Functionalized Collagen/Poly(ethylene glycol) Diacrylate Interpenetrating Network Hydrogel Enhances Beta Pancreatic Cell Sustenance.

Three-dimensional matrices are a new strategy used to tackle type I diabetes, a chronic metabolic disease characterized by the destruction of beta pancreatic cells. Type I collagen is an abundant extracellular matrix (ECM), a component that has been used to support cell growth. However, pure collagen possesses some difficulties, including a low stiffness and strength and a high susceptibility to cell-mediated contraction. Therefore, we developed a collagen hydrogel with a poly (ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN), functionalized with vascular endothelial growth factor (VEGF) to mimic the pancreatic environment for the sustenance of beta pancreatic cells. We analyzed the physicochemical characteristics of the hydrogels and found that they were successfully synthesized. The mechanical behavior of the hydrogels improved with the addition of VEGF, and the swelling degree and the degradation were stable over time. In addition, it was found that 5 ng/mL VEGF-functionalized collagen/PEGDA IPN hydrogels sustained and enhanced the viability, proliferation, respiratory capacity, and functionality of beta pancreatic cells. Hence, this is a potential candidate for future preclinical evaluation, which may be favorable for diabetes treatment.

Collagen-mesenchymal stem cell spheroids in suspension promote high adipogenic capacity.

Human mesenchymal stem cells (hMSC) represent a unique and promising platform because of their ability to promote soft tissue regeneration, particularly their ability to differentiate into adipocytes, which are important for adipose tissue regeneration. In this context, type I collagen is the most abundant extracellular matrix component of adipose tissue and can act as a natural spheroid source to support the differentiation process of stem cells. However, spheroids based on collagen and hMSCs without numerous pro-adipogenic factors that can induce adipogenesis have not yet been investigated. In this study, we focused on developing collagen-hMSC spheroids capable of differentiating into adipocyte-like cells in a short time (eight culture days) without adipogenic factors, with potential applications in adipose tissue repair. The physical and chemical properties of the spheroids indicated successful cross-linking of collagen. Upon spheroid development, stability, cell viability, and metabolic activity of the constructs were maintained. During adipogenesis, cell morphology shows significant changes, in which cells change from a fibroblast-like shape to an adipocyte-like shape, and adipogenic gene expression after eight days of cell culture. These results support the utility of collagen-hMSC 3 mg ml-1collagen concentration spheroids to differentiate into adipocyte-like cells in a short time without adverse effects on biocompatibility, metabolic activity, or cell morphology, suggesting that this construct may be used in soft tissue engineering.

Essential Oil of Carvone Chemotype Lippia alba (Verbenaceae) Regulates Lipid Mobilization and Adipogenesis in Adipocytes.

Obesity is characterized by an expansion of adipose tissue due to excessive accumulation of triglycerides in adipocytes, causing hypertrophy and hyperplasia, followed by hypoxia, alterations in adipocyte functionality, and chronic inflammation. However, current treatments require changes in lifestyle that are difficult to achieve and some treatments do not generate sustained weight loss over time. Therefore, we evaluated the effect of the essential oil (EO) of Lippia alba (Verbenaceae) carvone chemotype on viability, lipid mobilization, and adipogenesis of adipocytes in two normal and pathological cellular models in vitro. In 3T3-L1 adipocytes, a normal and a pathological model of obesity were induced, and then the cells were treated with L. alba carvone chemotype EO to evaluate cell viability, lipid mobilization, and adipogenesis. L. alba carvone chemotype EO does not decrease adipocyte viability at concentrations of 0.1, 1, and 5 µg/mL; furthermore, there was evidence of changes in lipid mobilization and adipogenesis, leading to a reversal of adipocyte hypertrophy. These results could be due to effects produced by EO on lipogenic and lipolytic pathways, as well as modifications in the expression of adipogenesis genes. L. alba carvone chemotype EO could be considered as a possible treatment for obesity, using the adipocyte as a therapeutic target.

Soluplus®-based dissolving microarray patches loaded with colchicine: towards a minimally invasive treatment and management of gout.

