AST-120 (Kremezin) initiated in early stage chronic kidney disease stunts the progression of renal dysfunction in type 2 diabetic subjects.

There is little clinical evidence when AST-120 should be prescribed for subjects with early stage overt diabetic nephropathy. We therefore designed a prospective, randomized, controlled study for subjects with type 2 diabetes (serum creatinine <1.5mg/dl and urinary protein >0.5g/day) in November, 2001. The primary end point was defined as exceeding 2mg/dl of serum creatinine, and the secondary end point was defined as introducing a hemodialysis. Twenty-two subjects were selected, and after excluding 6 drop-out subjects, 16 subjects (10 in the control group; 6 in the KRM group) finally entered the study. Mean follow-up periods were 37 and 34 months in the control and KRM groups, respectively. There was no difference in clinical characteristics including renal dysfunction at baseline between the two groups. There was a significant reduction in urinary indoxyl sulfate at month 12 in the KRM group than in the control group. A significant difference was observed in changes in mean levels of serum creatinine versus time between the two groups. The primary end points were counted in 7 (70%) of the control subjects, while only 1 (17%) of the KRM group, and the Kaplan-Meier analysis was statistically significant. Although 4 (40%) of the control group and 1 (17%) of the KRM group were initiated hemodialysis as the secondary end point, the difference did not reach a statistical significance. Thus, we concluded that administration of AST-120 initiated in early stage overt diabetic nephropathy stunts the progression of renal dysfunction.

Insulin responses to selective arterial calcium infusion under hyperinsulinemic euglycemic glucose clamps: case studies in adult nesidioblastosis and childhood insulinoma.

Selective arterial calcium stimulation and hepatic venous sampling (ASVS) for insulin secretion is used as a diagnostic procedure in patients with insulinomas or adult nesidioblastosis. In some of those patients, severe hypoglycemia requiring urgent glucose administration occurs during the procedure. Such glucose administration, however, may affect the results and damage the validity of the test. We report two cases of hyperinsulinemic hypoglycemia, in which ASVS tests were successfully performed under hyperinsulinemic euglycemic glucose clamps. A 40-year-old male with nesidioblastosis developed continual severe hypoglycemia several years after a Billroth II-Braun gastrectomy, and continuous glucose infusion could not be stopped even during ASVS tests. A 9-year-old girl with an insulinoma that showed atypical hypovascularity on imaging examinations had ASVS tests under a glucose clamp for safety. Hyperinsulinemic (approximately 100 microU/ml) euglycemic (approximately 90 mg/dl) clamps were achieved by an artificial endocrine pancreas. The insulin analogue lispro was utilized for clamps and endogenous insulin was measured with an assay that does not cross-react with the analogue. Diagnostically significant responses (more than twofold) of insulin secretion were observed under hyperinsulinemic clamps in both cases. The use of the hyperinsulinemic glucose clamp technique during the ASVS test should be considered for maintaining the safety of some hypoglycemic patients.

Relationship between impaired aldosterone response to adrenocorticotropic hormone and prevalence of hemodialysis in type 2 diabetic patients without azotemia.

The present prospective observational study was designed to assess the prevalence of hemodialysis in type 2 diabetic patients with an impairment of plasma aldosterone responsiveness to adrenocorticotropic hormone (ACTH). Sixty seven patients (43 men and 24 women) were selected. The inclusion criteria were age < 65 years; presence of normoalbuminemia (serum albumin > 3.6 g/dl); and absence of azotemia (serum creatinine < or = 1.2 mg/dl in males, and < or = 1.0 mg/dl in females). Soluble alpha(1-24)-ACTH was injected intramuscularly in a single dose of 0.25 mg after overnight recumbency. The area under the aldosterone curve (aldosterone AUC) was calculated. The diabetic patients were divided into two groups according to the levels of aldosterone AUC. Patients with an aldosterone AUC in the range of 0-381 were considered poor responders (n = 31) and those with an AUC of 397-1,007 were considered good responders (n = 36). The follow-up was performed during a 144-month period. The end point of the study was the introduction of hemodialysis. A total of 14 patients (12 poor responders and 2 good responders; p < 0.001) were introduced to hemodialysis. The prevalence of hemodialysis in the poor responders (5.74 per 100 patient-years) was significantly higher (p < 0.001, log-rank test) than that in the good responders (0.68 per 100 patient-years). One possible explanation is that an inappropriate level of salt intake relative to the impaired plasma aldosterone control may have contributed to the high prevalence of risks and hemodialysis in the poor responders.

