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BACKGROUND This study is the first to evaluate the short-term efficacy and long-term safety of AZD0585, a mixture of omega-3 free fatty acids, in Japanese patients with dyslipidemia.Methods and Results:In this randomized double-blind placebo-controlled Phase III study, 383 patients were randomized to 2 g AZD0585, 4 g AZD0585, or placebo once daily for 52 weeks. Eligible patients had low-density lipoprotein cholesterol (LDL-C) levels controlled regardless of statin use, and triglyceride levels between 150 and 499 mg/dL. The least-squares (LS) mean percentage changes in triglyceride concentrations from baseline to the 12-week endpoint (mean of measurements at Weeks 10 and 12) in the 2 and 4 g AZD0585 and placebo groups were -15.57%, -21.75%, and 11.15% respectively (P

BACKGROUND Previous studies showed higher risk of cardiovascular and cerebrovascular (CCV) events in primary aldosteronism compared with essential hypertension, but the patients of these studies were limited to primary aldosteronism patients with high plasma aldosterone concentration (PAC). The introduction of the aldosterone-renin ratio as the screening test for primary aldosteronism led to the recognition of primary aldosteronism patients with normal PAC (nPA). However, there is no information on the risk of primary aldosteronism including nPA. METHOD In this retrospectively and cross-sectional study, the clinical features and CCV event risk of primary aldosteronism at diagnosis including nPA were investigated and compared with essential hypertension. The study included 292 consecutive primary aldosteronism patients and 498 essential hypertension outpatients. All primary aldosteronism patients were diagnosed by autonomous aldosterone secretion using confirmatory tests, and then divided into nPA (n = 130) and primary aldosteronism patients with high PAC (hPA: n = 162) using a PAC cutoff level of less than 443 pmol/l (16 ng/dl), representing the normal upper limit of PAC. RESULTS nPA patients were significantly older at diagnosis of primary aldosteronism and at onset of hypertension compared with hPA patients. They had milder hypokalemia and easier-to-control blood pressure. The results suggested that nPA could be considered a mild type of primary aldosteronism but not an early-stage hPA. Moreover, the risk of all CCV events in nPA was significantly lower than that in hPA (odds ratio 0.42, 95% confidence interval 0.18-0.90, P

Excess intake of sucrose or fructose induces adiposity and insulin resistance syndrome, which subsequently lead to diabetes mellitus and hypertension. We evaluated the effects of Eucalyptus globulus leaf extract (ELE) on the postprandial changes in peripheral fructose concentrations during oral sucrose tolerance test in humans. When subjects (n = 18) ingested 25 g of sucrose, both fructose and glucose concentrations in peripheral blood increased transiently and peaked at 30 min after ingestion. Administration of ELE (3 g) 5 min before sucrose ingestion resulted in a significant decrease in the rise in fructose concentration, although no significant effect on glucose concentration was observed. These results suggest that, in humans, ELE ingestion reduces acute hyperfructosemia after sucrose consumption by inhibiting intestinal fructose absorption (but not the sucrase reaction).

本研究では，つつみ込む気流制御エアコンの健康維持および疲労に対する有用性を検証するため，健常女性を対象としたランダム化 2 試験区クロスオーバー試験を実施した．エアコン使用時に冷えを感じやすい女性 10 名に，2 時間のデスクワークによる疲労負荷を与えた後，つつみ込む気流または人あて気流でエアコン制御された部屋で 30 分間休息させた．Visual Analogue Scale (VAS) による主観評価，レーザ・ドップラ式血流計による血流測定，および体温測定を実施した結果，つつみ込む気流制御エアコンでの休息は，体温低下を抑制し，末梢血流を維持することにより，末梢の冷えおよび膝関節の疼痛を抑制し，疲労感の軽減および快適感の上昇を促進することが明らかとなった．本研究により，つつみ込む気流制御エアコンの健康維持および疲労に対する有用性が示された．

本研究では，エアーマッサージチェアの疲労に対する有用性を検証するため，健常成人を対象としたランダム化 2 試験区クロスオーバー試験を実施した．8 時間のデスクワーク負荷の条件下で，VAS, ATMT による作業効率評価，生化学的検査により検証した結果，エアーマッサージチェア群は対照群と比較して，VAS で疲労感の軽減，ATMT の作業効率評価でパフォーマンスの低下抑制がみられた．このことからエアーマッサージチェアがデスクワークによる疲労に対し効果を有することが明らかとなった．また，生化学的検査で唾液アミラーゼの上昇抑制がみられたことから，その作用機序は疲労時に起こる自律神経機能の乱れを調節することであると考えられた．エアーマッサージチェアは，デスクワークによる疲労をはじめとした日常生活において生じる疲労に対して有用であると考えられた．

We examined the effects of L-ornithine administration on physical fatigue. In a double-blind, placebo-controlled, 2-way crossover study, 17 healthy volunteers were randomized to L-ornithine (2000 mg/d for 7 days and 6000 mg/d for 1 day as L-ornithine hydrochloride) or placebo for 8 days. The fatigue-inducing physical task consisted of workload trials on a cycle ergometer at fixed workloads for 2 hours on 2 occasions. We found that oral L-ornithine administration promoted lipid metabolism and activated the urea cycle from serum triacylglycerol, ketone bodies, free fatty acids, and blood ammonia level changing. L-ornithine significantly attenuated the subjective feeling of fatigue (measured by visual analog scale at postrecovery) compared with postload (P < .01). Moreover, in female subjects, the subjective feeling of fatigue was significantly lower compared with the placebo group (P < .05). In the physical performance test in female subjects, the decrease in mean speed for 10 seconds maximum pedaling from 0.5- to 3.5-hour trials in the group receiving L-ornithine was smaller than that in the group receiving placebo (P < .05). These results suggest that L-ornithine has an antifatigue effect by increasing the efficiency of energy consumption and promoting the excretion of ammonia. L-ornithine is a free amino acid and is not rich in meats or fish, so it is difficult to obtain amounts of L-ornithine from ordinary meals that would be sufficient to promote the antifatigue effect. We recommend L-ornithine intake as a nutritional supplement in cases of physical fatigue.

Objective This study examined the effects of coenzyme Q10 administration on physical fatigue. Methods In a double-blinded, placebo-controlled, three crossover design, 17 healthy volunteers were randomized to oral coenzyme Q10 (100 or 300 mg/d) or placebo administration for 8 d. As a fatigue-inducing physical task, subjects performed workload trials on a bicycle ergometer at fixed workloads twice for 2 h and then rested for 4 h. During the physical tasks, subjects performed non-workload trials with maximum velocity for 10 s at 30 min (30-min trial) after the start of physical tasks and 30 min before the end of the tasks (210-min trial). Results The change in maximum velocity from the 30- to the 210-min trial in the 300-mg coenzyme Q10–administered group was higher than that in the placebo group. In addition, subjective fatigue sensation measured on a visual analog scale in the 300-mg coenzyme Q10–administered group after the fatigue-inducing physical task and recovery period was alleviated when compared with that in the placebo group. Conclusion Oral administration of coenzyme Q10 improved subjective fatigue sensation and physical performance during fatigue-inducing workload trials and might prevent unfavorable conditions as a result of physical fatigue.

