Metabolic-associated fatty liver disease: From simple steatosis toward liver cirrhosis and potential complications. Proceedings of the Third Translational Hepatology Meeting, organized by the Spanish Association for the Study of the Liver (AEEH).

This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC).

The role of nutrition in non-alcoholic fatty liver disease: Pathophysiology and management.

A healthy diet together with physical activity could induce weight loss and control the progression of non-alcoholic fatty liver disease (NAFLD). However, the composition of diet has not been clearly established. Macronutrients such as saturated fatty acids (SFA), trans-fats, simple sugars and animal proteins have a harmful effect on the liver. On the other hand, monounsaturated fats (MUFAs), polyunsaturated (PUFAs) omega-3-fats, plant-based proteins and dietary fibres are considered to be beneficial to the liver. The impact of specific micronutrients is less well-known. Nutrients are part of the food we eat. Food makes up our meals, which compose our dietary patterns. Non-alcoholic fatty liver disease patients usually follow Western diets which are rich in soda, frozen junk food, juice, red meat, lard, processed meats, whole fat dairy foods, fatty snack foods, take-away foods, cakes and biscuits and poor in cereals, whole grains, fruit, vegetables, extra virgin olive oil (EVOO) and fish. On the other hand, the Mediterranean diet (MD) is beneficial for NAFLD even when it is iso-caloric or there are no changes in body weight. A new approach, called 'nutritional geometry' considers the importance of integrating nutrition, animals and the environment. The goal of this approach is to combine nutrients and foods in a model to understand how food components interact to regulate the properties of diets affecting health and disease. The use of algorithms developed by artificial intelligence (AI) to create a personalized diet for patients can provide customized nutritional counselling to prevent and treat NAFLD.

Gene-Diet Interactions in Type 2 Diabetes: The Chicken and Egg Debate.

Consistent evidence from both experimental and human studies indicates that Type 2 diabetes mellitus (T2DM) is a complex disease resulting from the interaction of genetic, epigenetic, environmental, and lifestyle factors. Nutrients and dietary patterns are important environmental factors to consider in the prevention, development and treatment of this disease. Nutritional genomics focuses on the interaction between bioactive food components and the genome and includes studies of nutrigenetics, nutrigenomics and epigenetic modifications caused by nutrients. There is evidence supporting the existence of nutrient-gene and T2DM interactions coming from animal studies and family-based intervention studies. Moreover, many case-control, cohort, cross-sectional cohort studies and clinical trials have identified relationships between individual genetic load, diet and T2DM. Some of these studies were on a large scale. In addition, studies with animal models and human observational studies, in different countries over periods of time, support a causative relationship between adverse nutritional conditions during in utero development, persistent epigenetic changes and T2DM. This review provides comprehensive information on the current state of nutrient-gene interactions and their role in T2DM pathogenesis, the relationship between individual genetic load and diet, and the importance of epigenetic factors in influencing gene expression and defining the individual risk of T2DM.

Effect of thermal processing on the profile of bioactive compounds and antioxidant capacity of fermented orange juice.

Previously, we reported that alcoholic fermentation enhanced flavanones and carotenoids content of orange juice. The aim of this work was to evaluate the influence of pasteurization on the qualitative and quantitative profile of bioactive compounds and the antioxidant capacity of fermented orange juice. Ascorbic acid (203 mg/L), total flavanones (647 mg/L), total carotenoids (7.07 mg/L) and provitamin A (90.06 RAEs/L) values of pasteurized orange beverage were lower than those of fermented juice. Total phenolic remained unchanged (585 mg/L) and was similar to that of original juice. The flavanones naringenin-7-O-glucoside, naringenin-7-O-rutinoside, hesperetin-7-O-rutinoside, hesperetin-7-O-glucoside and isosakuranetin-7-O-rutinoside, and the carotenoids karpoxanthin and isomer, neochrome, lutein, ζ-carotene, zeaxanthin, mutatoxanthin epimers, ß-cryptoxanthin and auroxanthin epimers were the major compounds. Pasteurization produced a decrease in antioxidant capacity of fermented juice. However, TEAC (5.45 mM) and ORAC (6353 µM) values of orange beverage were similar to those of original orange juice. The novel orange beverage could be a valuable source of bioactive compounds with antioxidant capacity and exert potential beneficial effects.

