Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation.

Exendin-4 regulates the expression of the ATP-binding cassette transporter A1 via transcriptional factor PREB in the pancreatic ß cell line.

BACKGROUND: PRL regulatory element-binding (PREB) protein is a transcription factor that regulates insulin promoter activity in the rat anterior pituitary. The PREB protein is expressed not only in the anterior pituitary but also in pancreatic ß cells. Previously, we have reported that PREB plays an important role in glucose-mediated insulin gene expression in pancreatic ß cells. The ATP-binding cassette transporter A1 (ABCA1) in pancreatic ß cells influences insulin secretion and glucose homeostasis. Exendin-4 (Ex-4), a longacting agonist of the glucagon-like peptide 1, stimulates ABCA1 expression in pancreatic ß cells. AIMS: In this study, we examined the role played by PREB in Ex-4-induced ABCA1 expression in pancreatic ß cells. MATERIAL/SUBJECTS AND METHODS: PREB mRNA and protein expression were evaluated in pancreatic ß cell line (INS-1 cells) treated with Ex-4 (10 nM). RESULTS: Ex-4 stimulated PREB protein and mRNA expression in INS-1 cells. PREB stimulated the activity of the luciferase reporter protein that was under the control of the ABCA1 promoter. Chromatin immunoprecipitation assay showed that PREB mediates its transcriptional activity by directly binding to the ABCA1 promoter region. Finally, we used small interfering RNA to inhibit PREB expression in the cells and demonstrated that the knockdown of PREB expression attenuated the effects of Ex-4 on ABCA1 expression. CONCLUSION: PREB mediates Ex-4-stimulated transcription of the ABCA1 gene in pancreatic ß cells.

Suppression of prolactin expression by cabergoline requires prolactin regulatory element-binding protein (PREB) in GH3 cells.

The prolactin regulatory element-binding protein (PREB) is a transcriptional factor that regulates prolactin (PRL) promoter activity in the anterior pituitary. Prolactinomas are the most common pituitary tumors. Administration of cabergoline, a selective dopamine D2-receptor agonist, has become the initial therapy of choice for most patients with prolactinomas. Although activation of the D2 receptor results in the inhibition of PRL synthesis, the details of the underlying mechanisms remain unknown. Samples of ten prolactinomas and ten nonfunctioning pituitary adenomas were analyzed by immunohistochemistry to detect the expression of PREB. The effect of cabergoline on PREB expression was assessed by western blotting and real-time polymerase chain reaction (PCR) analysis. Reporter gene analysis of PRL was employed to examine the role of PREB on cabergoline-induced suppression of PRL transcription. Immunohistochemical analysis revealed strong positive PREB expression in the prolactinoma tissue, but extremely weak or undetected expression in the nonfunctioning pituitary tumor tissue. Western blots probed with a PREB-specific antiserum revealed that the relative abundance of the PREB protein in the GH3 cells decreased in a dose-dependent manner in response to cabergoline treatment, as did the relative abundance of PREB mRNA. Although cabergoline inhibited the activity of the PRL promoter, mutation of PREB-binding site within the promoter abrogated the ability of cabergoline to inhibit the PRL promoter activity. We have demonstrated that PREB is expressed in prolactinomas and that the suppression of PRL expression by cabergoline requires the transcriptional factor PREB.

Hyperglycemia suppresses ABCA1 expression in vascular smooth muscle cells.

Hyperglycemia is a major risk factor for atherosclerotic disease. The ATP-binding cassette transporter A1 (ABCA1) functions as a pivotal regulator of lipid efflux from cells to apolipoproteins and is thus involved in lowering the risk of atherosclerosis. In this study, we have examined the glucose-mediated regulation of the ABCA1 gene expression in vascular smooth muscle cells. ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and reporter gene assay. The results showed that the expression of the ABCA1 mRNA and protein decreased after the cells were treated with 22.4 mM glucose for 48 h. The transcriptional activity of the ABCA1 promoter paralleled the endogenous expression of the ABCA1 gene. Next, we used inhibitors of certain signal transduction pathways to demonstrate that the glucose-induced ABCA1 suppression is sensitive to the p38-mitogen-activated protein kinase (MAPK) inhibitors. The expression of a constitutively active form of p38-MAPK in the cells inhibited the ABCA1 promoter activity, irrespective of the presence of glucose. A dominant-negative mutant of p38-MAPK abrogated the inhibitory effect of glucose on the ABCA1 promoter activity. These results indicate that the glucose-induced suppression of ABCA1 expression is partially mediated by the activation of the p38-MAPK pathway.

