Type 1 diabetes mellitus in the context of high levels of rural deprivation: differences in demographic and anthropometric characteristics between urban and rural cases in NW Ethiopia.

Background: While there is increasing evidence for an altered clinical phenotype of Type 1 diabetes in several low-and middle-income countries, little is known about urban-rural differences and how the greater poverty of rural environments may alter the pattern of disease. Objective: Investigation of urban-rural differences in demographic and anthropometric characteristics of type 1 diabetes in a resource-poor setting. Research design and methods: Analysis of a unique case register, comprising all patients (rural and urban) presenting with Type 1 diabetes over a 20 yr. period in a poor, geographically defined area in northwest Ethiopia. The records included age, sex, place of residence, together with height and weight at the clinical onset. Results: A total of 1682 new cases of Type 1 diabetes were registered with a mean age of onset of 31.2 (SD 13.4) yr. The patients were thin with 1/3 presenting with a body mass index (BMI) <17kg/m2. There was a striking male predominance of cases when clinical onset was between 20 and 35 yr., this was more marked in the very poor rural dwellers compared to the urban population. While most patients with Type 1 diabetes presented with low BMIs and reduced height, stunting preferentially affected rural men. Conclusions: These data have led to the hypothesis that complex interactions among poor socioeconomic conditions in early life affect both pancreatic function and the development of autoimmunity and provide a possible explanation of the unusual phenotype of Type 1 diabetes in this very poor community.

Diabetic Retinopathy in Type 2 Diabetes Mellitus Patients Attending the Diabetic Clinic of the University of Gondar Hospital, Northwest Ethiopia.

OBJECTIVE: To investigate the level of diabetic retinopathy in type 2 diabetes (T2DM) patients attending the University of Gondar Hospital (UGH) Diabetic Clinic, Northwest Ethiopia. METHODS: An audit was carried out involving a total of 739 T2DM patients attending at the diabetic clinic of UGH. They represented approximately 90% and 50% of all T2DM patients under regular review at the urban and rural diabetic clinics of UGH, respectively. All were supervised by the same clinical team for a long period. Eye examinations were performed for visual acuity, cataract, and retinal changes (retinal photography and slit-lamp biomicroscopy). Body mass index (BMI) and HbA1c levels were measured. The presence or absence of hypertension was recorded. RESULTS: Men constituted 41.5% of the group, the mean age at diagnosis of T2DM was 50.4 years, and 50.2% were hypertensive. The BMI was 25.0 ± 4.1 kg/m2, and HbA1c was 7.75 ± 1.63% (61.2 ± 17.8 mmol/mol) (mean ± SD, for BMI and HbA1c)). Severe visual impairment/blindness was reported in 10.6%, 15.2% had cataract, 16.0% had retinopathy, and 11.1% had maculopathy. The prevalence of retinopathy increased with time from diagnosis of T2DM (chi-square for trend, p < 0.001) and with increasing HbA1c level (chi-square for trend, p=0.03). CONCLUSION: These results compare well with the most recent results in well-equipped, wealthier regions of the world and show the importance of stable healthcare infrastructure for chronic-disease management.

Type 1 diabetes in Africa: an immunogenetic study in the Amhara of North-West Ethiopia.

