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Delayed activation of nitric oxide synthase II in the aorta of streptozotocin-diabetic rats
Monday June 26, 2000: Poster Abstracts P:W1 Diabetes
16
nephropathy), their 36 non-diabetic siblings and 37 non-diabetic parents, and 3 control groups of healthy subjects. Were determined blood creatinine, glucose, HbAlc, cholesterol, triglycerides, Lp(a), fibrinogen, malondialdehyde (MDA), advanced oxidation protein products (AOPP). Erythrocyte response to oxidative stress was evaluated by RBC GSH, RBC MDA, haemolysis. Diabetics had higher blood glucose (p < 0.001), HbAlc (p < 0.001), Lp(a) (p < 0.01), fibrinogen (p < 0.05) than controls. Siblings had higher Lp(a) (p < 0.001). Parents had higher plasma glucose (p < 0.05) and Lp(a) (p < 0.01). Plasma and RBC MDA were elevated in diabetics and relatives than in controls. Basal RBC GSH was lower in diabetics (p < 0.01). Incubations of cells caused in diabetics a decrease in RBC GSH of lesser degree than in controls, while an increase in haemolysis. Among relatives, haemolysis was increased both at baseline and after incubation. Plasma MDA was associated with blood glucose, creatinine, and fibrinogen (R 0.5, p < 0.001); basal RBC MDA with plasma Lp(a), fibrinogen, and plasma MDA (R 0.6, p < 0.001). Our study shows that markers of fipoprotein metabolism, oxidative stress, and cellular fragility are abnormal in non diabetic relatives of type I diabetics suggesting the view that familial elements even precede diabetes.
MoP25:W1
I Moderately elevated triglycerides are associated to insulin resistance (evaluated by HOMA) in normal-weight subjects with normal glucose tolerance
I
P. Alessandrini, E. Morn, P. Gallina, M. Pals, G. BittoloBon. 2 na Department of Internal Medicine - Ospedale Civile - Venezia, Italy
Objective: To study whether plasma triglycerides (<400 mg/dl) affect insulin resistence estimated by homeostasis model assessment (HOMAIR) in subjects with normal body weight and normal glucose-tolerance. Methods: We retrospectively evaluated 440 normal weight subjects (BMI 20-25), who underwent an OGTT; subjects with age 20-64 and fasting triglycerides < 400 mg/dl were considered for analysis. For the 216 subjects with these characteristics we calculated two indexes of insulin resistance: Insulinogenic Index and HOMAm ((fasting 1RI (mU/ml) × FBG (rnmoL/l)/22.5). Data were analyzed both on the entire group by multiple regression analysis and by comparing HOMAm values in tertiles of triglycerides, by non parametric analysis of variance (Kruskal-Wallis test). Results: Subjects in the lower tertile of triglycerides levels (24--81 mg/dl) had a significantly lower HOMAm, as compared to the upper 2 tertiles, while no difference was observed between the upper 2 tertiles (p of K-W = 0.0011). There was no difference for Insulinogenic Index. Multiple regression analysis showed a highly significant independent direct correlation between HOMAIR and triglycerides (both in absolute values and log-transformed: p = 0.0007 and p = 0002 respectively) and an inverse association with HDL-C (p = 0.0003). Age just failed to reach statistical significance, and BMI was not an independent determinant in multiple regression analysis. Total and LDL-C had no relation to HOMAIR. Conclusions: Triglycerides levels are associated to insulin resistance as measured by HOMAm also in normal-weight subjects with normal glucose tolerance. Values of triglycerides 81-133 mg/dl are associated to insulin resistance, with no further increase in resistance as the values increase in the range 134-389 mg/dl. HDL-C is inversely and independently predictive of insulin resistance by multiple regression analysis2, while total and LDL-C do not interact with insulin activity, as measured by HOMAIR.
