Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of loss of beta cell mass.

The total mass of islets of Langerhans is reduced in individuals with type 2 diabetes, possibly contributing to the pathogenesis of this condition. Although the regulation of islet mass is complex, recent studies have suggested the importance of a signaling pathway that includes the insulin or insulin-like growth factor-1 receptors, insulin receptor substrate and phosphatidylinositol (PI) 3-kinase. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a serine-threonine kinase that mediates signaling downstream of PI 3-kinase. Here we show that mice that lack PDK1 specifically in pancreatic beta cells (betaPdk1-/- mice) develop progressive hyperglycemia as a result of a loss of islet mass. The mice show reductions in islet density as well as in the number and size of cells. Haploinsufficiency of the gene for the transcription factor Foxo1 resulted in a marked increase in the number, but not the size, of cells and resulted in the restoration of glucose homeostasis in betaPdk1-/- mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.

ALEX1 suppresses colony formation ability of human colorectal carcinoma cell lines.

Arm protein lost in epithelial cancers, on chromosome X (ALEX; also known as armadillo repeat containing, X-linked [ARMCX]) is a novel subgroup within the armadillo (ARM) family, which has several ARM repeat domains. The biological function of classical ARM family members such as ß-catenin is well understood, but that of the ALEX/ARMCX family members is largely unknown. Here we evaluate the effects of ALEX1 overexpression on in vitro colony formation ability and expression of ALEX1 mRNA in human colorectal tumor. Overexpression of ALEX1 suppressed the anchorage-dependent and -independent colony formation of human colorectal carcinoma cell lines by the study of stable clones of HCT116 cells expressing ALEX1 protein. Bisulfite genomic sequencing revealed that the promoter region of ALEX1 gene was highly methylated in both HCT116 and SW480 cells in comparison with PANC-1 and MCF-7 cells, which express endogenous ALEX1 mRNA, indicating the capability of promoter methylation to silence ALEX1 gene in HCT116 and SW480 cells. Our current findings suggest that overexpression of ALEX1 play a negative role in human colorectal tumorigenesis.

Reconstruction system of rat lymphatic tracts after total iliac lymphadenectomy.

BACKGROUND/AIMS: The lymphatic network is capable of reconstruction after the regional lymphadenectomy; however, the biological reconstruction system has not been fully elucidated. Our aim is to evaluate the reconstruction system after bilateral iliac lymphadenectomy using rat models and to establish and characterize rat lymphatic endothelial cells (rLECs) from the reconstructed tracts, compared with those from pre-existing vessels. METHODOLOGY: Under general anesthesia, we excised the dyed bilateral lymphatic vessels. We reopened the treated rats again after 2-10 weeks to observe the reconstruction system. We isolated and cultured rLECs from afferent, efferent and reconstructed lymphatic vessels. We characterized them morphologically and biologically, and examined their activity of proliferation, migration and invasion. RESULTS: We demonstrated that lymph vessels were reconstructed at 4-10 weeks at the site of surgical removal of iliac lymphatic channels, which occurred mainly at the left side of abdomen of the rat (86% on the left side). Isolated cells were all confirmed to be rLECs by expression of lymphatic vessel-specific markers. Proliferative activity of reconstructed rLECs was significantly higher than the other two strains of rLECs. CONCLUSIONS: We first established the reconstruction system after rat total iliac lymphadenectomy and characterized rLECs from the three strains of lymphatic vessels.

