Effects of troglitazone on in vitro oxidation of LDL and HDL induced by copper ions and endothelial cells

Summary Troglitazone is a new oral antidiabetic agent able to reduce lipid peroxidation. In this study we evaluated its effect on the susceptibility of LDL and HDL to in vitro oxidation induced by copper ions and endothelial cells. In Cu ⁺⁺ -induced LDL modification, different amounts of troglitazone were added to aliquots of the same pool of plasma with subsequent ultracentrifuge separation of LDL and HDL. Differences in LDL and HDL susceptibility to in vitro oxidation with Cu ⁺⁺ were studied by measuring the changes in fluorescence intensity (expressed as lag phase). LDL derived from plasma incubated with different amounts of troglitazone were also incubated with umbilical vein endothelial cells (HUVEC), the modification being monitored by LDL relative electrophoretic mobility and fluorescence. During Cu ⁺⁺ - and HUVEC-induced LDL oxidation, the decay rate of vitamin E, and the potency of troglitazone as a radical scavenger in comparison with vitamin E were also studied. Troglitazone determined a significant, dose-dependent decrease in Cu ⁺⁺ -induced LDL and HDL oxidation. Incubation with HUVEC was also followed by a progressive, significant decrease of LDL relative electrophoretic mobility and fluorescence intensity. During Cu ⁺⁺ - and HUVEC-induced-LDL modification, troglitazone significantly reduced the rate of vitamin E decay. In this study we also demonstrated that under the same oxidative stress, troglitazone was much more potent as a radical scavenger than vitamin E. In conclusion, the results demonstrate that troglitazone can reduce LDL and HDL in vitro oxidation and that, during this process, it can protect vitamin E. In addition to ensuring blood glucose control, the drug may therefore be useful in inhibiting lipoprotein peroxidation. [Diabetologia (1997) 40: 165–172] Received: 26 August 1996, and in revised form: 29 October 1996     

Insulin-induced vasodilatation and endothelial function in obesity/insulin resistance. Effects of troglitazone

Summary Insulin resistance is associated with a decreased vasodilator response to insulin. Because insulin's vasodilator effect is nitric oxide dependent, this impairment may reflect endothelial dysfunction. Troglitazone, an insulin-sensitiser, might thus improve insulin-dependent and/or endothelium-dependent vascular function in insulin resistant obese subjects. For 8 weeks, fifteen obese subjects were treated with either 400 mg troglitazone once daily or placebo, in a randomised, double-blind, cross-over design. At the end of each treatment period, we measured forearm vasodilator responses (plethysmography) to intra-arterial administered acetylcholine and sodium nitroprusside; insulin sensitivity and insulin-induced vascular and neurohumoral responses (clamp); vasoconstrictor responses to N ^G-monomethyl-L-arginine (L-NMMA) during hyperinsulinaemia; and ambulatory 24-h blood pressure (ABPM). Baseline data (placebo) of obese subjects were compared with those obtained in lean control subjects. Obese subjects were insulin resistant compared with leans (whole-body glucose uptake: 26.8 ± 3.0 vs. 53.9 ± 4.3 μmol · kg^–1· min^–1, p < 0.001). Troglitazone improved whole-body glucose uptake (to 31.9 ± 3.3 μmol · kg^–1· min^–1, p = 0.028), and forearm glucose uptake (from 1.09 ± 0.54 to 2.31 ± 0.69 μmol · dL^–1· min^–1, p = 0.006). Insulin-induced vasodilatation was blunted in obese subjects (percent increase in forearm blood flow (FBF) in lean 66.5 ± 23.0 %, vs. 10.1 ± 11.3 % in obese, p = 0.04), but did not improve during troglitazone. Vascular responses to acetylcholine, sodium nitroprusside and L-NMMA did not differ between the obese and lean group, nor between both treatment periods in the obese individuals. In conclusion, in insulin resistant obese subjects, endothelial vascular function is normal despite impaired vasodilator responses to insulin. Troglitazone improved insulin sensitivity but it had no effects on endothelium-dependent and -independent vascular responses. These data do not support an association between insulin resistance and endothelial function. [Diabetologia (1998) 41: 569–576] Received: 19 September 1997 and in revised form: 22 December 1997     

Troglitazone,an insulin action enhancer,improves glycaemic control and insulin sensitivity in elderly Type 2 diabetic patients