AIMS/HYPOTHESIS: We aimed to characterise the immunogenic background of insulin-dependent diabetes in a resource-poor rural African community. The study was initiated because reports of low autoantibody prevalence and phenotypic differences from European-origin cases with type 1 diabetes have raised doubts as to the role of autoimmunity in this and similar populations. METHODS: A study of consecutive, unselected cases of recently diagnosed, insulin-dependent diabetes (n = 236, ≤35 years) and control participants (n = 200) was carried out in the ethnic Amhara of rural North-West Ethiopia. We assessed their demographic and socioeconomic characteristics, and measured non-fasting C-peptide, diabetes-associated autoantibodies and HLA-DRB1 alleles. Leveraging genome-wide genotyping, we performed both a principal component analysis and, given the relatively modest sample size, a provisional genome-wide association study. Type 1 diabetes genetic risk scores were calculated to compare their genetic background with known European type 1 diabetes determinants. RESULTS: Patients presented with stunted growth and low BMI, and were insulin sensitive; only 15.3% had diabetes onset at ≤15 years. C-peptide levels were low but not absent. With clinical diabetes onset at ≤15, 16-25 and 26-35 years, 86.1%, 59.7% and 50.0% were autoantibody positive, respectively. Most had autoantibodies to GAD (GADA) as a single antibody; the prevalence of positivity for autoantibodies to IA-2 (IA-2A) and ZnT8 (ZnT8A) was low in all age groups. Principal component analysis showed that the Amhara genomes were distinct from modern European and other African genomes. HLA-DRB1*03:01 (p = 0.0014) and HLA-DRB1*04 (p = 0.0001) were positively associated with this form of diabetes, while HLA-DRB1*15 was protective (p < 0.0001). The mean type 1 diabetes genetic risk score (derived from European data) was higher in patients than control participants (p = 1.60 × 10-7). Interestingly, despite the modest sample size, autoantibody-positive patients revealed evidence of association with SNPs in the well-characterised MHC region, already known to explain half of type 1 diabetes heritability in Europeans. CONCLUSIONS/INTERPRETATION: The majority of patients with insulin-dependent diabetes in rural North-West Ethiopia have the immunogenetic characteristics of autoimmune type 1 diabetes. Phenotypic differences between type 1 diabetes in rural North-West Ethiopia and the industrialised world remain unexplained.

Type 1 Diabetes in a Resource-Poor Setting: Malnutrition Related, Malnutrition Modified, or Just Diabetes?

PURPOSE OF REVIEW: Very little is known about the occurrence of type 1 diabetes (T1DM) in resource-poor countries and particularly in their rural hinterlands. RECENT FINDINGS: Studies of the epidemiology of T1DM in Ethiopia and similar countries in sub-Saharan Africa show that the pattern of presenting disease differs substantially from that in the West. Typically, the peak age of onset of the disease is more than a decade later with a male excess and a low prevalence of indicators of islet-cell autoimmunity. It is also associated with markers of undernutrition. These findings raise the question as to whether the principal form of T1DM seen in these resource-poor communities has a different pathogenesis. Whether the disease is a direct result of malnutrition or whether malnutrition may modify the expression of islet-cell autoimmunity is unclear. However, the poor prognosis in these settings underlines the urgent need for detailed clinical and epidemiological studies.

Retinopathy in type 1 diabetes mellitus: Major differences between rural and urban dwellers in northwest Ethiopia.

AIM: To audit levels of diabetes-related eye disease in Type 1 diabetes mellitus (T1DM) patients in northwest Ethiopia. In particular to establish whether, despite identical clinical goals, major differences between the physically demanding life-style of rural subsistence farmers and the sedentary life-style of urban dwellers would influence the prevalence of diabetes-related eye complications. METHODS: A robust infrastructure for chronic disease management that comprehensively includes all rural dwellers was a pre-requisite for the investigation. A total of 544 T1DM were examined, representing 80% of all T1DM patients under regular review at both the urban and rural clinics and representative of patient age and gender (62.1% male, 37.9% female) of T1DM patients from this region; all were supervised by the same clinical team. Eye examinations were performed for visual acuity, cataract and retinal changes (retinal photography). HbA1c levels and the presence or absence of hypertension were recorded. RESULTS/CONCLUSIONS: Urban and rural groups had similar prevalences of severe visual impairment/blindness (7.0% urban, 5.2% rural) and cataract (7.3% urban, 7.1% rural). By contrast, urban dwellers had a significantly higher prevalence of retinopathy compared to rural patients, 16.1% and 5.0%, respectively (OR 2.9, p<0.02, after adjustment for duration, age, gender and hypertension). There was a 3-fold greater prevalence of hypertension in urban patients, whereas HbA1c levels were similar in the two groups. Since diabetic retinopathy is closely associated with microvascular disease and endothelial dysfunction, the possible influences of hypertension to increase and of sustained physical activity to reduce endothelial dysfunction are discussed.

