Interscapular brown adipose tissue is surrounded by a white fat depot, the interscapular WAT (isWAT), which has received little attention. Here, we have characterized isWAT and the impact of chronic intake of a very high-fat diet, the cafeteria diet, on isWAT browning capacity. The effect of high-fat diets on WAT browning is controversial, however, some groups have described increased WAT UCP1 expression, probably to counteract increased adiposity. We analyzed mRNA expression of key brown/brite markers and lipid metabolism genes, and of selected proteins in isWAT of control, cafeteria-obese and post-cafeteria rats. Morphological/immunohistochemical characterization was also performed. Results present isWAT as a depot with a molecular profile close to that of browning-prone white fat depots (inguinal and retroperitoneal), which also shares key features with BAT, including UCP1 expression (mRNA/protein). The cafeteria diet intake induced mRNA expression of browning/thermogenic genes, which was restored after the diet was withdrawn, and increased expression of key genes involved in lipid catabolism in isWAT. Moreover, basal mRNA expression of brown/brite markers was greater than in other white depots and higher UCP1 protein levels were observed compared to the browning-prone retroperitoneal depot. Thus, isWAT appears as an interesting depot to be taken into consideration for lipid research and to better characterize browning regulation.

Early-life metabolic environment significantly impacts long-term cognitive and metabolic health. This study investigates transcriptomic alterations in the hippocampus and peripheral blood mononuclear cells (PBMC) of young rats exposed to an isocaloric high-fat diet (HFD), resulting in the metabolically obese, normal-weight (MONW) phenotype. Rats were pair-fed either a standard (NW group) or HFD (MONW group) for 11 weeks after weaning. Another group (MONW-Lep) received leptin supplementation during lactation and subsequently HFD. Transcriptomic analysis of the hippocampus showed disruption of pathways linked to obesity and cognitive decline in the MONW group, which were attenuated by leptin intake. This was consistent with the results of working memory (T-maze test), impaired in MONW versus NW, but preserved in MONW-Lep animals. PBMC transcriptomics revealed overlapping genes with the hippocampus. Notably, Piwil1, a gene linked to neurodegeneration, metabolic syndrome and obesity, was up-regulated in PBMC of MONW but not of MONW-Lep animals, reflecting early hippocampal changes and leptin's preventive effect. These findings highlight the influence of early nutrition on cognitive health, the protective potential of leptin counteracting the effects of HFD intake and the usefulness of PBMC as a reliable source of biomarkers of brain health.

Background: Due to the continuous global rise in obesity prevalence, foods rich in bioactive compounds are increasingly recognised for the management of several diseases. Objective: The present study aims to investigate whether an Opuntia stricta var. dillenii fruit peel extract, rich in betalains and phenolic compounds, is able to prevent obesity induced by a high-fat high-fructose diet in rats, along with the potential mechanisms of action underlying this effect. Results: The supplementation with Opuntia stricta var. dillenii extract obtained from the peel fruit partially prevents obesity development by attenuating HFHF-induced fat accumulation. This effect was observed predominantly in visceral adipose tissue, rather than in the subcutaneous depot. The obesity prevention was accompanied by the improvement of serum lipid profile. The mechanisms underlying the extract anti-obesity effect which were analysed in epididymal adipose tissue, involve preventing the rise in the availability of triglyceride synthesis substrates induced by high-fat high-fructose feeding, the inhibition of triglyceride assembly, and in the case of the high dose, increased lipolysis. Conclusions: According to these results, the peel wastes of Opuntia stricta var. dillenii fruit represent a promising natural source of bioactive compounds for obesity prevention. Nevertheless, these preclinical effects should be replicated in further studies in human beings.