Objective We examined the effects of administering two different candidate antifatigue substances, caffeine and D-ribose, on mental fatigue. Methods In a double-blinded, placebo-controlled, three-way crossover design, 17 healthy volunteers were randomized to oral caffeine (200 mg/d), D-ribose (2000 mg/d), or placebo for 8 d. As fatigue-inducing mental tasks, subjects performed a 30-min Uchida-Kraepelin psychodiagnostic test and a 30-min advanced trail-making test on four occasions. Results During the tasks, the task performance of the caffeine group was better than that of the placebo group. However, after the fatigue-inducing tasks, although subjective perception of fatigue, motivation, or sleepiness was not significantly different, plasma branched-chain amino acid levels in the caffeine group were lower than those of the placebo group. Administration of D-ribose had no effect. Conclusion Because plasma branched-chain amino acid levels are decreased by mental fatigue, these results suggest that administration of caffeine improved task performance through the enhancement of central nervous system activity without increasing the sensation of fatigue. However, further decreases in branched-chain amino acid levels indicate that caffeine might promote deeper fatigue than placebo. Unfortunately, research subsequent to our study design has shown that D-ribose dosing higher than we used is needed to see a clinical effect and therefore no conclusions can be made from this study as to the efficacy of D-ribose.

The hypotensive effects and safety aspect of beverages containing Eucommia leaf glycosides were examined in a randomized double-blind, placebo-controlled, parallel group study. The subjects were high normotensive and mild hypertensive adult male and female subjects. Test or placebo meals were given one time per a day for 12 weeks.The results revealed that the systolic blood pressure (SBP) was significantly reduced after 4 weeks of intake in the test-meal group. The diastolic blood pressure (DBP) was also significantly reduced after 6 weeks of intake. In contrast, no changes in blood pressure were observed during the trial in the placebo group. Between the two groups, there was a significant difference in SBP at 8, 10, and 12 weeks and in DBP at 10 and 12 weeks. No significant abnormal changes were observed in the results of blood examination, urinalysis, and physical examination. Additionally, clinically relevant adverse effects such as digestive tract symptoms, dry cough, or allergic phenomena were not observed in association with test-meal intake. Thus, these results demonstrated the benefits and safety of beverages containing Eucommia leaf glycosides in high normotensive and mild hypertensive subjects.

Accumulating evidence shows that deterioration of vascular endothelial function underlies the pathophysiology of cardiovascular diseases following lifestyle-related diseases. Both Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which are tripeptides derived from proteolytic hydrolysate of milk casein, inhibit angiotensin-converting enzyme (ACE), suggesting that both VPP and IPP may improve vascular endothelial function, because many ACE inhibitors are known to improve endothelial function. We investigated the effects of ACE-inhibitory food component in humans with mild hypertension, since there has been no report on such effects. The study was conducted by the placebo-controlled, double-blind crossover method in 25 male subjects with mild hypertension. After casein hydrolysate containing both VPP and IPP were administered for 1 week, reactive hyperemia of the left upper forearm was measured using plethysmography as an index of vascular endothelial function. Since one subject dropped out, we analyzed the data of 24 subjects. The reactive hyperemia of the left upper forearm was produced by a 5 min occlusion using inflation of a cuff. The maximum blood flow during reactive hyperemia was 20.8+/-6.7 mL/min/100 mL tissue in the placebo group, whereas it increased remarkably to 30.0+/-10.4 mL/min/100 mL tissue in the group administered casein hydrolysate containing both VPP and IPP (p

We examined the effects of citric acid and l-carnitine administration on physical fatigue. In a double-blind, placebo-controlled, 3-way crossover study, 18 healthy volunteers were randomized to oral citric acid (2,700 mg/day), l-carnitine (1,000 mg/day), or placebo for 8 days. The fatigue-inducing physical task consisted of workload trials on a cycle ergometer at fixed workloads for 2 h on 2 occasions. Before the physical load, salivary chromogranin A, measured as a physiological stress marker, was lower in the group given citric acid than in the group given placebo. Also, after the physical load, the subjective feeling of fatigue assessed with a visual analogue scale was lower in the citric acid group than in the placebo group. In contrast, l-carnitine had no effect on chromogranin A or subjective fatigue. These results suggest that citric acid reduces physiological stress and attenuates physical fatigue, whereas l-carnitine does not.

Background:Lactobacillus casei strain Shirota (LcS) has been found to exert antiallergic effects in animal experiments, but there is little information about its clinical effects in human patients with allergy. Methods: We performed a randomized double-blind, placebo-controlled study to investigate the effects of LcS in patients with allergic rhinitis triggered by Japanese cedar pollen (JCP). Participants were asked to drink fermented milk containing LcS (LcS group) or placebo (control group) for 8 weeks. Clinical symptoms and immunological parameters were compared between the two groups. Results: Symptom-medication scores (SMS) worsened in accordance with the increase in the amount of scattered JCP. In terms of the nasal and ocular SMS, there was no significant difference between the LcS group and the placebo group during the ingestion period. In the subgroup of patients with moderate-to-severe nasal symptom scores before starting the ingestion of test samples, supplementation with LcS tended to reduce nasal SMS. Conclusion: These results indicate that fermented milk containing LcS does not prevent allergic symptoms in patients sensitive to JCP, but may delay the occurrence of allergic symptoms in patients with moderate-to-severe nasal symptom scores.

The transcription factor pancreatic and duodenal homeobox factor 1 (PDX-1) is expressed in pancreatic progenitor cells. In exocrine pancreas, PDX-1 is down-regulated during late development, while re-up-regulation of PDX-1 has been reported in pancreatic cancer and pancreatitis. To determine whether sustained expression of PDX-1 could affect pancreas development, PDX-1 was constitutively expressed in all pancreatic lineages by transgenic approaches. The transgenic pancreas was markedly small with the replacement of acinar cells by duct-like structures, accompanied by activated Stat3. Genetic ablation of Stat3 in the transgenic pancreas profoundly suppressed the metaplastic phenotype. These results provide a mechanism of pancreatic metaplasia by which persistent PDX-1 expression cell-autonomously induces acinar-to-ductal transition through Stat3 activation.

