Tyrosinemia type II: Mutation update, 11 novel mutations and description of 5 independent subjects with a novel founder mutation.

BACKGROUND: Tyrosinemia type II, also known as Richner-Hanhart Syndrome, is an extremely rare autosomal recessive disorder, caused by mutations in the gene encoding hepatic cytosolic tyrosine aminotransferase, leading to the accumulation of tyrosine and its metabolites which cause ocular and skin lesions, that may be accompanied by neurological manifestations, mostly intellectual disability. AIMS: To update disease-causing mutations and current clinical knowledge of the disease. MATERIALS AND METHODS: Genetic and clinical information were obtained from a collection of both unreported and previously reported cases. RESULTS: We report 106 families, represented by 143 individuals, carrying a total of 36 genetic variants, 11 of them not previously known to be associated with the disease. Variants include 3 large deletions, 21 non-synonymous and 5 nonsense amino-acid changes, 5 frameshifts and 2 splice variants. We also report 5 patients from Gran Canaria, representing the largest known group of unrelated families sharing the same P406L mutation. CONCLUSIONS: Data analysis did not reveal a genotype-phenotype correlation, but stressed the need of early diagnosis: All patients improved the oculocutaneous lesions after dietary treatment but neurological symptoms prevailed. The discovery of founder mutations in isolated populations, and the benefits of early intervention, should increase diagnostic awareness in newborns.

Insulin-requiring diabetes in Ethiopia: associations with poverty, early undernutrition and anthropometric disproportion.

BACKGROUND/OBJECTIVES: Most insulin-requiring diabetes patients in Ethiopia have an atypical form of the disease, which resembles previous descriptions of malnutrition-related diabetes. As so little is known about its aetiology, we have carried out a case-control study to evaluate its social and nutritional determinants. SUBJECTS/METHODS: Men and women with insulin-requiring diabetes (n=107), aged 18-40 years, were recruited in two centres, Gondar and Jimma, 750 km northwest and 330 km southwest of the capital, Addis Ababa, respectively. Controls of similar age and sex (n=110) were recruited from patients attending other hospital clinics. RESULTS: Diabetes was strongly associated with subsistence farming, odds ratio=3.5 (95% confidence interval: 1.5-7.8) and illiteracy/low levels of education, odds ratio=4.0 (2.0-8.0). Diabetes was also linked with a history of childhood malnutrition, odds ratio=5.5 (1.0-29.0) the mother's death during childhood, odds ratio=3.9 (1.0-14.8), and markers of poverty including poorer access to sanitation (P=0.004), clean water (P=0.009), greater overcrowding (P=0.04), increased distance from the clinic (P=0.01) and having fewer possessions (P=0.01). Compared with controls, people with diabetes had low mid upper arm circumference, body mass index (BMI) and fat/lean body mass (P<0.01). In addition, men with the disease tended to be shorter, were lighter (P=0.001), with reduced sitting height (P=0.015) and reduced biacromial (P=0.003) and bitrochanteric (P=0.008) diameters. CONCLUSIONS: Insulin-requiring diabetes in Ethiopia is strongly linked with poor education and markers of poverty. Men with the disease have associated disproportionate skeletal growth. These findings point towards a nutritional aetiology for this condition although the nature of the nutritional deficiency and its timing during growth and development remains obscure.

Insulin-requiring diabetes in rural Ethiopia: should we reopen the case for malnutrition-related diabetes?

