Sex hormone-binding globulin (SHBG) mitigates ER stress and improves viability and insulin sensitivity in adipose-derived mesenchymal stem cells (ASC) of equine metabolic syndrome (EMS)-affected horses.

BACKGROUND: Equine metabolic syndrome (EMS), which encompasses insulin resistance, low-grade inflammation and predisposition to laminitis is a critical endocrine disorder among the most prevalent conditions affecting horses from different breeds. According to the most recent research, low human sex hormone-binding globulin (SHBG) serum levels correlate with an increased risk of obesity, insulin resistance and diabetes, and may contribute to overall metabolic dysregulations. This study aimed to test whether exogenous SHBG could protect EMS affected adipose-derived stromal stem cells (EqASCEMS) from apoptosis, oxidative stress, ER stress and thus improve insulin sensitivity. METHODS: EqASCEMS wells were treated with two different concentrations (50 and 100 nM) of exogenous SHBG, whose biocompatibility was tested after 24, 48 and 72 h of incubation. Several parameters including cell viability, apoptosis, cell cycle, reactive oxygen species levels, ER stress, Pi3K/MAPK activation and insulin transducers expression were analysed. RESULTS: Obtained data demonstrated that exogenous SHBG treatment significantly promoted ASCs cells proliferation, cell cycle and survival with reduced expression of p53 and p21 pro-apoptotic mediators. Furthermore, SHBG alleviated the oxidative stress caused by EMS and reduced the overaccumulation of intracellular ROS, by reducing ROS + cell percentage and regulating gene expression of endogenous antioxidant enzymes (Sod 1, Cat, GPx), SHBG treatment exhibited antioxidant activity by modulating total nitric oxide (NO) levels in EMS cells as well. SHBG treatment dampened the activation of ER stress sensors and effectors in EqASCEMS cells via the upregulation of MiR-7a-5p, the decrease in the expression levels of ATF-6, CHOP and eiF2A and the restoration of PDIA3 chaperone protein levels. As a consequence, SHBG application substantially improved insulin sensitivity through the modulation of Pi3K/Akt/Glut4 insulin signalling cascades. CONCLUSION: Our results suggest that the SHBG is endowed with crucial beneficial effects on ASCs metabolic activities and could serve as a valuable therapeutic target for the development of efficient EMS treatment protocols. Video Abstract.

Equine Hoof Progenitor Cells Display Increased Mitochondrial Metabolism and Adaptive Potential to a Highly Pro-Inflammatory Microenvironment.

Medicinal signaling cells (MSC) exhibit distinct molecular signatures and biological abilities, depending on the type of tissue they originate from. Recently, we isolated and described a new population of stem cells residing in the coronary corium, equine hoof progenitor cells (HPCs), which could be a new promising cell pool for the treatment of laminitis. Therefore, this study aimed to compare native populations of HPCs to well-established adipose-derived stem cells (ASCs) in standard culture conditions and in a pro-inflammatory milieu to mimic a laminitis condition. ASCs and HPCs were either cultured in standard conditions or subjected to priming with a cytokines cocktail mixture. The cells were harvested and analyzed for expression of key markers for phenotype, mitochondrial metabolism, oxidative stress, apoptosis, and immunomodulation using RT-qPCR. The morphology and migration were assessed based on fluorescent staining. Microcapillary cytometry analyses were performed to assess the distribution in the cell cycle, mitochondrial membrane potential, and oxidative stress. Native HPCs exhibited a similar morphology to ASCs, but a different phenotype. The HPCs possessed lower migration capacity and distinct distribution across cell cycle phases. Native HPCs were characterized by different mitochondrial dynamics and oxidative stress levels. Under standard culture conditions, HPCs displayed different expression patterns of apoptotic and immunomodulatory markers than ASCs, as well as distinct miRNA expression. Interestingly, after priming with the cytokines cocktail mixture, HPCs exhibited different mitochondrial dynamics than ASCs; however, the apoptosis and immunomodulatory marker expression was similar in both populations. Native ASCs and HPCs exhibited different baseline expressions of markers involved in mitochondrial dynamics, the oxidative stress response, apoptosis and inflammation. When exposed to a pro-inflammatory microenvironment, ASCs and HPCs differed in the expression of mitochondrial condition markers and chosen miRNAs.

