Mahesh Ramalingam

Reactive oxygen/nitrogen species and their functional correlations in neurodegenerative diseases

The continuous production and efflux of reactive oxygen/nitrogen species from endogenous and exogenous sources can damage biological molecules and initiate a cascade of events. Mitochondria are pivotal in controlling cell survival and death. Cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage are related with various neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and others. Biochemical cascades of apoptosis are mediated in signaling molecules, including protein kinases and transcription factors. The expressions in the pro-apoptotic signal transduction networks may indeed promote cell death and degeneration in brain cells. The regulation of that protein phosphorylation by kinases and phosphatases is emerging as a prerequisite mechanism in the control of the apoptotic cell death program. In this review, we attempt to put forth the evidence for possible mechanistic explanations for involvement of free radicals in the pathogenesis of neurodegenerative diseases.

Insulin exerts neuroprotective effects via Akt/Bcl-2 signaling pathways in differentiated SH-SY5Y cells

Abstract In the present study, the changes in the cell viability at different concentrations of hydrogen peroxide (H2O2) for 3 h used to establish a model of oxidative stress. Further assays with 200 μM H2O2 induces significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), reactive oxygen species (ROS) and calcium ion (Ca2+) in neuronal cells, but insulin can effectively diminish the oxidative damages. Moreover, cells treated with insulin increased the H2O2-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of Akt, Bcl-2, Bax, IRβ, IGF-1Rβ, IRS-1 and IRS-2 showed that insulin treatment had a protective effect on H2O2-induced oxidative stress in RA-differentiated SH-SY5Y neuroblastoma cells.

The Neuroprotective Role of Insulin Against MPP+‐Induced Parkinson's Disease in Differentiated SH‐SY5Y Cells

Parkinsons disease (PD) is a common chronic neurodegenerative disorder associated with aging that primarily caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SN). Retinoic acid (RA)‐differentiated human neuroblastoma SH‐SY5Y cells (SH‐SY5Y+RA) have been broadly utilized in studies of mechanisms of the pathogenesis underlying 1‐Methyl‐4‐phenyl pyridinium (MPP+)‐induced PD models. Here, we investigated the neuroprotective mechanisms of insulin on MPP+‐induced neurotoxicity on SH‐SY5Y+RA cells. Recent studies suggest that insulin has a protective effect against oxidative stress but not been elucidated for PD. In this study, pretreatment of insulin prevented the cell death in a dose dependent manner and lowered nitric oxide (NO) release, reactive oxygen species (ROS), and calcium ion (Ca2+) influx induced by MPP+. Insulin also elevated tyrosine hydroxylase (TH) and insulin signaling pathways in dopaminergic neuron through activating PI3K/Akt/GSK‐3 survival pathways which in turn inhibits MPP+‐induced iNOS and ERK activation, and Bax to Bcl‐2 ratio. These results suggest that insulin has a protective effect on MPP+‐neurotoxicity in SH‐SY5Y+RA cells. J. Cell. Biochem. 117: 917–926, 2016.

Mechanisms of action of brain insulin against neurodegenerative diseases

Insulin, a pancreatic hormone, is best known for its peripheral effects on the metabolism of glucose, fats and proteins. There is a growing body of evidence linking insulin action in the brain to neurodegenerative diseases. Insulin present in central nervous system is a regulator of central glucose metabolism nevertheless this glucoregulation is not the main function of insulin in the brain. Brain is known to be specifically vulnerable to oxidative products relative to other organs and altered brain insulin signaling may cause or promote neurodegenerative diseases which invalidates and reduces the quality of life. Insulin located within the brain is mostly of pancreatic origin or is produced in the brain itself crosses the blood-brain barrier and enters the brain via a receptor-mediated active transport system. Brain Insulin, insulin receptor and insulin receptor substrate-mediated signaling pathways play important roles in the regulation of peripheral metabolism, feeding behavior, memory and maintenance of neural functions such as neuronal growth and differentiation, neuromodulation and neuroprotection. In the present review, we would like to summarize the novel biological and pathophysiological roles of neuronal insulin in neurodegenerative diseases and describe the main signaling pathways in use for therapeutic strategies in the use of insulin to the cerebral tissues and their biological applications to neurodegenerative diseases.

Insulin involved Akt/ERK and Bcl-2/Bax pathways against oxidative damages in C6 glial cells

Abstract Insulin, a hypoglycemic hormone, has multiple functions in the brain. The aim of this study to identify the mechanisms of insulin in hydrogen peroxide (H2O2)-induced toxicity in the C6 glial cells. Cytotoxicity, lactate dehydrogenase, nitric oxide, reactive oxygen species and calcium ion, lipid peroxidation, protein oxidation and glutathione levels were determined. Signaling pathway molecules were assessed by western blotting and RT-PCR. The results showed that treatment with insulin reduced the cell death and cell membrane damages against H2O2-induced toxicity. Furthermore, insulin interfered H2O2-induced intracellular generation of reactive oxygen species and calcium-ion transport, apoptosis, including lipid and protein oxidation products. Cells treated with insulin reverted H2O2-induced suppression of reduced glutathione levels by blocking oxidized glutathione. Moreover, insulin treatment activates Akt, restores ERK1/2 and Bcl-2 by preventing Bax and Bax/Bcl-2 ratio. Our results suggest that treatment of insulin exerts potential role against 24 h of H2O2-induced toxicity in C6 cells.

