2P-0418 Effects of pioglitazone treatment on postprandial lipid metabolism in type 2 diabetes

Tuesday September 30, 2003: Poster Session Diabetic dyslipidemia

130 2P-0414

Decreased plasma adiponectin concentrations in Japanese young women with normal body mass index but increased body fat, so-called masked obesity

N. Kishimoto 1 , I. Sakuma 1 , H. Chiba 2 , K. Ishii 3 , A. Kitabatake 1 . 1 Hokkaido University Graduate School of Medicine, Sapporo; 2 Hokkaido University Hospital Laboratory Medicine; 3 Hokkaido University Graduate School of Education, Japan Recently, decreased plasma Adiponectin(ADN is now thought to be one of the most important risk factors of atherosclerosis. In Asian countries many young women have a desire to be more slender and they tend to just modify food intake without practicing exercise. The muscle of these women may be exchanged to fat and they may have normal body mass index(BMI) but increased body fat. We investigated whether such women with so-called masked obesity(MO) have altered plasma ADN, lipid and lipoprotein profile. Whole body fat mass was measured in 157 Japanese female University students (21.1±1.9 yrs;mean+/-SD) using whole body dual energy X-ray absorptiometry and their fasting blood samples were taken during menstruation period. There were 82 women with normal BMI(18.5-25 kg/m2 ) and normal body fat mass(BFM)(22-30 percent of body weight): normal group, and 31 women with normal BMI but increased body fat mass (more than 31 percent of body weight): MO group. The MO group had significantly decreased ADN adjusted for BFM (0.44±0.18 vs 0.32±0.14%;p<0.01) compared with normal group. In the lipid profile, although no differences were found between two groups in total cholesterol(C), triglyceride, free fatty acid and C ester transfer protein, the MO group had significantly increased LDL-C (104.4±20.0 vs 91.9±21.2 mg/dl;p<0.01) and decreased HDL-C (73.5±14.5 vs 80.4±15.1mg/dl;p<0.05) levels compared with normal group. Thus, young women with MO already manifest with a decreased ADN production and a worse lipid profile compared with those with normal body architecture and composition. MO among young women may serve as an initiation of metabolic syndrome and underlie the future progression of atherosclerosis. These results indicate the necessity of promoting regular exercise to young women in order to prevent the development of metabolic syndrome leading to cardiovascular disease. 2P-0415

Rapid detection of uncoupling protein 2 -866G/A mutation by mutagenically separated polymerase chain reaction (MS-PCR)

B. Zhang, H. Tanaka, K. Saku. Fukuoka University, Japan Objectives: Uncoupling protein 2 (UCP2), a recently identified member of the mitochondrial transporter superfamily, is a candidate gene for obesity. A common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo. We developed a rapid and simple method, mutagenically separated polymerase chain reaction (MS-PCR), to genotype UCP2 -866G/A polymorphism. Methods and Results: Two reverse mutagenic allele-specific primers of different lengths for the UCP2 -866G/A polymorphic site were paired with the same forward primer in the same PCR reaction. Subsequent 2% agarose gel electrophoresis showed at least one of the two allelic products and provided a within-assay quality control to exclude false-negative results. The 203-bp fragment of the PCR products was A allele-specific and the 183-bp fragment was G allele-specific. The frequencies of UCP2 -866G/A genotypes in 72 Japanese subjects were AA: 21 (29.2%), AG: 32 (44.4%), and GG, 19 (26.4%). The results were confirmed by the PCR-RFLP genotyping method, in which a 360-bp fragment of PCR products was cut into 290-bp and 70-bp fragments by the restriction enzyme Mlu I when the G allele was present. This Japanese group showed higher frequencies of the AA genotype, which is associated with a low prevalence of obesity, than an Austria population (AA/AG/GG: 13.9%/48.6%/37.5%). Conclusions: The MS-PCR technique is a simple, rapid, and reliable method for UCP2 -866G/A genotyping. 2P-0416

A model for modulation of leptin activity by association with clusterin

T. Bajari, V. Strasser, J. Nimpf, W. Schneider. Institute of Medical Biochemistry, Department of Molecular Genetics, University & Biocenter of Vienna, Vienna, Austria Transport, biological action, and clearance of leptin are subject to modulation by plasma components responsible for the formation of the so-called “bound” fraction of serum leptin. Candidates for modulators have been identified previ-

ously, but mechanisms for their action, and thus their physiological roles, have remained unclear. Here we have obtained evidence for a role of serum-borne clusterin in leptin biology, and have delineated a possible mechanism for its action. We demonstrate complex formation between clusterin and leptin by several approaches, and show that the binary complex retains the ability to transduce the leptin signal via binding to the leptin receptor and activation of the JAK/STAT pathway. The interaction of leptin with clusterin does not require additional serum components. Furthermore, and importantly for modulation of leptin’s bioactivity, uptake of leptin present in the complex can be mediated by members of the LDL receptor family, i.e., apolipoprotein receptor type-2 and the VLDL receptor, which here are shown to efficiently endocytose both free and leptin-associated clusterin. Thus, bioavailability of leptin at a given tissue site may be determined by the levels of clusterin and/or by the relative distribution of certain relatives of the LDL receptor vis-à-vis active leptin receptors.