The hyperglycemia and adverse pregnancy outcome study: associations of GDM and obesity with pregnancy outcomes.

OBJECTIVE: To determine associations of gestational diabetes mellitus (GDM) and obesity with adverse pregnancy outcomes in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. RESEARCH DESIGN AND METHODS: Participants underwent a 75-g oral glucose tolerance test (OGTT) between 24 and 32 weeks. GDM was diagnosed post hoc using International Association of Diabetes and Pregnancy Study Groups criteria. Neonatal anthropometrics and cord serum C-peptide were measured. Adverse pregnancy outcomes included birth weight, newborn percent body fat, and cord C-peptide >90th percentiles, primary cesarean delivery, preeclampsia, and shoulder dystocia/birth injury. BMI was determined at the OGTT. Multiple logistic regression was used to examine associations of GDM and obesity with outcomes. RESULTS: Mean maternal BMI was 27.7, 13.7% were obese (BMI ≥33.0 kg/m(2)), and GDM was diagnosed in 16.1%. Relative to non-GDM and nonobese women, odds ratio for birth weight >90th percentile for GDM alone was 2.19 (1.93-2.47), for obesity alone 1.73 (1.50-2.00), and for both GDM and obesity 3.62 (3.04-4.32). Results for primary cesarean delivery and preeclampsia and for cord C-peptide and newborn percent body fat >90th percentiles were similar. Odds for birth weight >90th percentile were progressively greater with both higher OGTT glucose and higher maternal BMI. There was a 339-g difference in birth weight for babies of obese GDM women, compared with babies of normal/underweight women (64.2% of all women) with normal glucose based on a composite OGTT measure of fasting plasma glucose and 1- and 2-h plasma glucose values (61.8% of all women). CONCLUSIONS: Both maternal GDM and obesity are independently associated with adverse pregnancy outcomes. Their combination has a greater impact than either one alone.

Frequency of gestational diabetes mellitus at collaborating centers based on IADPSG consensus panel-recommended criteria: the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study.

OBJECTIVE: To report frequencies of gestational diabetes mellitus (GDM) among the 15 centers that participated in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study using the new International Association of the Diabetes and Pregnancy Study Groups (IADPSG) criteria. RESEARCH DESIGN AND METHODS: All participants underwent a 75-g oral glucose tolerance test between 24 and 32 weeks' gestation. GDM was retrospectively classified using the IADPSG criteria (one or more fasting, 1-h, or 2-h plasma glucose concentrations equal to or greater than threshold values of 5.1, 10.0, or 8.5 mmol/L, respectively). RESULTS: Overall frequency of GDM was 17.8% (range 9.3-25.5%). There was substantial center-to-center variation in which glucose measures met diagnostic thresholds. CONCLUSIONS: Although the new diagnostic criteria for GDM apply globally, center-to-center differences occur in GDM frequency and relative diagnostic importance of fasting, 1-h, and 2-h glucose levels. This may impact strategies used for the diagnosis of GDM.

Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study: associations of maternal A1C and glucose with pregnancy outcomes.

OBJECTIVE: To compare associations of maternal glucose and A1C with adverse outcomes in the multinational Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study and determine, based on those comparisons, if A1C measurement can provide an alternative to an oral glucose tolerance test (OGTT) in pregnant women. RESEARCH DESIGN AND METHODS: Eligible pregnant women underwent a 75-g OGTT at 24-32 weeks' gestation. A sample for A1C was also collected. Neonatal anthropometrics and cord serum C-peptide were measured. Associations with outcomes were assessed using multiple logistic regression with adjustment for potential confounders. RESULTS: Among 23,316 HAPO Study participants with glucose levels blinded to caregivers, 21,064 had a nonvariant A1C result. The mean ± SD A1C was 4.79 ± 0.40%. Associations were significantly stronger with glucose measures than with A1C for birth weight, sum of skinfolds, and percent body fat >90th percentile and for fasting and 1-h glucose for cord C-peptide (all P < 0.01). For example, in fully adjusted models, odds ratios (ORs) for birth weight >90th percentile for each measure higher by 1 SD were 1.39, 1.45, and 1.38, respectively, for fasting, 1-, and 2-h plasma glucose and 1.15 for A1C. ORs for cord C-peptide >90th percentile were 1.56, 1.45, and 1.35 for glucose, respectively, and 1.32 for A1C. ORs were similar for glucose and A1C for primary cesarean section, preeclampsia, and preterm delivery. CONCLUSIONS: On the basis of associations with adverse outcomes, these findings suggest that A1C measurement is not a useful alternative to an OGTT in pregnant women.

