Modulation of miR-29a and miR-29b Expression and Their Target Genes Related to Inflammation and Renal Fibrosis by an Oral Nutritional Supplement with Probiotics in Malnourished Hemodialysis Patients.

Malnutrition is prevalent in patients with chronic kidney disease (CKD), especially those on hemodialysis. Recently, our group described that a new oral nutritional supplement (ONS), specifically designed for malnourished (or at risk) hemodialysis patients with a "similar to the Mediterranean diet" pattern, improved caloric-protein intake, nutritional status and biomarkers of inflammation and oxidation. Our aim in this study was to evaluate whether the new ONS, associated with probiotics or not, may produce changes in miRNA's expression and its target genes in malnourished hemodialysis patients, compared to individualized diet recommendations. We performed a randomized, multicenter, parallel-group trial in malnourished hemodialysis patients with three groups (1: control (C) individualized diet (n = 11); 2: oral nutritional supplement (ONS) + placebo (ONS-PL) (n = 10); and 3: ONS + probiotics (ONS-PR) (n = 10)); the trial was open regarding the intake of ONS or individualized diet recommendations but double-blinded for the intake of probiotics. MiRNAs and gene expression levels were analyzed by RT-qPCR at baseline and after 3 and 6 months. We observed that the expression of miR-29a and miR-29b increased significantly in patients with ONS-PR at 3 months in comparison with baseline, stabilizing at the sixth month. Moreover, we observed differences between studied groups, where miR-29b expression levels were elevated in patients receiving ONS-PR compared to the control group in the third month. Regarding the gene expression levels, we observed a decrease in the ONS-PR group compared to the control group in the third month for RUNX2 and TNFα. TGFB1 expression was decreased in the ONS-PR group compared to baseline in the third month. PTEN gene expression was significantly elevated in the ONS-PR group at 3 months in comparison with baseline. LEPTIN expression was significantly increased in the ONS-PL group at the 3-month intervention compared to baseline. The new oral nutritional supplement associated with probiotics increases the expression levels of miR-29a and miR-29b after 3 months of intervention, modifying the expression of target genes with anti-inflammatory and anti-fibrotic actions. This study highlights the potential benefit of this oral nutritional supplement, especially associated with probiotics, in malnourished patients with chronic renal disease on hemodialysis.

A Network Comprised of miR-15b and miR-29a Is Involved in Vascular Endothelial Growth Factor Pathway Regulation in Thymus Adipose Tissue from Elderly Ischemic Cardiomyopathy Subjects.

As the human thymus ages, it undergoes a transformation into adipose tissue known as TAT. Interestingly, in previous research, we observed elevated levels of vascular endothelial growth factor A (VEGFA) in TAT from patients with ischemic cardiomyopathy (IC), particularly in those over 70 years old. Moreover, in contrast to subcutaneous adipose tissue (SAT), TAT in elderly individuals exhibits enhanced angiogenic properties and the ability to stimulate tube formation. This makes TAT a promising candidate for angiogenic therapies and the regeneration of ischemic tissues following coronary surgery. MicroRNAs (miRNAs) have emerged as attractive therapeutic targets, especially those that regulate angiogenic processes. The study's purpose is to determine the miRNA network associated with both the VEGFA pathway regulation and the enrichment of age-linked angiogenesis in the TAT. RT-PCR was used to analyze angiogenic miRNAs and the expression levels of their predicted target genes in both TAT and SAT from elderly and middle-aged patients treated with coronary artery bypass graft surgery. miRTargetLink Human was used to search for miRNAs and their target genes. PANTHER was used to annotate the biological processes of the predicted targets. The expression of miR-15b-5p and miR-29a-3p was significantly upregulated in the TAT of elderly compared with middle-aged patients. Interestingly, VEGFA and other angiogenic targets were significantly upregulated in the TAT of elderly patients. Specifically: JAG1, PDGFC, VEGFA, FGF2, KDR, NOTCH2, FOS, PDGFRA, PDGFRB, and RHOB were upregulated, while PIK3CG and WNT7A were downregulated. Our results provide strong evidence of a miRNA/mRNA interaction network linked with age-associated TAT angiogenic enrichment in patients with IC.

