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.

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.

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.

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.

Sex hormone binding globulin as a potential drug candidate for liver-related metabolic disorders treatment.

Sex hormone binding globulin (SHBG) is a hepatokine that binds to circulating steroid hormones (testosterone, oestradiol) to regulate their concentration in the bloodstream. Recently SHBG was recognized as an essential biomarker for metabolic syndrome (MetS) and hepatic steatosis development. At the hepatic level, the production of SHBG is mainly regulated by sex steroids and thyroxine. Studies of various research groups, including ours, showed that SHBG could be considered a reliable marker of insulin resistance and, therefore, can serve as a predictor of type 2 diabetes. Moreover, increased levels of circulating pro-inflammatory mediators strongly correlate with lowered serum levels of SHBG. This review paper emphasizes the role of SHBG as a potential drug candidate in the course of various metabolic dysfunctions, including non-alcoholic fatty liver disease (NAFLD), obesity, diabetes mellitus and insulin resistance. The studies related to SHBG and its role in the course of metabolic disorders are very limited. Here, we have summarized the most current knowledge about SHBG and its mechanism of action, indicating a novel concept for its possible therapeutic application in the management framework of commonly occurring metabolic dysfunctions.

Calystegines Improve the Metabolic Activity of Human Adipose Derived Stromal Stem Cells (ASCs) under Hyperglycaemic Condition through the Reduction of Oxidative/ER Stress, Inflammation, and the Promotion of the AKT/PI3K/mTOR Pathway.

Hyperglycaemia and its resulting glucotoxicity are among the most prominent hallmarks of diabetes mellitus (DM) development. Persistent hyperglycaemia further leads to oxidative stress via mitochondrial dysfunction and subsequent ER stress onset, while associated hyperlipidaemia triggers the adipose tissue to secrete pro-inflammatory cytokines. In this study, the effect of calystegines has been investigated in an experimental model of hyperglycaemia induced on human ASCs cells. Different cellular pathways including apoptosis, oxidative and ER stress, inflammation as well as Pi3K/AKT/mTOR metabolic-associated axis have been evaluated by means on RT-qPCR, western blot, and flow cytometry techniques. Treatment of HuASCs cells with calystegines strongly promoted the hyperglycaemic cells survival and significantly diminished oxidative stress, mitochondrial dynamics failure and ER stress, while improving the endogenous cellular antioxidant defenses. Interestingly, nortropane alkaloids efficiently prevented the hyperglycaemia-mediated inflammatory response, as evidenced by the regulation of the pro- and anti-inflammatory response in HuASCs cells. Finally, we evidenced that calystegines may exert their protective effect on HuASCs cells metabolic functions through the restoration of the defective PI3K/AKT/mTOR pathway. Overall, the present investigation demonstrated that calystegines possess important abilities to protect HuASCs against hyperglycaemia-induced cellular dysfunction, and it evidenced that the observed effects are associated to the promotion of PI3K/AKT/mTOR pathway.

Hepatic stellate cells role in the course of metabolic disorders development - A molecular overview.

Fibrosis is characterized by an abnormal accumulation of extracellular matrix (ECM) constituents in the liver parenchyma that lead to hepatic cirrhosis. After liver injury, the hepatic stellate cells (HSCs) undergo a response called "activation", transforming the cells into proliferative, fibrogenic and contractile myofibroblasts, representing the main collagen-producing cells in the injured tissue. Activated HSCs are considered as pro-inflammatory cells producing cytokines and several hepatomatogens; they are additionally involved in the recruitment of Kupffer cells, circulating monocytes and macrophages through the production of chemokines. Moreover, HSC have been proposed as being involved in the development of insulin resistance mainly mediated by their inflammatory properties, which undeniably links their activation to the development of diabetes and Non-alcoholic fatty liver disease. In addition, when the liver is injured, a complex interaction between hepatocytes and HSCs occurs, inducing mitochondrial dysfunction, which contributes to the accumulation of fats in hepatocytes that trigger to liver lipotoxicity. These mechanisms underlying the activation of HSC suggest their major role in the development of metabolic disorders. It turns out that several molecules including MicroRNAs and proteins have the ability to inhibit the activation and the proliferation of HSCs, which makes them interesting therapeutic targets for the subsequent management of metabolic conditions. This review focuses on the mechanisms and molecular pathways underlying the initiation and onset of metabolic disorders following HSCs activation, as well as on molecular therapeutic targets, which could limit their fibrogenic transdifferentiation and therefore improve the liver condition in the course of metabolic imbalance.

