Pathology Of Diabetic Retinopathy Research Articles

Palmitic Acid Induces M\xfcller Cell Inflammation that is Potentiated by Co-treatment with Glucose

Chronic hyperglycemia is thought to be the major stimulator of retinal dysfunction in diabetic retinopathy (DR). Thus, many diabetes-related systemic factors have been overlooked as inducers of DR pathology. Cell culture models of retinal cell types are frequently used to mechanistically study DR, but appropriate stimulators of DR-like factors are difficult to identify. Furthermore, elevated glucose, a gold standard for cell culture treatments, yields little to no response from many primary human retinal cells. Thus, the goal of this project was to demonstrate the effectiveness of the free fatty acid, palmitic acid and compare its use alone and in combination with elevated glucose as a stimulus for human Müller cells, a retinal glial cell type that is activated early in DR pathogenesis and uniquely responsive to fatty acids. Using RNA sequencing, we identified a variety of DR-relevant pathways, including NFκB signaling and inflammation, intracellular lipid signaling, angiogenesis, and MAPK signaling, that were stimulated by palmitic acid, while elevated glucose alone did not significantly alter any diabetes-relevant pathways. Co-treatment of high glucose with palmitic acid potentiated the expression of several DR-relevant angiogenic and inflammatory targets, including PTGS2 (COX-2) and CXCL8 (IL-8).

New Development and New Medicine in Treatment of Diabetic Retinopathy and Diabetic Macular Edema

The blood-retina barrier alteration is a hallmark feature of diabetic retinopathy. The vascular endothelial growth factor is an important vasopermeability factor that playing role in the pathogenesis of blood-retina barrier destruction. Inflammation is the second pathology in diabetic retinopathy. New researches are ongoing about both pathways for diabetic retinopathy and macular edema therapy.

Inhibition of Inflammation and Pyroptosis mitigate Diabetic Retinopathy via Suppressing Receptor Activated NF‐kB Ligand (RANK‐l)

Diabetic Retinopathy (DR) characterized by leaky blood vessels causing vision distortion. DR generally manifests as an asymptomatic condition on its onset, however; in most cases, it is usually diagnosed in its proliferative phase. Currently, there is no cure and the available therapeutic efforts are generally ineffective. Current therapeutic methods are palliative in nature and only a subset of patients benefited. Many studies have shown an association of inflammation with DR pathology. The vitreous and the retina of DR individuals exhibit the presence of inflammatory markers such as TNF‐a, iNOS, and IL‐1b. An increase in inflammation has been shown to impair the tight cellular junctions via degradations of connexins (43 & 26) and Occludin and ZO‐1. This eventually leads to BRB breakdown, thus the occurrence of leaky blood vessels that are responsible for vision loss in DR patients. Receptor‐activated of NF‐kB ligand (RANK‐l) is known to regulates survival of T‐cell, which can break down the BBB for non‐neural antigen activation. We hypothesize that inflammation induces an increase in T cells expression which remains activated via RANK‐l, gradually pyroptosis resulted in the breakdown of BRB. Akita mice were treated with 5 mg/kg body weight of JSH‐23 for two weeks, they were injected intraperitoneal every other day. The level of inflammatory mediators was measured (e.g. iNOS), T cells, RANK‐l, and pyroptosis of retinal cells. Fluorescence angiography was performed as well to analyze the leakiness of retinal vessels. Eye pressure was also measured in Akita mice. The preliminary result indicated a significantly higher pressure in the eye of Akita mice compared to control. Further analyses should indicate a decrease in inflammatory mediators, microvascular leakage, pyroptosis, and eye pressure in treated Akita mice relative to non‐treated, thus vision improvement.Support or Funding InformationMahavir Singh, DVM, Ph.DA.;Akash George, Ph.D.;Naira Metreveli;Suresh C Tyagi, Ph.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ultrastructure of neurovascular changes in human diabetic retinopathy.