MiR-221-3p/222-3p Cluster Expression in Human Adipose Tissue Is Related to Obesity and Type 2 Diabetes.

The progression of obesity and type 2 diabetes (T2D) is intricately linked with adipose tissue (AT) angiogenesis. Despite an established network of microRNAs (miRNAs) regulating AT function, the specific role of angiogenic miRNAs remains less understood. The miR-221/222 cluster has recently emerged as being associated with antiangiogenic activity. However, no studies have explored its role in human AT amidst the concurrent development of obesity and T2D. Therefore, this study aims to investigate the association between the miR-221-3p/222-3p cluster in human AT and its regulatory network with obesity and T2D. MiR-221-3p/222-3p and their target gene (TG) expression levels were quantified through qPCR in visceral (VAT) and subcutaneous (SAT) AT from patients (n = 33) categorized based on BMI as normoweight (NW) and obese (OB) and by glycemic status as normoglycemic (NG) and type 2 diabetic (T2D) subjects. In silico analyses of miR-221-3p/222-3p and their TGs were conducted to identify pertinent signaling pathways. The results of a multivariate analysis, considering the simultaneous expression of miR-221-3p and miR-222-3p as dependent variables, revealed statistically significant distinctions when accounting for variables such as tissue depot, obesity, sex, and T2D as independent factors. Furthermore, both miRNAs and their TGs exhibited differential expression patterns based on obesity severity, glycemic status, sex, and type of AT depot. Our in silico analysis indicated that miR-221-3p/222-3p cluster TGs predominantly participate in angiogenesis, WNT signaling, and apoptosis pathways. In conclusion, these findings underscore a promising avenue for future research, emphasizing the miR-221-3p/222-3p cluster and its associated regulatory networks as potential targets for addressing obesity and related metabolic disorders.

Serum vascular endothelial growth factor b and metabolic syndrome incidence in the population based cohort Di@bet.es study.

BACKGROUND/OBJECTIVES: Although vascular endothelial growth factor b (VEGFb) might have an impact on the development of obesity, diabetes and related disorders, the possible relationship between VEGFb serum levels and the incidence of these metabolic complications in humans is still unknown. The aim of our study was to evaluate the association between VEGFb serum levels and the new-onset of metabolic syndrome (MS) and its components in the Spanish adult population after 7.5 years of follow-up. SUBJECTS/METHODS: A total of 908 subjects from the Di@bet.es cohort study without MS at cross-sectional stage according to International Diabetes Federation (IDF) or Adult Treatment Panel III (ATP-III) criteria were included. Additionally, five sub-populations were grouped according to the absence of each MS component at baseline. Socio-demographic, anthropometric and clinical data were recorded. The Short Form of International Physical Activity Questionnaire (SF-IPAQ) was used to estimate physical activity. A fasting blood extraction and an oral glucose tolerance test were performed. Serum determinations of glucose, lipids, hsCRP and insulin were made. VEGFb levels were determined and categorized according to the 75th percentile of the variable. New cases of MS and its components were defined according to ATPIII and IDF criteria. RESULTS: A total of 181 or 146 people developed MS defined by IDF or ATP-III criteria respectively. Serum triglyceride levels, hs-CRP and systolic blood pressure at the baseline study were significantly different according to the VEGFb categories. Adjusted logistic regression analysis showed that the likelihood of developing MS and abdominal obesity was statistically reduced in subjects included in the higher VEGFb category. CONCLUSION: Low serum levels of VEGFb may be considered as early indicators of incident MS and abdominal obesity in the Spanish adult population free of MS, independently of other important predictor variables.

Metabolic endotoxemia promotes adipose dysfunction and inflammation in human obesity.