Analysis of time-dependent alterations of parameters related to erythrocytes after ipragliflozin initiation.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors often increase the hematocrit. It remains unclear whether this increase would be observed in all patients administered SGLT2 inhibitors, however. We therefore used the data from the previous study and investigated time-dependent alterations of various outcomes related to erythrocytes, erythropoiesis, and clinical outcome in type 2 diabetes subjects (n = 89) treated with ipragliflozin for 16 weeks. Among a total of 89 participants, 71 subjects (80.0% of total participants) showed the elevation of the hematocrit and 18 subjects (20.0% of total participants) did not at 16 weeks. Although the hematocrit levels at baseline were significantly lower in hematocrit-elevated group than non-elevated group, they reached the same levels 4 weeks after the onset of treatment. Binomial logistic regression analysis demonstrated that a lower baseline hematocrit level was related to the elevation of hematocrit at 16 weeks. Optimal cutoff hematocrit levels at baseline to predict hematocrit elevation were 46.9% (male) and 41.7% (female) in ROC analysis. Random intercept model analysis revealed the serum erythropoietin level increased in both hematocrit-elevated and non-elevated groups, whereas only the former group showed an increase in the percentage of reticulocytes during the first 4 weeks. These results suggest that the ipragliflozin-induced increase in hematocrit which is affected by the baseline hematocrit level is attributable to the responsiveness to, but not to the production of, erythropoietin. Collectively, Ht elevation observed in administration of SGLT2 inhibitors can result from erythropoietin-induced erythropoiesis, which is determined by the pre-treatment Ht level. Trial registration: This trial has been registered with University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR no. 000015478).

Human Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) gene is transcriptionally regulated by CREB and Wnt/beta-catenin signaling.

The aberrant activation of Wnt signaling is a key process in colorectal tumorigenesis. Canonical Wnt signaling controls transcription of target genes via beta-catenin and T-cell factor/lymphoid enhancer factor family transcription factor complex. Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) is a novel member of the Armadillo family which has two Armadillo repeats as opposed to more than six repeats in the classical Armadillo family members. Here we examine cis-regulatory elements and trans-acting factors involved in the transcriptional regulation of the ALEX1 gene. Site-directed mutations of a cyclic AMP response element (CRE) and an E-box impaired the basal activity of human ALEX1 promoter in colorectal and pancreatic cancer cell lines. Moreover, overexpression of CRE-binding protein (CREB) increased the ALEX1 promoter activity in these cell lines, whereas knockdown of CREB expression decreased the expression level of ALEX1 mRNA. Interestingly, luciferase reporter analysis and quantitative real-time RT-PCR demonstrated that the ALEX1 promoter was up-regulated in a CRE-dependent manner by continuous activation of Wnt/beta-catenin signaling induced by a glycogen synthase kinase-3 inhibitor and overexpression of beta-catenin. These results indicate that the CRE and E-box sites are essential cis-regulatory elements for ALEX1 promoter activity, and ALEX1 expression is regulated by CREB and Wntk/beta-catenin signaling.

Relationship between glycated hemoglobin level and duration of hypoglycemia in type 2 diabetes patients treated with sulfonylureas: A multicenter cross-sectional study.

AIMS/INTRODUCTION: Sulfonylurea-related hypoglycemia increases the risk of cardiovascular sequela, such as cardiac arrhythmia. This study aimed to clarify the relationship between the level of glycated hemoglobin (HbA1c ) and the duration of hypoglycemia in type 2 diabetes patients treated with sulfonylureas. MATERIALS AND METHODS: Glucose levels in the enrolled patients (n = 300) were investigated with a professional continuous glucose monitoring device in the outpatient setting at six diabetes centers in Japan. RESULTS: A total of 269 participants completed the study. The duration of hypoglycemia with glucose values of <54 mg/dL was significantly longer in patients with an HbA1c level of ≤6.4% than in those with an HbA1c level of ≥8.0%, and that of hypoglycemia with glucose values of <70 mg/dL was significantly longer in patients with an HbA1c level of ≤6.4%, 6.5-6.9% or 7.0-7.4% than in those with an HbA1c level of ≥8.0%. Patients with an HbA1c level of ≤6.4% were exposed to glucose values of <70 mg/dL for >10% of the time in daily life (6.8 ± 5.6 min/h). The duration of hypoglycemia with glucose values of <70 mg/dL was longer at night than during the daytime, and the nadir of glucose values occurred between 03.00 and 05.00 hours irrespective of HbA1c level. The duration of hypoglycemia was associated with the duration of diabetes and sulfonylurea dose. CONCLUSIONS: The duration of hypoglycemia was inversely correlated with HbA1c level and was longer during the night-time than daytime in type 2 diabetes patients treated with sulfonylureas.

Effects of ipragliflozin on glycemic control, appetite and its related hormones: A prospective, multicenter, open-label study (SOAR-KOBE Study).

