Tetsu Ebara

Lipoprotein lipase mRNA in white adipose tissue but not in skeletal muscle is increased by pioglitazone through PPAR-γ

Lipoprotein lipase (LPL), a key enzyme for triglyceride hydrolysis, is an insulin-dependent enzyme and mainly synthesized in white adipose tissue (WAT) and skeletal muscles (SM). To explore how pioglitazone, an enhancer of insulin action, affects LPL synthesis, we examined the effect of pioglitazone on LPL mRNA levels in WAT or SM of brown adipose tissue (BAT)-deficient mice, which develop insulin resistance and hypertriglyceridemia. Both LPL mRNA of WAT and SM were halved in BAT-deficient mice. Pioglitazone increased LPL mRNA in WAT by 8-fold, which was substantially associated with a 4-fold increase of peroxisome proliferator activated receptor (PPAR)-gamma mRNA (r=0.97, p<0.0001), whereas pioglitazone did not affect LPL mRNA in SM. These results suggest that pioglitazone exclusively increases LPL production in WAT via stimulation of PPAR-gamma synthesis. Since pioglitazone does not affect LPL production in SM, this would contribute to prevent the development of insulin resistance due to lipotoxicity.

High prevalence of small LDL particles in non-insulin-dependent diabetic patients with nephropathy

To determine whether small-sized low density lipoprotein (LDL) is associated with a high incidence of coronary heart disease in diabetic nephropathy, we measured the LDL particle size in non-insulin-dependent diabetes mellitus (NIDDM) patients with various degrees of albuminuria (n = 95) and age-, weight-matched non-diabetic control subjects (n = 31). The diabetic subjects were divided into three groups, normoalbuminuric, microalbuminuric and macroalbuminuric NIDDM, based on the amount of albuminuria. The average diameter of LDL particles was determined by non-denaturing polyacrylamide gradient (2-16%) gel electrophoresis. The plasma lipid and lipoprotein concentrations were comparable between the non-diabetic controls and normoalbuminuric NIDDM, whereas the plasma triglyceride, very-low-density lipoprotein (VLDL) or LDL concentration was significantly increased in diabetic nephropathy. The mean LDL particle size was significantly smaller in microalbuminuric NIDDM compared with the controls or normoalbuminuric NIDDM, and the LDL size was further decreased in macroalbuminuric NIDDM. The incidence of small LDL (diameter < 255 A) was remarkably increased in microalbuminuric (58%) and macroalbuminuric NIDDM (67%) compared to the control (13%) and normoalbuminuric NIDDM (27%). Corresponding to the decreased LDL size, the cholesterol content of the LDL was significantly depleted in NIDDM with nephropathy. The high prevalence of small LDL in diabetic nephropathy was also observed even when hypertriglyceridemic or hypertensive subjects were excluded from each group. The increment in triglyceride-rich lipoprotein (d < 1.006) after oral fat-loading was increased, and postheparin lipoprotein lipase activity was decreased significantly in diabetic nephropathy. These abnormalities were significantly associated with LDL particle size. Multivariate regression analysis revealed that the amount of albuminuria was closely associated with the average LDL particle size, and this association was independent of the plasma triglyceride level. Neither insulin resistance nor glycemic control was directly associated with LDL particle diameter. The present study indicates that LDL particles become smaller in diabetic nephropathy, and this may be associated primarily with abnormal triglyceride metabolism. However, in addition to hypertriglyceridemia, other metabolic abnormalities caused by diabetic nephropathy may also be involved in the pathogenesis of small LDL particles.

