O. Laurenti

Circulating endothelin-1 levels increase during euglycemic hyperinsulinemic clamp in lean NIDDM men.

OBJECTIVE To evaluate whether or not insulin stimulates endothelin (ET)-l secretion in vivo. RESEARCH DESIGN AND METHODS Plasma ET-1 levels were evaluated in 16 lean normotensive men with non-insulin-dependent diabetes mellitus (NIDDM) (mean age 50.3 ±4.1 years) during either a 2-h euglycemic hyperinsulinemic clamp (40 mU insulin · m−2 · min−1) or placebo infusion (50 ml isotonic saline) according to a single-blind randomized crossover protocol. RESULTS Circulating ET-1 levels increased during the euglycemic hyperinsulinemic clamp (from 0.88 ± 0.38 pg/ml at time 0 to 1.66 ± 0.22 pg/ml and 1.89 ± 0.99 pg/ml at 60 and 120 min, respectively [P < 0.05 vs. time 0]) and returned to baseline levels after the discontinuation of insulin infusion (0.71 ± 0.22 pg/ml after a 30-min period of recovery [NS]). Compared with placebo, the euglycemic hyperinsulinemic clamp induced a significant increase in plasma ET-1 levels at 60 min (P < 0.0001) and 120 min (P < 0.0001). Changes in basal insulin levels and corresponding changes in circulating ET-1 levels after a 2-h euglycemic hyperinsulinemic clamp were significantly correlated (r = 0.771, P < 0.0001). A possible unfavorable effect of ET-1 on the tissue sensitivity to insulin-stimulated glucose uptake was suggested by the presence of a negative correlation between total glucose uptake and baseline ET-1 levels (r = –0.498, P < 0.05). CONCLUSIONS Our findings indicate that circulating ET-1 levels significantly increase during euglycemic hyperinsulinemic clamp in men with NIDDM. The negative correlation between total glucose uptake and circulating ET-1 levels suggests that the peptide might exert negative effects on the insulin sensitivity of target tissues. The consequent increase in insulin secretion as well as the insulin-related ET-1 release from endothelial cells could favor the development of diabetes-related vascular lesions.

Reduction of oxidative stress by oral N-acetyl-L-cysteine treatment decreases plasma soluble vascular cell adhesion molecule-1 concentrations in non-obese, non-dyslipidaemic, normotensive, patients with non-insulin-dependent diabetes.

Summary To assess in vivo effects of antioxidants on vascular cell adhesion molecule (VCAM)-1 expression, circulating soluble VCAM-1 and intraerythrocytic reduced glutathione (GSH) and GSH disulphide (GSSG) concentrations were evaluated in non-insulin-dependent diabetic patients without complications (9 men, 6 women, 48 ± 6 years old) before and after 1 month of either oral N-acetyl-L-cysteine (1.200 mg/day) or placebo treatments, given in randomized, cross-over, double-blind fashion. Ten healthy subjects (7 men, 3 women, 52 ± 4 years old) served as control subjects. Baseline plasma VCAM-1 concentrations were higher (p = 0.007) in non-insulin-dependent diabetic patients (707.9 ± 52.5 ng/ml) than in control subjects (627.3 ± 84.6 ng/ml). Intraerythrocytic GSSG content was higher (non-insulin dependent diabetic patients: 0.618 ± 0.185 μmol/g Hb; control subjects: 0.352 ± 0.04 μmol/g Hb, p = 0.0002), whereas intraerythrocytic GSH concentrations were lower (p = 0.001) in non-insulin dependent diabetic patients (6.0 ± 0.7 μmol/g Hb) than in control subjects (7.1 ± 0.5 μmol/g Hb). The mean GSH:GSSG ratio was also lower (p = 0.0001) in the first (10.9 ± 4.5) than in the second group (20.2 ± 1.4). Circulating VCAM-1 and intraerythrocytic GSH concentrations were negatively correlated in non-insulin diabetic patients (r = 0.605, p = 0.01). Treatment with N-acetyl-L-cysteine decreased plasma VCAM-1 (p = 0.01) and intraerythrocytic GSSG (p = 0.006) but increased GSH concentrations (p = 0.04) and the GSH:GSSG ratio (p = 0.004) in non-insulin dependent diabetic patients. Our data indicate that the vascular endothelium is activated in non-insulin dependent diabetes. Antioxidant treatment counterbalanced such endothelial activation. Thus, antioxidant agents might protect against oxidant-related upregulation of endothelial adhesion molecules and slow down the progression of vascular damage in non-insulin dependent diabetes. [Diabetologia (1998) 41: 1392–1396]