The previous concept regarding diabetic retinopathy assigned a primary role to hyperglycemia-induced microvascular alterations, while neuronal and glial abnormalities were considered to be secondary to either ischemia or exudation. The aim of this study was to reveal the potential role of neuronal and glial cells in initial and advanced alterations of the retinopathy in human type 2 diabetes. Electron microscopy and histochemical studies were performed on 38 surgically removed human eyes (28 obtained from diabetic patients and 10 from non-diabetic patients). Morphometric analysis of basement membrane material and lipids was performed. An accumulation of metabolic by-products was found in the capillary wall with aging: this aspect was significantly more pronounced in diabetics. Müller glial cells were found to contribute to alterations of the capillary wall and to occlusion, as well as to the development of proliferative retinopathy and cystoid degeneration of the retina. Our results showed morphological evidence regarding the role of neuronal and glial cells in the pathology of diabetic retinopathy, prior and in addition to microangiopathy. These morphological findings support a neurovascular pathogenesis at the origin of diabetic retinopathy, thus the current treatment approach should be completed by neuroprotective measures.

Management of diabetic macular edema in Japan: a review and expert opinion.

Diabetic retinopathy is a frequent cause of visual impairment in working-age adults (≥30years) and in Japan is most commonly observed in those aged 50-69years. Diabetic macular edema (DME) is one of the main causes of vision disturbance in diabetic retinopathy, which is a clinically significant microvascular complication of diabetes. Anti-vascular endothelial growth factor (VEGF) therapy is becoming the mainstay of treatment for DME. However, to achieve sustained long-term improvement in visual acuity, conventional laser photocoagulation, vitrectomy and steroid therapy are also expected to play a role in the treatment of DME. This review summarizes the epidemiology and pathology of diabetic retinopathy and DME, evaluates the findings regarding the diagnosis and treatment of DME, and underscores the importance of systemic management of the disease in the context of the current health care situation in Japan. Finally, the unmet needs of patients with DME and prospects for research are discussed. The weight of evidence suggests that it is important to establish a multipronged treatment strategy centered on anti-VEGF therapy.

Immunohistochemical Characterization of Connexin43 Expression in a Mouse Model of Diabetic Retinopathy and in Human Donor Retinas.

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression.

Genetic predictors associated with diabetic retinopathy in patients with diabetic foot.

INTRODUCTION Early detection of diabetic retinopathy (DR) is crucial for preventing irreversible blindness. Recent studies identified some of the genetic factors involved in the pathology of DR, although their precise underlying mechanisms remain unclear. OBJECTIVES This pilot study aimed to determine genetic predictors of DR among patients with type 2 diabetes (T2D) and diabetic foot (DF) based on pathogenetic pathways. PATIENTS AND METHODS The study included 114 patients with T2D and DF (64 with DR, 50 without DR). Genetic analysis was performed for each patient and the following alterations were analyzed: rs759853 (AKR1B1), rs1800469 (TGFB1), rs2073618 and rs3134069 (TNFRSF11B), rs6330 and rs11466112 (NGF), rs1801133 (MTHFR), rs8192678 (PPARGC1A), rs1799983 (NOS3), rs1553005 (CALCA), and rs121917832 (CDKN1B). RESULTS Correlations with DR were identified for the following single nucleotide variants (SNVs): rs759853, rs2073618, and rs3134069. Carriers of the G allele of the rs759853 variant had a higher risk of DR in the dominant model (odds ratio [OR], 3.0; 95% confidence interval [CI], 1.15-7.81; P = 0.02). We analyzed 2 SNVs of the osteoprotegerin gene (rs3134069 and rs2073618), and found that the A allele of the rs3134069 variant decreased the risk of DR in both the recessive and additive models (OR, 3.33; 95% CI, 1.07-10.3; P = 0.04). Conversely, there were fewer carriers of the C allele of the rs2073618 variant in patients with DR in the dominant model (OR, 0.28; 95% CI, 0.09-0.92; P = 0.04). CONCLUSIONS The results of our study suggest that the SNVs rs759853, rs3134069, and rs2073618 may be involved in the development of DR in patients with T2D and DF.