Restoration of adipose function in obese glucose-tolerant men following pioglitazone treatment is associated with CCAAT enhancer-binding protein ß up-regulation.

Obese AT (adipose tissue) exhibits increased macrophage number. Pro-inflammatory CD16+ peripheral monocyte numbers are also reported to increase with obesity. The present study was undertaken to simultaneously investigate obesity-associated changes in CD16+ monocytes and ATMs (AT macrophages). In addition, a pilot randomized placebo controlled trial using the PPAR (peroxisome-proliferator-activated receptor) agonists, pioglitazone and fenofibrate was performed to determine their effects on CD14+/CD16+ monocytes, ATM and cardiometabolic and adipose dysfunction indices. Obese glucose-tolerant men (n=28) were randomized to placebo, pioglitazone (30 mg/day) and fenofibrate (160 mg/day) for 12 weeks. A blood sample was taken to assess levels of serum inflammatory markers and circulating CD14+/CD16+ monocyte levels via flow cytometry. A subcutaneous AT biopsy was performed to determine adipocyte cell surface and ATM number, the latter was determined via assessment of CD68 expression by IHC (immunohistochemistry) and real-time PCR. Subcutaneous AT mRNA expression of CEBPß (CCAAT enhancer-binding protein ß), SREBP1c (sterol-regulatory-element-binding protein 1c), PPARγ2, IRS-1 (insulin receptor substrate-1), GLUT4 (glucose transporter type 4) and TNFα (tumour necrosis factor α) were also assessed. Comparisons were made between obese and lean controls (n=16) at baseline, and pre- and post-PPAR agonist treatment. Obese individuals had significantly increased adipocyte cell surface, percentage CD14+/CD16+ monocyte numbers and ATM number (all P=0.0001). Additionally, serum TNF-α levels were significantly elevated (P=0.017) and adiponectin levels reduced (total: P=0.0001; high: P=0.022) with obesity. ATM number and percentage of CD14+/CD16+ monocytes correlated significantly (P=0.05). Pioglitazone improved adiponectin levels significantly (P=0.0001), and resulted in the further significant enlargement of adipocytes (P=0.05), without effect on the percentage CD14+/CD16+ or ATM number. Pioglitazone treatment also significantly increased subcutaneous AT expression of CEBPß mRNA. The finding that improvements in obesity-associated insulin resistance following pioglitazone were associated with increased adipocyte cell surface and systemic adiponectin levels, supports the centrality of AT to the cardiometabolic derangement underlying the development of T2D (Type 2 diabetes) and CVD (cardiovascular disease).

Linoleic acid increases monocyte chemotaxis and adhesion to human aortic endothelial cells through protein kinase C- and cyclooxygenase-2-dependent mechanisms.

