Takao Watarai

Alteration of Insulin-Receptor Kinase Activity by High-Fat Feeding

It has been demonstrated in in vivo and in vitro experiments that high-fat (HF) feeding causes insulin resistance. To elucidate the mechanism for this effect, we have measured the kinase activity of the insulin receptor purified from livers of HF-fed rats that showed impaired insulin action in isolated rat adipocytes. In adipocyte experiments, HF feeding led to a 65% decrease in the maximal response stimulated by insulin in a 2-deoxyglucose uptake study. Although insulin binding to adipocytes of HF-fed rats also decreased to 50% of control due to decreased binding affinity, the postbinding defect should be accounted for by decreased insulin action in view of the presence of spare receptor. In contrast to adipocytes, insulin binding to the lectin-purified insulin receptor from livers showed no difference in receptor-binding affinity between HF-fed and control rats. Insulin-stimulated phosphorylation of the β-subunit of the insulin receptor was decreased to almost 50% throughout the entire dose-response curve. The study of glutamine-tyrosine (4:1) phosphorylation by the insulin-receptor kinase showed results similar to those of the autophosphorylation study. These results suggest that an HF diet causes insulin resistance by affecting insulin-receptor kinase, which plays an important role in transmembrane signaling between insulin binding and insulin action.

Evaluation of gut motility in type II diabetes by the radiopaque marker method.

Background : The clinical usefulness of the radiopaque marker method for detecting diabetic gastrointestinal motility disturbances, was evaluated by examining 21 type II diabetes subjects who did not have any neuropathic symptoms.

Effect of strict metabolic control on glucose handling by the liver and peripheral tissues in non-insulin-dependent diabetes mellitus

To examine the effect of strict glycemic control on the insulin resistance of non-insulin-dependent diabetes mellitus (NIDDM), we applied euglycemic hyperinsulinemic clamp combined with an oral glucose load (OGL) to nine non-obese subjects with NIDDM and quantitated insulin-mediated glucose uptake by the liver (HGU) and peripheral tissues (PGU) simultaneously before and after 3 to 4 weeks of intimate glycemic control by preprandial regular insulin injections 3 times a day. The glucose infusion rate (GIR) required to maintain euglycemia during the clamp before OGL was considered as PGU. After OGL, the fraction of ingested glucose that is not extracted by the liver enters the systemic circulation and reduces the GIR required for the clamp. HGU was calculated from the difference between the amount of OGL and the cumulative decrements in GIR after OGL and was expressed as the ratio to the amount of OGL (%). Three to 4 weeks after initiation of strict metabolic control, FPG and HbA1c levels significantly improved (9.1 +/- 0.5 vs. 6.4 +/- 0.4 mmol/l, and 11.2 +/- 0.8 vs. 8.3 +/- 0.3%, P < 0.05). HGU significantly increased to 33.1 +/- 9.5 from 14.5 +/- 4.8%, while PGU did not change (38.2 +/- 5.2 vs. 37.4 +/- 3.9 mumol/kg.min). These data suggest that short-term strict metabolic control ameliorates insulin resistance in NIDDM mainly at the hepatic level.

Fasting Plus Prandial Insulin Supplements Improve Insulin Secretory Ability in NIDDM Subjects

To examine how insulin secretory ability is modified by strict glycemic control in non-insulin-dependent diabetes mellitus (NIDDM) subjects, basal and/or prandial insulin was supplemented for 4 wk in 24 diabetic subjects who were secondary failures to sulfonylurea treatment. One intermediate-acting insulin injection a day (n = 7) failed to suppress the rise in plasma C-peptide after meals and did not improve plasma C-peptide responses during a posttreatment oral glucose challenge. Continuous subcutaneous insulin infusion with a premeal bolus (n = 8) suppressed both fasting and meal-related rises in C-peptide and improved C-peptide response during the posttreatment oral glucose challenge. Daily insulin requirements during the 4 wk of treatment were reduced significantly by 52%. A short-acting insulin injection before each meal (n = 9) without basal supplementation suppressed the prandial rise in C-peptide and was associated with a significant reduction in daily insulin requirements during 4 wk of treatment by 28%. Diabetic subjects whose fasting and prandial hyperglycemia were <140 and <200 mg/dl, respectively, showed a significantly higher C-peptide response during oral glucose challenge after treatment than those whose insulin treatment only normalized (<200 mg/dl) prandial but not basal hyperglycemia (>140 mg/dl). These results suggest that a short-term period of meal-related insulin treatment (which normalized prandial glycemia) increases residual β-cell function in NIDDM subjects who failed long-term sulfonylurea administration. A basal insulin supplement alone was not effective. The effectiveness of a prandial insulin supplement may have been further improved by a combined basal and meal-related treatment program

