Polyphenols and metabolism: from present knowledge to future challenges.

A diet rich in polyphenols and other types of phytonutrients can reduce the occurrence of chronic diseases. However, a well-established cause-and-effect association has not been clearly demonstrated and several other issues will need to be fully understood before general recommendations will be carried out In the present review, some of the future challenges that the research on phenolic compounds will have to face in the next years are discussed: toxicological aspects of polyphenols and safety risk assessment; synergistic effects between different polyphenols; metabotype-based nutritional advice based on a differential gut microbial metabolism of polyphenols (precision nutrition); combination of polyphenols with other bioactive compounds; innovative formulations to improve the bioavailability of phenolic compounds; and polyphenols in sports nutrition and recovery.Other aspects related to polyphenol research that will have a boost in the next years are: polyphenol and gut microbiota crosstalk, including prebiotic effects and biotransformation of phenolic compounds into bioactive metabolites by gut microorganisms; molecular docking, molecular dynamics simulation, and quantum and molecular mechanics studies on the protein-polyphenol complexes; and polyphenol-based coating films, nanoparticles, and hydrogels to facilitate the delivery of drugs, nucleic acids and proteins.In summary, this article provides some constructive inspirations for advancing in the research of the applications, risk assessment and metabolic effects of dietary polyphenols in humans.

Editorial Special Issue: 2022 consortium for trans-pyrenean investigations on obesity and diabetes.

This Special Issue of the Journal of Physiology and Biochemistry contains 7 contributions that have been elaborated in the context of the mini-network "Consortium of Trans-Pyrenean Investigations on Obesity and Diabetes" (CTPIOD), which is on its 19th year of existence. This scientific community, mostly involving research groups from France and Spain, but also open to participants coming from other countries, is focused on investigating the molecular and physiological mechanisms implicated in the development of obesity, diabetes, non-alcoholic fatty liver disease, and other noncommunicable diseases, as well as new preventive and therapeutic strategies. This special issue covers novel nutritional, molecular, and physiological aspects related to these metabolic diseases. Some of these papers emerge from the lectures of the 19th Conference on Trans-Pyrenean Investigations in Obesity and Diabetes, organized by the University of Zaragoza and celebrated in the town of Jaca (Spain) on 17-18th October 2022, and have been prepared in collaboration between different groups of the network. Many lectures were focused on the preventive role of specific fatty acids, dietary phenolic compounds and other phytochemicals against metabolic disorders. Consequently, we encouraged submission of original research in this field for this special issue.

Plant miR6262 Modulates the Expression of Metabolic and Thermogenic Genes in Human Hepatocytes and Adipocytes.

BACKGROUND: Edible plants have been linked to the mitigation of metabolic disturbances in liver and adipose tissue, including the decrease of lipogenesis and the enhancement of lipolysis and adipocyte browning. In this context, plant microRNAs could be key bioactive molecules underlying the cross-kingdom beneficial effects of plants. This study sought to explore the impact of plant-derived microRNAs on the modulation of adipocyte and hepatocyte genes involved in metabolism and thermogenesis. METHODS: Plant miR6262 was selected as a candidate from miRBase for the predicted effect on the regulation of human metabolic genes. Functional validation was conducted after transfection with plant miRNA mimics in HepG2 hepatocytes exposed to free fatty acids to mimic liver steatosis and hMADs cells differentiated into brown-like adipocytes. RESULTS: miR6262 decreases the expression of the predicted target RXRA in the fatty acids-treated hepatocytes and in brown-like adipocytes and affects the expression profile of critical genes involved in metabolism and thermogenesis, including PPARA, G6PC, SREBF1 (hepatocytes) and CIDEA, CPT1M and PLIN1 (adipocytes). Nevertheless, plant miR6262 mimic transfections did not decrease hepatocyte lipid accumulation or stimulate adipocyte browning. CONCLUSIONS: these findings suggest that plant miR6262 could have a cross-kingdom regulation relevance through the modulation of human genes involved in lipid and glucose metabolism and thermogenesis in adipocytes and hepatocytes.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

Effect of a Diet Supplemented with Sphingomyelin and Probiotics on Colon Cancer Development in Mice.

