number AGL2014-5458-R) and Junta de Andalucía ́ıa (PAI-BIO311)Peer reviewe

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr −/−). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m2), diabetes mellitus and ALT were found to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears

Extra virgin olive oil improved body weight and insulin sensitivity in high fat diet-induced obese LDLr−/−.Leiden mice without attenuation of steatohepatitis

Dietary fatty acids play a role in the pathogenesis of obesity-associated non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance (IR). Fatty acid composition is critical for IR and subsequent NAFLD development. Extra-virgin olive oil (EVOO) is the main source of monounsaturated fatty acids (MUFA) in Mediterranean diets. This study examined whether EVOO containing high fat diets may prevent diet-induced NAFLD using Ldlr−/−. Leiden mice. In female Ldlr−/−.Leiden mice, the efects of the following high fat diets (HFDs) were examined: a lard-based HFD (HFD-L); an EVOO-based HFD (HFD-EVOO); a phenolic compounds-rich EVOO HFD (HFD-OL). We studied changes in body weight (BW), lipid profle, transaminases, glucose homeostasis, liver pathology and transcriptome. Both EVOO diets reduced body weight (BW) and improved insulin sensitivity. The EVOOs did not improve transaminase values and increased LDL-cholesterol and liver collagen content. EVOOs and HFD-L groups had comparable liver steatosis. The profbrotic efects were substantiated by an up-regulation of gene transcripts related to glutathione metabolism, chemokine signaling and NF-kappa-B activation and down-regulation of genes relevant for fatty acid metabolism. Collectivelly, EVOO intake improved weight gain and insulin sensitivity but not liver infammation and fbrosis, which was supported by changes in hepatic genes expression

Metabolic characterization of two different non-alcoholic fatty liver disease pre-clinical mouse models

Introduction: non-alcoholic fatty liver disease is one of the most prevalent liver disorders in the developed world. Currently, there is no approved pharmacological therapy except for lifestyle intervention. Therefore, there is a need to increase the knowledge of preclinical models in order to boost novel discoveries that could lead to a better therapeutic management. Material and methods: this study characterized the effects of two different diets, a long-term high-fat high-fructose diet (HF-HFD) and a choline-deficient, methionine supplemented high-fat diet (CDA-HFD) in C57BL/6J mice for 52 weeks or 16 weeks, respectively. Body weight, lipid and hepatic profile were analyzed and liver histology was subsequently evaluated. Results: HF-HFD animals showed an increased body weight and total cholesterol levels, whereas the opposite occurred in CDA-HFD. Both HF-HFD and CDA-HFD animals had higher ALT and AST levels. With regard to histology findings, HF-HFD and CDA-HFD diets induced an increased collagen deposit and intrahepatic steatosis accumulation. Conclusion: in conclusion, the comparison of these models helped us to decide if it is better to select a long-term but more physiological model for physiopathology studies or either a more rapid NASH model for novel molecules testing.The authors received funding from the Health Department of the Regional Government of Andalucía (Consejería de Salud de la Junta de Andalucía), project number PC-0148- 2016-0148 awarded to Dr. Francisco Martín Bermudo and Dr. Rocío Gallego-Durán; Instituto de Salud Carlos III, project number PI16/01842 awarded to Dr. Manuel Romero Gómez and Dr. Javier Ampuero Herrojo; and Health Department of the Regional Government of Andalucía , project number PAI-BIO311 awarded to Dr. Francisco Martín Bermudo

Extra virgin olive oil diet intervention improves insulin resistance and islet performance in diet-induced diabetes in mice

13 Páginas.-- 7 Figuras.-- 1 TablaDietary composition plays an important role in the pathophysiology of type 2 diabetes. Monounsaturated fatty acid consumption has been positively associated with improved insulin sensitivity and β-cell function. We examined whether an extra virgin olive oil (EVOO) high fat diet (HFD) can improve glucose homeostasis. C57BL/6J mice were fed a standard diet or a lard-based HFD to induce type 2 diabetes. Then, HFD mice were fed with three different based HFD (lard, EVOO and EVOO rich in phenolic compounds) for 24 weeks. HFD-EVOO diets significantly improved glycemia, insulinemia, glucose tolerance, insulin sensitivity and insulin degradation. Moreover, EVOO diets reduced β-cell apoptosis, increased β-cell number and normalized islet glucose metabolism and glucose induced insulin secretion. No additional effects were observed by higher levels of phenolic compounds. Thus, EVOO intake regulated glucose homeostasis by improving insulin sensitivity and pancreatic β-cell function, in a type 2 diabetes HFD animal model.We thank Dr. Raquel Araujo and Antonio Cardenas for their technical assistance. We thank Dr. Alex Rafacho for his help with the analysis of the glucose decay constant rate during the ITT test and for his comments on the figures of the manuscript. We thank Dr. Irene Cozar for her help with liver insulin degrading enzyme experiments.Peer reviewe