Inhibition of protein tyrosine phosphatase improves mitochondrial bioenergetics and dynamics, reduces oxidative stress, and enhances adipogenic differentiation potential in metabolically impaired progenitor stem cells.

BACKGROUND: Protein tyrosine phosphatase 1B (PTP1B) and low molecular weight protein tyrosine phosphatase (LMPTP) are implicated in the development of metabolic disorders. Yet, their role in progenitor stem cell adipogenic differentiation and modulation of mitochondrial dynamics remains elusive. METHODS: In this study, we decided to investigate whether inhibition of PTP1B and LMPTP enhance adipogenic differentiation of metabolically impaired progenitor stem cells via modulation of mitochondrial bioenergetics and dynamics. Cells were cultured under adipogenic conditions in the presence of PTP1B and LMPTP inhibitors, and were subjected to the analysis of the main adipogenic-related and mitochondrial-related genes using RT-qPCR. Protein levels were established with western blot while mitochondrial morphology with MicroP software. RESULTS: Selective inhibitors of both PTP1B and MPTP enhanced adipogenic differentiation of metabolically impaired progenitor stem cells. We have observed enhanced expression of PPARy and adiponectin in treated cells. What is more, increased antioxidative defence and alternations in mitochondrial bioenergetics were observed. We have found that inhibition of PTP1B as well as C23 activates oxidative phosphorylation and enhances mitochondrial fusion contributing to enhanced adipogenesis. CONCLUSIONS: The presented data provides evidence that the application of PTP1B and LMPTP inhibitors enhances adipogenesis through the modulation of mitochondrial dynamics. Video abstract.

Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway.

BACKGROUND: Chronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard. METHODS: In this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques. RESULTS: Treatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment. CONCLUSION: Overall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways. Video Abstract.

Cladophora glomerata enriched by biosorption with Mn(II) ions alleviates lipopolysaccharide-induced osteomyelitis-like model in MC3T3-E1, and 4B12 osteoclastogenesis.

Chronic osteomyelitis, a bone infectious disease, is characterized by dysregulation of bone homeostasis, which results in excessive bone resorption. Lipopolysaccharide (LPS) which is a gram-negative endotoxin was shown to inhibit osteoblast differentiation and to induce apoptosis and osteoclasts formation in vitro. While effective therapy against bacteria-induced bone destruction is quite limited, the investigation of potential drugs that restore down-regulated osteoblast function remains a major goal in the prevention of bone destruction in infective bone diseases. This investigation aimed to rescue LPS-induced MC3T3-E1 pre-osteoblastic cell line using the methanolic extract of Cladophora glomerata enriched with Mn(II) ions by biosorption. LPS-induced MC3T3-E1 cultures supplemented with C. glomerata methanolic extract were tested for expression of the main genes and microRNAs involved in the osteogenesis pathway using RT-PCR. Moreover, osteoclastogenesis of 4B12 cells was also investigated by tartrate-resistant acid phosphatase (TRAP) assay. Treatment with algal extract significantly restored LPS-suppressed bone mineralization and the mRNA expression levels of osteoblast-specific genes such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OCN), osteopontin (OPN), miR-27a and miR-29b. The extract also inhibited osteoblast apoptosis, significantly restored the down-regulated expression of Bcl-2, and decreased the loss of MMP and reactive oxygen spices (ROS) production in MC3T3-E1 cells induced by LPS. Furthermore, pre-treatment with algal extract strongly decreased the activation of osteoclast in MC3T3-E1-4B12 coculture system stimulated by LPS. Our findings suggest that C. glomerata enriched with Mn(II) ions may be a potential raw material for the development of drug for preventing abnormal bone loss induced by LPS in bacteria-induced bone osteomyelitis.

Artificial mitochondrial transplantation (AMT) reverses aging of mesenchymal stromal cells and improves their immunomodulatory properties in LPS-induced synoviocytes inflammation.