Metabolically obese, normal-weight (MONW) phenotype is characterized by visceral adiposity and obesity-related complications despite the absence of excess body weight. Early identification of this phenotype is crucial to establish preventive strategies. We aim to validate the utility of peripheral blood mononuclear cells (PBMC) transcriptome to detect metabolic risk related to the MONW phenotype at early life stages (young adulthood). Male Wistar rats were pair-fed either standard (NW group) or a high-fat diet (MONW group) after weaning, until 3.5 months. Global gene expression was examined by microarray in PBMC, and specific genes of interest by RT-qPCR in PBMC and liver. Results were validated in adult 6-month-old MONW rats. Young MONW animals had similar weight to controls (NW group) but greater adiposity, including liver fat content, and insulin resistance signs. PBMC transcriptome distinguished clearly MONW from NW rats. Neurological pathways were affected in line with impaired cognition in these animals. Most top-regulated genes were related to inflammation, including the top-up and down-regulated genes, Hpgds and Slfn4. Expression of fatty liver-related genes like Mkrn1 and Nampt was also affected in PBMC of the young MONW group mirroring liver alteration. Slfn4 and Mkrn1 appeared as especially relevant biomarkers with altered expression also in PBMC of adult 6-month-old MONW rats. In conclusion, PBMC transcriptomic analysis emerges as a tool for identifying early biomarkers of obesity-related metabolic risk in young and apparently healthy (lean) subjects, pointing towards increased inflammation, liver fat deposition, and cognitive alterations.

The intake of high-fat diets (HFDs) and obesity are linked to cognitive impairment. Here, we aimed to investigate whether an early metabolically obese, normal-weight (MONW) phenotype, induced with an HFD in young rats, also leads to cognitive dysfunction and to evaluate the potential cognitive benefits of neonatal intake of leptin. To achieve this, Wistar rats orally received physiological doses of leptin or its vehicle during lactation, followed by 11 weeks of pair-feeding with an HFD or control diet post-weaning. Working memory was assessed using a T-maze, and gene expression in the hippocampus and peripheral blood mononuclear cells (PBMCs) was assessed with real-time RT-qPCR to identify cognition biomarkers. Young MONW-like rats showed hippocampal gene expression changes and decreased working memory. Animals receiving leptin during lactation presented similar gene expression changes but preserved working memory despite HFD intake, partly due to improved insulin sensitivity. Notably, PBMC Syn1 expression appears as an accessible biomarker of cognitive health, reflecting both the detrimental effect of HFD intake at early ages despite the absence of obesity and the positive effects of neonatal leptin treatment on cognition. Thus, the MONW phenotype developed at a young age is linked to cognitive dysfunction, which is reflected at the transcriptomic level in PBMCs. Neonatal leptin intake can partly counteract this impaired cognition resulting from early HFD consumption.

(1) Background: Cold exposure induces metabolic adaptations that can promote health benefits, including increased energy disposal due to lipid mobilization in adipose tissue (AT). This study aims to identify easily measurable biomarkers mirroring the effect of cold exposure on AT. (2) Methods: Transcriptomic analysis was performed in peripheral blood mononuclear cells (PBMCs) and distinct AT depots of two animal models (ferrets and rats) exposed to cold, and in PBMCs of cold-exposed humans. (3) Results: One week of cold exposure (at 4 °C) affected different metabolic pathways and gene expression in the AT of ferrets, an animal model with an AT more similar to humans than that of rodents. However, only one gene, Tlcd4, was affected in the same way (overexpressed) in aortic perivascular and inguinal AT depots and in PBMCs, making it a potential biomarker of interest. Subsequent targeted analysis in rats showed that 1 week at 4 °C also induced Tlcd4 expression in brown AT and PBMCs, while 1 h at 4 °C resulted in reduced Tlcd4 mRNA levels in retroperitoneal white AT. In humans, no clear effects were observed. Nevertheless, decreased PBMC TLCD4 expression was observed after acute cold exposure in women with normal weight, although this effect could be attributed to short-term fasting during the procedure. No effect was evident in women with overweight or in normal-weight men. (4) Conclusions: Our results obtained for different species point toward TLCD4 gene expression as a potential biomarker of cold exposure/fat mobilization that could tentatively be used to address the effectiveness of cold exposure-mimicking therapies.

