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Aims It was recently reported that lactate acts as a metabolic mediator and rises in the diabetic state, but the physiological effects are as yet poorly understood. The objective of the current study was to evaluate the significance of serum lactate elevation in type 2 diabetes mellitus (T2DM) patients. Methods Fasting serum lactate levels, hematological and inflammatory serum markers and anthropometric parameters, obtained employing bioelectric impedance analysis, were measured in 103 patients with T2DM. Results Statistically significant correlations of serum lactate levels with C-reactive peptide, insulin, aspartate aminotransferase, alanine aminotransferase (ALT), serum lipids, total bilirubin, adiponectin, homeostasis model assessment-insulin resistance, body weight, body mass index and body fat (weight or percentage of subcutaneous fat, visceral fat or total body fat), but neither fasting plasma glucose nor HbA1c, were detected. Stepwise regression analysis showed ALT to be independently positively associated with total bilirubin, while being negatively associated with serum lactate levels. Furthermore, serum lactate levels were significantly higher in patients with ALT-predominant liver dysfunction. Conclusion We found fasting serum lactate elevation in T2DM patients to be associated with the serum levels of ALT and total bilirubin independently of blood glucose control. Trial registration: UMIN clinical trials registry (UMIN000029178).

Anti‐glutamic acid decarboxylase antibody (GADA) is an important islet cell‐associated autoantibody for the diagnosis of autoimmune type 1 diabetes mellitus. In Japan, the GADA assay kit was recently changed from radioimmunoassay (RIA) to enzyme‐linked immunosorbent assay (ELISA). Thereafter, a mismatched measurement between the two tests became apparent in clinical situations. The present study aimed to clarify the actual extent of mismatch between the two measurements on a larger‐scale real‐world clinical practice. In this cross‐sectional non‐local/non‐hospital‐based study, we collected anonymized data on GADA levels of 598 participants, who were simultaneously measured with GADA‐RIA and GADA enzyme‐linked immunosorbent assay tests. We found that 34% of the GADA‐RIA‐positive participants showed negative results in the GADA enzyme‐linked immunosorbent assay test; the mismatch was predominantly observed in participants with relatively low GADA‐RIA levels (

IntroductionGiven an increasing use of dipeptidyl peptidase-4 (DPP-4) inhibitors to treat patients with type 2 diabetes mellitus in the real-world setting, we conducted a prospective observational study (Japan-based Clinical Research Network for Diabetes Registry: J-BRAND Registry) to elucidate the safety and efficacy profile of long-term usage of alogliptin.Research design and methodsWe registered 5969 patients from April 2012 through September 2014, who started receiving alogliptin (group A) or other classes of oral hypoglycemic agents (OHAs; group B), and were followed for 3 years at 239 sites nationwide. Safety was the primary outcome. Symptomatic hypoglycemia, pancreatitis, skin disorders of non-extrinsic origin, severe infections, and cancer were collected as major adverse events (AEs). Efficacy assessment was the secondary outcome and included changes in hemoglobin A1c (HbA1c), fasting blood glucose, fasting insulin and urinary albumin.ResultsOf the registered, 5150 (group A: 3395 and group B: 1755) and 5096 (3358 and 1738) were included for safety and efficacy analysis, respectively. Group A patients mostly (>90%) continued to use alogliptin. In group B, biguanides were the primary agents, while DPP-4 inhibitors were added in up to ~36% of patients. The overall incidence of AEs was similar between the two groups (42.7% vs 42.2%). Kaplan-Meier analysis revealed the incidence of cancer was significantly higher in group A than in group B (7.4% vs 4.8%, p=0.040), while no significant incidence difference was observed in the individual cancer. Multivariate Cox regression analysis revealed that the imbalanced patient distribution (more elderly patients in group A than in group B), but not alogliptin usage per se, contributed to cancer development. The incidence of other major AE categories was with no between-group difference. Between-group difference was not detected, either, in the incidence of microvascular and macrovascular complications. HbA1c and fasting glucose decreased significantly at the 0.5-year visit and nearly plateaued thereafter in both groups.ConclusionsAlogliptin as a representative of DPP-4 inhibitors was safe and durably efficacious when used alone or with other OHAs for patients with type 2 diabetes in the real world setting.

To date, several clinical trials have compared differences in glucose fluctuation observed with dipeptidyl peptidase-4 inhibitor treatment in patients with type 2 diabetes mellitus. However, most patients were assessed for limited periods or during hospitalization. The aim of the present study was to evaluate the effects of switching from sitagliptin to vildagliptin, or vice versa, on 12-week glucose fluctuations using self-monitoring of blood glucose in the standard care setting. We conducted a multicenter, prospective, open-label controlled trial in Japanese patients with type 2 diabetes. Thirty-two patients were treated with vildagliptin (50 mg) twice daily or sitagliptin (50 mg) once daily and were allocated to one of two groups: vildagliptin treatment for 12 weeks before switching to sitagliptin for 12 weeks, or vice versa. Daily profiles of blood glucose were assessed several times during each treatment period, and the mean amplitude of glycemic excursions and M-value were calculated. Metabolic biomarkers such as hemoglobin A1c (HbA1c), glycated albumin, and 1,5-anhydroglucitol were also assessed. With vildagliptin treatment, mean amplitude of glycemic excursions was significantly improved compared with sitagliptin treatment (57.9 ± 22.2 vs. 68.9 ± 33.0 mg/dL; p=0.0045). M-value (p=0.019) and mean blood glucose (p=0.0021) were also lower with vildagliptin, as were HbA1c, glycated albumin, and 1,5-anhydroglucitol. There were no significant differences in other metabolic parameters evaluated. Reduction of daily blood glucose profile fluctuations by vildagliptin was superior to that of sitagliptin in Japanese patients with type 2 diabetes.

