Daniel Castellano-Castillo

Serum 25-Hydroxyvitamin D and Adipose Tissue Vitamin D Receptor Gene Expression: Relationship With Obesity and Type 2 Diabetes

CONTEXT The relationship between 25-hydroxyvitamin D [25(OH)D] and obesity and type 2 diabetes is not completely understood. Vitamin D receptor (VDR) expression in adipose tissue (AT) is related to obesity and might be regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. OBJECTIVE To analyze serum 25(OH)D and VDR gene expression in AT according to body mass index (BMI) and glycemic status and the effect of 1,25(OH)2D3 on AT according to BMI. DESIGN AND PATIENTS Two cohorts were studied: 1) 118 subjects classified according to their BMI (lean, overweight, obese, or morbidly obese [MO]) and their glycemic status (normoglycemic [NG] and prediabetic and diabetic [P&D]); and 2) 30 obese subjects (BMI > 30 kg/m(2)) classified as NG and P&D. VDR gene expression was analyzed during preadipocyte differentiation and in vitro stimulation with 1,25(OH)2D3 of AT explants from donors with different BMI values. SETTING University Hospital. MAIN OUTCOME MEASURES Serum 25(OH)D, parathyroid hormone (PTH), and AT VDR gene expression. RESULTS 25(OH)D levels were lower in P&D than NG subjects, significantly so in the lean and MO groups (P < .05). 25(OH)D levels correlated negatively with homeostasis model of assessment for insulin resistance (HOMA-IR) (r = -0.200; P = .032) and glucose (r = -0.295; P = .001), but not with BMI. VDR gene expression was higher in MO than in the other BMI groups (P < .05). 1,25(OH)2D3 increased VDR gene expression in AT from obese patients (P < .05) but not from lean subjects. CONCLUSIONS 25(OH)D levels are diminished in P&D compared to NG subjects, independently of BMI, and are closely related to glucose metabolism variables, suggesting that vitamin D deficiency is associated more with carbohydrate metabolism than with obesity. Moreover, AT has a different response to 1,25(OH)2D3 depending on the degree of obesity.

Adipose tissue infiltration in normal-weight subjects and its impact on metabolic function.

Discordant phenotypes, metabolically healthy obese and unhealthy normal-weight individuals, are always interesting to provide important insights into the mechanistic link between adipose tissue dysfunction and associated metabolic alterations. Macrophages can release factors that impair the proper activity of the adipose tissue. Thus, studying subcutaneous and visceral adipose tissues, we investigated for the first time the differences in monocyte/macrophage infiltration, inflammation, and adipogenesis of normal-weight subjects who differed in their degree of metabolic syndrome. The study included 92 normal-weight subjects who differed in their degree of metabolic syndrome. Their anthropometric and biochemical parameters were measured. RNA from subcutaneous and visceral adipose tissues was isolated, and mRNA expression of monocyte/macrophage infiltration (CD68, CD33, ITGAM, CD163, EMR-1, CD206, MerTK, CD64, ITGAX), inflammation (IL-6, tumor necrosis factor alpha [TNFα], IL-10, IL-1b, CCL2, CCL3), and adipogenic and lipogenic capacity markers (PPARgamma, FABP4) were measured. Taken together, our data provide evidence of a different degree of macrophage infiltration between the adipose tissues, with a higher monocyte/macrophage infiltration in subcutaneous adipose tissue in metabolically unhealthy normal-weight subjects, whereas visceral adipose tissue remained almost unaffected. An increased macrophage infiltration of adipose tissue and its consequences, such as a decrease in adipogenesis function, may explain why both the obese and normal-weight subjects can develop metabolic diseases or remain healthy.

