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Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

Background and aims: There is a lack of biliary epithelial molecular markers for primary sclerosing cholangitis (PSC). We analyzed candidates from disease susceptibility genes identified in recent genome-wide association studies. Methods: Expression was quantified using immunohistochemistry in biliary epithelia in liver biopsy samples from patients with PSC (N = 45) and controls (N = 12). Samples from patients with primary biliary cholangitis (PBC) were used as disease controls (N = 20). Results: The levels of hepatic expression of ATXN2, HHEX, PRDX5, MST1, and TNFRSF14 were significantly altered in the PSC group. We focused on the immune-related receptor, TNFRSF14. Immunohistochemistry revealed that TNFRSF14 positivity was significantly higher in biliary epithelia in the PSC group (96 %) than in the control (42 %) and PBC (55 %) groups. High expression of TNFRSF14 was observed only in patients with PSC. Moreover, the expression of LIGHT, which encodes a TNFRSF14-activating ligand, was increased in PSC liver. Immunohistochemistry showed that high expression of LIGHT was more common in PSC biliary epithelia (53 %) than in the PBC (15 %) or control (0 %) groups; moreover, it was positively associated with fibrotic progression. Conclusions: TNFRSF14 and LIGHT are attractive candidate markers for PSC.

Aging of society is accelerating in many countries. The purpose of this study was to describe the clinical features and sulfonylurea usage among diabetes outpatients aged ≥90 years (nonagenarians).This study was a retrospective observational study. The study population consisted of 69 nonagenarian diabetes outpatients and 857 diabetes outpatients aged90 years. Patients were classified into four groups: group 1,65 years; group 2, 65-74 years; group 3, 75-89 years; and group 4, ≥90 years. The presence of hypoglycemic episodes was defined as having self-reported symptoms, or self-monitored or clinically measured blood glucose level70 mg/dL.The median glycated hemoglobin (HbA1c) in group 1 and group 4 was 7.0% and 7.2%, respectively (P = 0.506). The proportion of sulfonylurea treatment in group 4 was 45.5%, which is significantly higher compared with the other three groups (20.0-27.8%, P 0.001). In group 4, there was no difference between patients with or without sulfonylurea in age, sex, body mass index, HbA1c and number of antihyperglycemic agents. Five out of 25 nonagenarian sulfonylurea-treated patients had hypoglycemic episodes within the last 2 years, their HbA1c were all 7.0 ≤ HbA1c 8.0, and sulfonylurea or insulin was tapered in all cases after confirming hypoglycemia. Tapering dosage was attempted in all 25 sulfonylurea-treated nonagenarian patients, but 15 needed to continue sulfonylurea for glycemic control, and 10 continued sulfonylurea with unknown reasons from their medical records.Although tapering the dosage of sulfonylurea was attempted in nonagenarian patients, sulfonylurea was widely continued for glycemic control. Reverse clinical inertia may exist in some sulfonylurea-treated nonagenarian patients.

The purpose of this retrospective observational cohort study was to compare outpatient diabetes care and glycated hemoglobin (HbA1c) level during the coronavirus disease 2019 pandemic in 2020 with 2019, and to compare the glucose-lowering effect of telemedicine and clinic visits during the state of emergency in Japan declared from 7 April to 25 May (inter-period) 2020.A total of 13 weeks before and after the inter-period were designated as the pre-period and post-period, respectively. The number of study participants who had clinic visits during the pre-period and the post-period were 3,333 in 2020 and 3,608 in 2019. Propensity score matching was carried out to compare the effect of telemedicine and clinic visits on diabetes control in 2020 among diabetes patients with insufficient glucose control (HbA1c ≥7%). The primary outcome was post-period HbA1c.The major difference between 2020 and 2019 was the use of telemedicine in 2020. After adjustment for age, sex, diabetes type, pre-period HbA1c and pre-period body mass index, glycemic control evaluated by HbA1c was significantly worse in the post-period of 2020 than 2019. In the propensity score-matched 618 pairs, the clinic visit group had significantly better post-period HbA1c than the telemedicine group (7.5% vs 7.4%, P = 0.023).Glycemic control was slightly, but significantly, worse in 2020 than 2019. Although telemedicine significantly improved glycemic control during the coronavirus disease 2019 pandemic in 2020, clinic visits improved HbA1c significantly more. The substitution of telemedicine for clinic visits appears to be a viable option under emergency conditions, but clinic visits might be a better option when possible.

Accurate diagnosis of the disease extension of cholangiocarcinoma (CCA) is often difficult in clinical practice. The diagnostic yield of conventional pre-operative imaging or endoscopic procedures is sometimes insufficient for the evaluation of longitudinal spreading of CCA. Here we investigated the usefulness of 5-aminolevulinic acid (5-ALA) for the pre- or intra-operative diagnosis of CCA, using patient-derived organoids.Four CCA- and two adjacent tissue-derived organoids were established. After 5-ALA treatment, we assessed their photodynamic activity using fluorescence microscopy.CCA organoids established from different patients showed diverse morphology in contrast to monolayer structures of non-tumor organoids, and had the ability to form subcutaneous tumors in immunodeficient mice. CCA organoids demonstrated remarkably high photodynamic activity based on higher accumulation of protoporphyrin IX as a metabolite of 5-ALA compared to non-tumor organoids (40-71% vs. 4%, respectively). Importantly, cancer cell-specific high photodynamic activity distinguished the organoids originated from biliary stenotic lesions from those of non-stenotic lesions in a CCA patient. The high photodynamic activity did not depend on the expression profile of heme biosynthesis genes.Distinct 5-ALA-based photodynamic activity could have diagnostic potential for the discrimination of CCA from non-tumor tissues.

Although adipocytes are major targets of insulin, the influence of impaired insulin action in adipocytes on metabolic homeostasis remains unclear. We here show that adipocyte-specific PDK1 (3'-phosphoinositide-dependent kinase 1)-deficient (A-PDK1KO) mice manifest impaired metabolic actions of insulin in adipose tissue and reduction of adipose tissue mass. A-PDK1KO mice developed insulin resistance, glucose intolerance, and hepatic steatosis, and this phenotype was suppressed by additional ablation of FoxO1 specifically in adipocytes (A-PDK1/FoxO1KO mice) without an effect on adipose tissue mass. Neither circulating levels of adiponectin and leptin nor inflammatory markers in adipose tissue differed between A-PDK1KO and A-PDK1/FoxO1KO mice. Lipidomics and microarray analyses revealed that leukotriene B4 (LTB4) levels in plasma and in adipose tissue as well as the expression of 5-lipoxygenase (5-LO) in adipose tissue were increased and restored in A-PDK1KO mice and A-PDK1/FoxO1KO mice, respectively. Genetic deletion of the LTB4 receptor BLT1 as well as pharmacological intervention to 5-LO or BLT1 ameliorated insulin resistance in A-PDK1KO mice. Furthermore, insulin was found to inhibit LTB4 production through down-regulation of 5-LO expression via the PDK1-FoxO1 pathway in isolated adipocytes. Our results indicate that insulin signaling in adipocytes negatively regulates the production of LTB4 via the PDK1-FoxO1 pathway and thereby maintains systemic insulin sensitivity.