The management of Type 2 diabetes mellitus with currently available oral agents may be complicated in the elderly by an increased frequency of side-effects. The effects of troglitazone, an insulin action enhancer, were studied in elderly patients with Type 2 diabetes in a double-blind, parallel-group, placebo-controlled trial. A total of 229 patients (41 % male), mean age 75 (range 69–85) years, with two fasting capillary blood glucose values ≥7 and ≤15 mmol l⁻¹ (and within 4.0 mmol l⁻¹ of each other) and previously treated with either diet alone (30 %) or oral hypoglycaemic agents, were randomized to placebo or troglitazone 400 mg once daily or 200 mg twice daily, or 800 mg once daily or 400 mg twice daily, for 12 weeks. After 12 weeks’ treatment, fasting serum glucose was significantly lower in troglitazone-treated patients (troglitazone, adjusted geometric mean 9.4–10.4 mmol l⁻¹ vs placebo 12.7 mmol l⁻¹, p<0.001). Adjusted geometric mean fructosamine was also lower in troglitazone-treated patients by 5 to 15 % compared to placebo (P <0.05 at all doses except 400 mg od). There was no significant difference between troglitazone doses for improvement in glycaemic control. Troglitazone lowered adjusted geometric mean fasting plasma insulin by 27–34 % compared to placebo (P<0.001) and insulin sensitivity (HOMA-S) improved by 9–15 % in all troglitazone dose groups (p<0.001). Troglitazone also lowered serum non-esterified fatty acids and triglyceride. Adverse event incidence in troglitazone-treated patients was similar to that in patients treated with placebo. No weight gain or symptomatic hypoglycaemia was recorded at any of the doses studied. Troglitazone is effective and well tolerated in elderly patients with Type 2 diabetes mellitus, providing improved glycaemic control in the absence of weight gain. © 1998 John Wiley & Sons, Ltd.     

Electronegative low density lipoprotein subform is increased in patients with short-duration IDDM and is closely related to glycaemic control

Summary We evaluated the effect of improving glycaemic control with intensive insulin therapy on LDL susceptibility to oxidation, electronegative LDL proportion, and LDL subfraction phenotype in a group of 25 patients with short-duration insulin-dependent diabetes mellitus (IDDM); 25 matched healthy control subjects were also studied. LDL susceptibility to oxidation was measured by continuous monitoring of conjugated diene formation. Electronegative LDL was isolated by anion exchange chromatography, and quantified as percentage of total LDL. Six LDL subfractions were isolated by density gradient ultracentrifugation and phenotype A or B classified as the quotient (LDL1-LDL3)/(LDL4-LDL6). Compared to the control group, IDDM subjects with poor glycaemic control showed higher electronegative LDL (19.03 ± 10.09 vs 9.59 ± 2.98 %, p < 0.001), similar LDL subfraction phenotype and lower susceptibility to oxidation (lag phase 45.6 ± 8.8 vs 41.2 ± 4.7 min, p < 0.05). After three months of intensive insulin therapy, HbA_{1 c} decreased from 10.88 ± 2.43 to 5.69 ± 1.54 % (p < 0.001), and electronegative LDL to 13.84 ± 5.15 % (p < 0.05). No changes in LDL susceptibility to oxidation or LDL subfraction phenotype were observed. Electronegative LDL appeared significantly correlated to HbA_{1 c} and fructosamine (p < 0.01 and p < 0.001) only in poorly controlled IDDM patients. These findings suggest that high electronegative LDL in IDDM subjects is related to the degree of glycaemic control, and could therefore be due to LDL glycation rather than to LDL oxidation or changes in LDL subfraction phenotype. [Diabetologia (1996) 39: 1469–1476] Received: 20 December 1995 and in final revised form: 19 June 1996     

Troglitazone,an insulin action enhancer,improves metabolic control in NIDDM patients

Summary The effects of troglitazone, a novel thiazolidinedione, in non-insulin-dependent diabetic (NIDDM) patients were studied in a double-blind, parallel-group, placebo-controlled, dose-ranging trial. A total of 330 patients (63% male), mean age 57 years (range 39–72), with two fasting capillary blood glucose values 7 and 15 mmol/l (within 2.5 mmol/l of each other) were randomised to treatment with placebo or troglitazone at doses of 200, 400, 600 or 800 mg once daily, or 200 or 400 mg twice daily, for 12 weeks. Prior to the study, treatment had been with diet alone (38% patients) or with oral hypoglycaemic agents which were stopped 3–4 weeks before study treatment started. During treatment, HbA_1c tended to rise in patients taking placebo (7.2–8.0%), but remained unchanged with all doses of troglitazone. After 12 weeks of treatment, HbA_1c was significantly lower in the troglitazone-treated (mean 7.0–7.4%) compared to the placebo-treated (8.0%) patients (p=0.055 to <0.001), as was fasting serum glucose concentration (troglitazone, 9.3–11.0 mmol/l vs placebo, 12.9 mmol/l, p<0.001). All doses of troglitazone were equally effective. Troglitazone also lowered fasting plasma insulin concentration, by 12–26% compared to placebo (p=0.074 to <0.001). Insulin sensitivity assessed by homeostasis model assessment (HOMA) was greater after 12 weeks of treatment in troglitazone-treated patients (troglitazone, 34.3–42.8% vs placebo, 29.9%, p<0.05). In addition, serum triglyceride and non-esterified fatty acid concentrations were significantly lower and HDL cholesterol higher at troglitazone doses of 600 and 800 mg/day. LDL cholesterol increased at 400 and 600 mg doses only (from 4.3 and 3.9 mmol/l at baseline to 4.8 and 4.5 mmol/l, respectively at 12 weeks, p<0.05), but not at doses of 800 mg once daily or 400 mg twice daily. LDL/HDL ratio did not change during treatment. All doses were well tolerated; incidence of adverse events in troglitazone-treated patients was no higher than in those treated with placebo. However, a tendency to reduced neutrophil counts was observed in patients taking the highest doses of troglitazone. We conclude that troglitazone is effective and well-tolerated and shows potential as a new therapeutic agent for the treatment of NIDDM.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - HDL high density lipoprotein - LDL low density lipoprotein - NEFA non-esterified fatty acids - HOMA homeostasis model assessment     

