Wilfredo Oliva-Olivera

Progression from High Insulin Resistance to Type 2 Diabetes Does Not Entail Additional Visceral Adipose Tissue Inflammation

Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.

Obesity Determines the Immunophenotypic Profile and Functional Characteristics of Human Mesenchymal Stem Cells From Adipose Tissue

Adipose tissue is a major source of mesenchymal stem cells (MSCs), which possess a variety of properties that make them ideal candidates for regenerative and immunomodulatory therapies. Here, we compared the immunophenotypic profile of human adipose‐derived stem cells (hASCs) from lean and obese individuals, and explored its relationship with the apparent altered plasticity of hASCs. We also hypothesized that persistent hypoxia treatment of cultured hASCs may be necessary but not sufficient to drive significant changes in mature adipocytes. hASCs were obtained from subcutaneous adipose tissue of healthy, adult, female donors undergoing abdominal plastic surgery: lean (n = 8; body mass index [BMI]: 23 ± 1 kg/m2) and obese (n = 8; BMI: 35 ± 5 kg/m2). Cell surface marker expression, proliferation and migration capacity, and adipogenic differentiation potential of cultured hASCs at two different oxygen conditions were studied. Compared with lean‐derived hASCs, obese‐derived hASCs demonstrated increased proliferation and migration capacity but decreased lipid droplet accumulation, correlating with a higher expression of human leukocyte antigen (HLA)‐II and cluster of differentiation (CD) 106 and lower expression of CD29. Of interest, adipogenic differentiation modified CD106, CD49b, HLA‐ABC surface protein expression, which was dependent on the donors BMI. Additionally, low oxygen tension increased proliferation and migration of lean but not obese hASCs, which correlated with an altered CD36 and CD49b immunophenotypic profile. In summary, the differences observed in proliferation, migration, and differentiation capacity in obese hASCs occurred in parallel with changes in cell surface markers, both under basal conditions and during differentiation. Therefore, obesity is an important determinant of stem cell function independent of oxygen tension.

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.

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.

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

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.

Effects of GLP‐1 on the differentiation and metabolism of human adipocytes

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.

Hypoxia is associated with a lower expression of genes involved in lipogenesis in visceral adipose tissue

BackgroundA key role for HIF-1α in the promotion and maintenance of dietary obesity has been proposed. We analyzed the association between hypoxia and de novo lipogenesis in human adipose tissue.MethodsWe studied HIF-1α mRNA and protein expression in fasting status in visceral adipose tissue (VAT) from non-obese and morbidly obese subjects, and in VAT from wild-type and ob/ob C57BL6J mice in both fasting and feeding status. We also analyzed the effect of hypoxia on the VAT mRNA expression of genes involved in lipogenesis.ResultsHIF-1α was increased in VAT from morbidly obese subjects. In fasting status, C57BL6J ob/ob mice had a higher VAT HIF-1α mRNA expression than C57BL6J wild-type mice. In feeding status, VAT HIF-1α mRNA expression significantly increased in C57BL6J wild-type, but not in C57BL6J ob/ob mice. In humans, HIF-1α mRNA expression correlated positively with body mass index and insulin resistance. VAT HIF-1α mRNA expression correlated negatively with ACC1, PDHB and SIRT3 mRNA expression, and positively with PPAR-γ. VAT explants incubated in hypoxia showed reduced SIRT3 and increased PPAR-γ, SREBP-1c, ACLY, ACC1 and FASN mRNA expression.ConclusionsMorbidly obese subjects have a higher level of VAT HIF-1α. Postprandial status is associated with an increase in HIF-1α mRNA expression in C57BL6J wild-type mice. Hypoxia alters the mRNA expression of genes involved in de novo lipogenesis in human VAT.

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.

Parathyroid hormone-related protein, human adipose-derived stem cells adipogenic capacity and healthy obesity

OBJECTIVE This study aimed to define the potential role of PTHrP on adipogenic regulation and to analyze its relationship with obesity and insulin resistance. DESIGN This was a cross-sectional study in which visceral (VAT) and subcutaneous (SAT) adipose tissue were extracted from 19 morbidly obese, 10 obese, and 10 lean subjects. PTHrP mRNA levels were measured in VAT and SAT. VAT mesenchymal stem cells and 3T3-L1 cells were differentiated into adipocytes in presence or absence of PTHrP siRNA. PTHrP mRNA and protein levels as well as adipogenic markers were evaluated by Western blotting or qPCR. Immunohistochemistry and immunofluorescence procedures were used for PTHrP intracellular localization. RESULTS Both human VAT and SAT express PTHrP protein mainly in the nucleolar compartment of stromal vascular fraction cells. The highest levels of PTHrP mRNA and protein expression were detected in undifferentiated mesenchymal cells and progressively decreased during adipogenesis. Remarkably, adipogenic differentiation in human mesenchymal stem cells (A-hMSC) was significantly impaired in a pthrp knockdown. PTHrP seems to be related to obesity-associated insulin resistance (IR), given that we found that PTHrP mRNA expression was higher in VAT from morbidly obese with a low IR degree (MO-L-IR) subjects than those from morbidly obese with a high IR degree (MO-H-IR) and lean subjects, and correlated positively with body mass index and hip circumference. We also found that A-hMSC from MO-L-IRs displayed higher adipogenic capacity than those from both MO-H-IRs and leans. In addition, adipogenesis was impaired in VAT from MO-H-IRs, given that mRNA expression levels of key adipogenic regulators were lower than those from MO-L-IR subjects. CONCLUSIONS PTHrP could be a potential new therapeutic target for the reprograming of adipogenesis and adipose tissue expansion, thus possibly ameliorating the metabolic syndrome in obese subjects.

Survivin, a key player in cancer progression, increases in obesity and protects adipose tissue stem cells from apoptosis

Adipose tissue (AT) has a central role in obesity-related metabolic imbalance through the dysregulated production of cytokines and adipokines. In addition to its known risk for cardiovascular disease and diabetes, obesity is also a major risk for cancer. We investigated the impact of obesity for the expression of survivin, an antiapoptotic protein upregulated by adipokines and a diagnostic biomarker of tumor onset and recurrence. In a cross-sectional study of 111 subjects classified by body mass index, circulating levels of survivin and gene expression in subcutaneous AT were significantly higher in obese patients and positively correlated with leptin. Within AT, survivin was primarily detected in human adipocyte-derived stem cells (hASCs), the adipocyte precursors that determine AT expansion. Remarkably, survivin expression was significantly higher in hASCs isolated from obese patients that from lean controls and was increased by proinflammatory M1 macrophage soluble factors including IL-1β. Analysis of survivin expression in hASCs revealed a complex regulation including epigenetic modifications and protein stability. Surprisingly, obese hASCs showed survivin promoter hypermethylation that correlated with a significant decrease in its mRNA levels. Nonetheless, a lower level of mir-203, which inhibits survivin protein translation, and higher protein stability, was found in obese hASCs compared with their lean counterparts. We discovered that survivin levels determine the susceptibility of hASCs to apoptotic stimuli (including leptin and hypoxia). Accordingly, hASCs from an obese setting were protected from apoptosis. Collectively, these data shed new light on the molecular mechanisms governing AT expansion in obesity through promotion of hASCs that are resistant to apoptosis, and point to survivin as a potential new molecular player in the communication between AT and tumor cells. Thus, inhibition of apoptosis targeting survivin might represent an effective strategy for both obesity and cancer therapy.