Nowadays, regenerative medicine techniques are usually based on the application of mesenchymal stromal cells (MSCs) for the repair or restoration of injured damaged tissues. However, the effectiveness of autologous therapy is limited as therapeutic potential of MSCs declines due to patient's age, health condition and prolonged in vitro cultivation as a result of decreased growth rate. For that reason, there is an urgent need to develop strategies enabling the in vitro rejuvenation of MSCs prior transplantation in order to enhance their in vivo therapeutic efficiency. In presented study, we attempted to mimic the naturally occurring mitochondrial transfer (MT) between neighbouring cells and verify whether artificial MT (AMT) could reverse MSCs aging and improve their biological properties. For that reason, mitochondria were isolated from healthy donor equine adipose-derived stromal cells (ASCs) and transferred into metabolically impaired recipient ASCs derived from equine metabolic syndrome (EMS) affected horses, which were subsequently subjected to various analytical methods in order to verify the cellular and molecular outcomes of the applied AMT. Mitochondria recipient cells were characterized by decreased apoptosis, senescence and endoplasmic reticulum stress while insulin sensitivity was enhanced. Furthermore, we observed increased mitochondrial fragmentation and associated PARKIN protein accumulation, which indicates on the elimination of dysfunctional organelles via mitophagy. AMT further promoted physioxia and regulated autophagy fluxes. Additionally, rejuvenated ASCs displayed an improved anti-inflammatory activity toward LPS-stimulated synoviocytes. The presented findings highlight AMT as a promising alternative and effective method for MSCs rejuvenation, for potential application in autologous therapies in which MSCs properties are being strongly deteriorated due to patients' condition.

Distinctive characteristics of extracellular vesicles from feline adipose and placenta stromal cells unveil potential for regenerative medicine in cats.

OBJECTIVE: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are promising avenues in regenerative medicine, offering unique immunomodulatory and regenerative properties with lower immunogenicity. This study delves into the distinctive features of EVs extracted from feline adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs). The tissues were collected from 11 female cats aged between 4 and 7 years old. SAMPLE: EVs extracted from MSCs from discarded fetal membranes from 7 female cats and SC adipose tissue from 11 cats. METHODS: We comprehensively explored morphological characteristics, mitochondrial density, surface markers, and pro- and anti-inflammatory mediators, uncovering notable differences between ASCs and PMSCs. RESULTS: Morphologically, ASCs exhibit a spindle-shaped form in contrast to the spherical morphology of PMSCs. Proliferation and clonogenic potential assessments reveal the faster proliferation and robust clonogenic nature of ASCs, suggesting their potential vital role in regenerative processes. Surface marker expression analysis indicates a significantly higher expression of multipotency-associated markers in ASCs, suggesting their superior proregenerative potential. Phenotyping of EVs demonstrates distinctive features, with CD9 expression suggesting varied EV secretion patterns. Notably, PMSCs exhibit superior CD81 expression, indicating their potential as preferred donors of mitochondria. Pro- and anti-inflammatory mediators analyzed at mRNA and microRNA levels reveal higher RNA content in EVs compared to source cells, emphasizing the potential of EVs in directing regenerative processes. Differential microRNA expression in EVs derived from ASCs hints at their regulatory roles in anti-inflammatory and immunometabolic processes. CLINICAL RELEVANCE: This study lays a foundation for understanding the nuances between ASCs and PMSCs, which is crucial for harnessing the full therapeutic potential of MSCs and their EVs in tissue repair and regeneration.

Sex hormone-binding globulin promotes the osteogenic differentiation potential of equine adipose-derived stromal cells by activating the BMP signaling pathway.

Background: Musculoskeletal injuries and chronic degenerative diseases pose significant challenges in equine health, impacting performance and overall well-being. Sex Hormone-Binding Globulin (SHBG) is a glycoprotein determining the bioavailability of sex hormones in the bloodstream, and exerting critical metabolic functions, thus impacting the homeostasis of many tissues including the bone. Methods: In this study, we investigated the potential role of SHBG in promoting osteogenesis and its underlying mechanisms in a model of equine adipose-derived stromal cells (ASCs). An SHBG-knocked down model has been established using predesigned siRNA, and cells subjected to osteogenic induction medium in the presence of exogenous SHBG protein. Changes in differentiation events where then screened using various analytical methods. Results: We demonstrated that SHBG treatment enhances the expression of key osteoconductive regulators in equine ASCs CD34+ cells, suggesting its therapeutic potential for bone regeneration. Specifically, SHBG increased the cellular expression of BMP2/4, osteocalcin (OCL), alkaline phosphatase (ALP), and osteopontin (OPN), crucial factors in early osteogenesis. Furthermore, SHBG treatment maintained adequate apoptosis and enhanced autophagy during osteogenic differentiation, contributing to bone formation and remodeling. SHBG further targeted mitochondrial dynamics, and promoted the reorganization of the mitochondrial network, as well as the expression of dynamics mediators including PINK, PARKIN and MFN1, suggesting its role in adapting cells to the osteogenic milieu, with implications for osteoblast maturation and differentiation. Conclusion: Overall, our findings provide novel insights into SHBG's role in bone formation and suggest its potential therapeutic utility for bone regeneration in equine medicine.