The effects of polyunsaturated n-6 linoleic acid on monocyte-endothelial interactions were investigated with particular emphasis on the expression of platelet/endothelial cell adhesion molecule (PECAM)-1 and the role of protein kinase C (PKC) and cyclooxygenase-2 (COX-2). As a diet rich in polyunsaturated fatty acids may favour atherosclerosis in hyperglycaemia, this study was performed in both normal and high-glucose media using human aortic endothelial cells (HAEC). The HAEC were preincubated with normal (5 mM) or high (25 mM) D-glucose for 3 days before addition of fatty acids (0.2 mM) for 3 days. Linoleic acid enhanced PECAM-1 expression independently of tumor necrosis factor (TNF)-α and significantly increased TNF-α-induced monocyte adhesion to HAEC in comparison to the monounsaturated n-9 oleic acid. Chronic glucose treatment (25 mM, 6 days) did not modify the TNF-α-induced or fatty acid-induced changes in monocyte binding. The increase in monocyte binding was accompanied by a significant increase in E-selectin and vascular cell adhesion molecule (VCAM)-1 expression and could be abrogated by an interleukin (IL)-8 neutralising antibody and by the PKC and COX inhibitors. Inhibition of PKC-δ reduced VCAM-1 expression regardless of experimental condition and was accompanied by a significant decrease in monocyte binding. Conditioned medium from linoleic acid-treated HAEC grown in normal glucose conditions significantly increased THP-1 chemotaxis. These results suggest that linoleic acid-induced changes in monocyte chemotaxis and subsequent binding are not solely mediated by changes in adhesion molecule expression but may be due to secreted factors such as IL-8, monocyte chemoattractant protein-1 or prostaglandins (PGs) such as PGE(2), as IL-8 neutralisation and COX-2 inhibition reduced monocyte binding without changes in adhesion molecule expression.

Hyperglycemia and adverse pregnancy outcome study: neonatal glycemia.

OBJECTIVE: The goal was to describe the temporal pattern of neonatal plasma glucose levels and associations with maternal glucose levels, cord serum C-peptide levels, and neonatal size and adiposity. METHODS: A total of 17,094 mothers and infants were included in the Hyperglycemia and Adverse Pregnancy Outcome Study (15 centers in 9 countries). Mothers underwent a 75-g, 2-hour, oral glucose tolerance test (OGTT) at 24 to 32 weeks of gestation. Cord blood and neonatal blood samples were collected. Biochemical neonatal hypoglycemia was defined as glucose levels of <10th percentile (2.2 mmol/L). Clinically identified hypoglycemia was ascertained through medical record review and associations were assessed. RESULTS: Plasma glucose concentrations were stable during the first 5 hours after birth. Maternal glucose levels were weakly positively associated with biochemical neonatal hypoglycemia (odds ratios: 1.07-1.14 for 1-SD higher OGTT glucose levels). Frequency of neonatal hypoglycemia was higher with higher cord C-peptide levels (odds ratio: 11.6 for highest versus lowest C-peptide category). Larger and/or fatter infants were more likely to have hypoglycemia (P < .001), and infants with hypoglycemia tended to have a higher frequency of cord C-peptide levels of >90th percentile. CONCLUSIONS: Mean neonatal plasma glucose concentrations varied little in the first 5 hours after birth, which suggests normal postnatal adjustment. Biochemical and clinical hypoglycemia were weakly related to maternal OGTT glucose measurements but were strongly associated with elevated cord serum C-peptide levels. Larger and/or fatter infants were more likely to develop hypoglycemia and hyperinsulinemia. These relationships suggest physiologic relationships between maternal glycemia and fetal insulin production.

Hyperglycemia and adverse pregnancy outcomes.

