The Systemic Redox Status Is Maintained in Non-Smoking Type 2 Diabetic Subjects Without Cardiovascular Disease: Association with Elevated Triglycerides and Large VLDL.

Decreased circulating levels of free thiols (R-SH, sulfhydryl groups) reflect enhanced oxidative stress, which plays an important role in the pathogenesis of cardiometabolic diseases. Since hyperglycemia causes oxidative stress, we questioned whether plasma free thiols are altered in patients with type 2 diabetes mellitus (T2DM) without cardiovascular disease or renal function impairment. We also determined their relationship with elevated triglycerides and very low density lipoproteins (VLDL), a central feature of diabetic dyslipidemia. Fasting plasma free thiols (colorimetric method), lipoproteins, VLDL (nuclear magnetic resonance spectrometry), free fatty acids (FFA), phospholipid transfer protein (PLTP) activity and adiponectin were measured in 79 adult non-smoking T2DM subjects (HbA1c 51 ± 8 mmol/mol, no use of insulin or lipid lowering drugs), and in 89 non-smoking subjects without T2DM. Plasma free thiols were univariately correlated with glucose (r = 0.196, p < 0.05), but were not decreased in T2DM subjects versus non-diabetic subjects (p = 0.31). Free thiols were higher in subjects with (663 ± 84 µmol/L) versus subjects without elevated triglycerides (619 ± 91 µmol/L; p = 0.002). Age- and sex-adjusted multivariable linear regression analysis demonstrated that plasma triglycerides were positively and independently associated with free thiols (ß = 0.215, p = 0.004), FFA (ß = 0.168, p = 0.029) and PLTP activity (ß = 0.228, p = 0.002), inversely with adiponectin (ß = -0.308, p < 0.001) but not with glucose (ß = 0.052, p = 0.51). Notably, the positive association of free thiols with (elevated) triglycerides appeared to be particularly evident in men. Additionally, large VLDL were independently associated with free thiols (ß = 0.188, p = 0.029). In conclusion, circulating free thiols are not decreased in this cohort of non-smoking and generally well-controlled T2DM subjects. Paradoxically, higher triglycerides and more large VLDL particles are likely associated with higher plasma levels of thiols, reflecting lower systemic oxidative stress.

Antibodies Against the C-Terminus of ApoA-1 Are Inversely Associated with Cholesterol Efflux Capacity and HDL Metabolism in Subjects with and without Type 2 Diabetes Mellitus.

BACKGROUND: We determined relationships of cholesterol efflux capacity (CEC), plasma cholesterol esterification (EST) and cholesteryl ester transfer (CET) with anti-c-terminus apoA-1 (Ac-terAA1) and anti-apolipoprotein (apo)-1 (AAA1) autoantibodies in subjects with and without Type 2 diabetes mellitus (T2D). METHODS: In 75 T2D subjects and 75 nondiabetic subjects, Ac-terAA1 and AAA1 plasma levels were measured by enzyme-linked immunosorbent assay. CEC was measured as [³H]-cholesterol efflux from human cultured fibroblasts to diluted individual subject plasma. Plasma EST and CET were assayed by isotope methods. RESULTS: Ac-terAA1 and AAA1 levels and were similar between T2D and control subjects. Univariate regression analysis (n = 150) demonstrated that Ac-terAA1 levels were inversely correlated with CEC, EST, CET, total cholesterol, non-HDL cholesterol, triglycerides and apolipoprotein B, (p < 0.05 to p < 0.01), but not with glucose and HbA1c. In separate multivariable linear regression models, CEC, EST and CET were inversely associated with Ac-terAA1 levels independently of age, sex, T2D and drug use (ß = -0.186, p = 0.026; ß = -0.261, p < 0.001; and ß = -0.321, p < 0.001; respectively). These associations were lost after additional adjustment for non-HDL cholesterol and triglycerides. No associations were observed for AAA1. CONCLUSIONS: CEC, plasma EST and CET are inversely associated with Ac-terAA1 autoantibodies, conceivably attributable to an inverse relationship of these autoantibodies with apolipoprotein B-containing lipoproteins.

Downregulation of cholesteryl ester transfer protein by glucocorticoids: a randomised study on HDL.

