High density lipoprotein structural changes and drug response in lipidomic profiles following the long-term fenofibrate therapy in the FIELD substudy.

In a recent FIELD study the fenofibrate therapy surprisingly failed to achieve significant benefit over placebo in the primary endpoint of coronary heart disease events. Increased levels of atherogenic homocysteine were observed in some patients assigned to fenofibrate therapy but the molecular mechanisms behind this are poorly understood. Herein we investigated HDL lipidomic profiles associated with fenofibrate treatment and the drug-induced Hcy levels in the FIELD substudy. We found that fenofibrate leads to complex HDL compositional changes including increased apoA-II, diminishment of lysophosphatidylcholines and increase of sphingomyelins. Ethanolamine plasmalogens were diminished only in a subgroup of fenofibrate-treated patients with elevated homocysteine levels. Finally we performed molecular dynamics simulations to qualitatively reconstitute HDL particles in silico. We found that increased number of apoA-II excludes neutral lipids from HDL surface and apoA-II is more deeply buried in the lipid matrix than apoA-I. In conclusion, a detailed molecular characterization of HDL may provide surrogates for predictors of drug response and thus help identify the patients who might benefit from fenofibrate treatment.

Macrophage cholesterol efflux to plasma and HDL in subjects with low and high homocysteine levels: a FIELD substudy.

OBJECTIVES: Increases of homocysteine (Hcy) by fenofibrate correlated inversely to changes in HDL-C and apoA-I in the FIELD study. This finding raised the question whether high Hcy may influence HDL function and counteract benefits of fenofibrate on cardiovascular outcomes. In a subset of the FIELD study we investigated whether fenofibrate therapy or high Hcy, separately or in concert, modulate: (1) ability of plasma or HDL to facilitate cholesterol efflux from THP-1 foam cells; (2) plasma potential to generate preß-HDL; (3) plasma phospholipid transfer protein (PLTP) activity, serum PON-1 mass and activity, HDL particle size and distribution. METHODS: We selected 33 subjects in the FIELD fenofibrate arm according to quartiles of Hcy at 5th year: 17 subjects were in the lowest (Low Hcy group) and 16 subjects were in the highest quartile (High Hcy group). In addition, 14 subjects allocated to placebo were matched by close-out Hcy levels to Low Hcy group. This design allowed us to examine the effects of both fenofibrate (comparison between placebo vs Low Hcy groups) and Hcy (comparison between close-out Low and High Hcy groups) on plasma and HDL ability to facilitate cellular cholesterol removal in the efflux assay in vitro using THP-1 foam cells. RESULTS: Hcy levels were 13.3±0.7 µmol/L (placebo), 13.2±2 µmol/L (Low Hcy) and 27.4±6.5 µmol/L (High Hcy). Cholesterol efflux values to HDL and plasma, percentage of plasma preß-HDL, PLTP activity, serum PON-1 mass and HDL particle size and distribution were similar in both fenofibrate groups and comparable to those of the placebo group. CONCLUSIONS: In the present study cohort fenofibrate and high Hcy levels did not modulate HDL and plasma functions in the first step of reverse cholesterol transport, cholesterol efflux from foam cells.

PPARalpha: an emerging therapeutic target in diabetic microvascular damage.

The global pandemic of diabetes mellitus portends an alarming rise in the prevalence of microvascular complications, despite advanced therapies for hyperglycemia, hypertension and dyslipidemia. Peroxisome proliferator-activated receptor alpha (PPARalpha) is expressed in organs affected by diabetic microvascular disease (retina, kidney and nerves), and its expression is regulated specifically in these tissues. Experimental evidence suggests that PPARalpha activation attenuates or inhibits several mediators of vascular damage, including lipotoxicity, inflammation, reactive oxygen species generation, endothelial dysfunction, angiogenesis and thrombosis, and thus might influence intracellular signaling pathways that lead to microvascular complications. PPARalpha has emerged as a novel target to prevent microvascular disease, via both its lipid-related and lipid-unrelated actions. Despite strong experimental evidence of the potential benefits of PPARalpha agonists in the prevention of vascular damage, the evidence from clinical studies in patients with diabetes mellitus remains limited. Promising findings from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study on microvascular outcomes are countered by elevations in participants' homocysteine and creatinine levels that might potentially attenuate the benefits of PPARalpha activation. This Review focuses on the role of PPARalpha activation in diabetic microvascular disease and highlights the available experimental and clinical evidence from studies of PPARalpha agonists.