Sex hormone binding globulin (SHBG) modulates mitochondrial dynamics in PPARγ-depleted equine adipose derived stromal cells.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that promotes adipogenesis, lipid uptake and storage, insulin sensitivity, and glucose metabolism. Hence, defects in PPARγ have been associated to the development of metabolic disorders. Sex hormone-binding globulin (SHBG) is a glycoprotein primarily produced in the liver that regulates the bioavailability of sex hormones. Alike PPARγ, low SHBG levels have been correlated with insulin resistance and associated endocrine abnormalities. Therefore, this study aimed to verify whether SHBG may restore depleted PPARγ functions and thus serve as a new candidate for the management of metabolic conditions. A model of equine adipose-derived stromal cells (EqASCs) has been used, in which a PPARγ silencing and SHBG treatment have been achieved to determine the changes in cell viability, premature senescence, oxidative stress, and mitochondrial functions. Obtained data demonstrated that loss in PPARγ triggers cell apoptosis which is not reversed by SHBG application. Moreover, PPARγ knockdown cells exhibited premature senescence, which has been substantially alleviated by SHBG concomitantly to increased BAX/BCL2 ratio, suggesting a possible effect on senescence-induced apoptosis resistance. Interestingly, PPARγ silencing induced a significant alteration in mitochondrial membrane potential as well as the expression of dynamics and metabolism-related markers. SHBG treatment enabled to ameliorate the transmembrane potential, to normalize the expression levels of key dynamics and metabolism mediators, and to restore the protein levels of PINK, which is critically involved in mitochondria recycling machinery. Presented data suggest that SHBG may provide new mechanistic insights into the regulation of PPARγ functions, and thus offers a preliminary picture on a possible SHBG-PPARγ metabolic crosstalk. KEY MESSAGES : PPARγ is a transcription factor that tightly regulates cell metabolism. Low SHBG levels correlate with insulin resistance and associated endocrine abnormalities. PPARγ silencing reduces cell viability, triggers premature senescence and profound mitochondrial failure in equine ASCs. SHBG protein reverses senescent phenotype and apoptosis resistance of PPARγ- ASCs. SHBG improves mitochondrial dynamics and metabolism following PPARγ knockdown. SHBG might serve as a PPARγ potential mimicking agent for the modulation of ASCs metabolic processes.

The PTP1B Inhibitor Trodusquemine (MSI-1436) Improves Glucose Uptake in Equine Metabolic Syndrome Affected Liver through Anti-Inflammatory and Antifibrotic Activity.

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.

The PTP1B selective inhibitor MSI-1436 mitigates Tunicamycin-induced ER stress in human hepatocarcinoma cell line through XBP1 splicing modulation.

Protein tyrosine phosphatase PTP1B is considered as a key metabolic enzyme that has been reported to be associated with insulin resistance onset, and underlying cellular metabolic malfunctions, including ER stress and mitochondrial failure. In this study, effects of selective PTP1B inhibition using MSI-1436 on cellular apoptosis, oxidative stress, mitochondrial dysfunction and ER stress have been assessed using an in vitro model of Tunicamycin induced ER stress in HepG2 cell line. Inhibition of PTP1B using MSI-1436 significantly increased cell viability and reduced the number of apoptotic cells as well as the expression of key apoptosis initiators and effectors. MSI-1436 further mitigated ER stress, by downregulating the expression of IRE1, ATF6 and PERK transcripts, all being key ER stress sensors. Interestingly, MSI-1436 inhibited the XBP1 splicing, and thus its UPR-associated transcriptional activity. PTP1B inhibition further enabled to restore proper mitochondrial biogenesis, by improving transmembrane potential, and diminishing intracellular ROS while restoring of endogenous antioxidant enzymes genes expression. PTP1B inhibition using MSI-1436 could improve cellular apoptosis and metabolic integrity through the mitigation of ER and mitochondrial stress signalling pathways, and excessive ROS accumulation. This strategy may be useful for the treatment of metabolic disorders including IR, NAFLD and diabetes.

