Culturing Conditions Dictate the Composition and Pathways Enrichment of Human and Rat Perirenal Adipose-Derived Stromal Cells' Secretomes.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly.

Immunometabolism in type 2 diabetes mellitus: tissue-specific interactions.

The immune system is frequently described in the context of its protective function against infections and its role in the development of autoimmunity. For more than a decade, the interactions between the immune system and metabolic processes have been reported, in effect creating a new research field, termed immunometabolism. Accumulating evidence supports the hypothesis that the development of metabolic diseases may be linked to inflammation, and reflects, in some cases, the activation of immune responses. As such, immunometabolism is defined by 1) inflammation as a driver of disease development and/or 2) metabolic processes stimulating cellular differentiation of the immune components. In this review, the main factors capable of altering the immuno-metabolic communication leading to the development and establishment of obesity and diabetes are comprehensively presented. Tissue-specific immune responses suggested to impair metabolic processes are described, with an emphasis on the adipose tissue, gut, muscle, liver, and pancreas.

Towards standardization of human adipose-derived stromal cells secretomes.

The secretome of adipose-derived stromal cells (ASC) is a heterogeneous mixture of components with a beneficial influence on cellular microenvironments. As such, it represents a cell-free alternative in regenerative medicine therapies. Pathophysiological conditions increase the therapeutic capacity of ASC and, with this, the benefits of the secretome. Such conditions can be partially mimicked in vitro by adjusting culturing conditions. Secretomics, the unbiased analysis of a cell secretome by mass spectrometry, is a powerful tool to describe the composition of ASC secretomes. In this proteomics databases review, we compared ASC secretomic studies to retrieve persistently reported proteins resulting from the most explored types of culturing conditions used in research, i.e., exposure to normoxia, hypoxia, or cytokines. Our comparisons identified only eight common proteins within ASC normoxic secretomes, no commonalities within hypoxic ASC secretomes, and only nine within secretomes of ASC exposed to proinflammatory cytokines. Within these, and regardless of the culturing condition that stimulated secretion, a consistent presence of extracellular matrix-related pathways associated with such proteins was identified. Confounders such as donors' age, sex, body mass index, the anatomical area where ASC were harvested, secretome collection method, data description, and how the data is shared with the scientific community are discussed as factors that might explain our outcomes. We conclude that standardization is imperative as the currently available ASC secretomic studies do not facilitate solid conclusions on the therapeutic value of different ASC secretomes.

Species-dependent impact of immunosuppressive squalene-gusperimus nanoparticles and adipose-derived stem cells on isolated human and rat pancreatic islets.

Transplantation of pancreatic islets is a promising approach to controlling glucose levels in type 1 diabetes mellitus (T1DM), but islet survival is still limited. To overcome this, islet co-culture with mesenchymal stromal cells (MSCs) together with safe immunosuppressive agents like squalene-gusperimus nanoparticles (Sq-GusNPs) may be applied. This could support islet survival and engraftment. Here, we studied how Sq-GusNPs and adipose-derived stem cells (ASCs) influence islets response under pro-inflammatory conditions. Through qRT-PCR, we studied the expression of specific genes at 24 hours in human and rat islets and ASCs in co-culture under indirect contact with or without treatment with Sq-GusNPs. We characterized how the response of islets and ASCs starts at molecular level before impaired viability or function is observed and how this response differs between species. Human islets and ASCs responses showed to be principally influenced by NF-κB activation, whereas rat islet and ASCs responses showed to be principally mediated by nitrosative stress. Rat islets showed tolerance to inflammatory conditions due to IL-1Ra secretion which was also observed in rat ASCs. Human islets induced the expression of cytokines and chemokines with pro-angiogenic, tissue repair, and anti-apoptotic properties in human ASCs under basal conditions. This expression was not inhibited by Sq-GusNPs. Our results showed a clear difference in the response elicited by human and rat islets and ASCs in front of an inflammatory stimulus and Sq-GusNPs. Our data support the use of ASCs and Sq-GusNP to facilitate engraftment of islets for T1DM treatment.

Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in ß-Cells.

How immune tolerance is lost to pancreatic ß-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered ß-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing ß-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 µM), or exposed to proinflammatory cytokines interleukin (IL)-1ß or interferon gamma (IFNγ) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1ß expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in ß-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1ß cellular levels declined, coupled with increased secretion of mature IL-1ß. Our results suggest that limiting ß-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize ß-cells to immune-attack.

In silico approach to predict pancreatic ß-cells classically secreted proteins.