DIABETIC DYSLIPIDEMIA 2P-0417

Statins: A double weapon in treating dyslipidaemic osteoporotic menopausal type 2 diabetics

F. Amara 1 , N. Lachine 1 , A. Hassab 2 , M. Melees 3 , T. Abdel-Aaty 1 , E. Youssef 1 , S. El-Shiekh 1 , M.A.-R. Korani 4 . 1 Department of Internal Medicine, Alexandria University; 2 Department of Clinical Pathology, Alexandria University; 3 Department of Gynaecology, Alexandria University; 4 Department of Internal Medicine, Menoufia University, Egypt Objective: To study the effect of two brands of 3-hydroxy-3 methyl gluterile coenzyme A reductase inhibitors (Statins) Simvastatin and Pravastatin on bone mineral density (BMD) of dyslipidaemic postmenopausal females with type 2 diabetes mellitus and suffering from osteoporosis. Methods: Thirty patients aged above 50 years with no history of diseases of drugs that affect bone metabolism were enrolled in two groups: I) 15 patients received 40 mg simvastatin daily and II) 15 patients 40 mg pravastatin daily, both for three months. Laboratory investigations were done before and after the trial and included: fasting and post-prandial plasma glucose, glycosylated haemoglobin, alanine aminotransferase (ALT), serum cholesterol and triglycerides, serum calcium (total and ionized), and phosphorus, serum osteocalcin (as a marker of bone formation) and urinary deoxypyridinoline (DPD; as a marker of bone resorption). Dual energy X-ray absorptiometry (DEXA) was used to assess BMD of forearm and L2-L3 lumbar vertebrae. Results: Serum osteocalcin and BMD revealed significant increase and urinary DPD significant decrease after simvastatin in group I. A mild change in osteocalcin, DPD and BMD were noticed after pravastatin in group II, yet it did not reach significance. There was significant reduction of serum cholesterol and triglycerides after both statins as expected. None showed any abnormal change in ALT levels. Conclusion: Our results prove the beneficial role of the lipophyllic statin, simvastatin, in prevention and treatment of osteoporosis. Further studies are needed to reach the best effective dose and mode of administration of statins. Also, the possibility of using statins in combination with other currently used drugs in this domain. 2P-0418

Effects of pioglitazone treatment on postprandial lipid metabolism in type 2 diabetes

K. Al Majali 1 , M. Cooper 1 , M. Adiseshiah 1 , S. Hurel 1 , P. Harry 2 , R. Urquhart 2 , D.J. Betteridge 1 . 1 Royal Free and University College Medical School, Middlesex Hospital, London; 2 Takeda UK Ltd., United Kingdom Type 2 diabetes is associated with abnormal postprandial lipid metabolism. Pioglitazone (P) is an insulin sensitiser used in this study to investigate effects of enhanced sensitivity to insulin on postprandial lipid metabolism compared with treatment with Glibenclamide (G). In a double-blind trial, patients were allocated to regimes incorporating P (45 mg/day) or G designed to achieve the same glycaemic control over 20 weeks. Patients received a high-fat meal, containing retinyl palmitate (RP) as a marker of intestinal lipoproteins, after 4 weeks treatment with diet alone and again after 20 weeks treatment. A group of controls also received the test meal. Blood was sampled over 8 hours following the meal and plasma triglyceride (TG) together with chylomicon (Chy) and chylomicron remnant (CR) RP were measured. There were no differences in glycaemic control between G or P groups (p=NS). In the baseline test the area-under-the-curve (AUC) for postprandial plasma TG was the same in both treated groups and were significantly greater than the control

XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan

Tuesday September 30, 2003: Poster Session Diabetic dyslipidemia subjects (p<0.05). After treatment, the P group showed a significant fall in the magnitude and duration of postprandial lipaemia (p<0.05) and no longer differed from the controls but G treated showed no significant improvement. In the baseline test, the RP-AUC of Chy and CR were higher in the diabetic subjects than control (p<0.05). After treatment, there was a significant reduction to control level in the Chy RP-AUC in the P group (p<0.01) that did not occur in the G group. CR-RP AUC showed a significant reduction in the P group (p<0.05); G group remained significantly higher than the controls (p<0.05). In summary, important effects on postprandial TG metabolism are observed in type 2 diabetic patients treated with P, compared to patients treated with G at the same level of glycaemic control. 2P-0419