Peroxisome Proliferator-Activated Receptor (PPAR) γ and Retinoid X Receptor (RXR) agonists have complementary effects on glucose and lipid metabolism in human skeletal muscle

Aims/hypothesis. To determine the independent and potentially synergistic effects of agonists for PPARγ and RXR on glucose and lipid metabolism, as well as gene expression, in human skeletal muscle cell cultures. Methods. Fully differentiated myotubes from non-diabetic subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus were chronically (2 days) treated with LG100 268 (4 μmol/l), an RXR agonist, or troglitazone (4.6 μmol/l), a PPARγ agonist or both, to determine the effects on glucose uptake, activity of glycogen synthase and palmitate oxidation. Results. The combination of both agents increased glucose uptake (60 ± 9 % compared to control subjects) but not either agent alone (16 ± 9 and 26 ± 6 % for LG100 268 and troglitazone, p < 0.01, respectively). The agent LG100 268 alone had little effect on the activity of glycogen synthase but the effect of troglitazone increased with LG100 268 (p < 0.05). With chronic exposure, LG100 268 upregulated palmitate oxidation (53 ± 12 % increase, p < 0.005), in a way similar to troglitazone (68 ± 23 %, p < 0.005). Synergism was observed when both agonists were combined (146 ± 38 %, p < 0.005 vs either agent alone). Treatment with either agent led to about a twofold increase in the expression of fatty acid transporter (FAT/CD36). Troglitazone upregulated PPARγ protein expression, whereas LG100 268 had no effect. Furthermore, neither LG100 268 nor troglitazone had any effect on the protein expression of RXR isoforms or PPARα. Conclusion/interpretation. Co-activation of PPARγ and RXR results in additive or synergistic effects on glucose and lipid metabolism in skeletal muscle, but unlike troglitazone, LG100 268 does not alter expression of its own receptor. [Diabetologia (2001) 44: 444–452] Received: 5 October 2000 and in revised form: 8 December 2000     

E-Selectin plasma concentration is influenced by glycaemic control in NIDDM patients: possible role of oxidative stress

Summary Although elevated levels of soluble E-selectin and intercellular cell adhesion molecules-1 (ICAM-1) have been reported in non-insulin-dependent diabetes mellitus (NIDDM), it is not clear by what mechanism this elevation occurs and whether or not it is related to glycaemic control. In this study we analyse: 1) the relation of glycaemic control with the concentrations of E-selectin, vascular cell adhesion molecules-1 (VCAM-1) and ICAM-1 in NIDDM patients; 2) whether metabolic control can affect the oxidative stress (as measured by plasma hydroperoxide concentration and susceptibility of LDL to in vitro oxidation) and hence the adhesion molecule plasma concentrations. Thirty-four (19 males and 15 females) poorly controlled NIDDM patients were studied. All parameters were evaluated at the beginning of the study and after 90 days of dietary and pharmacological treatment. The treatment decreased HbA_1C (p < 0.001), E-selectin (p < 0.001), plasma hydroperoxides (p < 0.003) and the susceptibility of LDL to in vitro oxidation (lag phase) (p < 0.0001). Before treatment HbA_1C, lag phase and lipid hydroperoxides correlated with E-selectin plasma concentration (r = 0.51, –0.57 and 0.54, respectively, p < 0.01). There was also a correlation between HbA _1C and lag phase (p < 0.01) and between HbA _1C and lipid hydroperoxides (p < 0.01). In addition, the variations of HbA _1C, lag phase and lipid hydroperoxide values correlated with those for E-selectin concentration after 90 days' treatment (r = 0.54, –0.64 and 0.61, respectively, p < 0.01). In multiple linear correlation analysis, however, the partial correlation coefficients of HbA _1C (basal and variations) with E-selectin concentration (basal and variations) fell to non-significant values (r = 0.12 and 0.25, respectively) when LDL lag phase and plasma hydroperoxides were kept constant. The results indicate that the improvement of metabolic control in NIDDM patients is associated with a decrease of E-selectin plasma levels; they also suggest that glycaemic control per se is not directly implicated in determining E-selectin plasma concentration; glycaemic control could affect E-selectin concentration through its effect on oxidative stress. [Diabetologia (1997) 40: 584–589] Received: 31 October 1996 and in revised form 23 January 1997     