Glucocorticoids are widely used in clinical practice, while the chronic administration at a high dose often leads to the progression of steroid-induced diabetes. This hyperglycemic state results mainly from the suppression of insulin secretion due to pancreatic β-cell dysfunction and the increment of systemic insulin resistance. However, the mechanisms which underlie glucocorticoid action on expression and secretion of insulin in pancreatic β-cells have not been clarified. The aim of this study is to elucidate the effects of glucocorticoid on insulin synthesis and secretion in pancreatic β-cells. Insulin gene expression was significantly increased in 30nM of dexamethasone and decreased in 300 and 3000 nM of dexamethasone. These changes in the expression were canceled by RU486, a GR antagonist, at any concentrations of dexamethasone. Meanwhile, insulin and proinsulin secretion were significantly reduced at any concentrations of dexamethasone. Interestingly, the proinsulin/insulin ratio was significantly decreased at 30 nM, and increased at 300 and 3000 nM. The changes were completely restored by RU486 at 30 nM of dexamethasone, while the reduction of insulin and proinsulin secretion by 300 or 3000 nM of dexamethasone was not released by RU486. In addition, similar results were obtained in experiments using knockdown of GR expression. Interestingly, the short-term induction of insulin secretion by membrane depolarization treated with 30 mM of KCl was rather enhanced by 3000 nM of dexamethasone, and this enhancement was not canceled by RU486. In conclusion, glucocorticoid regulates insulin gene expressions via the genomic pathway containing GR in pancreatic β-cells, while particularly high-dose glucocorticoid regulates insulin secretion principally via non-genomic pathway. Since the proinsulin/insulin ratio increased under chronic administration of high-dose glucocorticoid, it was assumed that the non-genomic pathway was involved in insulin processing. Disclosure T. Murashima: None. T. Kondo: None. K. Kondo: None. N. Morita: None. A. Kitahara: None. Y. Sumitani: None. K. Takahashi: None. T. Tanaka: None. T. Hosaka: Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Ono Pharmaceutical Co., Ltd. H. Ishida: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim International GmbH, Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Kowa pharmaceutical Co. Ltd., MSD, Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho. Funding Japan Ministry of Education, Culture, Sports, Science and Technology

Most cases of type 1 diabetes mellitus are considered to be caused by pancreatic β-cell destruction through islet cell autoimmunity. Anti-glutamic acid decarboxylase antibody (GADA) is an important islet cell-associated autoantibody in the diagnosis of autoimmune type 1 diabetes mellitus. In Japan, the GADA assay kit was changed from radioimmunoassay (RIA) to enzyme-linked immunosorbent assay (ELISA) in 2015. Thereafter, a mismatch of measurements between the two tests became apparent in clinical situations. The aim of this study was to clarify the extent of the mismatch between the two tests on a larger scale. In this cross-sectional non-local/non-hospital-based study, we collected anonymized data on GADA levels of 598 randomly selected participants in whom GADA levels were simultaneously measured by GADA-RIA and GADA-ELISA tests. We found that 34% (= 162/477) of the GADA-RIA-positive participants showed negative results in the GADA-ELISA test; the mismatch was predominantly observed in participants with relatively low GADA-RIA levels. Concretely, when considering lower GADA-RIA levels alone (1.5-32 U/mL), GADA-ELISA-negative participants accounted for 44.8% (= 162/362) of the GADA-RIA-positive participants. These findings suggest that a single application of the GADA-ELISA test may affect the precise diagnosis of type 1 diabetes mellitus, and cause physicians’ confusion on the diagnosis. Therefore, the cause for the mismatch should be promptly identified. Disclosure Y. Oikawa: None. T. Kondo: None. A. Shimada: Advisory Panel; Self; Terumo Medical Corporation. Speaker's Bureau; Self; Abbott, Eli Lilly and Company, Novo Nordisk A/S, Sanofi-Aventis. Y. Seino: Research Support; Self; Arkray, Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. M. Kitaoka: Research Support; Self; AstraZeneca, Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd.

In Bacillus subtilis , the dl -endopeptidase LytE is responsible for lateral peptidoglycan hydrolysis during cell elongation. We found that σ I -dependent transcription of lytE is considerably enhanced in a strain with a mutation in ltaS , which encodes a major lipoteichoic acid (LTA) synthase. Similar enhancements were observed in mutants that affect the glycolipid anchor and wall teichoic acid (WTA) synthetic pathways. Immunofluorescence microscopy revealed that the LytE foci were considerably increased in these mutants. The localization patterns of LytE on the sidewalls appeared to be helix-like in LTA-defective or WTA-reduced cells and evenly distributed on WTA-depleted or -defective cell surfaces. These results strongly suggested that LTA and WTA affect both σ I -dependent expression and localization of LytE. Interestingly, increased LytE localization along the sidewall in the ltaS mutant largely occurred in an MreBH-independent manner. Moreover, we found that cell surface decorations with LTA and WTA are gradually reduced at increased culture temperatures and that LTA rather than WTA on the cell surface is reduced at high temperatures. In contrast, the amount of LytE on the cell surface gradually increased under heat stress conditions. Taken together, these results indicated that reductions in these anionic polymers at high temperatures might give rise to increases in SigI-dependent expression and cell surface localization of LytE at high temperatures. IMPORTANCE The bacterial cell wall is required for maintaining cell shape and bearing environmental stresses. The Gram-positive cell wall consists of mesh-like peptidoglycan and covalently linked wall teichoic acid and lipoteichoic acid polymers. It is important to determine if these anionic polymers are required for proliferation and environmental adaptation. Here, we demonstrated that these polymers affect the expression and localization of a peptidoglycan hydrolase LytE required for lateral cell wall elongation. Moreover, we found that cell surface decorations with teichoic acid polymers are substantially decreased at high temperatures and that the peptidoglycan hydrolase is consequently increased. These findings suggest that teichoic acid polymers control lateral peptidoglycan hydrolysis by LytE, and bacteria drastically change their cell wall content to adapt to their environment.