The purpose of this study was to investigate the association of glycemic control and diabetes treatment to gastric residue observed during an esophagogastroduodenoscopy. Among 6,592 individuals who had esophagogastroduodenoscopy at our clinic between 2003 and 2019, we retrospectively and longitudinally identified those who had gastric residue during an esophagogastroduodenoscopy. Other data collected were age, sex, diagnosis of diabetes, glycated hemoglobin and diabetes medication. Cox proportional hazards models were used to assess the association of these data with the occurrence of gastric residue. To the best of our knowledge, this is the first retrospective cohort study finding that undergoing insulin treatment is a risk factor for gastric residue independent of age, sex and diabetes or glycated hemoglobin.

Immunoprecipitation with autoantibodies to the insulin receptor derived from patients with extreme insulin resistance and acanthosis nigricans revealed that the receptor is comprised of two subunits of 135 kDa (α subunit) and 95 kDa (β subunit) and that insulin induces the rapid phosphorylation of the β subunit in intact cells. Incubation of a highly purified insulin receptor preparation with [γ-32P]ATP also resulted in tyrosine phosphorylation of the β subunit in an insulin-dependent manner, suggesting that the receptor itself is a tyrosine-specific protein kinase. Furthermore, a Japanese boy with insulin resistance and acanthosis nigricans was found to be heterozygous for a mutation of the insulin receptor gene that resulted in the replacement of glycine-996 with valine in the ATP binding site of the receptor. Expression of the mutant receptor in cultured cells revealed it to be deficient in tyrosine kinase activity and mediation of insulin action, suggesting that the tyrosine kinase activity of the insulin receptor is essential for insulin action in vivo.

Sodium-glucose cotransporter 2 inhibitor (SGLT2i) consistently reduces blood glucose levels in type 2 diabetes mellitus but increases hepatic gluconeogenic gene expression and glucose production, offsetting its glucose-lowering effect. This study aimed to elucidate the effect of SGLT2i on hepatic gluconeogenic response and its mechanism in both insulin-sensitive and insulin-resistant states. A hepatic mouse model was generated to show liver-specific expression of Gaussia luciferase (GLuc) driven by the gluconeogenic enzyme gene G6pc promoter. Hepatic gluconeogenic response was evaluated by measuring plasma GLuc activity. SGLT2i was given to lean and obese mice in single gavage administration or 4-week dietary administration with controlled feeding every 3 hours. In lean mice, single-dose SGLT2i increased plasma GLuc activity from 2 hours after administration, decreasing blood glucose and plasma insulin from 1 to 2 hours after administration. In obese mice, which had higher plasma GLuc activity than lean ones, SGLT2i did not further increase GLuc activity despite decreased blood glucose and plasma insulin. Hepatic Akt and GSK3β phosphorylation was attenuated by single-dose SGLT2i in lean mice in accordance with the plasma insulin decrease, but not in obese mice. Long-term SGLT2i administration, which increased plasma GLuc activity in lean mice, decreased it in obese mice from 3 weeks after initiation, with increased hepatic Akt and GSK3β phosphorylation. In conclusion, single SGLT2i administration increases hepatic gluconeogenic response in lean insulin-sensitive mice, but not in obese insulin-resistant mice. Long-term SGLT2i administration relieves obesity-induced upregulation of the hepatic gluconeogenic response by restoring impeded hepatic insulin signaling in obese insulin-resistant mice.

Aims To analyze the duration and extent of the legacy effect on diabetic complications in real-world patients with type 2 diabetes. Methods This was a retrospective cohort study. We included the following three cohorts of patients: diabetic retinopathy (DR) (n = 1107), diabetic kidney disease (DKD) (n = 1486), and cardiovascular disease (CVD) (n = 1485). Patients were enrolled from 1995 to 1999 and followed up to 2017. Endpoints were DR incidence, ≥40% decrease in estimated glomerular filtration rate, and CVD incidence. The relationships between HbA1c as a time-dependent variable and the risk of reaching each endpoint were analyzed using multivariate Cox regression models. Results A total of 313 patients developed DR, 316 developed DKD, and 177 developed CVD. Hazard ratios as a function of time-dependent HbA1c (moving mean) accumulated over time. This accumulation was largest for DR, followed by DKD and CVD. The hazard ratios for each endpoint reached a plateau during the preceding 14–19 years. Conclusions The effect of past glycemic control may continue during 14–19 years, with a greater effect during ≤10 years. Therefore, the end of the legacy effect could be 15–20 years. This effect may be the greatest for DR, followed by DKD, and the smallest for CVD.

To ensure that experiences and lessons learned from the unprecedented 2011 Great East Japan Earthquake are used to improve future disaster planning, the Japan Diabetes Society (JDS) launched the “Research and Survey Committee for Establishing Disaster Diabetes Care Systems Based on Relevant Findings from the Great East Japan Earthquake” under the supervision of the Chairman of the JDS. The Committee conducted a questionnaire survey among patients with diabetes, physicians, disaster medical assistance teams (DMATs), nurses, pharmacists, and nutritionists in disaster areas about the events they saw happening, the situations they found difficult to handle, and the needs that they felt required to be met during the 2011 Great East Japan Earthquake. A total of 3,481 completed questionnaires were received. Based on these and other experiences and lessons reported following the 2011 Great East Japan Earthquake and the 2004 Niigata‐Chuetsu Earthquakes, the current “Manual for Disaster Diabetes Care” has been developed by the members of the Committee and other invited authors from relevant specialties. To our knowledge, the current Manual is the world's first to focus on emergency diabetes care, with this digest English version translated from the Japanese original. It is sincerely hoped that patients with diabetes and healthcare providers around the world will find this manual helpful in promoting disaster preparedness and implementing disaster relief.

Although adipocytes are major targets of insulin, the influence of impaired insulin action in adipocytes on metabolic homeostasis remains unclear. We here show that adipocyte-specific PDK1 (3′-phosphoinositide–dependent kinase 1)-deficient (A-PDK1KO) mice manifest impaired metabolic actions of insulin in adipose tissue and reduction of adipose tissue mass. A-PDK1KO mice developed insulin resistance, glucose intolerance, and hepatic steatosis, and this phenotype was suppressed by additional ablation of FoxO1 specifically in adipocytes (A-PDK1/FoxO1KO mice) without an effect on adipose tissue mass. Neither circulating levels of adiponectin and leptin nor inflammatory markers in adipose tissue differed between A-PDK1KO and A-PDK1/FoxO1KO mice. Lipidomics and microarray analyses revealed that leukotriene B(4) (LTB(4)) levels in plasma and in adipose tissue as well as the expression of 5-lipoxygenase (5-LO) in adipose tissue were increased and restored in A-PDK1KO mice and A-PDK1/FoxO1KO mice, respectively. Genetic deletion of the LTB(4) receptor BLT1 as well as pharmacological intervention to 5-LO or BLT1 ameliorated insulin resistance in A-PDK1KO mice. Furthermore, insulin was found to inhibit LTB(4) production through down-regulation of 5-LO expression via the PDK1−FoxO1 pathway in isolated adipocytes. Our results indicate that insulin signaling in adipocytes negatively regulates the production of LTB(4) via the PDK1−FoxO1 pathway and thereby maintains systemic insulin sensitivity.

