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.

Association of novel promoter single nucleotide polymorphisms in vasopressin V1a receptor gene with essential hypertension in nonobese Japanese.

We studied the association between four novel single nucleotide polymorphisms (SNPs) in the promoter region of V1aR gene and essential hypertension in 620 Japanese subjects (365 hypertensives and 255 healthy). A significant association was found between one of the genotypes and alleles at SNP -6951 and hypertension in a subsample of nonobese individuals. This association demonstrated an independent risk for nonobese hypertension.

A case of thyrotropin-producing pituitary adenoma, accompanied by an increase in anti-thyrotropin receptor antibody after tumor resection.

We describe a rare, but interesting, case of TSH-producing adenoma (TSHoma), accompanied by increases in both anti-TSH receptor antibody (TRAb) and thyroid-stimulating antibody (TSAb) after tumor resection. A 21-yr-old woman was referred to our department for further evaluation of pituitary tumor. In a nearby hospital, she had been diagnosed as having pituitary tumor. Her serum free T4, free T3, and TSH levels were all elevated concomitantly. On the basis of a diagnosis of pituitary adenoma with TSH production, transsphenoidal resection of the pituitary adenoma was performed. Two weeks after the operation, the blood concentrations of TSH were undetectable, whereas both TRAb and TSAb levels were elevated. TSAb levels gradually increased further from 2 weeks to 3 months after the operation, accompanied by an increase in TSH and free T4 levels. TSH is an important hormone in maintaining physiology and regulating immunomodulators in thyrocytes, as it can influence a variety of immune-regulating cytokine-like activities and inhibit expressions of Fas antigen, intracellular adhesion molecule-1, and class II trans-activator. Changes in TSH would modulate the immune circumstances in the thyroid, and then induce TRAb and TSAb. Autoimmune parameters with thyroid function should be observed carefully when managing patients with TSHoma.

Gene analysis of the calcium channel 1 subunit and clinical studies for two patients with hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (HypoPP) is a skeletal muscle disorder in which episodic attacks of muscle weakness occur; they are associated with decreased serum potassium (K+) levels. Recent molecular approaches have clarified that the condition is caused by mutations in the skeletal muscle voltage-gated calcium channel 1 subunit (CACNA1S). We describe two unrelated patients with HypoPP, followed by their relevant clinical studies and gene analysis. Clinical studies included an oral glucose tolerance test (OGTT), food-loading and insulin tolerance tests (ITT). For Case 1, serum K+ levels were extremely decreased following insulin tolerance testing compared with levels for controls. These results support the hypothesis that no efflux of K+ ion occurs in patients because of low activity of adenosine triphosphate (ATP)-sensitive K+ channel (KATP) channels. Mutational analysis of the CACNA1S gene showed a duplicate insertion of 14 base pairs (bp) from 52 to 65 in intron 26, present in the heterozygous state in both patients. No other mutations were detected in the CACNA1S gene, the muscle sodium channel gene (SCN4A) or the voltage-gated K+ channel gene (KCN3) of either patient. Further analysis showed that this duplicate insertion of 14 bp in intron 26 of the CACNA1S gene was found in 23.7% of healthy subjects. K+ dynamics studies are useful for confirming this syndrome, while further gene analysis for various ion channels using amplification and direct sequencing are required to evaluate the molecular basis of the disorder in the individual patient.

A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome.

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.

Diagnosis of a case of Gitelman's syndrome based on renal clearance studies and gene analysis of a novel mutation of the thiazide-sensitive Na-Cl cotransporter.

Gitelman's syndrome is a recessively inherited renal tubular disorder characterized by low plasma potassium and magnesium levels, reduced calcium excretion, metabolic alkalosis, and increased plasma renin activity and plasma aldosterone concentration with normal blood pressure levels. A 23-yr-old man was referred to our department for further evaluation of hypokalemia. The patient also had hypomagnesemia and markedly reduced urinary calcium excretion. Renal clearance studies and gene analysis of the thiazide-sensitive Na-Cl cotransporter (TSC) were performed in the patient. In response to an iv injection of furosemide, chloride clearance (CCl) increased markedly, while distal fractional chloride reabsorption CH2O/(CH2O+CCl) was considerably reduced. In contrast, thiazide ingestion had no significant effects on these parameters. The patient had compound heterozygous mutations in the alleles encoding the TSC gene, one of which has not been formerly reported. Renal clearance studies and TSC gene analysis by amplification and direct sequencing are useful diagnostic tools for confirming a diagnosis of Gitelman's syndrome.

Hypertension in a patient with Gitelman's syndrome.

Gitelman's syndrome is an autosomal recessive disorder characterized by sodium wasting and hypotension. A middle-aged woman was diagnosed with Gitelman's syndrome because of typical clinical manifestations in the youth and homozygous mutations of 18-base-pair insertion in exon 6 of thiazide-sensitive NaCl-cotransporter gene. It was unusual that she showed hypertension with advancing age. Her serum potassium levels remained low at around 3.5 mEq/l despite potassium supplementation. This case demonstrates that hypertension could result in spite of the extremely decreased sodium reabsorption in Gitelman's syndrome and that essential hypertension is genetically heterogeneous, and abnormality of all genes may not be necessarily required to cause blood pressure rise.