Considered as one of the most common inflammatory arthritis, gout is characterised by a sudden onset of severe joint pain. As the first-line drug of choice used in treating acute gout, colchicine (CLC) is hindered by poor gastrointestinal permeability as well as unfavourable gastrointestinal side effects. Herein, we present, for the first time, the preparation of microarray array patches (MAPs) made of a polymeric solubiliser, Soluplus®, loaded with CLC for its systemic delivery. The fabricated MAPs displayed acceptable mechanical properties and were capable of being inserted into the skin to a depth of ≈500 µm in full thickness ex vivo neonatal porcine skin, as evidenced by optical coherence tomography. In vitro dermatokinetic studies utilising full thickness neonatal porcine skin demonstrated that the CLC-loaded MAPs delivered CLC across all skin strata, resulting in a delivery efficiency of 73% after 24 hours. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell proliferation assays along with LIVE/DEAD™ staining on the 3T3-L1 cell line showed that the MAP formulation displayed minimal toxicity, with acceptable biocompatibility. Lastly, the anti-inflammatory properties of the formulation were evaluated using a THP-1 macrophage cell line. It was shown that treatment of THP-1 macrophages that are exposed to lipopolysaccharide (LPS) with CLC-loaded MAPs caused a significant (p < 0.05) reduction of TNF-α production, a pro-inflammatory cytokine typically associated with the early onset of acute gout. Accordingly, CLC-loaded MAPs could represent a new minimally-invasive alternative strategy for management of acute gout.

Hipertrofia y resistencia a la insulina en un modelo in vitro de obesidad y DMT2 inducida por alta glucosa e insulina / Hypertrophy and insulin resistance in an in vitro model of obesity and T2DM induced by high glucose and insulin

Resumen Introducción: La obesidad es considerada un factor de riesgo para desarrollar resistencia a la insulina. La expansión del tejido adiposo se ha relacionado con el aumento de la producción de citoquinas proinflamatorias que, junto a los ácidos grasos son responsables, al menos en parte, del desarrollo de la resistencia a la insulina y esta a su vez, facilita el desarrollo de diabetes mellitus tipo 2 (DMT2). Objetivo: El propósito de este estudio fue realizar y caracterizar un modelo in vitro de obesidad empleando concentraciones altas de glucosa e insulina en una línea de células adipocitarias. Métodos: Se indujo modelo de hipertrofia celular realizando un estímulo en adipocitos maduros con una concentración de glucosa (450 mg/dL) e insulina (106 pmol/L) (modelo HGHI). Tras estímulo se realizaron ensayos de viabilidad celular, diámetro celular, movilización de lípidos y marcadores de señalización de insulina. Resultados: Tras el tratamiento con HGHI, se evidencia hipertrofia adipocitaria, incremento en la acumulación de lípidos, reducción de la ruptura de éstos, alteración de la señalización de insulina y tendencia a modificación de proteínas de marcadores de estrés de retículo y estrés oxidativo. Conclusión: Estos resultados demuestran la validez del modelo in vitro que simula al menos en parte la obesidad asociada a insulino resistencia, siendo una herramienta útil para estudiar los mecanismos de susceptibilidad a obesidad y resistencia a la insulina inducida in vitro con diferentes moléculas.

Abstract Introduction: Obesity is considered a risk factor for developing insulin resistance. The increase in adipose tissue has been related to the increase in the production of pro-inflammatory cytokines, which together with fatty acids are responsible, at least in part, for the development of insulin resistance, and this in turn facilitates the development of T2 diabetes mellitus type 2 (DMT2). Objective: The purpose of this study was to perform and characterize an in vitro model of obesity using high concentrations of glucose and insulin on an adipocyte cell line. Methods: A cell hypertrophy model was induced by stimulating mature adipocytes with a concentration of glucose (450 mg/dL) and insulin (106 pmol/L) (HGHI model). The cell viability, cell diameter, lipid mobilization and insulin signalling markers were evaluated. Results: After HGHI treatment, adipocytes show hypertrophy, increase in lipid accumulation, reduction of lipid breakdown, alteration of insulin signalling, a tendency to modify proteins of reticulum stress markers and, oxidative stress. Conclusion: These results demonstrate a new in vitro model that simulates, at least in part, obesity associated with insulin resistance being a useful tool to study the mechanisms of susceptibility to obesity and insulin resistance induced in vitro by different molecules.