Previous studies have reported that dietary sphingomyelin could inhibit early stages of colon cancer. Lactic acid-producing bacteria have also been associated with an amelioration of cancer symptoms. However, little is known about the potential beneficial effects of the combined administration of both sphingomyelin and lactic acid-producing bacteria. This article analyzes the effect of a diet supplemented with a combination of the probiotics Lacticaseibacillus casei and Bifidobacterium bifidum (108 CFU/ml) and sphingomyelin (0.05%) on mice with 1,2-dimethylhydrazine (DMH)-induced colon cancer. Thirty-six BALB/c mice were divided into 3 groups: one healthy group (group C) and two groups with DMH-induced cancer, one fed a standard diet (group D) and the other fed a diet supplemented with sphingomyelin and probiotics (DS). The number of aberrant crypt foci, marker of colon cancer development, was lower in the DS. The dietary supplementation with the synbiotic reversed the cancer-induced impairment of galactose uptake in enterocyte brush-border-membrane vesicles. These results confirm the beneficial effects of the synbiotic on the intestinal physiology of colon cancer mice and contribute to the understanding of the possible mechanisms involved.

Microencapsulated Bifidobacterium bifidum and Lactobacillus gasseri in Combination with Quercetin Inhibit Colorectal Cancer Development in ApcMin/+ Mice.

Recent studies have suggested that flavonoids such as quercetin and probiotics such as Bifidobacterium bifidum (Bf) and Lactobacillus gasseri (Lg) could play a relevant role in inhibiting colon cancer cell growth. Our study investigated the role of dietary supplementation with microencapsulated probiotics (Bf and Lg) along with quercetin in the development of mouse colorectal cancer (CRC). Methods: Adenomatous polyposis coli/multiple intestinal neoplasia (ApcMin/+) mice were fed a standard diet or the same diet supplemented with microencapsulated probiotics (Bf and Lg strains, 107 CFU/100 g food) or both probiotics strains plus microencapsulated quercetin (15 mg/100 g food) for 73 days. Changes in body and organ weights, energy metabolism, intestinal microbiota, and colon tissue were determined. The expression of genes related to the Wnt pathway was also analyzed in colon samples. Results: Dietary supplementation with microencapsulated probiotics or microencapsulated probiotics plus quercetin reduced body weight loss and intestinal bleeding in ApcMin/+ mice. An improvement in energy expenditure was observed after 8 weeks but not after 10 weeks of treatment. A supplemented diet with microencapsulated Bf and Lg reduced the number of aberrant crypt foci (ACF) and adenomas by 45% and 60%, respectively, whereas the supplementation with Bf, Lg and quercetin decreased the number of ACF and adenomas by 57% and 80%, respectively. Microencapsulated Bf and Lg in combination with quercetin could exert inhibition of the canonical Wnt/ß-catenin signaling pathway in the colon of ApcMin/+ mice Conclusions: The administration of microencapsulated Bf and Lg, individually or in combination with quercetin, inhibits the CRC development in ApcMin/+ mice.

Azoxymethane-Induced Colorectal Cancer Mice Treated with a Polyphenol-Rich Apple Extract Show Less Neoplastic Lesions and Signs of Cachexia.