I MoP26:W1
] Delayed activation of nitric oxide synthase II in the aorta of streptozotocin-diabetic rats
I
E Nardi, C. Bolego, A. Cignarella, L. Puglisi. Institute of Pharmacological Sciences, Via Balzaretti, 9, 20133 Milan, Italy
After tissue damage or inflammatory stimuli, smooth muscle cells express the inducible isoform of nitric oxide synthase (NOS II), which has also been detected within the atherosclerotic plaque. In diabetes, NOS II induction has been postulated to be involved in /%cell destruction but could also be a mechanism for counteracting diabetes-related enhanced NO inactivation by free radicals. The aim of the present work was to investigate the expression and activity of this enzyme after incubation with proinflammatory stimuli of i) aortic vascular smooth muscle cells (VSMC) or ii) aorta homogenates from streptozotocin-diabetic rats and iii) after in vivo treatment of diabetic rats with lipopolysaccharide (LPS). NOS II protein and activity were measured by immunoblotting and Griess reaction, respectively. After incubation with a cocktail of cytokines (IL-1/~, INF-y, TNF-o~, and LPS) for 24 or 48 h, NOS II protein content was maximal at 24 h and then decreased in control VSMC. In contrast, the enzyme was detectable after 24 h but reached the synthesis
peak after 48 h in diabetic VSMC. When measuring NOS II activity after 24-h stimulation, the levels of nitrite significantly increased in the culture medium of control but not in that of diabetic VSMC. After a 48-h challenge, however, nitrite production was considerably enhanced in both control and diabetic VSMC. The addition of L-arginine to cells treated for 24 h with cytokines did not influence nitrite accumulation in the medium. Similarly, after a 24-h challenge of cells with cytokines in the presence of antioxidant compounds such as ascorbic acid, lercanidipine or leucoselect, nitrite production was unchanged. Interestingly, however, nitrite formation in diabetic VSMC after 24-h cytokine treatment was similar to that in control cells when the incubation was carried out in medium deprived of any estrogen activity. Similarly to what observed in primary cultures, the peak of NOS II production in aortic rings incubated with either cytokine mix or LPS alone occurred after 24 h in control and after 48 h in diabetic tissues. Accordingly, in vivo treatment with LPS for 24 h significantly reduced aortic production of NOS II in diabetic with respect to control animals. In conclusion, a delayed NO production by NOS II was observed in the aorta of diabetic rats. In the case of VSMC, this delay was reversed by removal of estrogen-like compounds from the culture medium. While the nature of this block is currently being investigated, these results, together with the well known impairment in endothelial NOS III activity, point to a general impairment of the NO pathway in diabetes.
I MoP27:W1
I Homocysteine and lipid metabolism in children with type 1 diabetes
E. Wiltshire, J. Couper, D. Thomas. Women's and Children's Hospital, North Adelaide, South Australia Adults with micro- and macro-vascular complications of type 1 and type 2 diabetes have increased Hcy and plasma lipids. As the processes leading to diabetes complications begin early in life, we aimed to investigate Hcy and lipid metabolism in children with type 1 diabetes. Fasting blood samples were collected from 75 children (aged 13.6 + 2.6 years) with type 1 diabetes for 5.4 + 3.7 years and 53 healthy controls (aged 13.4 + 2.5 years). Total plasma Hcy; MTHFR genotype (677 and 1298 polymorphisms); HbAlc; serum vitamin B12, serum folate, and red cell folate; plasma triclycerides, total and HDL-cholesterol; and plasma creatinine were measured. LDL-cholesterol and creadnine clearance were calculated. Fasting plasma total Hcy was reduced in patients compared with controls, whereas serum vitamin B12, serum folate and red cell folate were increased. Using stepwise regression analysis, after correction of Hcy levels for age, serum folate, serum BI2 and creatinine clearance, a significant difference between patients with type 1 diabetes and controls persisted (p = 0.001). There was no difference in the frequency of MTHFR polymorphisms. Plasma total Hcy did not correlate with triglycerides, LDL-cholesterol or HDL-cholesterol. LDL-cholesterol was significantly higher in patients than controls, HDL cholesterol was also higher, bordering on significance. HbAlc correlated significantly with LDL choesterol (r = 0.412, p < 0.001) and triglycerides (r = 0.474, p < 0.001) but did not correlate with Hcy (r = 0.1, p = 0.4) or HDL-cholesterol (r = 0.l, p = 0.39). Hcy metabolism is altered in children with type 1 diabetes. Higher values for serum folate and vitamin B12, reflecting differences in dietary intake between children with diabetes and controls, partially account for this difference. Our data do not suggest an interaction between Hcy and lipid metabolism in children with type 1 diabetes.