Glucagon-mediated gene transcription in the liver is critical for maintaining glucose homeostasis. Promoting the induction of gluconeogenic genes and blocking that of insulin receptor substrate (Irs)2 in hepatocytes contributes to the pathogenesis of type 2 diabetes. However, the molecular mechanism by which glucagon signalling regulates hepatocyte metabolism is not fully understood. We previously showed that a fasting-inducible signalling module consisting of general control non-repressed protein 5, co-regulator cAMP response element-binding protein binding protein/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2, and protein kinase A is required for glucagon-induced transcription of gluconeogenic genes. The present study aimed to identify the downstream effectors of this module in hepatocytes by examining glucagon-induced potential target genes. One of these genes was prolyl hydroxylase domain (PHD)3, which suppressed stress signalling through inhibition of the IκB kinase–nuclear factor-κB pathway in a proline hydroxylase-independent manner to maintain insulin signalling. PHD3 was also required for peroxisome proliferator–activated receptor γ coactivator 1α-induced gluconeogenesis, which was dependent on proline hydroxylase activity, suggesting that PHD3 regulates metabolism in response to glucagon as well as insulin. These findings demonstrate that glucagon-inducible PHD3 regulates glucose metabolism by suppressing stress signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

Introduction To examine the long‐term efficacy and safety of duloxetine in the treatment of Japanese patients with diabetic neuropathic pain, we carried out a 52‐week, randomized, open‐label extension of a 12‐week, double‐blind, placebo‐controlled study. Materials and Methods Japanese adults with diabetic neuropathic pain who completed the double‐blind study were eligible for this long‐term study, carried out at 71 sites in Japan (March 2008 to March 2010). Participants (n = 258) were re‐randomized (1:1) to 40 mg/day or 60 mg/day duloxetine. Pain (Brief Pain Inventory severity and interference), quality of life (Patient's Global Impression of Improvement), and safety (primary outcome; adverse events, vital signs, metabolic measures) were measured. Results Significant (P

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 13 disease genes have been identified. However, the pathogenesis of MODY is not fully understood, because the pancreatic β-cells of the patients are inaccessable. Therefore, we attempted to establish MODY patient-derived induced pluripotent stem cells (MODY-iPS) cells to investigate the pathogenic mechanism of MODY by inducing pancreatic β-cells. We established MODY5-iPS cells from a Japanese patient with MODY5 (R177X), and confirmed that MODY5-iPS cells possessed the characteristics of pluripotent stem cells. In the course of differentiation from MODY5-iPS cells into pancreatic β-cells, we examined the disease gene, HNF1B messenger ribonucleic acid. We found that the amount of R177X mutant transcripts was much less than that of wild ones, but they increased after adding cycloheximide to the medium. These results suggest that these R177X mutant messenger ribonucleic acids are disrupted by nonsense-mediated messenger ribonucleic acid decay in MODY-iPS cells during the developmental stages of pancreatic β-cells.

The liver has robust regenerative potential in response to damage, but hepatic steatosis (HS) weakens this potential. We found that the enhanced integrated stress response (ISR) mediated by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) impairs regeneration in HS and that growth arrest and DNA damage-inducible 34 (Gadd34)-dependent suppression of ISR plays a crucial role in fatty liver regeneration. Although mice fed a high-fat diet for 2 weeks developed moderate fatty liver with no increase in eIF2α phosphorylation before 70% hepatectomy, they showed impaired liver regeneration as a result of reduced proliferation and increased death of hepatocytes with increased phosphorylation of eIF2α and ISR. An increased ISR through Gadd34 knockdown induced C/EBP homologous protein (CHOP)-dependent apoptosis and receptor-interacting protein kinase 3-dependent necrosis, resulting in increased hepatocyte death during fatty liver regeneration. Furthermore, Gadd34 knockdown and increased phosphorylation of eIF2α decreased cyclin D1 protein and reduced hepatocyte proliferation. In contrast, enhancement of Gadd34 suppressed phosphorylation of eIF2α and reduced CHOP expression and hepatocyte apoptosis without affecting hepatocyte proliferation, clearly improving fatty liver regeneration. In more severe fatty liver of leptin receptor-deficient db/db mice, forced expression of hepatic Gadd34 also promoted hepatic regeneration after hepatectomy. Conclusion: Gadd34-mediated regulation of ISR acts as a physiological defense mechanism against impaired liver regeneration resulting from steatosis and is thus a possible therapeutic target for impaired regeneration in HS. (Hepatology 2015;61:1343–1356)

Children born with low birth weight have a high risk of developing type 2 diabetes mellitus later in life. In this study, we developed a mouse model for low birth weight induced by maternal caloric restriction and investigated its effects on pancreatic β-cells. At birth, the pancreatic β-cell mass in the restricted diet group (RG) offspring was significantly lower than in the control group (CG) offspring. At 8 weeks of age, the pancreatic β-cell mass was greater in the RG offspring than in the CG offspring. RG offspring showed upregulated expression of insulin receptor substrate 2 in islets at 10 weeks of age. Moreover, the activity of insulin signaling molecules in the pancreatic β-cell mass was increased during the period of catch-up growth during the early stage of life. To investigate the effect of insulin signaling on the regulation of pancreatic β-cell mass, we generated β-cell-specific 3-phosphoinositide-dependent protein kinase 1 (PDK1) heterozygous knockout (βPDK1+/−) mice. We detected delayed catch-up growth in the β-cell mass of βPDK1+/− mice that were undernourished as fetuses. Moreover, the insulin signaling pathway was impaired in the islets of βPDK1+/− mice exposed to fetal undernutrition. These findings indicate that fetal undernutrition affects the regulation of pancreatic β-cell mass through altered insulin signaling.

In recent years, diabetes has been shown to be associated with cancer risk, and this has led to a joint committee being formed, enlisting experts from the Japan Diabetes Society (JDS) and the Japanese Cancer Association (JCA) to address this issue. Epidemiological data in Japan provides evidence to demonstrate that diabetes is associated with increased risk for cancers, especially colorectal, liver, and pancreatic cancers. The mechanisms through which diabetes is assumed to promote oncogenesis include insulin resistance and associated hyperinsulinemia, hyperglycemia and inflammation. Common risk factors for type 2 diabetes and cancer include aging, male sex, obesity, physical inactivity, inappropriate diet (excessive red/processed meat intake, inadequate vegetable/fruit/dietary fiber intake), excessive alcohol drinking, and smoking. Given that inappropriate diet/exercise, smoking and excessive alcohol drinking are common risk factors for diabetes and cancer, diet/exercise therapy, smoking cessation and alcohol moderation may be associated with decreased risk for cancer in diabetic patients. There is as yet limited evidence as to whether any particular anti-diabetic agents may influence cancer risk.

Chemerin, a recently discovered adipocytokine plays an important role in obesity and obesity-associated metabolic complications. However, the role of chemerin in the pathogenesis of type 2 diabetes mellitus (T2DM) has not fully been elucidated. We compared the serum chemerin levels and metabolic parameters between 88 control subjects, 86 patients with metabolic syndrome (MS), and 147 patients with T2DM in a Japanese population and further analyzed their correlation. Enzyme-linked immunosorbent assay was used to measure the serum chemerin levels. The chemerin levels were significantly higher in male than in female control subjects (p < 0.005), with significant decreases in patients with T2DM compared with those with MS and control subjects (164.9 ± 6.3 ng/mL vs. 209.8 ± 7.7 and 218.7 ± 7.3 ng/mL; p < 0.0001 vs. p < 0.0001, respectively) but no significant differences in female subjects. The multiple regression analysis revealed that the chemerin levels negatively correlated with the fasting glucose and HbA1c levels in total and male subjects. In the patients with T2DM, the chemerin levels negatively correlated with fasting glucose and high-density lipoprotein cholesterol but positively correlated with body mass index (BMI), and total cholesterol and triglyceride levels. The negative correlation between the chemerin and fasting glucose levels remained significant after adjustment for age, sex, and BMI in the total and male subjects and those with T2DM. These results suggest the role of chemerin in sex dimorphism and a potential link between chemerin levels and T2DM pathogenesis in a Japanese population.