Obesity is considered a risk factor for the development of colorectal cancer. In rodents, high-fat (HF) diets are able to increase the formation of azoxymethane (AOM)-induced polyps. Polyphenol-rich apple extracts have antioxidant and anti-inflammatory activities and may induce an amelioration of the manifestations of colorectal cancer. Twenty-seven male Crl:CD-1 mice received AOM during four weeks and were subsequently divided into three groups fed a HF diet (n = 9 each group): a non-supplemented group, a second group supplemented with apple extract at 1%, and a third group supplemented with the same apple extract at 1.5%. Energy metabolism and the respiratory quotient were not affected by the supplementation with the apple extract. Although body weight was not affected by the treatment, the mice supplemented with the apple extract showed less signs of cachexia than the non-treated mice. In the intestine, the mice supplemented with the apple extract showed lower sucrase, dipeptidyl-peptidase IV, and aminopeptidase N activities, and less intestinal lesions (aberrant crypt foci and polyps). Administration of a polyphenol-rich apple extract reduces the number of neoplastic lesions in mice with AOM-induced colorectal cancer and contributes to preserve adipose tissue mass.

Epigenetic signatures underlying inflammation: an interplay of nutrition, physical activity, metabolic diseases, and environmental factors for personalized nutrition.

AIM AND OBJECTIVE: Emerging translational evidence suggests that epigenetic alterations (DNA methylation, miRNA expression, and histone modifications) occur after external stimuli and may contribute to exacerbated inflammation and the risk of suffering several diseases including diabetes, cardiovascular diseases, cancer, and neurological disorders. This review summarizes the current knowledge about the harmful effects of high-fat/high-sugar diets, micronutrient deficiencies (folate, manganese, and carotenoids), obesity and associated complications, bacterial/viral infections, smoking, excessive alcohol consumption, sleep deprivation, chronic stress, air pollution, and chemical exposure on inflammation through epigenetic mechanisms. Additionally, the epigenetic phenomena underlying the anti-inflammatory potential of caloric restriction, n-3 PUFA, Mediterranean diet, vitamin D, zinc, polyphenols (i.e., resveratrol, gallic acid, epicatechin, luteolin, curcumin), and the role of systematic exercise are discussed. METHODS: Original and review articles encompassing epigenetics and inflammation were screened from major databases (including PubMed, Medline, Science Direct, Scopus, etc.) and analyzed for the writing of the review paper. CONCLUSION: Although caution should be exercised, research on epigenetic mechanisms is contributing to understand pathological processes involving inflammatory responses, the prediction of disease risk based on the epigenotype, as well as the putative design of therapeutic interventions targeting the epigenome.

A combination of borage seed oil and quercetin reduces fat accumulation and improves insulin sensitivity in obese rats.

The metabolic properties of omega-6 fatty acid consumption are being increasingly accepted. We had previously observed that supplementation with a borage seed oil (BSO), as a source of linoleic (18:2n-6; LA) and gamma-linolenic (18:3n-6; GLA) acids, reduces body weight and visceral adiposity and improves insulin sensitivity in a diet-induced obesity model of Wistar rats. Here, it was investigated whether the anti-obesogenic properties of BSO could be maintained in a pre-obese model of rats, and if these effects are enhanced by a combination with low doses of quercetin, together with its potential role in the regulation of the adipocyte biology. The combination of BSO and quercetin during 8 weeks was able to ameliorate glucose intolerance and insulin resistance, and to improve liver steatosis. Although no effects were observed on body weight, animals supplemented with this combination exhibited a lower proportion of visceral adiposity. In addition, in vitro differentiation of epididymal adipose-precursor cells of the BSO-treated animals exhibited a down-regulation of Fasn, Glut4, Pparg and Srebp1 genes, in comparison with the control group. Finally, in vitro evaluation of the components of BSO demonstrated that the anti-adipogenic activity of quercetin was significantly potentiated by the combination with both LA and GLA through the down-regulation of different adipogenesis-key genes in 3T3-L1 cells. All these data suggest that omega-6 fatty acids LA and GLA, and their natural sources such as BSO, could be combined with quercetin to potentiate their effects in the prevention of the excess of adiposity and the insulin resistance.