AIMS/HYPOTHESIS: We evaluated the incidence of insulin-requiring diabetes in a rural area of sub-Saharan Africa. METHODS: Health surveillance data from a chronic disease programme in two zones of Ethiopia, Gondar and Jimma, were studied. The two zones have a population of more than 5,000,000 people. RESULTS: In Gondar Zone (1995-2008) and Jimma Zone (2002-2008) 2,280 patients presented with diabetes, of whom 1,029 (45%) required insulin for glycaemic control at diagnosis. The annual incidence of insulin-requiring diabetes was 2.1 (95% CI 2.0-2.2) per 100,000 and was twice as high in men (2.9 per 100,000) as in women (1.4 per 100,000). In both sexes incidence rates peaked at the age of 25 to 29 years. Incidence rates in the urban areas of Gondar and Jimma were five times higher than in the surrounding rural areas. Patients with insulin-requiring diabetes from rural and urban areas had a very low BMI and most were subsistence farmers or unemployed. CONCLUSIONS/INTERPRETATION: The typical patient with diabetes in rural Ethiopia is an impoverished, young adult male with severe symptoms requiring insulin for glycaemic control. The low incidence rates in rural compared with urban areas suggest that many cases of this disease remain undiagnosed. The disease phenotype encountered in this area of Africa is very different from the classical type 1 diabetes seen in the West and most closely resembles previous descriptions of malnutrition-related diabetes, a category not recognised in the current WHO Diabetes Classification. We believe that the case for this condition should be reopened.

Glucose lowers the threshold for human aortic vascular smooth muscle cell migration: inhibition by protein phosphatase-2A.

AIMS/HYPOTHESIS: Atherosclerosis, which occurs prematurely in individuals with diabetes, incorporates vascular smooth muscle cell (VSMC) chemotaxis. Glucose, through protein kinase C-beta(II) signalling, increases chemotaxis to low concentrations of platelet-derived growth factor (PDGF)-BB. In VSMC, a biphasic response in PDGF-beta receptor (PDGF-betaR) level occurs as PDGF-BB concentrations increase. The purpose of this study was to determine whether increased concentrations of PDGF-BB and raised glucose level had a modulatory effect on the mitogen-activated protein kinase/extracellular-regulated protein kinase pathway, control of PDGF-betaR level and chemotaxis. METHODS: Cultured aortic VSMC, exposed to normal glucose (NG) (5 mmol/l) or high glucose (HG) (25 mmol/l) in the presence of PDGF-BB, were assessed for migration (chemotaxis chamber) or else extracted and immunoblotted. RESULTS: At concentrations of PDGF-BB <540 pmol/l, HG caused an increase in the level of PDGF-betaR in VSMC (immunoblotting) versus NG, an effect that was abrogated by inhibition of aldose reductase or protein kinase C-beta(II). At higher concentrations of PDGF-BB (>540 pmol/l) in HG, receptor level was reduced but in the presence of aldose reductase or protein kinase C-beta(II) inhibitors the receptor levels increased. It is known that phosphatases may be activated at high concentrations of growth factors. At high concentrations of PDGF-BB, the protein phosphatase (PP)2A inhibitor, endothall, caused an increase in PDGF-betaR levels and a loss of biphasicity in receptor levels in HG. At higher concentrations of PDGF-BB in HG, the chemoattractant effect of PDGF-BB was lost (chemotaxis chamber). Under these conditions inhibition of PP2A was associated with a restoration of chemotaxis to high concentrations of PDGF-BB. CONCLUSION/INTERPRETATION: The biphasic response in PDGF-betaR level and in chemotaxis to PDGF-BB in HG is due to PP2A activation.

Fenofibrate and pioglitazone improve endothelial function and reduce arterial stiffness in obese glucose tolerant men.

Obesity is a low grade inflammatory state associated with premature cardiovascular morbidity and mortality. Along with traditional risk factors the measurement of endothelial function, insulin resistance, inflammation and arterial stiffness may contribute to the assessment of cardiovascular risk. We conducted a randomised placebo controlled trial to assess the effects of 12 weeks treatment with a PPAR alpha agonist (fenofibrate) and a PPAR gamma agonist (pioglitazone) on these parameters in obese glucose tolerant men. Arterial stiffness was measured using augmentation index and pulse wave velocity (PWV). E-selectin, VCAM-1 and ICAM-1 were used as markers of endothelial function. Insulin sensitivity improved with pioglitazone treatment (p=0.001) and, in keeping with this, adiponectin increased by 85.2% (p<0.001). Pro-inflammatory cytokine levels (TNFalpha, IL-6 and IL-1 beta) fell with both treatments (p<0.01 for TNFalpha and IL-1 beta, p<0.001 for IL-6). VCAM-1 and ICAM-1 were reduced with both treatments (p<0.001 for VCAM-1, p<0.05 for ICAM-1) and E-selectin improved with pioglitazone treatment (p=0.05). Both treatments resulted in a fall in augmentation index. PWV fell by 17.4% with fenofibrate treatment (p<0.001) and 16.3% with pioglitazone treatment (p<0.001). Pioglitazone and fenofibrate treatment of obese, glucose tolerant men reduces inflammation, improves markers of endothelial function and reduces arterial stiffness. These results suggest that treatment with PPAR agonists has potential to reduce the incidence of premature cardiovascular disease associated with obesity.