Background Physical activity (PA) provides health benefits across the lifespan and improves many established cardiovascular risk factors that have a significant impact on overall mortality. However, discrepancies between self-reported and device-based measures of PA make it difficult to obtain consistent results regarding PA and its health effects. Moreover, PA may produce different health effects depending on the type, intensity, duration, and frequency of activities and individual factors such as age, sex, body weight, early life conditions/exposures, etc. Appropriate biomarkers relating the degree of PA level with its effects on health, especially in children and adolescents, are required and missing. The main objective of the INTEGRActiv study is to identify novel useful integrative biomarkers of PA and its effects on the body health in children and adolescents, who represent an important target population to address personalized interventions to improve future metabolic health. Methods/design The study is structured in two phases. First, biomarkers of PA and health will be identified at baseline in a core cohort of 180 volunteers, distributed into two age groups: prepubertal ( n = 90), and postpubertal adolescents ( n = 90). Each group will include three subgroups ( n = 30) with subjects of normal weight, overweight, and obesity, respectively. Identification of new biomarkers will be achieved by combining physical measures (PA and cardiorespiratory and muscular fitness, anthropometry) and molecular measures (cardiovascular risk factors, endocrine markers, cytokines and circulating miRNA in plasma, gene expression profile in blood cells, and metabolomics profiling in plasma). In the second phase, an educational intervention and its follow-up will be carried out in a subgroup of these subjects (60 volunteers), as a first validation step of the identified biomarkers. Discussion The INTEGRActiv study is expected to provide the definition of PA and health-related biomarkers (PA-health biomarkers) in childhood and adolescence. It will allow us to relate biomarkers to factors such as age, sex, body weight, sleep behavior, dietary factors, and pubertal status and to identify how these factors quantitatively affect the biomarkers’ responses. Taken together, the INTEGRActiv study approach is expected to help monitor the efficacy of interventions aimed to improve the quality of life of children/adolescents through physical activity. Clinical Trial Registration ClinicalTrials.gov , Identifier NCT05907785.

SCOPE:Peripheral blood mononuclear cells (PBMC) provide a useful and minimally invasive source of biomarkers. Here to identify PBMC transcriptomic biomarkers predictive of metabolic impairment related to increased adiposity is aimed.METHODS AND RESULTS:The study analyzed the global PBMC transcriptome in metabolically healthy (normoglycemic) volunteers with overweight-obesity (OW-OB, n = 12), and in subjects with metabolically obese normal-weight (MONW, n = 5) phenotype, in comparison to normal-weight (NW, n = 12) controls. The study identifies 1072 differentially expressed genes (DEGs) in OW-OB versus NW and 992 in MONW versus NW. Hierarchical clustering of the top 100 DEGs clearly distinguishes OW-OB and MONW from NW. Remarkably, the OW-OB and MONW phenotypes share 257 DEGs regulated in the same direction. The top up-regulated gene CXCL8, coding for interleukin 8, with a role in obesity-related pathologies, is of special interest as a potential marker for predicting increased metabolic risk. CXCL8 expression is increased mainly in the MONW group and correlated directly with C-reactive protein levels.CONCLUSIONS:PBMC gene expression analysis of CXCL8 or a pool of DEGs may be used to identify early metabolic risk in an apparently healthy population regardless of their BMI, i.e., subjects with OW-OB or MONW phenotype and to apply adequate and personalized nutritional preventive strategies.

Perinatal nutrition has a well-known influence on obesity susceptibility. We previously demonstrated the protective anti-obesity effects of perinatal leptin administration. Celastrol is a natural compound acting as a leptin sensitizer with anti-obesity effects when administered in adult animals. Here, we aimed to determine if perinatal treatment with leptin, celastrol, or their combination was able to improve metabolic health in animals fed an isocaloric high-fat (HF) diet. Leptin and/or celastrol or their vehicle were administered orally to rats during the suckling period. After weaning, animals were chronically pair-fed with an HF diet provided isocaloric to the intake of a normal-fat diet by control animals to avoid obesity. Isocaloric HF feeding in vehicle-treated animals resulted in metabolic features characteristic of the metabolically obese, normal-weight (MONW) phenotype, i.e., obesity-related disturbances without increased body weight. Leptin treatment prevented liver fat deposition and insulin resistance, induced greater insulin and leptin signaling capacity, decreased gene expression of orexigenic signals at the hypothalamic level, and induced browning in retroperitoneal adipose tissue. However, celastrol treatment did not provide any protective effect and resulted in greater size of the retroperitoneal adipose depot, higher circulating glucose and insulin levels, and decreased leptin sensitivity capacity in adipose tissue. The co-administration of leptin ameliorated the negative effects of celastrol on the retroperitoneal depot, inducing browning and decreasing its size. In conclusion, the perinatal administration of leptin, but not celastrol, provided protection against the consequences of dietary unbalances leading to an MONW phenotype in adulthood.

