Conditioned medium of adipose derived Mesenchymal Stem Cells reverse insulin resistance through downregulation of stress induced serine kinases

Abstract:

Insulin resistance (IR) is a constituent part of Type 2 Diabetes Mellitus (T2DM). Conditioned medium from Adipose derived Mesenchymal Stem Cells (ADMSCs–CM) has been shown to reverse IR. However, its effect on cellular stress is not well established. The objective of this study was to explore the effect of ADMSCs-CM on reactive oxygen species, mitochondrial membrane potential (ΔΨm), endoplasmic reticulum (ER) stress and expression of oxidative and inflammatory stress induced serine kinases (SISK) which are pathophysiologically linked to IR. In insulin resistant, 3T3-L1 adipocytes and C2C12 myoblast cell culture models, glucose uptake was assayed by 2-NBDG uptake. Immunomodulatory cytokines, intracellular reactive oxygen species generation, ΔΨm and protein expression of JNK1, IKKβ and phospho-IRS1 (307) were analyzed using FACS. mRNA expression of ER stress markers (CHOP1 and IRE1) and SISK (JNK1, IKKβ, ERK1 and S6K1) were analyzed using RT-PCR. ADMSCs-CM effectively improve glucose uptake as evidenced by 2-NBDG uptake assay. FACS analysis showed that ADMSCs-CM possessed significantly higher levels of IL-6 and IL-10. ADMSCs-CM decreased intracellular generation of reactive oxygen species where it restored ΔΨm in C2C12 cells. ADMSCs-CM mediated reduction in ER stress was confirmed by down-regulation in CHOP1 and IRE1 mRNA expression. ADMSCs-CM treatment showed significant down-regulation of SISK mRNA expression including IKKβ, JNK, ERK and S6K1. Our results unequivocally demonstrate for the first time the mechanism of action of ADMSCs-CM in amelioration IR by reducing oxidative and inflammatory cellular stress. This study identifies SISK as potential therapeutic targets for T2DM therapy.

Introduction

The global epidemic of Type 2 Diabetes Mellitus (T2DM) has progressed exponentially, unhindered due to lack of effective therapies(World Health Organization, 2016). Kidney failure, neuropathy, retinopathy and vascular complications that lead to ischemic heart disease are among the many co-morbidities associated with T2DM(Jeanie et al., n.d). Impaired insulin signaling and dysfunctional β cells are hallmarks of T2DM which accounts for 90–95% of all Diabetes Mellitus cases(Raz et al., 2013). Pharmacological intervention become necessary as life style modifications proved to be difficult in the management of T2DM in the general population(Deborah M Muoio and Newgard, 2008a, Muoio and Newgard, 2008b). A combination of improvement in insulin sensitivity with simultaneous enhancement in β-cell function is believed to be ideal strategy to tackle T2DM pathogenesis. Currently available therapeutics temporarily ameliorate IR, however in a long run they can neither improve insulin sensitivity nor β-cell dysfunction warranting new therapeutic approach(Inzucchi, 2002).

Mesenchymal Stem Cells (MSCs) based therapies have emerged as potential candidate for the treatment of metabolic and immune disorders(Zang et al., 2017). MSCs are considered advantageous as they are easy to isolate and culture, lack immunogenicity and ethical issues, possess multi-lineage differentiation potential(Lalu et al., 2012). Also, MSCs possess unique secretome containing potent immunomodulatory and anti-inflammatory bioactive proteins and metabolites(Ranganath et al., 2016).

In the context of T2DM, the ability of MSCs to differentiate into Insulin Producing Cells (IPCs) was believed to be primary mechanisms to ameliorate hyperglycemia in T2DM. However, recent evidences suggest that paracrine effect of MSCs can improve insulin sensitivity by activating the insulin receptor substrate 1 (IRS1) signaling pathwaySi et al., 2012. Our group has demonstrated that conditioned medium from adipose derived MSCs (ADMSCs-CM) ameliorate IR through improved insulin signaling, Akt phosphorylation and GLUT4 expression in adipose and skeletal muscle cell models(Shree and Bhonde, 2017). Umbilical cord MSCs (UC-MSCs) have been reported to reprogram classically activated macrophage (M1, pro-inflammatory) into alternately activated phenotype (M2, anti-inflammatory) alleviating IR in T2DM rats(Xie et al., 2016). UCMSCs are also reported to alleviate IR through regulation of NLRP3 inflammasome in T2DM rats(Sun et al., 2017). However, knowledge on cellular and molecular mechanisms through which MSCs improves insulin sensitivity remains obscure.

IR arises from multiple etiological factors, of them excessive nutrient overload, accumulation of fatty acids derivatives, pro-inflammatory milieu, endoplasmic reticulum (ER) stress, mitochondrial dysfunction and oxidative stress are major drivers of insulin resistance(Hotamisligil, 2006). At molecular level, insulin signaling pathway negatively regulated by serine/threonine kinases activated by inflammatory cytokines, fatty acids derivatives, such as ceramides and diacylglycerol, reactive oxygen species(Czech, 2017; Deborah M Muoio and Newgard, 2008a; Tangvarasittichai, 2015). Kinases, including inhibitory-kB kinase β (IKKβ), c-Jun N-terminal kinases (JNK), extracellular signal regulated kinases (ERK) and p70S6 kinase (S6K1) when activated by metabolic and inflammatory stresses promote inhibitory serine phosphorylation on Insulin Receptor Substrate 1 (IRS1) promoting IR(Tanti and Jager, 2009a). High fat diet induced pre-diabetes exhibit decreased ΔΨm and ATP production in skeletal muscle suggest key role of mitochondrial dysfunction in IR(Xu et al., 2018).

In this study, we have examined effect of ADMSCs-CM on reactive oxygen species generation, mitochondrial membrane potential, ER stress markers, expression of stress induced serine kinases and its downstream effect on levels of inhibitory phosphorylation at ser-307 at IRS1 in differentiated and insulin resistant 3T3-L1 adipocytes and C2C12 myoblast cell culture models.