Influence of reduced glutathione infusion on glucose metabolism in patients with non—insulin-dependent diabetes mellitus

To evaluate the relationship between oxidative stress and glucose metabolism, insulin sensitivity and intraerythrocytic reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio were measured in 10 non-insulin-dependent diabetes mellitus (NIDDM) patients and 10 healthy subjects before and after the intravenous administration of GSH. In particular, after baseline insulin sensitivity was assessed by a 2-hour euglycemic hyperinsulinemic clamp, either glutathione (1.35 g x m2 x min(-1)) or placebo (saline) were infused over a period of 1 hour. The same protocol was repeated at a 1-week interval, in cross-over, according to a randomized, single-blind design. In healthy subjects, baseline intraerythrocytic GSH/GSSG ratio (P < .0005) and total glucose uptake (P < .005) were significantly higher than in NIDDM patients. In the same subjects, GSH infusion significantly increased total glucose uptake (from 37.1 +/- 6.7 micromol kg(-1) x min(-1) to 39.5 +/- 7.7 micromol x kg(-1) x min(-1), P < .05), whereas saline infusion was completely ineffective. In addition, the mean intraerythrocytic GSH/GSSG ratio significantly increased after GSH infusion (from 21.0 +/- 0.9 to 24.7 +/- 1.3, P < .05). Similar findings were found in diabetic patients, in whom GSH infusion significantly increased both total glucose uptake (from 25.3 +/- 9.0 micromol x kg(-1) x min(-1) to 31.4 +/- 10.0 micromol x kg(-1) x min(-1), P < .001) and intraerythrocytic GSH/GSSG ratio (from 14.8 +/- 4.1 to 21.7 +/- 6.7, P < .01). Pooling diabetic patients and controls, significant correlations were found between intraerythrocytic GSH/GSSG ratio and total glucose uptake (r = .425, P < .05), as well as between increments of the same variables after GSH infusion (r = .518, P < .05). In conclusion, our data support the hypothesis that abnormal intracellular GSH redox status plays an important role in reducing insulin sensitivity in NIDDM patients. Accordingly, intravenous GSH infusion significantly increased both intraerythrocytic GSH/GSSG ratio and total glucose uptake in the same patients.

Polyol pathway activation and glutathione redox status in non-insulin-dependent diabetic patients.

The current study aimed to evaluate whether nicotinamide adenine dinucleotide phosphate (NADPH) alteration in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM) is responsible for the impaired glutathione (GSH) redox status, and to assess if short-term inhibition of the polyol pathway normalizes NADPH levels and GSH redox status via an amelioration of the NADPH/total NADP (tNADP) ratio. For this purpose, erythrocyte NADPH and GSH levels were measured in 18 NIDDM patients at baseline and then after 1 week of random double-blind assignment to treatment with either tolrestat (an aldose reductase inhibitor, 200 mg daily) (n = 12) or placebo (n = 6). A group of 16 healthy volunteers served as the control. In the basal condition, mean GSH (P < .0001) and NADPH (P < .0001) levels and NADPH/tNADP (P < .0001) and GSH/ glutathione disulfide (GSSG) (P < .005) ratios were lower in NIDDM patients than in control subjects. Tolrestat treatment increased GSH levels (P < .05 v placebo and baseline) and the NADPH/tNADP ratio (P < .05 v placebo and baseline). Interestingly, tolrestat-induced changes in GSH and NADPH levels and in GSH/GSSG and NADPH/tNADP ratios were significant only in patients who showed a decreased NADPH/tNADP ratio at baseline (n = 8). In these latter patients, we also found a direct correlation between percentage increments in GSH levels and NADPH/tNADP ratios after tolrestat treatment (r = .71, P < .05). In conclusion, our findings support the hypothesis that polyol pathway activation decreases NADPH and GSH levels. Accordingly, short-term inhibition of this enzymatic route increased both the GSH level and the NADPH/tNADP ratio. These changes were observable only in the subgroup of patients with an abnormal NADPH/tNADP ratio at baseline. Polyol pathway inhibition could be useful for decreasing oxidative stress in NIDDM.

Gliclazide improves anti‐oxidant status and nitric oxide‐mediated vasodilation in Type 2 diabetes

Aims To evaluate the effects of gliclazide on oxidative status and vascular response to systemic administration of L‐arginine, the natural precursor of nitric oxide (NO), in Type 2 diabetic patients.