Acute exposure to rosiglitazone does not affect glucose transport in intact human skeletal muscle.

Thiazolidinediones (TZDs) such as rosiglitazone are widely used as antidiabetic drugs. Animal studies suggest that TZDs may have direct metabolic actions in skeletal muscle. Here, we examined if acute exposure to rosiglitazone stimulates glucose transport rate and affects proximal insulin signaling in isolated skeletal muscle strips from nondiabetic men. Open muscle biopsies were obtained from musculus vastus lateralis from 15 nondiabetic men (50 +/- 3 years old, 26.9 +/- 1.1 kg/m(2)). Skeletal muscle strips were isolated and exposed to rosiglitazone (1 or 10 micromol/L), 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (1 mmol/L), insulin (120 nmol/L), or a combination of insulin (120 nmol/L) and rosiglitazone (10 micromol/L) in vitro for 1 hour. Glucose transport was analyzed by accumulation of intracellular 3-O-methyl [(3)H] glucose; phosphorylation of Akt-Ser(473) and Akt-Thr(308) and phosphorylation of acetyl coenzyme A carboxylase beta were determined using phosphospecific antibodies. 5-Aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside and insulin increased glucose transport rate 1.5-fold (P < .05) and 1.7-fold (P < .01) in isolated muscle strips, respectively. Exposure to rosiglitazone transiently increased phosphorylation of acetyl coenzyme A carboxylase beta, with a maximum effect at 15 minutes and return to baseline at 60 minutes. However, rosiglitazone did not affect basal or insulin-stimulated glucose transport rate, or phosphorylation of Akt-Ser(473) or Akt-Thr(308) in isolated muscle strips. In conclusion, acute exposure to rosiglitazone does not affect glucose transport in human skeletal muscle.

Effects of long-term fenofibrate treatment on markers of renal function in type 2 diabetes: the FIELD Helsinki substudy.

OBJECTIVE: Although fenofibrate was associated with less progression of albuminuria in the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, it is unknown if it has any effect on renal function. We explored if there were changes in commonly available markers of renal function during fenofibrate treatment in the FIELD Helsinki cohort excluding statin users. RESEARCH DESIGN AND METHODS: One hundred and seventy subjects with type 2 diabetes were randomly assigned to micronized fenofibrate (200 mg/day) or placebo for 5 years. In this substudy, we measured several markers of albumin excretion and renal function. RESULTS: After intensified treatment, blood pressure and fasting glucose decreased in both groups while A1C remained at 7.2%. Plasma creatinine increased with fenofibrate while urine creatinine remained comparable between the groups, resulting in significant decreases in both creatinine clearance and estimated glomerular filtration rate (eGFR) by the Modification of Diet in Renal Disease (MDRD)-4 and Cockroft-Gault equations in the fenofibrate group. Cystatin C increased during fenofibrate treatment. Urinary albumin-to-creatinine ratio and diurnal urine protein remained unchanged, whereas overnight urinary albumin excretion rate showed minor decreases in both groups. CONCLUSIONS: We report concomitant decreases in creatinine clearance and eGFR by fenofibrate. These changes complicate the clinical surveillance during fenofibrate treatment. We could not demonstrate the beneficial effects of fenofibrate on albumin excretion. A novel finding is the increase of cystatin C in type 2 diabetic patients during fenofibrate treatment. The clinical relevance of the changes needs to be assessed in a long-term outcome study of renal function.

ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan.