Objective. A growing body of evidence indicates that AMP-activated protein kinase (AMPK) contributes to not only energy metabolic homeostasis but also the inhibition of inflammatory responses. However, the underlying mechanisms remain unclear. To elucidate the role of AMPK, in this study, we observed the effects of AMPK activation on monocyte chemoattractant protein-1 (MCP-1) release in mature 3T3-L1 adipocytes.Methods. We observed signal transduction pathways regulating MCP-1, which increased in obese adipocytes, in anin vitromodel of hypertrophied 3T3-L1 adipocytes preloaded with palmitate.Results. Palmitate-preloaded cells exhibited significant increase in MCP-1 release and triglyceride (TG) deposition. Increased MCP-1 release and TG deposition were significantly decreased by an AMPK activator. In addition, the AMPK activator not only markedly diminished MCP-1 secretion but also augmented phosphorylation of nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK) 1/2. In contrast, MCP-1 release suppression was abolished by the AMPK inhibitor compound C and the MEK inhibitor U0126.Conclusions. MCP-1 release from hypertrophied adipocytes is suppressed by AMPK activation through the NF-κB and ERK pathways. These findings provide evidence that AMPK plays a crucial role in ameliorating obesity-induced inflammation.

We aimed to examine the association between impaired proinsulin processing in pancreatic beta cells and type 2 diabetes mellitus in non-obese Japanese patients. Participants were divided into groups for normal glucose tolerance, prediabetes, and type 2 diabetes based on the oral glucose tolerance test (OGTT). Activities of prohormone convertase (PC) 1/3 and PC2 in fasting states were estimated. Multiple regression analysis was undertaken to ascertain if alteration of the activities of these enzymes contributes to the development of impaired glucose tolerance by comparison with HOMA-β and the oral disposition index (DI(O)). Overall, 452 subjects were included. PC1/3 activity tended to decrease in type 2 diabetes compared with normal glucose tolerance. PC2 activity showed no difference among the three groups. Decreased estimated PC1/3 activity was significantly associated with type 2 diabetes after adjustment for sex, age, creatinine, triglycerides, HOMA-β and DI(O). Odds ratios (95% CI) of PC1/3, HOMA-β, and DI(O) were 2.16 (1.12-4.19), 3.44 (1.82-6.52) and 14.60 (7.87-27.11), respectively. Furthermore, decreased PC1/3(≤1.7) combined with decreased HOMA-β (≤30) had a sensitivity of 73% and specificity of 62%. Decreased PC1/3 activity may be a useful measurement of beta-cell function alongside decreased HOMA-β or DI(O). A combined decrease in estimated fasting PC1/3 activity and HOMA-β measurement led to suspicion of type 2 diabetes in the non-obese Japanese population studied.

Astaxanthin, an antioxidant agent, can protect pancreatic β-cells of db/db mice from glucotoxicity and resolve chronic inflammation in adipose tissue. Nonetheless, the effects of astaxanthin on free-fatty-acid-induced inflammation and cellular stress in β-cells remain to be demonstrated. Meanwhile, palmitate enhances the secretion of pro-inflammatory adipokines monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF120). We therefore investigated the influence of astaxanthin on palmitate-stimulated MCP-1 and VEGF120 secretion in mouse insulinoma (MIN6) pancreatic β-cells. Furthermore, whether astaxanthin prevents cellular stress in MIN6 cells was also assessed. Pre-treatment with astaxanthin or with N-acetyl-cysteine (NAC) which is an antioxidant drug, significantly attenuated the palmitate-induced MCP-1 release through downregulation of phosphorylated c-Jun NH2-terminal protein kinase (JNK) pathways, and suppressed VEGF120 through the PI3K/Akt pathways relative to the cells stimulated with palmitate alone. In addition, palmitate significantly upregulated homologous protein (CHOP) and anti-glucose-regulated protein (GRP78), which are endoplasmic reticulum (ER) stress markers, in MIN6 cells. On the other hand, astaxanthin attenuated the increased CHOP content, but further up-regulated palmitate-stimulated GRP78 protein expression. By contrast, NAC had no effects on either CHOP or GRP78 enhancement induced by palmitate in MIN6 cells. In conclusion, astaxanthin diminishes the palmitate-stimulated increase in MCP-1 secretion via the downregulation of JNK pathways in MIN6 cells, and affects VEGF120 secretion through PI3K/Akt pathways. Moreover, astaxanthin can prevent not only oxidative stress caused endogenously by palmitate but also ER stress, which NAC fails to attenuate, via upregulation of GRP78, an ER chaperon.