Oxidative stress is induced under diabetic conditions through various pathways, including the electron transport chain in mitochondria and the nonenzymatic glycosylation reaction, and is likely involved in progression of pancreatic beta-cell dysfunction developing in diabetes. beta-Cells are vulnerable to oxidative stress, possibly due to low levels of antioxidant enzyme expression. When oxidative stress was induced in vitro in beta cells, the insulin gene promoter activity and mRNA levels were suppressed, accompanied by the reduced activity of pancreatic and duodenal homeobox factor-1 (PDX-1) (also known as IDX-1/STF-1/IPF1), an important transcription factor for the insulin gene. The suppression of oxidative stress by a potent antioxidant, N-acetyl-l-cysteine or probucol, led to the recovery of insulin biosynthesis and PDX-1 expression in nuclei and improved glucose tolerance in animal models for type 2 diabetes. As a possible cause of this, we recently found that PDX-1 was translocated from the nucleus to the cytoplasm in response to oxidative stress. Furthermore, the addition of a dominant-negative form of c-Jun N-terminal kinase (JNK) inhibited the oxidative stress-induced PDX-1 translocation, suggesting an essential role of JNK in mediating the phenomenon. Taken together, the oxidative stress-mediated activation of the JNK pathway leads to nucleocytoplasmic translocation of PDX-1 and thus is likely involved in the progression of beta-cell dysfunction found in diabetes.

An open randomized prospective study was performed to elucidate the effect of an alpha-glucosidase inhibitor, voglibose, on the progression of atherosclerosis in subjects with type 2 diabetes.Voglibose at a dose of 0.4-0.6 mg/day was added on 51 subjects out of 101 type 2 diabetic patients being treated with diet, sulphonylurea (SU) or insulin injections, and the average (AveIMT) and maximum intima-media thickness (MaxIMT) of their carotid arteries were examined for 3 years.Irrespective of the differences in treatments, addition of voglibose reduced the progression of AveIMT and MaxIMT to -0.024 +/- 0.047 (+/-S.D.) and -0.021 +/- 0.144 mm/year, respectively. Without voglibose, diabetic patients showed significant (P0.0001) progression of AveIMT and MaxIMT (0.056 +/- 0.046 and 0.098 +/- 0.122 mm/year, respectively). The administration of voglibose resulted in a significant reduction of hemoglobin A1c (HbA1c), total cholesterol and triglyceride concentrations, as well as an increase in HDL cholesterol concentration. Multivariate regression analysis showed that administration of voglibose independently reduced the progression of AveIMT by 0.069 mm/year (P0.0001).These results suggest that voglibose reduces the progression of IMT and may be a candidate for an anti-atherosclerotic drug for type 2 diabetic patients.

We investigated the effect of consumption of a catechin-containing drink on body fat level and its safety in healthy adults. The beverage (250 ml/bottle) contained 215.3 mg of tea catechins mostly possessing a galloyl moiety, which included (-)-epigallocatechin gallate 74.6 mg, (-)-epicatechin gallate 34.1 mg, (-)-gallocatechin gallate 77.8 mg, (-)-catechin gallate 24.5 mg. We conducted a double-blind study with three parallel groups. Healthy subjects (98 men and 97 women) aged from 20 to 65 years old with 22.5 < body mass index (BMI) ≤ 30 kg/m2 were assigned to consume 3 bottles of placebo drink (control group), 2 bottles of catechin-containing drink and 1 bottle of placebo drink (low-dose group), or 3 bottles of catechin-containing drink (high-dose group), per day at mealtimes for 12 week (daily consumption of catechins was 41.1, 444.3 or 665.9 mg respectively). Compared to the value at 0 week, consumption of two or three bottles of catechin-containing drink results in significant decrease in body weight and BMI at 8 and 12 or 4, 8 and 12 week, respectively. Body weight and BMI was significantly decreased in both catechin groups compared with the control group from 4 to 12 week. The measurements of abdominal fat areas indicated significant reduction of total fat area and visceral fat area in both catechin groups compared with the control group at 12 week. Thus our present observations suggest that consumption of a catechin-containing drink may be useful for the prevention of obesity-related disorders.

The proliferation of vascular smooth muscle cells (VSMCs) and alterations of their phenotype are implicated in the pathogenesis of atherosclerosis. Arterial wall injury induces the expression of proto-oncogenes, leading to the proliferation of VSMCs. In particular, c-Myc and c-Myb play a central role in cell cycle progression and are essential for VSMC replication. The protooncogene Pim-1 cooperates with c-Myc and enhances the transcriptional activity of c-Myb in hematopoietic cells, suggesting that Pim-1 is involved in cell cycle regulation. The aim of this study was to examine the possible involvement of Pim-1 in VSMC proliferation. Pim-1 was substantially induced in neointimal VSMCs of balloon-injured rat carotid arteries, and in vivo infection with a dominant negative Pim-1-expressing adenovirus (Ad-DN-Pim-1) markedly suppressed neointima formation and cell cycle progression in the balloon-injured arteries. In cultured VSMCs, treatment with serum or H(2)O(2) induced Pim-1 expression, and H(2)O(2)- or serum-stimulated cell cycle progression and DNA synthesis were almost completely inhibited by DN-Pim-1 overexpression. Furthermore, we performed immunohisto-chemical staining for Pim-1 in human thoracic aortas and coronary arteries obtained from six individuals at autopsy and found that Pim-1-positive cells are observed predominantly in the thickened intima of the aortas and coronary arteries. To the best of our knowledge, this is the first report showing Pim-1 expression in rodent and human arterial walls. To summarize, Pim-1 expression was observed in the neointima of balloon-injured rat carotid arteries and in human thoracic aortas and coronary arteries showing intimal thickening, and the specific inhibition of Pim-1 function markedly suppressed neointima formation after balloon injury and the proliferation of cultured VSMCs, suggesting that Pim-1 plays a role in VSMC proliferation.

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic β-cell dysfunction found in diabetes. Possibly caused by low levels of antioxidant enzyme expressions, pancreatic β-cells are vulnerable to oxidative stress. When β-cell-derived HIT-T15 cells or isolated rat islets were exposed to oxidative stress, insulin gene expression was markedly decreased. To investigate the significance of oxidative stress in the progression of pancreatic β-cell dysfunction in type 2 diabetes, we evaluated the effects of antioxidants in diabetic C57BIL/KsJ-db/db mice. According to an intraperitoneal glucose tolerance test, the treatment with antioxidants retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Histological analyses of the pancreata revealed that the β-cell mass was significantly larger in the mice treated with the antioxidants, and the antioxidant treatment suppressed apoptosis in β-cells without changing the rate of β-cell proliferation. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident As possible mechanism underlying the phenomena, expression of pancreatic and duodenal homeobox factor-1 (also known as IDX-l/STF-1/ IPF1), an important transcription factor for the insulin gene, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. Under diabetic conditions, JNK is activated by oxidative stress and involved in the suppression of insulin gene expression. This JNK effect appears to be mediated in part by nucleocytoplasmic translocation of PDX-1, which is also downstream of JNK activation. Taken together, oxidative stress and consequent activation of the JNK pathway are involved in progression of β-cell dysfunction found in diabetes. Antioxidants may serve as a novel mechanism-based therapy for type 2 diabetes.

