RESUMO
Equine metabolic syndrome (EMS) is a steadily growing endocrine disorder representing a real challenge in veterinary practice. As a multifactorial condition, EMS is characterized by three main metabolic abnormalities including insulin resistance, increased adiposity or obesity and hoof laminitis. Adipose tissue dysfunction is recognized as a core pathophysiological determinant of EMS, as it strongly participates to lipotoxicity and systemic metaflammation, both of which have been closely linked to the development of generalized insulin resistance. Besides, sex hormone binding globulin (SHBG) is an important sex steroids transporters that has been recently proposed as an important metabolic mediator. Therefore, the aim of this study was to verify whether SHBG treatment may ameliorate subcutaneous adipose tissue metabolic failure under EMS condition in terms of lipidome homeostasis, lipid metabolism programs, insulin signalling and local inflammation. Subcutaneous adipose tissue (SAT) biopsies were collected post-mortem from healthy (n = 3) and EMS (n = 3) slaughtered horses. SHBG protein has been applied to SAT samples from EMS horses for 24 h at a final concentration of 50 nM, while control groups (healthy and untreated EMS) were cultured in the presence of SHBG-vehicle only. Tissues from all groups were afterwards secured for downstream analysis of gene expression using RT-qPCR, protein levels by Western blot and ELISA assay and lipidomics through GC-MS technique. Obtained results showcased that SHBG intervention efficiently normalized the altered fatty acids (FAs) profiles by lowering the accumulation of saturated and trans FAs, as well as the pro-inflammatory arachidonic and linoleic acids. Moreover, SHBG showed promising value for the regulation of adipocyte lipolysis and engorgement by lowering the levels of perilipin-1. SHBG exerted moderated effect toward SCD1 and FASN enzymes expression, but increased the LPL abundance. Interestingly, SHBG exhibited a negative regulatory effect on pro-adipogenic stimulators and induced higher expression of KLF3, IRF3 and ß-catenin, known as strong adipogenesis repressors. Finally, SHBG protein showed remarkable ability in restoring the insulin signal transduction, IR/IRS/Pi3K/AKT phosphorylation events and GLUT4 transporter abundance, and further attenuate pro-inflammatory response by lowering IL-6 tissue levels and targeting the PDIA3/ERK axis. Overall, the obtained data clearly demonstrate the benefice of SHBG treatment in the regulation of adipose tissue metabolism in the course of EMS and provide new insights for the development of molecular therapies with potential translational application to human metabolic disorders.
RESUMO
Background: Hyperactivation of protein tyrosine phosphatase (PTP1B) has been associated with several metabolic malfunctions ranging from insulin resistance, metaflammation, lipotoxicity, and hyperglycaemia. Liver metabolism failure has been proposed as a core element in underlying endocrine disorders through persistent inflammation and highly fibrotic phenotype. Methods: In this study, the outcomes of PTP1B inhibition using trodusquemine (MSI-1436) on key equine metabolic syndrome (EMS)-related alterations including inflammation, fibrosis, and glucose uptake have been analyzed in liver explants collected from EMS-affected horses using various analytical techniques, namely, flow cytometry, RT-qPCR, and Western blot. Results: PTP1B inhibition using trodusquemine resulted in decreased proinflammatory cytokines (IL-1ß, TNF-α, and IL-6) release from liver and PBMC affected by EMS and regulated expression of major proinflammatory microRNAs such as miR-802 and miR-211. Moreover, MSI-1436 enhanced the anti-inflammatory profile of livers by elevating the expression of IL-10 and IL-4 and activating CD4+CD25+Foxp3+ regulatory T cells in treated PBMC. Similarly, the inhibitor attenuated fibrogenic pathways in the liver by downregulating TGF-ß/NOX1/4 axis and associated MMP-2/9 overactivation. Interestingly, PTP1B inhibition ameliorated the expression of TIMP-1 and Smad7, both important antifibrotic mediators. Furthermore, application of MSI-1436 was found to augment the abundance of glycosylated Glut-2, which subsequently expanded the glucose absorption in the EMS liver, probably due to an enhanced Glut-2 stability and half-life onto the plasma cell membranes. Conclusion: Taken together, the presented data suggest that the PTP1B inhibition strategy and the use of its specific inhibitor MSI-1436 represents a promising option for the improvement of liver tissue integrity and homeostasis in the course of EMS and adds more insights for ongoing clinical trials for human MetS management.
RESUMO
BACKGROUND: Sex hormone binding globulin (SHBG) deteriorated expression has been recently strongly correlated to increased level of circulating pro-inflammatory cytokines and insulin resistance, which are typical manifestations of equine metabolic syndrome (EMS). Despite previous reports demonstrated the potential therapeutic application of SHBG for liver-related dysfunctions, whether SHBG might modulate equine adipose-derived stem/stromal cells (EqASCs) metabolic machinery remains unknown. Therefore, we evaluated for the first time the impact of SHBG protein on metabolic changes in ASCs isolated from healthy horses. METHODS: Beforehand, SHBG protein expression has been experimentally lowered using a predesigned siRNA in EqASCs to verify its metabolic implications and potential therapeutic value. Then, apoptosis profile, oxidative stress, mitochondrial network dynamics and basal adipogenic potential have been evaluated using various molecular and analytical techniques. RESULTS: The SHBG knockdown altered the proliferative and metabolic activity of EqASCs, while dampening basal apoptosis via Bax transcript suppression. Furthermore, the cells treated with siRNA were characterized by senescent phenotype, accumulation of reactive oxygen species (ROS), nitric oxide, as well as decreased mitochondrial potential that was shown by mitochondrial membrane depolarization and lower expression of key mitophagy factors: PINK, PARKIN and MFN. The addition of SHBG protein reversed the impaired and senescent phenotype of EMS-like cells that was proven by enhanced proliferative activity, reduced apoptosis resistance, lower ROS accumulation and greater mitochondrial dynamics, which is proposed to be related to a normalization of Bax expression. Crucially, SHBG silencing enhanced the expression of key pro-adipogenic effectors, while decreased the abundance of anti-adipogenic factors namely HIF1-α and FABP4. The addition of exogenous SHBG further depleted the expression of PPARγ and C/EBPα and restored the levels of FABP4 and HIF1-α evoking a strong inhibitory potential toward ASCs adipogenesis. CONCLUSION: Herein, we provide for the first time the evidence that SHBG protein in importantly involved in various key metabolic pathways governing EqASCs functions, and more importantly we showed that SHBG negatively affect the basal adipogenic potential of tested ASCs through a FABP4-dependant pathway, and provide thus new insights for the development of potential anti-obesity therapeutic approach in both animals and humans.