The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system that controls responses to stress, and has an important function in the regulation of various body processes. We previously created a mouse line deficient in the melanocortin 2 receptor (MC2R). MC2R-deficient mice (MC2R−/− mice) have high adrenocorticotropic hormone (ACTH) levels because of undetectable corticosterone levels. Increased neuromedin B (NMB) expression was recently reported in the pituitary gland of adrenalectomized mice, a model for acute adrenal insufficiency. To investigate gene expression in the pituitary gland under chronic adrenal deficiency, we examined the pituitary gland of MC2R−/− mice, a model of chronic adrenal insufficiency. To understand the molecular background of pituitary cells under chronic adrenal deficiency, we first performed DNA microarray analyses using the pituitary glands of the MC2R−/− mice. The DNA microarray analysis and real-time polymerase chain reaction showed that NMB expression was higher in the MC2R−/− than in the wild-type (WT) mice. We detected NMB expression in the MC2R−/− pituitary corticotrophs by immunohistochemistry using the specific antibodies for ACTH and NMB. In addition, the plasma NMB concentration was significantly higher in the MC2R−/− mice than in the WT mice. Subcutaneous implantation of a sustained-release corticosterone pellet decreased the expression of NMB mRNA as well as pituitary proopiomelanocortin mRNA. In isolated anterior pituitary cells, NMB mRNA expression was increased by the administration of corticotropin-releasing hormone (CRH) and was suppressed by dexamethasone treatment. In this study, we first demonstrate NMB expression in corticotrophs and its regulation by CRH and glucocorticoids. Furthermore, corticotrophs seemed to secrete NMB into the systemic circulation.

Aims/Introduction The global prevalence of type 2 diabetes has been increasing in all age groups in recent years. Some studies have suggested that perinatal outcomes in these women are worse than in women with type 1 diabetes. We evaluated pregnancy outcomes in Japanese women with type 2 and type 1 diabetes.

Sodium‐glucose cotransporter 2 inhibitors (SGLT2i) exert their antidiabetic effects by promoting urinary glucose excretion. Nutrition therapy is obviously important, but little is known about the interactions between SGLT2i agents and carbohydrate restriction. Therefore, we studied these interactions using an obese diabetic animal model. KK‐A y mice were pair‐fed normal chow [NC; carbohydrate: fat: protein = 65:15:20], low carbohydrate [LC; 43:42:15] or severely carbohydrate restricted diets [SR; 12:45:43] for 12 weeks. Tofogliflozin (Tofo) was administered as the SGLT2i in the NC and LC diet groups. Blood glucose levels were significantly increased in the SR group. Tofo reduced blood glucose levels significantly in the NC group during the experiment and in the LC group at 2‐6 weeks. Plasma triglycerides were markedly elevated in the SR group without Tofo, but decreased in response to Tofo administration. Hepatic triglyceride contents were not changed by the LC or the SR diet alone. However, Tofo ameliorated hepatosteatosis in NC‐fed animals. Consistent with the downregulation of stearoyl‐CoA desaturase 1, the ratio of plasma monounsaturated to saturated fatty acids was significantly reduced in the LC with Tofo and in the SR alone groups, but was not altered in the NC with Tofo group. In summary, metabolism of glucose and lipids was improved by Tofo but not by the SR diet. Furthermore, Tofo improved these parameters more effectively in the NC than in the LC diet group. These data suggest that the effects of SGLT2i are distinct from those of carbohydrate restriction and that a nonrestricted dietary carbohydrate composition is essential for SGLT2i treatment to be effective.

Objective Gitelman's syndrome, recognized as a variant of Bartter's syndrome, is characterized by hypokalaemic metabolic alkalosis in combination with hypomagnesaemia and hypocalciuria. Overlapping biochemical features in Gitelman's syndrome and Bartter's syndrome has been observed. Here, we investigated the clinical, biochemical, and genetic characteristics of five, chronic, nonhypertensive and hypokalaemic Japanese patients. Methods Serum and urinary electrolytes, plasma renin activity and plasma aldosterone concentration were measured in five patients (four males and one female) with hypokalaemia. Renal clearance tests were performed and distal fractional chloride reabsorption calculated. Finally, mutational analysis of the thiazide-sensitive Na-Cl co-transporter gene was performed. Results Symptoms in patients varied from mild (muscle weakness and numbness) to severe (tetany and foot paralysis). All patients were normotensive or hypotensive, and all had hypokalaemia, hypocalciuria, and hyperreninaemic hyperaldosteronism. However, two male patients had normomagnesaemia, while the remainder was hypomagnesaemic. Renal clearance tests showed that the administration of furosemide decreased distal fractional chloride reabsorption, while thiazide ingestion failed to decrease it. Genetic analysis identified six thiazide-sensitive Na-Cl co-transporter gene mutations, including two novel ones. Therefore, on the basis of the confirmatory renal clearance tests and mutational analysis, a diagnosis of Gitelman's syndrome was made in these patients. Conclusions Two of the five patients diagnosed with Gitelman's syndrome were normomagnesaemic, which is uncommon in this syndrome. Our study indicates that renal clearance tests and mutation analysis can play an important role in diagnosing Gitelman's syndrome more precisely.

Mammalian MafA/RIPE3b1 is an important glucose-responsive transcription factor that regulates function, maturation, and survival of beta-cells. Increased expression of MafA results in improved glucose-stimulated insulin secretion and beta-cell function. Because MafA is a highly phosphorylated protein, we examined whether regulating activity of protein kinases can increase MafA expression by enhancing its stability. We demonstrate that MafA protein stability in MIN6 cells and isolated mouse islets is regulated by both p38 MAPK and glycogen synthase kinase 3. Inhibiting p38 MAPK enhanced MafA stability in cells grown under both low and high concentrations of glucose. We also show that the N-terminal domain of MafA plays a major role in p38 MAPK-mediated degradation; simultaneous mutation of both threonines 57 and 134 into alanines in MafA was sufficient to prevent this degradation. Under oxidative stress, a condition detrimental to beta-cell function, a decrease in MafA stability was associated with a concomitant increase in active p38 MAPK. Interestingly, inhibiting p38 MAPK but not glycogen synthase kinase 3 prevented oxidative stress-dependent degradation of MafA. These results suggest that the p38 MAPK pathway may represent a common mechanism for regulating MafA levels under oxidative stress and basal and stimulatory glucose concentrations. Therefore, preventing p38 MAPK-mediated degradation of MafA represents a novel approach to improve beta-cell function.