Objective: This study was conducted to examine whether tissue characterization of the carotid artery wall by determining integrated backscatter (IBS) can identify subjects with a recent history of acute coronary syndrome (ACS) or atherothrombotic infarction (ATI). Research design and methods: The maximum thickness (Max-IMT) and IBS value (corrected-IBS) of the carotid artery intima–media complex were measured ultrasonographically for 132 type 2 diabetic subjects (62.9±8.5 (±S.D.) years old, 87 men and 45 women) with or without cardiovascular diseases. Results: The diabetic patients with recent ACS or ATI had a significantly lower corrected-IBS and higher Max-IMT than those with Max-IMT ≧ 1.1mm but without cardiovascular diseases. The product of Max-IMT and corrected-IBS (IMT-IBS product) of the patients with recent ACS was significantly lower than that of the patients with chronic stable angina. The multiple logistic model showed that only the IMT-IBS product was associated with a recent history of ACS (odds ratio 0.94, P P Conclusions: Diabetic patients who had a recent history of ACS or ATI showed a lower IBS value and higher IMT for their carotid artery. Both the size and tissue characteristics of the carotid plaque may identify subjects with a high risk of cardiovascular disease.

WHO/ISHの血圧の定義・分類で正常高値に該当する者を対象として,FMGのプラセボを対照とした12週間の2重盲検長期摂取試験を実施したところ,以下のことが明らかとなった.被験飲料群では,収縮期血圧が摂取8週間後に有意に低下し,摂取12週間後まで継続して安定した降圧が認められた.また,拡張期血圧は摂取12週間後に有意に低下した.試験期間中,血液および尿検査値の異常はみられず,また,診察所見および自覚的所見において,被験飲料摂取によると思われる重篤な副次的作用は認められなかった.したがって,FMGは,正常高値血圧者に対して,収縮期血圧および拡張期血圧の降下作用を有し,かつ高い安全性を有することが明らかとなった.

To date, the potency of pancreatic and duodenal homeobox gene 1 (PDX-1) in inducing differentiation into insulin-producing cells has been demonstrated in some cells and tissues. In order to carry out efficient screening of somatic tissues and cells that can transdifferentiate into beta-cell-like cells in response to PDX-1, we generated CAG-CAT-PDX1 transgenic mice carrying a transgene cassette composed of the chicken beta-actin gene (CAG) promoter and a floxed stuffer DNA sequence (CAT) linked to PDX-1 cDNA. When the mice were crossed with Alb-Cre mice, which express the Cre recombinase driven by the rat albumin gene promoter, PDX-1 was expressed in more than 50% of hepatocytes and cholangiocytes. The PDX-1 (+) livers expressed a variety of endocrine hormone genes such as insulin, glucagon, somatostatin, and pancreatic polypeptide. In addition, they expressed exocrine genes such as elastase-1 and chymotrypsinogen 1B. However, the mice exhibited marked jaundice due to conjugated hyperbilirubinemia, and the liver tissue displayed abnormal lobe structures and multiple cystic lesions. Thus, the in vivo ectopic expression of PDX-1 in albumin-producing cells was able to initiate but not complete the differentiation of liver cells into pancreatic cells. The conditional PDX-1 transgenic mouse system developed in this study appeared to be useful for efficient screening of PDX-1 responsive somatic tissues and cells.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, has been reported to ameliorate hyperglycemia in obese diabetic animal models. To elucidate the mechanism of BDNF on glucose metabolism, we determined the glucose turnover under basal and euglycemic hyperinsulinemic (insulin infusion rate, 54 pmol · kg−1 · min−1) clamp conditions in obese insulin-resistant rats, male Zucker fatty rats, which had been acutely administered a subcutaneous injection of BDNF (20 mg/kg) (n = 9, BDNF) or vehicle (n = 8, vehicle). Under the basal condition, acute administration of BDNF did not affect the blood glucose level, plasma insulin level, rate of glucose disappearance (Rd), and endogenous glucose production (EGP). Under the clamp condition, the glucose infusion rate (GIR) was significantly higher in BDNF than in vehicle (mean ± SD, 61.4 ± 19.1 v 41.4 ± 4.9 μmol · kg−1 · min−1, P

Blood cholesterol-lowering effect and safety of tea catechins were examined in a placebo-controlled double-blind study in which a beverage containing tea catechins (a 250ml beverage containing 197.4mg of catechins) was given twice a day at breakfast and dinner for 12 weeks. The subjects used were adult males and females with mild and borderline hypercholesterolemia with the range of serum total cholesterol levels 180-259mg/dl (male/ female, 38/22; age, 48±9 years; total cholesterol, 222±20mg/dl), who were not taking drugs that may affect lipid metabolism. The study was initiated with a 2-week pre-observation period followed by a 12-week intake period and a 2-week post-observation period. The results revealed that the serum total cholesterol levels in the test beverage group (initial intake level) was significantly decreased at 8 weeks (228±23mg/dl to 220±21mg/dl, p

A homeodomain containing transcription factor PDX-1 can induce beta-cell-specific gene expressions in some non-beta-cells and may therefore be useful for future diabetes gene/cell therapy. Among the potential target organs or tissues for transcription factor-mediated induction of beta-cell-like differentiation are the intestinal epithelial cells. They have certain merits over other tissues and organs in terms of accessibility for gene delivery and of similarity in developmental background to the pancreatic primordium. In this study, we used an intestinal epithelium-derived cell line, IEC-6 cells, and investigated the possible effects of PDX-1 expression in those cells. By exogenous expression of the PDX-1 gene, IEC-6 cells started expressing multiple beta-cell-specific genes such as amylin, glucokinase, and Nkx6.1, which were not found in the original IEC-6 cells. Insulin gene expression, which was missing initially even in the PDX-1-transfected IEC-6 cells, became detectable when the cells were transplanted under the renal capsule of a rat. When the PDX-1(+) IEC-6 cells were kept in vitro, treatment with betacellulin could also confer insulin gene expression to them. Although insulin secretory granules became visible by electron microscopy, they were secreted regardless of glucose concentration. The in vivo or in vitro inductions of the insulin gene expression were not observed in the PDX-1(-) IEC-6 cells. Thus, our present observations demonstrate the potency of intestinal epithelial cells as a tool for diabetes gene/cell therapy and provide further support for the potency of PDX-1 in driving beta-cell-like differentiation in non-beta-cells.

