Interaction of glucose and long chain fatty acids (C18) on antioxidant defences and free radical damage in porcine vascular smooth muscle cells in vitro.

Aims/hypothesis: Abnormalities of glucose and fatty acid metabolism in diabetes are believed to contribute to the development of oxidative stress and the long term vascular complications of the disease therefore the interactions of glucose and long chain fatty acids on free radical damage and endogenous antioxidant defences were investigated in vascular smooth muscle cells. Methods: Porcine vascular smooth muscle cells were cultured in 5 mmol/l or 25 mmol/l glucose for ten days. Fatty acids, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and gamma-linolenic acid (18:3) were added with defatted bovine serum albumin as a carrier for the final three days. Results. Glucose (25 mmol/l) alone caused oxidative stress in the cells as evidenced by free radical-mediated damage to DNA, lipids, and proteins. The addition of fatty acids (0.2 mmol/l) altered the profile of free radical damage; the response was J-shaped with respect to the degree of unsaturation of each acid, and oleic acid was associated with least damage. The more physiological concentration (0.01 mmol/l) of gamma-linolenic acids was markedly different in that, when added to 25 mmol/l glucose it resulted in a decrease in free radical damage to DNA, lipids and proteins. This was due to a marked increase in levels of the antioxidant, glutathione, and increased gene expression of the rate-limiting enzyme in glutathione synthesis, gamma-glutamylcysteine synthetase. Conclusion/Interpretation: The results clearly show that glucose and fatty acids interact in the production of oxidative stress in vascular smooth muscle cells.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Effect of a COL1A1 Sp1 Binding Site Polymorphism on Arterial Pulse Wave Velocity: An Index of Compliance.

Reduced arterial compliance precedes changes in blood pressure, which may be mediated through alterations in vessel wall matrix composition. We investigated the effect of the collagen type I-1 gene (COL1A1) +2046G>T polymorphism on arterial compliance in healthy individuals. We recruited 489 subjects (251 men and 238 women; mean age, 22.6±1.6 years). COL1A1 genotypes were determined using polymerase chain reaction and digestion by restriction enzyme Bal1. Arterial pulse wave velocities were measured in 3 segments, aortoiliac (PWVA), aortoradial (PWVB), and aorto-dorsalis-pedis (PWVF), as an index of compliance using a noninvasive optical method. Data were available for 455 subjects. The sample was in Hardy-Weinberg equilibrium with genotype distributions and allele frequencies that were not significantly different from those reported previously. The T allele frequency was 0.22 (95% confidence interval, 0.19 to 0.24). Two hundred eighty-three (62.2%) subjects were genotype GG, 148 (35.5%) subjects were genotype GT, and 24 (5.3%) subjects were genotype TT. A comparison of GG homozygotes with GT and TT individuals demonstrated a statistically significant association with arterial compliance: PWVF 4.92±0.03 versus 5.06±0.05 m/s (ANOVA, P=0.009), PWVB 4.20±0.03 versus 4.32±0.04 m/s (ANOVA, P=0.036), and PWVA 3.07±0.03 versus 3.15±0.03 m/s (ANOVA, P=0.045). The effects of genotype were independent of age, gender, smoking, mean arterial pressure, body mass index, family history of hypertension, and activity scores. We report an association between the COL1A1 gene polymorphism and arterial compliance. Alterations in arterial collagen type 1A deposition may play a role in the regulation of arterial compliance

Higher incidence of childhood-onset type 1 diabetes mellitus in remote areas: a UK regional small-area analysis

Aims/hypothesis: We investigated the association between the incidence of type 1 diabetes mellitus and remoteness (a proxy measure for exposure to infections) using recently developed techniques for statistical analysis of small-area data.

Subjects, materials and methods: New cases in children aged 0 to 14 years in Northern Ireland were prospectively registered from 1989 to 2003. Ecological analysis was conducted using small geographical units (582 electoral wards) and area characteristics including remoteness, deprivation and child population density. Analysis was conducted using Poisson regression models and Bayesian
hierarchical models to allow for spatially correlated risks that were potentially caused by unmeasured explanatory variables.

Results: In Northern Ireland between 1989 and 2003, there were 1,433 new cases of type 1 diabetes, giving a directly standardised incidence rate of 24.7 per 100,000 personyears. Areas in the most remote fifth of all areas had a significantly (p=0.0006) higher incidence of type 1 diabetes mellitus (incidence rate ratio=1.27 [95% CI 1.07, 1.50]) than those in the most accessible fifth of all areas. There was also a higher incidence rate in areas that were less deprived (p<0.0001) and less densely populated (p=0.002). After adjustment for deprivation and additional adjustment for child population density the association between diabetes and remoteness remained significant (p=0.01 and p=0.03, respectively).

Conclusions/interpretation: In Northern Ireland, there is evidence that remote areas experience higher rates of type 1 diabetes mellitus. This could reflect a reduced or delayed exposure to infections, particularly early in life, in these areas.