During fasting, induction of hepatic gluconeogenesis is crucial to ensure proper energy homeostasis. Such induction is dysregulated in type 2 diabetes, resulting in the development of fasting hyperglycemia. Hormonal and nutrient regulation of metabolic adaptation during fasting is mediated predominantly by the transcriptional coactivator peroxisome proliferative activated receptor γ coactivator 1α (PGC-1α) in concert with various other transcriptional regulators. Although CITED2 (CBP- and p300-interacting transactivator with glutamic acid- and aspartic acid-rich COOH-terminal domain 2) interacts with many of these molecules, the role of this protein in the regulation of hepatic gluconeogenesis was previously unknown. Here we show that CITED2 is required for the regulation of hepatic gluconeogenesis through PGC-1α. The abundance of CITED2 was increased in the livers of mice by fasting and in cultured hepatocytes by glucagon-cAMP-protein kinase A (PKA) signaling, and the amount of CITED2 in liver was higher in mice with type 2 diabetes than in non-diabetic mice. CITED2 inhibited the acetylation of PGC-1α by blocking its interaction with the acetyltransferase general control of amino acid synthesis 5-like 2 (GCN5). The consequent downregulation of PGC-1α acetylation resulted in an increase in its transcriptional coactivation activity and an increased expression of gluconeogenic genes. The interaction of CITED2 with GCN5 was disrupted by insulin in a manner that was dependent on phosphoinositide 3-kinase (PI3K)-thymoma viral proto-oncogene (Akt) signaling. Our results show that CITED2 functions as a transducer of glucagon and insulin signaling in the regulation of PGC-1α activity that is associated with the transcriptional control of gluconeogenesis and that this function is mediated through the modulation of GCN5-dependent PGC-1α acetylation. We also found that loss of hepatic CITED2 function suppresses gluconeogenesis in diabetic mice, suggesting it as a therapeutic target for hyperglycemia.

The phosphatidylinositol 3-kinase signaling pathway in vascular endothelial cells is important for systemic angiogenesis and glucose metabolism. In this study, we addressed the precise role of the 3-phosphoinositide-dependent protein kinase 1 (PDK1)-regulated signaling network in endothelial cells in vivo, using vascular endothelial PDK1 knockout (VEPDK1KO) mice. Surprisingly, VEPDK1KO mice manifested enhanced glucose tolerance and whole-body insulin sensitivity due to suppression of their hepatic glucose production with no change in either peripheral glucose disposal or even impaired vascular endothelial function at 6 months of age. When mice were fed a standard diet at 6 months of age and a high-fat diet at 3 months of age, hypertrophy of epididymal adipose tissues was inhibited, adiponectin mRNA was significantly increased, and mRNA of MCP1, leptin, and TNFα was decreased in the white adipose tissue of VEPDK1KO mice in comparison with controls. Consequently, both the circulating adiponectin levels and the activity of hepatic AMP-activated protein kinase were significantly increased, subsequently enhancing whole-body insulin sensitivity and energy expenditure with increased hepatic fatty acid oxidation in VEPDK1KO mice. These results provide the first in vivo evidence that lowered angiogenesis through the deletion of PDK1 signaling not only interferes with the growth of adipose tissue but also induces increased energy expenditure due to amelioration of the adipocytokine profile. This demonstrates an unexpected role of PDK1 signaling in endothelial cells on the maintenance of proper glucose homeostasis through the regulation of adipocyte development.

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic β cells mediated through modulation of oxidative stress.

Diabetes mellitus is a group of diseases associated with various metabolic disorders, the main feature of which is chronic hyperglycemia due to insufficient insulin action. Its pathogenesis involves both genetic and environmental factors. The long-term persistence of metabolic disorders can cause susceptibility to specific complications and also foster arteriosclerosis. Diabetes mellitus is associated with a broad range of clinical presentations, from being asymptomatic to ketoacidosis or coma, depending on the degree of metabolic disorder.

Aims/Introduction: Duloxetine has been suggested to exert analgesic effects by activating the descending inhibitory system through inhibition of serotonin (5-HT) and noradrenaline (NA) reuptake. This randomized controlled trial investigated the efficacy and safety of duloxetine in Japanese patients with diabetic neuropathic pain (DNP). Materials and Methods: Duloxetine 40 or 60 mg/day or placebo was given orally once daily for 12 weeks. The primary efficacy measure was weekly mean 24-h average pain severity score on the 11-point Numerical Rating Scale. Results: At 12 weeks vs baseline, the 24-h average pain score (adjusted mean ± SE) was significantly improved in the combined duloxetine (−2.47 ± 0.18) and duloxetine 40 mg (−2.41 ± 0.21) and 60 mg groups (−2.53 ± 0.21) as compared with the placebo group (−1.61 ± 0.18). Duloxetine also exerted significant improvements over the placebo in nearly all secondary outcome measures including 24-h worst pain, night pain, Brief Pain Inventory (BPI) pain scores, Patient’s Global Impression of Improvement (PGI-I) and health outcome measures, namely, various BPI interference scores. The incidence of adverse events (AE) was higher in the duloxetine groups than in the placebo group (duloxetine overall, 84.8%; duloxetine 40 mg, 84.7%; duloxetine 60 mg, 84.9%; placebo, 73.7%). Most AE were mild or moderate in severity, and resolved or relieved. There were no clinically significant safety concerns. Conclusions: Duloxetine 40 or 60 mg/day showed superiority over the placebo at reducing pain scores in patients with DNP. Duloxetine is safe, efficacious and clinically useful in the management of DNP. This trial was registered with ClinicalTrials.gov (no. NCT-00552175). (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00073.x, 2010)

Concept of Diabetes Mellitus: Diabetes mellitus is a group of diseases associated with various metabolic disorders, the main feature of which is chronic hyperglycemia due to insufficient insulin action. Its pathogenesis involves both genetic and environmental factors. The long‐term persistence of metabolic disorders can cause susceptibility to specific complications and also foster arteriosclerosis. Diabetes mellitus is associated with a broad range of clinical presentations, from being asymptomatic to ketoacidosis or coma, depending on the degree of metabolic disorder.

We conducted a genome-wide association study of type 2 diabetes (T2D) using 459,359 SNPs in a Japanese population with a three-stage study design (stage 1, 4,470 cases and 3,071 controls; stage 2, 2,886 cases and 3,087 controls; stage 3, 3,622 cases and 2,356 controls). We identified new associations in UBE2E2 on chromosome 3 and in C2CD4A-C2CD4B on chromosome 15 at genome-wide significant levels (rs7612463 in UBE2E2, combined P = 2.27 × 10⁻⁹; rs7172432 in C2CD4A-C2CD4B, combined P = 3.66 × 10⁻⁹). The association of these two loci with T2D was replicated in other east Asian populations. In the European populations, the C2CD4A-C2CD4B locus was significantly associated with T2D, and a combined analysis of all populations gave P = 8.78 × 10⁻¹⁴, whereas the UBE2E2 locus did not show association to T2D. In conclusion, we identified two new loci at UBE2E2 and C2CD4A-C2CD4B associated with susceptibility to T2D.

