Effects of combined PPARgamma and PPARalpha agonist therapy on reverse cholesterol transport in the Zucker diabetic fatty rat.

AIM: We investigated the effects of the combined therapy of PPARgamma and PPARalpha agonists on HDL metabolism, especially concerning reverse cholesterol transport (RCT), using Zucker diabetic fatty rats (ZDF/Crl-Lepr fa rats) that are the rodent model for type 2 diabetes with obesity, hyperlipidaemia and insulin resistance. METHODS: The ZDF rats were divided into four medicated groups as follows: pioglitazone as a PPARgamma agonist (5 mg/kg/day; P group, n = 6), fenofibrate as a PPARalpha agonist (30 mg/kg/day; F group, n = 6), both these medications (P + F group, n = 6) and no treatment (UNT group, n = 6). Non-diabetic rats (ZDF/GmiCrl-lean, CON group, n = 6) served as controls. We evaluated HDL particle size and messenger RNA (mRNA) levels of the following factors: liver X receptor alpha (L x R alpha), ATP-binding cassette A1 (ABCA1) and ABCG1 which are regulated by PPARs and are related to early stage RCT. RESULTS: The significant increase in HDL particle size was demonstrated in rats of the F and P + F groups, although changes in plasma HDL-cholesterol levels were not significant. In accordance with this finding, mRNA levels of ABCG1 in the liver increased significantly. CONCLUSIONS: These findings suggest the efficacy of combined therapy with PPARgamma and PPARalpha in improving lipid metabolism, partly through the enhanced RCT, and insulin resistance in type 2 diabetes mellitus.

Influence of apolipoprotein E genotype on the response to caloric restriction in type 2 diabetic patients with hyperlipidaemia.

AIMS: Hyperlipidaemia is a major predisposing factor to atherosclerosis in patients with type 2 diabetes. Apolipoprotein (apo) E polymorphism influences lipoprotein metabolism, and the present study was undertaken to explore the relation, in type 2 diabetics, between apo E genotype and the plasma lipid response to dietary therapy. METHODS: The subjects were 104 patients with type 2 diabetes and hyperlipidaemia, and the difference, due to apo E genotype, in dietary response was followed for 4-6 weeks. The caloric intake was maintained in the range 20-25 kcal/kg, and the medications for diabetes were not changed during the follow-up period. RESULTS: Plasma total cholesterol (TC) and triglyceride (TG) levels were significantly lowered by the dietary treatment in patients with apo E genotypes of epsilon3/3, epsilon2/3 and epsilon3/4; however, the lipid levels in patients with epsilon2/4 did not respond to the diet. CONCLUSIONS: apo E genotype should be taken into consideration in the treatment of hyperlipidaemia in diabetic patients.

Plasma levels of soluble vascular adhesion molecule-1 and cholesterol oxidation product in type 2 diabetic patients with nephropathy.

Functional impairment of the vascular endothelium is an early event in the development of atherosclerosis, and soluble adhesion molecules in plasma are regarded as an indicator of the endothelial damage in diabetes mellitus. We compared the soluble vascular adhesion molecule levels in the patients with diabetic nephropathy in concerning with plasma 7-ketocholesterol levels, which is major cholesterol auto-oxidation products. Average value of plasma VCAM-1 in 31 patients with type 2 diabetes mellitus was 297.6+/-10.2 ng/ml (mean+/-SE), and the value was significantly higher than that in 8 age-matched healthy controls (231.9+/-15.0 ng/ml). Among the 31 diabetic patients, the group with macroalbuminuria (n = 8) had the higher levels of plasma VCAM-1 (349.5+/-26.0 ng/ml) than the levels in the group with normoalbuminuria (n=15; 280.6+/-12.3 ng/ml). The levels of plasma 7-ketocholesterol in diabetes (26.9+/-1.5 ng/ml) or the patients with macroalbuminuria (31.4+/-3.3 ng/ml) were significantly higher than the control (22.5+/-1.8 ng/ml). The level of soluble VCAM-1 showed significant correlation between the values of 7-ketocholesterol (r=0.42, p=0.024), TC (r=0.42, p=0.014) and LDL-C (r=0.38, p=0.044). However no correlation was demonstrated with HbA1c nor creatinine level. We conclude that soluble VCAM-1 in plasma may be an indicator of oxidative stress and vascular injury in diabetic nephropathy.

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture.

Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.

Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes.

We and others have recently identified mutations in the ABCA1 gene as the underlying cause of Tangier disease (TD) and of a dominantly inherited form of familial hypoalphalipoproteinemia (FHA) associated with reduced cholesterol efflux. We have now identified 13 ABCA1 mutations in 11 families (five TD, six FHA) and have examined the phenotypes of 77 individuals heterozygous for mutations in the ABCA1 gene. ABCA1 heterozygotes have decreased HDL cholesterol (HDL-C) and increased triglycerides. Age is an important modifier of the phenotype in heterozygotes, with a higher proportion of heterozygotes aged 30-70 years having HDL-C greater than the fifth percentile for age and sex compared with carriers less than 30 years of age. Levels of cholesterol efflux are highly correlated with HDL-C levels, accounting for 82% of its variation. Each 8% change in ABCA1-mediated efflux is predicted to be associated with a 0.1 mmol/l change in HDL-C. ABCA1 heterozygotes display a greater than threefold increase in the frequency of coronary artery disease (CAD), with earlier onset than unaffected family members. CAD is more frequent in those heterozygotes with lower cholesterol efflux values. These data provide direct evidence that impairment of cholesterol efflux and consequently reverse cholesterol transport is associated with reduced plasma HDL-C levels and increased risk of CAD.

Effect of doxazosin on the size of LDL particle in the type 2 diabetic patients with hypertension.

Hypertension is common in patients with type 2 diabetes mellitus (DM), and contributes to the progression of its complications in patients with diabetes. Doxazosin is a selective alpha1-adrenoceptor-blocking anti-hypertensive agent and has a favorable impact upon lipid metabolism. We investigated the effect of doxazosin on the lipid metabolism in hypertensive patients with type 2 diabetes, especially low-density lipoprotein (LDL) particle size that is associated with many elements of the insulin resistance syndrome. Cross-sectional study (n=19) was designed to determine whether doxazosin, administered with an angiotensin II-converting enzyme inhibitor (ACEI) and a Ca antagonist, affects LDL particle size. As a follow-up study (n=6), lipid and glucose metabolism and LDL particle size were followed for 12 weeks before and after the initiation of doxazosin administration (1-4 mg/day). The average size of LDL particle was significantly larger in the patients treated with doxazosin (LDL-migration index (LDL-MI): 0.348+/-0.027) than those in the patient treated without doxazosin (0.378+/-0.035), although LDL cholesterol levels did not differ between the two groups. The plasma glucose and HbA1c levels remained unchanged. Lipid profile showed normolipemia throughout the period of the study. However, LDL particle size was demonstrated to become larger during the following period. Small LDL fraction (LDL3-7) diminished remarkably and large LDL (LDL1-2) increased on the polyacrylamide gel electrophoresis (PAGE) LDL system (LipoPrint). From this pilot study, it was concluded that doxazosin is a useful anti-hypertensive agent for hypertensive type 2 diabetic patients in improving the size of LDL particle.

Plasma oxysterols and tocopherol in patients with diabetes mellitus and hyperlipidemia.

The plasma levels of free oxysterols (7-ketocholesterol; 7alpha-hydroxy-, 7beta-hydroxy-, 25-hydroxy-, and 27-hydroxycholesterol; and 5alpha,6alpha-epoxycholestanol) in patients with diabetes mellitus and hypercholesterolemia were determined using gas chromatography-mass spectrometry with selective ion monitoring. We studied 39 patients with diabetes mellitus, 20 nondiabetic patients with hypercholesterolemia, and 37 normal controls. Plasma cholesterol levels in diabetic and hypercholesterolemic patients showed no statistical difference. Plasma 7-ketocholesterol was significantly higher in patients with diabetes (31.6+/-2.8 ng/mL) or hypercholesterolemia (52.3+/-5.9) than in the control group (22.4+/-1.2). The increased plasma cholesterol can be regarded as an oxidation substrate for the oxidant stress and the higher absolute levels of oxysterols in hypercholesterolemic plasma compared with the control plasma. This difference disappeared when 7-ketocholesterol was expressed in proportion to total cholesterol. The oxidizability of plasma cholesterol was evaluated by comparing the increased ratio of 7-ketocholesterol after CuSO4 oxidation to the ratio before. We demonstrated that the patients with diabetes showed increased oxidizability (77.5%) compared with the control (36.6%) or hyperlipemic group (45.3%), which is likely due to the lower amounts of alpha-tocopherol in the diabetics. Measurement of oxysterols may serve as a marker for in vivo oxidized lipoproteins in diabetes and hyperlipemia.