I MoP28:W1
[ Predictors of LDL particle size in type 2 diabetes
A.M. W~gner, A. Ptrez, O. Jorba I , M. Rigla, J.L. S~nchez I , J. Ord6fiez I . 1Endocrinology and Biochemistry; Deps. Hospital Sant Pau, Barcelona, Spain
Objective: To evaluate LDLc/apolipoproteinB ratio and other easily measured biological and clinical parameters, previously described in non-diabetic subjects as predictors of LDL particle size, in a group of type 2 diabetic patients. Methods: 45 type 2 diabetic patients (34 male, age 56.8:1:11.6 years, body mass index 28.8:1:4.0 kg/m 2, diabetes duration 11.7 4- 8.6 years and HbAlc 8.3 4- 1.8%) were studied. Samples were obtained after a 10-12-hours fast. Total cholesterol (c) and triglyceride (tg) (enzymatic), HDLc (direct), LDLc (Friedewald/ultracentrifugation), apolipoproteinB (apoB) (immunoturbidimetric, standardized against WHO/IFCC standard SP3-07) and LDL size (electroforesis on gradient polyacrylamide gel) were measured. LDLc/apo(B) ratio and non-HDLc were calculated. Univariate correlations and multiple regression analysis were applied.
Xlhh International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
16
nephropathy), their 36 non-diabetic siblings and 37 non-diabetic parents, and 3 control groups of healthy subjects. Were determined blood creatinine, glucose, HbAlc, cholesterol, triglycerides, Lp(a), fibrinogen, malondialdehyde (MDA), advanced oxidation protein products (AOPP). Erythrocyte response to oxidative stress was evaluated by RBC GSH, RBC MDA, haemolysis. Diabetics had higher blood glucose (p < 0.001), HbAlc (p < 0.001), Lp(a) (p < 0.01), fibrinogen (p < 0.05) than controls. Siblings had higher Lp(a) (p < 0.001). Parents had higher plasma glucose (p < 0.05) and Lp(a) (p < 0.01). Plasma and RBC MDA were elevated in diabetics and relatives than in controls. Basal RBC GSH was lower in diabetics (p < 0.01). Incubations of cells caused in diabetics a decrease in RBC GSH of lesser degree than in controls, while an increase in haemolysis. Among relatives, haemolysis was increased both at baseline and after incubation. Plasma MDA was associated with blood glucose, creatinine, and fibrinogen (R 0.5, p < 0.001); basal RBC MDA with plasma Lp(a), fibrinogen, and plasma MDA (R 0.6, p < 0.001). Our study shows that markers of fipoprotein metabolism, oxidative stress, and cellular fragility are abnormal in non diabetic relatives of type I diabetics suggesting the view that familial elements even precede diabetes.