BACKGROUND: High density lipoprotein (HDL) cholesterol is not decreased in hypercortisolism despite high triglycerides, which may be ascribed to effects on the cholesteryl ester transfer protein (CETP) pathway. We explored if CETP mRNA expression is modulated by glucocorticoid treatment in vitro. Effects of doubling the hydrocortisone (HCT) replacement dose on plasma CETP activity, and HDL characteristics were tested in patients with secondary adrenal insufficiency. MATERIALS AND METHODS: Human THP-1 macrophages were incubated with corticosterone in vitro in the presence or absence of a liver X receptor (LXR) agonist, followed by determination of CETP mRNA levels by quantitative real-time PCR. In addition, a randomised double-blind cross-over study was performed in 47 patients with secondary adrenal insufficiency (university medical setting; 10 weeks exposure to a higher HCT dose (0·4-0·6 mg/kg body weight) vs. 10 weeks of a lower HCT dose (0·2-0·3 mg/kg body weight). RESULTS: Corticosterone dose dependently decreased CETP mRNA in THP-1 macrophages. Corticosterone also decreased CETP mRNA expression after LXR pretreatment. In patients, CETP activity decreased with doubling of the HCT dose (P = 0·049), coinciding with an increase in HDL cholesterol, apolipoprotein A-I and the HDL cholesterol/apolipoprotein A-I ratio (reflecting HDL size; P < 0·01 for each). The increase in the HDL cholesterol/apolipoprotein A-I ratio was correlated with the decrease in plasma CETP activity (r = -0·442, P = 0·002). CONCLUSION: Glucocorticoids downregulate CETP gene expression in a human macrophage cell system. In line, a higher glucocorticoid replacement dose decreases plasma CETP activity in patients, thereby contributing to higher HDL cholesterol and an increase in estimated HDL size.

Pre-ß-HDL formation relates to high-normal free thyroxine in type 2 diabetes mellitus.

OBJECTIVES: Low-normal thyroid function within the euthyroid range may influence plasma lipoprotein levels. Associations between variation in thyroid function and pre-ß-high density lipoproteins (pre-ß-HDL), i.e. lipid-poor or lipid free HDL particles that act as initial acceptor of cell-derived cholesterol, are unknown. We determined relationships of plasma pre-ß-HDL with thyroid function in euthyroid subjects with and without type 2 diabetes mellitus (T2DM). DESIGN AND SUBJECTS: TSH, free T4, plasma (apo)lipoproteins, pre-ß-HDL, pre-ß-HDL formation (pre-ß-HDL generation during incubation with lecithin:cholesterol acyltransferase being inhibited) and phospholipid transfer protein (PLTP) activity were measured in fasting plasma from 72 T2DM and 82 non-diabetic subjects. RESULTS: TSH was similar and free T4 was slightly higher (P < 0.05) in T2DM vs. non-diabetic subjects. HDL cholesterol and apoA-I were lower, whereas pre-ß-HDL (expressed as % of apoA-I), triglycerides and PLTP activity were higher in T2DM (P < 0.05 to P < 0.001). In T2DM, pre-ß-HDL formation (in apoA-I concentration and in % of apoA-I) was positively related to free T4, PLTP activity, total cholesterol and triglycerides (P < 0.05 for each). Multivariable linear regression analyses, adjusted for age, sex, PLTP activity, total cholesterol and triglycerides, demonstrated that pre-ß-HDL formation was positively related to free T4 (in apoA-I concentration: ß = 0.278, P = 0.014; in % of apoA-I: ß = 0.343, P = 0.003) in T2DM, but not in non-diabetic subjects (both P > 0.30; interaction terms: both P < 0.05). CONCLUSIONS: Variations in thyroid function within the euthyroid range may influence the metabolism of pre-ß-HDL in T2DM.

Low normal thyroid function enhances plasma cholesteryl ester transfer in Type 2 diabetes mellitus.