To assess the efficacy of different modalities and frequencies of physical exercise on glycaemic control in adults with prediabetes. A two-phase, parallel, randomised, controlled clinical trial will be carried out, in 210 participants. In phase 1, 120 participants will be randomized into four arms: (1) aerobic exercise, (2) aerobic exercise combined with resistance, (3) high-intensity intervallic exercise and (4) control group. In phase 2, 90 new participants will be randomized into three arms, using the exercise modality that showed the best glycaemic control in phase 1 in the following manner: (1) frequency of 5 days/week, (2) frequency of 3 days/week and (3) frequency of 2 days/week. The control group (n = 30) will be included in phase 1 to evaluate the effect of any type of intervention versus no intervention. Data collection will be performed at baseline and after 15 weeks of follow up. Sociodemographic data, medication, comorbidity, blood biochemical parameters, blood pressure, anthropometric measurements, body composition, physical activity, sedentary lifestyle, diet, smoking, alcohol consumption, quality of life and sleep questionnaires will be collected. Physical activity, sedentary behaviour and sleep will be further determined with an accelerometer, and continuous glycaemia will be determined with a glycaemic monitor, both during seven days, at two time points. The main dependent variable will be the reduction in the mean amplitude of glycaemic excursions. The impact of these interventions on health will also be evaluated through gene expression analysis in peripheral blood cells. The results of this study will contribute to a better understanding of the mechanisms behind the glucose response to physical exercise in a population with prediabetes as well as improve physical exercise prescriptions for diabetes prevention. Increasing glycaemic control in people with prediabetes through physical exercise offers an opportunity to prevent diabetes and reduce associated comorbidities and health costs.

Peripheral blood mononuclear cells (PBMC) are widely used as a biomarker source in nutrition/obesity studies because they reflect gene expression profiles of internal tissues. In this pilot proof-of-concept study we analysed in humans if, as we previously suggested in rodents, PBMC could be a surrogate tissue to study overweight/obesity impact on lipid metabolism. Pre-selected key lipid metabolism genes based in our previous preclinical studies were analysed in PBMC of normoglycemic normal-weight (NW), and overweight-obese (OW-OB) subjects before and after a 6-month weight-loss plan. PBMC mRNA levels of CPT1A, FASN and SREBP-1c increased in the OW-OB group, according with what described in liver and adipose tissue of humans with obesity. This altered expression pattern was related to increased adiposity and early signs of metabolic impairment. Greater weight loss and/or metabolic improvement as result of the intervention was related to lower CPT1A, FASN and SREBP-1c gene expression in an adjusted linear mixed-effects regression analysis, although no gene expression recovery was observed when considering mean comparisons. Thus, human PBMC reflect lipid metabolism expression profile of energy homeostatic tissues, and early obesity-related alterations in metabolic at-risk subjects. Further studies are needed to understand PBMC usefulness for analysis of metabolic recovery in weigh management programs.