MoP25:W1
I Moderately elevated triglycerides are associated to insulin resistance (evaluated by HOMA) in normal-weight subjects with normal glucose tolerance
I
P. Alessandrini, E. Morn, P. Gallina, M. Pals, G. BittoloBon. 2 na Department of Internal Medicine - Ospedale Civile - Venezia, Italy
Objective: To study whether plasma triglycerides (<400 mg/dl) affect insulin resistence estimated by homeostasis model assessment (HOMAIR) in subjects with normal body weight and normal glucose-tolerance. Methods: We retrospectively evaluated 440 normal weight subjects (BMI 20-25), who underwent an OGTT; subjects with age 20-64 and fasting triglycerides < 400 mg/dl were considered for analysis. For the 216 subjects with these characteristics we calculated two indexes of insulin resistance: Insulinogenic Index and HOMAm ((fasting 1RI (mU/ml) × FBG (rnmoL/l)/22.5). Data were analyzed both on the entire group by multiple regression analysis and by comparing HOMAm values in tertiles of triglycerides, by non parametric analysis of variance (Kruskal-Wallis test). Results: Subjects in the lower tertile of triglycerides levels (24--81 mg/dl) had a significantly lower HOMAm, as compared to the upper 2 tertiles, while no difference was observed between the upper 2 tertiles (p of K-W = 0.0011). There was no difference for Insulinogenic Index. Multiple regression analysis showed a highly significant independent direct correlation between HOMAIR and triglycerides (both in absolute values and log-transformed: p = 0.0007 and p = 0002 respectively) and an inverse association with HDL-C (p = 0.0003). Age just failed to reach statistical significance, and BMI was not an independent determinant in multiple regression analysis. Total and LDL-C had no relation to HOMAIR. Conclusions: Triglycerides levels are associated to insulin resistance as measured by HOMAm also in normal-weight subjects with normal glucose tolerance. Values of triglycerides 81-133 mg/dl are associated to insulin resistance, with no further increase in resistance as the values increase in the range 134-389 mg/dl. HDL-C is inversely and independently predictive of insulin resistance by multiple regression analysis2, while total and LDL-C do not interact with insulin activity, as measured by HOMAIR.
I MoP26:W1
] Delayed activation of nitric oxide synthase II in the aorta of streptozotocin-diabetic rats
I
E Nardi, C. Bolego, A. Cignarella, L. Puglisi. Institute of Pharmacological Sciences, Via Balzaretti, 9, 20133 Milan, Italy
After tissue damage or inflammatory stimuli, smooth muscle cells express the inducible isoform of nitric oxide synthase (NOS II), which has also been detected within the atherosclerotic plaque. In diabetes, NOS II induction has been postulated to be involved in /%cell destruction but could also be a mechanism for counteracting diabetes-related enhanced NO inactivation by free radicals. The aim of the present work was to investigate the expression and activity of this enzyme after incubation with proinflammatory stimuli of i) aortic vascular smooth muscle cells (VSMC) or ii) aorta homogenates from streptozotocin-diabetic rats and iii) after in vivo treatment of diabetic rats with lipopolysaccharide (LPS). NOS II protein and activity were measured by immunoblotting and Griess reaction, respectively. After incubation with a cocktail of cytokines (IL-1/~, INF-y, TNF-o~, and LPS) for 24 or 48 h, NOS II protein content was maximal at 24 h and then decreased in control VSMC. In contrast, the enzyme was detectable after 24 h but reached the synthesis
peak after 48 h in diabetic VSMC. When measuring NOS II activity after 24-h stimulation, the levels of nitrite significantly increased in the culture medium of control but not in that of diabetic VSMC. After a 48-h challenge, however, nitrite production was considerably enhanced in both control and diabetic VSMC. The addition of L-arginine to cells treated for 24 h with cytokines did not influence nitrite accumulation in the medium. Similarly, after a 24-h challenge of cells with cytokines in the presence of antioxidant compounds such as ascorbic acid, lercanidipine or leucoselect, nitrite production was unchanged. Interestingly, however, nitrite formation in diabetic VSMC after 24-h cytokine treatment was similar to that in control cells when the incubation was carried out in medium deprived of any estrogen activity. Similarly to what observed in primary cultures, the peak of NOS II production in aortic rings incubated with either cytokine mix or LPS alone occurred after 24 h in control and after 48 h in diabetic tissues. Accordingly, in vivo treatment with LPS for 24 h significantly reduced aortic production of NOS II in diabetic with respect to control animals. In conclusion, a delayed NO production by NOS II was observed in the aorta of diabetic rats. In the case of VSMC, this delay was reversed by removal of estrogen-like compounds from the culture medium. While the nature of this block is currently being investigated, these results, together with the well known impairment in endothelial NOS III activity, point to a general impairment of the NO pathway in diabetes.