Both insulin and leptin signaling converge on phosphatidylinositol 3-OH kinase [PI( 3 )K]/3-phosphoinositide-dependent protein kinase-1 (PDK-1)/protein kinase B (PKB, also known as Akt) in proopiomelanocortin (POMC) neurons. Forkhead box-containing protein-O1 (FoxO1) is inactivated in a PI( 3 )K-dependent manner. However, the interrelationship between PI( 3 )K/PDK-1/Akt and FoxO1, and the chronic effects of the overexpression of FoxO1 in POMC neurons on energy homeostasis has not been elucidated. To determine the extent to which PDK-1 and FoxO1 signaling in POMC neurons was responsible for energy homeostasis, we generated POMC neuron-specific Pdk1 knockout mice ( POMCPdk1−/−) and mice selectively expressing a constitutively nuclear (CN)FoxO1 or transactivation-defective (Δ256)FoxO1 in POMC neurons ( CNFoxO1POMCor Δ256FoxO1POMC). POMCPdk1−/−mice showed increased food intake and body weight accompanied by decreased expression of Pomc gene. The CNFoxO1POMCmice exhibited mild obesity and hyperphagia compared with POMCPdk1−/−mice. Although expression of the CNFoxO1 made POMCPdk1−/−mice more obese due to excessive suppression of Pomc gene, overexpression of Δ256FoxO1 in POMC neurons had no effects on metabolic phenotypes and Pomc expression levels of POMCPdk1−/−mice. These data suggest a requirement for PDK-1 and FoxO1 in transcriptional regulation of Pomc and food intake.

We collated and analysed data from hospital records regarding the cause of death of 18,385 patients with diabetes who died in 282 medical institutions throughout Japan over the 10-year period between 1991 and 2000. Autopsy was carried out in 1750 cases. The most frequent cause of death in all 18,385 cases was malignant neoplasia, accounting for 34.1% of cases, followed by vascular diseases (including diabetic nephropathy, ischemic heart diseases and cerebrovascular diseases) in 26.8%, infections in 14.3%, and then diabetic coma in 1.2%. The most common malignancy was liver cancer, accounting for 8.6% of all the deaths. Of the deaths from vascular diseases, diabetic nephropathy was the cause of death in 6.8% of cases, and the frequency as cause of death for ischemic heart diseases and cerebrovascular diseases were similar at 10.2% and 9.8%, respectively. Myocardial infarction accounted for almost all the deaths from ischemic heart diseases, whereas deaths from cerebral infarction were 2.2-fold as common as those from cerebral hemorrhage. In the analyses of the relationship between age and causes of death in diabetic patients who underwent autopsy, the overall mortality rate as a result of vascular diseases increased with age, although the mortality rates from diabetic nephropathy and cerebrovascular diseases increased little from the fifth decade of life. The mortality rate from ischemic heart diseases increased with age, however, and was higher than the other forms of vascular diseases from the sixth decade of life, accounting for approximately 50% of vascular deaths in the eighth decade. Malignant neoplasia was the most frequent cause of death from the fifth decade of life, and was extremely common in the seventh decade, accounting for 46.3% of all the deaths. The mortality rate from infections varied little between age groups from the fifth decade of life. In the analyses of glycemic control and the age at the time of death, lifespans were 2.5 years shorter in males, and 1.6 years shorter in female diabetics with poor glycemic control than in those with good or fair glycemic control. This difference was greater for deaths as a result of infections and vascular diseases, particularly diabetic nephropathy, than for malignant neoplasia. Analysis of the relationship between glycemic control and the duration of diabetes and deaths as a result of vascular diseases showed no correlation between the level of glycemic control and death from diabetic nephropathy, ischemic heart diseases or cerebrovascular diseases. In diabetics with disease durations of less than 10 years, the mortality rate from macroangiopathy was higher than that as a result of diabetic nephropathy, a form of microangiopathy. Treatment for diabetes comprised of diet alone in 21.5%, oral hypoglycemic agents in 29.5%, and insulin with or without oral hypoglycemic agents in 44.2%, which was the most common. In particular, 683/1170 (58.4%) diabetics who died from diabetic nephropathy were on insulin therapy, a higher proportion than the 661/1687 (39.2%) who died from ischemic heart diseases, or the 659/1622 (40.6%) who died from cerebrovascular diseases. The average age at the time of death in the survey population was, 68 years for males and 71.6 years for females. These were 9.6 and 13 years, respectively, short of the average life expectancy for the Japanese general population. In comparison with the previous survey (1981–1990), the average age at the time of death had increased 1.5 years for males, and 3.2 years for females. The average life expectancy for the Japanese general population had also increased 1.7 and 2.7 years, respectively, over that period, showing that advances in the management and treatment of diabetes have not led to any improvement in patients’ life expectancies. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00019.x, 2010)

Pancreatic beta cell failure is thought to underlie the progression from glucose intolerance to overt diabetes, and ER stress is implicated in such beta cell dysfunction. We have now shown that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) accumulated in the islets of diabetic animal models as a result of ER stress before the onset of hyperglycemia. Transgenic overexpression of C/EBPbeta specifically in beta cells of mice reduced beta cell mass and lowered plasma insulin levels, resulting in the development of diabetes. Conversely, genetic ablation of C/EBPbeta in the beta cells of mouse models of diabetes, including Akita mice, which harbor a heterozygous mutation in Ins2 (Ins2WT/C96Y), and leptin receptor-deficient (Lepr-/-) mice, resulted in an increase in beta cell mass and ameliorated hyperglycemia. The accumulation of C/EBPbeta in pancreatic beta cells reduced the abundance of the molecular chaperone glucose-regulated protein of 78 kDa (GRP78) as a result of suppression of the transactivation activity of the transcription factor ATF6alpha, thereby increasing the vulnerability of these cells to excess ER stress. Our results thus indicate that the accumulation of C/EBPbeta in pancreatic beta cells contributes to beta cell failure in mice by enhancing susceptibility to ER stress.

In order to investigate the risk-reducing effects of coffee in metabolic syndrome, we performed a study in mice fed a high-fat diet with added coffee and analyzed gene expression in liver and adipose tissues using cDNA microarray. Male C57BL/6J mice were raised for 8 weeks on either a normal diet (N group), a high-fat diet (HF group), or a high-fat diet with 1.1% decaffeinated (HF+DC group) or 1.1% caffeine-containing instant coffee (HF+CC group). The body weights of mice in the HF+DC and HF+CC groups were mostly intermediate between the N and HF groups, even if there were no difference in the amount of diet consumption in each group. Mesenteric fat weight was lower in the HF+DC group than in the HF group (p < 0.05) and tended to become lower in the HF+CC group than in the HF group. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly lower in the HF+DC and HF+CC groups than in the HF group (p < 0.05). Inflammatory cytokine interleukin (IL)-1beta gene expression in liver was up-regulated in the HF group and significantly down-regulated in the HF+DC and HF+CC groups (p < 0.01), while MCP-1 gene expression in white adipose tissue was also significantly suppressed in the HF+DC group (p < 0.01). The induction of these anti-inflammatory responses by coffee consumption may contribute to reducing the risks of metabolic syndrome.

