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

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

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

Abstract 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 Abstrac

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

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