Impaired adipose tissue (AT) lipid handling and inflammation is associated with obesity-related metabolic diseases. Circulating lipopolysaccharides (LPSs) from gut microbiota (metabolic endotoxemia), proposed as a triggering factor for the low-grade inflammation in obesity, might also be responsible for AT dysfunction. Nevertheless, this hypothesis has not been explored in human obesity. To analyze the relationship between metabolic endotoxemia and AT markers for lipogenesis, lipid handling, and inflammation in human obesity, 33 patients with obesity scheduled for surgery were recruited and classified according to their LPS levels. Visceral and subcutaneous AT gene and protein expression were analyzed and adipocyte and AT in vitro assays performed. Subjects with obesity with a high degree of metabolic endotoxemia had lower expression of key genes for AT function and lipogenesis ( SREBP1, FABP4, FASN, and LEP) but higher expression of inflammatory genes in visceral and subcutaneous AT than subjects with low LPS levels. In vitro experiments corroborated that LPS are responsible for adipocyte and AT inflammation and downregulation of PPARG, SCD, FABP4, and LEP expression and LEP secretion. Thus, metabolic endotoxemia influences AT physiology in human obesity by decreasing the expression of factors involved in AT lipid handling and function as well as by increasing inflammation.

Au@16-pH-16/miR-21 mimic nanosystem: An efficient treatment for obesity through browning and thermogenesis induction.

Despite the abundance of registered clinical trials worldwide, the availability of effective drugs for obesity treatment is limited due to their associated side effects. Thus, there is growing interest in therapies that stimulate energy expenditure in white adipose tissue. Recently, we demonstrated that the delivery of a miR-21 mimic using JetPEI effectively inhibits weight gain in an obese mouse model by promoting metabolism, browning, and thermogenesis, suggesting the potential of miR-21 mimic as a treatment for obesity. Despite these promising results, the implementation of more advanced delivery system techniques for miR-21 mimic would greatly enhance the advancement of safe and efficient treatment approaches for individuals with obesity in the future. Our objective is to explore whether a new delivery system based on gold nanoparticles and Gemini surfactants (Au@16-ph-16) can replicate the favorable effects of the miR-21 mimic on weight gain, browning, and thermogenesis. We found that dosages as low as 0.2 µg miR-21 mimic /animal significantly inhibited weight gain and induced browning and thermogenic parameters. This was evidenced by the upregulation of specific genes and proteins associated with these processes, as well as the biogenesis of beige adipocytes and mitochondria. Significant increases in miR-21 levels were observed in adipose tissue but not in other tissue types. Our data indicates that Au@16-ph-16 could serve as an effective delivery system for miRNA mimics, suggesting its potential suitability for the development of future clinical treatments against obesity.

miR-21 mimic blocks obesity in mice: A novel therapeutic option.

MicroRNAs (miRNAs) are promising drug targets for obesity and metabolic disorders. Recently, miRNA mimics are providing a unique mechanism of action that guides the process for drug development and sets out the context of their therapeutic application. miRNA (miR)-21 expression in white adipose tissue (WAT) has been associated with obesity. We aimed to analyze miR-21 expression levels in relation to diabetes and obesity to determine the effect that miR-21 mimic has on processes involved in WAT functionality, to dissect the underlying molecular mechanisms, and to study the potential therapeutic application of the miR-21 mimic against obesity. We found higher miR-21 levels in WAT from non-diabetic obese compared to normoweight humans and mice. Moreover, in 3T3-L1 adipocytes, miR-21 mimic affect genes involved in WAT functionality regulation and significantly increase the expression of genes involved in browning and thermogenesis. Interestingly, in vivo treatment with the miR-21 mimic blocked weight gain induced by a high-fat diet in obese mice, without modifying food intake or physical activity. This was associated with metabolic enhancement, WAT browning, and brown adipose tissue (AT) thermogenic programming through vascular endothelial growth factor A (VEGF-A), p53, and transforming growth factor ß1 (TGF-ß1) signaling pathways. Our findings suggest that miR-21 mimic-based therapy may provide a new opportunity to therapeutically manage obesity and consequently, its associated alterations.

miR-20b, miR-296, and Let-7f Expression in Human Adipose Tissue is Related to Obesity and Type 2 Diabetes.