Platelet activation in diabetic patients with asymptomatic atherosclerosis

We studied 27 non-insulin-dependent diabetics without apparent atherosclerosis (AS) to investigate whether abnormal platelet function is related to asymptomatic atherosclerosis in diabetes mellitus. The degree of AS was quantitatively evaluated by determining the intimal plus medial thickness (IMT) of the carotid artery wall with ultrasound high-resolution B-mode imaging. Based on our previous finding that the upper threshold of the IMT was 1.1 mm in healthy subjects, the patients were divided into the AS-positive group with the IMT > 1.1 mm, (n = 17) and the AS-negative group with the IMT < 1.1 mm (n = 10). Among five variables measured as the factors concerned with thrombogenesis, only plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) were significantly higher in the AS-positive group than in the AS-negative group. Chronic administration of pentoxifylline (300 mg/day) significantly reduced the abnormally high plasma levels of beta-TG and PF4 in 7 patients of the AS-positive group to normal levels, without lowering the normal plasma beta-TG and PF4 levels in the remaining 10 patients. Pentoxifylline treatment did not affect the plasma levels of the 3 other variables, von Willebrand factor, 6-keto prostaglandin F1 alpha and thromboxane B2. This study suggests that the progress of atherosclerosis in diabetes mellitus is associated with in vivo platelet activation and platelet activation does not occur in diabetics without carotid atherosclerosis. Pentoxifylline may impede the vicious cycle in which atherosclerosis is accelerated by platelet activation.

Sensitive, selective gas chromatographic—mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol-producing activity

One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[13C]sorbitol, 13C6H14O6, 98.7%) as an internal standard. Gas chromatography-mass spectrometry (GC-MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[13C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1-8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r = 0.999, r = 0.997 and r = 0.997, respectively. The precision of the GC-MS measurement of standard solution was C.V. = 4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, Km and Vmax values of the reductase in erythrocytes were 115 +/- 19 mmol/l and 4.42 +/- nmol/min/g of hemoglobin. In human liver, on the other hand, they were 755 +/- 132 mmol/l and 0.773 +/- 0.090 nmol/min/mg of protein, respectively. This difference of Km values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.

Effect of dexamethasone on the synthesis and degradation of' insulin receptor mRNA in cultured IM-9 cells

SummaryThe effect of glucocorticoid (dexamethasone) on insulin receptor mRNA was examined in the IM-9 cell line by dot hybridization analysis using insulin receptor c-DNA probe (phINSR 13-1). Insulin receptor mRNA was found in a dose- and time-dependent manner, to increase during a 24-h culture with glucocorticoid. Although there has been some dispute about the effect of glucocorticoid on degradation of the insulin receptor itself, we have found glucocorticoid had no significant effect on the degradation rate of insulin receptor mRNA. These results suggest that glucocorticoid has the effect of increasing de novo synthesis of insulin receptor mRNA. But the molecular mechanism of glucocorticoids effect still needs to be investigated.

LP(A) IN JAPANESE DIABETIC CHILDREN

the characteristics of the study protocol and the small numbers of subjects recruited from most centers, these observations should be interpreted with caution. Differences in the risk of severe hypoglycemia between centers are well recognized. We have repeatedly tried to draw attention to this phenomenon (1-3). Differences in patient characteristics referred to the various centers may be one explanation. On the other hand, the quality of diabetes care, especially with respect to patient education, may be crucial in this context (4). Rational insulin substitution and training of the patients to prevent and effectively treat hypoglycemia appear to be the most important determinants of the risk of severe hypoglycemia. If it were true—as Dr. Stephenson suggests—that any effect of insulin species might be obscured by (these) other differences that exist between centers, and that such bias cannot be overcome even by large numbers of patients investigated, then a clinically relevant effect of insulin species on the risk of severe hypoglycemia definitely can be ruled out.