Prolactin regulatory element binding protein as a potential transcriptional factor for the insulin gene in response to glucose stimulation.

AIMS/HYPOTHESIS: Prolactin regulatory element binding (PREB) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas; however its role in the pancreas is not known. We therefore examined the role of PREB in insulin gene expression. MATERIALS AND METHODS: To analyse the effects of PREB on insulin gene transcription, we employed the luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA). In cells expressing or knocked down for PREB, insulin expression and secretion were determined. RESULTS: PREB was located mainly in nuclei of rat pancreatic beta cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognised by PREB antiserum. This nuclear extract also showed insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter, mediated the transcriptional effect of PREB. In addition, synthesised PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Cells expressing or knocked down for PREB exhibited increased or decreased insulin expression, respectively. CONCLUSIONS/INTERPRETATION: These results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.

Extensive primary cardiac liposarcoma with multiple functional complications.

Cardiac liposarcoma in a patient manifested multiple cardiac functional complications. Three dimensional reconstruction of the heart with multidetector row computed tomography made apparent each of the cardiac complications that resulted from the tumour's invasion. On the basis of these findings, the floating mass in the pulmonary artery and the compressing mass around the superior vena cava and interatrial septum were successfully resected. Pathological examination of the mass was consistent with well differentiated liposarcoma.

Negative air ions created by water shearing improve erythrocyte deformability and aerobic metabolism.

UNLABELLED: To elucidate a potential mechanism by which negative air ions improve aerobic metabolism, changes in venous blood lactate levels, pH, erythrocyte deformability, and plasma superoxide dismutase activity and ceruloplasmin levels were examined during a 1-h exposure to negative air ions created by water shearing or corona discharge in nine adult healthy volunteers. The blood lactate level decreased from 1.3 +/- 0.3 to 1.0 +/- 0.2 mmol/l, pH increased from 7.388 +/- 0.025 to 7.417 +/- 0.036, and erythrocyte deformability improved from 37.0 +/- 2.2 to 35.1 +/- 3.0 s, expressed as the mean +/- s.d., when exposed to negative air ions created by water shearing, but did not change when exposed to negative air ions created by corona discharge. Other variables did not change in either exposure. The results obtained suggest that negative air ions created by water shearing improve aerobic metabolism by improving erythrocyte deformability. PRACTICAL IMPLICATIONS: The paper shows that negative air ions created by water shearing method improve aerobic metabolism only during a 1-h exposure, which may be caused by improvement of erythrocyte deformability, but negative air ions created by corona discharge have no effects. A potential mechanism is that negative air ions enter the circulating blood via the lungs and electrons of these ions are delivered to the plasma protein. Why negative air ions created by corona discharge have no effects is considered that water binding does not exist so that the lifetime of these ions is markedly short, by which the ions cannot reach the alveoli of the lungs sufficiently.

Spinal anesthesia hypotension in elective cesarean section in parturients wearing extra-strong compression stockings.

We have routinely applied an extra-strong graduated compression stocking to cesarean section patients to reduce the incidence of spinal anesthesia hypotension. Because bupivacaine has recently become available in Japan, we compared the incidence of spinal hypotension using either 2.0 ml of hyperbaric 0.3% dibucaine or 0.5% bupivacaine. There were 98 full-term parturients wearing the stocking who received 2.0 ml injection of dibucaine or bupivacaine for elective cesarean section. When systolic blood pressure decreased to 90-100 mm Hg or to less than 70% of the pre-anesthesia value, ephedrine was injected intravenously. There was no significant difference in systolic blood pressure or heart rate during spinal anesthesia between the dibucaine and bupivacaine groups. Although the demographic data and various data related to anesthesia or surgery were similar in the groups, the fluid volume and the dose and incidence of ephedrine injection during anesthesia showed significant differences: the mean dose was 3.6 and 1.5 mg and the incidence was 41% and 19% in the dibucaine and bupivacaine groups, respectively. Spinal anesthesia using bupivacaine results in a lower incidence of spinal hypotension compared with dibucaine and, in combination with fitting the extra-strong stockings onto both legs, is clinically useful for cesarean sections.