OBJECTIVE: Apolipoprotein CIII (apoCIII) is an independent risk factor for cardiovascular disease, but the molecular mechanisms involved are poorly understood. We investigated potential proatherogenic properties of apoCIII-containing LDL from hypertriglyceridemic patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: LDL was isolated from control subjects, subjects with type 2 diabetes, and apoB transgenic mice. LDL-biglycan binding was analyzed with a solid-phase assay using immunoplates coated with biglycan. Lipid composition was analyzed with mass spectrometry. Hydrolysis of LDL by sphingomyelinase was analyzed after labeling plasma LDL with [(3)H]sphingomyelin. ApoCIII isoforms were quantified after isoelectric focusing. Human aortic endothelial cells were incubated with desialylated apoCIII or with LDL enriched with specific apoCIII isoforms. RESULTS: We showed that enriching LDL with apoCIII only induced a small increase in LDL-proteoglycan binding, and this effect was dependent on a functional site A in apoB100. Our findings indicated that intrinsic characteristics of the diabetic LDL other than apoCIII are responsible for further increased proteoglycan binding of diabetic LDL with high-endogenous apoCIII, and we showed alterations in the lipid composition of diabetic LDL with high apoCIII. We also demonstrated that high apoCIII increased susceptibility of LDL to hydrolysis and aggregation by sphingomyelinases. In addition, we demonstrated that sialylation of apoCIII increased with increasing apoCIII content and that sialylation of apoCIII was essential for its proinflammatory properties. CONCLUSIONS: We have demonstrated a number of features of apoCIII-containing LDL from hypertriglyceridemic patients with type 2 diabetes that could explain the proatherogenic role of apoCIII.

Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity.

The main antiatherogenic function of HDL is to promote the efflux of cholesterol from peripheral cells and transport it to the liver for excretion in a process termed reverse cholesterol transport. The aim of this study was to evaluate the cholesterol efflux capacity in low- and high-HDL subjects by utilizing monocytes and serum from 18 low-HDL and 15 high-HDL subjects. Low and high HDL levels were defined, respectively, as HDL < or =10(th) and HDL > or =90(th) Finnish age/sex-specific percentile. Cholesterol efflux from [(3)H]cholesterol-oleate-acetyl-LDL-loaded monocyte-derived macrophages to standard apolipoprotein A-I (apoA-I), HDL(2), and serum was measured. In addition, cholesterol efflux from acetyl-LDL-loaded human THP-1 macrophages to individual sera (0.5%) derived from the study subjects was evaluated. Cholesterol efflux to apoA-I, HDL(2), and serum from macrophage foam cells derived from low- and high-HDL subjects was similar. The relative ABCA1 and ABCG1 mRNA expression levels in unloaded macrophages, as well as their protein levels in loaded macrophage foam cells, were similar in the two study groups. Cholesterol efflux from THP-1 foam cells to serum recovered from high-HDL subjects was slightly higher than that to serum from low-HDL subjects (P = 0.046). Cholesterol efflux from THP-1 macrophages to serum from study subjects correlated with serum apoB (P = 0.033), apoA-I (P = 0.004), apoA-II (P < 0.0001), and the percentage of apoA-I present in the form of prebeta-HDL (P = 0.0001). Our data reveal that macrophages isolated from either low- or high-HDL subjects display similar cholesterol efflux capacity to exogenous acceptors. However, sera from low-HDL subjects have poorer cholesterol acceptor ability as compared with sera from high-HDL subjects.

Long-term effects of fenofibrate on carotid intima-media thickness and augmentation index in subjects with type 2 diabetes mellitus.

OBJECTIVES: The aim of this substudy was to ascertain whether long-term treatment with fenofibrate reduces surrogate measures of atherosclerosis, biomarkers of inflammation, and endothelial activation in patients with type 2 diabetes. BACKGROUND: Some fibrates may decrease cardiovascular events, improve endothelial function, and reduce levels of acute-phase proteins. In the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study, fenofibrate failed to decrease the primary end point of coronary events in patients with type 2 diabetes. METHODS: A total of 170 patients with type 2 diabetes of the FIELD Helsinki cohort were randomly assigned to micronized fenofibrate 200 mg/day or placebo in a double-blind design. Carotid intima-media thickness (IMT) and the augmentation index (a measure of large artery stiffness) were measured at baseline and at second- and fifth-year visits. Plasma levels of interleukin (IL)-6, C-reactive protein (CRP), serum amyloid A (SAA), secretory phospholipase A2 IIA (SPLA2), E-selectin, vascular cellular adhesion molecule (VCAM)-1, and intercellular adhesion molecule (CAM)-1 were determined by commercial enzyme-linked immunosorbent assay kits at the same visits. RESULTS: IMT and the augmentation index increased similarly in both treatment groups during the study. Plasma levels of CRP, IL-6, SPLA2, SAA, VCAM-1, ICAM-1, and E-selectin remained unchanged in both groups. CONCLUSIONS: Fenofibrate treatment was not associated with beneficial changes in IMT, augmentation index, or biomarkers of inflammation and endothelial function. (Fenofibrate Intervention and Event Lowering in Diabetes; NCT00132886).