[Hypertension and gene polymorphisms].

For our understanding of the genetic factors of human essential hypertension, gene polymorphisms have played a significant role as DNA markers in association and linkage studies. We found positive linkages between hypertension and 4 gene polymorphisms including angiotensinogen Met235Thr, angiotensin converting enzyme I/D, aldosterone synthase CYP11B2 T-344C, and endothelial nitric oxide synthase Glu298Asp in the Aomori population. These results suggest that the 4 gene polymorphisms might be genetic risk factors for hypertension in this district. However, there has been a frustration with the inconsistencies of accumulated evidence. Because, the genetic associations tend to vary across race, ethnicity, and ecological states. Thus, the rates of racial inter-mixture can explain regional differences in disease susceptibility. We emphasize that human lineage based analysis across populations may lead to the better understanding of the variability.

[New techniques and laboratory examinations in the detection and evaluation of hypertension].

Monitoring of 24-hour ambulatory blood pressure(ABPM), measurements of circulating vasoactive substances and microalbuminuria, and assessment of gene polymorphisms as genetic markers are introduced to detect and evaluate hypertension. Classifications of ABPM based on impact on risks of cardiovascular diseases have been currently available. Plasma level of brain natriuretic peptide(BNP), a cardiac hormone, increases markedly in congestive heart failure, in proportion to its severity, and is evaluated as a potential index of severity of heart failure. In addition, serum level of hepatocyte growth factor(HGF), a member of endothelium specific growth factors, in hypertension might be useful for evaluating the presence of complications and degree of endothelial dysfunction. In diabetes mellitus, onset of microalbuminuria appeared as an important sign of early nephropathy. There is growing evidence that microalbuminuria is an independent predictor of atherosclerosis and premature death in the general population. Current studies have shown that gene polymorphisms including components of the renin-angiotensin-aldosterone system may be possible genetic markers for hypertension and its associated cardiovascular diseases. Our data suggest positive linkages between hypertension and 4 gene polymorphisms including angiotensinogen Met235Thr, angiotensin converting enzyme I/D, aldosterone synthase CYP11B2 T-344C, and endothelial nitric oxide synthase Glu298Asp in the Aomori population.

[Activities of antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. Levofloxacin--Surveillance Group].

The surveillance study was conducted to determine the antimicrobial activity of fluoroquinolones (ofloxacin, levofloxacin, ciprofloxacin, tosufloxacin) and other 20 antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. The resistance to fluoroquinolones was remarkable in Enterococci, methicillin-resistant staphylococci and Pseudomonas aeruginosa from UTI. However, many of the common pathogens such as Streptococcus pneumoniae including penicillin-resistant isolates, methicillin-susceptible Stahylococcus aureus, Moraxella catarrhalis, the family of Enterobacteriaceae, Haemophilus influenzae including ampicillin-resistant isolates have been kept to be susceptible to fluoroquinolones. About 90% of P. aeruginosa isolates from RTI were susceptible to fluoroquinolones. In conclusion, the results from this surveillance study suggest that fluoroquinolones are useful in the treatment of various bacterial infections including respiratory infections.

Reliable differential diagnosis between osteosarcoma and regenerative bone cells in rats through simultaneous analysis of nuclear DNA content and size.

OBJECTIVE: To evaluate the difference between benign proliferative lesions of regenerative bone calluses and malignant tumors in rats by simultaneous analysis of nuclear DNA content and size. STUDY DESIGN: Nuclei were stained with DAPI and measured by cytofluorometry. Five regenerative bones and five osteosarcoma samples were used. Two-dimensional dispersion diagrams between relative nuclear DNA content and nuclear size were drawn up and their regression lines were compared. RESULTS: It was possible to distinguish between osteosarcomas and regenerative bones by simultaneous analysis of nuclear size and DNA content during the cell cycle. The results yielded coefficients of correlation and regression. All five regression lines of osteosarcomas were situated dramatically higher on dispersiongrams than those of regenerative bones. CONCLUSION: Differential diagnosis between regenerative lesions and malignant tumors may now be possible using simultaneous measurement of nuclear DNA content and size.

Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan.

Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Although Endothelial NO synthase (eNOS) gene polymorphisms have been shown to have a positive association with coronary artery disease, the linkage between eNOS gene polymorphisms and hypertension has been controversial. In the present study, therefore, we identified genotypes for Glu298Asp and variable number tandem repeats in intron 4 (4b/a) in 183 hypertensive and 193 normotensive populations. The Glu298Asp variant had a significant association with hypertension (odds ratio, 1.8; 95% confidence interval, 1.1-3.0). The allele frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subjects (0.128 vs 0.080, p<0.05). Diastolic and mean arterial blood pressures were significantly higher in hypertensive subjects with the 298Asp allele than those without the variant allele (p<0.05). However, disequilibrium of 4b/a polymorphism was absent between these two groups. These results suggest that the Glu298Asp variant may be a genetic susceptibility factor for hypertension.

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.

Renal-protective effect of nondepressor dose of cicletanine in diabetic rats with hypertension.