A prognostic role of mean 24-h pulse pressure level for cardiovascular events in type 2 diabetic subjects under 60 years of age.

OBJECTIVE: To assess the prognostic role of ambulatory 24-h pulse pressure (PP) on various vascular events in relatively young type 2 diabetic subjects under 60 years of age. RESEARCH DESIGN AND METHODS: In this prospective study, 237 type 2 diabetic subjects without any history of vascular complications were analyzed. After excluding 9 dropout subjects, 228 subjects (mean age, 46 years; 69% men; mean follow-up period, 100 months) entered the study. RESULTS: Distribution of 24-h PP for all subjects showed left skewed data, indicating that there may be a diabetic subgroup that had a wide PP. Therefore, further analysis was performed by stratifying the diabetic subjects by quartile of 24-h PP. Outcomes for the widest quartile (n = 58; cut point = 53.3 mmHg) was then compared with those from the other narrower quartiles (n = 170). In the diabetic subjects with a wide PP, cardiovascular events occurred more frequently than those in the diabetic subjects with a narrow one (20.7 vs. 4.1%; P < 0.001), resulting in the significant difference in the cumulative incidence of cardiovascular events (P < 0.001, log-rank test), but not cerebrovascular events, between the two subgroups. The Cox model revealed that a wide 24-h PP at baseline independently predicted subsequent cardiovascular events but not cerebrovascular events. By contrast, only duration of diabetes was the risk factor for cerebrovascular events. CONCLUSIONS: This study showed that a wide 24-h PP is predictive for cardiovascular events in relatively young diabetic subjects.

Implication of steady state concentrations of nitrite and nitrate metabolites of nitric oxide in plasma and whole blood in healthy human subjects.

1. The aim of the present study was to determine whether the steady state NOx concentration reflects NOx formation in vivo. 2. A NO3- load study was performed after achieving NOx steady state. Chronological changes in NOx concentrations in plasma and whole blood samples from nine healthy subjects were determined by the HPLC-Griess system and NOx concentrations in erythrocytes were estimated as a possible NOx compartment influential in regulating plasma NOx concentrations. 3. Analysis was performed using the first-order one-compartment open model and the NOx formation rate was subsequently calculated. 4. The mean (+/-SEM) steady state NOx concentration of plasma (15.5 +/- 1.6 micromol/L), whole blood (12.8 +/- 1.2 micromol/L) and erythrocytes (11.9 +/- 0.7 micromol/L) did not correlate with the NOx formation rate in the compartments (0.50 +/- 0.05, 0.61 +/- 0.04 and 0.91 +/- 0.17 micromol/kg per h, respectively), whereas a significant correlation was found between the steady state NOx concentration and NOx elimination rate (Kel) in plasma (r=-0.69; P=0.04) and whole blood (r=-0.79; P=0.01). 5. Although there was no direct correlation between steady state NOx concentrations and serum creatinine levels, the correlation between half-life and serum creatinine levels was significant (plasma: r=0.60, P=0.02; whole blood: r=0.49, P=0.04). 6. Plasma NOx concentrations correlated significantly with erythrocyte NOx concentrations (r=0.92, P <0.01; erythrocyte NOx=0.66 x plasma NOx). 7. The results of the present study indicate that NOx does not accumulate excessively into erythrocytes at steady state and during a NO3- load and that the steady state NOx concentration in whole blood and plasma preferentially implies NOx elimination (mainly depending on renal function) rather than NOx formation.

Receptor gene expression of glucagon-like peptide-1, but not glucose-dependent insulinotropic polypeptide, in rat nodose ganglion cells.

We previously reported that afferent signals of the rat hepatic vagus increased upon intraportal appearance of insulinotropic hormone glucagon-like peptide-1(7-36) amide (GLP-1), but not glucose-dependent insulinotropic polypeptide (GIP). To obtain molecular evidence for the vagal chemoreception of GLP-1, the concept derived from those electrophysiological observations, receptor gene expressions of GLP-1 and GIP in the rat nodose ganglion were examined by means of reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and Northern blot analysis. Gene expression of the GLP-1 receptor was clearly detected by both RT-PCR and Northern blot analysis. In situ hybridization study confirmed that the expression occurs in neuronal cells of the ganglion. As to the GIP receptor, RT-PCR amplified the gene transcript faintly though Northern blot analysis failed to detect any messages. However, semi-quantitative RT-PCR revealed that the ratio of the gene expression level of the GIP receptor to that of the GLP-1 receptor was less than 1:250, indicating that receptor gene expression of GIP is practically negligible in the ganglion. Additionally, an equal level of GLP-1 receptor gene expressions between left- and right-side ganglia was evidenced by semi-quantitative RT-PCR, implying possible extrahepatic occurrence of vagal GLP-1 reception in addition to the reception through the hepatic vagus (originating from the left-side ganglion). The present results offer, for the first time, the molecular basis for the vagal chemoreception of GLP-1 via its specific receptor.