BACKGROUND:Obesity was consistently associated with a poor prognosis in patients with COVID-19. Epigenetic mechanisms were proposed as the link between obesity and comorbidities risk.AIM:To evaluate the methylation levels of angiotensin-converting enzyme 2 (ACE2) gene, the main entry receptor of SARS-CoV-2, in different depots of adipose tissue (AT) and leukocytes (PBMCs) in obesity and after weight loss therapy based on a very-low-calorie ketogenic diet (VLCKD), a balanced hypocaloric diet (HCD) or bariatric surgery (BS).MATERIALS AND METHODS:DNA methylation levels of ACE2 were extracted from our data sets generated by the hybridization of subcutaneous (SAT) (n = 32) or visceral (VAT; n = 32) adipose tissue, and PBMCs (n = 34) samples in Infinium HumanMethylation450 BeadChips. Data were compared based on the degree of obesity and after 4-6 months of weight loss either by following a nutritional or surgical treatment and correlated with ACE2 transcript levels.RESULTS:As compared with normal weight, VAT from patients with obesity showed higher ACE2 methylation levels. These differences were mirrored in PBMCs but not in SAT. The observed obesity-associated methylation of ACE2 was reversed after VLCKD and HCD but not after BS. Among the studied CpG sites, cg16734967 and cg21598868, located at the promoter, were the most affected and correlated with BMI. The observed DNA methylation pattern was inversely correlated with ACE2 expression.CONCLUSION:Obesity-related VAT shows hypermethylation and downregulation of the ACE2 gene that is mirrored in PBMCs and is restored after nutritional weight reduction therapy. The results warrant the necessity to further evaluate its implication for COVID-19 pathogenesis.

BACKGROUND:Imbalanced dietary intake is related to increased adiposity, which is linked to increased metabolic risk even in the absence of obesity. BMI is traditionally used to classify body fatness and weight range, but it only considers body weight and height. The Clínica Universidad de Navarra-Body Adiposity Estimator (CUN-BAE) equation has appeared as an additional tool to estimate adiposity considering also other relevant parameters, i.e., sex and age.OBJECTIVES:We aimed to determine whether the CUN-BAE index could estimate adiposity-related metabolic risk in apparently healthy, normoglycemic adults.METHODS:In this case-control study, men and women (18-45 y old) were classified as normal-weight (NW) [n = 20; BMI (in kg/m2) <25] or overweight-obese (OW-OB) (n = 34; BMI ≥25). The primary outcome was body fat content and clinical circulating parameters to assess by correlation analysis CUN-BAE's usefulness as a predictor of metabolic risk. In addition, transcriptomic biomarkers of lipid metabolism were analyzed in peripheral blood mononuclear cells (PBMCs) as secondary outcome indicators of metabolic impairment. Data were analyzed by correlation analysis and comparison of means.RESULTS:CUN-BAE values correlated directly with body fatness obtained by DXA (r = 0.89, P < 0.01), with classical molecular biomarkers of metabolic risk, and with PBMC gene expression of carnitine palmitoyltransferase 1A (CPT1A), sterol regulatory element binding transcription factor 1c (SREBP-1c), and fatty acid synthase (FASN), early markers of metabolic impairment (P < 0.05). Moreover, CUN-BAE allowed identification of NW individuals with excessive body fatness, who were not yet presenting obesity-related molecular alterations. In these subjects, visceral fat correlated directly with circulating glucose, triglycerides, and total and LDL cholesterol, and with triglyceride-glucose and fatty liver indexes (P < 0.05). This is indicative of a metabolically obese NW phenotype.CONCLUSIONS:Data obtained in our cohort of young normoglycemic volunteers support the use of the CUN-BAE index as a tool to estimate accurately body fat mass, but also as a first easy/effective screening tool to identify lean people with increased fat mass and increased metabolic risk.This trial was registered at clinicaltrials.gov as NCT04402697.