Oxidative stress is induced under diabetic conditions and causes various forms of tissue damage in patients with diabetes. Recently, pancreatic beta-cells have emerged as a putative target of oxidative stress-induced tissue damage and this seems to explain in part the progressive deterioration of beta-cell function in type 2 diabetes. As a step toward clinical trial of antioxidant for type 2 diabetes, we investigated the possible anti-diabetic effects of probucol, an antioxidant widely used as an anti-hyperlipidemic agent, on preservation of beta-cell function in diabetic C57BL/KsJ-db/db mice. Probucol-containing diet was given to mice from 6 to 16 weeks of age. Immunostaining for oxidative stress markers such as 4-hydroxy-2-nonenal (HNE)-modified proteins and heme oxygenase-1 revealed that probucol treatment decreased reactive oxygen species (ROS) in pancreatic islets of diabetic animals. Oxidative stress is known to enhance apoptosis of beta-cells and to suppress insulin biosynthesis, but probucol treatment led to preservation of beta-cell mass and the insulin content. According to intraperitoneal glucose tolerance tests, the probucol treatment preserved glucose-stimulated insulin secretion and improved glucose tolerance at 10 and 16 weeks: insulin, 280+/-82 vs. 914+/-238 pmol/l (120 min, at 16 weeks; P

This study aimed to evaluate the effect of a phosphodiesterase inhibitor, cilostazol, on the prevention of silent brain infarction in diabetic patients without symptoms of vascular events.A total of 89 subjects were allocated at random to the cilostazol group ( n = 43) or the control group ( n = 46).After the study period (3.2 +/- 0.5 years), carotid intima-media thickness (IMT) (means +/- SD) had increased ( p0.01) by 0.18 +/- 0.19 mm in the control group. In the cilostazol group, intima-media thickness showed almost no change (-0.00 +/- 0.16 mm). In the control group, 2 out of 46 subjects showed symptomatic brain infarctions and 10 out of 34 subjects without infarct-like region assessed by standard brain MRI examination showed silent brain infarctions after the observation period. On the other hand, no subjects in the cilostazol group showed silent brain infarction or strokes during the study period. Both at the beginning and end of the study period, the number of infarct-like regions positively correlated with IMT ( r = 0.335, p0.001 or r = 0.347, p0.001 respectively). The progression of infarct-like regions was directly related to the increase in IMT during the study period ( r = 0.299, p = 0.004).These data demonstrated that cilostazol could prevent the onset of silent brain infarction in Japanese subjects with Type II (non-insulin-dependent) diabetes mellitus. Also, an increase in intima-media thickness of the carotid artery wall could be able to predict the onset of silent brain infarction.

Pax4 is one of the transcription factors that play an important role in the differentiation of islet β-cells. We scanned the Pax4 gene in 200 unrelated Japanese type 2 diabetic patients and found a missense mutation (R121W) in 6 heterozygous patients and 1 homozygous patient (mutant allele frequency 2.0%). The mutation was not found in 161 nondiabetic subjects. The R121W mutation was located in the paired domain and was thought to affect its transcription activity through lack of DNA binding. Six of seven patients had family history of diabetes or impaired glucose tolerance, and four of seven had transient insulin therapy at the onset. One of them, a homozygous carrier, had relatively early onset diabetes and slowly fell into an insulin-dependent state without an autoimmune-mediated process. This is the first report of a Pax4 gene mutation that exhibits loss of function and seems to be associated with type 2 diabetes. This work provides significant implications for the Pax4 gene as one of the predisposing genes for type 2 diabetes in the Japanese.

To investigate the role of heart rate (HR) and blood pressure (BP) for diabetic retinopathy, 24-h ambulatory HR and BP were monitored for 162 in patients with type 2 diabetes and normoalbuminuria. The fundus was assessed as no retinopathy, simple diabetic retinopathy (SDR) and proliferative retinopathy (PDR). Comparing the highest with the lowest quartile of diabetic duration, the relative risk for retinopathy was 9.3 and for nocturnal HR, it was 3.6. Comparison among three retinopathy groups (no retinopathy, group 1, n=122; SDR, group 2, n=24; Pre-PDR or PDR, group 3, n=16) showed that 24-h and nocturnal HR were significantly higher in group 3 (80+/-9 and 71+/-9 beats per min) than in group 2 (73+/-8 and 64+/-8) and group 1 (72+/-7 and 60+/-7). In multiple logistic analysis, the odds ratio of diabetic duration and nocturnal HR to the existence of retinopathy was 1.17 (95% CI, 1.10-1.25, P=0.00001) and 1.11 (95% CI, 1.05-1.17, P=0.0002). We concluded that diabetic retinopathy is related to diabetic duration and high heart rate in type 2 diabetes mellitus with normoalbuminuria. Heart rate elevation may be a predictor of advanced retinopathy.

OBJECTIVE: To examine carotid intima-media thickness (IMT), predictors of its progression, and its relationship with incident coronary heart disease (CHD) in type 2 diabetic Japanese patients. RESEARCH DESIGN AND METHODS: Carotid IMT of 287 subjects with type 2 diabetes (mean age 61.6 years) without CHD or cerebrovascular disease was examined at baseline and after a mean follow-up of 3.1 years. RESULTS: The annual progression of IMT (means +/- SEM) was 0.04+/-0.004 mm/year. Stepwise multivariate analysis demonstrated that independent risk factors for progress of IMT were the initial IMT (P

The intima-media thickness of the carotid artery has been established as a surrogate of definite atherosclerosis in subjects with high risk of vascular events. This study was done to evaluate the effectiveness of long-term antiplatelet therapy in attenuating progression of the intima-media thickness of the carotid artery of subjects with type 2 diabetes. Subjects who had an intima-media thickness over the threshold of the normal subjects but showed no symptoms of vascular events were randomly divided into groups given antiplatelet drugs [ticlopidine (n = 34) or a small dose of aspirin (n = 40)] or no drugs (n = 74). For the follow-up period (3.0+/-0.06 years), the subjects not given antiplatelet drugs showed a significantly higher progression of intima-media thickness (0.067+/-0.009 mm/year) than those given ticlopidine (0.034+/-0.013 mm/year) or aspirin (0.033+/-0.010 mm/year). Stepwise multivariant regression analysis showed that long-term administration of ticlopidine or aspirin significantly reduced the progression of intima-media thickness of diabetic subjects by 0.041 mm/year or 0.032 mm/ year, respectively. These data indicated that despite differences of their pharmacological mechanisms, antiplatelet drugs could attenuate the progression of intima-media thickness of the carotid artery wall of asymptomatic type 2 diabetics who had early-stage carotid atherosclerosis.