OBJECTIVE: This study aimed to analyze the potential association of different microRNA (miRNA) molecules with both type 2 diabetes (T2D) and obesity and determine their target genes. METHODS: Quantitative PCR was used to analyze the miR-20b, miR-296, and Let-7f levels in human visceral and subcutaneous adipose tissues (ATs) in relation to obesity and T2D, miRTarBase 4.0 was used for validation of target genes, and the Protein Analysis Through Evolutionary Relationships (PANTHER) Classification System and the Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to annotate the biological processes of the predicted targets. RESULTS: In AT, miR-20b, miR-296, and Let-7f levels were significantly different between normoglycemic subjects and those with T2D. In visceral adipose tissue, miRNA levels were higher in normoglycemic/obesity samples than in T2D/obesity samples. miR-20b-miR-296 and Let-7f target genes that showed significant differences in both ATs in relation to obesity and T2D were CDKN1A, CX3CL1, HIF1A, PPP2R1B, STAT3, and VEGFA. These genes are known to be principally involved in the vascular endothelial growth factor (VEGF) and WNT pathways. CONCLUSIONS: This study provides experimental evidence of the possible correlation between AT miR-20b-miR-296-Let-7f with obesity and T2D, which might involve vascular endothelial growth factor and WNT-dependent pathways that are regulated by six different genes, suggesting a novel signaling pathway that could be important for understanding the mechanisms underlying the AT dysfunction associated with obesity and T2D.

Involvement of acetyl-CoA-producing enzymes in the deterioration of the functional potential of adipose-derived multipotent cells from subjects with metabolic syndrome.

OBJECTIVE: The expansion capacity of white adipose tissue influences the distribution of fat depots in the body, the visceral accumulation of which is linked to metabolic syndrome, regardless of the degree of obesity of the subjects. Alterations in the adipose tissue-derived mesenchymal stem cells (ASCs) may contribute to the adipose tissue remodeling associated with metabolic syndrome and impact the regional distribution of adipose tissue by generating inherently dysfunctional adipocytes. Here we examine the expression levels of acetyl-CoA-producing enzymes and their relationship with the lipogenic, antioxidant and oxidative potential of adipocytes generated from visceral ASCs (adipo-visASCs) and subcutaneous ASCs (adipo-subASCs) from subjects with different metabolic profiles. MATERIALS/METHODS: Paired samples of visceral and subcutaneous adipose tissue were processed to isolate the respective ASCs from normal-weight (Nw) subjects and obese patients with metabolic syndrome (METS) and without METS (NonMETS). qPCR was used to quantify the expression levels of the genes studied in both adipo-ASCs from the patient groups and those generated after silencing by small interfering RNA of acetyl-CoA-producing enzymes. The accumulation of lipids was quantified by absorbance. RESULTS: No significant differences in cell yield or CD34+CD31-CD45- ASC percentage were observed between the different patient groups. Unlike adipo-visASCs, adipo-subASCs from METS patients showed a decrease in expression levels of acetyl-CoA-producing enzymes as well as proteins linked to lipogenesis, antioxidant defense and fatty acid oxidation. Transcriptional silencing of acetyl-CoA-producing enzymes in adipo-subASCs reduced lipid accumulation and affected transcription levels of lipogenic and antioxidant defense proteins. CONCLUSIONS: Adipo-subASCs may be more susceptible than adipo-visASCs to deterioration of the lipogenic, oxidative and antioxidant potential associated with metabolic syndrome. Intrinsic alterations in transcription levels of acetyl-CoA-producing enzymes may contribute to the metabolic reprogramming of adipo-subASCs from METS patients.

Differences in the neovascular potential of thymus versus subcutaneous adipose-derived stem cells from patients with myocardial ischaemia.