Globular adiponectin stimulates glucose transport in type 2 diabetic muscle.

BACKGROUND: Adiponectin acts as an insulin sensitizer in rodent models. The direct effect of adiponectin in intact type 2 diabetic muscle is unknown. We examined whether adiponectin stimulates glucose transport in isolated skeletal muscle strips from type 2 diabetic men. METHODS: We obtained open muscle biopsies from 12 men with type 2 diabetes (56 +/- 1 years, 30.5 +/- 1.1 kg/m(2)), and from 15 non-diabetic men (59 +/- 1 years, 28.0 +/- 1.0 kg/m(2)). Skeletal muscle strips were isolated and exposed to globular adiponectin (2.5 microg/mL), insulin (120 nM) and/or AICAR (1 mM) in vitro for 1 h. Glucose transport was analysed by accumulation of intracellular 3-O-methyl [(3)H] glucose, phosphorylation of Akt-Ser(473) and Akt-Thr(308) was determined using phosphospecific antibodies, and adiponectin receptor 1 and 2 content was measured using specific antibodies. RESULTS: Globular adiponectin increased glucose transport rate by 1.3-fold (P < 0.01) in type 2 diabetic, but not in non-diabetic muscle. Insulin-stimulated glucose transport rate was unaltered by exposure to globular adiponectin in either group. AICAR increased glucose transport and enhanced insulin-stimulated glucose transport in type 2 diabetic and non-diabetic muscles. Insulin-stimulated phosphorylation of Akt-Ser(473) or Akt-Thr(308) was comparable in type 2 diabetic and non-diabetic muscles, and unaltered by the addition of globular adiponectin in either group. Adiponectin receptor expression was similar in skeletal muscle from type 2 diabetic and non-diabetic men. CONCLUSIONS: Globular adiponectin directly increases glucose transport in skeletal muscle from type 2 diabetic patients. This may occur via Akt-independent signalling routes.

Common ABCA1 variants, HDL levels, and cellular cholesterol efflux in subjects with familial low HDL.

HDL promotes cholesterol efflux from peripheral cells via ABCA1 in the first step of reverse cholesterol transport (RCT). We investigated whether the early steps of RCT were disturbed in subjects with familial low HDL and an increased risk for early atherosclerosis. Cholesterol efflux from monocyte-derived macrophages to lipid-free apolipoprotein A-I (apoA-I; %) was measured in 22 patients with familial low HDL without Tangier disease mutations and in 21 healthy controls. In addition, we defined the different alleles of ABCA1 using single-nucleotide polymorphism haplotypes and measured ABCA1 and ABCG1 mRNA transcript levels in cholesterol-loaded macrophages. Similar ABCA1-mediated cholesterol efflux levels were observed for macrophages derived from control subjects and from low-HDL subjects. However, when efflux of cholesterol was estimated as cholesterol efflux to apoA-I (%)/relative ABCA1 mRNA expression level, cholesterol removal was significantly (P = 0.001) lower in the low-HDL group. Cholesterol-loaded macrophages from low-HDL subjects showed significantly increased levels of ABCA1 mRNA but not of ABCG1 mRNA and were more often carriers of the rare ABCA1 alleles L158 and R219K. These results suggest that defective ABCA1 function in cholesterol-loaded macrophages is one potential contributor to the impaired RCT process and the increased coronary heart disease risk in subjects with familial low HDL.

Decreased high-density lipoprotein (HDL) particle size, prebeta-, and large HDL subspecies concentration in Finnish low-HDL families: relationship with intima-media thickness.