We assessed the renal and cardiac benefits of cicletanine (CIC), a furopyridine derivative drug with diuretic and antihypertensive properties, in diabetic spontaneously hypertensive rats with renal impairment. Uninephrectomized streptozotocin (STZ)-diabetic spontaneously hypertensive Izmo rats (SHRIzm) (10 weeks old) were randomly assigned to receive vehicle or CIC (100 mg/kg/day, orally), and age-matched, uninephrectomized STZ diabetic Wistar-Kyoto Izmo rats (WKYIzm) were assigned to receive vehicle for up to 12 weeks. Blood pressure increased progressively in diabetic SHRIzm but not in diabetic WKYIzm. Urinary albumin excretion increased significantly in both diabetic SHRIzm and diabetic WKYIzm throughout the experiment. The antihypertensive effect of CIC was not significantly observed in diabetic SHRIzm. However, the subdepressor doses of CIC significantly decreased urinary albumin excretion, serum creatinine, and blood urea nitrogen in diabetic SHRIzm. These results were confirmed by morphological analysis of kidneys in each group of rats. The index of focal glomerular sclerosis (FGS) in diabetic SHRIzm was significantly higher than that in diabetic WKYIzm. The CIC treatment significantly and effectively protected against an increase in the index of FGS in diabetic SHRIzm. Moreover, CIC treatment significantly attenuated the increase in the heart weight to body weight ratio in diabetic SHRIzm. Treatment with CIC did not affect urinary and blood glucose concentrations at this dose. These results suggest that CIC has a renal-protective action, which is not related to improvement of diabetes or improvement of high blood pressure in diabetic rats with hypertension. The action might be due to the reduction of intraglomerular capillary pressure or protection of the renal glomerular vascular endothelial cell injury and mesangial cell injury through stimulation of PGI2 generation or elimination of free radicals, although the mechanism remains to be further investigated.

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis.

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.

Analysis of aldehyde dehydrogenase 2 gene polymorphism and ethanol patch test as a screening method for alcohol sensitivity.

To assess clinical availability of the aldehyde dehydrogenase (ALDH) 2 gene polymorphism to detect alcohol sensitivity among a Japanese population, we determined the ALDH 2 genotypes and also compared to an ethanol patch test in 119 young Japanese. Their alcohol sensitivity was evaluated by a questionnaire on the frequency of alcohol-associated symptoms when they drink. Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction (PCR). PCR primers were flanking the polymorphic region in exon 12 of the ALDH 2 gene. The distribution of the typical homozygote, the heterozygote and the atypical homozygote was 63.9, 31.9 and 4.2%, respectively. Gene frequencies of the typical and atypical alleles calculated from the genotype frequencies were 0.80 and 0.20. The atypical genotypic homozygotes were positively associated with facial flushing symptom, but not with positive response for ethanol patch test. These results indicate that ALDH 2 genotypes determination is essential to detect alcohol sensitivity whereas the ethanol patch test has some limitations.

Renal-protective effect of non-depressor dose of cicletanine in streptozotocin diabetic rats.

OBJECTIVE: To assess the renal benefits of cicletanine (CIC) in diabetic rats with renal impairment. METHODS: Hemi-nephrectomized streptozotocin-diabetic Wistar-Kyoto Izmo rats (WKYIzm) (10 weeks old) were randomly assigned to receive vehicle or a low or high dose of CIC (30 or 100 mg/kg per day, orally) for 12 weeks. RESULTS: The blood pressure was raised slightly but not significantly in this model. An anti-hypertensive effect of CIC was not significantly observed. However, the sub-depressor doses of CIC significantly and dose-dependently decreased urinary albumin excretion. These results were confirmed by morphological analysis of kidneys in each group of rats. CIC treatment significantly and effectively protected against an increase in the percentage of focal glomerular sclerosis. CIC did not affect urinary and blood glucose concentrations at either dose. CONCLUSIONS: These results suggest that CIC has a renal-protective action, which is not related to improvement of diabetes or of high blood pressure in this model. The action might be due to the reduction of intraglomerular capillary pressure, although the mechanism remains to be further investigated.

Glucocorticoidal regulation of pituitary vasopressin content in rats.

Although glucocorticoids are known to attenuate vasopressin (AVP) secretion, it is still controversial whether glucocorticoids act on the hypothalamo-neurohypophysial system. We report here glucocorticoidal regulation of pituitary AVP content, which is a specific indicator for the system. The hypothalamic AVP mRNA content and the pituitary AVP content were measured in rats given dexamethasone (2 mg/kg, 2 times over the course of 5 d) or the glucocorticoid receptor antagonist RU-38486 (20 mg/kg, 3 times over the course of 3 d) during euhydration or dehydration. In dexamethasone-treated rats, both the hypothalamic AVP mRNA content and pituitary AVP content decreased after dehydration. In contrast, in the RU-38486 group the hypothalamic AVP mRNA content and pituitary AVP content increased in both euhydrated and dehydrated rats. These results suggest that glucocorticoids may act on hypothalamo-neurohypophysial vasopressinergic system and attenuate its activity under both basal and dehydrated states.