Correction: Double burden of maternal and child malnutrition and socioeconomic status in urban Sri Lanka.

[This corrects the article DOI: 10.1371/journal.pone.0224222.].

Double burden of maternal and child malnutrition and socioeconomic status in urban Sri Lanka.

Child malnutrition and maternal obesity are serious public health issues in Sri Lanka. This study explores the associations between socioeconomic status and the double burden of malnutrition among school-aged children and within their household. A total of 543 primary school children aged 5-10 years (204 boys and 339 girls) in Gampaha District, Sri Lanka, were included in the analysis. The nutritional statuses of thinness, normal, overweight, and obesity for children and mothers were defined according to WHO growth references and body mass index. Maternal education, household equivalent income, and maternal employment were used as socioeconomic status indicators. The proportion of child thinness and overweight was 19.3% and 13.4%, respectively, and that of maternal overweight (body mass index ≥ 25 kg/m2) was 36.5%. A positive correlation was found between maternal body mass index and the child's body mass index for age z-score in older boys and younger girls. A multivariate stepwise logistic regression analysis showed that lower education of mothers posed a higher association with child thinness (adjusted odds ratio = 2.33, 95% confidence interval: 1.08-5.00). Mothers with overweight and obesity were less likely to have a child with thinness (adjusted odds ratio = 0.30, 95% confidence interval: 0.16-0.58). Maternal employment status and household equivalent income were not significantly, but marginally, associated with child overweight and obesity. Socioeconomic inequality combined with maternal nutritional status affected child malnutrition. These findings suggest that the underlying circumstances within households should be considered to improve child malnutrition.

Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 ß-cells.

Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes the post-translational methylation of C-terminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of heterotrimeric G-proteins. It is widely felt that carboxymethylation promotes efficient membrane association of the methylated proteins and specific protein-protein interactions. In the current study, we tested the hypothesis that ICMT-mediated carboxymethylation of specific proteins (e.g., Rac1) plays a regulatory role in glucose-stimulated insulin secretion (GSIS). Western blot analysis indicated that lCMT is expressed and predominantly membrane associated in INS 832/13 ß-cells. siRNA-mediated knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin secretion. These findings were further supported by pharmacological observations, which suggested a marked reduction in glucose-, but not KCl-stimulated insulin secretion by acetyl farnesyl cysteine (AFC), a selective inhibitor of ICMT. In addition, glucose-induced Rac1 activation, a hallmark signaling step involved in glucose-stimulated insulin secretion, was markedly inhibited following pharmacological (AFC) or molecular biological (siRNA-ICMT) inhibition of ICMT. Lastly, we also noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen species in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these data suggest that ICMT regulates glucose-induced Rac1 activation, generation of reactive oxygen species and insulin secretion in pancreatic ß-cells.

Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic ß-cells: evidence for regulation by Rac1.

Reactive oxygen species (ROS) are important mediators of cellular signal transduction cascades such as proliferation, migration, and apoptosis. Chronic exposure of isolated ß-cells to proinflammatory cytokines elevates intracellular oxidative stress leading to the demise of pancreatic ß-cells culminating in the onset of diabetes. Although the mitochondrial electron transport chain is felt to be the primary source of ROS, several lines of recent evidence suggest that phagocyte-like NADPH oxidase plays a central role in cytokine-mediated ROS generation and apoptosis of ß-cells. However, the precise mechanisms underlying the regulation of NADPH oxidase remain unknown. To address this, insulin-secreting INS 832/13 cells were treated with cytomix (IL-1ß, IFN-γ, and TNF-α; 10 ng/ml each) for different time intervals (0-24 h). A significant, time-dependent increase in NADPH oxidase activation/intracellular ROS production, p47(phox) subunit, but not p67(phox) subunit, expression of the phagocyte-like NADPH oxidase were demonstrable under these conditions. Furthermore, siRNA-p47(phox) transfection or exposure of INS 832/13 cells to apocynin, a selective inhibitor of NADPH oxidase, markedly attenuated cytomix-induced ROS generation in these cells. Cytomix-mediated mitochondrial dysfunction in INS 832/13 cells was evident by a significant loss of mitochondrial membrane potential (MMP) and upregulated caspase 3 activity. Cytomix treatment also caused a transient (within 15 min) activation of Rac1, a component of the NADPH oxidase holoenzyme. Furthermore, GGTI-2147 and NSC23766, known Rac1 inhibitors, not only attenuated the cytomix-induced Rac1 activation but also significantly prevented loss of MMP (NSC23766 > GGTI-2147). However, NSC23766 had no effect on cytomix-induced NO generation or caspase 3 activation, suggesting additional regulatory mechanisms might underlie these signaling steps. Together, these findings suggested that Rac1-mediated regulation of phagocyte-like NADPH oxidase contributes to cytokine-mediated mitochondrial dysfunction in the ß-cell.

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic beta-cells.

The phagocytic NADPH oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic beta-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic beta-cells. Incubation of isolated beta-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic beta-cells.

Zinc-activated C-peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia-induced phosphatidylserine externalization and reversed by metformin.

