High-density lipoproteins (HDL) plays a key role in the protection against oxidative damage of lipoprotein and biological membranes. The aim of the present study was to investigate the relationship between the antioxidant role of HDL and the HDL-paraoxonase (PON) activity in healthy subjects and in type 1 diabetic patients. Moreover, the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage was studied. HDL were isolated from 31 type 1 diabetic patients and 31 sex- and age-matched healthy subjects and immediately used to evaluate lipid hydroperoxides and HDL-PON activity. Erythrocyte membranes obtained from healthy subjects were oxidized with 2,2-azo-bis(2-aminidinopropane)dihydrochloride and then incubated in the presence of HDL isolated from healthy or type 1 diabetic subjects, with measurements of membrane lipid hydroperoxides before and after the incubation. HDL from type 1 diabetic patients showed higher levels of lipid hydroperoxides and a lower activity of HDL-PON than healthy subjects. Moreover, HDL of type 1 diabetic patients protected less efficiently erythrocyte membranes against oxidative damage compared with HDL from healthy subjects. A negative correlation was found between HDL-PON activity and the levels of hydroperoxides of HDL, confirming the relationship between PON and lipid peroxidation and suggesting that subjects with low PON activity are more exposed to oxidative damage than subjects with high PON activity. The ability of HDL to protect erythrocyte membranes was positively correlated with HDL-PON activity and negatively correlated with the levels of lipid hydroperoxides of HDL of healthy subjects. These results confirm a linkage between PON activity and lipid peroxidation of lipoproteins and suggest that the ability of HDL to protect erythrocyte membranes might be related to the PON activity. It might be hypothesized that the decrease of PON activity in diabetic patients and the lower HDL protective action against membrane peroxidation could contribute to acceleration of arteriosclerosis in type 1 diabetes mellitus.
Nitric oxide (NO), an important mediator of both physiological and pathological processes [1], is derived from l-arginine by a family of enzymes termed NO synthases [2]. At least three isoforms of NO synthase (NOS) have been detected. Of these, endothelial (eNOS) and neuronal (nNOS) enzymes are constitutive and regulated by Ca 2+ /calmodulin. The inducible NOS (iNOS) originally detected in macrophages and in the endothelium is produced in response to cytokines and cellular debris of microbial origin. This inducible form can produce 10-to 50-fold more NO than the constitutive NOS. In addition, Diabetologia (1999) Abstract Aims/hypothesis. The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. Methods. Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting.Results. In the presence of extracellular l-arginine (100 mmol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of larginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of l-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. Conclusion/interpretation. Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes. [Diabetologia (1999) 42: 539± 544]
A fraction from normal human plasma inhibiting Na(+)-K(+)-ATPase has been recently identified as lysophosphatidylcholine (LPC). The aim of this study was to investigate the existence of a relationship between the activity of the cellular membrane Na(+)-K(+)-ATPase and plasma LPC in human diabetes. We studied 10 patients with insulin-dependent-diabetes mellitus (IDDM), 14 patients with non-insulin-dependent diabetes mellitus (NIDDM), and 10 sex- and age-matched control subjects. Plasma LPC concentrations were increased in both IDDM and NIDDM patients compared with control subjects. Na(+)-K(+)-ATPase activity was reduced in both groups of patients in erythrocyte and platelet membranes. There was a significant correlation between the concentrations of plasma LPC and Na(+)-K(+)-ATPase activity in both erythrocyte and platelet membranes (P < 0.01). To investigate the effect of LPC on the enzyme, Na(+)-K(+)-ATPase activity was determined in erythrocyte membranes obtained from six healthy subjects after in vitro incubation with increasing concentrations of LPC (1-10 microM). Enzymatic activity was significantly reduced by in vitro LPC at a concentration of 2.5 microM, with a further decrease at 5 microM. These data suggest that the decrease in Na(+)-K(+)-ATPase activity in diabetes might be due to increased LPC concentrations.
Nitric oxide (NO) has been shown to inhibit platelet activation through an increase in cytoplasmic cGMP levels [1,2]. Recent evidence suggests that the cGMP increase induced by insulin in human platelets, which accounts for the antiaggregatory effect of the hormone, is mediated by NO [3]. NO production within the platelet is made possible by the presence of a constitutive isoform of the enzyme nitric oxide synthase (NOS) , which has been recently isolated from human platelets [4]. Platelet NOS has a distinct molecular weight (150 kDa) and its amino acid sequence has been deduced from its mRNA sequence [4].Low levels of intraplatelet cGMP have been described in insulin-dependent diabetic (IDDM) subjects and it has been hypothesized that this alteration might account for the platelet hyperreactivity during the disease [5]. However, no data are available at present in the literature directly concerning the study of platelet NOS in human diabetes, which is accompanied by an enhanced platelet activation.The aim of the present study was to investigate directly the platelet NOS activity in patients affected by IDDM and NIDDM and to look for possible relations with metabolic control. Moreover, we determined in the same subjects the platelet Na + /K + ATPase activity, as a relation has been hypothesized between this enzymatic activity and NO production in endothelial cells from diabetic rats [6]. Subjects and methodsThe study was performed on 19 IDDM outpatients (9 men, 10 women, age 34 ± 8 years, duration of disease 7 ± 5 years, fasting glycaemia = 9.5 ± 2.9 mmol/l, HbA 1c 9.0 ± 1.3 %, range 6.3--11.3 %), 21 NIDDM outpatients (10 men, 11 women, age Diabetologia (1998) Summary Nitric oxide (NO) produced by platelet nitric oxide synthase (NOS) inhibits platelet activation by increased cytoplasmic cGMP levels. The aim of this study was to investigate platelet NOS activity in insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), which are characterized by enhanced platelet activation. HbA 1 c levels, platelet NOS and platelet membrane Na + /K + ATPase activity were determined in 19 IDDM patients, 21 NIDDM patients and 31 healthy control subjects. NOS activity was measured by a spectrophotometric method based on NO-dependent oxidation of oxyhaemoglobin to met-haemoglobin. Na + /K + ATPase activity was measured by the method of Kitao and Hattori. Both NOS and Na + /K + ATPase activity were significantly reduced in diabetic subjects compared with control subjects. NOS showed a significant negative relation with HbA 1 c levels and a positive relation with Na + /K + ATPase activity in diabetic patients. It is hypothesized that the decreased NOS activity might play a role in the pathogenesis of diabetic vascular complications. [Diabetologia (1998)
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