Autoantibodies to oxidized low density lipoprotein: the relationship to low density lipoprotein fatty acid composition in diabetes

Autoantibodies to oxidized low density lipoprotein have been shown to be an independent predictor of the progression of carotid atherosclerosis. This study examines the relationship between low density lipoprotein fatty acid composition and autoantibodies to both malondialdehyde-modified and copper-oxidized low density lipoprotein in non-diabetic patients with (n = 17), and without (n = 18), definite evidence of previous myocardial infarction. The third group were non-insulin-dependent diabetic patients with no evidence of atherosclerosis (n = 15) and the fourth group were patients with non-insulin-dependent diabetes (n = 17) who had definite evidence of previous myocardial infarction. Fatty acids were measured by gas-liquid chromatography. Antibodies to malondialdehyde-modified low density lipoprotein and copper-oxidized low density lipoprotein were determined by an ELISA method. Autoantibodies to copper-oxidized low density lipoprotein were significantly higher in the non-diabetic patients with heart disease when compared to any other group (p < 0.05). Autoantibodies to malondialdehyde-modified low density lipoprotein were significantly higher in the non-diabetic subjects with heart disease and in both diabetic groups compared to non-diabetic subjects without coronary heart disease (p < 0.05). Lineolic acid (%) in low density lipoprotein did not differ between groups but arachidonic acid (%) was significantly lower in both diabetic and non-diabetic patients with coronary heart disease (p < 0.05). The diabetic patients with low antibodies had 39.6 ± 2.2 % polyunsaturated fatty acids in their low density lipoprotein while diabetic patients with high antibodies had 46.7 ± 1.2 % polyunsaturates in their low density lipoprotein (p < 0.01). This study confirms the association between antibodies to oxidized low density lipoprotein and coronary heart disease and shows raised low density lipoprotein antibody levels in diabetic patients with and without demonstrable atherosclerosis. In the diabetic patients, those with high antibody levels had high polyunsaturated fatty acid levels in their LDL suggesting a possible role for dietary intervention. © 1997 John Wiley & Sons, Ltd.     

Alterations in serum lipids and apolipoproteins in male Type 1 (insulin-dependent) diabetic patients with microalbuminuria

Summary The relationships between serum lipid, apolipoprotein levels and urinary albumin excretion were investigated in 20 male Type 1 (insulin-dependent) diabetic patients with microalbuminuria (overnight urinary albumin excretion between 10 and 200 g/min), in 18 male Type 1 diabetic patients without microalbuminuria and in 18 male control subjects. In the microalbuminuric patients low density lipoprotein cholesterol was higher than in the control subjects (p<0.05); the high density lipoprotein/low density lipoprotein cholesterol ratio was lower than in the normoalbuminuric diabetic patients (p<0.05), and in the control subjects (p<0.01); apolipoprotein B was higher than in the normoalbuminuric patients (p<0.05); the apolipoprotein A₁/B ratio was lower than in the normoalbuminuric diabetic patients (p<0.05). Serum triglyceride was higher in the microalbuminuric diabetic patients and in the control subjects than in the normoalbuminuric diabetic patients (p<0.05, for both), but was not different between the microalbuminuric diabetic patients and the control subjects. No significant differences between the 3 groups were present with respect to serum cholesterol, high density lipoprotein cholesterol and apolipoprotein A₁. In the 2 combined Type 1 diabetic groups there were significant correlations between urinary albumin excretion and the high density lipoprotein/low density lipoprotein cholesterol ratio (R -0.40, p<0.02), apolipoprotein B (R0.35, p<0.05) and the apolipoprotein A₁/B ratio (R -0.44, p<0.01). These results indicate microalbuminuria related differences in lipid and apolipoprotein levels in male Type 1 diabetic patients, which may contribute to an increased risk of cardiovascular disease.     