BACKGROUND: Plasma cholesteryl ester transfer (CET), reflecting endogenous transfer of cholesteryl esters from HDL to very low and low density lipoproteins, is elevated in Type 2 diabetes mellitus (T2DM), and may predict (subclinical) cardiovascular disease. Low normal thyroid function may adversely affect lipoprotein metabolism and atherosclerosis development. We tested whether plasma CET is related to thyroid function in euthyroid T2DM and non-diabetic subjects. SUBJECTS AND METHODS: Plasma CET was measured in 74 T2DM and 82 non-diabetic subjects with thyroid-stimulating hormone (TSH) and free thyroxine levels within the reference range. RESULTS: Plasma CET was 20% higher in T2DM (P = 0.003) coinciding higher cholesteryl ester transfer protein (CETP) mass (P = 0.009) and triglycerides (P = 0.02). In univariate analysis, plasma CET was correlated positively with TSH in T2DM only (r = 0.330, P = 0.004). Multiple linear regression analysis revealed a positive interaction between the presence of T2DM and TSH on plasma CET after controlling for age, sex, body mass index, non-HDL cholesterol, triglycerides and CETP mass (ß = 0.167, P = 0.030). The relationship of plasma CET with TSH was also positively modified by plasma glucose and HbA1c (interaction terms: ß = 0.119, P = 0.036, ß = 0.170, P = 0.001, respectively). Additionally, plasma triglycerides interacted positively with TSH on plasma CET in T2DM (ß = 0.198, P = 0.011). CONCLUSIONS: Low normal thyroid function, as inferred from higher TSH, confers increased plasma CET in the context of chronic hyperglycemia. Effects of thyroid function on plasma CET may be particularly relevant in hypertriglyceridemic T2DM. Low normal thyroid function could influence atherosclerosis susceptibility in T2DM by affecting the plasma cholesteryl ester transfer process.

Increased LCAT activity and hyperglycaemia decrease the antioxidative functionality of HDL.

BACKGROUND: Type 2 diabetes mellitus increases the risk of atherosclerotic cardiovascular disease. Antioxidative properties of high density lipoprotein (HDL) are important for atheroprotection. This study investigated whether the antioxidative functionality of HDL is altered in type 2 diabetes mellitus and aimed to identify potential determinants of this parameter. MATERIALS AND METHODS: In a cross-sectional study, we investigated 74 patients with type 2 diabetes and 75 control subjects. Antioxidative properties of HDL were measured and expressed as either (i) HDL antioxidative capacity or (ii) HDL antioxidation index after multiplying HDL antioxidative capacity results with individual plasma HDL cholesterol concentrations. Lecithin:cholesterol acyltransferase (LCAT) and paraoxonase-1 (PON-1) activities were determined. RESULTS: HDL antioxidative capacity was similar in patients with diabetes and controls, while the HDL antioxidation index was decreased in patients with diabetes (P = 0.005) owing to lower plasma HDL cholesterol (P < 0.001). LCAT activity was higher and PON-1 activity lower in type 2 diabetes mellitus (each P < 0.001). In the combined subjects, HDL antioxidative capacity was inversely related to LCAT activity (P < 0.01). The HDL antioxidation index correlated negatively with blood glucose (P < 0.001), HbA1c and LCAT activity (each P < 0.01), and positively with PON-1 activity (P < 0.01). Multiple linear regression analysis demonstrated that high LCAT activity was associated with both decreased HDL antioxidation capacity (P < 0.05) and index (P < 0.001) independent of diabetes status, glycaemic control and PON-1. CONCLUSIONS: Overall, the antioxidative functionality of HDL is impaired in type 2 diabetes mellitus mostly because of lower HDL cholesterol. Hyperglycaemia, increased LCAT activity and lower PON-1 activity likely contribute to impaired antioxidative functionality of HDL.

Carotid intima media thickness is related positively to plasma pre ß-high density lipoproteins in non-diabetic subjects.

BACKGROUND: Lipid-poor or lipid-free high density lipoprotein (HDL) particles, designated pre ß-HDL, stimulate removal of cell-derived cholesterol to the extracellular compartment, which is an initial step in the reverse cholesterol transport pathway. Pre ß-HDL levels may be elevated in subjects with established cardiovascular disease. We determined the relationship of carotid intima media thickness (IMT), a marker of subclinical atherosclerosis, with pre ß-HDL in subjects without clinically manifest cardiovascular disease. METHODS: IMT and plasma pre ß-HDL, assayed by crossed immuno-electrophoresis, were determined in 70 non-diabetic subjects (aged 56±9 years; non-smokers only; 27 women). RESULTS: IMT was correlated positively with pre ß-HDL, both expressed as plasma apolipoprotein (apo) A-I concentration (r=0.271, p=0.023) and as% of apo A-I (r=0.341, p=0.004). In contrast, IMT was correlated inversely with HDL cholesterol (r=-0.253, p=0.035). IMT was also related positively to pre ß-HDL after adjustment for age, sex, systolic blood pressure (in apoA-I concentration, ß=0.203, p=0.043; in% of plasma apoA-I, ß=0.235, p=0.023). IMT remained associated with pre ß-HDL after additional adjustment for either body mass index, plasma glucose, cholesterol, triglycerides, HDL cholesterol, apoA-I and apoB. CONCLUSION: Subclinical atherosclerosis may relate to higher plasma pre ß-HDL independently of apoA-I and HDL cholesterol levels.