Role of Gut Microbiota on Cardio-Metabolic Parameters and Immunity in Coronary Artery Disease Patients with and without Type-2 Diabetes Mellitus

Gut microbiota composition has been reported as a factor linking host metabolism with the development of cardiovascular diseases (CVD) and intestinal immunity. Such gut microbiota has been shown to aggravate CVD by contributing to the production of trimethylamine N-oxide (TMAO), which is a pro-atherogenic compound. Treg cells expressing the transcription factor Forkhead box protein P3 (FoxP3) play an essential role in the regulation of immune responses to commensal microbiota and have an atheroprotective role. However, the aim of this study was to analyze the role of gut microbiota on cardio-metabolic parameters and immunity in coronary artery disease (CAD) patients with and without type-2 diabetes mellitus (DM2). The study included 16 coronary CAD-DM2 patients, and 16 age, sex, and BMI matched CAD patients without DM2 (CAD-NDM2). Fecal bacterial DNA was extracted and analyzed by sequencing in a GS Junior 454 platform followed by a bioinformatic analysis (QIIME and PICRUSt). The present study indicated that the diversity and composition of gut microbiota were different between the CAD-DM2 and CAD-NDM2 patients. The abundance of phylum Bacteroidetes was lower, whereas the phyla Firmicutes and Proteobacteria were higher in CAD-DM2 patients than those in the CAD-NDM2 group. CAD-DM2 patients had significantly less beneficial or commensal bacteria (such as Faecalibacterium prausnitzii and Bacteroides fragilis) and more opportunistic pathogens (such as Enterobacteriaceae, Streptococcus, and Desulfovibrio). Additionally, CAD-DM2 patients had significantly higher levels of plasma zonulin, TMAO, and IL-1B and significantly lower levels of IL-10 and FOXP3 mRNA expression than CAD-NDM2. Moreover, in the CAD-MD2 group, the increase in Enterobacteriaceae and the decrease in Faecalibacterium prausnitzii were significantly associated with the increase in serum TMAO levels, while the decrease in the abundance of Bacteroides fragilis was associated with the reduction in the FOXP3 mRNA expression, implicated in the development and function of Treg cells. These results suggest that the presence of DM2 is related to an impaired regulation of the immune system in CAD patients, mediated in part by the gut microbiota composition and functionality and the production and effects of their gut microbiota derived molecules.

Neovascular deterioration, impaired NADPH oxidase and inflammatory cytokine expression in adipose-derived multipotent cells from subjects with metabolic syndrome

OBJECTIVE Expansion of adipose tissue depends on the growth of its vascular network and it has been shown that adipose tissue dysfunction in obese subjects with the metabolic syndrome is associated with decreased angiogenesis. However, some subjects with a high body mass index do not develop metabolic abnormalities associated with obesity. In this study we examined the neovascular properties, expression levels of proteins involved in cellular redox balance and inflammatory cytokines in adipose-derived multipotent mesenchymal cells (ASCs) of subjects with different metabolic profiles. MATERIALS/METHODS We applied cell culture, flow cytometry, RT-qPCR and ELISA techniques to characterize the ASCs isolated from paired biopsies of visceral (visASCs) and subcutaneous (subASCs) adipose tissue from 39 subjects grouped into normal weight (Nw), obese without metabolic syndrome (NonMS) and with metabolic syndrome (MS). RESULTS VisASCs and subASCs from MS subjects showed a decrease in tubules formation capacity compared to ASCs from NonMS subjects as well as changes in the expression levels of proteins involved in cell redox balance and secretion levels of proteins linked to the senescence-associated secretory phenotype. Deterioration in the neovascular properties of subASCs from the MS subjects was also evident in the decreased levels of VEGF secretion during adipogenesis and in the effects of the conditioned medium on endothelial cell tubule formation. CONCLUSIONS Our findings suggest a redox imbalance status in ASCs from subjects with metabolic syndrome and decreased their neovascular function that probably contributes to the vascular insufficiency of adipose depots.

Identification of an episignature of human colorectal cancer associated with obesity by genome-wide DNA methylation analysis