The role of dietary cholesterol in the regulation of postprandial apolipoprotein B48 levels in diabetes

The atherogenicity of intestinally derived postprandial lipoproteins has been confirmed in a number of recent studies. We have shown abnormalities in postprandial lipoprotein metabolism in diabetic patients, a group with an increased susceptibility to atherosclerosis. This study examined the relationship between dietary cholesterol and the postprandial, intestinally derived, apolipoprotein B48 and apolipoprotein B100 from the liver. We compared 10 non-insulin-dependent (Type 2, NIDDM) diabetic patients and 10 age-matched non-diabetic control subjects. Fasting blood was taken and subjects were fed a cholesterol-free, high fat meal. Blood samples were repeated at 2 h, 4 h, 6 h, and 8 h postprandial. The following week fasting blood was collected and subjects were given the same meal with 1g of added cholesterol. Blood was collected at the same time points. Chylomicrons and very low density lipoprotein were isolated by sequential ultracentrifugation and their lipoprotein composition determined. Apolipoproteins B48 and B100 were separated by gradient gel electrophoresis and quantified by densitometric scanning using a low density lipoprotein apolipoprotein B100 standard. Post prandial chylomicron cholesterol and triglyceride increased after the high cholesterol meal in both groups (p < 0.001). The postprandial chylomicron apolipoprotein B48 response of both diabetic and control subjects to the cholesterol meal was less than to the cholesterol-free meal (p < 0.001). Fasting very low density lipoprotein apolipoprotein B48 was higher in diabetic patients compared to control subjects and their postprandial increase following the cholesterol-free meal was significantly greater (p < 0.001). There was a 10-fold increase in the incremental postprandial VLDL apolipoprotein B48 area under the curve after the cholesterol-rich meal in the diabetic patients compared to a 3-fold increase in control subjects. The postprandial very low density lipoprotein apolipoprotein B100 was similar in the two groups with both meals. The study demonstrates a very significant increase in the amount of intestinally derived small apolipoprotein B48-associated particles in the very low density lipoprotein fraction following a cholesterol-rich meal in diabetic patients. Synthesis rather than clearance may be the major cause of the increase in these atherogenic postprandial particles. © 1997 John Wiley & Sons, Ltd.     

Effects of troglitazone on atherogenic lipoprotein phenotype in coronary patients with insulin resistance.

Insulin resistance is associated with atherogenic lipoprotein phenotype, including small dense LDL particle, hypertriglycemia and low HDL cholesterol levels. Troglitazone, a novel insulin sensitizing agent, may improve the associated lipid profile in patients with insulin resistance. We examined the effects of troglitazone (400 mg daily for 12 weeks) in 12 non-diabetic coronary patients (60+/-10 years), all of whom had hyperinsulinemic response to an oral glucose load. Troglitazone markedly reduced the insulin response. After the treatment, plasma triglycerides decreased by 32% (P<0.05), HDL cholesterol increased by 11%, (P<0.05) and LDL peak particle diameter increased from 24.7+/-0.3 to 25.5+/-0.5 nm (P<0.01). These lipidic improvements were associated with a significant rise in postheparin lipoprotein lipase levels (175+/-52 to 217+/-69 ng/ml, P<0.01). In patients with insulin resistance syndrome, troglitazone improved the atherogenic lipoprotein phenotype as well as hyperinsulinemia. Our data suggest that troglitazone therapy could reduce the atherosclerotic risk due to insulin resistance even in non-diabetic patients.     

Effect of Combination Therapy of Troglitazone and Sulphonylureas in Patients with Type 2 Diabetes Who Were Poorly Controlled by Sulphonylurea Therapy Alone

The clinical efficacy of troglitazone, a new oral hypoglycaemic agent was investigated in Type 2 diabetes in combination with sulphonylureas. Two hundred and ninety-one patients with Type 2 diabetes (age 21–81 years) whose previous glycaemic control by sulphonylureas was judged stable but unsatisfactory (fasting plasma glucose (FPG) > 8.3 mmol l⁻¹) were randomly allocated into the troglitazone treatment group (troglitazone group, n = 145) or the placebo treatment group (placebo group, n = 146). They were treated by test drugs for 12 weeks in combination with the same dose of sulphonylureas before the trial. One hundred and twenty-two patients who received troglitazone and 126 patients who received placebo were evaluated for efficacy. The baseline characteristics did not differ significantly between the two groups. In the troglitazone group, FPG and HbA_1C decreased significantly after the treatment (before vs after, FPG: 10.8 ± 2.0 mmol l⁻¹ vs 9.2 ± 2.5 mmol l⁻¹, p < 0.001; HbA_1C: 9.2 ± 1.4 % vs 8.5 ± 1.5 %, p < 0.001). FPG and HbA_1C did not change after the treatment in the placebo group (before vs after, FPG: 10.5 ± 1.7 mmol l⁻¹ vs 10.7 ± 2.2 mmol l⁻¹; HbA_1C: 9.0 ± 1.5 % vs 9.2 ± 1.6 %). Serum total cholesterol and HDL-cholesterol did not change in either group, however, serum triglyceride significantly decreased in the troglitazone group. No serious adverse events occurred in either group. In conclusion, troglitazone 400 mg day⁻¹ had a significant hypoglycaemic effect in combination with sulphonylureas without any serious adverse events. Troglitazone, developed as an insulin action enhancer, can be a useful hypoglycaemic agent in the treatment of patients with Type 2 diabetes who are not well controlled by sulphonylureas alone.     