We are what we eat: The role of lipids in metabolic diseases.

Lipids play a fundamental role, both structurally and functionally, for the correct functioning of the organism. In the last two decades, they have evolved from molecules involved only in energy storage to compounds that play an important role as components of cell membranes and signaling molecules that regulate cell homeostasis. For this reason, their interest as compounds involved in human health has been gaining weight. Indeed, lipids derived from dietary sources and endogenous biosynthesis are relevant for the pathophysiology of numerous diseases. There exist pathological conditions that are characterized by alterations in lipid metabolism. This is particularly true for metabolic diseases, such as liver steatosis, type 2 diabetes, cancer and cardiovascular diseases. The main issue to be considered is lipid homeostasis. A precise control of fat homeostasis is required for a correct regulation of metabolic pathways and safe and efficient energy storage in adipocytes. When this fails, a deregulation occurs in the maintenance of systemic metabolism. This happens because an increased concentrations of lipids impair cellular homeostasis and disrupt tissue function, giving rise to lipotoxicity. Fat accumulation results in many alterations in the physiology of the affected organs, mainly in metabolic tissues. These alterations include the activation of oxidative and endoplasmic reticulum stress, mitochondrial dysfunction, increased inflammation, accumulation of bioactive molecules and modification of gene expression. In this chapter, we review the main metabolic diseases in which alterations in lipid homeostasis are involved and discuss their pathogenic mechanisms.

Contribution of Liver and Pancreatic Islet Crosstalk to ß-Cell Function/Dysfunction in the Presence of Fatty Liver.

Tissue-to-tissue crosstalk regulates organ function, according to growing data. This phenomenon is relevant for pancreatic ß-cells and the liver, as both tissues are involved in glucose homeostasis and lipid metabolism. The ability to fine-tune regulation and adaptive responses is enabled through communication between pancreatic ß-cells and the liver. However, the crosstalk between both tissues changes when metabolic dysregulation is present. Factors and cargo from extracellular vesicles (EVs) released by liver and pancreatic ß-cells that reach the circulation form the words of this interaction. The molecules released by the liver are called hepatokines and are usually secreted in response to the metabolic state. When hepatokines reach the pancreatic islets several mechanisms are initiated for their protection or damage. In the case of the crosstalk between pancreatic ß-cells and the liver, only one factor has been found to date. This protein, pancreatic derived factor (PANDER) has been proposed as a novel linker between insulin resistance (IR) and type 2 diabetes mellitus (T2D) and could be considered a biomarker for non-alcoholic fatty liver disease (NAFLD) and T2D. Furthermore, the cargo released by EVs, mainly miRNAs, plays a significant role in this crosstalk. A better knowledge of the crosstalk between liver and pancreatic ß-cells is essential to understand both diseases and it could lead to better prevention and new therapeutic options.

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.

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

Dietary 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 ß-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 ß-cell apoptosis, increased ß-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 ß-cell function, in a type 2 diabetes HFD animal model.

An extra-virgin olive oil rich in polyphenolic compounds has antioxidant effects in OF1 mice.