ABSTRACTBackgroundObesity was established as a relevant modifiable risk factor in the onset and progression of colorectal cancer (CRC). This relationship could be mediated by an epigenetic regulation.ObjectivesThe current work aimed to explore the effects of excess body weight on the DNA methylation profile of CRC using a genome-wide DNA methylation approach and to identify an epigenetic signature of obesity-related CRC.MethodsFifty-six CRC-diagnosed patients (50 years) were included in the study and categorized according to their body mass index (BMI) as non-obese (BMI ≤ 25 kg/m2) or overweight/obese (BMI > 25 kg/m2). Data from Infinium 450k array-based methylomes of 28 CRC tumor samples were coupled with information on BMI categories. Additionally, DNA methylation results were validated in 28 CRC tumor samples.ResultsThe analysis revealed statistically significant differences at 299 CpG sites, and they were mostly characterized as changes towards CpG hypermethylation occurring in the obese group. The 152 identified genes were involved in inflammatory and metabolic functional processes. Among these genes, novel genes were identified as epigenetically regulated in CRC depending on adiposity. ZNF397OS and ZNF543 represented the top scoring associated events that were further validated in an independent cohort and exhibited strong correlation with BMI and excellent and statistically significant efficiency in the discrimination of obese from non-obese CRC patients (area under the curve >0.80; p < 0.05).ConclusionsThe present study identifies a potential epigenome mark of obesity-related CRC that could be useful for precision medicine in the management of this disease taking into account adiposity as a relevant risk factor.

Adipose Tissue LPL Methylation is Associated with Triglyceride Concentrations in the Metabolic Syndrome

BACKGROUND DNA methylation is one of the epigenetic mechanisms that regulate gene expression. DNA methylation may be modified by environmental and nutritional factors. Thus, epigenetics could potentially provide a mechanism to explain the etiology of metabolic disorders, such as metabolic syndrome (MetS). The aim of this study was to analyze the level of DNA methylation of several lipoprotein lipase (LPL)-promoter-CpG dinucleotides in a CpG island region and relate this to the gene and protein expression levels in human visceral adipose tissue (VAT) from individuals with and without MetS. METHODS VAT samples were collected from laparoscopic surgical patients without and with MetS, and levels of LPL mRNA, LPL protein, and LPL DNA methylation were measured by qPCR, western blot, and pyrosequencing. Biochemical and anthropometric variables were analyzed. Individuals included in a subset underwent a dietary fat challenge test, and levels of postprandial triglycerides were determined. RESULTS We found higher levels of DNA methylation in MetS patients but lower gene expression and protein levels. There was a negative association between LPL methylation and LPL gene expression. We found a positive association between LPL methylation status and abnormalities of the metabolic profile and basal and postprandial triglycerides, whereas LPL gene expression was negatively associated with these abnormalities. CONCLUSIONS We demonstrate that LPL methylation may be influenced by the degree of metabolic disturbances and could be involved in triglyceride metabolism, promoting hypertriglyceridemia and subsequent associated disorders, such as MetS.

IGFBP-3 Interacts with the Vitamin D Receptor in Insulin Signaling Associated with Obesity in Visceral Adipose Tissue

Adipose tissue has traditionally only been considered as an energy storage organ. Nevertheless, the importance of this tissue in systemic physiology and, especially, in systemic inflammation has been highlighted in recent years. Adipose tissue expresses proteins related to vitamin D (VD) metabolism, and it has been proposed that it can act as a VD storage tissue. The active form of VD, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is able to modify adipocyte and adipose tissue physiology via the VD receptor (VDR), decreasing the expression of pro-inflammatory cytokines in adipose tissue. Moreover, VD deficiency and VDR has been reported to be associated with obesity and diabetes. However, the results of the different studies are not conclusive. Insulin growth binding proteins (IGFBPs) have been identified in adipose tissue, but their roles are poorly understood. Therefore, the objective of this study was to analyze the plasma levels of VD and the gene expression of VDR in the adipose tissue of subjects with morbid obesity (MO) and with different degrees of insulin resistance (IR), as well as the functionality of direct interaction between IGFBP-3 and VDR, which could explain its inhibitory role in adipogenesis. Our results show a novel role of the VD system in the regulation and activation of IGFBP-3 in visceral adipose tissue (VAT) of patients with MO, as a new and alternative mechanism proposed in the insulin signaling associated with obesity.

Chromatin immunoprecipitation improvements for the processing of small frozen pieces of adipose tissue.