Astaxanthin enhances catalase activity in retinal tissue of experimental diabetic rats

It has been shown that diabetes causes a decrease in antioxidant capacity. Since the decrease of antioxidant capacities are related to the pathology of diabetic retinopathy; we aimed to evaluate the effect of Astaxanthin (ASTA), a powerful natural antioxidant, on catalase content in retinal tissue. Forty-eight rats were randomly divided into four groups (n =8-12 for each group): Control, Control-treated, Diabetic and Diabetic-treated groups. Diabetes was induced by a single injection of streptozotocin (50 mg/kg body weight, intravenously). Animals with a blood glucose more than 350 mg were selected as diabetic animals. Treatment with ASTA, (20 mg/kg) was administrated orally once a day by gavages over six weeks. After six weeks the final blood glucose concentration and body weight were measured. The animals were then euthanized by a lethal dose of Ketamine and Diazepam, the eyeballs removed and the retinas quickly extracted for catalase (CAT) measurement and histopathological assessments. Hyperglycemia decreased the enzymatic activity of CAT in retinal tissue of the Diabetic group but, with ASTA treatment, it returned toward normal value and ASTA treatment in Control-treated group increased the CAT capacity significantly. Retinal ganglion cell layer (RGC) in both Diabetic and Diabetic-treated groups showed fluid retention and edema but in the Diabetic-treated group, it was lower than inthe Diabetic group. We suggest that the increase of CAT activity and decrease of fluid retention in the Diabetic-treated group compared with the Diabetic group are related to the ability of ASTA to balance excess ROS production.

Müller Cell-Microglia Cross Talk Drives Neuroinflammation in Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common complication of diabetes and a leading cause of vision loss worldwide (1). Unfortunately, there are no treatments targeting early stages of the disease prior to the onset of sight-threatening vascular defects such as macular edema or neovascularization. A better understanding of the etiology of DR is needed to identify therapeutic targets to halt early disease progression. To this end, numerous studies demonstrated that a low-grade inflammation occurs in retinas of diabetic animal models and suggest that inflammation contributes a role in DR progression. Various mechanisms leading to retinal inflammation in DR have been described, with the majority of studies implicating retinal Muller glial cells and microglia as the initiators of retinal inflammation (for review, see ref. 2). However, seldom does a study make a strong connection between these two cell types. In this issue of Diabetes , Portillo et al. (3) describe a mechanism in which retinal inflammation in diabetic mice is dependent upon expression of the cluster of differentiation gene 40 (CD40) receptor by Muller cells (Fig. 1). The study suggests that CD40 activation induces Muller cells to release ATP, leading to activation of P2X7 purinergic receptors on retinal microglia and their subsequent expression of inflammatory cytokines. Importantly, the requirement of Muller cell–specific CD40 expression to recapitulate the appearance of acellular capillaries in diabetic retinas also suggests that inflammation is necessary for the loss of vascular cells associated with DR pathology. Figure 1 Proposed …

Improvement in distance and near visual acuities using low vision devices in diabetic retinopathy

Purpose:The aim of this study is to elucidate the causes and level of visual impairment (VI) in patients with different pathologies of diabetic retinopathy (DR) who presented to a low vision care (LVC) clinic, to analyze the type of distant and near devices prescribed to them and the visual benefits thereof.Methods:A retrospective chart review was done for 100 consecutive patients with DR who were referred to the LVC clinic from June 2015 to June 2016. The reason for referral was assessed from the electronic medical records and available fundus photographs, fundus fluorescein angiograms, and optical coherence tomography images by a retina specialist. The details of low-vision devices and subsequent improvements were noted.Results:Of the 100 patients, 52% had moderate VI, 19% mild VI, 16% severe VI, and 13% had profound VI or blindness. The most commonly prescribed low vision device was half-eye spectacles (38.4%). The pathologies which had statistically significant improvement (P < 0.05) in distance vision with low vision devices were DR with disc pallor (4.4% improvement), ischemic maculopathy (11.9% improvement), and plaque of hard exudate (10.1% improvement). However, in all pathologies, there was statistically significant improvement (P < 0.05) in near vision.Conclusion:Usually, the patients with DR presented to the LVC clinic with moderate VI. The use of low vision devices can help these patients in cases where medical and surgical treatment have no or a limited role in restoring useful vision.

