Full length articleConditioned medium of adipose derived Mesenchymal Stem Cells reverse insulin resistance through downregulation of stress induced serine kinases
Graphical abstract
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.
Section snippets
Adipose derived Mesenchymal Stem Cells culture
ADMSCs were obtained from Manipal School of Regenerative Medicine, Bengaluru (India). ADMSCs were cultured in α-MEM medium (Gibco) with 10% FBS and 1% Antibiotic-antimycotic solution (Gibco). Further, passage 4 cells were characterized for expression of CD90, CD73, CD105 (PE Tagged), CD34, CD45 and HLADR (FITC Tagged) (BD Biosciences) as previously described by us(Shree and Bhonde, 2017). Cells were further characterized for its chondrogenic (Stem-pro, A1007101), adipogenic (Stempro, A1007001)
Characterization of ADMSCs
Passage 4 cells subjected to CD antigen expression analysis using FACS were found to be positive for CD90, CD73, CD105 (>95%) and negative for CD34, CD45 and HLADR (MHC class II) (<2%). Further, cells were able to differentiate into adipogenic, osteogenic and chondrogenic lineage confirming its tri-lineage potential as depicted in Fig. 1.
Cytokines analysis of ADMSCs-CM
MSCs exert its immunomodulatory action through secretion of cytokines and bioactive proteins(Zang et al., 2017, Zhang et al., 2017). We performed cytokine
Discussion
IR is the major driver in the pathogenesis of T2DM which arises from metabolic overload, physical inactivity, hypoxia, psychological stress and environmental pollutants. These multi-etiological factors induce a network of cellular stress, stress responses and stress response dysregulations that inhibits downstream insulin signaling in insulin target cells(Onyango, 2018). Control of T2DM with conventional pharmaceutical agents is proving insufficient to tackle this multi-factorial etiology and
Conclusion
In conclusion, our data for the first time reveals that ADMSCs-CM rescues oxidative, inflammatory and ER stress and downregulates expression of stress induced serine kinases asserting its use as a prospective therapy for T2DM. The results obtained are attributed to the cumulative Secretome of ADMSCs-CM.
CRediT authorship contribution statement
Avinash Sanap: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft. Ramesh Bhonde: Conceptualization, Methodology, Writing - review & editing, Visualization, Resources. Kalpana Joshi: Formal analysis, Writing - review & editing, Supervision, Validation.
Declaration of competing interest
All authors declare no conflict of interest.
Acknowledgements
We would like to thank Council of Scientific and Industrial Research (CSIR), Government of India for providing research fellowship to the first author. We are thankful to Sinhgad College of Engineering, Pune (India) and Dr. D. Y. Patil Vidyapeeth, Pune (India) for the extended support provided to carry out the work. Thanks are also due to Dr. Nitya Shree and Mr. Avinash Kharat for their guidance and assistance in manuscript editing.
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