Identification of homozygous lipoprotein lipase gene mutation in a woman with recurrent aggravation of hypertriglyceridaemia induced by pregnancy.

We herein report a case of a 40-year-old Japanese woman (patient IT) with a history of recurrent aggravation of hypertriglyceridaemia, pancreatitis and miscarriages in three previous pregnancies. However, strict dietary intervention was applied during a fourth pregnancy. As a result, acute pancreatitis was avoided, and the patient gave birth to a healthy infant. In patient IT, the underlying etiology of the recurrent aggravation of hypertriglyceridaemia during pregnancy was a lipoprotein lipase (LPL) gene aberration. She was homozygous for LPL deficiency due to a nonsense mutation (TGG1401 --> TGA/Trp382 --> Stop) in exon 8 of the LPL gene, which resulted in the absence of LPL activity and immunoreactive LPL mass. Our findings indicate that, in LPL deficiency, pregnancy seriously exacerbates hypertriglyceridaemia and increases the risk of acute pancreatitis, which endangers both the mother and fetus. Early diagnosis of LPL deficiency and appropriate management thereof are essential for normal childbirth.

Effects of advanced glycation endproducts on the generation of macrophage-mediated oxidized low-density lipoprotein.

Macrophages have a receptor that recognizes advanced glycation endproducts (AGE). In this study, we evaluated the effect of AGEs on the generation of macrophage-mediated oxidized low-density lipoprotein (LDL) by measuring the electrophoretic mobilities and lipid hydroperoxide (LHPO) levels of LDL. In the absence of the macrophage monolayer, the differences of the electrophoretic mobilities or LHPO levels of native (n) LDL did not differ significantly between control (c) bovine serum albumin (BSA) and those with AGE-BSA. In the presence of the macrophage monolayer, however, the difference was significant with higher levels after the incubation with AGE-BSA than with c-BSA. In the case of cLDL and glycated (g) LDL, the electrophoretic mobilities and LHPO levels of LDL after 20 h incubation with AGE-BSA in the presence of the macrophage monolayer were significantly higher than those with c-BSA. There were no significant differences, however between the electrophoretic mobilities and LHPO levels of cLDL and of gLDL. These results suggest that AGEs stimulate the generation of macrophage-mediated oxidized LDL, but do not directly stimulate the oxidative modification of gLDL.

Reduction of oxysterol levels up-regulates HMG-CoA reductase activity in rat liver.

Cholesterol regulates hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity by feedback inhibition. It has been suggested that oxidized derivatives of cholesterol (oxysterols) play an important role, as an intracellular mediator, in the feedback inhibition of cholesterol biosynthesis. We, therefore, investigated the role of intracellular oxysterols in the regulation of HMG-CoA reductase activity. Rats were fed with food (control), cholesterol, clofibrate as a potentiator of the microsomal monooxygenase cytochrome P-450 enzyme system, ketoconazole as a strong inhibitor of the system, or butylated hydroxytoluene (BHT) as an antioxidant. We analyzed and compared hepatic microsomal oxysterol levels among the groups. The results of this study indicated that the oxysterol level, especially 7beta-hydroxycholesterol and 7-ketocholestrol, in the liver was lowered by the administration of ketoconazole and BHT, and HMG-CoA reductase activity was increased in response to these agents. However, there was no change in the HMG-CoA reductase activity, after the administration of clofibrate. We conclude that reduced levels of oxysterol may release the inhibitory effect on the HMG-CoA reductase enzyme and lead to up-regulation of the enzyme.

Central neuropeptide Y enhances bile secretion through vagal and muscarinic but not nitric oxide pathways in rats.