MiR-15a/16 reduces retinal leukostasis through decreased pro-inflammatory signaling.

BackgroundHyperglycemia is a significant risk factor for diabetic retinopathy and induces increased inflammatory responses and retinal leukostasis, as well as vascular damage. Although there is an increasing amount of evidence that miRNA may be involved in the regulation in the pathology of diabetic retinopathy, the mechanisms by which miRNA mediate cellular responses to control onset and progression of diabetic retinopathy are still unclear. The purpose of our study was to investigate the hypothesis that miR-15a/16 inhibit pro-inflammatory signaling to reduce retinal leukostasis.MethodsWe generated conditional knockout mice in which miR-15a/16 are eliminated in vascular endothelial cells. For the in vitro work, human retinal endothelial cells (REC) were cultured in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). Statistical analyses were done using unpaired Student t test with two-tailed p value. p < 0.05 was considered significant. Data are presented as mean ± SEM.ResultsWe demonstrated that high glucose conditions decreased expression of miR-15a/16 in cultured REC. Overexpression of miR-15a/16 with the mimic significantly decreased pro-inflammatory signaling of IL-1β, TNFα, and NF-κB in REC. In vivo data demonstrated that the loss of miR-15a/16 in vascular cells led to increased retinal leukostasis and CD45 levels, together with upregulated levels of IL-1β, TNFα, and NF-κB.ConclusionsThe data indicate that miR-15a/16 play significant roles in reducing retinal leukostasis, potentially through inhibition of inflammatory cellular signaling. Therefore, we suggest that miR-15a/16 offer a novel potential target for the inhibition of inflammatory mediators in diabetic retinopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0771-8) contains supplementary material, which is available to authorized users.

P75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy.

In many diseases, expression and ligand-dependent activity of the p75(NTR) receptor can promote pericyte and vascular dysfunction, inflammation, glial activation, and neurodegeneration. Diabetic retinopathy (DR) is characterized by all of these pathological events. However, the mechanisms by which p75(NTR) may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75(NTR) is upregulated very early in glia and in pericytes to mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. In a mouse model of oxygen-induced retinopathy, mimicking proliferative DR, p75(NTR)-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75(NTR) or antagonists of the ligand proNGF suppresses each distinct phase of pathology, ameliorate disease, and prevent disease progression. Thus, our study documents novel disease mechanisms and validates druggable targets for diabetic retinopathy. Diabetic retinopathy (DR) affects an estimated 250 million people and has no effective treatment. Stages of progression comprise pericyte/vascular dysfunction, inflammation, glial activation, and neurodegeneration. The pathophysiology of each stage remains unclear. We postulated that the activity of p75NTR may be implicated. We show that p75NTR in glia and in pericytes mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood-retina barrier breakdown, edema, and neuronal death. p75NTR-promoted inflammation leads to ischemia and angiogenesis through Semaphorin 3A. Antagonists of p75NTR or antagonists of proNGF suppress each distinct phase of pathology, ameliorate disease, and prevent disease progression. Our study documents novel mechanisms in a pervasive disease and validates druggable targets for treatment.

Automated Diabetic Retinopathy Screening and Monitoring Using Retinal Fundus Image Analysis.