AIMS/INTRODUCTION: Sodium-glucose cotransporter 2 (SGLT-2) inhibitors improve blood glucose control, as well as reducing bodyweight by promoting urinary glucose excretion. The weight loss is less than expected from urinary glucose loss, however, likely because of an increase in food intake. To investigate whether SGLT-2 inhibitors might increase appetite by affecting related hormones, we examined the effects of the SGLT-2 inhibitor, ipragliflozin, including those on appetite-regulating hormones, in individuals with suboptimally controlled type 2 diabetes. MATERIALS AND METHODS: The present prospective, multicenter, open-label study was carried out with 96 patients with a body mass index of ≥22 kg/m2 who were treated with ipragliflozin (50 mg/day) for 16 weeks. Parameters including glycated hemoglobin level, bodyweight, circulating leptin and active ghrelin concentrations, and appetite as assessed with a visual analog scale were measured before and during treatment. RESULTS: Both glycated hemoglobin level (from 7.9 ± 0.8 to 7.1 ± 0.7%) and bodyweight (from 75.2 ± 12.6 to 72.6 ± 12.4 kg) were significantly decreased after treatment for 16 weeks. The fasting serum leptin level was significantly decreased after 2 weeks (from 19.5 ± 13.1 to 18.1 ± 12.4 ng/mL) and remained decreased up to 16 weeks, even after adjustment for bodyweight, whereas the plasma active ghrelin level showed no significant change. The visual analog scale score for hunger was significantly increased at 2 and 8 weeks. CONCLUSIONS: The present results suggest that ipragliflozin improved glycemic control and reduced bodyweight, but also reduced serum leptin levels and might thereby have increased appetite.

Preclinical assessment and pilot study using anti-CEA monoclonal antibody 1B2 for colorectal carcinoma imaging.

BACKGROUND/AIMS: Preclinical assessment of radio-labeled monoclonal antibodies is essential to the understanding of target-specific tumor localization. The purpose of this study is to prepare for fundamental evaluation of antibodies and to validate the clinical usefulness of the candidate considered useful for practice through preclinical assessment. METHODOLOGY: The immunoreactivity and affinity constant of three kinds of monoclonal antibody (1A4, 1B2, 4H11: CEA-specific) were evaluated with the method of cell binding assay. Tumor localization and biodisrtibution were performed in tumor-bearing athymic mice. Five patients of colorectal cancer underwent radioimmunodetection with 99mTc. Histological analysis was performed to evaluate the tumor localization of injected antibody. RESULTS: The immunoreactive fraction value of 1B2 is the highest than the other antibodies. The difference between the antibody affinities among three antibodies although were rather small. In an animal model, 1B2 obtained more highly tumor targeting and cleared more rapidly from the blood than the other two antibodies did. Pilot study using 1B2 was successful in all cases without background imaging. Visualization of tumor sites surgically removed revealed positive CEA and IgG immunostaining. CONCLUSION: The preparation for preclinical assessment of the characteristic parameters is practically valuable for clinical application of radiolabeled antibodies.

Effects of Insulin Degludec and Insulin Glargine U300 on Day-to-Day Fasting Plasma Glucose Variability in Individuals with Type 1 Diabetes: A Multicenter, Randomized, Crossover Study (Kobe Best Basal Insulin Study 2).