One-Carbon Metabolism and Nonalcoholic Fatty Liver Disease: The Crosstalk between Nutrients, Microbiota, and Genetics.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Its etiology includes nutritional, genetic, and lifestyle factors. Several mechanisms may link one-carbon metabolism - the associated metabolic pathways of folate, methionine, and choline - to the onset of NAFLD. In this review, we attempted to assess how choline, folate, methionine, and betaine affect NAFLD development, mainly through their role in the secretion of very low-density lipoproteins (VLDL) from the liver. We also reviewed recent articles that have described the relation between microbiota metabolism and NAFLD progression. Moreover, we describe the effect of single-nucleotide polymorphisms (SNP) in genes related to one-carbon metabolism and disease prevalence. We additionally seek SNP identified by genome-wide associations that may increase the risk of this disease. Even though the evidence available is not entirely consistent, it seems that the concentrations of choline, methionine, folate, and betaine may affect the progression of NAFLD. Since there is no effective therapy for NAFLD, further investigations into the link between nutrition, gut microbiota, genetic factors, and NAFLD are still necessary, with a particular emphasis on methyl donors.

The regulation of inflammation-related genes after palmitic acid and DHA treatments is not mediated by DNA methylation.

Fatty acids (FAs) are known to participate in body inflammatory responses. In particular, saturated FAs such as palmitic acid (PA) induce inflammatory signals in macrophages, whereas polyunsaturated FAs, including docosahexaenoic acid (DHA), have been related to anti-inflammatory effects. Several studies have suggested a role of fatty acids on DNA methylation, epigenetically regulating gene expression in inflammation processes. Therefore, this study investigated the effect of PA and DHA on the inflammation-related genes on human macrophages. In addition, a second aim was to study the epigenetic mechanism underlying the effect of FAs on the inflammatory response. For these purposes, human acute monocytic leukaemia cells (THP-1) were differentiated into macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA), followed by an incubation with PA or DHA. At the end of the experiment, mRNA expression, protein secretion, and CpG methylation of the following inflammatory genes were analysed: interleukin 1 beta (IL1B), tumour necrosis factor (TNF), plasminogen activator inhibitor-1 (SERPINE1) and interleukin 18 (IL18). The results showed that the treatment with PA increased IL-18 and TNF-α production. Contrariwise, the supplementation with DHA reduced IL-18, TNF-α and PAI-1 secretion by macrophages. However, the incubation with these fatty acids did not apparently modify the DNA methylation status of the investigated genes in the screened CpG sites. This research reveals that PA induces important pro-inflammatory markers in human macrophages, whereas DHA decreases the inflammatory response. Apparently, DNA methylation is not directly involved in the fatty acid-mediated regulation of the expression of these inflammation-related genes.

Low doses of cocoa extract supplementation ameliorate diet-induced obesity and insulin resistance in rats.

Cocoa polyphenols exhibit high antioxidant activity and have been proposed as a potential adjuvant for the treatment of metabolic disturbances. Here, we demonstrate that supplementation with low doses (14 and 140 mg per kg per rat) of a complete cocoa extract induces metabolic benefits in a diet-induced obesity (DIO) model of Wistar rats. After 10 weeks, cocoa extract-supplemented animals exhibited significantly lower body weight gain and food efficiency, with no differences in energy intake. Cocoa significantly reduced visceral (epididymal and retroperitoneal) and subcutaneous fat accumulation accompanied by a significant reduction in the adipocyte size, which was mediated by downregulation of the adipocyte-specific genes Cebpa, Fasn and Adipoq. Additionally, cocoa extract supplementation reduced the triacylglycerol/high density lipoprotein (TAG/HDL) ratio, decreased hepatic triglyceride accumulation, improved insulin sensitivity by reducing HOMA-IR, and significantly ameliorated glucose tolerance after an intraperitoneal glucose tolerance test. Finally, no adverse effect was observed in an in vivo toxicity evaluation of our cocoa extract at doses up to 500 mg kg-1 day-1. Our data demonstrate that low doses of cocoa extract supplementation (14 and 140 mg kg-1 day-1) are safe and sufficient to counteract obesity and type-2 diabetes in rats and provide new insights into the potential application of cocoa supplements in the management of the metabolic syndrome.