BACKGROUND: It is controversial whether maternal hyperglycemia less severe than that in diabetes mellitus is associated with increased risks of adverse pregnancy outcomes. METHODS: A total of 25,505 pregnant women at 15 centers in nine countries underwent 75-g oral glucose-tolerance testing at 24 to 32 weeks of gestation. Data remained blinded if the fasting plasma glucose level was 105 mg per deciliter (5.8 mmol per liter) or less and the 2-hour plasma glucose level was 200 mg per deciliter (11.1 mmol per liter) or less. Primary outcomes were birth weight above the 90th percentile for gestational age, primary cesarean delivery, clinically diagnosed neonatal hypoglycemia, and cord-blood serum C-peptide level above the 90th percentile. Secondary outcomes were delivery before 37 weeks of gestation, shoulder dystocia or birth injury, need for intensive neonatal care, hyperbilirubinemia, and preeclampsia. RESULTS: For the 23,316 participants with blinded data, we calculated adjusted odds ratios for adverse pregnancy outcomes associated with an increase in the fasting plasma glucose level of 1 SD (6.9 mg per deciliter [0.4 mmol per liter]), an increase in the 1-hour plasma glucose level of 1 SD (30.9 mg per deciliter [1.7 mmol per liter]), and an increase in the 2-hour plasma glucose level of 1 SD (23.5 mg per deciliter [1.3 mmol per liter]). For birth weight above the 90th percentile, the odds ratios were 1.38 (95% confidence interval [CI], 1.32 to 1.44), 1.46 (1.39 to 1.53), and 1.38 (1.32 to 1.44), respectively; for cord-blood serum C-peptide level above the 90th percentile, 1.55 (95% CI, 1.47 to 1.64), 1.46 (1.38 to 1.54), and 1.37 (1.30 to 1.44); for primary cesarean delivery, 1.11 (95% CI, 1.06 to 1.15), 1.10 (1.06 to 1.15), and 1.08 (1.03 to 1.12); and for neonatal hypoglycemia, 1.08 (95% CI, 0.98 to 1.19), 1.13 (1.03 to 1.26), and 1.10 (1.00 to 1.12). There were no obvious thresholds at which risks increased. Significant associations were also observed for secondary outcomes, although these tended to be weaker. CONCLUSIONS: Our results indicate strong, continuous associations of maternal glucose levels below those diagnostic of diabetes with increased birth weight and increased cord-blood serum C-peptide levels.

Integration of local and central laboratory functions in a worldwide multicentre study: Experience from the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study.

BACKGROUND: Measurement of glucose in multiple Field Laboratories requires rigorous standardization when patients and caregivers are masked, unless predefined thresholds are met. Local misclassification of participants at the thresholds can introduce recruitment bias and adversely affect the integrity of study findings. PURPOSE: To describe the challenges and the approach to meeting them in measuring glucose, HbA1c, and C-peptide in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. HAPO is an observational epidemiologic study of 25 000 pregnant women from 15 centres in 10 countries, designed to clarify unanswered questions on associations of maternal glycemia, less severe than overt diabetes mellitus, with risks of adverse pregnancy outcome. METHODS: Glucose tolerance (75 g two-hour OGTT) is assessed locally at 24-32 weeks' gestation, with results masked if fasting and two-hour plasma glucose are

Activation of peripheral blood CD14+ monocytes occurs in diabetes.

Blood levels of inflammatory markers associated with endothelial dysfunction and atherosclerosis are increased in diabetic patients; the highest levels occur in poorly controlled diabetes. We investigated the activation state of peripheral blood monocytes in diabetes with respect to scavenger receptor (CD36) expression and monocyte chemoattractant protein-1, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and peroxisome proliferator-activated receptors mRNA expression. CD14(+) monocytes were isolated from peripheral blood of type 1 and type 2 diabetic patients with good (HbA(1c) <7.0%) or poor (>9.4%) glycemic control and a group of nondiabetic subjects. Monocytes from diabetic subjects displayed increased CD36 cell surface expression (P < 0.0005) and increased uptake of oxidized LDL (P < 0.05). Monocyte chemoattractant protein-1 gene expression was increased in monocytes from both groups of diabetic subjects (P < 0.05). Both CD68 and peroxisome proliferator-activated receptor-gamma gene expression were increased in the poorly controlled diabetic group (P < 0.05 for each), whose monocytes also displayed increased attachment to endothelial monolayers (P < 0.0005 vs. nondiabetic control subjects). In poorly controlled diabetes, CD14(+) monocytes are functionally activated and show some of the differentiation markers associated with macrophages. These monocytes also demonstrate an increased ability for attachment to normal endothelial cells, one of the early stages in atherogenesis.

Modification of PI3K- and MAPK-dependent chemotaxis in aortic vascular smooth muscle cells by protein kinase CbetaII.

Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKCalpha and PKCbetaII levels were increased in 25 mmol/L glucose. However, only PKCbetaII inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKCbetaII were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKCbetaII inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKCbetaII was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKCbetaII-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.

High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells.

Diabetes is associated with oxidative stress and increased concentrations of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with increased glucose concentrations on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High-glucose (25 mmol/l) conditions reduced glutathione (GSH) concentrations in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant, alpha-lipoic acid, resulted in the restoration of GSH concentrations to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid, and completely blocked with inhibition of nuclear factor kappa B (NFkappaB) activity. Stimulation of cytokines (interleukin-1 beta, tumour necrosis factor-alpha, interferon-gamma) induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappaB activity decreased, but did not completely inhibit, cytokine-induced iNOS promoter activity and subsequent production of NO. In conclusion, iNOS promoter activity induced by high concentrations of glucose is mediated in part through intracellular GSH and NFkappaB.

Glucose-potentiated chemotaxis in human vascular smooth muscle is dependent on cross-talk between the PI3K and MAPK signaling pathways.

Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110beta isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110beta-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.

Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-beta receptor potentiates vascular smooth muscle cell chemotaxis.

The aim of this study was to investigate the effects of elevated D-glucose concentrations on vascular smooth muscle cell (VSMC) expression of the platelet-derived growth factor (PDGF)beta receptor and VSMC migratory behavior. Immunoprecipitation, immunofluorescent staining, and RT-PCR of human VSMCs showed that elevated D-glucose induced an increase in the PDGFbeta receptor that was inhibited by phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors. Exposure to 25 mmol/l D-glucose (HG) induced increased phosphorylation of protein kinase B (PKB) and extracellular-regulated kinase (ERK). All HG chemotaxis assays (with either 10 days' preincubation in HG or no preincubation) in a FCS or PDGF-BB gradient showed positive chemotaxis, whereas those in 5 mmol/l D-glucose did not. Assays were also run with concentrations ranging from 5 to 25 mmol/l D-glucose. Chemotaxis was induced at concentrations > or =9 mmol/l D-glucose. An anti-PDGFbeta receptor antibody inhibited glucose-potentiated VSMC chemotaxis, as did the inhibitors for the PI3K and MAPK pathways. This study has shown that small increases in D-glucose concentration, for a short period, increase VSMC expression of the PDGFbeta receptor and VSMC sensitivity to chemotactic factors in serum, leading to altered migratory behavior in vitro. It is probable that similar processes occur in vivo with glucose-enhanced chemotaxis of VSMCs, operating through PDGFbeta receptor-operated pathways, contributing to the accelerated formation of atheroma in diabetes.

Rapid isolation of VLDL subfractions: assessment of composition and susceptibility to copper-mediated oxidation.

VLDLs, synthesized and released by the liver, are a heterogeneous group of particles of varying composition and metabolic fates. A method is described for the rapid isolation of VLDL into four subfractions (A-D) and assessment of their susceptibility to oxidation. The total isolation procedure required less than 3.5 h, and was achieved by gradient ultracentrifugation. Each subfraction was assessed for triglyceride, cholesterol, and apolipoprotein B (apoB) composition and for the presence of contaminants such as albumin and urate. The oxidation potential, in the presence of copper ions, of each subfraction was also assessed. This rapid procedure produced VLDL fractions analogous to those produced by a previously reported but more prolonged isolation method. Comparison of the two procedures demonstrated that lipid and apoB were similar, while the rapid procedure produced subfractions void of albumin and urate contamination and lower in preformed hydroperoxides. Compositional changes were found between the subfractions: as the subfractions became smaller and more dense (A-->D), there was a decrease in the ratio of triglyceride to apoB and an increase in the ratio of cholesterol to apoB, also arachidonic acid was increased in subfraction D compared with subfractions A, B, and C. The smaller subfractions were more susceptible to oxidation, a trend similar to that reported previously for the oxidation of LDL subfractions.