VLDL Triglyceride Kinetics in Wistar Fatty Rats, An Animal Model of NIDDM: Effects of Dietary Fructose Alone or in Combination With Pioglitazone

The effects of dietary fructose alone or in combination with a new oral agent, pioglitazone, on VLDL-triglyceride (TG) turnover were studied in genetically obese Wistar fatty rats characterized by hyperinsulinemia (7,488 ± 954 pmol/l), hyperglycemia (22.5 ± 1.4 mmol/l), and hypertriglyceridemia (4.39 ± 0.54 mmol/l). They had an increased hepatic TG production (16.2 ± 0.1 μmol/min; lean rats, 5.4 ± 0.3 μmol/min) as well as a longer half-life of VLDL-TG from lean donors (8.8 ± 1.4 min, lean recipients; 2.3 ± 0.9 min). In addition, in lean recipients, the half-life of VLDL-TG from fatty donors was longer than that from lean donors (4.80 ± 0.56 vs. 3.14 ± 0.23 min). Although feeding fructose into fatty rats did not change plasma glucose and insulin levels, it produced a twofold increase in TG levels (8.74 ± 1.15 mmol/l). This was associated with a 1.7-fold increase in TG production to 27.5 ± 1.2 μmol/min, while no significant change was found in the half-life of lean VLDL-TG in fructose-fed fatty recipients (10.9 ± 2.4 min) or in that of VLDL-TG from fructose-fed fatty donors in lean recipients (4.46 ± 0.76 min). Daily administration of pioglitazone (3 mg/kg body weight) in fructose-fed fatty rats ameliorated glycemia and triglyceridemia to the level of lean rats (8.1 ± 0.7 and 1.18 ± 0.05 mmol/l, respectively) and insulinemia to a lesser extent (2,712 ± 78 pmol/l). A fall in TG levels was associated with improvement of an impairment in the ability of fructose-fed fatty rats to remove lean VLDL-TG (half-life: 2.6 ± 0.6 min). Pioglitazone, however, produced no change in TG production (25.9 ± 2.7 μmol/min), the half-life of VLDL-TG from fructose-fed fatty donors in lean recipients (4.17 ± 0.38 min), or the activity of lipoprotein lipase and hepatic lipase in postheparin plasma. We conclude that in Wistar fatty rats 1) hypertriglyceridemia is attributed to TG overproduction and impaired TG catabolism, and the latter is due to changes in both VLDL, such that they are less able to be removed, and changes in the nature of Wistar fatty rats, such that they are less able to remove VLDL-TG; 2) fructose further increases hepatic TG production with a resultant deterioration in hypertriglyceridemia; 3) pioglitazone normalizes TG levels by altering the physiology of the Wistar fatty rats in a manner that increases their ability to remove VLDL-TG from the circulation.

No evidence of accelerated atherosclerosis in a 66-yr-old chylomicronemia patient homozygous for the nonsense mutation (Tyr61→Stop) in the lipoprotein lipase gene

Whether chylomicronemia is atherogenic or not has yet to be determined in humans. We investigated a 66-yr-old female with severe chylomicronemia resulting from a lipoprotein lipase (LPL) deficiency. The patients plasma triglyceride level was approximately 2000 mg/dl. Both LPL activity and the mass of postheparin plasma in this patient were virtually absent. A nonsense mutation in exon 3 (Tyr61-->Stop) was identified in the patients LPL gene, and a restriction fragment length polymorphism analysis established that the patient was homozygous for this mutation. The patient was neither a diabetic nor a smoker. Clinically, the patient had never experienced pancreatitis or angia pectoris. An examination of her carotid, femoral and coronary arteries by ultrasonogram and electrocardiogram after exercise-tolerance testing showed no accelerated atherosclerosis. This case suggests that atherosclerosis may not occur despite massive hyperlipidemia, when LPL bridging was not present due to the absence of LPL secretion and circulating mass.

Molecular analysis of the AGL gene: heterogeneity of mutations in patients with glycogen storage disease type III from Germany, Canada, Afghanistan, Iran, and Turkey

AbstractGlycogen storage disease type III (GSD III) is an autosomal recessive disorder characterized by excessive accumulation of abnormal glycogen in the liver and/or muscles and caused by deficiency in the glycogen debranching enzyme (AGL). Previous studies have revealed that the spectrum of AGL mutations in GSD III patients depends on ethnic grouping. We investigated nine GSD III patients from Germany, Canada, Afghanistan, Iran, and Turkey and identified six novel AGL mutations: one nonsense (W255X), three deletions (1019delA, 3202-3203delTA, and 1859-1869del11-bp), and two splicing mutations (IVS7 + 5G > A and IVS21 + 5insA), together with three previously reported ones (R864X, W1327X, and IVS21 + 1G > A). All mutations are predicted to lead to premature termination, which abolishes enzyme activity. Our molecular study on GSD III patients of different ethnic ancestry showed allelic heterogeneity of AGL mutations. This is the first AGL mutation report for German, Canadian, Afghan, Iranian and Turkish populations.