Sex Hormone-Binding Globulin (SHBG) Maintains Proper Equine Adipose-Derived Stromal Cells (ASCs)' Metabolic Functions and Negatively Regulates their Basal Adipogenic Potential.

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.

In Vitro Generated Equine Hepatic-Like Progenitor Cells as a Novel Potent Cell Pool for Equine Metabolic Syndrome (EMS) Treatment.

Equine metabolic syndrome (EMS) is recognized as one of the leading cause of health threatening in veterinary medicine worldwide. Recently, PTP1B inhibition has been proposed as an interesting strategy for liver insulin resistance reversion in both equines and humans, however as being a multifactorial disease, proper management of EMS horses further necessities additional interventional approaches aiming at repairing and restoring liver functions. In this study, we hypothesized that in vitro induction of Eq_ASCs hepatogenic differentiation will generate a specialized liver progenitor-like cell population exhibiting similar phenotypic characteristics and regenerative potential as native hepatic progenitor cells. Our obtained data demonstrated that Eq_ASCs-derived liver progenitor cells (Eq_HPCs) displayed typical flattened polygonal morphology with packed fragmented mitochondrial net, lowered mesenchymal CD105 and CD90 surface markers expression, and significant high expression levels of specific hepatic lineage genes including PECAM-1, ALB, AFP and HNF4A. therewith, generated Eq_HPCs exhibited potentiated stemness and pluripotency markers expression (NANOG, SOX-2 and OCT-4). Hence, in vitro generation of hepatic progenitor-like cells retaining high differentiation capacity represents a promising new approach for the establishment of cell-based targeted therapies for the restoration of proper liver functions in EMS affected horses.

The PTP1B inhibitor MSI-1436 ameliorates liver insulin sensitivity by modulating autophagy, ER stress and systemic inflammation in Equine metabolic syndrome affected horses.

Background: Equine metabolic syndrome (EMS) is a multifactorial pathology gathering insulin resistance, low-grade inflammation and past or chronic laminitis. Among the several molecular mechanisms underlying EMS pathogenesis, increased negative insulin signalling regulation mediated by protein tyrosine phosphatase 1 B (PTP1B) has emerged as a critical axis in the development of liver insulin resistance and general metabolic distress associated to increased ER stress, inflammation and disrupted autophagy. Thus, the use of PTP1B selective inhibitors such as MSI-1436 might be considered as a golden therapeutic tool for the proper management of EMS and associated conditions. Therefore, the present investigation aimed at verifying the clinical efficacy of MSI-1436 systemic administration on liver metabolic balance, insulin sensitivity and inflammatory status in EMS affected horses. Moreover, the impact of MSI-1436 treatment on liver autophagy machinery and associated ER stress in liver tissue has been analysed. Methods: Liver explants isolated from healthy and EMS horses have been treated with MSI-1436 prior to gene and protein expression analysis of main markers mediating ER stress, mitophagy and autophagy. Furthermore, EMS horses have been intravenously treated with a single dose of MSI-1436, and evaluated for their metabolic and inflammatory status. Results: Clinical application of MSI-1436 to EMS horses restored proper adiponectin levels and attenuated the typical hyperinsulinemia and hyperglycemia. Moreover, administration of MSI-1436 further reduced the circulating levels of key pro-inflammatory mediators including IL-1ß, TNF-α and TGF-ß and triggered the Tregs cells activation. At the molecular level, PTP1B inhibition resulted in a noticeable mitigation of liver ER stress, improvement of mitochondrial dynamics and consequently, a regulation of autophagic response. Similarly, short-term ex vivo treatment of EMS liver explants with trodusquemine (MSI-1436) substantially enhanced autophagy by upregulating the levels of HSC70 and Beclin-1 at both mRNA and protein level. Moreover, the PTP1B inhibitor potentiated mitophagy and associated expression of MFN2 and PINK1. Interestingly, inhibition of PTP1B resulted in potent attenuation of ER stress key mediators' expression namely, CHOP, ATF6, HSPA5 and XBP1. Conclusion: Presented findings shed for the first time promising new insights in the development of an MSI-1436-based therapy for proper equine metabolic syndrome intervention and may additionally find potential translational application to human metabolic syndrome treatment.