Plasma Metabolome Profiling by High-Performance Chemical Isotope-Labelling LC-MS after Acute and Medium-Term Intervention with Golden Berry Fruit (Physalis peruviana L.), Confirming Its Impact on Insulin-Associated Signaling Pathways.

PURPOSE: Golden berry (Physalis peruviana L.) is an exotic fruit exported from Colombia to different countries around the world. A review of the literature tends to demonstrate a hypoglycaemic effect with an improvement in insulin sensitivity after oral ingestion of fruit extracts in animal models. However, little is known about their potential effects in humans, and very little is known about the mechanisms involved. This study aimed at identifying discriminant metabolites after acute and chronic intake of golden berry. METHOD: An untargeted metabolomics strategy using high-performance chemical isotope-labelling LC-MS was applied. The blood samples of eighteen healthy adults were analysed at baseline, at 6 h after the intake of 250 g of golden berry (acute intervention), and after 19 days of daily consumption of 150 g (medium-term intervention). RESULTS: Forty-nine and 36 discriminant metabolites were identified with high confidence, respectively, after the acute and medium-term interventions. Taking into account up- and downregulated metabolites, three biological networks mainly involving insulin, epidermal growth factor receptor (EGFR), and the phosphatidylinositol 3-kinase pathway (PI3K/Akt/mTOR) were identified. CONCLUSIONS: The biological intracellular networks identified are highly interconnected with the insulin signalling pathway, showing that berry intake may be associated with insulin signalling, which could reduce some risk factors related to metabolic syndrome. Primary registry of WHO.

Effects of oleic (18: 1n-9) and palmitic (16: 0) fatty acids on the metabolic state of adipocytes / Efectos de los ácidos grasos oleico (18: 1n-9) y palmítico (16: 0) en el estado metabólico del adipocito

Abstract Background: Elevated serum-free fatty acid (FFA) levels induce insulin resistance (IR) or a protective mechanism to IR development in humans; it depends on FFA type. Objetive: This study explores the effects of oleic (OLA - unsatured) and palmitic (PAM - saturated) fatty acids on insulin action in mature adipocytes effect. Methods: Cells were incubated 18 h with or without OLA and PAM at 250 μM, and 500 μM. After the culture period, were measured: adipocyte viability, size, fatty acids mobilisation, insulin signalling proteins, and glucose uptake. Results: Adipocytes exhibited optimal viability tolerances regardless of the kinds of fatty acids used for treatment. However, adipocytes were hypertrophic after OLA and PAM stimuli. Additionally, lipogenesis (lipid synthesis), and lipolysis (lipid breakdown) were significantly increased by treatment with OLA, or PAM (500 μM) compared to control. Moreover, OLA results showed that there was no significant reduction in signalling cascades, except for a downstream proinflammatory response. Instead, PAM hypertrophic adipocytes were insulin resistant with alteration of proinflammatory and stress markers. Conclusions: Current findings suggest that PAM induces insulin resistance, mitochondrial and reticulum stress on fat cells compared to those treated with OLA that, protects adipocytes to all those alterations.