Background & Aims: Nonalcoholic steatohepatitis (NASH) is an emerging progressive hepatic disease and demonstrates steatosis, inflammation, and fibrosis. Insulin resistance is a common feature in the development of NASH. Molecular pathogenesis of NASH consists of 2 steps: triglyceride accumulation in hepatocytes with insulin resistance and an enhanced oxidative stress caused by reactive oxygen species. Interestingly, NASH demonstrates a striking similarity to the pathologic conditions observed in adult growth hormone deficiency (AGHD). AGHD is characterized by decreased lean body mass, increased visceral adiposity, abnormal lipid profile, and insulin resistance. Moreover, liver dysfunctions with hyperlipidemia and nonalcoholic fatty liver disease (NAFLD) are frequently observed in patients with AGHD, and it is accompanied by metabolic syndrome. Methods: We studied a case diagnosed as NASH with hyperlipidemia in AGHD. The effect of GH-replacement therapy on the patient was analyzed. Results: Six months of GH-replacement therapy in the patient drastically ameliorated NASH and the abnormal lipid profile concomitant with a marked reduction in oxidative stress. Conclusions: These results suggest that GH plays an essential role in the metabolic and redox regulation in the liver.

The large-Maf transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) has been found to be crucial for insulin transcription and synthesis and for pancreatic beta-cell function and maturation. However, insights about the effects of small Maf factors on beta-cells are limited. Our goal was to elucidate the function of small-Maf factors on beta-cells using an animal model of endogenous small-Maf dysfunction. Transgenic (Tg) mice with beta-cell-specific expression of dominant-negative MafK (DN-MafK) experiments, which can suppress the function of all endogenous small-Mafs, were fed a high-fat diet, and their in vivo phenotypes were evaluated. Phenotypic analysis, glucose tolerance tests, morphologic examination of beta-cells, and islet experiments were performed. DN-MafK-expressed MIN6 cells were also used for in vitro analysis. The results showed that DN-MafK expression inhibited endogenous small-Maf binding to insulin promoter while increasing MafA binding. DN-MafK Tg mice under high-fat diet conditions showed improved glucose metabolism compared with control mice via incremental insulin secretion, without causing changes in insulin sensitivity or MafA expression. Moreover, up-regulation of insulin and glucokinase gene expression was observed both in vivo and in vitro under DN-MafK expression. We concluded that endogenous small-Maf factors negatively regulates beta-cell function by competing for MafA binding, and thus, the inhibition of small-Maf activity can improve beta-cell function.

The transcription factor MafA/RIPE3b1 is an important regulator of insulin gene expression. MafA binds to the insulin enhancer element RIPE3b (C1-A2), now designated as insulin MARE (Maf response element). The insulin MARE element shares an overlapping DNA-binding region with another insulin enhancer element A2. A2.2, a β-cell-specific activator, like the MARE-binding factor MafA, binds to the overlapping A2 element. Our previous results demonstrated that two nucleotides in the overlapping region are required for the binding of both factors. Surprisingly, instead of interfering with each other's binding activity, the MafA and the A2-binding factors co-operatively activated insulin gene expression. To understand the molecular mechanisms responsible for this functional co-operation, we have determined the nucleotides essential for the binding of the A2.2 factor. Using this information, we have constructed non-overlapping DNA-binding elements and their derivatives, and subsequently analysed the effect of these modifications on insulin gene expression. Our results demonstrate that the overlapping binding site is essential for maximal insulin gene expression. Furthermore, the overlapping organization is critical for MafA-mediated transcriptional activation, but has a minor effect on the activity of A2-binding factors. Interestingly, the binding affinities of both MafA and A2.2 to the overlapping or non-overlapping binding sites were not significantly different, implying that the overlapping binding organization may increase the activation potential of MafA by physical/functional interactions with A2-binding factors. Thus our results demonstrate a novel mechanism for the regulation of MafA activity, and in turn β-cell function, by altering expression and/or binding of the A2.2 factor. Our results further suggest that the major downstream targets of MafA will in addition to the MARE element have a binding site for the A2.2 factor.

Interleukin 3 (IL-3)-dependent proliferation of haematopoietic cells is specifically inhibited by p130, a member of the pRB-family proteins. p130 interacts with the cell-cycle regulatory E2F transcription factors, notably E2F-4 and E2F-5, and affects promoters containing E2F-binding sites through two distinct mechanisms. First, upon complex formation with E2F, it blocks transcriptional activation by E2F. Second, the formed p130-E2F complex binds to E2F sites and actively represses transcription by inhibiting the activity of surrounding enhancer elements on the promoter. To pursue the relative contributions of each mechanism in the p130-mediated inhibition of IL-3-dependent cell proliferation, we employed a dominant-negative DP-1, which suppresses both E2F-dependent transactivation and the formation of active transcriptional repressors. Ectopic expression of the dominant negative DP-1 in the IL-3-dependent BaF3 lymphoid cells gave rise to an inhibition of cell proliferation, which was concomitantly associated with a decrease in levels of cyclin E, an indispensable molecule for G1 to S-phase cell-cycle progression. Our results indicate that blocking E2F-dependent transactivation, but not the formation of p130-E2F transcriptional repressor complexes, is responsible for the inhibition of IL-3-dependent cell growth by p130.