Circulatory, respiratory and metabolic changes after thigh tourniquet release in combined epidural-propofol anaesthesia with preservation of spontaneous respiration.

Twelve elderly patients undergoing total knee arthroplasty received lumbar epidural anaesthesia and propofol infusion at 5 mg.kg(-1).h(-1) following a 1.5-2.0 mg.kg(-1) bolus dose with preservation of spontaneous respiration via a laryngeal mask airway. Circulatory, respiratory and metabolic variables were measured before and 1, 3, 5, 15 and 30 min after release of a pneumatic thigh tourniquet. The blood pressure was decreased at all time-points and the respiratory rate increased at 1 min. The P(a)CO(2) was increased only at 1 min. Arterial blood pH and base excess were decreased at 1 and 3 min and 1, 3 and 5 min, respectively. Arterial blood lactate levels were increased at all times. These characteristics were considered to be identical to those under regional anaesthesia with conscious spontaneous respiration, showing that spontaneous respiration under this anaesthetic regimen has a similar respiratory capacity to that of conscious spontaneous respiration.

Telomere dynamics in myelodysplastic syndromes and acute leukemic transformation.

Myelodysplastic syndromes (MDS) are characterized by cytopenias in the blood and dysplastic features in the hematopoietic cells. Although the impact of cytogenetic abnormalities is considerable for prognosis, the exact genetic mechanism of MDS remains undetermined. In this study we assessed cytogenetic changes, microsatellite alterations, and telomere dynamics in order to obtain further insight into the pathogenesis of MDS. Thirty-three percentage of MDS patients and 60% of post-MDS acute leukemia (post-MDS AML) had de novo microsatellite changes. In the MDS phase, however, > 60% of patients showed reduction of telomere lengths without microsatellite changes, indicating that telomere reduction in most MDS patients does not seem to be directly linked to genome instability, or that reduction of telomere length does not induce microsatellite changes in the MDS phase. Some MDS patients had microsatellite changes without telomerase elevation, indicating that genome instability might accumulate during the disease progression in some MDS patients, and this condition (cellular senescence) may be related to ineffective hemopoiesis in MDS patients. In contrast, 40% of post-MDS AML patients had elevated telomerase activity with microsatellite changes, indicating that approximately 40% of patients with post-MDS AML patients had accumulation of genome instability resulting in elevated telomerase activity in an attempt to obtain genetic stability. However, the remaining MDS patients had microsatellite changes without telomerase up-regulation, suggesting that some MDS had genome instability even after leukemic transformation. Most MDS patients with elevated telomerase activity in the AML phase had elevated telomerase activity even in the MDS phase without apparent change in telomere length before and after leukemic transformation. These findings indicate that telomerase activity in the MDS phase may be independent of telomere length, although telomere shortening seems to be related to genomic instability, and this process may be linked to apoptosis of MDS cells.

Bradykinin-associated reactions in white cell-reduction filter.

PURPOSE: The purpose of this study was to examine the effect of temperature on bradykinin generation during blood transfusion using positively charged (positive filter), negatively charged (negative filter), and neutral (neutral filter) filters. MATERIALS AND METHODS: Whole blood collected from six volunteers at 4 degrees C or 37 degrees C was passed through the positive or negative filter. In six surgical patients during surgery, autologous blood transfusion at 37 degrees C was initiated through the positive filter, and the same transfusion was reintroduced through the negative filter. Whole blood from another six volunteers at 4 degrees or 37 degrees C was passed through the neutral filter. RESULTS: The positive filter did not generate bradykinin at any temperature, whereas the negative filter generated bradykinin by approximately 4,000-fold when warm blood was used but did not at cool blood. Blood pressure decreased and heart rate increased during warm blood transfusion using the negative filter but did not change using the positive filter. Plasma bradykinin levels increased in patients with use of the negative filter. The neutral filter generated bradykinin when warm blood was used but at levels lower than for the negative filter. CONCLUSIONS: Use of negative filter results in the temperature-dependent generation of bradykinin, which becomes a potential anaphylatoxin when warm blood is used.

