Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy

Objective: Protein glutathionylation is considered an important post-translational modification in the pathogenesis of complex diseases. The aim of this study was to examine whether hemoglobin (Hb) is modified by reduced glutathione (GSH) via oxidation of the thiol groups present in diabetes and its associated microangiopathy and to determine whether oxidative imbalance has any correlation with glutathionylated Hb (HbSSG) levels. Methods: The study group consisted of a total of 130 subjects which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. All subjects were assessed for glycemic and lipidemic status, while diabetic subjects were also assessed for the diagnosis of retinopathy and nephropathy. RBC lysates from all the subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for HbSSG β-globin chains. Levels of GSH and thiobarbituric acid substances (TBARS) levels were measured by spectrophotometric and fluorimetric methods, respectively. Results: The positivity for HbSSG in diabetic subjects with microangiopathy was significantly higher (69%) compared to diabetics without microangiopathy (22%) and control subjects (14%). In univariate regression analysis, HbSSG levels were significantly associated with the duration of diabetes, HbA1c, and TBARS levels. GSH levels were negatively correlated (r = -0.57, P < 0.001) with HbSSG in diabetic subjects. A significant inverse correlation (r = -0.42, P < 0.001) between the GSH levels and HbA1c levels was also seen in diabetic subjects. Conclusions: This is perhaps the largest LC-MS-based study to demonstrate that HbSSG levels are markedly increased in diabetic subjects with microangiopathy. Since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased HbSSG concentrations and altered reduction-oxidation (redox) signaling

Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy

Abstract Objective: Protein glutathionylation is considered an important post-translational modification in the pathogenesis of complex diseases. The aim of this study was to examine whether hemoglobin (Hb) is modified by reduced glutathione (GSH) via oxidation of the thiol groups present in diabetes and its associated microangiopathy and to determine whether oxidative imbalance has any correlation with glutathionylated Hb (HbSSG) levels. Methods: The study group consisted of a total of 130 subjects which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. All subjects were assessed for glycemic and lipidemic status, while diabetic subjects were also assessed for the diagnosis of retinopathy and nephropathy. RBC lysates from all the subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for HbSSG h-globin chains. Levels of GSH and thiobarbituric acid substances (TBARS) levels were measured by spectrophotometric and fluorimetric methods, respectively. Results: The positivity for HbSSG in diabetic subjects with microangiopathy was significantly higher (69%) compared to diabetics without microangiopathy (22%) and control subjects (14%). In univariate regression analysis, HbSSG levels were significantly associated with the duration of diabetes, HbA1c, and TBARS levels. GSH levels were negatively correlated (r = À0.57, P < 0.001) with HbSSG in diabetic subjects. A significant inverse correlation (r = À0.42, P < 0.001) between the GSH levels and HbA1c levels was also seen in diabetic subjects. Conclusions: This is perhaps the largest LC-MS-based study to demonstrate that HbSSG levels are markedly increased in diabetic subjects with microangiopathy. Since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased HbSSG concentrations and altered reduction -oxidation (redox) signaling

Congenital myasthenic syndrome caused by a frameshift insertion mutation in

Objective: Description of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families. Methods: Muscle biopsies, EMG, and whole-exome sequencing were performed. Results: All 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the GFPT1 gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pathology. Conclusions: These results expand on the spectrum of known loss-of-function GFPT1 mutations in CMS12 and in one family demonstrate a novel mode of inheritance due to UPD