Prediction of the disease status is one of the most important objectives of genetic studies. To select the genes with strong evidence of the association with type 2 diabetes mellitus, we validated the associations of the seven candidate loci extracted in our earlier study by genotyping the samples in two independent sample panels. However, except for KCNQ1, the association of none of the remaining seven loci was replicated. We then selected 11 genes, KCNQ1, TCF7L2, CDKAL1, CDKN2A/B, IGF2BP2, SLC30A8, HHEX, GCKR, HNF1B, KCNJ11 and PPARG, whose associations with diabetes have already been reported and replicated either in the literature or in this study in the Japanese population. As no evidence of the gene-gene interaction for any pair of the 11 loci was shown, we constructed a prediction model for the disease using the logistic regression analysis by incorporating the number of the risk alleles for the 11 genes, as well as age, sex and body mass index as independent variables. Cumulative risk assessment showed that the addition of one risk allele resulted in an average increase in the odds for the disease of 1.29 (95% CI=1.25-1.33, P=5.4 x 10(-53)). The area under the receiver operating characteristic curve, an estimate of the power of the prediction model, was 0.72, thereby indicating that our prediction model for type 2 diabetes may not be so useful but has some value. Incorporation of data from additional risk loci is most likely to increase the predictive power.

Objectives: FOLFIRI is one of the current standard first-line regimens for advanced colorectal cancer, but its administration is onerous. Because the replacement of 2-day-infusional 5-fluorouracil (5-FU) of FOLFIRI with oral tegafur-uracil/leucovorin (UFT/LV) would be highly beneficial for clinical management, we performed a phase I trial using oral UFT/LV and a pharmacokinetic evaluation. Methods: Treatment consisted of infusional irinotecan (100 mg/m 2 )/l-LV (15 mg/m 2 ) and a bolus injection of 5-FU (500 mg/m 2 ) on day 1, and oral UFT (300 mg as tegafur/m 2 /d)/LV (75 mg/d) on days 1-5 (level 1), days 1-7 (level 2), or days 1-10 (level 3). Cycles were repeated every 14 days. After determination of the recommended UFT/LV administration period, irinotecan was dose-escalated (level 4: 125 mg/m 2 ; level 5: 150 mg/m 2 ). Results: Nineteen patients were enrolled. One dose-limiting toxicity (DLT), grade 4 neutropenia lasting for ≥4 days was observed at level 2. At level 3, one DLT of treatment delay of ≥8 days occurred due to prolonged neutropenia, and 2 patients refused to continue the treatment because of prolonged grade 2 anorexia. Therefore, a 7-day administration of UFT/LV was recommended. No DLT was observed at levels 4 and 5. Pharmacokinetic evaluation suggested continuous exposure to 5-FU by means of oral UFT/LV administration in this combination. Conclusions: The recommended administration period was 7 days for oral UFT/LV, and the recommended dose of irinotecan was 150 mg/m 2 . A phase II study is ongoing to validate the clinical outcome.

In addition to ligation of the T cell antigen receptor (TCR), activation of the CD28 coreceptor by the costimulatory molecule B7 is required for induction of the transcription factor NF-kappaB and robust T cell activation, although the contribution of CD28 to this process remains incompletely understood. We show here that phosphoinositide-dependent kinase 1 (PDK1) is essential for integrating the TCR and CD28 signals. After we deleted PDK1 from T cells, TCR-CD28 signals were unable to induce activation of NF-kappaB or phosphorylation of protein kinase C-theta, although T cell survival and pathways dependent on the kinases p38 and Jnk or the transcription factor NFAT were unaffected. CD28 facilitated NF-kappaB activation by regulating recruitment and phosphorylation of PDK1, which are necessary for efficient binding of PDK1 to protein kinase C-theta and the adaptor CARMA1 and thus for NF-kappaB induction.

Empty sella syndrome is frequently accompanied with pituitary dysfunction. Most of the patients with empty sella syndrome demonstrate primary pituitary or stalk dysfunction and few cases show hypothalamic dysfunction. A 71-year-old man manifested appetite loss, nausea and vomiting with hyponatremia and adrenal insufficiency. Hormonal evaluation and cranial MRI revealed a panhypopituitarism with empty sella. Intriguingly, while the response of ACTH to CRH administration was exaggerated, the response to insulin hypoglycemia was blunted. Serum PRL levels were normal. Further, decreased level of fT4, slightly elevated basal levels of TSH, and delayed response of TSH to TRH administration were observed. These findings strongly suggest that the panhypopituitarism is caused by hypothalamic dysfunction. The presence of autoantibodies to pituitary and cerebrum in the patient's serum implies an autoimmune mechanism as a pathogenesis.

In patients with various catabolic conditions, glucocorticoid excess induces skeletal muscle wasting by accelerating protein degradation via the ubiquitin-proteasome pathway. Although the transcriptional coactivator p300 has been implicated in this pathological process, regulatory mechanisms and molecular targets of its action remain unclear. Here we show that CREB-binding protein (CBP)/p300-interacting transactivator with ED-rich tail 2 (Cited2), which binds to the cysteine-histidine-rich region 1 of p300 and CBP, regulates muscle mass in vitro. Adenovirus-mediated overexpression of wild-type Cited2 significantly blocked morphological alterations of C2C12 myotubes with a concomitant decrease in myosin heavy chain protein in response to synthetic glucocorticoid dexamethasone, which were attributable to the reduced induction of atrophy-related ubiquitin ligases MuRF1 and MAFbx. These myotube-sparing effects were less pronounced, however, with a carboxyl-terminally truncated mutant of Cited2 that lacked the ability to bind p300. These results suggest that the gain of Cited2 function counteracts glucocorticoid-induced muscle atrophy through inhibition of proteolysis mediated by p300-dependent gene transcription.

KLF15 (Kruppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one of these genes, that for adrenomedullin, with the use of a luciferase reporter assay in 3T3-L1 preadipocytes and adipocytes. Such analysis revealed that the most proximal CACCC element in the promoter of the human adrenomedullin gene (located in the region spanning nucleotides −70 and −29) is required for trans-inhibition by KLF15. Furthermore, chromatin immunoprecipitation showed that KLF15 binds to this region of the human adrenomedullin gene promoter in cultured human adipocytes. These results thus implicate KLF15 in the regulation of adrenomedullin expression in adipose tissue.

Bisphosphonate is an effective drug to reduce fracture risk in osteoporotic patients; however, factors affecting the efficacy of bisphosphonate treatment are not fully known, especially in Japanese patients. In the present study, we examined the relationships between an increase in lumbar spine bone mineral density (BMD) by bisphosphonates and several pretreatment parameters, including biochemical, bone/mineral, and body composition indices, in 85 postmenopausal osteoporotic patients treated with alendronate or risedronate. BMD increase was measured by dual-energy X-ray absorptiometry at the lumbar spine before and 2 years after treatment. BMD increase at the lumbar spine was observed as independent of age, height, weight, body mass index, and fat mass, although lean body mass seemed slightly related. On the other hand, fasting plasma glucose (FPG) levels were significantly and positively related to BMD increase at the lumbar spine. In multiple regression analysis, FPG levels were not significantly related to BMD increase at the lumbar spine when lean body mass was considered. As for bone/mineral parameters, BMD increase at the lumbar spine was not significantly related to serum levels of calcium, parathyroid hormone (PTH), and alkaline phosphatase or urinary levels of deoxypiridinoline and calcium excretion. As for BMD parameters, Z-scores of BMD at any site and bone geometry parameters obtained by forearm peripheral quantitative computed tomography were not significantly related to BMD increase at the lumbar spine. BMD increases at the lumbar spine were similar between groups with or without vertebral fractures. In conclusion, BMD increase at the lumbar spine by bisphosphonate treatment was not related to any pretreatment parameters, including body size, body composition, and bone/mineral metabolism in postmenopausal Japanese women with primary osteoporosis, although FPG correlated partly to BMD through lean body mass.