Adipose tissue-derived multipotent mesenchymal cells (ASCs) participate in the information of blood vessels under hypoxic conditions. It is probable that the susceptibility of ASCs to the influence of age and ageing-associated pathologies compromises their therapeutic effectiveness depending on the adipose tissue depot. Our aim was to examine the neovascular potential under hypoxic conditions of ASCs-derived from thymic (thymASCs) and subcutaneous (subASCs) adipose tissue from 39 subjects with and without type 2 diabetes mellitus (T2DM) and of different ages who were undergoing coronary bypass surgery. We confirmed a significant decrease in the percentage of CD34+ CD31- CD45- subASCs in the cell yield of subASCs and in the survival of cultured endothelial cells in the medium conditioned by the hypox-subASCs with increasing patient age, which was not observed in thymASCs. Whereas the length of the tubules generated by hypox-subASCs tended to correlate negatively with patient age, tubule formation capacity of the hypoxic thymASCs increased significantly. Compared with subASCs, thymASCs from subjects over age 65 and without T2DM showed higher cell yield, tubule formation capacity, vascular endothelial growth factor secretion levels, and ability to promote endothelial cell survival in their conditioned medium. Deterioration in subASCs neovascular potential relative to thymASCs derived from these subjects was accompanied by higher expression levels of NOX4 mRNA and fibrotic proteins. Our results indicate that thymASCs from patients over age 65 and without T2DM have a higher angiogenic potential than those from the other patient groups, suggesting they may be a good candidate for angiogenic therapy in subjects undergoing coronary bypass surgery.

Inflammatory gene expression in adipose tissue according to diagnosis of anxiety and mood disorders in obese and non-obese subjects.

Psychiatric disorders have been widely reported to be associated with systemic inflammation upregulation and adiposity. However, there are no data that link adipose tissue inflammation to these mental disorders. The analysis of adipokines and inflammation-related markers in adipose tissue could help to elucidate the potential association between obesity and mental health. An observational study was conducted in samples of patients consisting of non-obese and obese subjects, who were diagnosed with anxiety or mood disorders. Gene expression of adiponectin (ADIPOQ), leptin (LEP) and inflammatory markers (IL6, IL1B, TNF, CCL2, CSF3, ITGAM, and PLAUR) were determined in visceral (VAT) and subcutaneous (SAT) adipose tissues. Our results showed that the gene expression of adipokines and inflammation-related markers was higher in the VAT and SAT of obese subjects compared with non-obese subjects. Regarding mental disorders, all the inflammatory genes in the VAT were significantly higher in non-obese subjects with anxiety or mood disorders than in subjects without mental disorders, except for TNF and ITGAM. Additionally, IL6 expression was significantly lower in SAT. In contrast, obese patients diagnosed with anxiety or mood disorders only showed significantly lower expression levels of IL1B in VAT and ADIPOQ in SAT when compared with obese subjects without mental disorders. These data suggest the potential involvement of VAT inflammation in anxiety and mood disorders, involving complex mechanisms which are strongly affected by obesity.

Different response to hypoxia of adipose-derived multipotent cells from obese subjects with and without metabolic syndrome.

BACKGROUND/OBJECTIVES: Multiple studies suggest that hypoxia, together with inflammation, could be one of the phenomena involved in the onset and progression of obesity-related insulin resistance. In addition, dysfunction of adipose tissue in obese subjects with metabolic syndrome is associated with decreased angiogenesis. However, some subjects with a high body mass index do not develop metabolic abnormalities associated with obesity. The aim of the current study was to examine the neovascular properties of visceral adipose tissue-derived multipotent mesenchymal cells subjected to hypoxia (hypox-visASCs) from normal-weight subjects (Nw) and obese patients with metabolic syndrome (MS) and without metabolic syndrome (NonMS). METHODS: This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. Eight patients who underwent either bariatric surgery or cholecystectomy (27 patients) participated in the study. Visceral adipose tissue samples from Nw, MS and NonMS subjects were processed by enzymatic digestion. VisASCs cultured under hypoxic conditions were characterized by tubule formation assay, ELISA, flow cytometry, migration rate, and qRT-PCR, and the effects of visASCs-conditioned medium on survival and endothelial cell tubule formation were evaluated. RESULTS: Hypox-visASCs from NonMS subjects showed a greater capacity for tubule formation than hypox-visASCs from Nw and MS subjects. The lower percentage of CD140b+/CD44+ and CD140b+/CD184+ cells observed in hypox-visASCs from NonMS subjects compared to MS subjects was accompanied not only by a lower migration rate from the chemotactic effects of stromal cell derived factor 1α, but also by lower levels of NOX5 mRNA expression. While the levels of monocyte chemoattractant protein 1 mRNA expressed by hypox-visASCs correlated positively with the body mass index and waist circumference of the subjects, the concentration of vascular endothelial growth factor present in hypox-visASC-conditioned culture medium decreased significantly with increasing plasma glucose. The survival rate and tubules formed by endothelial cells cultured in hypox-visASC-conditioned medium decreased significantly with increasing homeostasis model assessment to quantify insulin resistance. CONCLUSIONS: Our results suggest that hypox-visASCs from NonMS subjects could promote healthy adipose tissue expansion, while hypox-visASCs from MS subjects appear to contribute to the decreased angiogenic potential and increased inflammation underlying adipose tissue dysfunction in obesity. Our results emphasize the importance of taking into account not only the BMI but also the metabolic profile of the subjects during the implementation of ASCs-based therapy to promote neovascularization.