Plasma cholesteryl ester transfer, but not cholesterol esterification, is related to lipoprotein-associated phospholipase A2: possible contribution to an atherogenic lipoprotein profile.

CONTEXT: Plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) predicts incident cardiovascular disease and is associated preferentially with negatively charged apolipoprotein B-containing lipoproteins. The plasma cholesteryl ester transfer (CET) process, which contributes to low high-density lipoprotein cholesterol and small, dense low-density lipoproteins, is affected by the composition and concentration of apolipoprotein B-containing cholesteryl ester acceptor lipoproteins. OBJECTIVE: We tested relationships of CET with Lp-PLA(2) in subjects with and without metabolic syndrome (MetS). DESIGN AND SETTING: In 68 subjects with MetS and 74 subjects without MetS, plasma Lp-PLA(2) mass, cholesterol esterification (EST), lecithin:cholesterol acyltransferase (LCAT) activity level, CET, CET protein (CETP) mass, and lipoproteins were measured. RESULTS: EST, LCAT activity, CET (P < 0.001 for all), and CETP (P = 0.030) were increased, and Lp-PLA(2) was decreased (P = 0.043) in MetS. CET was correlated positively with Lp-PLA(2) in subjects with and without MetS (P < 0.05 for both). EST and LCAT activity were unrelated to Lp-PLA(2), despite a positive correlation between EST and CET (P < 0.001). After controlling for age, sex, and diabetes status, CET was determined by Lp-PLA(2) in the whole group (ß = 0.245; P < 0.001), and in subjects with (ß = 0.304; P = 0.001) and without MetS (ß = 0.244; P = 0.006) separately, independently of triglycerides and CETP. CONCLUSIONS: Plasma CET is related to Lp-PLA(2) in subjects with and without MetS. The process of CET, but not EST, may be influenced by Lp-PLA(2). These findings provide a rationale to evaluate whether maneuvers that inhibit Lp-PLA(2) will reduce CET, and vice versa to document effects of CETP inhibition on Lp-PLA(2).

Hepatic lipid accumulation in apolipoprotein C-I-deficient mice is potentiated by cholesteryl ester transfer protein.

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoC-I deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP.

Plasma cholesteryl ester transfer is a determinant of intima-media thickness in type 2 diabetic and nondiabetic subjects: role of CETP and triglycerides.

We tested whether carotid artery intima-media thickness (IMT) is associated with plasma cholesteryl ester transfer (CET) and/or the plasma cholesteryl ester transfer protein (CETP) concentration in type 2 diabetic and control subjects. In 87 male and female subjects with type 2 diabetes (nonsmokers, no insulin or lipid-lowering drug treatment) and 82 control subjects, IMT, plasma CET, CETP mass, and lipids were determined. HDL cholesterol was lower, whereas IMT, pulse pressure, plasma triglycerides, and plasma CET and CETP concentration were higher in diabetic patients versus control subjects. In diabetic patients, plasma CET was positively determined by triglycerides (P < 0.001), non-HDL cholesterol (P < 0.001), CETP (P = 0.002), and the interaction between CETP and triglycerides (P = 0.004). In control subjects, plasma CET was positively related to triglycerides (P < 0.001) and non-HDL cholesterol (P < 0.001). HDL cholesterol was inversely related to plasma CET in each group (P < 0.01 for both). IMT was positively associated with plasma CET in diabetic (P = 0.05) and control (P < 0.05) subjects after adjustment for age, sex, and pulse pressure. No independent relationship with plasma CETP mass was found. Plasma CET is a positive determinant of IMT. Plasma CETP mass, in turn, is a determinant of CET with an increasing effect at higher triglycerides. These data, therefore, provide a rationale to evaluate the effects of CETP inhibitor treatment on plasma CET and on cardiovascular risk in diabetes-associated hypertriglyceridemia.