The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.

Background Proinflammatory (M1) macrophages and anti-inflammatory (M2) macrophages have been identified in atherosclerotic plaques. While these macrophages have been speculated to be related to plaque vulnerability, there are limited studies investigating this relationship. Therefore, we examined the association between macrophage phenotype (M1 versus M2) and plaque vulnerability and clinical events. Methods Patients undergoing carotid endarterectomy received an ultrasound of the carotid artery before surgery. Plaques were processed for analysis by immunohistochemistry, Western blotting, and real-time polymerase chain reaction studies. Medical history and clinical data were obtained from medical records. Results Patients were divided into 2 groups: those suffering from acute ischemic attack (symptomatic, n=31) and those that did not present with symptoms (asymptomatic, n=34). Ultrasound analysis revealed that plaque vulnerability was greater in the symptomatic group ( P = .033; Chi-square test). Immunohistochemistry revealed that plaques from the symptomatic group had a greater concentration of M1 macrophages (CD68-, CD11c-positive) while plaques from the asymptomatic group had more M2 macrophages (CD163-positive). This observation was confirmed by Western blotting. Characterization by real-time polymerase chain reaction studies revealed that plaques from the symptomatic group had increased expression of the M1 markers CD68 and CD11c, as well as monocyte chemoattractive protein-1, interleukin-6, and matrix metalloproteinase-9. In addition, more M1 macrophages expressed in unstable plaques were defined by ultrasound analysis, while more M2 macrophages were expressed in stable plaques. Conclusions Our data show that M1 macrophage content of atherosclerotic plaques is associated with clinical incidence of ischemic stroke and increased inflammation or fibrinolysis. We also show the benefits of using ultrasound to evaluate vulnerability in the plaques.

Aims/Introduction: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder including polycystic ovary morphology (PCOM), ovulatory dysfunction and hyperandrogenism. PCOS is frequently associated with type 2 diabetes mellitus; however, it is unknown whether PCOM and PCOS are prevalent in Japanese patients with type 1 diabetes mellitus. The purpose of our study was to determine the frequency of PCOM and PCOS in women with type 1 diabetes mellitus. Materials and Methods: We evaluated clinical, hormonal and ovarian ultrasound data from 21 type 1 diabetes mellitus patients whose average glycated hemoglobin levels were 7.9 – 1.5%. Results: Ultrasound identified PCOM in 11 patients (52.4%) and these patients also had higher levels of the androgen dehydroepiandrosterone sulfate (DHEA-S) than those without PCOM (P < 0.05). Of the patients with PCOM, five presented menstrual irregularities (45.5%) and three met the Japanese criteria for PCOS (27.2%); whereas all patients without PCOM had a normal menstrual cycle (P < 0.05). Conclusions: Japanese premenopausal women with type 1 diabetes mellitus had a high frequency of PCOM as well as PCOS. This is the first research of this area carried out in an Asian population. (J Diabetes Invest doi: 10.1111/jdi.12040, 2013)

Familial hypocalciuric hypercalcemia (FHH) is a benign disorder with heterozygous inactivating mutations in the calcium-sensing receptor (CASR) gene. The present study describes the identification and functional analysis of a novel CASR gene mutation leading to FHH. The proband is a 33-yr-old woman (Ca 11.0 mg/dL, intact-PTH 68 pg/mL, FECa 0.17 %). Leukocyte DNA was isolated in four family members and a novel heterozygous mutation (D190G, GAT>GGT) in exon 4 of CASR gene was identified by direct sequence analysis. The mutant CASR expression vector was constructed by mutagenesis procedure and its response to Ca(2+) was characterized by transient transfection into human embryonic kidney (HEK) 293 cells and treatment with increasing extracellular Ca(2+) concentrations. HEK cells didn't activate intracellular signaling (MAPK activation) in response to increases of extracellular Ca(2+) concentrations when the mutant receptor was expressed normally at the cell surface. The novel heterozygous mutation (D190G) identified in the present study showed that the reduction of activity of CASR to extracellular Ca(2+) caused FHH in patients and our study demonstrated the importance of Asp-190 participated in response to Ca(2+) in CASR.

The following information was missing from the Funding section: Dr. Greenberg’s (ASG) work was supported by grants American Diabetes Association Grant: 7-08-RA-57; by the USDA, Agricultural Research Service under Contract no. 58-1950-7-707; and NIH RO1-DK0822574 and 1RC2ES018781.

Recent observations have suggested that the pathological mutations in human P-450(C21) deficiency are generated through gene conversion-like events between the functional gene [P-450(21)B] and the pseudogene [P-450(C21)A]. To address this point more extensively, we investigated the effects of the base changes in the A pseudogene on the P-450(21) activity by using the COS cell expression system. In addition to the defective mutations found previously in the pseudogene, four single base changes with amino acid substitutions of Pro(30), Ile(172), Val(282), or Arg(356) were further identified as causing complete [Arg(356)] or partial [Pro(30), Ile(172), and Val(282)] inactivation of P-450(C21). Blot hybridization analysis of patient DNAs using oligonucleotide probes specific for these mutations revealed that the splicing mutation in the 2nd intron was distributed most frequently in both simple-virilizing and salt-wasting forms. The mutation Ile(172) seemed to be frequent in patients with the less severe simple-virilizing form, whereas the mutation Arg(356), together with other most serious mutations reported previously, was preferentially associated with salt-wasting, the most severe form of the disease. In combination with the present results of the effects of various mutations on the P-50(C21) activity, a survey of the distribution of the various mutations in the patient genomes so far reported suggests that the heterogeneous clinical symptoms of this genetic disease are somehow related to the degree of attenuation of the activities of the mutated gene products.