Phenolic Compounds Inhibit 3T3-L1 Adipogenesis Depending on the Stage of Differentiation and Their Binding Affinity to PPARγ.

Phenolic compounds might modulate adiposity. Here, we report our observation that polyphenols and phenolic acids inhibit adipogenesis in 3T3-L1 with different intensity depending on the family and the stage of differentiation. While quercetin and resveratrol inhibited lipid accumulation along the whole process of differentiation, apigenin and myricetin were active during the early and latest stages, but not intermediate, contrary to hesperidin. The activity of phenolic acids was limited to the early stages of the differentiation process, except p-coumaric and ellagic acids. This anti-adipogenic effect was accompanied by down-regulation of Scd1 and Lpl. Molecular docking analysis revealed that the inhibitory activity of these phenolic compounds over the early stages of adipogenesis exhibits a significant correlation (r = 0.7034; p = 0.005) with their binding affinity to the ligand-binding domain of PPARγ. Results show that polyphenols and phenolic acids would interact with specific residues of the receptor, which could determine their potential anti-adipogenic activity during the early stages of the differentiation. Residues Phe264, His266, Ile281, Cys285 and Met348 are the most frequently involved in these interactions, which might suggest a crucial role for these amino acids modulating the activity of the receptor. These data contribute to elucidate the possible mechanisms of phenolic compounds in the control of adipogenesis.

DNA methylation markers in obesity, metabolic syndrome, and weight loss.

The fact that not all individuals exposed to the same environmental risk factors develop obesity supports the hypothesis of the existence of underlying genetic and epigenetic elements. There is suggestive evidence that environmental stimuli, such as dietary pattern, particularly during pregnancy and early life, but also in adult life, can induce changes in DNA methylation predisposing to obesity and related comorbidities. In this context, the DNA methylation marks of each individual have emerged not only as a promising tool for the prediction, screening, diagnosis, and prognosis of obesity and metabolic syndrome features, but also for the improvement of weight loss therapies in the context of precision nutrition. The main objectives in this field are to understand the mechanisms involved in transgenerational epigenetic inheritance, and featuring the nutritional and lifestyle factors implicated in the epigenetic modifications. Likewise, DNA methylation modulation caused by diet and environment may be a target for newer therapeutic strategies concerning the prevention and treatment of metabolic diseases.

Association of Methylation Signatures at Hepatocellular Carcinoma Pathway Genes with Adiposity and Insulin Resistance Phenotypes.

Obesity and type 2 diabetes mellitus are independent risk factors for the onset and progression of hepatocellular carcinoma (HCC). This study aimed to analyze the association of DNA methylation signatures at HCC pathway genes with obesity and related metabolic disturbances. A population of 474 adults within the Methyl Epigenome Network Association (MENA) project was included. DNA methylation levels were measured in white blood cells by microarray. The identification and discrimination of HCC pathway genes were performed using KEGG and PathDIP databases. Anthropometry measurements, the blood metabolic profile, and clinical data were analyzed. The methylation patterns of 20 CpG sites at HCC pathway genes strongly correlated with BMI (FDR <0.0001). These genes encompassed GADD45A, MTOR, FRAT2, E2F3, WNT7B, FRAT1, LRP5, DPF3, GSTA2, APC, MYC, WNT10B, ARID1B, AKT1, GSTA1, WNT5A, CDK4, GAB1, TCF7, which statistically contributed to the regulation of the HCC pathway (P = 2.10e-07). The main biological process where these genes were implicated included uncontrolled cell proliferation, DNA damage, increased survival, and altered oncogenic expression. Interestingly, 9 out of 20 BMI-associated CpGs also correlated with waist circumference and HOMA-IR index. In conclusion, pathway analysis revealed potential associations of DNA methylation signatures at HCC pathway genes with adiposity and insulin resistance phenotypes.