Extra-virgin olive oil (OO) is becoming more important in daily diets due to its beneficial effects on health, most of which are because of its antioxidant content. We studied the antioxidant activity and mechanisms of an extra-virgin OO that is rich in phenolics on pancreatic islets and liver in control mice (CTL) fed a nonpurified diet and in mice supplemented with 50 microL/d sunflower oil (SO) or 50 microL/d extra-virgin OO for 4 d. Plasma hydroxytyrosol concentration was determined by HPLC-diode array detector. Plasma antioxidant capacity, enzymatic activities, and lipid peroxidation were measured by spectrophotometry. Islet function was studied by measuring insulin release. Islet cell gene expression was examined using quantitative RT-PCR. The plasma hydroxytyrosol concentration was greater in OO mice than in CTL or SO mice (P < 0.05) and was greater in SO mice than in CTL mice. The ratio of reduced:oxidized glutathione and the antioxidant capacity in plasma was greater in OO mice than in CTL or SO mice (P < 0.05) and higher in SO mice than in CTL mice. Glucose-stimulated insulin secretion was greater in OO mice than in CTL or SO mice (P < 0.05) and was also higher in SO mice than in CTL mice. Protection against liver cell and beta cell membrane lipid peroxidation was greater in OO mice than in CTL or SO mice (P < 0.05) and was greater in SO mice than in CTL mice. Catalase (CAT) expression in the islet of Langerhans was higher in OO mice than in CTL mice and SO mice (P < 0.05). The CAT and glutathione peroxidase 1 activities in the islet of Langerhans were 25% greater in OO mice than in CTL mice and higher than in SO mice (P < 0.05) and they were greater in SO mice than in CTL mice. These results indicate that, in metabolic tissues, protection by extra-virgin OO against oxidative stress occurs primarily through a direct antioxidant effect as well as through an indirect mechanism that involves greater expression and activity of certain enzymes with antioxidant activities.

Zn2+ chelation by serum albumin improves hexameric Zn2+-insulin dissociation into monomers after exocytosis.

ß-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K+-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of "slow" Zn2+-insulin into "fast" insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.

An extra virgin olive oil rich diet intervention ameliorates the nonalcoholic steatohepatitis induced by a high-fat "Western-type" diet in mice.

SCOPE: We evaluated the protective effect of extra virgin olive oil (EVOO) in high-fat diets (HFDs) on the inflammatory response and liver damage in a nonalcoholic fatty liver disease (NAFLD) mouse model. METHODS AND RESULTS: C57BL/6J mice were fed a standard diet or a lard-based HFD (HFD-L) for 12 wk to develop NAFLD. HFD-fed mice were then divided into four groups and fed for 24 wk with the following: HFD-L, HFD-EVOO, HFD based on phenolics-rich EVOO, and reversion (standard diet). HFD-L-induced metabolic disorders were alleviated by replacement of lard with EVOO. EVOO diets improved plasma lipid profile and reduced body weight, plasma and epididymal fat INF-γ, IL-6 and leptin levels, and macrophage infiltration. Moreover, NAFLD activity scores were reduced. The liver lipid composition showed an increase in MUFAs, especially oleic acid, and a decrease in saturated fatty acids. Hepatic adiponutrin and Cd36 gene expression was upregulated in the EVOO groups. Liver ingenuity pathway analysis revealed in EVOO groups regulation of proteins involved in lipid metabolism, small molecule biochemistry, gastrointestinal disease, and liver regeneration. CONCLUSION: Dietary EVOO could repair HFD-induced hepatic damage, possibly via an anti-inflammatory effect in adipose tissue and modifications in the liver lipid composition and signaling pathways.

Consumption of orange fermented beverage reduces cardiovascular risk factors in healthy mice.

The consumption of fruits prevents the risk of cardiovascular diseases. Alcoholic fermentation has been carried out in fruits resulting in products which provide high concentration of bioactive compounds and variable alcohol content. The aim of this study was to assess the potential beneficial effect of an orange beverage obtained by alcoholic fermentation and pasteurization of orange juice on cardiovascular risk biomarkers. For this purpose, four mice groups (n = 8) ingested orange beverage (equivalent volume to 250 mL/day in human), orange juice, alcoholic solution (at the proportional amount of orange beverage) or water during 12 weeks. The equivalent amount to double serving of orange beverage (500 mL/day) was administered to mice in a subsequent intervention, and a control group was also evaluated. Orange beverage consumption increased levels of glutathione and uric acid, improved lipid profile, decreased oxidized LDL and maintained levels of IL-6 and C-reactive protein. Synergistic effects between the bioactive compounds and the alcohol content of orange beverage may occur. The intake of double serving also increased antioxidant enzyme activities, bilirubin content and plasma antioxidant capacity. These results suggest that orange beverage may produce greater protection against cardiovascular risk factors than orange juice in healthy mice.