Water-diluted local anesthetic for trigger-point injection in chronic myofascial pain syndrome: evaluation of types of local anesthetic and concentrations in water.

BACKGROUND AND OBJECTIVES: We have recently demonstrated that a mixture of 1% lidocaine with water in a 1:3 ratio has less injection pain and is more effective than unaltered 1% lidocaine in treating chronic myofascial pain syndromes. Yet, the most suitable local anesthetic and the most effective dilution in water have not been evaluated. METHODS: Various mixtures of local anesthetics and water or saline were injected intramuscularly into the shoulder of 40 female volunteers, and pain scores on injection were evaluated in a randomized and double-blinded manner. In another portion of the study, 0.25% or 0.2% lidocaine in water were injected randomly into 1 side of 21 outpatients with chronic neck, shoulder, or lumbar myofascial pain to the same degree in both sides. The other solution was injected into the other side of the same patients. RESULTS: Less injection pain was experienced with the water-diluted 0.25% lidocaine and water-diluted 0.25% mepivacaine than the saline-diluted 0.25% lidocaine and water-diluted 0.0625% bupivacaine. Also, less injection pain was experienced with the water-diluted 0.25% and 0.2% lidocaine than the water-diluted 0.3% and 0.15% lidocaine. In the other study, there were no differences in either the effectiveness or duration of analgesia between the 0.25% and 0.2% water-diluted lidocaine. CONCLUSIONS: The suitable type of local anesthetic may be lidocaine or mepivacaine, and the most effective water-diluted concentration is considered to be 0.2% to 0.25%.

Extra-strong graduated compression stocking reduces usage of vasopressor agents during spinal anesthesia for cesarean section.

Because a standard type of graduated compression stocking is not effective for the prevention of spinal anesthesia hypothension during cesarean section, we have used an extra-strong type of graduated compression stocking. This study examined whether the extra-strong stocking reduces usage of vasopressor agents. Forty-eight and 47 full term parturients were fitted with the standard stocking and the extra-strong stocking, respectively. Spinal anesthesia was performed by injecting 2.0 ml 0.3% dibucaine hyperbaric solution. When systolic blood pressure decreased to 90-100 mmHg or to less than 70% of the pre-anesthesia value, ephedrine was injected. There was no significant difference in systolic blood pressure or heart rate during spinal anesthesia between the groups. The mean dose of ephedrine injected during anesthesia was 12.2 and 4.3 mg, and the incidence of ephedrine injected was 85 and 49%, in the standard and extra-strong stocking groups, respectively. The extra-strong stocking group showed significantly lower values. Fitting the extra-strong stocking on both legs for cesarean sections undergoing spinal anesthesia reduced usage of vasopressor agents, suggesting a promising use of this stocking for non-invasive prophylaxis.

Impaired telomere regulation mechanism by TRF1 (telomere-binding protein), but not TRF2 expression, in acute leukemia cells.

Telomere regulation is suggested to be an important mechanism in cellular proliferation and cellular senescence not only in normal diploid cells but also in neoplastic cells, including human leukemia cells. We studied the possible correlation among telomere length, telomerase (a ribonuclear protein that synthesizes the telemeres de novo) activity, hTERT (a catalytic subunit of telomerase) expression, and TRF1 and TRF2 (telomere DNA binding proteins) expression in human acute leukemia cells. The hTERT expression level was strongly associated with telomerase activity (P=0.0001), indicating that the expression level of the catalytic subunit (hTERT) regulates telomerase activity in human acute leukemia cells. TRF1 expression, which is believed to control telomere length, was significantly elevated in patients with acute lymphoblastic leukemia (ALL) (P=0.0232) compared to those in acute myeloid leukemia (AML); TRF1 expression tended to be higher in patients without telomere shortening (P=0.077) and in those with hTERT expression (P=0.055). This indicates that TRF1 may act to monitor telomere length under the condition of up-regulated telomerase activity in some neoplastic cells. In contrast, TRF2 expression in acute leukemia did not show any correlation with telomere parameters in this study. Although the precise regulation mechanism of telomere length is still uncertain, these results may suggest that regulation of telomere length is partially associated with TRF1 expression, whereas dysfunction of TRF1 expression may be speculated in a subset of acute leukemia.