Ex vivo human peripheral blood mononuclear cell (PBMC) systems offer the possibility to test transcriptomic effects of food bioactive compounds with potential health effects. We investigated all-trans retinoic acid (ATRA) effect on mRNA expression of key lipid metabolism and inflammatory genes in PBMCs from normal-weight (NW) and overweight-obese (OW-OB) men with different metabolic syndrome-related features. PBMCs were incubated with 10 µM ATRA and mRNA levels of selected genes were analyzed using real-time RT-qPCR. Human ex vivo PBMCs responded to ATRA treatment, but the response for some genes was dependent on body mass index (BMI), with a lower response in PBMC from OW-OB than from NW donors. Moreover, gene expression response was affected by circulating high-density lipoprotein (HDL)-cholesterol levels. Particularly, the response to ATRA of CPT1A, previously reported as a sensitive metabolic risk predictive biomarker, was dependent on HDL levels and not on BMI, being impaired in those individuals with lower HDL levels, specifically in OW-OB. Thus, PBMCs’ insensitivity to ATRA, which can be considered as indicative of impaired metabolism, was observed in individuals with higher metabolic risk (OW-OB with low HDL levels). In conclusion, an ex vivo human PBMC system indicates that ATRA response could be influenced by metabolic syndrome features. Moreover, our study reinforces the role of CPT1A as a marker of metabolic risk and points to plasmatic HDL-cholesterol levels as a parameter to take into consideration when the effects of nutritional factors and/or dietary interventions on humans are under study. Further studies including women are required to detect potential gender differences in the observed effects.

III Congreso de Alimentación, Nutrición y Dietética. Combinar la nutrición comunitaria y personalizada: nuevos retos.

Male ferrets, aged 3 months, were divided into two group: one group remained at 22 degrees Celsius, while the other group was acclimatized to 4 degrees Celsius for one week. After sacrification, inguinal and periaortic white adipose tissues were dissected, and peripheral blood mononuclear cells (PBMC) were isolated. The three tissues are used for RNA isolation and subsequent global gene expression profiling using custom Agilent ferret-specific 2x400K microarrays. Data analysis indicated that the cold exposure induce a clear gene expression response of some genes in the inguinal and periaortic white adipose tissue as in PBMC. These genes could be defined as a biomakers of the effect of cold exposure in these cells.

Animal studies, mostly performed in rodents, show the beneficial anti-obesity effects of cold studies. This is due to thermogenic activation of brown adipose tissue (BAT), a tissue also recently discovered in adult humans. Studies in humans, however, are hampered by the accessibility of most tissues. In contrast, peripheral blood mononuclear cells (PBMC) are accessible and share the expression profile of different sets of genes with other tissues, including those that reflect metabolic responses. Ferrets are an animal model physiologically closer to humans than rodents. Here, we investigated the effects on ferrets of one-week acclimation to 4 °C by analysing the PBMC transcriptome. Cold exposure deeply affected PBMC gene expression, producing a widespread down-regulation of genes involved in different biological pathways (cell cycle, gene expression regulation/protein synthesis, immune response, signal transduction, and genes related to extracellular matrix/cytoskeleton), while thermogenic and glycogenolysis-related processes were increased. Results obtained in PBMC reflected those of adipose tissue, but hardly those of the liver. Our study, using ferret as a model, reinforce PBMC usefulness as sentinel biological material for cold-exposure studies in order to deepen our understanding of the general and specific pathways affected by cold acclimation. This is relevant for future development of therapies to be used clinically.

Omega-3 rich diets have been shown to improve inflammatory status. However, in an ex vivo system of human blood cells, the efficacy of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) modulating lipid metabolism and cytokine response is attenuated in overweight subjects and shows high inter-individual variability. This suggests that obesity may be exerting a synergistic effect with genetic background disturbing the anti-inflammatory potential of omega-3 long-chain polyunsaturated fatty acids (PUFA). In the present work, a genetic score aiming to explore the risk associated to low grade inflammation and obesity (LGI-Ob) has been elaborated and assessed as a tool to contribute to discern population at risk for metabolic syndrome. Pro-inflammatory gene expression and cytokine production as a response to omega-3 were associated with LGI-Ob score; and lower anti-inflammatory effect of PUFA was observed in subjects with a high genetic score. Furthermore, overweight/obese individuals showed positive correlation of both plasma C-Reactive Protein and triglyceride/HDLc-index with LGI-Ob; and high LGI-Ob score was associated with greater hypertension (p = 0.047), Type 2 diabetes (p = 0.026), and metabolic risk (p = 0.021). The study shows that genetic variation can influence inflammation and omega-3 response, and that the LGI-Ob score could be a useful tool to classify subjects at inflammatory risk and more prone to suffer metabolic syndrome and associated metabolic disturbances.