Mutations of DSS (dosage sensitive sex reversal)-AHC critical region on the X chromosome, gene 1 DAX-1(NROB1)] results in X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). Here we report four Japanese patients with AHC and HHG caused by the mutations of the DAX-1 gene. All patients manifested adrenal crisis at early childhood. Three patients did not show any pubertal sign and were diagnosed as having HHG. One patient manifested spontaneous pubertal development at 17 years of age. Nevertheless, his puberty did not develop further and his gonadotropin and testosterone levels decreased thereafter. Therefore, he was also diagnosed as having HHG. We performed testicular biopsy in another patient with HHG. Histological examination demonstrated Sertoli cell hypoplasia and no sperm formation in the seminiferous tubules. Molecular analysis demonstrated two novel point mutations (V269D and L278R) in two patients. Transient transfection assays showed that all these mutations (V269D, L271X, L278R, and Q395X) abolished the repression activity to both StAR and LHbeta gene promoter activation. In conclusion, we reported patients with AHC and HHG caused by the loss of function mutations of the DAX-1 gene.

pRB family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G1 to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G1 restriction point, they are hyperphosphorylated by G1 cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G1regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G1 to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G1 cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells.

Genomic DNA from 19 Japanese patients with congenital lipoid adrenal hyperplasia (lipoid CAH) representing 16 different families was examined to identify the genetic alterations of steroidogenic acute regulatory protein (StAR). Ten of 19 patients had a 46,XX karyotype and nine had a 46,XY karyotype. Six of the 46,XX patients have experienced spontaneous pubertal changes including breast development and irregular menstruation whereas none of the 46,XY subjects displayed pubertal changes. Eight different mutations were identified. Sixteen patients were either homozygotes or compound heterozygotes for the Q258X mutation. The seven other mutations identified were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T. The 189delG, 246insG, 546del13bp and Q212X mutants encode truncated proteins. COS-1 cells transfected with expression vectors encoding cDNAs for the mutant StAR proteins which affect the C-terminus, 838delA, A218V and Q258X, exhibited no steroidogenesis enhancing activity. However, the M225T mutant retained some steroidogenic activity. The patient with the M225T mutation had late onset of this disorder and some capacity to secrete testosterone in response to hCG. These findings suggest: (i) that the Q258X mutation can be used as a genetic marker for the screening of Japanese for lipoid CAH, (ii) that the C-terminus of StAR plays an important role in the protein's activity and (iii) that there are differences in the extent of functional impairment of the testis and ovaries in lipoid CAH.

Background: Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model. Methodology and Principal Findings: When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size. Conclusions: These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.

Nine female and 20 male hypogonadotropic GH-deficient patients were studied for sexual development by hCG/hMG. In the female patients, gonadotropin therapy was started at the mean age of 22.7 +/- 2.1 years. The administration of progesterone induced withdrawal bleeding at an average of 2.77 +/- 1.94 years after the initiation of hMG/hCG therapy in 8 of the 9 patients studied. Of 6 patients who had been confirmed as positive in a gestagen test, induction of ovulation by hMG/hCG was observed in 5 patients at an average of 5.58 +/- 1.23 years after the onset of therapy, but not in the remaining patient who had been given estrogen and progesterone 4 years 9 months prior to the initiation of the gonadotropin therapy. In male patients, gonadotropin therapy was started at the mean age of 23.6 +/- 5.7 years. Seminal fluid was obtained by masturbation and brought to our clinic in the morning. Of the 20 patients, 19 patients could be observed once a month regularly. Of the 19 patients, spermatozoa could be detected at a mean period of 2.19 +/- 0.87 years after initiation of hCG/hMG therapy in 18, but not in the remaining patient, after 5 years of therapy, who did not receive hCG/hMG regularly. The sperm count exceeded 20 x 10(6)/ml and more in 12 and was lower than that in 8 patients after 3 years of the therapy. No side effects were observed in female patients, but gynecomastia developed in 2 of the 20 male patients. These data suggest that gonadotropin therapy for hypogonadotropic GH-deficient patients is effective in promoting ovulation and spermatogenesis despite the initial replacement therapy with sex hormones.

The binding properties of the angiotensin II receptors of the adrenal cortex have been studied in isolated cells prepared by collagenase dispersion of the zona glomerulosa of the canine adrenal gland. Such cell preparations are responsive to physiological concentrations of angiotensin II, and permit correlation of binding of angiotensin II and its analogues with aldosterone production in vitro. Uptake of 125I-angiotensin II (5 X 10(-11) M) by glomerulosa cells at 37 degrees C reached a steady state at 45 minutes, with a subsequent plateau for at least 60 minutes. Angiotensin II binding was also dependent upon the hormone and cell concentrations employed during uptake studies. Bound angiotensin II was rapidly dissociated from canine adrenal cells after addition of the unlabeled octapeptide. High affinity sites with equilibrium association constant (Ka) of 3.3 X 10(9) M-1 comprised 25-33% of the receptor population and the remainder of the sites were of lower affinity, 2.5 X 10(8)M-1. Binding of angiotensin II analogues and antagonists was found to be consistent with their biological activities. The analogue most extensively evaluated was [Sar-1]angiotensin II, which exhibited enhanced binding activity when compared to angiotensin II, and had a higher equilibrium association constant by kinetic analysis and direct binding studies. Direct binding of labeled angiotensin II to the adrenal glomerulosa receptor has been correlated with a progressive response in aldosterone production. The steroidogenic response to angiotensin II was maximal when 25% of the receptor population was occupied; this fraction corresponds to the proportion of high affinity receptor sites measured by binding analysis. In addition, inhibition of angiotensin II binding to receptor sites by the competitive antagonist [Sar-1, Ala-8]angiotensin II has been correlated with inhibition of aldosterone production. These findings serve to demonstrate the biological significance of the angiotensin II binding sites of the adrenal cortex, and confirm their role as receptors which mediate the steroidogenic responses to angiotensin II.