Role of Plasma and Urinary Endothelin-1 in Early Diabetic and Hypertensive Nephropathy

To evaluate the role of circulating and renal endothelin-1 (ET-1) in early diabetic nephropathy, plasma ET-1 levels and urinary ET-1 excretion were evaluated in lean, normotensive patients affected by non-insulin-dependent diabetes (NIDDM) either with (n = 9, NIDDM+) or without microalbuminuria (n = 18, NIDDM-); in never-treated, lean, essential hypertensive patients with (n = 12, EH+) or without microalbuminuria (n = 10, EH-); and in healthy volunteers (n = 12). Results showed higher plasma ET-1 levels in NIDDM+ (1.97 +/- 0.58 pg/mL) than in NIDDM- (1.59 +/- 0.14 pg/mL, P = .013), EH+ (1.40 +/- 0.21 pg/mL, P = .005), EH- (0.91 +/- 0.19 pg/mL, P < .0001), and controls (0.60 +/- 0.10 pg/mL, P < .0001). The circulating ET-1 concentration was also higher in EH+ than EH- and controls (P < .0001). Urinary ET-1 excretion did not differ (P = .387, NS) between NIDDM+ (48.5 +/- 20.1 pg/min) and NIDDM- (40.9 +/- 21.6 pg/min), but was significantly reduced (P < .0001) in both groups compared with controls (70.0 +/- 15.5 pg/min). Similar findings were observed in hypertensive subgroups. No correlations were found between urinary ET-1 and other variables, including plasma ET-1 levels, in all groups. In conclusion, NIDDM+ is accompanied by a significant increase in plasma ET-1 levels. A significant elevation of circulating ET-1 concentration was evident also in NIDDM-, suggesting that early abnormalities of ET-1 production might precede the microalbuminuric phase of diabetes-related renal damage.

Effect of aldose reductase inhibition on glutathione redox status in erythrocytes of diabetic patients

Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in non-insulin-dependent diabetic patients. For this purpose, we investigated the effect of polyol pathway inhibition on the glutathione redox status in these patients. We measured GSH and GSSG levels in erythrocytes of non-insulin-dependent diabetic patients (n = 15) before and after 1 week of treatment with placebo, followed by 1 week of treatment with an aldose reductase inhibitor (tolrestat 200 mg/dl). We found lower GSH levels (7.7 +/- 1.4 mumol/g hemoglobin [Hb]), higher GSSG levels (0.35 +/- 0.09 mumol/g Hb), and lower GSH/GSSG ratios (23.9 +/- 7.7) in diabetics compared with controls (n = 15; 9.8 +/- 0.8 mumol/g Hb, P < .001; 0.17 +/- 0.02, P < .001; and 58.3 +/- 9.1, P < .001, respectively). We did not demonstrate any statistical difference after 1 week of treatment with placebo. In contrast, the treatment with tolrestat induced a significant increase in GSH (8.9 +/- 0.7 mumol/g Hb, P < .01), a decrease in GSSG (0.25 +/- 0.06 mumol/g Hb, P < .02), and an increase in the GSH/GSSG ratio (37.3 +/- 8.4, P < .01). These data strongly support the hypothesis that the polyol pathway plays an important role in the impairment of the glutathione redox status in diabetic patients.

Diabetic endothelial dysfunction: Effect of free radical scavenging in Type 2 diabetic patients

Diabetes mellitus is characterized by oxidative stress, which in turn determines endothelial dysfunction. It has been recently demonstrated that gliclazide, a second-generation sulfonylurea with antioxidant properties, is able to protect endothelial function in animal models of diabetes. In streptozotocin-induced diabetic rats, gliclazide prevented endothelial dysfunction when given orally and improved the impaired relaxations to exogenous nitric oxide (NO) when applied on aortic segments. Moreover, gliclazide was able to inhibit glycosylated oxyhemoglobin-induced endothelial dysfunction both in animal and human microvessels. All these effects were not shared by glibenclamide, but were mimicked by vitamin C or superoxide dismutase (SOD), thus suggesting that gliclazides action on endothelium-dependent vasodilation is mediated by its antioxidant properties. Thus far, there are no clinical studies that describe the influence of gliclazide on both oxidative status and NO-mediated vasodilation. We therefore evaluated the effects of gliclazide on plasma lipid peroxides, plasma total radical trapping antioxidant parameter (TRAP), and NO-mediated vasodilation assessed by blood pressure modifications following intravenous L-arginine in 30 subjects with Type 2 diabetes mellitus. The patients received glibenclamide (n=15) or gliclazide (n=15) in a 12-week, randomized, observer-blinded, parallel study, and were studied pre- and post-treatment. At 12 weeks, gliclazide-treated patients had lower plasma lipid peroxides (13.3+/-3.8 vs. 19.2+/-4.3 micromol/l; P=.0001, respectively) and higher plasma TRAP (1155.6+/-143.0 vs. 957.7+/-104.3 micromol/l; P=.0001, respectively) than the glibenclamide-treated patients. Gliclazide, but not glibenclamide, significantly reduced the systolic and diastolic blood pressure (P=.0199 and P=.00199, respectively, two-way repeated-measures analysis of variance) in response to intravenous L-arginine. In conclusion, our results demonstrate that glicazide treatment improves both antioxidant status and NO-mediated vasodilation in diabetic patients.