Ambulatory blood pressure level rather than dipper/nondipper status predicts vascular events in type 2 diabetic subjects.

To clarify which parameter, diurnal pattern of blood pressure (BP) or level of BP variability, has the stronger predictive value for fatal and nonfatal vascular events, vital status after a mean (+/-SD) follow-up period of 86+/-46 months was determined in 392 type 2 diabetic subjects without any history of vascular disease, in whom the 24-h BP profile had been monitored between 1988 and 1998. After the exclusion of 28 subjects who died during the follow-up period of causes unrelated to diabetes, 364 subjects were recruited for further analysis. A total of 147 first events, including 50 fatal vascular events and 97 nonfatal vascular events, were recorded during the follow-up period. The rates of various vascular events increased with both reduced nocturnal falls in systolic BP (SBP) and levels of all ambulatory BP parameters. The ambulatory BP parameter showing the largest area under the receiver operating characteristic curve (ROCAUC) for fatal events was the mean 24-h pulse pressure (PP), and that for nonfatal events was the mean nighttime SBP; both exceeded the respective values of nocturnal fall in SBP. Furthermore, when dipper and nondipper diabetic subjects were divided into subgroups based on the 24-h PP (54.3 mmHg) and the nighttime SBP (116.5 mmHg) cut-off points derived from the ROC analyses, Kaplan-Meier plots showed that the diabetic subgroups with high ambulatory BP levels had worse outcomes, independent of dipper/nondipper status. Finally, these parameters were applied to the Cox model with the values of nocturnal fall in SBP and other confounding factors, and results showed that mean 24-h PP and mean nighttime SBP predicted fatal and nonfatal vascular events, respectively, more strongly than nocturnal fall in SBP in type 2 diabetic subjects. These findings therefore suggest that ambulatory BP levels in type 2 diabetic subjects have a higher predictive value for organ damage and death compared with diurnal BP patterns or dipper/nondipper status.

Insulin resistant state in type 2 diabetes is related to advanced autonomic neuropathy.

To elucidate the relationships between obesity, glycemic control, dyslipidemia, hypertension, microvascular complications, and insulin resistance assessed using an euglycemic hyperinsulinemic clamp technique, we studied 54 hospitalized type 2 diabetic subjects (DM) and 10 age- and sex-matched normotensive, nonobese control subjects (C). Glucose infusion rate (GIR) derived from the clamp study was used as an index of insulin resistance. Body mass index (BMI), the prevalence of hypertension, HbA1c and serum nonesterified fatty acids (NEFA) were significantly higher, and serum high-density-lipoprotein (HDL)-cholesterol was significantly lower in DM than in C (p < 0.05 or less). The median GIR level was significantly lower inDM than in C (p = 0.038). The difference in GIR between the two groups wasstill statistically significant even after adjustment for BMI, mean BP, HbA1c, NEFA, and HDL-cholesterol. However, after simultaneous adjustment for these factors, there was no difference in GIR between the two groups. Body mass index, mean BP, HbA1c, and NEFA showed negative correlations, and serum HDL-cholesterol showed a positive correlation with GIR, but neither age nor duration of diabetes correlated with GIR. When GIR values in DM were divided according to the degree of neuropathy, retinopathy, and nephropathy, and compared to those in C, GIR levels tended to be decreased with increasing severity of each microvascular complication, but there was no difference in median GIR levels among the diabetic subgroups. Relationships between the GIR levels and confounding factors such as age, sex, BMI, mean BP, HbA1c, serum NEFA, and serum HDL-cholesterol, were examined simultaneously with a multiple regression analysis. This analysis revealed that HbA1c and serum NEFA may affect the GIR level. Furthermore, together with these two factors, the relationships between the GIR levels and the severity of each microvascular complication were explored with the same analysis. This model clearly demonstrated that both the decreased CVR-R and pronounced orthostatic fall in systolic BP were independent factors for the decreased GIR. These findings suggest that marked autonomic dysfunction, rather than other confounding factors, is related to increased insulin resistance in DM.