Acute respiratory distress syndrome in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies.

Inborn errors of metabolism have not previously been recognized as a risk factor for acute respiratory distress syndrome (ARDS). We report this complication in four patients with defects of the mitochondrial trifunctional protein (MTP). This enzyme catalyses three steps in the beta-oxidation of long-chain fatty acids. Three of the patients were homozygous for the 'common' 1528G>C mutation in the alpha-subunit of the MTP, giving rise to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The fourth patient did not carry this mutation but had severely decreased activities of long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-ketoacyl-CoA thiolase. One patient died and histology in this patient showed severe interstitial pulmonary fibrosis. The other three patients recovered after being ventilated for up to 6 months. The high frequency of ARDS in patients with MTP defects suggests that this inborn error may be a risk factor for ARDS.

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.

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 10 days. Fatty acids, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and alpha-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. At a lower concentration alpha-linolenic acid (0.01 mmol/l) 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 accompanied by a marked increase in antioxidant and glutathione concentrations as well as by increased gene expression is of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. CONCLUSIONS/INTERPRETATION: The results clearly show that glucose and fatty acids interact in the production of oxidative stress in vascular smooth muscle cells.

Follow up of fetal outcome in cases of maternal phenylketonuria in Northern Ireland.

BACKGROUND: Maternal phenylketonuria (PKU) can result in multiple congenital anomalies. In Northern Ireland, the prevalence of PKU is relatively high at 1 in 4000. OBJECTIVE: To assess the outcome of 39 pregnancies in 20 mothers. RESULTS: Dietary control was established before conception in 17 pregnancies (44%). Five mothers with hyperphenylalaninaemia had 11 pregnancies. There were no congenital anomalies in this group, and all appear to be developing normally. Fifteen women with classical PKU had 28 pregnancies. One pregnancy ended in a first trimester miscarriage. Twelve out of 27 (44%) completed pregnancies produced babies with a congenital anomaly and/or developmental delay. CONCLUSIONS: Most problems occurred when dietary control was not established until after the 2nd trimester. As the cohort of young women with treated PKU is growing steadily, maternal PKU is going to become an even greater cause for concern.

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions.

Hyperglycemia-induced oxidative stress may play a key role in the pathogenesis of diabetic vascular disease. The purpose of this study was to determine the effects of glucose on levels of glutathione (a major intracellular antioxidant), the expression of gamma-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione de novo synthesis), and DNA damage in human vascular smooth muscle cells in vitro. High glucose conditions and buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, reduced intracellular glutathione levels in vascular smooth muscle cells. This reduction was accompanied by a decrease in the mRNA expression of both subunits of gamma-glutamylcysteine synthetase as well as an increase in DNA damage. In high glucose conditions, incubation of the vascular smooth muscle cells with alpha-lipoic acid and L-cystine restored glutathione levels. We suggest that the decrease in GSH levels seen in high glucose conditions is mediated by the availability of cysteine (rate-limiting substrate in de novo glutathione synthesis) and the gene expression of the gamma-glutamylcysteine synthetase enzyme. Glutathione depletion is associated with an increase in DNA damage, which can be reduced when glutathione levels are restored.

Lactulose-mannitol intestinal permeability test: a useful screening test for adult coeliac disease.