Sex hormone-binding globulin improves lipid metabolism and reduces inflammation in subcutaneous adipose tissue of metabolic syndrome-affected horses.

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.

The Hepatic Stellate Cells (HSTCs) and Adipose-derived Mesenchymal Stem Cells (ASCs) Axis as a Potential Major Driver of Metabolic Syndrome - Novel Concept and Therapeutic Implications.

Herein, we would like to introduce a novel concept for the prevention and treatment of metabolic syndrome, which is based on molecular relationship between liver and adipose tissue. Particularly, we believe, that unravelling the molecular crosstalk between hepatokines and adipokines will allow to better understand the pathophysiology of metabolic diseases and allow to develop novel, effective therapeutic solutions against obesity and metabolic syndrome. Inter-organ communication on the level of stem progenitor cells-hepatic stellate cells (HSTCs) and adipose-derived progenitors (ASCs) could represents a key mechanism involved in controlling glucose tolerance as well as insulin sensitivity.

Astaxanthin Carotenoid Modulates Oxidative Stress in Adipose-Derived Stromal Cells Isolated from Equine Metabolic Syndrome Affected Horses by Targeting Mitochondrial Biogenesis.

Astaxanthin is gaining recognition as a natural bioactive component. This study aimed to test whether astaxanthin could protect adipose-derived stromal stem cells (ASCs) from apoptosis, mitochondrial dysfunction and oxidative stress. Phaffia rhodozyma was used to extract astaxanthin, whose biocompatibility was tested after 24, 48 and 72 h of incubation with the cells; no harmful impact was found. ASCs were treated with optimal concentrations of astaxanthin. Several parameters were examined: cell viability, apoptosis, reactive oxygen levels, mitochondrial dynamics and metabolism, superoxide dismutase activity, and astaxanthin's antioxidant capacity. A RT PCR analysis was performed after each test. The astaxanthin treatment significantly reduced apoptosis by modifying the normalized caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Furthermore, by regulating the expression of related master factors SOD1, SOD2, PARKIN, PINK 1, and MFN 1, astaxanthin alleviated the oxidative stress and mitochondrial dynamics failure caused by EMS. Astaxanthin restored mitochondrial oxidative phosphorylation by stimulating markers associated with the OXPHOS machinery: COX4I1, COX4I2, UQCRC2, NDUFA9, and TFAM. Our results suggest that astaxanthin has the potential to open new possibilities for potential bio-drugs to control and suppress oxidative stress, thereby improving the overall metabolic status of equine ASCs suffering from metabolic syndrome.

Postbiotics as potential new therapeutic agents for metabolic disorders management.

The prevalence of obesity, diabetes, non-alcoholic fatty liver disease, and related metabolic disorders has been steadily increasing in the past few decades. Apart from the establishment of caloric restrictions in combination with improved physical activity, there are no effective pharmacological treatments for most metabolic disorders. Many scientific-studies have described various beneficial effects of probiotics in regulating metabolism but others questioned their effectiveness and safety. Postbiotics are defined as preparation of inanimate microorganisms, and/or their components, which determine their safety of use and confers a health benefit to the host. Additionally, unlike probiotics postbiotics do not require stringent production/storage conditions. Recently, many lines of evidence demonstrated that postbiotics may be beneficial in metabolic disorders management via several potential effects including anti-inflammatory, antibacterial, immunomodulatory, anti-carcinogenic, antioxidant, antihypertensive, anti-proliferative, and hypocholesterolaemia properties that enhance both the immune system and intestinal barrier functions by acting directly on specific tissues of the intestinal epithelium, but also on various organs or tissues. In view of the many reports that demonstrated the high biological activity and safety of postbiotics, we summarized in the present review the current findings reporting the beneficial effects of various probiotics derivatives for the management of metabolic disorders and related alterations.