Diabetic retinopathy (DR)-a common complication of diabetes-is the leading cause of vision loss among the working-age population in the western world. DR is largely asymptomatic, but if detected at early stages the progression to vision loss can be significantly slowed. With the increasing diabetic population there is an urgent need for automated DR screening and monitoring. To address this growing need, in this article we discuss an automated DR screening tool and extend it for automated estimation of microaneurysm (MA) turnover, a potential biomarker for DR risk. The DR screening tool automatically analyzes color retinal fundus images from a patient encounter for the various DR pathologies and collates the information from all the images belonging to a patient encounter to generate a patient-level screening recommendation. The MA turnover estimation tool aligns retinal images from multiple encounters of a patient, localizes MAs, and performs MA dynamics analysis to evaluate new, persistent, and disappeared lesion maps and estimate MA turnover rates. The DR screening tool achieves 90% sensitivity at 63.2% specificity on a data set of 40 542 images from 5084 patient encounters obtained from the EyePACS telescreening system. On a subset of 7 longitudinal pairs the MA turnover estimation tool identifies new and disappeared MAs with 100% sensitivity and average false positives of 0.43 and 1.6 respectively. The presented automated tools have the potential to address the growing need for DR screening and monitoring, thereby saving vision of millions of diabetic patients worldwide.

Microglia-Derived Interleukin-6 Increases Retinal Endothelial Cell Permeability through STAT3 Activation in Diabetic Retinopathy

Diabetic retinopathy (DR) accompanies an inflammatory response associated with an increase in inflammatory cytokines. Among cytokines elevated in the diabetic retina, the mechanism by which IL-6 induces DR pathologies, more specifically endothelial permeability, is not well understood. Thus, we focused on the cellular source of the elevated IL-6 in high glucose condition and the molecular mechanism by which IL-6 impairs endothelial tight junctions. In the present study, we found that the expression of IL-6 and TNF-α was elevated in the streptozotocin-induced diabetic retina and that IL-6, but not TNF-α, expression was increased in microglia exposed to high glucose condition in vitro. Interestingly, in co-culture condition with microglia and endothelial cell, microglia-derived IL-6 increased retinal endothelial permeability, which was exaggerated by high glucose exposure. We demonstrated that the IL-6 activated STAT3, through which led to the reduced expression of tight junction protein ZO-1 and occludin. Taken together, we propose that IL-6-induced STAT3 activation is important for the induction of endothelial permeability through the reduction of ZO-1 and occludin levels in retinal endothelial cells. In addition, microglia-derived IL-6 and IL-6/STAT3 signaling could be a therapeutic target to prevent the DR pathology.

Diabetic Retinopathy: Animal Models, Therapies, and Perspectives.

Diabetic retinopathy (DR) is one of the major complications of diabetes. Although great efforts have been made to uncover the mechanisms underlying the pathology of DR, the exact causes of DR remain largely unknown. Because of multifactor involvement in DR etiology, currently no effective therapeutic treatments for DR are available. In this paper, we review the pathology of DR, commonly used animal models, and novel therapeutic approaches. Perspectives and future directions for DR treatment are discussed.

Clinical Pathology of Diabetic Retinopathy and Macular Edema

Clinical Pathology of Diabetic Retinopathy and Macular Edema

Automated detection of diabetic retinopathy in retinal images.

Diabetic retinopathy (DR) is a disease with an increasing prevalence and the main cause of blindness among working-age population. The risk of severe vision loss can be significantly reduced by timely diagnosis and treatment. Systematic screening for DR has been identified as a cost-effective way to save health services resources. Automatic retinal image analysis is emerging as an important screening tool for early DR detection, which can reduce the workload associated to manual grading as well as save diagnosis costs and time. Many research efforts in the last years have been devoted to developing automatic tools to help in the detection and evaluation of DR lesions. However, there is a large variability in the databases and evaluation criteria used in the literature, which hampers a direct comparison of the different studies. This work is aimed at summarizing the results of the available algorithms for the detection and classification of DR pathology. A detailed literature search was conducted using PubMed. Selected relevant studies in the last 10 years were scrutinized and included in the review. Furthermore, we will try to give an overview of the available commercial software for automatic retinal image analysis.