The effect of green tea on glucose, cholesterol and triglyceride level of plasma in diabetic rats

M. Eskandari, A. Shirpoor. Department of Physiology, Faculty of Medicine, University of Medical Sciences, Urmia, Iran

2P-0420

Influence of fluvastatin administration on serum Lp(a) levels in NIDDM patients

M. Djeric 1 , M. Ubavic 2 , S. Kojic-Damjanov 1 , E. Stokic 1 , P. Pantelinac 1 . 1 Clinical Center Novi Sad, Novi Sad; 2 Medlab Novi Sad, Serbia Objective: We studied the effect of the cholesterol synthesis inhibitor fluvastatin on serum levels of lipoprotein (a). So far no efficient drug is available for treatment of elevated Lp(a) levels, a significant and independent risk factor for premature atherosclerosis. Methods: The serum Lp(a) levels were measured by imunoturbidimetric method (test kit Menarini) on automatic random access analyzer Chiron model “EXPRESS 550”. Research was performed in 14 patients with NIDDM (8 women and 6 men age 59 7 years) and hyperlipoproteinemia (serum levels of LDL cholesterol above 4.1 mmol/l and triglyceride levels below 4.5 mmol/l) who were on diet alone or concomitantly on derivates of sulfanylurea. The effect was evaluated after 4 and 8 weeks with 20 mg/day and after 12 weeks on the same doses in 5 patients and with additionally 20 mg/day (total 40 mg/day) of fluvastatin in 9 patients. Results: Baseline values of Lp(a) showed a wide range (42.9 49.1 mg/dl; 4.5-162.8 mg/dl). Fluvastatin significantly reduced serum Lp(a) levels even after 4 weeks (p<0.05) and was more effective after 12 weeks (p<0.005). Conclusion: Although the decrease in Lp(a) during fluvastatin therapy has been reported earlier, there is still a great need to confirm such data. 2P-0421

Novel ELISA for Apo B-48 revealed chylomicron remnants (CR) are accumulated in the serum of diabetic patients despite normolipidemia

N. Sakai 1 , K. Ohashi 1 , T. Hibuse 1 , K. Kishida 1 , H. Hiraoka 1 , T. Nakamura 1 , T. Funahashi 1 , S. Yamashita 1 , Y. Matsuzawa 1 , Y. Uchida 2 . 1 Dept. of Internal Medicine and Molecular Science, Osaka University Graduate School of Medicine, Osaka; 2 Fujirebio Inc., Japan Objectives and Subjects: Recent studies have demonstrated that impaired clearance of chylomicron remnants (CR) causes postprandial hyperlipidemia, one of risk factors for coronary heart disease (CHD). The present study was designed to evaluate the metabolism of chylomicron and CR in diabetic

patients, by measuring fasting serum apo B-48 levels in 588 healthy volunteers (335 normolipidemic [NL] and 253 hyperlipidemic [HL] subjects), using a novel ELISA system. We measured and correlated fasting serum apo B-48 to fasting plasma glucose (FPG), hemoglobin A1c (HbA1c), body mass index (BMI) and lipids concentrations. Results: The distribution of fasting serum apo B-48 levels in NL subjects varied widely, ranging from less than 1 to over 24 µg/ml (mean, 5.2 ± 3.8, median, 3.9, and 95 percentile was less than 13). In total 588 subjects, FPG (102.5±31.9 mg/dl vs 95.5±17.6, p<0.05), HbA1c (5.4±1.0% vs 5.0±0.7, p<0.01), BMI (24.4±2.8 kg/m2 vs 23.4±3.0, p<0.01), total cholesterol, triglycerides, and HDL-C levels were significantly higher in apo B-48>13 µg/ml group than those in apo B-48 ≤13 µg/ml group. Serum apo B-48 levels were significantly higher in FPG>110 mg/dl and BMI≥25 kg/m2 groups than those in FPG≤110 mg/dl and BMI<25 kg/m2 groups, respectively, and tended to be higher (p=0.08) in subjects with HbA1c>5.8% than subjects with HbA1c≤5.8%. In 335 NL subjects, HbA1c level was significantly higher in subjects with apo B-48>8.5 µg/ml (85 percentile) than those with apo B-48≤8.5 µg/ml. Serum apo B-48 levels were significantly higher in those with FPG>110 mg/dl than those with FPG≤110 mg/dl. Conclusions: In conclusion, the levels of CR were increased in diabetic patients even if they were normolipidemic, predisposing the patients to CHD. 2P-0422 ∗