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract Exogenous hydrogen peroxide (H2O2) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H2O2-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H2O2 for 24 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100 µM H2O2 was selected to establish a model of H2O2-induced oxidative stress. Further assays showed that 24 h of 100 µM H2O2-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca2+) in neuronal cells, but insulin can effectively diminish the H2O2-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H2O2-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H2O2-induced oxidative stress related to the Akt/Bcl-2 pathways.

Pharmacological Activities and Applications of Spicatoside A

Liriopogons (Liriope and Opiopogon) species are used as a main medicinal ingredient in several Asian countries. The Liriopes Radix (tuber, root of Liriope platyphylla) has to be a promising candidate due to their source of phytochemicals. Steroidal saponins and their glycosides, phenolic compounds, secondary metabolites are considered of active constituents in Liriopes Radix. Spicatoside A, a steroidal saponin, could be more efficacious drug candidate in future. In this review, we summarized the available knowledge on phytochemical and pharmacological activities for spicatoside A. It significantly suppressed the level of NF-κB, NO, iNOS, Cox-2, IL-1β, IL-6 and MAPKs in LPS-stimulated inflammation. The production of MUC5AC mucin was increased. MMP-13 expression was down-regulated in IL-1β-treated cells and reduced glycosaminoglycan release from IL-1α-treated cells. The neurite outgrowth activity, PI3K, Akt, ERK1/2, TrkA and CREB phosphorylation and neurotropic factors such as NGF and BDNF were upregulated with increased latency time. It also showed cell growth inhibitory activity on various carcinoma cells. From this, spicatoside A exerts anti-inflammation, anti-asthma, anti-osteoclastogenesis, neurite outgrowth, memory consolidation and anticancer activities. Further studies are needed on spicatoside A in order to understand mechanisms of action to treat various human diseases.

Protective effects of activated signaling pathways by insulin on C6 glial cell model of MPP+-induced Parkinson’s disease

Abstract Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD) associated with mitochondrial dysfunction mediated by oxidative stress. Astrocytes regulate neuronal function via the modulation of synaptic transmission and plasticity, secretion of growth factors, uptake of neurotransmitters, and regulation of extracellular ion concentrations and metabolic support of neurons. Therefore, this study was undertaken to investigate the mechanism of action of insulin on a 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity of events associated in cell viability and toxicity to the expression profile of cell signaling pathway proteins and genes in rat C6 glial cells. The various concentrations of MPP+ alone inhibited cell viability in a dose-dependent manner. Pretreatment of insulin prevented the cell death and lowered the intracellular reactive oxygen species and calcium ion influx by MPP+. Insulin also suppressed the α-synuclein and elevated the insulin signaling pathway molecules IR, IGF-1R, IRS-1 and IRS-2 in C6 cells through phosphorylation of Akt/ERK survival pathways. Moreover, insulin inhibits MPP+-induced Bax to Bcl-2 ratio. These results suggest that insulin has a protective effect on the MPP+-toxicity in C6 glial cells.

Insulin as a Potent Stimulator of Akt, ERK and Inhibin-βE Signaling in Osteoblast-Like UMR-106 Cells

Insulin is a peptide hormone of the endocrine pancreas and exerts a wide variety of physiological actions in insulin sensitive tissues, such as regulation of glucose homeostasis, cell growth, differentiation, learning and memory. However, the role of insulin in osteoblast cells remains to be fully characterized. In this study, we demonstrated that the insulin (100 nM) has the ability to stimulate the phosphorylation of protein kinase B (Akt/PKB) and extracellular signal-regulated kinase (ERK) and the levels of inhibin-βE in the osteoblast-like UMR-106 cells. This insulin-stimulated activities were abolished by the PI3K and MEK1 inhibitors LY294002 and PD98059, respectively. This is the first report proving that insulin is a potential candidate that enables the actions of inhibin-βE subunit of the TGF-β family. The current investigation provides a foundation for the realization of insulin as a potential stimulator in survival signaling pathways in osteoblast-like UMR-106 cells.

Insulin suppresses MPP+-induced neurotoxicity by targeting integrins and syndecans in C6 astrocytes

Abstract Parkinson’s disease (PD) is the second most common neurodegenerative disease in the elderly. In central nervous system, astrocytes regulates neuronal function via the modulation of synaptic transmission and plasticity, secretion of growth factors, uptake of neurotransmitters and regulation of extracellular ion concentrations and metabolic support of neurons. Therefore, C6 astroglial cells have been used to study the in vitro PD model induced by 1-methyl-4-phenyl pyridinium (MPP+). In this study, pre-treatment of insulin inhibited MPP+-induced cell membrane damages on LDH and NO releases, which also inhibited the iNOS and Cox-2 levels. Insulin also up-regulated the PI3K and p-GSK-3β protein expressions in C6 cells. In addition, MPP+ and/or insulin enhanced the autophagy by increasing LC3-I to LC3-II conversion. Furthermore, MPP+-induced toxicity diminished the integrin β3, αV, syndecan-1 and -3. Insulin pre-treatment enhanced the phosphorylation of integrin-linked kinase and further induced the integrin and syndecan molecules. These findings suggest that insulin prevents MPP+-induced toxicity through activation of PI3K, p-GSK-3β, autophagy, integrins and syndecans pathways in C6 glial cells.