Circulating endothelin-1 levels in lean non-insulin-dependent diabetic patients: Influence of ACE inhibition

To evaluate the effect of captopril on plasma endothelin-1 (ET-1) levels and insulin sensitivity, 15 lean normotensive men (51.6 +/- 3.8 years) affected by non-insulin-dependent diabetes mellitus (NIDDM) underwent 2-h euglycemic hyperinsulinemic clamp. Each patient was then assigned to receive either captopril (25 mg twice daily for 1 week) or placebo, in a double-blind randomized fashion, before repeating clamp. At baseline, plasma ET-1 levels were 0.77 +/- 0.25 pg/mL in captopril (n = 10) and 0.83 +/- 0.3 pg/mL in placebo patients (n = 5). A twofold increase in plasma ET-1 levels occurred during the 2-h insulin infusion in both groups (P < .05 after 60 and 120 min), with a rapid return to baseline after 30 min from insulin withdrawal. After 1 week of therapy, total glucose uptake significantly increased in captopril (from 3.71 +/- 1.70 mg/kg/min to 4.24 +/- 1.72 mg/kg/min, P < .03) but not in placebo patients. Plasma ET-1 levels significantly decreased after captopril therapy (0.48 +/- 0.25 pg/mL at time 0, P < .03 v pretreatment levels), but were unaffected by placebo. Moreover, captopril slightly reduced the magnitude of ET-1 increment during insulin infusion (0.65 +/- 0.28 pg/mL and 0.88 +/- 0.48 pg/mL at 60 and 120 min, respectively, P < .05 v time 0). As a consequence, during the second insulin infusion circulating ET-1 levels were significantly lower in captopril- than in placebo-treated patients at time 0 (P < .02), 60 (P < .002), 120 (P < .004), and 150 min (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin on in vitro vascular cell adhesion molecule-1 expression and in vivo soluble VCAM-1 release

Aims/hypothesis. To evaluate the effects of insulin on vascular cell adhesion molecule-1 expression by cultured human vascular endothelial cells and soluble vascular cell adhesion molecule-1 release in vivo. Methods. Human vascular endothelial cells derived from umbilical cord veins were incubated with either insulin (from 10–6 to 10–9 mol/l) or tumour necrosis factor-α (5 ng/ml) for 6 to 24 h. Plasma soluble vascular cell adhesion molecule-1 concentrations were evaluated in 12 non-insulin-dependent diabetic patients (8 men, 4 women, mean age 47.1 ± 7.7 years) and 12 healthy volunteers matched for age, sex and weight (7 men, 5 women, mean age 42.2 ± 7.2 years) before and after a 2-h euglycaemic hyperinsulinaemic clamp. Results. Transcriptional activities of nuclear factor-ϰB luciferase and vascular adhesion molecule-1 luciferase statistically significantly increased after incubation with tumour necrosis factor-α. By contrast, a slight increment of nuclear factor-ϰB luciferase (mean: 1.8 ± 0.3 fold) but not of vascular cell adhesion molecule-1 luciferase transcriptional activities were detected in cells stimulated with insulin. Soluble vascular cell adhesion molecule-1 concentrations in cell supernatants increased after tumour necrosis factor-α but not insulin stimulation. In vivo, baseline plasma soluble vascular cell adhesion molecule-1 concentrations were higher (p = 0.03) in non-insulin-dependent patients (708.7 ± 97.4 μg/l) than controls (632.1 ± 65.2 μg/l) but were not related to fasting insulin concentrations and did not change during insulin infusion. Conclusion/interpretation. The increased concentrations of circulating soluble vascular cell adhesion molecule-1 indicates that the vascular endothelium is activated in non-insulin dependent diabetic patients. Our in vitro and in vivo findings show that vascular cell adhesion molecule-1 activation cannot be due to hyperinsulinaemia. [Diabetologia (1999) 42: 1235–1239]