Acyl-coenzyme A:cholesterol acyltransferase-2 (ACAT-2) is responsible for elevated intestinal ACAT activity in streptozotocin (STZ)-induced diabetic rats

M. Hori 1 , M. Satoh 1 , Y. Sakamoto 1 , M. Sakai 1 , A. Miyazaki 2 , S. Horiuchi 1 . 1 Kumamoto University, Kumamoto; 2 Showa University, Japan Objective: STZ-induced diabetic rats are known to exhibit dyslipidemia due largely to an elevated intestinal ACAT activity. Since two ACAT isozymes (ACAT-1 and ACAT-2) were identified, this study was designed to determine which isozyme is involved in the elevated intestinal ACAT activity in diabetic rats. Methods: We previously cloned rat ACAT1 cDNA and confirmed that antihuman ACAT-1 antibody (DM10) cross-reacted with rat ACAT-1. Rat ACAT-2 cDNA was cloned by RACE-PCR. ACAT-deficient CHO (AC29) cells were transfected with pcDNA3.1/rACAT-2. Tissue distribution of rat ACAT-2 at mRNA was determined by RT-PCR. DM10 and anti-human ACAT-2 antibody (DM54) were used for analysis of intestinal ACAT in STZ-induced diabetic rats. Results: Rat ACAT-2 cDNA possesses a cording region of 1572 bp which shows 84% and 91% identity with human and mouse ACAT-2, respectively. Stable expression of rat ACAT-2 cDNA in AC29 cells resulted in a 5-25-fold increase in the ACAT activity. Immunoblotting revealed a DM54-reactive 45 kDa band in these cells, indicating a cross-reactivity of DM54 to rat ACAT-2. Detection of ACAT-2 mRNA by RT-PCR was limited to intestine and liver. The intestinal ACAT activity of diabetic rats was 6-times higher than that of control. Immunoblotting demonstrated 2-fold increase in ACAT-2 over control, whereas ACAT-1 was detected in neither intestine. Moreover, upon immunodepletion assay, DM54 immunodepleted >80% of the intestinal ACAT activity in both rats, while DM10 had no effect, indicating that the major ACAT isozyme in the rat intestine is ACAT-2. Conclusion: The elevated intestinal ACAT activity in diabetic rats is explained by an increase ACAT-2 at mRNA and protein level, suggesting a major role of ACAT-2 in diabetes-associated dyslipidemia. 2P-0423

LDL subfraction profile at baseline in a double-blind clinical trial with atorvastatin in patients with type 2 diabetes on hemodialysis (Die Deutsche Diabetes Dialysis Study)

K. Winkler 1 , G. Ruf 2 , V. Krane 3 , H. Wieland 1 , E. Ritz 4 , C. Wanner 3 , W. März 5 . 1 University of Freiburg, Freiburg; 2 Pfizer Ltd., Karlsruhe; 3 University of Würzburg, Würzburg; 4 University of Heidelberg, Heidelberg, Germany; 5 University of Graz, Graz, Austria Objective: Patients with type 2 diabetes mellitus (T2DM) on long-term hemodialysis (HD) suffer from cerebro-/cardiovascular (CV) morbidity and mortality. In general, lipid metabolism in T2DM is characterized by the atherogenic lipoprotein phenotype: elevated triglycerides, low HDL cholesterol (CH) and, on the basis of moderately elevated LDL-CH, a preponderance of atherogenic dense LDL (dLDL) in about 70%. Methods: The 4D-Study is a prospective, randomized, double-blind study investigating the potential of atorvastatin to reduce cerebro-/CV events in patients with T2DM on HD. In 690 screened patients the LDL subfraction

XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan

TUESDAY

The purpose of this study was to investigate the effect of green tea on plasma glucose(GLU), cholesterol(Chol) and triglycerid(TG) in the rats which diabetized by streptozocin(STZ). Twenty wistar male rats with an average age of 8 month, were chosen. The STZ was injected with on dose of 60 mg/kg intraperitoneally. Blood sampling was done from the tail of the rats before injecting STZ, plasma level of GLU, Chol and TG were measured. After STZ injection, another blood sampling was done after 72 hours to measure the GLU level of plasma. In 16 rats the level of GLU was higher than 300 mg/dl. The diabetic rats were divided into two groups, experimental group(n=8) and control group(n=8). The rats of experimental group were eaten boiled green tea with the normal diet. The amount of the green tea was about 6.25 percent of the daily diet. The control group was eaten only the normal daily diet. Blood sampling was done again after 4 weeks from both of the control and experimental groups. The plasma level of the GLU, Chol and TG was measured. The difference of GLU in the two groups was not significant, but Chol and TG level of plasma showed a significant decrease in experimental group comparing control group. Our result sugest that threatment with green tea may reduce the risk of cardiovascular complication in diabetes.

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