The Regulation of Post-prandial Cellular Cholesterol Metabolism in Type 2 Diabetic and Non-diabetic Subjects

The purpose of the study was to determine the effect of diabetes on the regulation of postprandial cholesterol metabolism. Four groups of patients (n = 8 for each group) were examined: Type 2 diabetic patients with and without hypercholesterolemia and nondiabetic subjects with and without hypercholesterolaemia. Serum lipoproteins, lipoprotein composition, cellular cholesterol, and cellular cholesterol synthesis were measured before and 4 h after a high calorie meal. The BMI for the hypercholesterolaemic diabetic patients of 31.5 ± 0.95 (SEM) was significantly higher than that for the control group of 26.2 ± 1.0 (p < 0.01). Fasting triglyceride levels were significantly higher in the normocholesterolaemic and hypercholesterolaemic diabetic patients and in the hypercholesterolaemic non-diabetic subjects (1.45 ± 0.22, 2.27 ± 0.34, and 1.58 ± 0.18 mmol l^?1, respectively) compared with normocholesterolaemic non-diabetic subjects (0.75 ± 0.12 mmol l^?1: p < 0.01). The normocholesterolaemic and hypercholesterolaemic diabetic subjects had significantly lower fasting serum high density lipoprotein (HDL) (1.06 ± 0.08 and 1.04 ± 0.06 mmol l^?1) compared to the corresponding non-diabetic groups (1.29 ± 0.11 and 1.45 ± 0.17 mmol l^?1, p < 0.05). The esterified/free cholesterol ratio of very low density lipoprotein (including chylomicrons VLDL-C) decreased postprandially in all groups with an overall decrease of 1.33 to 0.83 (p < 0.01). Fasting cellular cholesterol in mononuclear leucocytes from normocholesterolaemic diabetic patients was similar to that for hypercholesterolaemic diabetic (36.8 ± 1.2 vs 40.6 ± 5.5 mg g^?1 protein) and non-diabetic subjects (36.7 ± 6.8 mg g^?1 protein) and significantly greater than cholesterol in cells from control subjects (29.7 ± 1.5 mg g^?1 protein, p < 0.05). In cells from control subjects only, there was a significant postprandial increase in cholesterol to 39.4 ± 5.2 mg g^?1 protein (p < 0.05) and a corresponding postprandial reduction in cholesterol synthesis from 149 ± 34 to 102 ± 33 nmol g^?1 cell protein (p < 0.05). These results demonstrate a lack of correlation between serum and cellular cholesterol in diabetic patients and an inability to suppress cellular cholesterol synthesis postprandially in these patients. The differences may, in part, explain the increased deposition of cholesterol in atheromatous plaques in normocholesterolaemic diabetic patients.     

The metabolic effects of troglitazone in patients with diabetes and end-stage renal disease

Thiazolidinediones (TZD) are effective agents for the treatment of hyperglycemia, and appear ideal in diabetic patients with progressive or end-stage renal disease because of its predominant hepatic clearance. Troglitazone, the first available TZD for clinical use, was withdrawn due to safety concerns; however, studies completed with this agent can provide a better understanding of the class effect of TZDs. This study was an open-label, controlled clinical trial examining the safety and efficacy of troglitazone in type 2 diabetic patients with end-stage renal disease (ESRD). Twelve subjects were randomized to parallel study groups and treated for 6 mo with or without troglitazone at a maximum dose of 600 mg/d in addition to continuing their previous diabetes medications (insulin or sulfonylurea). The results showed no significant differences in glycemic control with or without troglitazone treatment for 6 mo. However, there was a significant reduction in insulin dosage with troglitazone treatment (22.9±7.3 units/d) than without troglitazone treatment (54±12.9 units/d) (p<0.05), as well as the change in the insulin dosage from baseline between the two groups (troglitazone, −8.4 units vs control, +4.3 units, p<0.05). Weight changes and aspartate aminotransferase levels greater than 1.5 times the upper limit of normal were not observed in participants of either treatment group. This study demonstrates that troglitazone was safe and effective for the treatment of hyperglycemia in patients requiring dialysis, and strongly supports the clinical use of currently available TZDs in diabetic patients with renal failure.     