Prediction of Blood Lipid Phenotypes Using Obesity-Related Genetic Polymorphisms and Lifestyle Data in Subjects with Excessive Body Weight.

BACKGROUND AND AIM: Individual lipid phenotypes including circulating total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and triglycerides (TG) determinations are influenced by gene-environment interactions. The aim of this study was to predict blood lipid level (TC, LDL-c, HDL-c, and TG) variability using genetic and lifestyle data in subjects with excessive body weight-for-height. METHODS: This cross-sectional study enrolled 304 unrelated overweight/obese adults of self-reported European ancestry. A total of 95 single nucleotide polymorphisms (SNPs) related to obesity and weight loss were analyzed by a targeted next-generation sequencing system. Relevant genotypes of each SNP were coded as 0 (nonrisk) and 1 (risk). Four genetic risk scores (GRS) for each lipid phenotype were calculated by adding the risk genotypes. Information concerning lifestyle (diet, physical activity, alcohol drinking, and smoking) was obtained using validated questionnaires. Total body fat (TFAT) and visceral fat (VFAT) were determined by dual-energy X-ray absorptiometry. RESULTS: Overall, 45 obesity-related genetic variants were associated with some of the studied blood lipids. In addition to conventional factors (age, sex, dietary intakes, and alcohol consumption), the calculated GRS significantly contributed to explain their corresponding plasma lipid trait. Thus, HDL-c, TG, TC, and LDL-c serum concentrations were predicted by approximately 28% (optimism-corrected adj. R 2 = 0.28), 25% (optimism-corrected adj. R 2 = 0.25), 24% (optimism-corrected adj. R 2 = 0.24), and 21% (optimism-corrected adj. R 2=0.21), respectively. Interestingly, GRS were the greatest contributors to TC (squared partial correlation (PC2) = 0.18) and LDL-c (PC2 = 0.18) features. Likewise, VFAT and GRS had a higher impact on HDL-c (PC2 = 0.09 and PC2 = 0.06, respectively) and TG levels (PC2 = 0.20 and PC2 = 0.07, respectively) than the rest of variables. CONCLUSIONS: Besides known lifestyle influences, some obesity-related genetic variants could help to predict blood lipid phenotypes.

Do the Effects of Resveratrol on Thermogenic and Oxidative Capacities in IBAT and Skeletal Muscle Depend on Feeding Conditions?

The aim of this study was to compare the effects of mild energy restriction and resveratrol on thermogenic and oxidative capacity in interscapular brown adipose tissue (IBAT) and in skeletal muscle. Rats were fed a high-fat high-sucrose diet for six weeks, and divided into four experimental groups fed a standard diet: a control group, a resveratrol-treated group, an energy-restricted group and an energy-restricted group treated with resveratrol. Weights of IBAT, gastrocnemius muscle and fat depots were measured. Activities of carnitine palmitoyltransferase (CPT) and citrate synthase (CS), protein levels of sirtuin (SIRT1 and 3), uncoupling proteins (UCP1 and 3), glucose transporter (GLUT4), mitochondrial transcription factor (TFAM), nuclear respiratory factor (NRF1), peroxisome proliferator-activated receptor (PPARα) and AMP activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator (PGC1α) activation were measured. No changes in IBAT and gastrocnemius weights were found. Energy-restriction, but not resveratrol, decreased the weights of adipose depots. In IBAT, resveratrol enhanced thermogenesis activating the SIRT1/PGC1α/PPARα axis. Resveratrol also induced fatty acid oxidation and glucose uptake. These effects were similar when resveratrol was combined with energy restriction. In the case of gastrocnemius muscle, the effects were not as clear as in the case of IBAT. In this tissue, resveratrol increased oxidative capacity. The combination of resveratrol and energy restriction seemingly did not improve the effects induced by the polyphenol alone.