Aims/hypothesis. Chronic hyperglycaemia in patients with Type II (non-insulin-dependent) diabetes mellitus often leads to a decline in glucose-responsive insulin secretion from pancreatic beta cells, a phenomenon called glucose toxicity. Upon hyperglycaemia, glycation reaction occurs in the beta cells and induces oxidative stress. To understand the molecular basis of the beta-cell glucose toxicity, we investigated the possible effects of glycation on the expression and enzymatic activity of glucokinase, which plays a crucial part in glucose-responsive insulin secretion. Methods. Glycation and reactive oxygen species were induced in HIT-T15 cells by treatment with d-ribose and effects on glucokinase gene transcription, glucokinase protein amount, glucose phosphorylation activity, and DNA-binding activities of putative glucokinase gene transcription factors were evaluated. Results. When glycation was induced in HIT-T15 cells, the activity of the human glucokinase gene beta-cell-type promoter was suppressed substantially (83 % reduction at 60 mmol/l d-ribose). Also, similar reductions in mRNA and protein amounts of glucokinase and in the Vmax of its enzymatic activity were observed. In agreement with the reduction in the promoter activity, the two major transcription factors of the glucokinase gene, the Pal-binding factor and PDX-1, reduced their binding to their target sequences in the glucokinase gene promoter in glycation-induced HIT cells. Because these effects of d-ribose were counteracted by aminoguanidine or N-acetylcysteine, reactive oxygen species, generated by the glycation reaction, appears to be involved in the phenomena. Conclusion/interpretation. The induction of the glycation reaction, which is known to occur in pancreatic beta cells in chronic hyperglycaemia, suppresses the glucokinase gene transcription and its enzymatic activity. Thus, hyperglycaemia-dependent inhibition of glucokinase activity could in part explain beta-cell glucose toxicity. [Diabetologia (1999) 42: 1417‐1424]

To determine the role of the polyol metabolizing pathway under hyperglycemic conditions, the effects of aldose reductase (AR) on the cellular functions of pancreatic beta-cells were examined. Stable transfectants of rat AR cDNA were obtained with a pancreatic beta-cell line, HIT, in which a negligible amount of AR was originally expressed. Overproduction of AR triggered DNA fragmentation, as judged with the TUNEL method and agarose gel electrophoresis. Morphological analysis by electron microscopy also clearly showed apoptosis of the AR-overexpressing HIT cells. Induction by interleukin-1beta of gene expression such as those of an inducible form of nitric oxide synthase (NOS-II) and Mn-superoxide dismutase (Mn-SOD), was much lower in the transfectants than in the control cells, while the expression of constitutively expressed genes such as those for Cu,Zn-superoxide dismutase and insulin was not changed. The susceptibility to interleukin-1beta stimulation of the expression of the NOS II and Mn-SOD genes was due to suppressed NF-kappaB activity, which is essential for the expression of these genes. In addition, the intracellular NADPH/NADP+ ratio was considerably lower in the AR-transfected cells than in control cells. Thus, the overexpression of AR in pancreatic beta-cells induced apoptosis that may be caused by a redox imbalance.

The development of the pancreas appears to be regulated by various growth factors. We report here the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) in the developing pancreas. Immunostaining of fetal and neonatal rat pancreata, in which endocrine cells are visible as cell clusters often associated with primitive ducts or ductular cells, revealed that most of the cluster-forming cells and primitive ducts or ductular cells express HB-EGF protein. In contrast, the exocrine pancreas lacked HB-EGF expression. Based on findings that the expression pattern was similar to that of the homeodomain-containing transcription factor PDX-1 (IDX-1/STF-1/IPF1) and that the regulatory region of the HB-EGF gene contained sequences similar to the PDX-1-binding A element, we examined whether PDX-1 could be a potential activator of HB-EGF gene expression. The results of reporter gene analyses suggested that the HB-EGF gene promoter is PDX-1-responsive and that the activity of the promoter in pancreatic beta cell-derived βTC1 cells depends on the PDX-1 binding site-like sequences. Gel-mobility shift analyses using an anti-PDX-1 antibody indicated that PDX-1 is a specific and dominant binding factor for an A element-like sequence in the HB-EGF gene. These observations suggest the possible involvement of HB-EGF in pancreas development. While PDX-1 is essential for pancreas development, HB-EGF may function as a mediator of PDX-1 and thus be involved in the development of the endocrine pancreas.

The insulin gene transcription factor PDX-1/IPF1/STF-1/ IDX-1 plays a key role in directing beta cell-specific gene expressions. Recently, impairment of PDX-1 expression or activity has been observed in beta cell-derived HIT cells cultured under high glucose concentrations, and this has been suggested as a possible cause of the decrease in insulin gene transcription. To investigate the pathophysiological significance of PDX-1 as a determinant of the rate of insulin gene transcription, we suppressed its expression in beta cell-derived MIN6 cells using an antisense oligodeoxynucleotide (ODN) and searched for possible changes in the beta cell-specific gene expression. Treatment of MIN6 cells with an 18-mer phosphorothioate ODN complementary to a sequence starting at the translation initiation codon of PDX-1 caused a potent, concentration-dependent reduction in PDX-1 expression; addition of 2 microM antisense ODN could reduce PDX-1 expression to 14+/-4% of the control. There was also a decrease in its DNA binding to the insulin gene A element. Despite such suppression of PDX-1, Northern blot analysis revealed no decrease in the amount of insulin mRNA in the MIN6 cells. Similarly, no changes were detected in the transcription of the glucokinase or islet amyloid polypeptide gene, for which PDX-1 was shown to function as a transcription factor. Thus, our findings dispute the physiological significance of PDX-1 in determining the rate of insulin gene transcription. This means that other components constituting the transcription-controlling machinery need to be evaluated in order to understand the molecular basis of impaired insulin biosynthesis such as that observed due to glucose toxicity.

To clarify the regeneration process of pancreatic beta-cells, we established a new mouse model of diabetes induced by selective perfusion of alloxan after clamping the superior mesenteric artery. In this model, diabetes could be induced by the destruction of beta-cells in alloxan-perfused segments, while beta-cells in nonperfused segments were spared. Intraperitoneal glucose tolerance tests showed glucose intolerance, which gradually ameliorated and was completely normalized in 1 year with a concomitant increase of insulin content in the pancreas. Histological examination showed neo-islet formation in the alloxan-perfused segment and the proliferation of spared beta-cells in the nonperfused segment. In the alloxan-perfused segment, despite a marked reduction of islets in size and number at an early stage, both the number of islets, including islet-like cell clusters (ICCs), and the relative islet area significantly increased at a later stage. Increased single beta-cells and ICCs were located in close contact with duct cell lining, suggesting that they differentiated from duct cells and that such extra-islet precursor cells may be important for beta-cell regeneration in beta-cell-depleted segment. In addition to beta-cells, some nonhormone cells in ICCs were positive for nuclear insulin promoter factor 1, which indicated that most, if not all, nonhormone cells positive for this factor were beta-cell precursors. In the nonperfused segment, the islet area increased significantly, and the highest 5-bromo-2-deoxyuridine-labeling index in beta-cells was observed at day 5, while the number of islets did not increase significantly. This indicated that the regeneration of islet endocrine cells occurs mostly through the proliferation of preexisting intra-islet beta-cells in the nonperfused segment. In conclusion, the regeneration process of beta-cells varied by circumstance. Our mouse model is useful for studying the mechanism of regeneration, since differentiation and proliferation could be analyzed separately in one pancreas.