The aim of this study was to determine the value of the lactulose mannitol intestinal permeability test in screening the general adult population for unrecognized enteropathy and latent coeliac disease. Subjects with positive serology (identified by screening carried out by the Belfast MONICA Project) along with controls were followed-up after 3 years and classified as having transient serology, persistent serology or coeliac disease. A 5-h urine collection was performed following the ingestion of 5 g lactulose, 2 g mannitol and glucose as an osmotic filler. Urinary concentrations of lactulose and mannitol were measured by enzymatic analysis. Percentage lactulose excretion (%LE) (0.94 versus 0.31, P<0.001) and lactulose mannitol excretion ratio (LMER) (0.12 versus 0.02, P<0.001) were significantly higher in screening-detected coeliac disease subjects compared with MONICA controls. The sensitivity of the permeability test was 87% in the screening situation compared with 81% in the clinical situation. In subjects with persistent and transient serology the LMER did not differ significantly from that of controls. The lactulose-mannitol test is a useful test for screening the general adult population for coeliac disease. Subjects with persistent and transient serology did not differ from MONICA controls and are unlikely to have latent coeliac disease.

Very low density lipoprotein subfractions in Type II diabetes mellitus: alterations in composition and susceptibility to oxidation.

AIMS/HYPOTHESIS: Type II (non-insulin-dependent) diabetes mellitus is associated with raised triglycerides and increased very low density lipoprotein cholesterol. The aim of this study was to assess if very low density lipoprotein subfraction composition and potential to oxidise were altered in this condition. METHODS: Very low density lipoprotein was separated into four subfractions (A-->D) by a novel, rapid ultracentrifugation procedure. Analysis of each subfraction included lipid and fatty acid composition. Preformed peroxides were measured spectrophotometrically and conjugated dienes were used as an indicator of in vitro lipid oxidation. RESULTS: In all results we compared patient and control subfractions. Mean fasting plasma glucose was 8.9 +/- 2.0 mmol/l in patients vs 5.1 +/- 0.4 mmol/l in control subjects (p < 0.001); patient HbA1c was 7.6 +/- 1.4%. Patient total lipid standardised for apo B was higher than controls in subfractions A, B and C; A, 201 vs 60; B, 191 vs 40; C, 63 vs 21; D, 29 vs 34 micromol lipid per mg apo B (p < 0.05). Preformed peroxides were higher in all patient subfractions compared with controls: A, 340 vs 48; B, 346 vs 42; C, 262 vs 28; D, 54 vs 16 nmol per mg apo B (p < 0.001). Patient subfractions A and D were more susceptible to in vitro oxidation. Monounsaturated fatty acids were lower in patients subfractions, 35.2 vs 36.7; B, 35.1 vs 38.7; C, 34.4 vs 36.5; D, 33.0 vs 35.5 as per cent total (p < 0.05). CONCLUSIONS/INTERPRETATION: These results indicate abnormalities in very low density lipoprotein subfraction composition and oxidation profile in Type II diabetic subjects, which are characteristic of more atherogenic particles and that may contribute to the development of cardiovascular disease in these patients.

Effect of genotype on changes in intelligence quotient after dietary relaxation in phenylketonuria and hyperphenylalaninaemia.

BACKGROUND: Associations between genotype and intellectual outcome in patients with phenylketonuria are complicated because intelligence is influenced by many variables, including environmental factors and other genetic determinants. Intellectual changes with age, both on and after relaxation of diet, vary within the patient population. This study aims to determine whether a significant association exists between genotype and change in intelligence after relaxation of diet. METHODS: 125 patients with hyperphenylalaninaemia and phenylketonuria whose diet was relaxed after 8 years of age. Verbal, performance, and full scale intelligence quotients at 8, 14, and 18 years were expressed as standard deviation scores (IQ-SDS), and genotype as predicted residual enzyme activity (PRA) of phenylalanine hydroxylase. RESULTS: IQ-SDS at 8, 14, and 18 years were significantly below normal; no association was found between PRA and IQ-SDS. Significant reductions in verbal and full scale IQ-SDS occurred between 8 and 14 years and 8 and 18 years. There was a significant association between PRA and the reduction in verbal, performance, and full scale IQ between these years. Multiple regression analysis of 18 year results, using 8 year results as covariates, supported the association between PRA and IQ-SDS; after adjustment for phenylalanine control, both up to and after the age of 8 years, the full scale IQ-SDS at 14 and 18 years was 0.15 higher for each 10% increase in PRA. CONCLUSIONS: Genotype might be useful in predicting the likelihood of intellectual change in patients with hyperphenylalaninaemia and phenylketonuria whose diet is relaxed after the age of 8 years.