Autoantibodies to oxidised low density lipoproteins in IDDM are inversely related to metabolic control and microvascular complications

Summary Diabetes mellitus is associated with an increased risk of atherosclerosis. The oxidation of low-density lipoproteins (LDL) is considered a key event in the initiation of atherosclerosis. To investigate LDL oxidation in vivo we measured autoantibodies to oxidised LDL (oxLDL) in 94 patients with insulin-dependent diabetes mellitus (IDDM), compared to 27 age-matched, healthy control subjects. Patients and control subjects were screened for autoantibodies using a solid phase ELISA, comparing the binding to oxLDL with that to native LDL (nLDL). In patients with IDDM the oxLDL/nLDL antibody ratio was significantly higher than in control subjects (means ± SEM: 2.24 ± 0.26 vs 1.17 ± 0.17, p < 0.03). Antibody-negative patients had a longer diabetes duration (13.5 ± 1.3 vs 9.1 ± 1.1 years, p < 0.01) and higher actual and mean HbA_{1 c} levels compared to antibody-positive patients (8.8 ± 0.2 vs 7.9 ± 0.2 %, p < 0.005 and 8.3 ± 0.2 vs 7.7 ± 0.2 %, p < 0.03; respectively). In patients with a high microangiopathy score, the antibody ratio was lower than in patients without complications (1.04 ± 0.10 vs 2.40 ± 0.29, p < 0.01). OxLDL specific immune complexes were found exclusively in antibody-negative as compared to antibody-positive patients (18.3 vs 0 %; p < 0.01). Our data demonstrate an inverse relationship between free oxLDL antibodies and the severity of the disease. This apparent paradox can be explained in part by our demonstration of oxLDL immune complexes, masking free antibodies. [Diabetologia (1998) 41: 350–356] Received: 18 June 1997 and in final revised form: 30 September 1997     

Effect of ciprofibrate on lipoprotein metabolism and oxidative stress parameters in patients with type 2 diabetes mellitus and atherogenic lipoprotein phenotype

The effect of ciprofibrate therapy on plasma lipids and lipoproteins, HDL and LDL subfraction profile, fractional esterification rate of HDL cholesterol (FER_HDL) and the resistance of LDL and serum lipids to oxidation was studied in 24 males with type 2 diabetes and atherogenic lipoprotein phenotype (ALP). We also examined the effect of ciprofibrate therapy on oxidative DNA damage in peripheral lymphocytes. No differences in glucose, HbA1C and BMI levels were found after three months of ciprofibrate therapy. Ciprofibrate significantly decreased total cholesterol and triglyceride levels by 5.5% and 50% (p = 0.05; 0.001, respectively) and increased HDL-cholesterol levels by 8.5% (p = 0.05). FER_HDL and LDL subfraction profile were also favorably affected, However, no effect on HDL subclasses was found. There were no statistically significant differences in lipid resistance to oxidation measured in serum and in LDL (lag time and V_max) before and after therapy. No significant effect of ciprofibrate was found on oxidative DNA damage. The evaluation of the relationship between oxidative damage purines with lag time in LDL and maximal rate of serum lipid oxidation showed significant correlations after therapy (r = −0.58; 0.47, p = 0.01; 0.05, respectively), but only trends before starting ciprofibrate treatment. Type 2 diabetes mellitus represents a complex metabolic disorder expressed in glucose and lipoprotein disturbances and increased oxidative stress. Ciprofibrate therapy favorably affected major features of lipid abnormalities of diabetic patients, but the level of oxidative stress assessed by in vitro and in vivo methods was not changed. The evaluation of expected logical correlations between the parameters of lipoprotein metabolism, lipid resistance in serum and LDL, and oxidative DNA damage showed that those correlations were more relevant and significant after ciprofibrate treatment and were not related with glucose homeostasis. Received: 13 March 2000 / Accepted in revised form: 7 November 2000     

What dose of vitamin E is required to reduce susceptibility of LDL to oxidation?

Background: Oxidative modification of low density lipoprotein (LDL) may play a role in the pathogenesis of atherosclerosis. Ingestion of vitamin E in high dosage has been shown to reduce the susceptibility of LDL to copper-induced oxidation, as assessed ex vivo .
Aim: To determine a minimum dose of supplementary vitamin E which will significantly reduce the susceptibility of LDL to oxidation.
Methods: A single centre, double-blind, parallel placebo-controlled trial. Healthy volunteers (total n =42) were randomised to receive placebo, 500, 1000 or 1500 IU/day of vitamin E (D-α-tocopherol) for a period of six weeks. Primary outcomes were change in lag time or oxidation rate to copper-induced LDL oxidation. Secondary outcomes were changes in plasma vitamin E levels and clinical tolerance.
Results: Lag time to LDL oxidation was significantly prolonged and oxidation rate significantly slowed at all dose levels of vitamin E, indicating a threshold effect from 500 IU/day. Compared to placebo, the median prolongation in lag time on 500 IU/day was 26%, on 1000 IU/day 24% and on 1500 IU/day 35%. The corresponding slowing in oxidation rates was 14%, 19% and 25% respectively. The per cent change in plasma vitamin E concentration was highly correlated with the change in lag time ( r =0.61, p<0.001) and oxidation rate (r=−0.55, p <0.001). Vitamin E was generally well tolerated.
Conclusions: Vitamin E in a dose of 500 IU/day will significantly reduce the susceptibility of LDL to oxidation. Whether or not this treatment will consistently reduce the future incidence of coronary artery disease will only be answered by further clinical trials.     