A practical procedure for achieving a steady state of NOx concentration in plasma: with special reference to the NOx content of Japanese daily food.

To establish a concrete procedure to achieve a steady state plasma NOx concentration with Japanese daily food, NOx contents of about 200 types of food and beverages consumed daily were measured and NOx concentration in plasma was monitored till steady state after various degree of intake of NOx restricted food. The NOx content was found to be high in dark green leaved vegetables and low in grains, processed food, fresh and processed seafood. Tap water and mineral water were found to contain various amounts of NOx that were drastically reduced by treatment with a reverse osmosis column and remained in trace amounts after ion exchange column treatment. NOx content was low in drinks such as cola, but was extremely high in vegetable juice containing dark green leaved vegetables. The intake of high NOx drinks resulted in elevated plasma NOx concentration, but intake of low NOx drinks did not change the plasma NOx concentration. Based on these findings, a steady state could be achieved by 18 hours fasting after the intake of a moderately NOx-restricted diet (about 370, micromoles/day) and by 13 hours fasting after the intake of an extremely NOx-restricted diet (< 100 micromoles/day). NOx concentrations in randomly collected blood samples without these conditions were sometimes ten times higher than that at steady state. This procedure can be undertaken under normal Japanese daily life and is expected to be applicable even to outpatients.

Testosterone inhibits tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in human aortic endothelial cells.

We investigated the effect of testosterone (T) on tumor necrosis factor-alpha (TNF-alpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells. Incubation of these cells with T resulted in a dose-dependent reduction in the expression, with this reduction completely abolished by a selective androgen receptor blocker. Electrophoretic mobility shift assay demonstrated that T inhibited TNF-alpha-induced activation of the transcriptional nuclear factor-kappaB, which is critical for the inducible expression of VCAM-1, probably through the suppression of the nuclear translocation. Our results may suggest an inhibitory effect of T on atherogenesis, providing a novel insight into the consideration of the pathogenesis of atherosclerosis.

Thrombospondin expression in aldosterone-producing adenomas.

Thrombospondin (TSP) 1 and 2 are extracellular matrix proteins that appear to play a role in cell adhesion and cell migration. It has been demonstrated that the pattern of TSP expression is shifted from TSP1 to TSP2 under adrenocorticotrophic hormone treatment in bovine adrenocortical cells. We investigated the expression in human adrenal tissues by Northern blot analysis and correlated these data with the expression of the adrenocorticotrophic hormone-receptor (ACTH-R). All adrenal tissues (control adrenals, nonfunctional adenomas and ACTH-dependent aldosterone-producing adenomas (APA)) expressed both TSP1 and TSP2 mRNAs. Compared to control adrenals (TSP1 and TSP2 expression = 100 +/- 12%, respectively), TSP1 expression was negatively (51 +/- 10%, p < 0.01) and TSP2 expression was positively (289 +/- 36%, p < 0.01) regulated in APA. No significant differences in TSP1 and TSP2 expressions were found between control adrenals and nonfunctional adenomas. In APA, TSP1 (r = -0.86, p<0.01) and TSP2 (r = 0.88, p < 0.01) expressions correlated closely with the expression of ACTH-R. These results suggest that ACTH activity plays an important role in regulating the expression of TSPs in human adrenal tissues. We speculate that the shift of expression observed in APA may be associated with the phenotype of the tumors.

Insulin resistance is associated with reduced nocturnal falls of blood pressure in normotensive, nonobese type 2 diabetic subjects.

To assess the relationship between insulin resistance and ambulatory blood pressure (BP) pattern, we determined glucose infusion rate (GIR) as a marker of insulin resistance using a glucose clamp method, and measured 24-h BPs in 25 normotensive, nonobese type 2 diabetic subjects. They were divided into two groups: 11 dippers and 14 nondippers. Clinical characteristics were similar in the two groups except for orthostatic fall in systolic BP. The median GIR level was significantly lower in nondippers than in dippers (P < 0.05). Spearman's rank correlation revealed that the GIRs were negatively correlated with the systolic, diastolic and mean BPs during nighttime (P < 0.05 or less), but not with daytime or whole day BPs. Moreover, based on a logistic regression analysis, the GIR as well as orthostatic fall in systolic BP discriminated independently between dippers and nondippers. Thus, our results suggest that insulin resistance is associated with decreased nocturnal BP fall in type 2 diabetic subjects.