Effect of glucose on endothelin-1-induced calcium transients in cultured bovine retinal pericytes.

Published work has shown that endothelin-1-induced contractility of bovine retinal pericytes is reduced after culture in high concentrations of glucose. The purpose of the present study was to establish the profile of endothelin-1-induced calcium transients in pericytes and to identify changes occurring after culture in high concentrations of glucose. Glucose had no effect on basal levels of cytosolic calcium or on endothelin-1-induced calcium release from intracellular stores. However, influx of calcium from the extracellular medium after endothelin-1 stimulation was reduced in pericytes that had been cultured in 25 mM D-glucose. L-type Ca(2+) currents were identified by patch clamping. The L-type Ca(2+) channel agonist, (-)-Bay K8644, caused less influx of calcium from the extracellular medium in pericytes that had been cultured in 25 mM D-glucose than in those cultured with 5 mM D-glucose. However, 3-O-methylglucose, a nonmetabolizable analogue of glucose which can cause glycation, had similar effects to those of high concentrations of glucose. The results suggest that reduced function of the L-type Ca(2+) channel that occurs in bovine retinal pericytes after culture in high concentrations of D-glucose is probably due to glycation of a channel protein.

The effects of glucose-induced oxidative stress on growth and extracellular matrix gene expression of vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy and this may include abnormalities in growth characteristics and the extracellular matrix. As the actual mechanisms by which glucose induces VSMC dysfunction remain unclear, the aim of this study was to assess the potential role of glucose-induced oxidative stress. Porcine aortic VSMCs were cultured for 10 days in either 5 mmol/l normal glucose or 25 mmol/l D-glucose (high glucose). There was evidence of oxidative stress as indicated by a 50% increase in intracellular malondialdehyde (p < 0.05), increased mRNA expression of CuZn superoxide dismutase and Mn superoxide dismutase (by 51% and 37% respectively, p < 0.01) and a 50% decrease in glutathione in 25 mmol/l D-glucose (p < 0.001). Growth was increased by 25.0% (p < 0.01). mRNA expression of extracellular matrix proteins (collagens I, III, IV and fibronectin) was not altered by high glucose in these experimental conditions. Repletion of glutathione with N-acetyl L-cysteine (1 mmol/l) in VSMC grown in high glucose was associated with reduction in malondialdehyde and restored growth to that of normal glucose. The water soluble analogue of vitamin E, Trolox (200 mumol/l), reduced malondialdehyde concentrations, but had no effect on glutathione depletion or the increased growth rate seen with high glucose. The addition of buthionine sulphoximine (10 mumol/l) to VSMC cultured in normal glucose reduced glutathione, increased malondialdehyde and increased growth to a similar extent as that found in high glucose alone. These results suggest that thiol status, rather than lipid peroxides, is a key factor in modulating VSMC growth and that mRNA expression of extracellular matrix proteins is not increased in VSMC under conditions of glucose-induced oxidative stress.

A simple method for assessing copper-mediated oxidation of very-low-density lipoprotein isolated by rapid ultracentrifugation.

The association of very-low-density lipoprotein (VLDL) with atherosclerosis remains controversial. However, studies have shown that oxidative modification of VLDL can promote foam cell formation, leading to the development of atherosclerosis. A rapid method is described which will allow the significance of VLDL oxidation to be assessed in clinical studies. VLDL was isolated from heparinized plasma by a 1-h, single spin ultracentrifugation. Total protein was standardized to 25 mg/L. Oxidation was promoted by the addition of copper ions (17.5 mumol/L, final concentration) incubated at 37 degrees C. Conjugated diene production was followed at 234 nm. Total assay preparation time was 2 h. Urate greatly inhibited the oxidation of VLDL and was successfully removed by size exclusion chromatography. VLDL isolated from frozen plasma (-70 degrees C) was stable for 15 weeks. This simple, rapid method for the isolation of VLDL may be applied to assess the significance of VLDL oxidation in disease.