Role of IGF-1 Signaling in the Pathology of Diabetic Retinopathy

Diabetic retinopathy (DR) is a common eye disease caused by diabetic mellitus and can lead to blindness of eye. Currently, researches on the mechanisms and clinical interventions of DR are at an early stage. Under the condition of DR, insulin is usually used for the control of blood glucose. Similar to insulin, Insulin-like growth factor-1 (IGF-1), a protein which has been reported to lower the level of blood glucose, may also promote cell survival and proliferation in various tissues. However, effects of IGF-1 on DR are complex and controversial. The effect of IGF-1 on the normalization of blood glucose is beneficial for the patients, especially those with severe insulin resistance. However, IGF-1 mediates a strong mitogenic signaling and long term application of IGF-1 may aggravate retinal deterioration through promoting the proliferation of retinal endothelial cells. IGF-1 enhances the expression of vascular endothelial growth factor and erythropoietin. The binding of IGF-I to IGF-IR also promotes the activation of multiple signaling pathways, including phosphatidylinositol 3-kinase/protein kinase B pathway, mitogen activated protein kinase pathway and nuclear factor-κB signaling pathway. All of these have been shown to be associated with the pathology of DR. In this review, we discuss the signaling pathway of IGF-1, evidence for a functional relationship between the IGF-I and DR, as well as a possible role of IGF-1 signaling pathway in the process of DR.

Diabetic retinopathy: loss of neuroretinal adaptation to the diabetic metabolic environment

Diabetic retinopathy (DR) impairs vision of patients with type 1 and type 2 diabetes, associated with vascular dysfunction and occlusion, retinal edema, hemorrhage, and inappropriate growth of new blood vessels. The recent success of biologic treatments targeting vascular endothelial growth factor (VEGF) demonstrates that treating the vascular aspects in the later stages of the disease can preserve vision in many patients. It would also be highly desirable to prevent the onset of the disease or arrest its progression at a stage preceding the appearance of overt microvascular pathologies. The progression of DR is not necessarily linear but may follow a series of steps that evolve over the course of multiple years. Abundant data suggest that diabetes affects the entire neurovascular unit of the retina, with an early loss of neurovascular coupling, gradual neurodegeneration, gliosis, and neuroinflammation occurring before observable vascular pathologies. In this article, we consider the pathology of DR from the point of view that diabetes causes measurable dysfunctions in the complex integral network of cell types that produce and maintain human vision.

Effect of hyperglycemia on insulin receptor signaling in the cultured retinal Müller glial cells

Hyperglycemia and impaired insulin signaling are considered as major factors in the retinal pathology in diabetic retinopathy (DR). Numerous reports support that these two factors damage retinal glial as well as neuronal cells early in diabetes. However, it is not known whether diabetic induced hyperglycemia causes a depression to the insulin signaling. In this study we utilized a well characterized cultured Muller cells (TR-MUL) where we found a high expression of insulin receptor molecules. TR-MUL Cells were treated with high glucose, glutamate and hydrogen peroxide, and activated with insulin. Following treatments, cell lysates were analyzed by immunoblotting experiments for insulin receptor (IRβ) and insulin receptor substrate (IRS1). In addition, cell lysates were immunoprecipitated using antibodies against insulin receptor proteins to analyze tyrosine phosphorylation and serine phosphorylation of insulin receptor proteins. Results indicate that hyperglycemia did not affect the expression of insulin receptor proteins in cultured TR-MUL cells. Although, hyperglycemia seems to inhibit the interaction between IRS1 and IRβ. Hydrogen peroxide increased the tyrosine phosphorylation of insulin receptor proteins but excess glutamate could not affect the insulin receptor proteins indicating that glutamate may not cause oxidative stress in TR-MUL cells. Hyperglycemia lowered serine phosphorylation of IRSser632 and IRSser1101 however, IRSser307 was not affected. Thus, hyperglycemia may not affect insulin signaling through tyrosine phosphorylation of insulin receptor proteins but may inhibit the interactions between insulin receptor proteins. Hyperglycemia induced phosphorylation of various serine residues of IRS1 and their influence on insulin signaling needs further investigation in TR-MUL cells.