I MoP27:W1
I Homocysteine and lipid metabolism in children with type 1 diabetes
E. Wiltshire, J. Couper, D. Thomas. Women's and Children's Hospital, North Adelaide, South Australia Adults with micro- and macro-vascular complications of type 1 and type 2 diabetes have increased Hcy and plasma lipids. As the processes leading to diabetes complications begin early in life, we aimed to investigate Hcy and lipid metabolism in children with type 1 diabetes. Fasting blood samples were collected from 75 children (aged 13.6 + 2.6 years) with type 1 diabetes for 5.4 + 3.7 years and 53 healthy controls (aged 13.4 + 2.5 years). Total plasma Hcy; MTHFR genotype (677 and 1298 polymorphisms); HbAlc; serum vitamin B12, serum folate, and red cell folate; plasma triclycerides, total and HDL-cholesterol; and plasma creatinine were measured. LDL-cholesterol and creadnine clearance were calculated. Fasting plasma total Hcy was reduced in patients compared with controls, whereas serum vitamin B12, serum folate and red cell folate were increased. Using stepwise regression analysis, after correction of Hcy levels for age, serum folate, serum BI2 and creatinine clearance, a significant difference between patients with type 1 diabetes and controls persisted (p = 0.001). There was no difference in the frequency of MTHFR polymorphisms. Plasma total Hcy did not correlate with triglycerides, LDL-cholesterol or HDL-cholesterol. LDL-cholesterol was significantly higher in patients than controls, HDL cholesterol was also higher, bordering on significance. HbAlc correlated significantly with LDL choesterol (r = 0.412, p < 0.001) and triglycerides (r = 0.474, p < 0.001) but did not correlate with Hcy (r = 0.1, p = 0.4) or HDL-cholesterol (r = 0.l, p = 0.39). Hcy metabolism is altered in children with type 1 diabetes. Higher values for serum folate and vitamin B12, reflecting differences in dietary intake between children with diabetes and controls, partially account for this difference. Our data do not suggest an interaction between Hcy and lipid metabolism in children with type 1 diabetes.
I MoP28:W1
[ Predictors of LDL particle size in type 2 diabetes
A.M. W~gner, A. Ptrez, O. Jorba I , M. Rigla, J.L. S~nchez I , J. Ord6fiez I . 1Endocrinology and Biochemistry; Deps. Hospital Sant Pau, Barcelona, Spain
Objective: To evaluate LDLc/apolipoproteinB ratio and other easily measured biological and clinical parameters, previously described in non-diabetic subjects as predictors of LDL particle size, in a group of type 2 diabetic patients. Methods: 45 type 2 diabetic patients (34 male, age 56.8:1:11.6 years, body mass index 28.8:1:4.0 kg/m 2, diabetes duration 11.7 4- 8.6 years and HbAlc 8.3 4- 1.8%) were studied. Samples were obtained after a 10-12-hours fast. Total cholesterol (c) and triglyceride (tg) (enzymatic), HDLc (direct), LDLc (Friedewald/ultracentrifugation), apolipoproteinB (apoB) (immunoturbidimetric, standardized against WHO/IFCC standard SP3-07) and LDL size (electroforesis on gradient polyacrylamide gel) were measured. LDLc/apo(B) ratio and non-HDLc were calculated. Univariate correlations and multiple regression analysis were applied.
Xlhh International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000