Metabolic characterization of two different non-alcoholic fatty liver disease pre-clinical mouse models.

non-alcoholic fatty liver disease is one of the most prevalent liver disorders in the developed world. Currently, there is no approved pharmacological therapy except for lifestyle intervention. Therefore, there is a need to increase the knowledge of preclinical models in order to boost novel discoveries that could lead to a better therapeutic management. this study characterized the effects of two different diets, a long-term high-fat high-fructose diet (HF-HFD) and a choline-deficient, methionine supplemented high-fat diet (CDA-HFD) in C57BL/6J mice for 52 weeks or 16 weeks, respectively. Body weight, lipid and hepatic profile were analyzed and liver histology was subsequently evaluated. HF-HFD animals had an increased body weight and total cholesterol levels, whereas the opposite occurred in CDA-HFD. Both HF-HFD and CDA-HFD animals had higher ALT and AST levels. With regard to histology findings, HF-HFD and CDA-HFD diets induced an increased collagen deposit and intrahepatic steatosis accumulation. in conclusion, the comparison of these models aided in the selection of a long-term, more physiological model for physiopathology studies or a more rapid NASH model for novel molecule testing

Extra virgin olive oil improved body weight and insulin sensitivity in high fat diet-induced obese LDLr-/-.Leiden mice without attenuation of steatohepatitis

Dietary fatty acids play a role in the pathogenesis of obesity-associated non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance (IR). Fatty acid composition is critical for IR and subsequent NAFLD development. Extra-virgin olive oil (EVOO) is the main source of monounsaturated fatty acids (MUFA) in Mediterranean diets. This study examined whether EVOO-containing high fat diets may prevent diet-induced NAFLD using Ldlr-/-. Leiden mice. In female Ldlr-/-.Leiden mice, the effects of the following high fat diets (HFDs) were examined: a lard-based HFD (HFD-L); an EVOO-based HFD (HFD-EVOO); a phenolic compounds-rich EVOO HFD (HFD-OL). We studied changes in body weight (BW), lipid profile, transaminases, glucose homeostasis, liver pathology and transcriptome. Both EVOO diets reduced body weight (BW) and improved insulin sensitivity. The EVOOs did not improve transaminase values and increased LDL-cholesterol and liver collagen content. EVOOs and HFD-L groups had comparable liver steatosis. The profibrotic effects were substantiated by an up-regulation of gene transcripts related to glutathione metabolism, chemokine signaling and NF-kappa-B activation and down-regulation of genes relevant for fatty acid metabolism. Collectivelly, EVOO intake improved weight gain and insulin sensitivity but not liver inflammation and fibrosis, which was supported by changes in hepatic genes expression.This work was supported by the grants AGL2014-54585-R, AGL-2017-86927-R, CP14/ 00105, PI18/01590 (Ministerio de Economía y Competitividad), PAI-BIO311 (Junta de Andalucía), CB07/08/0006 (Instituto de Salud Carlos III) and by the TNO research programs “Body Brain Interactions ERP 020” and “Functional Biomarkers PMC13”.Ye

Contemporary use of cefazolin for MSSA infective endocarditis: analysis of a national prospective cohort

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P <0.01) and previous renal failure (57.9%, P <0.01). Patients treated with cloxacillin presented higher rates of septic shock (25%, P = 0.033) and new-onset or worsening renal failure (47.3%, P = 0.024) with significantly higher rates of in-hospital mortality (38.5%, P = 0.017). One-year IE-related mortality and rate of relapses were similar between treatment groups. None of the treatments were identified as risk or protective factors. Conclusion: Our results suggest that cefazolin is a valuable option for the treatment of MSSA IE, without differences in 1-year mortality or relapses compared with cloxacillin, and might be considered equally effective