Diabetic Retinopathy and Inflammation: Novel Therapeutic Targets

Most anti-vascular endothelial growth factor (VEGF) therapies in diabetic macular edema are not as robust as in proliferative diabetic retinopathy. Although the VEGF appears to be a good target in diabetic macular edema, the anti-VEGF therapies appear to be of transient benefit as the edema recurs within a few weeks, and repeated injections are necessary. There is new evidence that indicates ‘retinal inflammation’ as an important player in the pathogenesis of diabetic retinopathy. There are common sets of inflammatory cytokines that are upregulated in both the serum and vitreous and aqueous samples, in subjects with diabetic retinopathy, and these cytokines can have multiple interactions to impact the pathogenesis of the disease. The key inflammatory events involved in the blood retinal barrier (BRB) alteration appear to be: (1) Increased expression of endothelial adhesion molecules such as ICAM1, VCAM1, PECAM-1, and P-selectin, (2) adhesion of leukocytes to the endothelium, (3) release of inflammatory chemokines, cytokines, and vascular permeability factors, (4) alteration of adherens and tight junctional proteins between the endothelial cells, and (5) infiltration of leukocytes into the neuro-retina, resulting in the alteration of the blood retinal barrier (diapedesis). VEGF inhibition itself may not achieve neutralization of other inflammatory molecules involved in the inflammatory cascade of the breakdown of the BRB. It is possible that the novel selective inhibitors of the inflammatory cascade (like angiopoietin-2, TNFα, and chemokines) may be useful therapeutic agents in the treatment of diabetic macular edema (DME), either alone or in combination with the anti-VEGF drugs

Advanced glycation index and its association with severity of diabetic retinopathy in type 2 diabetic subjects

Background: This study investigates the association of advanced glycation index (AGI), a simple assay to detect advanced glycation endproducts (AGEs) in serum, with severity of diabetic retinopathy (DR) in type 2 diabetic subjects. Methods: The study included 188 type 2 diabetic subjects without DR, 153 subjects with nonproliferative DR, 41 subjects with proliferative DR, and 188 control participants. Serum levels of AGEs were monitored with a spectrofluorimeter by recording Maillard-specific fluorescence. Results: AGI values increased with severity of DR (analysis of variance, P&lt;.0001). Among diabetic subjects, AGI (mean±S.E.) was higher among subjects with nonproliferative diabetic retinopathy (NPDR; 6.7±0.1 U) and proliferative diabetic retinopathy (PDR; 9.1±0.3 U) than among subjects without DR (P&lt;.0001). By arranging the levels of serum AGI in quartiles, the proportion of PDR subjects increased with increasing AGI values, with maximum subjects in the last quartile (trend χ<SUP>2</SUP>=60.239, P&lt;.0001). AGI was associated with NPDR even after adjusting for age, gender, duration of diabetes, and glycated hemoglobin [odds ratio (OR)=1.33; 95% confidence interval (95% CI)=1.12-1.57; P=.001]. Similarly, AGI showed a significant association with PDR even after adjusting for various risk factors (OR=2.47; 95% CI=1.75-3.47; P&lt;.0001). Receiver-operating-characteristics curve analysis revealed that the threshold level of 8.07 U had a 78% sensitivity, an 83.6% specificity, and an 86.1% accuracy for detecting PDR. Conclusion: AGI showed a significant association with the severity of DR and, hence, could be used as a prognostic tool to predict the development and progression of DR

Association of leukocyte count with varying degrees of glucose intolerance in Asian Indians: the Chennai Urban Rural epidemiology study (CURES-26)

Objective: This study assessed the association of leukocyte count with different grades of glucose intolerance in Asian Indian subjects. Methods: Three groups of subjects were recruited from the Chennai Urban Rural Epidemiology Study (CURES), a population-based study, representative of Chennai (formerly Madras), a city in southern India. Group 1 represented normal glucose tolerance (NGT) (n = 840), group 2 included impaired glucose tolerance (IGT) (n = 180), and group 3 included type 2 diabetes (n = 1170). Anthropometric measurements including weight, height, and waist measurements were obtained using standardized techniques. Leukocyte count was measured by an automated flow cytometry instrument (Sysmex SF-3000, Japan). Fasting insulin was measured by enzyme-linked immunosorbent assay and insulin resistance was calculated using the homeostasis model assessment (HOMA-IR). Results: Subjects with diabetes (8.0 ± 1.5 × 10<SUP>3</SUP>/μL) and IGT (7.9 ± 1.3 × 10<SUP>3</SUP>/μL) had a significantly higher mean leukocyte count compared to the NGT group (7.4 ± 1.5 × 10<SUP>3</SUP>/μL) (P &lt; 0.001). Leukocyte count was significantly increased in NGT subjects with insulin resistance (IR) as measured by HOMA-IR (7.5 ± 1.5 × 10<SUP>3</SUP>/μL; P &lt; 0.001) compared to NGT subjects without IR (7.0 ± 1.4 × 10<SUP>3</SUP>/μL). Regression analysis showed that there was a linear increase in mean leukocyte count with increasing severity of glucose intolerance, even after adjusting for age, waist circumference, and HOMA-IR. Conclusions: Among Asian Indians who are known to have high risk of premature coronary artery disease and diabetes, a significant association exists between leukocyte count and glucose intolerance