INTRODUCTION: Administered basal insulin markedly influences the fasting plasma glucose (FPG) level of individuals with type 1 diabetes. Insulin degludec (IDeg) and insulin glargine U300 (IGlar U300) are now available as ultra-long-acting insulin formulations, but whether or how their glucose-stabilizing effects differ remains unclear. We will compare the effects of these basal insulins on parameters related to blood glucose control, with a focus on day-to-day glycemic variability, in individuals with type 1 diabetes treated with multiple daily injections. METHODS: A multicenter, randomized, open-label, crossover, comparative study (Kobe Best Basal Insulin Study 2) will be performed at 13 participating institutions in Japan. A total of 46 C-peptide-negative adult outpatients with type 1 diabetes will be randomly assigned 1:1 by a centralized allocation process to IGlar U300 (first period)/IDeg (second period) or IDeg (first period)/IGlar U300 (second period) groups, in which subjects will be treated with the corresponding basal insulin for consecutive 4-week periods. The basal insulin will be titrated to achieve an FPG of less than 130 mg/dL initially and then less than 110 mg/dL if feasible. In the last week of each period, plasma glucose will be determined seven times a day by self-monitoring of blood glucose (SMBG) and intraday and day-to-day glucose excursions will be determined by flash glucose monitoring (FGM). The primary end point is comparison of day-to-day glycemic variability as evaluated by the standard deviation (SD) of FPG during the last week of each treatment period. Secondary end points include the coefficient of variance of FPG, the frequency of severe hypoglycemia as evaluated by SMBG, the duration of hypoglycemia as evaluated by FGM, intraday glycemic variability calculated from both SMBG and FGM data, and the administered insulin dose. PLANNED OUTCOMES: The results of the study will be submitted for publication in a peer-reviewed journal to report differences in the effects of two ultra-long-acting basal insulins, IDeg and IGlar U300. CONCLUSION: This head-to-head comparison will be the first study to compare the effects of IDeg and IGlar U300 on day-to-day FPG variability in C-peptide-negative individuals with type 1 diabetes. TRIAL REGISTRATION: Registered in University Hospital Medical Information Network (UMIN) Clinical Trials Registry as 000029630 on 20 June 2017. FUNDING: Novo Nordisk Pharma Ltd.

Evaluation of the hepatic blood flow increase in the cases with liver metastasis and prediction of patent cases of liver metastasis using dynamic CT.

BACKGROUND/AIMS: This study aimed to measure hepatic blood flow increase in cases with liver metastasis and to diagnose minimal metastasis which cannot be visualized by imaging modalities. METHODOLOGY: The evaluation of hepatic arterial flow increase was performed quantitatively by newly devised index ELR (early-late-ratio) using dynamic computed tomographic (CT) scanning with contrast media. RESULTS: The ratio of the cases with liver metastasis was significantly higher than that of normal liver control. It was revealed that the ratio was correlated with microvessel proliferation in the liver around the metastasis by examination of surgical specimen. Moreover, the patent group which proved to have metastasis within 6 months also had a significant higher ELR value. CONCLUSIONS: ELR not only was useful to evaluate hepatic blood flow increase in the cases with liver metastasis but also could be applied to predict the patent group with micrometastasis.

[A case of Epstein-Barr virus (EBV) associated remnant gastric carcinoma arising 7 years after distal gastrectomy for EBV associated gastric carcinoma].

A 52-year-old man undergoing distal gastrectomy for gastric cancer in July 1998 was found to have a 0-IIa type gastric tumor near EC junction in January 2005. Histological examination showed the tumor was moderately differentiated adenocarcinoma. As the tumor was diagnosed as mucosal cancer, endoscopic mucosal dissection was performed. But pathological findings showed the depth of cancer cell invasion into deep submucosal layer. Then total resection of remnant stomach was performed. Both tumors were diagnosed as EBV-associated carcinoma. It is speculated that the mucosa changing after initial operation would give risk to a new occurrence of EBV-associated remnant gastric carcinoma. And then follow up after operation is important. Although some cases of EBV-associated remnant gastric carcinoma is found for short period after the primary surgery, our case second primary cancer was found 7 year after primary surgery. Long term follow-up by Endoscopy seems to be important.

The Improvement of the Hepatic Histological Findings in a Patient with Non-alcoholic Steatohepatitis with Type 2 Diabetes after the Administration of the Sodium-glucose Cotransporter 2 Inhibitor Ipragliflozin.

The patient was a 67-year-old woman with type 2 diabetes and non-alcoholic steatohepatitis (NASH). The administration of the sodium-glucose cotransporter 2 (SGLT2) inhibitor, ipragliflozin improved her liver dysfunction clinically and histologically. The serum alanine aminotransferase (ALT) and ferritin levels decreased to normal limits after treatment for four months. Type IV collagen and hyaluronic acid, both of which were serum fibrotic markers, decreased after treatment. Ultrasonography and computed tomography showed a decrease in the fat deposits in her liver. Her liver sample showed marked improvement, especially in steatosis, inflammation, and ballooning. The SGLT2 inhibitor ipragliflozin may be useful as a specific therapeutic drug for NASH.