OBJECTIVE: High-density lipoprotein (HDL) cholesterol correlates inversely with the risk of coronary heart disease (CHD). The precise antiatherogenic mechanisms of HDL subspecies are not thoroughly elucidated. We studied the relationship between carotid intima-media thickness (IMT) and HDL subspecies distribution in Finnish families with low HDL cholesterol and premature CHD. METHODS AND RESULTS: Altogether, 148 members of Finnish low-HDL families and 133 healthy control subjects participated in our study. HDL particle size was significantly smaller in affected family members (HDL < or =10th Finnish age-sex specific percentile) compared with unaffected family members and control subjects (9.1+/-0.04 nm versus 9.5+/-0.05 nm, P<0.0001, versus 9.8+/-0.03 nm, P<0.0001 [mean+/-SE]). Large HDL2b particles as well as prebeta-HDL concentration were significantly decreased among the affected family members. Mean IMT was significantly higher in the affected family members than in the control subjects (0.85+/-0.01 mm versus 0.79+/-0.01 mm; P<0.0001). Age, HDL2b, systolic blood pressure, and prebeta-HDL were significant independent determinants of mean IMT. CONCLUSIONS: The decreased levels of HDL2b and prebeta-HDL reflect the potentially efflux-deficient HDL subspecies profile in the affected low-HDL family members. Decreased HDL particle size caused by the decrease of plasma concentration of HDL2b and decreased prebeta-HDL levels correlate with increased IMT.

Alterations of lipids and apolipoprotein CIII in very low density lipoprotein subspecies in type 2 diabetes.

AIMS/HYPOTHESIS: Very low density lipoprotein (VLDL) particles are heterogeneous, comprising two main subspecies, VLDL 1 (Sf 60-400) and VLDL 2 (Sf 20-60). The aim of the study was to examine the distribution and composition of VLDL subspecies in type 2 diabetes. SUBJECTS, MATERIALS AND METHODS: We studied the composition and concentration of triglyceride-rich lipoproteins (TRLs) in 217 type 2 diabetic patients and 93 control subjects between 50 and 75 years of age. Lipoprotein subspecies were separated by density-gradient ultracentrifugation. Apolipoprotein (apo) CIII and apo E in plasma and apo CIII in TRL subspecies were measured by nephelometry and apo CII in serum by a commercial kit using a single radial immunodiffusion method. RESULTS: The concentrations of VLDL 1, VLDL 2 and intermediate density lipoprotein were significantly increased in type 2 diabetes subjects, the change being most marked for VLDL 1. There was a strong linear correlation between VLDL 1 triglycerides and plasma triglycerides in both groups (r = 0.879, p < 0.001 and r = 0.899, p < 0.001). Diabetic subjects had markedly higher plasma ratios of apo CII:apo CIII and apo CIII:apo E. Despite elevated plasma apo CIII, type 2 diabetic subjects had a relative deficiency of apo CIII in all TRL subspecies, suggesting profound disturbances of apo CIII metabolism. CONCLUSIONS/INTERPRETATION: The elevation of VLDL 1 triglycerides is the major determinant of plasma triglyceride concentration in normal subjects and in type 2 diabetic individuals. Both apo CIII and apo E metabolism are disturbed in type 2 diabetes.

Insulin resistance and adiposity correlate with acute-phase reaction and soluble cell adhesion molecules in type 2 diabetes.

OBJECTIVE: To investigate the relationship of inflammation and endothelial activation with insulin resistance and adiposity in type 2 diabetes. METHODS AND RESULTS: Hundred and thirty-four (45 female) type 2 diabetic subjects aged 50-75 in the Fenofibrate Intervention and Event Lowering in Diabetes Study in Helsinki were examined before fenofibrate intervention. Fasting levels of circulating intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) (vascular cell adhesion molecule), ultra-sensitive C-reactive protein (CRP), human serum amyloid A (hSAA), interleukin-6 (IL-6), macrophage colony-stimulating factor (M-CSF), secretory phospholipase A(2) IIA (PLA(2)), total, HDL and LDL cholesterol, triglycerides, P-glucose, HbA1c, and serum free insulin were determined. Insulin resistance was assessed by the homeostasis model. HOMA IR correlated significantly with all measures of adiposity and markers of inflammation and endothelial dysfunction. BMI was significantly associated with inflammation and endothelial activation, but with neither lipoproteins nor glycaemic control. After controlling for age, gender and BMI, HbA1c correlated significantly with CRP, hSAA, ICAM-1, E-selectin, and HOMA IR. HDL cholesterol correlated inversely with IL-6, M-CSF, E-selectin, and HOMA IR. HbA1c, phospholipase A(2), VCAM-1, and HDL cholesterol remained independent determinants of HOMA IR in the linear regression analysis controlled for age, gender, and BMI. CONCLUSION: Endothelial activation and acute-phase reaction correlate with insulin resistance and obesity in type 2 diabetic patients.