Neuropeptide Y (NPY) acts in the central nervous system to regulate gastrointestinal functions in rats and dogs. The effects of intracisternal injection of NPY on bile secretion and biliary components were investigated in urethane-anesthetized rats with bile duct cannula. Intracisternal NPY (0.02-0.12 nmol) dose-dependently increased bile secretion by 9.2-19.5%. The secretory response occurred within the first 20-40 min and lasted for the 120-min observation period. Intravenous injection of NPY (0.12 nmol) did not modify bile secretion under identical conditions. Biliary bile acid, phospholipid, and cholesterol secretion were not modified by intracisternal injection of NPY (0.12 nmol), whereas bicarbonate was increased by 19.0 +/- 1.7% from 40 to 120 min after NPY injection. Cervical cord transection at the C6 level, acute bilateral adrenalectomy (-120 min), or injection of NG-nitro-L-arginine methyl ester (10 mg/kg, IV, -15 min), an inhibitor of nitric oxide biosynthesis, did not alter intracisternal NPY (0.12 nmol)-induced stimulation of bile secretion. Atropine (2.0 mg/kg, IP, -30 min) and bilateral cervical vagotomy (-120 min) completely abolished the stimulatory effect of intracisternal NPY (0.12 nmol) on bile secretion. These findings indicate that NPY acts in the brain to stimulate bicarbonate-dependent bile secretion through vagal and muscarinic pathways and suggest that peptides in the central nervous system may be involved in the vagal regulation of bile secretion.

Conditions for solid-phase extraction of urinary bile acids with octadecylsilane-bonded silica.

Fast atom bombardment mass spectrometry was used as a detection method in a study of the extraction of urinary bile acids with octadecylsilane-bonded silica. The procedure commonly used in many laboratories was found to result in significant losses of sulfated taurine-conjugated bile acids. Losses of other double conjugates and of bile acid and bile alcohol glucuronides were also seen. The losses were avoided by addition of an equal volume of 0.5 M triethylamine sulfate to the urine before passing it through the sorbent bed. Quantitative elution was then achieved with methanol. Batch variations were observed with sorbents from two different manufacturers.

The effect of dietary 7-ketocholesterol, inhibitor of sterol synthesis, on hepatic microsomal cholesterol 7 alpha-hydroxylase activity in rat.

A group of oxygenated sterols has been identified as physiological regulators of hepatic cholesterol biosynthesis. However, the regulatory effects of these oxysterols on cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, is not clearly elucidated. We administered 0.1% 7-ketocholesterol (15 mg/day), a strong inhibitor of sterol synthesis, to rats orally for 6 days. Then, the levels of accumulated oxysterols in liver microsomes and microsomal 7 alpha-hydroxylase activity were determined. The results were compared to those in the groups of rats treated with either control diet or diets containing 0.1 or 1% cholesterol, 0.1% butylated hydroxytoluene, 3% cholestyramine or 1% taurocholate. 7-Ketocholesterol feeding resulted in significant increase of both 7-ketocholesterol and 7 beta-hydroxycholesterol in microsomal fraction (449.4 +/- 36.8 and 438.2 +/- 46.8 ng/mg protein, respectively; mean +/- S.E.). Hepatic microsomal 7 alpha-hydroxylase activity in the rats fed 7-ketocholesterol was significantly elevated as compared with those of control rats; 44.70 +/- 5.97 vs. 16.57 +/- 2.46 pmol/min per mg protein. Addition of BHT to 7-ketocholesterol reduced the accumulation of 7 beta-hydroxycholesterol, and the stimulatory effect of 7-ketocholesterol on 7 alpha-hydroxylase activity was suppressed. Our results demonstrate that oxysterols do not inhibit but rather stimulate hepatic microsomal 7 alpha-hydroxylase.

Pheochromocytoma with multiple islet cell carcinoma.

We reported a rare case of a 17-year-old female with pheochromocytoma associated with multiple islet cell carcinoma. Pheochromocytoma was identified in the right adrenal gland. Multiple pancreas tumours were demonstrated unpredictably in the diagnostic imaging of the pheochromocytoma. No other endocrinological neoplasm was observed in the pituitary, thyroid and parathyroid gland. The patient underwent right adrenalectomy and total pancreatectomy. Pheochromocytoma was benign, however, pancreas tumours were non-functioning islet cell tumours and histologically malignant. This combination is assumed to represent a mixed form of multiple endocrine neoplasia (MEN) 1 and MEN 2.

The effect of biliary bile acid concentration and composition on the calcium level in human gallbladder bile.