Implication of miR-612 and miR-1976 in the regulation of TP53 and CD40 and their relationship in the response to specific weight-loss diets.

BACKGROUND: Non-coding RNAs (i.e., miRNAs) play a role in the development of obesity and related comorbidities and the regulation of body weight. OBJECTIVE: To identify candidate miRNA biomarkers throughout omics approaches in order to predict the response to specific weight-loss dietary treatments. DESIGN: Genomic DNA and cDNA isolated from white blood cells of a subset from the RESMENA nutritional intervention study (Low-responders (LR) vs High-responders (HR)) was hybridized in Infinium Human Methylation450 BeadChip and in Illumina Human HT-12 v4 gene expression BeadChips arrays respectively. A bioinformatic prediction of putative target sites of selected miRNAs was performed by applying miRBase algorithms. HEK-293T cells were co-transfected with expression vectors containing the 3'-UTR of candidate genes to validate the binding of miRNAs to its target sites. RESULTS: 134 miRNAs were differentially methylated between HR and LR in the methylation array, whereas 44 miRNAs were differentially expressed between both groups in the expression array. Specifically, miR-1237, miR-1976, miR-642, miR-636, miR-612 and miR-193B were simultaneously hypomethylated and overexpressed in HR. miR-612 and miR-1976 showed greatest differences in methylation and expression levels, respectively. The bioinformatic prediction revealed that TP53 was a putative target gene of miR-612 and CD40 of miR-1976. Moreover, TP53 was downregulated in the expression array when comparing HR vs LR expression levels adjusted by sex, diet, age and baseline weight, and CD40 showed a statistical trend. Furthermore, gene expression levels of TP53 and CD40 in white blood cells, when measured by qPCR, were also downregulated in HR. Finally, miR-612 and miR-1976 potently repressed TP53 and CD40 respectively by targeting its 3'-UTR regions. CONCLUSION: miR-612 and miR-1976 levels could be prospective biomarkers of response to specific weight-loss diets and might regulate the gene expression of TP53 and CD40.

Effects of exosomes from LPS-activated macrophages on adipocyte gene expression, differentiation, and insulin-dependent glucose uptake.

Obesity is usually associated with low-grade inflammation, which determines the appearance of comorbidities like atherosclerosis and insulin resistance. Infiltrated macrophages in adipose tissue are partly responsible of this inflammatory condition. Numerous studies point to the existence of close intercommunication between macrophages and adipocytes and pay particular attention to the proinflammatory cytokines released by both cell types. However, it has been recently described that in both, circulation and tissue level, there are extracellular vesicles (including microvesicles and exosomes) containing miRNAs, mRNAs, and proteins that can influence the inflammatory response. The objective of the present research is to investigate the effect of exosomes released by lipopolysaccharide (LPS)-activated macrophages on gene expression and cell metabolism of adipocytes, focusing on the differential exosomal miRNA pattern between LPS- and non-activated macrophages. The results show that the exosomes secreted by the macrophages do not influence the preadipocyte-to-adipocyte differentiation process, fat storage, and insulin-mediated glucose uptake in adipocytes. However, exosomes induce changes in adipocyte gene expression depending on their origin (LPS- or non-activated macrophages), including genes such as CXCL5, SOD, TNFAIP3, C3, and CD34. Some of the pathways or metabolic processes upregulated by exosomes from LPS-activated macrophages are related to inflammation (complement activation, regulation of reactive oxygen species, migration and activation of leukocyte, and monocyte chemotaxis), carbohydrate catabolism, and cell activation. miR-530, chr9_22532, and chr16_34840 are more abundant in exosomes from LPS-activated macrophages, whereas miR-127, miR-143, and miR-486 are more abundant in those secreted by non-activated macrophages.