We carried out a multistage genome-wide association study of type 2 diabetes mellitus in Japanese individuals, with a total of 1,612 cases and 1,424 controls and 100,000 SNPs. The most significant association was obtained with SNPs in KCNQ1, and dense mapping within the gene revealed that rs2237892 in intron 15 showed the lowest P value (6.7 x 10(-13), odds ratio (OR) = 1.49). The association of KCNQ1 with type 2 diabetes was replicated in populations of Korean, Chinese and European ancestry as well as in two independent Japanese populations, and meta-analysis with a total of 19,930 individuals (9,569 cases and 10,361 controls) yielded a P value of 1.7 x 10(-42) (OR = 1.40; 95% CI = 1.34-1.47) for rs2237892. Among control subjects, the risk allele of this polymorphism was associated with impairment of insulin secretion according to the homeostasis model assessment of beta-cell function or the corrected insulin response. Our data thus implicate KCNQ1 as a diabetes susceptibility gene in groups of different ancestries.

Background: In Europeans and populations of European origin, several groups have recently identified novel type 2 diabetes susceptibility genes, including FTO, SLC30A8, HHEX, CDKAL1, CDKN2B, and IGF2BP2, none of which were in the list of functional candidates. Objective and Design: The aim of this study was to replicate in a Japanese population previously identified associations of single nucleotide polymorphisms (SNPs) within 10 candidate loci with type 2 diabetes using a relatively large sample size: 1921 subjects with type 2 diabetes and 1622 normal controls. Results: A total of 15 SNPs were genotyped. Eight SNPs in five loci were found to be associated with type 2 diabetes: rs3802177 [odds ratio (OR) = 1.16 (95% confidence interval (CI) 1.05–1.27); P = 4.5 × 10−3] in SLC30A8; rs1111875 [OR = 1.27 (95% CI 1.14–1.40); P = 1.4 × 10−5] and rs7923837 [OR = 1.27 (95% CI 1.13–1.43); P = 1.0 × 10−4] in HHEX; rs10811661 [OR = 1.27 (95% CI 1.15–1.40); P = 1.9 × 10−6] in CDKN2B; rs4402960 [OR = 1.23 (95% CI 1.11–1.36); P = 8.1 × 10−5] and rs1470579 [OR = 1.18 (95% CI 1.07–1.31); P = 8.3 × 10−4] in IGF2BP2; and rs7754840 [OR = 1.28 (95% CI 1.17–1.41); P = 4.5 × 10−7] and rs7756992 [OR = 1.27 (95% CI 1.15–1.40); P = 9.8 × 10−7] in CDKAL1. The first and second strongest associations were found at variants in CDKAL1 and CDKN2B, both of which are involved in the regenerative capacity of pancreatic β-cells. Conclusion: Some of these variants represent common type 2 diabetes-susceptibility genes in both Japanese and Europeans.

Background and Aim: Metallothionein (MT) has a proven relationship with various kinds of cancer and reduces tissue damage. Helicobacter pylori (H. pylori) infection is associated with the alteration of gastric epithelial cell cycle events, a condition implicated in the initiation and development of gastric cancer. This study investigates the role of MT in H. pylori-induced gastritis with or without early gastric cancer (ECG) and evaluates the effect on MT expression after eradication therapy. Methods: Gastric biopsy samples were immunohistochemically examined for MT expression in 36 H. pylori-negative patients without ECG and 98 positive patients with or without ECG. Real time polymerase chain reaction was performed in 14 antral biopsy samples with or without H. pylori. The severity of gastritis was also evaluated according to the updated Sydney System. In 31 successfully eradicated patients, the above assessment was repeated for two consecutive years. Results: MT expression was higher in H. pylori-negative patients than in positive patients (P

According to the expression of gastric (claudin-18) and intestinal claudins (claudin-3 and claudin-4), the authors have previously proposed a new phenotypic classification of gastric carcinoma (GC): the gastric (G-CLDN), intestinal (I-CLDN) and unclassified claudin (U-CLDN) phenotypes. The aim of the present study was to examine the role of Cdx2, the caudal-related transcription factor, on the regulation of intestinal claudins expression in vitro and in vivo. It was confirmed on immunohistochemistry that non-neoplastic gastric mucosa with intestinal metaplasia (IM) expressed Cdx2 with increased levels of intestinal claudin expression. In addition, Cdx2 expression was detected in 28 (30%) of 94 GC at the invasive front. Interestingly, Cdx2 expression had a significant association with the I-CLDN phenotype (P < 0.001), which was almost identical to the established gastric and intestinal mucin-based GC classification. Furthermore, the transfection of a recombinant human CDX2-expressing vector into TMK-1 (Cdx2-negative) GC cells specifically elevated the expression of claudin-3 and claudin-4 at the mRNA (CLDN3, 3.9-fold; CLDN4, 2.8-fold) and protein levels (claudin-3, 8.6-fold; claudin-4, 9.8-fold), whereas no induction of the other claudins was detected. These findings suggest that Cdx2 plays an important role in the regulation of intestinal claudin expression not only in gastric mucosa with IM but also GC.

Aims. A missense mutation in the low density lipoprotein receptor-related protein 6 gene (LRP6) was recently shown to be responsible for a disorder characterized by early-onset coronary artery disease as well as diabetes mellitus (DM), hyperlipidemia, hypertension, and osteoporosis. Mice deficient in LRP5, a closely related paralog of LRP6, manifest a marked impairment in glucose tolerance. The aim of the present study was to examine whether common variants of LRP5 and LRP6 are associated with Type 2 DM or dyslipidemia in Japanese individuals. Methods. Thirteen single nucleotide polymorphisms (SNPs) of LRP6 and nine SNPs of LRP5 were genotyped in a total of 608 Type 2 DM patients and 366 nondiabetic control subjects (initial study). An association analysis was then performed for each SNP and for haplotypes. For some of the SNPs, we provided another sample panel of 576 cases and 576 controls for the replication study. The relation to clinical characteristics was also examined in diabetic subjects. Results. In the initial study, three SNPs of LRP6 were found to be associated with susceptibility to Type 2 DM. However, this association was not detected in the replication panel. None of SNPs in LRP5 were associated with Type 2 DM in the initial panel. Neither LRP6 nor LRP5 was associated with body mass index, HOMA-β, HOMA-IR or serum lipid concentrations. Conclusions. We found no evidence for a substantial effect of LRP5 or LRP6 SNPs on susceptibility to type 2 diabetes or clinical characteristics of diabetic subjects in Japanese population.