A novel advanced glycation index and its association with diabetes and microangiopathy

Formation of advanced glycation end products (AGEs) is an important mechanism by which chronic exposure to high glucose levels leads to vascular complications. Measurement of AGEs is hence of great importance for clinicians and researchers concerned with the management and prevention of diabetic vascular disease. The aim of this study was to evaluate a simple methodology to detect AGEs in the serum and to correlate their levels with diabetes and microangiopathy, specifically retinopathy and nephropathy. We studied 157 subjects, which included nondiabetic control subjects (n = 38), type 2 diabetic patients without microangiopathy (n = 65), and type 2 diabetic subjects with retinopathy (n = 29) or both retinopathy and nephropathy (n = 25). All subjects were assessed for their glycemic and lipid status. Serum AGEs were monitored by recording the Maillard-specific fluorescence that resulted from sequential addition of serum into the buffer. The resultant linear regression was modeled to yield the slope values that were termed advanced glycation index (AGI) in arbitrary units. The serum levels of AGI (mean &#177; SD) were higher in diabetic subjects without complications (6.0 &#177; 1.6 units) compared with nondiabetic subjects (4.6 &#177; 1.0 units), still higher among diabetic subjects with retinopathy (7.6 &#177; 1.2 units) and highest in diabetic subjects with both retinopathy and nephropathy (8.3 &#177; 2.0 units). Among diabetic subjects, AGI had a significant positive correlation with duration of diabetes (r = 0.25, P = .006), glycated hemoglobin (r = 0.27, P = .004), cholesterol (r = 0.24, P = .009), triglycerides (r = 0.23, P = .014), and serum creatinine (r = 0.30, P = .001), and a significant negative correlation with creatinine clearance (r = -0.27, P = .003). Logistic regression analysis using diabetic microangiopathy as the dependent variable showed an association with AGI even after including age, duration of diabetes, and glycated hemoglobin (P &lt; .001) into the model. Advanced glycation index is a simple method to detect AGEs, and it correlates well with diabetes, particularly with microangiopathy

Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model [version 1; referees: 2 approved]

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene, encoding methyl-CpG-binding protein 2. We have created a mouse model (Mecp2 A140V “knock-in” mutant) expressing the recurrent human MECP2 A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN) cultures from mutant (Mecp2A140V/y) and wild type (Mecp2+/y) male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele) and a wild type Mecp2 gene linked to an eGFP transgene (paternal allele). In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V) allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1) treatment of neuronal cells from Mecp2 mutant mice rescued the soma size phenotype. We also found that Mecp2  mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i) reduced neuronal size is an important in vitro cellular phenotype of Mecp2 mutation in mice, and ii) MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, in vitro assay to screen for compounds that rescue small neuronal phenotype (“phenotypic assay”)

Do Genomic Factors Play a Role in Diabetic Retinopathy?

Although there is strong clinical evidence that the control of blood glucose, blood pressure, and lipid level can prevent and slow down the progression of diabetic retinopathy (DR) as shown by landmark clinical trials, it has been shown that these factors only account for 10% of the risk for developing this disease. This suggests that other factors, such as genetics, may play a role in the development and progression of DR. Clinical evidence shows that some diabetics, despite the long duration of their diabetes (25 years or more) do not show any sign of DR or show minimal non-proliferative diabetic retinopathy (NPDR). Similarly, not all diabetics develop proliferative diabetic retinopathy (PDR). So far, linkage analysis, candidate gene studies, and genome-wide association studies (GWAS) have not produced any statistically significant results. We recently initiated a genomics study, the Diabetic Retinopathy Genetics (DRGen) Study, to examine the contribution of rare and common variants in the development of different phenotypes of DR, as well as their responsiveness to anti-VEGF treatment in diabetic macular edema (DME). Our preliminary findings reveal a novel set of genetic variants involved in the angiogenesis and inflammatory pathways that contribute to DR progression or protection. Further investigation of variants can help to develop novel biomarkers and lead to new therapeutic targets in DR