Pancreatic ß-cells, residents of the islets of Langerhans, are the unique insulin-producers in the body. Their physiology is a topic of intensive studies aiming to understand the biology of insulin production and its role in diabetes pathology. However, investigations about these cells' subset of secreted proteins, the secretome, are surprisingly scarce and a list describing islet/ß-cell secretome upon glucose-stimulation is not yet available. In silico predictions of secretomes are an interesting approach that can be employed to forecast proteins likely to be secreted. In this context, using the rationale behind classical secretion of proteins through the secretory pathway, a Python tool capable of predicting classically secreted proteins was developed. This tool was applied to different available proteomic data (human and rodent islets, isolated ß-cells, ß-cell secretory granules, and ß-cells supernatant), filtering them in order to selectively list only classically secreted proteins. The method presented here can retrieve, organize, search and filter proteomic lists using UniProtKB as a central database. It provides analysis by overlaying different sets of information, filtering out potential contaminants and clustering the identified proteins into functional groups. A range of 70-92% of the original proteomes analyzed was reduced generating predicted secretomes. Islet and ß-cell signal peptide-containing proteins, and endoplasmic reticulum-resident proteins were identified and quantified. From the predicted secretomes, exemplary conservational patterns were inferred, as well as the signaling pathways enriched within them. Such a technique proves to be an effective approach to reduce the horizon of plausible targets for drug development or biomarkers identification.

Resveratrol Improves Glycemic Control in Type 2 Diabetic Obese Mice by Regulating Glucose Transporter Expression in Skeletal Muscle and Liver.

Insulin resistance participates in the glycaemic control disruption in type 2 diabetes mellitus (T2DM), by reducing muscle glucose influx and increasing liver glucose efflux. GLUT4 (Slc2a4 gene) and GLUT2 (Slc2a2 gene) proteins play a fundamental role in the muscle and liver glucose fluxes, respectively. Resveratrol is a polyphenol suggested to have an insulin sensitizer effect; however, this effect, and related mechanisms, have not been clearly demonstrated in T2DM. We hypothesized that resveratrol can improve glycaemic control by restoring GLUT4 and GLUT2 expression in muscle and liver. Mice were rendered obese T2DM in adult life by neonatal injection of monosodium glutamate. Then, T2DM mice were treated with resveratrol for 60 days or not. Glycaemic homeostasis, GLUT4, GLUT2, and SIRT1 (sirtuin 1) proteins (Western blotting); Slc2a4, Slc2a2, and Pck1 (key gluconeogenic enzyme codifier) mRNAs (RT-qPCR); and hepatic glucose efflux were analysed. T2DM mice revealed: high plasma concentration of glucose, fructosamine, and insulin; insulin resistance (insulin tolerance test); decreased Slc2a4/GLUT4 content in gastrocnemius and increased Slc2a2/GLUT2 content in liver; and increased Pck1 mRNA and gluconeogenic activity (pyruvate tolerance test) in liver. All alterations were restored by resveratrol treatment. Additionally, in both muscle and liver, resveratrol increased SIRT1 nuclear content, which must participate in gene expression regulations. In sum, the results indisputably reveals that resveratrol improves glycaemic control in T2DM, and that involves an increase in muscle Slc2a4/GLUT4 and a decrease in liver Slc2a2/GLUT2 expression. This study contributes to our understanding how resveratrol might be prescribed for T2DM according to the principles of evidence-based medicine.

Resveratrol improves glycemic control in insulin-treated diabetic rats: participation of the hepatic territory.

BACKGROUND: Resveratrol is a natural polyphenol that has been proposed to improve glycemic control in diabetes, by mechanisms that involve improvement in insulin secretion and activity. In type 1 diabetes (T1D), in which insulin therapy is obligatory, resveratrol treatment has never been investigated. The present study aimed to evaluate resveratrol as an adjunctive agent to insulin therapy in a T1D-like experimental model. METHODS: Rats were rendered diabetic by streptozotocin (STZ) treatment. Twenty days later, four groups of animals were studied: non-diabetic (ND); diabetic treated with placebo (DP); diabetic treated with insulin (DI) and diabetic treated with insulin plus resveratrol (DIR). After 30 days of treatment, 24-hour urine was collected; then, blood, soleus muscle, proximal small intestine, renal cortex and liver were sampled. Specific glucose transporter proteins were analyzed (Western blotting) in each territory of interest. Solute carrier family 2 member 2 (Slc2a2), phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) mRNAs (qPCR), glycogen storage and sirtuin 1 (SIRT1) activity were analyzed in liver. RESULTS: Diabetes induction increased blood glucose, plasma fructosamine concentrations, and glycosuria. Insulin therapy partially recovered the glycemic control; however, resveratrol as adjunctive therapy additionally improved glycemic control and restored plasma fructosamine concentration to values of non-diabetic rats. Resveratrol did not alter the expression of the glucose transporters GLUT2 and SGLT1 in the intestine, GLUT2 and SGLT2 in kidney and GLUT4 in soleus, suggesting that fluxes of glucose in these territories were unaltered. Differently, in liver, resveratrol promoted a reduction in Slc2a2, Pck1, and G6pc mRNAs, as well as in GLUT2 protein (P < 0.05, DIR vs. DI); besides, it increased (P < 0.01, DIR vs. DI) the hepatic glycogen content, and SIRT1 protein. CONCLUSIONS: Resveratrol is able to improve glycemic control in insulin-treated T1D-like rats. This effect seems not to involve changes in glucose fluxes in the small intestine, renal proximal tubule, and soleus skeletal muscle; but to be related to several changes in the liver, where downregulation of Slc2a2/GLUT2, Pck1, and G6pc expression was observed, favoring reduction of glucose production and efflux. Besides, resveratrol increased SIRT1 nuclear protein content in liver, which may be related to the observed gene expression regulations.