Mechanism of hypertriglyceridemia in dahl salt-sensitive rats, an animal model of spontaneous nephrotic syndrome

It has been reported that focal and segmental glomerulosclerosis (FSGS) with pronounced proteinuria rapidly develop in Dahl salt-sensitive hypertensive (DS) rats fed a high-salt diet. We found that even when they are fed a standard rat chow (0.3% NaCl), DS rats, especially males, exhibit marked proteinuria, hypoalbuminemia, and hypertriglyceridemia without marked hypertension at 32 to 38 weeks of age. The nephrosis was associated with spontaneously developed FSGS. We therefore investigated the mechanism of hypertriglyceridemia in nephrotic animals. Plasma triglyceride (TG) and apoprotein (apo) B levels were markedly increased in DS rats compared with Sprague-Dawley (SD) rats, and this was mainly attributable to an increase in the concentration of very-low-density lipoprotein (VLDL). The TG secretion rate estimated by the Triton WR1339 method was significantly greater in DS rats. VLDL-TGs isolated from both the DS and SD rats were endogenously radiolabeled with different isotopes, and a mixture of these was then injected into DS and SD recipients. The half-life of VLDL-TG was about three times longer in DS recipients, regardless of the source of VLDL. In SD recipients, VLDL from DS rats was cleared at a slower rate than VLDL from SD rats. The activity of lipoprotein lipase in postheparin plasma was substantially decreased in DS rats. Isoelectric focusing gel electrophoresis (IEF) showed that the ratio of apo E/C or apo C-II/C-III in VLDL was markedly decreased and the ratio of apo E or apo C to apo A1 in high-density lipoprotein (HDL) was slightly decreased in DS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of elevated serum lipoprotein (a) concentrations on the risk of coronary heart disease in patients with type 2 diabetes mellitus

Type 2 diabetes mellitus is associated with a marked increase of coronary heart disease (CHD). We aimed to assess the impact of elevated serum lipoprotein (a) (Lp[a]) concentrations on the risk of CHD in patients with type 2 diabetes mellitus. A consecutive series of 352 outpatients was investigated. We determined the serum lipid profile and checked the patients for a history of CHD and of its traditional risk factors. Furthermore, the patients were divided into 3 groups according to the degree of elevation of the serum Lp(a) concentration: serum Lp(a) concentrations greater than 50 mg/dL, between 30 and 50 mg/dL, and less than 30 mg/dL, a presumed high normal value; and the prevalence of CHD was compared among the 3 groups. The serum Lp(a) concentrations in the subjects varied widely from 0.4 to 163.6 mg/dL. Patients with CHD had significantly higher serum Lp(a) concentrations than those without CHD (P = .0045). Logistic regression analysis to identify factors associated with the presence of CHD revealed that elevated serum Lp(a) is a significant risk factor (P = .0246). The prevalence of CHD increased with increasing serum Lp(a) concentrations (P = .048). Patients with serum Lp(a) concentrations greater than 50 mg/dL had a significantly higher prevalence of CHD than those with serum Lp(a) concentrations less than 30 mg/dL: the odds ratio of an elevated serum Lp(a) concentration was 3.346 (P = .039). In conclusion, elevated serum Lp(a) is a significant risk factor; and the risk of CHD appears to increase with increasing serum Lp(a) concentrations. Serum Lp(a) concentration of 50 mg/dL might represent a threshold level in relation to the risk of CHD in patients with type 2 diabetes mellitus.