Resumen Introducción: Los niveles elevados de ácidos grasos libres (AGL) en suero inducen resistencia a insulina (RI) o un mecanismo de protección del desarrollo de RI en humanos, esto depende del tipo de AGL. Objetivo: Este estudio explora los efectos de los ácidos grasos oleico (insaturados - OLA) y palmítico (saturados - PAM) sobre la insulina en adipocitos maduros. Métodos: Las células se incubaron 18 h con o sin OLA y PAM a 250 μM y 500 μM. Después del período de cultivo, se evaluó en adipocitos: viabilidad, tamaño, movilización de ácidos grasos, proteínas de señalización de insulina y absorción de glucosa. Resultados: Los adipocitos mostraron viabilidad óptima independientemente de los tipos de ácidos grasos utilizados en el tratamiento. Los adipocitos eran hipertróficos tras estimulo con OLA y PAM. La lipogénesis (síntesis de lípidos) y la lipólisis (degradación de lípidos) aumentaron significativamente con el tratamiento con OLA o PAM (500 μM) en comparación con el control. En los resultados de OLA no se evidenció una reducción significativa en las cascadas de señalización de insulina, a excepción de una respuesta proinflamatoria posterior. En cambio, los adipocitos hipertróficos tratados con PAM presentaron resistencia a la insulina y alteración de los marcadores proinflamatorios y de estrés. Conclusiones: Nuestros hallazgos sugieren que PAM induce resistencia a la insulina, estrés mitocondrial y del retículo en las células grasas en comparación con aquellos tratados con OLA, AGL que, en cambio, protegen a los adipocitos de todas esas alteraciones.

Cambios en el perfil proteómico de preadipocitos con diferentes condiciones metabólicas / Changes in the proteomic pro/ile of preadipocytes with different metabolic status

Resumen Introducción y objetivos: El tejido adiposo subcutáneo se considera un depósito con un papel protector desde un punto de vista metabólico. El exceso de tejido adiposo desencadena en obesidad, la cual, está acompañada típicamente por resistencia a insulina, dislipidemia, e hipertensión arterial. No obstante, se conoce que existe un subgrupo de obesos que parecen estar protegidos de dichas complicaciones. Estos individuos son definidos como obesos sanos metabólicamente. A pesar de los avances en el conocimiento de las alteraciones que suceden en el tejido adiposo en obesidad, aún se desconocen los mecanismos que subyacen en el desarrollo de resistencia a insulina. Por lo tanto, en este trabajo, se estudió la asociación entre obesidad y desarrollo de enfermedad metabólica identificando factores y procesos que determinan la transición desde el fenotipo obeso sano y no sano, empleando preadipocitos provenientes de tejido adiposo subcutáneo. Metodología: Se emplearon datos de un estudio de proteómica comparada de preadipocitos de tejido subcutáneo obtenidos de pacientes obesos normoglucémicos no resistentes a insulina y de pacientes obesos con diabetes mellitus de tipo 2. El estudio proteómico, se llevó a cabo utilizando la técnica de iTRAQ combinada con LC-MSMS. Resultados y conclusiones: Las diferencias entre preadipocitos de tejido adiposo subcutáneo en sujetos normoglucémicos y con diabetes, afectan sobre todo a proteínas citosólicas y, en particular, a proteínas relacionadas con procesos metabólicos mientras que, las membranales no cambian entre fenotipos obesos. En el estudio se identificaron importantes diferencias en el perfil proteómico de los preadipocitos de tejido adiposo subcutáneo en obesidad, tanto en sujetos normoglucémicos como diabéticos, apoyando la importancia de estas células en el mantenimiento de la identidad del depósito graso. También se encontró que, la transición desde el fenotipo obeso sano hacia el no sano conlleva un mayor desarrollo de estrés oxidativo e inflamación en las células precursoras adipocitarias.

Abstract Introduction and objectives: The subcutaneous adipose tissue is considered as a depot with a protective role from a metabolic point of view. An excess of adipose tissue is triggered in obesity, which is accompanied by insulin resistance, dyslipidemia and arterial hypertension. However, it is known that, there is a subgroup of obese people who seem to be protected from obese complications. These individuals are defined as metabolically healthy obese. Despite the advances in the knowledge of the alterations that occur in adipose tissue during obesity, the mechanisms underlying the development of insulin resistance are still unknown. Therefore, in this work, we studied the association between obesity and the development of metabolic disease, we identified factors and processes that determined the transition of healthy and unhealthy obesity phenotype, using preadipocytes from subcutaneous adipose tissue. Methods: Data obtained from a comparative proteomics study of subcutaneous adipose tissue preadipocytes from normoglycemic obese patients-not resistant to insulin and from obese patients with type 2 diabetes mellitus were used. The proteomic study was carried out using the iTRAQ combined with LC -MSMS. Results and conclusions: The differences between pre-adipocytes of subcutaneous adipose tissue in normoglycemic subjects and with diabetes affect mainly cytosolic proteins and, in particular, proteins related to metabolic processes while, membrane proteins do not change between obese phenotypes. In this study, we identified significant differences in the proteomic profile of preadipocytes from subcutaneous adipose tissue in obesity in both, normoglycemic and diabetic subjects, supporting the importance of these cells in the maintenance of the fat depot identity. We also found that, the transition from unhealthy to healthy phenotype in obesity, leads to further development of oxidative stress and inflammation in adipocyte precursor cells.