Increase of peak expiratory flow by atropine is dependent on circadian rhythm.

PURPOSE: To examine whether the bronchodilatory effect of atropine differs in the evening from the morning. METHODS: Thirteen adult healthy volunteers with no oral medication intake were studied. At 1600, peak expiratory flow (PEF) was measured three times, and the highest value recorded. Subsequently, the volunteer received 0.01 mg(-1) x kg(-1) atropine im, and the PEF was measured every 30 min for 180 min. On a different day, at 0400, the effect of atropine on the PEF was measured again in the same way. RESULTS: The PEF values before atropine at 1600 and 0400 were 485 +/- 92 (350-730) and 458 +/- 76 (340-600) 1 min(-1),(P < 0.05). There was no difference in PEF values between the 1600 and 0400 time courses after atropine. The PEF value was increased only at 90 min at 1600 (P = 0.0012), but at 30, 60, 90 and 120 min at 0400 (P = 0.0001). CONCLUSION: Atropine administration has a weak bronchodilatory effect in the evening, but a stronger effect in the morning. Airways are narrower in the morning than in the evening, and this change is inhibited by atropine such that the PEF values are restored to those observed in the evening.

Measurement of soluble Fas antigen and ligand in circulating serum and intra-abdominal or cerebrospinal fluid during gastrointestinal or cerebrovascular surgery.

PURPOSE: Soluble Fas antigen (sFas) and ligand (sFasL), which are associated with apoptosis, have not been evaluated in gastrointestinal or cerebrovascular surgery. The aim of this study was to measure these substances in serum, intra-abdominal fluid, or cerebrospinal fluid, and to speculate a pathophysiologic role for Fas-FasL apoptosis in surgery. MATERIALS AND METHODS: Arterial blood and intraabdominal or cerebrospinal fluid were collected at intervals from 27 gastrointestinal surgical patients, 10 cerebrovascular surgical patients, and 10 spinal anesthesia patients. RESULTS: Serum sFas levels did not change during and after surgery. Intra-abdominal and cerebrospinal sFas levels were identical to and lower than those in serum. Serum sFasL levels did not change during surgery, but decreased after surgery. Intra-abdominal and cerebrospinal sFasL levels were higher than and identical to those in serum. In spinal anesthesia, cerebrospinal sFas and sFasL levels were lower than those in serum. CONCLUSIONS: Serum sFasL decreases after surgery, whereas intra-abdominal or cerebrospinal sFasL increases, although sFas in each site does not change, suggesting redistribution of activated lymphocytes into local surgical wounds and induction of apoptosis in this site.

Chronic lymphocytic leukemia complicated by plasmacytoma originating from different clones.

In a woman with chronic lymphocytic leukemia (CLL), a plasmacytoma developed on the back region after four years. CLL cases complicated with plasmacytoma are rare. In the present case, the plasmacytoma showed kappa cytoplasmic immunoglobulin (Ig), and the CLL showed gamma lambda surface Ig. To reveal the clonal origin of CLL and plasmacytoma, we analyzed Ig gene rearrangements in the patient's peripheral blood and plasmacytoma. Ig gene DNA analysis confirmed the presence of different rearrangements in the heavy and light chain genes of CLL and plasmacytoma. These findings suggest that in this patient, the two B cell malignancies arose from expansion of two phenotypically and genotypically distinct clones.

The superiority of water-diluted 0.25% to neat 1% lidocaine for trigger-point injections in myofascial pain syndrome: a prospective, randomized, double-blinded trial.

Trigger-point injection with a mixture of commercially available 1% lidocaine in sterile distilled water at a ratio of 1:3 compared with 1% lidocaine alone resulted in better efficacy and less injection pain. This simple procedure may be suitable for treatments of a wide range of myofascial pain syndromes.