The pancreatic beta- and alpha-cells are developmentally related to each other but reveal diverse gene expression patterns. Among the two important transcription factors for insulin gene expression, IEF1 is present both in alpha- and beta-cells, but PDX-1/IPF1/STF-1/IDX-1, a homeodomain-containing transcription factor, is present in beta-cells but not in alpha-cells. To elucidate the function of PDX-1 in the expression of beta-cell-specific genes, we established stable alphaTC1 clone 6 (alphaTC1.6)-derived transfectants expressing PDX-1 and examined the changes in the gene expression patterns in them. The exogenous expression of PDX-1 in alphaTC1.6 cells alone could induce islet amyloid polypeptide (IAPP) mRNA expression in the cells but not the expression of insulin, glucokinase, or GLUT2 gene. However, when betacellulin was added to the medium, the PDX-1-expressing alphaTC1.6 cells, but not the control alphaTC1.6 cells, came to express insulin and glucokinase mRNAs. This did not occur with other growth factors such as epidermal growth factor, transforming growth factor alpha, and insulin-like growth factor I. GLUT2 mRNA remained undetectable in the PDX-1--expressing alphaTC1.6 cells. These observations demonstrate the potency of PDX-1 for the expression of the insulin, glucokinase, and IAPP genes and suggest that certain regulatory factors, which can partially be modified by betacellulin, also contribute to the beta-cell specificity of gene expression.

The glycolytic enzyme glucokinase plays a primary role in the glucose-responsive secretion of insulin, and defects of this enzyme can cause NIDDM. As a step toward understanding the molecular basis of glucokinase (GK) gene regulation, we assessed the structure and regulation of the human GK gene β-cell-type promoter. The results of reporter gene analyses using HIT-T15 cells revealed that the gene promoter was comprised of multiple cis-acting elements, including two primarily important cis-motifs: a palindrome structure, hPal-1, and the insulin gene cis-motif A element-like hUPE3. While both elements were bound specifically by nuclear proteins, it was the homeodomain-containing transcription factor insulin promoter factor 1 (IPF1)/STF-1/PDX-1 that bound to the hUPE3 site: IPF1, when expressed in CHO-K1 cells, became bound to the hUPE3 site and activated transcription. An anti-IPF1 antiserum used in gel-mobility shift analysis supershifted the DNA protein complex formed with the hUPE3 probe and nuclear extracts from HIT-T15 cells, thus supporting the involvement of IPF1 in GK gene activation in HIT-T15 cells. In contrast to the insulin gene, however, neither the synergistic effect of the Pan1 expression on the IPF1-induced promoter activation nor the glucose responsiveness of the activity was observed for the GK gene promoter. These results revealed some conservative but unique features for the transcriptional regulation of the β-cell-specific genes in humans. Being implicated in insulin and GK gene regulations as a common transcription factor, IPF1/STF-1/PDX-1 is likely to play an essential role in maintaining normal β-cell functions.

Cyclic ADP-ribose (cADPR), a well-known stimulator of ca(2+) release from the intracellular Ca(2+) pool, has recently emerged as a potential regulator of insulin secretion in pancreatic beta cells. As recently described, BST-1 is a glycosyl-phosphatidylinositol (GPI)-anchored surface molecule that exhibits homology with CD38 and Aplysia ADP-ribosyl cyclase. Like CD38, BST-1 has both ADP-ribosyl cyclase and cADPR hydrolase activities. As a step toward elucidating the physiological role of cADPR in insulin secretion we examined whether BST-1 is expressed in pancreatic islet cells. Sensitive reverse transcription-polymerase chain reaction detected almost as abundant expression of BST-1 mRNA in pancreatic islets as CD38 mRNA. Immunohistochemical analyses utilizing mirror image sections revealed that BST-1 protein is expressed in a majority of the cells in pancreatic islets and that at least beta cells and, to an even greater extent, alpha cells express BST-1. These observations suggest the involvement of multiple enzymes in the regulation of cADPR concentrations in pancreatic islet cells.

Goto-Kakizaki (GK) rat, a rodent model of spontaneously occurring non-insulin dependent diabetes mellitus (NIDDM), exhibits impaired glucose-stimulated insulin secretion. To explore the background of the β-cell dysfunction in NIDDM, we investigated whether and how the expression pattern of factors that would potentially be involved in the glucose-stimulated insulin secretion machinery is changed in GK rats. Using quantitative reverse transcription-PCR (RT-PCR) method, we found that the gene expression of CD38, a type 2 membrane protein which has ADP-ribosyl cyclase activity, is reduced by approximately 50% in islets of GK rats. Despite previous studies showing reduction in the FAD-linked mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) activity in GK rats, the mGPDH mRNA amounts were equal to those in the control Wistar rats, suggesting a difference that arose post-transcriptionally. These observations support the idea that multiple defects of the glucose-responsive insulin secreting machinery are involved in the development of diabetes in GK rats.

Atherosclerotic changes have not been demonstrated directly in asymptomatic hyperglycaemic non-diabetic subjects, although high mortality due to coronary heart disease has been reported. We measured arterial wall thickness non-invasively, in order to directly demonstrate atherosclerosis of the carotid arteries of hyperglycaemic non-diabetic subjects and to evaluate its risk factors. The thicknesses of the intimal plus medial complex (IMT) of the carotid arteries of 112 asymptomatic hyperglycaemic non-diabetic subjects (aged 22-81, 95 males and 17 females) were compared with those of 55 healthy male subjects and 211 non-insulin-dependent NIDDM male diabetic patients. The subjects were subgrouped into impaired glucose-tolerant (IGT) subjects who had a 2-h glycaemic level of more than 7.8 mmol/l, and non-IGT subjects whose 2-h glycaemic levels were within 6.7-7.7 mmol/l. Non-IGT and IGT subjects showed significantly greater IMTs than age-matched healthy males and showed no significant differences compared to age-matched NIDDM patients. Multivariate analysis demonstrated that the risk factors for IMT of non-IGT and IGT subjects were age and systolic blood pressure. According to data on the accumulation of atherogenic risks (hypertension, dyslipidaemia, and smoking), IMT increased linearly in non-IGT and IGT subjects. However, non-IGT and IGT subjects without hyperlipidaemia, hypertension, or smoking risk still had significantly greater IMT than age-matched normal males (1.019 +/- 0.063 vs 0.770 +/- 0.111 mm, p0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Two basic helix-loop-helix (bHLH) transcription factors, E47 and E12, are involved in cell-specific gene expression as part of dimeric complexes which interact with the cis-acting motif E-box. Although both generated from a single gene (E2A) by means of alternative splicing, the structural difference in these bHLH regions between the two suggests that the two bHLH proteins may differ in some of their functions. As a step toward elucidating the individual implications of E47 and E12, we investigated the mRNA expression ratios of their homologues (A1 and kA1, respectively) in mouse tissues and cell lines. Both the A1 and kA1 mRNAs were ubiquitously expressed in all tissues examined. However, their ratios varied: e.g., skeletal muscle, 2.2 ±0.3 (mean ±SE); spleen, 2.0±0.2; pancreatic islet cells, 1.2±0.2. The A1 kA1 ratios in the cell lines investigated were similar to those of their original tissues. In conclusion, the ubiquity in mRNA expression observed for both the E47 and E12 homologues in mouse provides support for their involvement in a broad range of transcriptional regulation. The variation in the A1 kA1 expression ratios, on the other hand, supports the idea that A1 (E47) and kA1 (E12) each have some unique roles in the functions of these E2A gene-encoded bHLH proteins.