Characteristics of peripheral arterial disease in Japanese type 2 diabetic patients

To the Editor: It is well known that diabetes is an important factor in the progression of peripheral arterial disease [1, 2]. Indeed, peripheral arterial disease in diabetic patients often leads to extremity amputation and has a poorer prognosis and higher mortality [3, 4], suggesting that vessel lesions are even worse in diabetes. There have been few reports, however, that directly compare the characteristics of peripheral arterial disease in type 2 diabetic and nondiabetic subjects as observed by angiography. The aim of this study was to directly compare the characteristics of peripheral arterial disease in type 2 diabetic and nondiabetic subjects according to the TransAtlantic InterSociety Consensus (TASC) [1, 5], which is thought to provide strong worldwide consensus. The TASC classifies vessel lesions into four categories based on severity of the lesions from a therapeutic point of view. The categories are as follows: A, an intravascular intervention is recommended; B, an intravascular intervention tends to be recommended; C, a surgical operation tends to be recommended; D, a surgical operation is recommended. To examine the characteristics of peripheral arterial disease in type 2 diabetic subjects, we screened patients in Kansai Rosai Hospital from January 2000 to July 2004, and recruited 211 patients who had significant stenosis in the lower limb arteries upon observation by aortography and who were thought to need revascularisation. We believe that revascularisation is necessary when patients meet the following criteria: (1) patients have symptoms such as rest pain, foot ulcer, foot gangrene (Fontaine classification III or IV) and significant stenosis upon observation by aortography; or (2) subjects have symptoms such as psychroesthesia, numbness, intermittent claudication (Fontaine classification I or II), significant stenosis, and are resistant to 6-month exercise and internal medication. Clinical characteristics and features of the vessel lesions such as severity, length, calcification, and distribution were compared between type 2 diabetic (n=122) and nondiabetic subjects (n=89). Coronary artery disease was diagnosed when there was clinical evidence of angina or myocardial infarction, abnormalities on electrocardiography, or significant stenosis or obstruction of the coronary artery on angiography. Diabetes was diagnosed when the fasting glucose level was 7 mmol/l or higher, HbA1c was 6.5% or greater, or the patient was currently on antidiabetic treatment. In this study, hypertension was provisionally diagnosed when systolic blood pressure was 140 mmHg or higher and/or diastolic blood pressure was 90 mmHg or higher, or when the patient was taking anti-hypertensive medication. Hyperlipidaemia was provisionally diagnosed when total cholesterol was 5.7 mmol/l or more, or when the patient was taking anti-hyperlipidaemic medication. The criteria for diagnosis of nephropathy were continuous macro-albuminuria or a serum creatinine level of 140 μmol/l or higher. We evaluated the results of aortography according to TASC. A total of 122 subjects (58%) had diabetes with a duration of 15.±12.5 years (mean±SD). Type 2 diabetic and non-diabetic subjects showed similar age (68.2±8.5 and 69.7±9.9 years) and sex distribution (male: 74.6 and 84.3%). There was no difference in the proportion of patients with a smoking habit (41.7 and 46.4%), hypertenS. Gorogawa . O. Iida . M. Ikeda . S. Nanto Department of Internal Medicine, Kansai Rosai Hospital, 3-1-69 Inabasco, Amagasaki, Hyogo, Japan

Inhibitory effect of methylguanidine on insulin binding to its receptor. Mechanism underlying insulin resistance in uremia

To elucidate the mechanism responsible for the decreased insulin binding to erythrocytes in uremic patients, the effects of incubation with sera obtained from uremic patients or with methylguanidine, respectively, on insulin binding were examined. Insulin binding to erythrocytes from uremic patients was lower than that from normal subjects (3.1 +/- 0.19% vs 6.6 +/- 0.33%, Mean +/- SEM, P less than .005), being due mainly to decreased binding affinity (58% of control). Incubation of erythrocytes with 1:5 diluted sera of uremic patients resulted in decreased insulin binding (65 +/- 5% of control) and this decrease was restored to the level of 78 +/- 3% of the controls after incubation with buffer for 12 h. Methylguanidine inhibited insulin binding to erythrocytes in a dose-dependent manner. Post-dialyzed serum with 100 ng/ml of methylguanidine (as seen in pre-dialyzed uremic patients) inhibited insulin binding to erythrocytes as much as pre-dialyzed serum (54.3 +/- 3% vs 47 +/- 1% of control). Incubation of IM-9 lymphocytes with 100 ng/ml of methylguanidine did not alter the insulin receptor mRNA level. These results suggest that methylguanidine inhibits insulin binding to its receptor, resulting in decreased insulin binding to erythrocytes.