Laurus nobilis ethanolic extract attenuates hyperglycemia and hyperinsulinemia-induced insulin resistance in HepG2 cell line through the reduction of oxidative stress and improvement of mitochondrial biogenesis - Possible implication in pharmacotherapy.

The aim of this study was to establish the potential effect of Laurus nobilis ethanolic extract on improving insulin sensitivity and protecting liver cells from apoptosis, mitochondrial dysfunction, oxidative stress (OS), and inflammation; all of which considered as major alterations occurring during insulin resistance (IR) as well as diabetes onset, in hyperinsulinemic and hyperglycemic-induced HepG2 cell line. Thereby, L. nobilis ethanolic extract has been first chemically characterized using LC-MS/MS technique. Subsequently, HepG2 cells were pre-treated with an optimal concentration of L. nobilis ethanolic extract for 24 h, and then, subjected to 30 mM D-glucose and 500 nM insulin mixture for another 24 h in order to induce hyperinsulinemia and hyperglycaemia (HI/HG) status. Several parameters such as biocompatibility, hepatotoxicity, reactive oxygen species (ROS), mitochondrial transmembrane potential, dynamics, and metabolism, multicaspase activity, glucose uptake, in addition to genes and proteins expression levels were investigated. The obtained results showed that the bioactive extract of Laurus nobilis increased the number of living cells and their proliferation rate, significantly attenuated apoptosis by modulating pro-apoptotic pathways (p21, p53 and Bax genes), allowed a relative normalization of caspases-activity, and decreased the expression of inflammatory markers including c-Jun, NF-κB and Tlr4 transcripts. L. Nobilis ethanolic extract reduced considerably total intracellular ROS levels in challenged HepG2 cells, and regulated the mitochondrial OXPHOS pathway, demonstrating the potential antioxidant effect of the plant. Ethanolic plant extract increased insulin sensitivity, since an elevated expression of master transcripts responsible for insulin sensitivity including IRS1, IRS2, INSR was found. Taken together, obtained data suggest that L. nobilis ethanolic extract offers new insights in the development of potential antioxidant, insulin sensitizing as well as hepatoprotective drugs.

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.

Inhibition of Protein-tyrosine Phosphatase PTP1B and LMPTP Promotes Palmitate/Oleate-challenged HepG2 Cell Survival by Reducing Lipoapoptosis, Improving Mitochondrial Dynamics and Mitigating Oxidative and Endoplasmic Reticulum Stress.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is considered a well-known pathology that is determined without using alcohol and has emerged as a growing public health problem. Lipotoxicity is known to promote hepatocyte death, which, in the context of NAFLD, is termed lipoapoptosis. The severity of NAFLD correlates with the degree of hepatocyte lipoapoptosis. Protein-tyrosine phosphatases (PTP) including PTP1B and Low molecular weight PTP (LMPTP), are negative regulators of the insulin signaling pathway and are considered a promising therapeutic target in the treatment of diabetes. In this study, we hypothesized that the inhibition of PTP1B and LMPTP may potentially prevent hepatocyte apoptosis, mitochondrial dysfunction and endoplasmic reticulum (ER) stress onset, following lipotoxicity induced using a free fatty acid (FFA) mixture. METHODS: HepG2 cells were cultured in the presence or absence of two PTP inhibitors, namely MSI-1436 and Compound 23, prior to palmitate/oleate overloading. Apoptosis, ER stress, oxidative stress, and mitochondrial dynamics were then evaluated by either MUSE or RT-qPCR analysis. RESULTS: The obtained data demonstrate that the inhibition of PTP1B and LMPTP prevents apoptosis induced by palmitate and oleate in the HepG2 cell line. Moreover, mitochondrial dynamics were positively improved following inhibition of the enzyme, with concomitant oxidative stress reduction and ER stress abrogation. CONCLUSION: In conclusion, PTP's inhibitory properties may be a promising therapeutic strategy for the treatment of FFA-induced lipotoxicity in the liver and ultimately in the management of the NAFLD condition.