Cyclooxygenase-2 is up-regulated by interleukin-1 beta in human colorectal cancer cells via multiple signaling pathways.

Overexpression of cyclooxygenase-2 (COX-2) has been observed in human colorectal cancer. COX-2 expression in human tumors can be induced by growth factors, cytokines, oncogenes, and other factors. The mechanisms regulating COX-2 expression in human colon cancer have not been completely elucidated. We hypothesized that the proinflammatory cytokine interleukin-1 beta (IL-1 beta) mediates COX-2 expression in HT-29 human colon cancer cells. Treatment of HT-29 cells with IL-1 beta induced expression of COX-2 mRNA and protein in a time- and dose-dependent manner. Inhibitors of the extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, P38 mitogen-activated protein kinase, and nuclear factor-kappa B (NF-kappa B) signaling pathways blocked the ability of IL-1 beta to induce COX-2 mRNA. In contrast, Wortmannin, a phosphoinositide 3-kinase inhibitor upstream of protein kinase B/Akt, led to a slight increase in COX-2 mRNA expression after IL-1 beta treatment. Electrophoretic mobility shift assay on nuclear extracts demonstrated that IL-1 beta induced NF-kappa B DNA binding activity in HT-29 cells, and the activated NF-kappa B complex was eliminated after treatment with an inhibitor of NF-kappa B. Supershift assay indicated that the two NF-kappa B subunits, p65 and p50, were involved in activation of NF-kappa B complex by IL-1 beta stimulation. The stability of COX-2 mRNA was not altered by IL-1 beta treatment. These data demonstrate that IL-1 beta induces COX-2 expression in HT-29 cells through multiple signaling pathways and NF-kappa B.

Isolated home hypertension in the morning is associated with target organ damage in patients with type 2 diabetes.

To investigate the relationship between the blood pressure control level and cardiovascular risk in type 2 diabetic patients, we evaluated home blood pressure, office blood pressure, biochemical data, and carotid echographic and echocardiographic findings in 148 patients with type 2 diabetes. According to the criteria for hypertension in the guidelines of the Japanese Society of Hypertension, we classified patients into a normotensive group with home systolic blood pressure in the morning (morning HSBP)<135 mmHg and office systolic blood pressure (OSBP)<140 mmHg, an office hypertension group with a morning HSBP<135 mmHg and OSBP>or=140 mmHg, an isolated home hypertension in the morning group with morning HSBP>or=135 mmHg and OSBP<140 mmHg, and a sustained hypertension group with morning HSBP>or=135 mmHg and OSBP>or=140 mmHg. In the isolated home hypertension in the morning group, the fasting insulin level, urinary albumin excretion, maximum carotid artery intima-media complex thickness, and left ventricular posterior wall thickness were significantly higher and the coefficient of variation for RR intervals was significantly lower than in the normotensive group. These results suggest that isolated home hypertension in the morning is a risk factor for target organ damage in type 2 diabetic patients.

[Ruptured aneurysm of pancreaticoduodenal artery, report of two cases].

We describe two cases of ruptured areurysm of pancreaticoduodenal artery (PDA), whose chief complaints were right upper abdominal pain. They were diagnosed to retroperitoneal hematoma around the duodenum and the uncus of the pancreas by Computed tomography and aneurysm of pancreaticoduodenal artery by Angiography. The patients underwent a coil embolization of the eneurysms, one ending to successful, the other to unsuccessful, so, underwent pancreatioduodenectomy. In our both cases, no history of chronic pancreatitis, trauma, infection and connective tissue disorders, and celiac axis stenosis or occulusion, were found. We reviewed 71 case repotrs of PDA aneurysm in Japanese literature and the treatment of PDA aneurysm.

Biology of angiogenesis in tumors of the gastrointestinal tract.