Congenital adrenocortical unresponsiveness to ACTH is characterized by hyperpigmentation, muscular weakness, and episodes of hypoglycemia, with lethargy or coma and convulsion, and without signs or symptoms of salt wasting. Endocrinological evaluation reveals low levels of serum and urinary glucocorticoids, elevated levels of plasma ACTH and unresponsiveness to exogenous ACTH. On the other hand, aldosterone secretion or excretion is normal and is elevated during a low sodium diet. We reported on two patients with this syndrome. Case A, a 4 year-old-girl, showed skin hyperpigmentation, hypoglycemia, convulsion and coma. She was tall wlth a marked advance of bone age. Case B, a 4 year-old-boy, showed skin hyperpigmentation, fatigability and muscular weakness. Both of them revealed low urinary excretion of THE, THF, cortolone, and beta-cortolone and low levels of plasma cortisol. The levels of urinary glucocorticoids and plasma cortisol did not respond to ACTH administration. During a low sodium diet, urinary aldosterone excretion was elevated, urinary sodium excretion ws decreased, and the patients were able to conserve sodium normally. We also summerized other reported cases and discussed the etiology of this syndrome.

Specific receptors for angiotensin II (A II) were demonstrated in membrane fractions and collagenase-dispersed cells from the zona glomerulosa of the rat adrenal gland. The equilibrium association constant (Ka) of the A II binding sites was similar in particulate fractions (2.0 +/- 0.4 (SE) X 10(9) M-1) and intact glomerulosa cells (1.8 +/- 0.3 X 10(9) M-1). Specific binding of [125I]iodo-A II was enhanced by increasing sodium concentration, and in the presence of dithiothreitol, EDTA, and EGTA. Plasma membrane fractions prepared by density gradient centrifugation showed increased binding of [125I]iodo-A II, and were correspondingly enriched in adenylate cyclase and sodium-potassium-dependent ATPase. Steroid production by collagenase-dispersed adrenal glomerulosa cells was highly responsive to A II and ACTH. Significant increases in aldosterone and corticosterone production were elicited by A II concentrations as low as 3 X 10(-11) M, equivalent to normal blood levels of A II in rats (5 X 10(-11) M). The maximum increase in aldosterone production, of 6--7 times the basal value, was obtained at 10(-9) M A II. Dispersed capsular cells were also highly sensitive to ACTH, responding to concentrations down to 3 X 10(-12) M with increased aldosterone production, reaching a maximum aldosterone response of 20-fold above the basal value. The magnitudes of the aldosterone and corticosterone responses to A II in capsular and fasciculata-reticularis cells were commensurate with the distribution of A II receptors, which were 11-fold more concentrated in capsular cells. The ability of A II to evoke aldosterone production at physiological concentrations, and the correspondence between A II binding and steroidogenesis in capsular cells, demonstrate the functional importance of A II receptor sites in the zona glomerulosa of the rat adrenal cortex.

Dog and rat adrenal glomerulosa cells and subcellular fractions have been utilized to evaluate the mechanism of angiotensin II- and angiotensin III-induced aldosterone production. The effects of angiotensin, ACTH, and potassium have been compared on cyclic AMP and cyclic GMP in isolated glomerulosa cells and adenylate cyclase activity in subcellular fractions. The effect of angiotensin II has also been assessed on Na+-K+-activated ATPase of plasma membrane enriched fractions of dog and rat adrenals. We have demonstrated no effect of angiotensin II or angiotensin III on either adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-dependent ATPase activity over a wide range of concentrations. Potassium ion in concentrations that stimulate significant aldosterone production was also without effect. The negative effects of angiotensin and potassium were contrasted against a positive correlation between an ACTH-induced effect on aldosterone production, adenylate cyclase, and cyclic AMP accumulation. These studies have served to demonstrate that neither adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-activated ATPase seem to be directly involved in the mechanism of action of angiotensins on aldosterone production in the rat and dog adrenal glomerulosa.

The influence of extracellular potassium concentration on adrenal sensitivity to angiotensin II and ACTH was studied in isolated canine adrenal glomerulosa cells. When potassium was absent from the incubation medium, the aldosterone response to angiotensin II or ACTH was completely abolished. At physiologic angiotensin II concentrations (2.5 x 10(-11) M), aldosterone formation increased 4-fold when potassium concentration was increased from 2.5 to 5.0 mM, and rose 6-fold as potassium was increased from 2.5 to 7.5 mM. In the absence of angiotensin II, the same changes in potassium concentration increased aldosterone production only to 2-fold and 3.5-fold, respectively. The effect of potassium concentration upon the aldosterone response to ACTH was similar but less marked. The concentration and binding affinity of angiotensin II receptor sites in glomerulosa cells were not changed by increasing potassium concentrations from 0 to 7.5 mM. These observations demonstrate that the aldosterone response to the glomerulosa cell to angiotensin II is potassium-dependent within the physiological range for each of these stimuli. Such an interaction suggests that the in vivo effect of potassium upon aldosterone secretion includes a significant modulating action upon adrenal sensitivity to angiotensin II, as well as a direct action of potassium upon the adrenal glomerulosa cell.