Glucose-induced oxidative stress in vascular contractile cells: comparison of aortic smooth muscle cells and retinal pericytes.

Free radical-mediated damage to vascular cells may be involved in the pathogenesis of diabetic vasculopathy. The aim of this study was to compare the extent of glucose-induced oxidative stress in both vascular smooth muscle cells (VSMCs) and pericytes and the effect on antioxidant enzyme gene expression and activities. Porcine aortic VSMC and retinal pericytes were cultured in either 5 or 25 mmol/l glucose for 10 days. Intracellular malondialdehyde (MDA) was measured as a marker of peroxidative damage, and mRNA expression of CuZn-SOD, MnSOD, catalase, and glutathione peroxidase (GPX) were measured by Northern analysis. Glutathione (GSH) was also measured. There was a significant increase in MDA in VSMCs in 25 mmol/l glucose (1.34 +/- 0.11 vs. 1.88 +/- 0.24 nmol/mg protein, 5 vs. 25 mmol/l D-glucose, mean +/- SE, n = 15, P < 0.01), but not in pericytes (0.38 +/- 0.05 vs. 0.37 +/- 0.05 nmol/mg protein, n = 11). There was a significant decrease in GSH in both cell types (VSMC, 1.40 +/- 0.13 vs. 0.69 +/- 0.12 nmol/mg protein, n = 15, P < 0.001; pericytes, 1.97 +/- 0.17 vs. 0.94 +/- 0.16 nmol/mg protein, n = 11, P < 0.001). mRNA expression of CuZnSOD and MnSOD was increased only in VSMCs (by 58.5 +/- 8.1 and 41.0 +/- 6.9%, respectively, n = 8, P < 0.01). CuZnSOD protein was increased by approximately 120% (P < 0.00001). None of the antioxidant enzyme activities was altered between 5 and 25 mmol/l glucose in either cell type. Both MnSOD activities and GSH concentrations were higher in pericytes compared with VSMC under basal (5 mmol/l) conditions (P < 0.05 and P < 0.02, respectively). These results demonstrate glucose-induced reduction of GSH in both cells, but only in VSMC is there evidence of oxidant damage in the form of lipid peroxidation, implying significant differences in intracellular responses to glucose between contractile cells in the macro- and microvasculature.

Exercise responses in patients with IDDM.

OBJECTIVE: The hemodynamic, respiratory, and metabolic responses to exercise were studied in IDDM patients and control subjects to detect diabetic cardiomyopathy. RESEARCH DESIGN AND METHODS: Eight subjects aged 25-40 years with diabetes of at least 10 years' duration were compared with eight control subjects aged 21-46 years. All subjects underwent a progressive incremental bicycle exercise test with measurement of gas exchange, blood glucose, lactate, fat metabolite, and catecholamine levels and two steady-state exercise tests with measurement of cardiac output by a CO2 rebreathing method. A new first-pass radionuclide method was used to measure cardiac ejection fractions (EFs) at rest, peak exercise, and steady-state exercise. RESULTS: The peak achieved oxygen consumption was similar in the diabetic and control subjects (29.9 [25.1-34.6] and 31.4 [26.9-35.9] ml.min-1.kg-1, respectively; mean [95% CI]). There were no significant differences in heart rate, double product, ventilation, respiratory exchange ratio, or ventilatory equivalents for oxygen and CO2 during the incremental test. Glucose levels were higher in the diabetic subjects, but there were no significant differences in levels of lactate, catecholamines, free fatty acids, glycerol, or beta-hydroxybutyrate. Left ventricular EF fell from rest to peak exercise within the diabetic group (66.0% [59.6-72.4] at rest; 53.6% [45.6-61.6] at peak; P < 0.05) but this did not differ significantly from the control group (58.7% [52.3-65.1] at rest; 60.3% [48.9-71.7] at peak). Right ventricular EFs were similar in each group, and there was no reduction in peak filling rate to suggest diastolic dysfunction. The cardiac output responses to exercise were also similar in the two groups. CONCLUSIONS: There is no evidence of impairment of the exercise response in subjects with long-standing diabetes, and the apparent fall in left ventricular EF at peak exercise could be related to hemodynamic adaptation.