We saw a 57-yr-old female non-insulin dependent diabetes mellitus (NIDDM) patient with antiinsulin antibody, in whom insulin-like growth factor- I (IGF-I) was shown to be effective as a blood glucose-lowering agent. Due to the presence of the antibody, the patient suffered from postprandial hyperglycemia and frequent hypoglycemia, which were uncontrollable even by multiple insulin injection therapy. In contrast to insulin injection (0.24IU/kg body wt) which showed a delayed glucoselowering effect, subcutaneous injection of recombinant human IGF-I (0.05-0.1mg/kg body wt) caused a quick fall in plasma glucose levels. Prolongation of the glucose-lowering effect of “rapid-acting” insulin frequently caused subsequent hypoglycemia in this patient, but the effect of IGF-I seemed to have disappeared within the first three hours.

To elucidate the mechanism of impaired pancreatic A cell function in hypoglycemia in diabetics, the effect of long-term strict glycemic regulations on hypoglycemia-induced glucagon secretion was studied. Firstly, the effect of plasma insulin concentrations on suppressing A cell was studied in healthy volunteers by injecting insulin at doses of 0.1 U/kg and 0.3 U/kg. With 0.3 U/kg of insulin, the rate of fall in glycemia and the nadir of blood glucose were made similar to those with 0.1 U/kg of insulin by glucose infusion with artificial endocrine pancreas. Plasma glucagon response after 0.3 U/kg of insulin was significantly suppressed as compared to that after 0.1 U/kg of insulin, demonstrating that not only hypoglycemic stimulus but also plasma insulin concentration were important determinants responsible for glucagon secretion in insulin-induced hypoglycemia. Secondly, effect of strict glycemic control was studied. Short-acting insulin at a dose of 0.1 U/kg was injected in an intravenous bolus form into 12 insulin-dependent (IDDM) and 9 non-insulin-dependent (NIDDM) diabetics before and 1–3 months after strict glycemic control with multiple insulin injections therapy. Before strict glycemic regulations in IDDM, no significant rise in plasma glucagon concentrations was observed during the insulin-induced hypoglycemia. In NIDDM, a rise in plasma glucagon concentrations was observed, though the response was delayed. After strict glycemic regulations, in patients with residual endogenous insulin secretion, the glucagon response to hypoglycemia improved considerably in IDDM and normalized in NIDDM. In IDDM and NIDDM, improvement in glucagon response to hypoglycemia related positively to daily urinary secretion rate of C-peptide. From these experiments, it is clearly shown that blunted hypoglycemia induced glucagon secretion in diabetics is secondary to poor glycemic control, and that the existence of endogenous insulin secretion is essential to glucagon response to hypoglycemia.

As a step to elucidate a role of protein kinase C(PKC) pathways in the regulation of insulin-like growth factor I(IGF-I) gene, we sought to determine whether the IGF-I gene promoter of chicken can be a target of regulation by PKC. An initial gene transfer study showed that, in a human cell line HepG2, the IGF-I gene promoter directs accurate transcription of IGF-I-luciferase fusion gene and enhances luciferase activity. Treatment of transfected cells with 12-o-tetradecanoylphorbol 13-acetate(TPA) increased promoter activity of 2100 and 600bp 5′-flanking sequence 4.9-and 3.6-fold, respectively. Site-directed mutagenesis in the AP-l-like sequence located within the 600bp resulted in 91% loss of its TPA-induced promoter activity, and a gel mobility-shift analysis revealed that TPA-stimulation of HepG2 cells caused a dramatic increase in specific protein-binding to the AP-1-like sequence, suggesting that the sequence functions as an AP-l enhancer. These observations support a direct role for PKC pathways in activating the IGF-I gene promoter in chicken.

This study examined the effect of acetate on endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) by immunoblotting assay and the ability of acetic acid to upregulate flow-mediated vasodilatation in humans. In HUVECs, acetate induced a biphasic increase in the phosphorylated form of eNOS. The amount of phosphorylated eNOS was significantly increased by exposure to 200 mumol/l acetate for 20 min (early phase) and for 4 h (late phase). The inhibitors of cAMP-dependent protein kinase (PKA) and AMP-activated protein kinase (AMPK) blocked acetate-induced eNOS phosphorylation in the early and the late phase respectively. Furthermore, in postmenopausal women, maximum forearm blood flow (FBF) in response to shear stress increased in the vinegar (acetic acid) administered group compared to the placebo group. These results suggest that acetic acid-induced eNOS phosphorylation contributes to upregulation of flow-mediated vasodilatation in humans.

To ascertain whether any routine practices or clinical manipulations in a neonatal intensive care unit could induce intraventricular hemorrhage (IVH) in preterm infants, we performed ultrasonic monitoring of the germinal layer continuously for 48 hours in 33 extremely premature infants with respiratory distress. Intraventricular hemorrhage developed in 16 of these infants. In four infants the timing of the germinal layer hemorrhage was confirmed with ultrasonic monitoring. Three of the four cases were apparently associated with clinical events occurring at the moment of IVH: manual ventilation for improvement of hypercapnia associated with primary pulmonary hypertension of the newborn; correction of hyperkalemia, which was causing an arrhythmia, with administration of calcium gluconate and sodium bicarbonate; and administration of surfactant-TA to improve respiratory failure caused by pulmonary hemorrhage. In these three infants it appeared that one of the basic factors inducing IVH might be an increase in blood pressure with or without hypercapnia, causing cerebral reperfusion after ischemic damage of the germinal layer.