Troglitazone inhibits atherosclerosis in apolipoprotein E-knockout mice: pleiotropic effects on CD36 expression and HDL

Atherosclerotic coronary heart disease is a common complication of the insulin resistance syndrome that can occur with or without diabetes mellitus. Thiazolidinediones (TZDs), which are insulin-sensitizing antidiabetic agents, can modulate the development of atherosclerosis not only by changing the systemic metabolic conditions associated with insulin resistance but also by exerting direct effects on vascular wall cells that express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a nuclear receptor for TZDs. Here we show that troglitazone, a TZD, significantly inhibited fatty streak lesion formation in apolipoprotein E-knockout mice fed a high-fat diet (en face aortic surface lesion areas were 6.9+/-2.5% vs 12.7+/-4.7%, P<0.05; cross-sectional lesion areas were 191 974+/-102 911 micrometer(2) vs 351 738+/-175 597 micrometer(2), P<0.05; n=10). Troglitazone attenuated hyperinsulinemic hyperglycemia and increased high density lipoprotein cholesterol levels. In the aorta, troglitazone markedly increased the mRNA levels of CD36, a scavenger receptor for oxidized low density lipoprotein, presumably by upregulating its expression, at least in part, in the macrophage foam cells. These results indicate that troglitazone potently inhibits fatty streak lesion formation by modulating both metabolic extracellular environments and arterial wall cell functions.     

Resistance training improves the metabolic profile in individuals with type 2 diabetes

Aerobic endurance exercise has traditionally been advocated in the treatment of type 2 diabetes, while the potential role of resistance training has often been overlooked. The aim of the present study was to determine the effect of circuit-type resistance training on blood pressure, lipids and long-term glycaemic control (HbA_1c) in type 2 diabetic subjects. Thirty-eight type 2 diabetic subjects were enrolled in the study; 18 participated in a 5-month individualized progressive resistance training programme (moderate intensity, high volume) twice a week, while the remaining 20 served as controls. The exercise group showed improvements in total cholesterol (6.0±.3 vs 5.3±.3 mM; P<0.01), low density lipoprotein (LDL)-cholesterol (3.90±.22 vs 3.35±.21 mM; P<0.01) and triglycerides (1.91±.25 vs 1.53±.22 mM; P<0.01). Also, the difference in the change in HbA_1c between the groups (0.5%) achieved statistical significance (P<0.01). Circuit-type resistance training seems to be feasible in moderately obese, sedentary type 2 diabetic subjects and the inclusion of circuit-type resistance training in exercise training programmes for type 2 diabetic subjects seems appropriate. Received: 14 April 1997 / Accepted in revised form: 25 July 1997     

Decreased oxidation susceptibility of plasma low density lipoproteins in patients with Gilbert's syndrome

Background and Aim: The association of hyperbilirubinemia in Gilbert's syndrome (GS) with a decrease in prevalence of coronary artery disease is a well‐known phenomenon. In this study, the state of low‐density lipoprotein (LDL) oxidation which has been postulated to be a significant determinant at the etiopathogenesis of atherosclerotic disorders was investigated among individuals with GS. Methods: For this purpose, serum cholesterol, LDL cholesterol, high‐density lipoprotein cholesterol, triglycerides, uric acid, apolipoprotein A and B, bilirubins, thiobarbituric acid‐reactive substances, and the sensitivity of LDL oxidation levels, as well as serum alanine aminotransferase, aspartate aminotranserfase, gamma glutamil transferase, and alkaline phosphatase activities, were determined in 17 patients with Gilbert's syndrome and 15 healthy adults. Results: There was no significant difference between the groups except the indirect bilirubin parameter (P < 0.001). In comparison with the healthy individuals, LDL oxidation levels between 75 and 120 min were significantly lower (P < 0.005) along with prolonged lag‐phase in GS patients, indicating a delay in oxidation susceptibility. Conclusion: It is suggested that the chronic hyperbilirubinemia leading to a lag‐phase prolongation in LDL oxidation and a decrease in LDL oxidation may be reason for the low percentage of coronary artery disease.