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.

Adipogenic Impairment of Adipose Tissue-Derived Mesenchymal Stem Cells in Subjects With Metabolic Syndrome: Possible Protective Role of FGF2.

Context: The decreased expansion capacity of adipose tissue plays a crucial role in the onset of disorders associated with metabolic syndrome. Objective: The aim of this study was to examine the state of adipose tissue-derived mesenchymal stem cells (ASCs) from obese subjects with different metabolic profiles. Design: This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. Setting: University Hospital. Patients and Intervention: Patients who underwent either bariatric surgery (20 morbidly obese) or cholecystectomy (40 subjects) participated in the study. Main Outcome Measures: ASCs were obtained from both visceral and subcutaneous adipose tissue. Adipogenic, fibrotic gene expression was quantified by quantitative polymerase chain reaction; Smad7 and fibroblast growth factor 2 were quantified by western blotting and enzyme-linked immunosorbent assay, respectively. The susceptibility of ASCs to apoptosis, their population doubling time, and their clonogenic potential were evaluated. Results: The worsening metabolic profile of the patients was accompanied by a decrease in the intrinsic levels of adipogenic gene expression, reduced proliferation rate, clonogenic potential, and exportation of fibroblast growth factor 2 to the cell surface of the ASCs derived from both tissues. In addition, the ASCs from patients without metabolic syndrome showed differences in susceptibility to apoptosis and expression of TGFß-signaling inhibitory protein Smad7 with respect to the ASCs from patients with metabolic syndrome. Conclusion: Our results suggest that the decrease in adipogenic-gene mRNA and clonogenic potential, as well as the accumulation of fibrotic proteins with metabolic alterations, could be a relevant mechanism controlling the number and size of neogenerated adipocytes and involved in alteration of adipose-tissue expansion.

The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice.

The endocannabinoid system (ECS) is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the ß-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1) signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1) receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS) at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6) within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight), which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic ß-cell diseases.

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.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

Effects of glucagon-like peptide-1 on the differentiation and metabolism of human adipocytes.

BACKGROUND AND PURPOSE: Glucagon-like peptide-1 (GLP-1) analogues improve glycaemic control in type 2 diabetic (T2D) patients and cause weight loss in obese subjects by as yet unknown mechanisms. We recently demonstrated that the GLP-1 receptor, which is present in adipocytes and the stromal vascular fraction of human adipose tissue (AT), is up-regulated in AT of insulin-resistant morbidly obese subjects compared with healthy lean subjects. The aim of this study was to explore the effects of in vitro and in vivo administration of GLP-1 and its analogues on AT and adipocyte functions from T2D morbidly obese subjects. EXPERIMENTAL APPROACH: We analysed the effects of GLP-1 on human AT and isolated adipocytes in vitro and the effects of GLP-1 mimetics on AT of morbidly obese T2D subjects in vivo. KEY RESULTS: GLP-1 down-regulated the expression of lipogenic genes when administered during in vitro differentiation of human adipocytes from morbidly obese patients. GLP-1 also decreased the expression of adipogenic/lipogenic genes in AT explants and mature adipocytes, while increasing that of lipolytic markers and adiponectin. In 3T3-L1 adipocytes, GLP-1 decreased free cytosolic Ca2+ concentration ([Ca2+]i). GLP-1-induced responses were only partially blocked by GLP-1 receptor antagonist exendin (9­39). Moreover, administration of exenatide or liraglutide reduced adipogenic and inflammatory marker mRNA in AT of T2D obese subjects. CONCLUSIONS AND IMPLICATIONS: Our data suggest that the beneficial effects of GLP-1 are associated with changes in the adipogenic potential and ability of AT to expand, via activation of the canonical GLP-1 receptor and an additional, as yet unknown, receptor.