The realization that the growth and spread of tumors are dependent on angiogenesis has created new avenues of research designed to help us to better understand cancer biology and to facilitate the development of new therapeutic strategies. However, the process of angiogenesis consists of multiple, sequential, and interdependent steps with a myriad of positive and negative regulators of angiogenesis being involved. The survival of tumors and thus their metastases are dependent upon the balance of endogenous angiogenic and anti-angiogenic factors such that the outcome favors increased angiogenesis. Several growth factors have been identified that regulate angiogenesis in cancers of the gastrointestinal tract. These include pro-angiogenic factors like vascular endothelial growth factor (VEGF) and anti-angiogenic factors, i.e., thrombospondin. The following review provides a brief overview about the most important factors that are involved in the angiogenic process in tumors derived from colon, stomach, and pancreas. A thorough understanding of the role these factors play in the angiogenic process may lead to the development of novel therapeutic antineoplastic strategies.

Prognostic significance of serum p53 antibody in patients with esophageal squamous cell carcinoma.

BACKGROUND: The p53 protein overexpression that usually results from genetic alterations has been reported to induce serum antibodies against p53. There is little information about the clinicopathologic and prognostic significance of preoperative serum p53 antibody in patients with esophageal cancer. METHODS: A highly specific enzyme-linked immunosorbent assay was used to analyze serum p53 antibodies in 105 patients with esophageal squamous cell carcinoma. The cutoff level of 1.3 U/mL was used to indicate seropositive patients, and the cutoff level of 10 U/mL was used to identify high titer patients. At 3 months after surgery, seropositive patients were examined again. RESULTS: A total of 28 patients (26.7%) were positive for serum p53 antibodies. The patients who remained seropositive were more likely to develop tumor recurrence (P =.025). Seropositive patients had worse outcome than seronegative patients. The high titer group had significant association with advanced tumor stages and worse outcomes than the low titer group. High serum p53 antibody titer was an independent prognostic factor (P <.001). CONCLUSIONS: We found that serum p53 antibody was useful to detect esophageal cancer and to identify those with a high risk of tumor recurrence and a poor prognosis.

Thrombocytosis associated with poor prognosis in patients with esophageal carcinoma.

BACKGROUND: Thrombocytosis in patients with cancer has been reported to be associated with increased expression of angiogenic factors and with a poor prognosis in some types of cancer. The aim of this study is to analyze the clinicopathologic significance and prognostic value of platelet counts in patients with esophageal cancer. STUDY DESIGN: Platelet counts were measured before surgery in 374 patients diagnosed between 1987 and 1999 with primary esophageal squamous cell carcinoma. We used the cut-off level of 293 x 10(9)/L (mean of 80 healthy controls +/- standard deviation) to define thrombocytosis. We analyzed the relationship between platelet counts, TNM factors, and white blood cell counts. Among 374 patients, the levels of C-reactive protein were analyzed in 170 patients and serum thymidine phosphorylase concentration was analyzed in 91 patients. The multivariate prognostic value of platelet counts, tumor size, and TNM factors were determined using Cox's proportional hazards model. RESULTS: Platelet counts were significantly increased in patients with large tumors (p < 0.001), deep tumors, nodal involvement, and distant metastasis in univariate analysis. C-reactive protein level, white blood cell count, and serum thymidine phosphorylase concentration were also significantly increased in patients analyzed with thrombocytosis in univariate analysis. Adjusting for tumor size and TNM factors, multivariate analysis indicated that thrombocytosis as defined in this study was an independent prognostic factor (hazard ratio = 1.52, 95% CI = 1.11 to 2.08, p = 0.009). CONCLUSIONS: A high platelet count is associated with tumor progression and poor survival in patients with esophageal carcinoma.

Prognostic significance of thymidine phosphorylase and p53 co-expression in esophageal squamous cell carcinoma.

This study investigated the prognostic significance of thymidine phosphorylase (TP) and p53 expression in 96 surgically resected primary esophageal squamous cell carcinomas. Expression was assessed using immunohistochemical staining methods. Prognostic values were determined by multivariate analysis using Cox's proportional hazards model. Although TP expression was not correlated with p53 expression, it was associated with genetic alteration of the latter. In univariate analysis, TP expression emerged as a significant prognostic factor but p53 expression did not. However, in multivariate analysis, despite TP not being a significant independent predictor of survival, co-expression of TP together with p53 did represent a statistically significant prognostic factor.