Chromatin immunoprecipitation (ChIP) has gained importance to identify links between the genome and the proteome. Adipose tissue has emerged as an active tissue, which secretes a wide range of molecules that have been related to metabolic and obesity-related disorders, such as diabetes, cardiovascular failure, metabolic syndrome, or cancer. In turn, epigenetics has raised the importance in discerning the possible relationship between metabolic disorders, lifestyle and environment. However, ChIP application in human adipose tissue is limited by several factors, such as sample size, frozen sample availability, high lipid content and cellular composition of the tissue. Here, we optimize the standard protocol of ChIP for small pieces of frozen human adipose tissue. In addition, we test ChIP for the histone mark H3K4m3, which is related to active promoters, and validate the performance of the ChIP by analyzing gene promoters for factors usually studied in adipose tissue using qPCR. Our improvements result in a higher performance in chromatin shearing and DNA recovery of adipocytes from the tissue, which may be useful for ChIP-qPCR or ChIP-seq analysis.

Complement Factor C3 Methylation and mRNA Expression Is Associated to BMI and Insulin Resistance in Obesity

Epigenetic marks, and especially DNA methylation, are becoming an important factor in obesity, which could help to explain its etiology and associated comorbidities. Adipose tissue, now considered as an important endocrine organ, produces complement system factors. Complement component 3 (C3) turns out to be an important protein in metabolic disorders, via either inflammation or the C3 subproduct acylation stimulating protein (ASP) which directly stimulates lipid storage. In this study, we analyze C3 DNA methylation in adipose tissue from subjects with a different grade of obesity. Adipose tissue samples were collected from subjects with a different degree of obesity determined by their body mass index (BMI) as: Overweight subjects (BMI ≥ 25 and <30), obese class 1/2 subjects (BMI ≥ 30 and <40) and obese class 3 subjects (BMI ≥ 40). C3 DNA methylation was measured for 7 CpGs by pyrosequencition using the Pyromark technology (Qiagen, Madrid Spain). C3 messenger RNA (mRNA) levels were analyzed by pre-designed Taqman assays (Applied biosystems, Foster City, CA, USA) and ASP/C3a was measured using a ELISA kit. The data were analyzed using the statistic package SPSS. C3 DNA methylation levels were lower in the morbid obese group. Accordingly, C3 methylation correlated negatively with BMI and leptin. However, C3 mRNA levels were more associated with insulin resistance, and positive correlations with insulin, glucose and homeostasis model assessment-estimated insulin resistance (HOMA-IR) existed. ASP correlated negatively with high density lipoprotein (HDL) cholesterol. C3 methylation levels were associated to adiposity variables, such as BMI and leptin, while the C3 mRNA levels were associated to glucose metabolism.

Gut Microbiota Differs in Composition and Functionality Between Children With Type 1 Diabetes and MODY2 and Healthy Control Subjects: A Case-Control Study

OBJECTIVE Type 1 diabetes is associated with compositional differences in gut microbiota. To date, no microbiome studies have been performed in maturity-onset diabetes of the young 2 (MODY2), a monogenic cause of diabetes. Gut microbiota of type 1 diabetes, MODY2, and healthy control subjects was compared. RESEARCH DESIGN AND METHODS This was a case-control study in 15 children with type 1 diabetes, 15 children with MODY2, and 13 healthy children. Metabolic control and potential factors modifying gut microbiota were controlled. Microbiome composition was determined by 16S rRNA pyrosequencing. RESULTS Compared with healthy control subjects, type 1 diabetes was associated with a significantly lower microbiota diversity, a significantly higher relative abundance of Bacteroides, Ruminococcus, Veillonella, Blautia, and Streptococcus genera, and a lower relative abundance of Bifidobacterium, Roseburia, Faecalibacterium, and Lachnospira. Children with MODY2 showed a significantly higher Prevotella abundance and a lower Ruminococcus and Bacteroides abundance. Proinflammatory cytokines and lipopolysaccharides were increased in type 1 diabetes, and gut permeability (determined by zonulin levels) was significantly increased in type 1 diabetes and MODY2. The PICRUSt analysis found an increment of genes related to lipid and amino acid metabolism, ABC transport, lipopolysaccharide biosynthesis, arachidonic acid metabolism, antigen processing and presentation, and chemokine signaling pathways in type 1 diabetes. CONCLUSIONS Gut microbiota in type 1 diabetes differs at taxonomic and functional levels not only in comparison with healthy subjects but fundamentally with regard to a model of nonautoimmune diabetes. Future longitudinal studies should be aimed at evaluating if the modulation of gut microbiota in patients with a high risk of type 1 diabetes could modify the natural history of this autoimmune disease.