Myocardial Ischemic Subject's Thymus Fat: A Novel Source of Multipotent Stromal Cells.

OBJECTIVE: Adipose Tissue Stromal Cells (ASCs) have important clinical applications in the regenerative medicine, cell replacement and gene therapies. Subcutaneous Adipose Tissue (SAT) is the most common source of these cells. The adult human thymus degenerates into adipose tissue (TAT). However, it has never been studied before as a source of stem cells. MATERIAL AND METHODS: We performed a comparative characterization of TAT-ASCs and SAT-ASCs from myocardial ischemic subjects (n = 32) according to the age of the subjects. RESULTS: TAT-ASCs and SAT-ASCs showed similar features regarding their adherence, morphology and in their capacity to form CFU-F. Moreover, they have the capacity to differentiate into osteocyte and adipocyte lineages; and they present a surface marker profile corresponding with stem cells derived from AT; CD73+CD90+CD105+CD14-CD19-CD45-HLA-DR. Interestingly, and in opposition to SAT-ASCs, TAT-ASCs have CD14+CD34+CD133+CD45- cells. Moreover, TAT-ASCs from elderly subjects showed higher adipogenic and osteogenic capacities compared to middle aged subjects, indicating that, rather than impairing; aging seems to increase adipogenic and osteogenic capacities of TAT-ASCs. CONCLUSIONS: This study describes the human TAT as a source of mesenchymal stem cells, which may have an enormous potential for regenerative medicine.

Differences in the Osteogenic Differentiation Capacity of Omental Adipose-Derived Stem Cells in Obese Patients With and Without Metabolic Syndrome.

Multiple studies have suggested that the reduced differentiation capacity of multipotent adipose tissue-derived mesenchymal stem cells (ASCs) in obese subjects could compromise their use in cell therapy. Our aim was to assess the osteogenic potential of omental ASCs and to examine the status of the isolated CD34(negative)-enriched fraction of omental-derived ASCs from subjects with different metabolic profiles. Omental ASCs from normal-weight subjects and subjects with or without metabolic syndrome were isolated, and the osteogenic potential of omental ASCs was evaluated. Additionally, osteogenic and clonogenic potential, proliferation rate, mRNA expression levels of proteins involved in redox balance, and fibrotic proteins were examined in the CD34(negative)-enriched fraction of omental-derived ASCs. Both the omental ASCs and the CD34(negative)-enriched fraction of omental ASCs from subjects without metabolic syndrome have a greater osteogenic potential than those from subjects with metabolic syndrome. The alkaline phosphatase and osteonectin mRNA were negatively correlated with nicotinamide adenine dinucleotide phosphate oxidase-2 mRNA and the mRNA expression levels of the fibrotic proteins correlated positively with nicotinamide adenine dinucleotide phosphate oxidase-5 mRNA and the homeostasis model assessment. Although the population doubling time was significantly higher in subjects with a body mass index of 25 kg/m(2) or greater, only the CD34(negative)-enriched omental ASC fraction in the subjects with metabolic syndrome had a higher population doubling time than the normal-weight subjects. The osteogenic, clonogenic, fibrotic potential, and proliferation rate observed in vitro suggest that omental ASCs from subjects without metabolic syndrome are more suitable for therapeutic osteogenic applications than those from subjects with metabolic syndrome.