Insulin resistance can broadly be defined as the diminished ability of cells to respond to the action of insulin in transporting glucose from the bloodstream into cells and tissues. Here, we report that erythrocytes (ERYs) obtained from type 2 diabetic rats display an apparent resistance to Zn(2+)-activated C-peptide. Thus, the aims of this study were to demonstrate that Zn(2+)-activated C-peptide exerts potentially beneficial effects on healthy ERYs and that these same effects on type 2 diabetic ERYs are enhanced in the presence of metformin. Incubation of ERYs (obtained from type 2 diabetic BBZDR/Wor-rats) with Zn(2+)-activated C-peptide followed by chemiluminescence measurements of ATP resulted in a 31.2 +/- 4.0% increase in ATP release from these ERYs compared to a 78.4 +/- 4.9% increase from control ERYs. Glucose accumulation in diabetic ERYs, measured by scintillation counting of (14)C-labeled glucose, increased by 35.8 +/- 1.3% in the presence of the Zn(2+)-activated C-peptide, a value significantly lower than results obtained from control ERYs (64.3 +/- 5.1%). When Zn(2+)-activated C-peptide was exogenously added to diabetic ERYs, immunoassays revealed a 32.5 +/- 8.2% increase in C-peptide absorbance compared to a 64.4 +/- 10.3% increase in control ERYs. Phosphatidylserine (PS) externalization and metformin sensitization of Zn(2+)-activated C-peptide were examined spectrofluorometrically by measuring the binding of FITC-labeled annexin to PS. The incubation of diabetic ERYs with metformin prior to the addition of Zn(2+)-activated C-peptide resulted in values that were statistically equivalent to those of controls. Summarily, data obtained here demonstrate an apparent resistance to Zn(2+)-activated C-peptide by the ERY that is corrected by metformin.

Personalized metabolic assessment of erythrocytes using microfluidic delivery to an array of luminescent wells.

The metabolic syndrome is often described as a group of risk factors associated with diabetes. These risk factors include, but are not limited to, such conditions as insulin resistance, obesity, high blood pressure, and oxidant stress. Here, we report on a tool that may provide some clarity on the relationship between some of these associated risk factors, especially oxidant stress and hypertension. Specifically, we describe the ability to simultaneously monitor nicotinamide dinucleotide phosphate (NADPH), reduced glutathione (GSH), and shear-induced adenosine triphosphate (ATP) release from erythrocytes using luminescence detection on a microfabricated device. The measurements are performed by delivering erythrocyte lysate (for the NADPH and GSH measurements, two analytes indicative of oxidative stress) or whole red blood cells (RBCs) (for the determination of ATP release from the cells) to an array of wells that contain the necessary reagents to generate a luminescence emission that is proportional to analyte concentration. A fluorescence macrostereomicroscope enables whole-chip imaging of the resultant emission. The concentrations of each NADPH and GSH contained within a 0.7% erythrocyte solution were determined to be 31.06 +/- 4.12 and 22.55 +/- 2.47 microM, respectively, and the average ATP released from a nonlysed 7% erythrocyte solution was determined to be 0.54 +/- 0.04 microM. Collectively, the device represents a precursor to subsequent merging of microfluidics and microtiter-plate technology for high-throughput assessment of metabolite profiles in the diabetic erythrocyte.

Simultaneous determination of cell aging and ATP release from erythrocytes and its implications in type 2 diabetes.

Glucose-6-phosphate dehydrogenase (G6PD) is a determinant in the antioxidant status of the red blood cell (RBC) and is also used as an indicator of cell age. However, it is unknown if the relationship among antioxidant status, cell age, and RBC-derived adenosine triphosphate (ATP) occurs immediately or over a period of time. Therefore, the development of a simultaneous determination of G6PD activity (via the determination of nicotinamide adenine dinucleotide phosphate (NADPH)) in RBCs and the determination of deformation-induced RBC-derived ATP is described. The NADPH and ATP were determined while undergoing a chemically induced aging process via inhibition of G6PD with dehydroepiandroesterone (DHEA). Upon incubation with DHEA for 30 min, NADPH levels measured in a flow stream decreased to 7.96+/-1.10 microM from an original value of 13.20+/-1.80 microM in a 0.02% solution of RBCs. In order to demonstrate a direct relationship between G6PD activity and deformation-induced ATP release from RBCs, a simultaneous microflow determination of G6PD activity and ATP release was performed. Upon inhibition with DHEA, NADPH levels decreased to 8.62+/-0.29 microM from its original value of 12.73+/-0.50 microM while ATP release decreased from 0.21+/-0.07 microM to 0.06+/-0.02 microM. These values were validated by an examination of NADPH levels in, and ATP release from, RBC fractions containing younger and older cells (separated by cell density centrifugation). This determination provides evidence that antioxidant status in the RBC and its ability to release ATP, a known stimulus of nitric oxide production, are closely related.

An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP.

A novel microflow technique is used to demonstrate that a weakened oxidant defense system found in diabetic erythrocytes leads to decreased levels of deformation-induced release of adenosine triphosphate (ATP) from erythrocytes. Addition of an oxidant to rabbit erythrocytes resulted in a 63% decrease in deformation-induced ATP release before eventually recovering to a value that was statistically equivalent to the initial value. Inhibition of glucose-6-phosphate dehydrogenase prevents recovery from the oxidant attack. Finally, results indicated that the ATP release from the erythrocytes of type II diabetics (91 nM +/- 10 nM) was less than half of that measured from the erythrocytes of healthy controls (190 +/- 10 nM). These data suggest that the antioxidant status of erythrocytes is a critical determinant in the ability of these cells to release ATP, a known nitric oxide stimulus.