The Effects of Bariatric Surgery-Induced Weight Loss on Adipose Tissue in Morbidly Obese Women Depends on the Initial Metabolic Status.

BACKGROUND: Adipose tissue (AT) dysfunction in obesity is commonly linked to insulin resistance and promotes the development of metabolic disease. Bariatric surgery (BS) represents an effective strategy to reduce weight and to improve metabolic health in morbidly obese subjects. However, the mechanisms and pathways that are modified in AT in response to BS are not fully understood, and few information is still available as to whether these may vary depending on the metabolic status of obese subjects. METHODS: Abdominal subcutaneous adipose tissue (SAT) samples were obtained from morbidly obese women (n = 18) before and 13.3 ± 0.37 months after BS. Obese women were stratified into two groups: normoglycemic (NG; Glu < 100 mg/dl, HbA1c <5.7 %) or insulin resistant (IR; Glu 100-126 mg/dl, HbA1c 5.7-6.4 %) (n = 9/group). A multi-comparative proteomic analysis was employed to identify differentially regulated SAT proteins by BS and/or the degree of insulin sensitivity. Serum levels of metabolic, inflammatory, and anti-oxidant markers were also analyzed. RESULTS: Before surgery, NG and IR subjects exhibited differences in AT proteins related to inflammation, metabolic processes, the cytoskeleton, and mitochondria. BS caused comparable weight reductions and improved glucose homeostasis in both groups. However, BS caused dissimilar changes in metabolic enzymes, inflammatory markers, cytoskeletal components, mitochondrial proteins, and angiogenesis regulators in NG and IR women. CONCLUSIONS: BS evokes significant molecular rearrangements indicative of improved AT function in morbidly obese women at either low or high metabolic risk, though selective adaptive changes in key cellular processes occur depending on the initial individual's metabolic status.

Surgery-Induced Weight Loss Is Associated With the Downregulation of Genes Targeted by MicroRNAs in Adipose Tissue.

CONTEXT: Molecular mechanisms associated with physiological variations in adipose tissue (AT) are not fully recognized. The most recent reports highlight the critical relevance of microRNAs (miRNAs) found in AT. OBJECTIVE: To identify changes in messenger RNA (mRNA) and miRNA expressions and their interaction in human AT before and after surgery-induced weight loss. Research Design and Setting: Genome-wide mRNA and miRNA expressions were assessed by microarrays in abdominal subcutaneous AT of 16 morbidly obese women before and 2 years after laparoscopic Roux-en-Y gastric bypass. The association of changes in miRNAs with their respective mRNA targets was studied. The results were replicated in publicly available microarray datasets. Validation was made by real-time polymerase chain reaction in additional fat samples from 26 age-matched lean women and in isolated human adipocytes. RESULTS: A total of 5018 different mRNA probe sets and 15 miRNAs were differentially expressed after surgery-induced weight loss. The clustering of similar expression patterns for gene products with related functions revealed molecular footprints that elucidate significant changes in cell cycle, development, lipid metabolism, and the inflammatory response. The participation of inflammation was demonstrated by results assessed in isolated adipocytes. Interestingly, when transcriptomes were analyzed taking into account the presence of miRNA target sites, miRNA target mRNAs were upregulated in obese AT (P value = 2 × 10(-181)) and inflamed adipocytes (P value = 4 × 10(-61)), according to the number of target sites harbored by each transcript. CONCLUSIONS: Current findings suggest impaired miRNA target gene expression in obese AT in close association with inflammation, both improving after weight loss.