Molecular features of 23 patients with glycogen storage disease type III in Turkey: a novel mutation p.R1147G associated with isolated glucosidase deficiency, along with 9 AGL mutations

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by deficiency in the glycogen debranching enzyme (gene symbol: AGL) with two enzyme activities: transferase and glucosidase. A missense mutation causing isolated glucosidase deficiency has never been reported. In this study, we examined 23 patients of Turkish ancestry and identified a novel missense mutation p.R1147G with isolated glucosidase deficiency, along with nine AGL mutations: six nonsense mutations (p.W373X, p.R595X, p.Q667X, p.Q1205X, p.W1327X and p.Q1376X), one deletion (c.1019delA) and two splicing mutation (c.293+2T>G and c.958+1G>A). As p.R1147G impaired glucosidase activity, but maintained transferase activity in vitro, a 12-year-old girl homozygous for p.R1147G was diagnosed with having isolated glucosidase deficiency. Of nine other mutations, p.W1327X and c.1019delA were recurrent, whereas seven mutations were novel. Six patients with p.W1327X were all from two nearby cities on the East Black Sea and shared the same AGL haplotype, indicating a founder effect in Turkish patients. Patients with the same mutations had identical haplotypes. Our results provide the first comprehensive overview of clinical and molecular features of Turkish GSD III patients and the first description of the missense mutation associated with isolated glucosidase deficiency.

Molecular characterization of Egyptian patients with glycogen storage disease type IIIa

AbstractGlycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder characterized by excessive accumulation of abnormal glycogen in the liver and muscles and caused by a deficiency in the glycogen debranching enzyme. The spectrum of AGL mutations in GSD IIIa patients depends on ethnic group—prevalent mutations have been reported in the North African Jewish population and in an isolate such as the Faroe islands, because of the founder effect, whereas heterogeneous mutations are responsible for the pathogenesis in Japanese patients. To shed light on molecular characteristics in Egypt, where high rate of consanguinity and large family size increase the frequency of recessive genetic diseases, we have examined three unrelated patients from the same area in Egypt. We identified three different individual AGL mutations; of these, two are novel deletions [4-bp deletion (750-753delAGAC) and 1-bp deletion (2673delT)] and one the nonsense mutation (W1327X) previously reported. All are predicted to lead to premature termination, which completely abolishes enzyme activity. Three consanguineous patients are homozygotes for their individual mutations. Haplotype analysis of mutant AGL alleles showed that each mutation was located on a different haplotype. Our results indicate the allelic heterogeneity of the AGL mutation in Egypt. This is the first report of AGL mutations in the Egyptian population.

Fluvastatin, a New Inhibitor of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, Suppresses very Low-Density Lipoprotein Secretion in Puromycin Aminonucleoside-Nephrotic Rats

The effects of fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the hyperlipidemia associated with nephrosis were studied. Nephrotic rats, induced by a single intraperitoneal injection of puromycin aminonucleoside (100 mg/kg body weight), had significantly higher plasma triglyceride (TG), total cholesterol and apoprotein (apo) B concentrations than controls. Fluvastatin was administrated as a 0.01% solution in drinking water for 14 days to either normal control or nephrotic rats. Concentrations of TG and apo B in plasma, and very low-density lipoprotein (VLDL) in nephrosis were completely normalized by the treatment with fluvastatin, but concentrations of cholesterol in plasma and each lipoprotein fraction were not altered by the treatment. The ratio of apo E to C in VLDL was significantly decreased in nephrotic rats, but the fluvastatin treatment increased this ratio. TG secretion rate estimated by the Triton WR1339 method was significantly increased in nephrotic rats, but was normalized by fluvastatin. Percent composition of TG in newly secreted VLDL particles in post-Triton plasma was not decreased by fluvastatin treatment, suggesting that the number of newly secreted VLDL particles was reduced by the treatment. Postheparin plasma lipolytic activities were not affected by the fluvastatin treatment. These results demonstrate that fluvastatin can effectively ameliorate the high concentration of VLDL by suppressing the hepatic secretion in nephrotic rats, and suggest that an inhibition of cholesterol biosynthesis suppresses VLDL secretion from the liver.