Background: Previously, MDR1 T-129C polymorphism, encoding multidrug resistant transporter MDR1/P-glycoprotein, was reported to be predictive of poorly-differentiated colorectal adenocarcinomas. Here, VEGF T-1498C, C-634G and C-7T polymorphisms, encoding vascular endothelial growth factor (VEGF), were investigated in terms of their association with differentiation grade. Methods: VEGF genotypes were determined by TaqManR MGB probe based polymerase chain reaction and evaluated were confirmed by direct sequencing in 36 Japanese patients. Results: VEGF T-1498C, but not C-634G or C-7T, was predictive of poorly-differentiated ones, and thereby a poor prognosis (p = 0.064 for genotype, p = 0.037 for allele), and this effect can be explained by that on VEGF expression. Treatment of a colorectal adenocarcinoma cell line, HCT-15, with sodium butyrate, a typical differentiating agent, resulted in an increase of alkaline phosphatase activity and MDR1 mRNA expression, but in a decrease of VEGF mRNA expression. The transfection of VEGF small interfering RNA (siRNA) induced the expression of MDR1 mRNA to 288-332% of the control level, whereas MDR1 siRNA had no effect on VEGF mRNA expression. Conclusions: VEGF T-1498C polymorphism is also a candidate marker predictive of poorly-differentiated colorectal adenocarcinomas, but further investigations with a large number of patients should be addressed to draw a conclusion.

Transcription factor 7-like 2 (TCF7L2) has been shown to be associated with type 2 diabetes mellitus in multiple ethnic groups. Regarding the Asian population, Horikoshi et al. (Diabetologia 50:747-751, 2007) and Hayashi et al. (Diabetologia 50:980-984, 2007) reported that single nucleotide polymorphisms (SNPs) in TCF7L2 were associated with type 2 diabetes in the Japanese population, while contradictory results were reported for Han Chinese populations. The aim of this study was to investigate the associations of the TCF7L2 gene with type 2 diabetes using a relatively large sample size: 2,214 Japanese individuals with type 2 diabetes and 1,873 normal controls. The minor alleles of rs7903146, rs11196205, and rs12255372 showed significant associations with type 2 diabetes (OR=1.48, P=2.7 x 10(-4); OR=1.39, P=4.6 x 10(-4); OR=1.70, P=9.8 x 10(-5), respectively) in the combined sample sets. However, neither rs11196218 nor rs290487 showed a significant association. These results indicate that TCF7L2 is an important susceptibility gene for type 2 diabetes in the Japanese population.

This study was performed to find the genetic factors predictive of clinical outcome to a 5-fluorouracil (5-FU)/cisplatin (CDDP)-based chemoradiotherapy (CRT) in Japanese patients with locally advanced esophageal squamous cell carcinoma (ESCC).Thirty-one patients with stage I-IVa ESCC (I/II/III/IVa = 7/7/14/3) were enrolled in this study. One course of treatment consisted of protracted venous infusions (PVIs) of 5-FU (400 mg/m2/24 hours for days 1-5 and 8-12), CDDP (40 mg/m2/3 hours on days 1 and 8) and radiation (2 Gy/d on days 1-5, 8-12, and 15-19), and a 2nd course was successively repeated after a 2-week interval. A total of 8 measurements of the plasma concentration of 5-FU were made using high performance liquid chromatography. Genetic polymorphisms examined herein included those in the genes coding thymidylate synthase (TS), glutathione S-transferase P1 (GSTP1), multidrug resistant transporter MDR1/P-glycoprotein, and intercellular adhesion molecule-1, and in a circadian rhythm-relating gene, CLOCK.The CR rate depended on stage (P = 0.001), but the analysis was not sufficiently powered to reach a level of statistical significance for the 2-year survival rate (P = 0.061). For stage II/III patients, to have 2 or 3 polymorphisms of 3R/3R of 5'-TSER, a 6 bp of 3'-TSUTR, and GSTP1-Ile105Val resulted in an extensively longer survival (P = 0.020), although no difference was found between 2 groups, with respect to the plasma concentrations of 5-FU and clinicopathologic characteristics.The prognostic index may allow predictions of the clinical outcome of a 5-FU/CDDP-based CRT in stage II/III ESCC patients.

Gastrointestinal (GI) hormones play an important role in GI secretion, motility, and eating behaviors. It was recently suggested that GI hormones may have a trophic role in GI tract. Here we demonstrate that two principal GI hormones, anorexigenic peptide YY (PYY) and orexigenic ghrelin, affect neural tube development. Chronic administration into the pregnant mice or transgenic overexpression of PYY led to a neural tube defect (NTD) in the embryos that was blocked by ghrelin. PYY Y1 receptor antagonist prevented the occurrence of NTD induced not only by PYY but also by vitamin A, a well-known teratogen in humans and animals. Y1 receptor deficiency also engendered NTDs, indicating the need to maintain normal Y1 receptor signaling. The present study is the first linking GI hormones to the leading cause of infant mortality and provides a novel insight for neurogenesis in which materno-fetal communication through GI hormones appears to be important.

The Solomon Islands is a multi-ethnic nation with a high rate of hepatitis B virus (HBV) infection. The prevalence relative to ethnicity was examined in relation to HBV infection, genotypes, and mutations. Asymptomatic populations (n = 564, 308 Melanesian and 118 Micronesian) from the Western Province were enrolled. Positive samples for Hepatitis B surface antigen (HBsAg) were examined for serological status, genotyping, viral load, and mutations of the basic core promoter (BCP) and pre-core (Pre-C) regions. The positive rate for HBsAg was 21.5%. The major Melanesian genotype was C (HBV/C), whereas the major Micronesian genotype was D (HBV/D). The prevalence of Hepatitis B e antigen (HBeAg) in serum was lower in carriers of HBV/D than of HBV/C. While the prevalence of the BCP mutation (T(1762)A(1764)) tended to be higher in HBV/C, that of the Pre-C mutation (T(1846)) was significantly higher in HBV/D (P < 0.0001). Genetic distance and phylogenetic analyses based on complete genome sequences were also carried out for two strains of HBV/C and two strains of HBV/D, and the findings were compared with those in the DDBJ/EMBL/GenBank database. The full-length sequence revealed that strains from the Solomon Islands were classified into subgenotype C3 (HBV/C3) and D4 (HBV/D4), and that the HBV/D strains were related closely to those from Papua New Guinea. HBV infection in the Solomon Islands is hyperendemic, and the genotype is ethnicity-specific. HBeAg appears to clear from the serum in young adulthood in HBV/D infection, which may be influenced by genotype-dependent features in relation to viral mutations.

Objective: Claudin-4 plays a key role in constructing the tight junction (TJ), and altered claudin-4 expression has been documented in various human malignancies; however, little is known about the biological significance of claudin-4 in colorectal cancers (CRCs). The aim of this study is to investigate the significance of claudin-4 expression in CRC and its association with clinicopathological factors. Methods: The levels of claudin-4 expression in a total of 129 CRCs and 44 metastatic tumors were examined by immunohistochemistry. A small interfering RNA (siRNA)-mediated claudin-4 knockdown examination was also conducted to assess the biological role(s) of claudin-4 in cultured cells. Results: Expression of claudin-4 at the intercellular membrane was well preserved at the surface of the tumor; however, decreased claudin-4 expression was detected in 57% of CRCs, particularly at the invasive front. Interestingly, decreased claudin-4 expression was detected in metastatic lesions of CRC. The siRNA-mediated claudin-4 knockdown in SW480 claudin-4-positive CRC cells upregulated cell motility, whereas no significant change was detected in cell proliferation. Conclusions: These observations suggested that disruption of claudin-4-mediated TJ construction enhances cancer cell invasion and metastasis in human CRC. Claudin-4 might be a good biomarker for diagnosing the risk of distant metastasis.