Nutrigenetics and nutrigenomics insights into diabetes etiopathogenesis.

Diabetes mellitus (DM) is considered a global pandemic, and the incidence of DM continues to grow worldwide. Nutrients and dietary patterns are central issues in the prevention, development and treatment of this disease. The pathogenesis of DM is not completely understood, but nutrient-gene interactions at different levels, genetic predisposition and dietary factors appear to be involved. Nutritional genomics studies generally focus on dietary patterns according to genetic variations, the role of gene-nutrient interactions, gene-diet-phenotype interactions and epigenetic modifications caused by nutrients; these studies will facilitate an understanding of the early molecular events that occur in DM and will contribute to the identification of better biomarkers and diagnostics tools. In particular, this approach will help to develop tailored diets that maximize the use of nutrients and other functional ingredients present in food, which will aid in the prevention and delay of DM and its complications. This review discusses the current state of nutrigenetics, nutrigenomics and epigenomics research on DM. Here, we provide an overview of the role of gene variants and nutrient interactions, the importance of nutrients and dietary patterns on gene expression, how epigenetic changes and micro RNAs (miRNAs) can alter cellular signaling in response to nutrients and the dietary interventions that may help to prevent the onset of DM.

Fermented orange juice: source of higher carotenoid and flavanone contents.

The intake of bioactive compounds and moderate alcohol decreases the risk of cardiovascular diseases. These effects could be joined in a beverage created by a controlled alcoholic fermentation of orange juice. The influence of controlled alcoholic fermentation on the bioactive compound profile of orange juice has not been previously evaluated, and this is the purpose of the present study. Total and individual flavanones and carotenoids significantly increased throughout the fermentation. The reason for this was an enhanced extraction of these compounds from the pulp. Besides, the potential bioavailability of flavanones increased due to a higher content of hesperetin-7-O-glucoside (2-fold higher at the end of the fermentation process). Ascorbic acid did not undergo a significant change, and only total phenolics decreased. Antioxidant capacity was also evaluated. TEAC and FRAP values remained constant throughout the process. However, ORAC and DPPH values significantly increased. Correlation analysis concluded that the increase in ORAC and DPPH values could be due to enhancement of flavanones.

Changes in antioxidant endogenous enzymes (activity and gene expression levels) after repeated red wine intake.

The antioxidant properties of wine have been largely related to the reactive oxygen species (ROS) scavenging ability of phenolic compounds. Polyphenolic compounds are hardly absorbed and quickly transformed into metabolites. Their antioxidant activities just as radical scavenging properties are therefore limited, but it is worth looking to other mechanisms. This study intended to test whether wine consumption affects antioxidant enzyme activity and gene expression. For this purpose, eight subjects drank 300 mL of red wine every day for a week and ate a low phenolic diet (LPD + W) specifically designed to avoid interferences from other polyphenols in the diet. The control period was a week with this diet, and volunteers refrained from drinking wine (LPD). Blood samples were taken at 0, 1, and 7 days. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities were determined in erythrocytes (SOD), plasma (CAT and GR), and blood (GPx). Gene expression was determined in macrophages. Oxidative stress caused by LPD reduced SOD, CAT, and GR activities. After wine consumption, these activities significantly increased (P < 0.05), and this overcame the effect of oxidative stress on enzyme activity. The modulation of CAT activity may be independent of changes in their gene expression, which significantly increased after LPD. However, SOD gene expression increased only during the LPD + W week. Enzyme activities are not all regulated in the same way. The results show that subacute moderate wine ingestion modulated antioxidant enzyme expression and activity, which is important for the prevention of ROS-associated diseases.

Taurine supplementation modulates glucose homeostasis and islet function.

Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells.

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.

Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors.

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/betageo/phosphoglycerate kinase-hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day-16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bro-modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription-polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering beta cells from stem cells.