We analyzed total and ionized calcium concentrations in gallbladder bile of 34 humans in four groups: 8 patients with no gallstone, 11 gallstone patients treated with no gallstone dissolution agents, 8 gallstone patients treated with chenodeoxycholic acid (CDCA) and 7 gallstone patients treated with ursodeoxycholic acid (UDCA). We found that total calcium level ranged from 1.40 to 8.01 mmol/liter, closely related to total bile acid concentration (r = 0.759). However, ionized calcium level was maintained in a narrow range of 0.25 to 1.23 mmol/liter and had no relation to total bile acid concentration. UDCA-rich bile showed relatively high level of ionized calcium. We performed ultrafiltration of bile with cut-off molecular weight 1,000 to investigate the interaction between biliary calcium and bile acid aggregates. The proportion of ultrafiltrated bile acid level to that in original bile in the UDCA group was statistically higher than the other groups. Relatively large percentage of smaller bile acid aggregates in UDCA-rich bile may impair its calcium solubility.

[Phenylketonuria with adult-onset neurological manifestation].

We report a male patient with phenylketonuria (PKU) who developed multisystem neurological manifestation in his fourth decade. He was born in 1957 when a neonatal mass screening had not been available. His neuropsychological development was entirely normal and he was a good athlete during his high school days. He was in good health until the age of 32, when his vision was blurred. In four months his gait progressively deteriorated to bind him to a wheel chair. On physical examination he had red hair and gray eyes. IQ was 68. Visual field showed concentric narrowing and his visual acuity was 0.2/0.3 (2.0/2.0). The limbs were spastic and weakened. He complained of pain in the extremities. He suffered from pollakisuria. Routine blood tests and CSF findings were normal. He was also found to be normal in peripheral nerve conduction studies and central conduction studies of SEP and VEP. EEG showed diffuse slowing in background activities. T2-weighted MRI of the head revealed widespread high-intensity areas in the deep white matter especially in bilateral occipital lobes. Serum aminogram disclosed the remarkably elevated phenylalanine (Phe) level to 1663 nmol/ml (normal range 50-90) and reduced tyrosine. Urinary secretion of endogenous tetrahydroxy-biopterin (BH4; coenzyme of Phe hydroxylase) remained in a normal range, and oral administration of 100 mg/kg of BH4 failed to normalize the serum Phe level. Despite a strict dietary control (oral intake of Phe less than 0.5 g/day), the serum Phe level remained high around 500 nmol/ml and his neurological deficits still deteriorated.(ABSTRACT TRUNCATED AT 250 WORDS)

Higher levels of plasma cholesterol sulfate in patients with liver cirrhosis and hypercholesterolemia.

An analytical method for the determination of cholesterol sulfate (CS) in plasma using gas-liquid chromatography was developed. We measured plasma CS concentrations in patients with liver cirrhosis and hypercholesterolemia as examples of disorders that involve aberrations in cholesterol metabolism. Patients with liver cirrhosis had plasma CS concentrations that were significantly higher than those of control subjects (444.6 +/- 51.7 vs. 253.0 +/- 24.6 micrograms/dL, mean +/- SE). The levels of other lipids were lower in cirrhotics, although the differences were not significant. There was no correlation between the levels of CS and sulfated bile acids in cirrhotic patients. CS levels in plasma were also higher in subjects with hypercholesterolemia (413.7 +/- 44.5 micrograms/dL); however, the ratio of CS to total cholesterol (TC) clearly differed between cirrhotics and hypercholesterolemic subjects (1.44 +/- 0.11 x 10(-3) vs. 3.31 +/- 0.63 x 10(-3); P < 0.05). Both in subjects with hypercholesterolemia and in healthy controls, the CS/TC ratio was similar and CS accounted for roughly 0.14% of the TC concentration.

Analytical examination of oxidized free and esterified 7-ketocholesterol and related oxysterols in human plasma incubated with copper.

Oxygenated derivatives of cholesterol (oxysterols) have been demonstrated to possess a wide variety of biological properties and evaluated for their abilities to inhibit cholesterol biosynthesis. We investigated a method to analyze copper-catalyzed oxidation products of human plasma cholesterol. Free and esterified oxysterols produced were mainly 7-ketocholesterol, and small amounts of 7 beta-hydroxycholesterol and 5, 6 alpha-epoxy-cholesterol were also identified. Quantitatively, the sterol nucleus of ester was less susceptible to oxidation than that of the free form. This finding suggested that the cholesterol nucleus of ester form was more resistant against oxidative stress than free form. Additionally we demonstrated that the addition of probucol, a powerful antioxidant used clinically to lower blood cholesterol, inhibited this copper-catalyzed oxidation of cholesterol.