Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30-40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

Reduction of insulin signaling upregulates angiopoietin-like protein 4 through elevated free fatty acids in diabetic mice.

BACKGROUND: Angiopoietin-like protein 4 (Angptl4) is thought to cause an increase in serum triglyceride levels. In the present study, we elucidated Angptl4 expression in the mouse models of type 1 and type 2 diabetes mellitus, and investigated the possible mechanisms involved. METHODS: Type 1 diabetes was induced in C57BL/6 J mice by treating them with streptozotocin (STZ). Type 2 diabetes was induced by feeding the mice a high-fat diet (HFD) for 18 weeks. RESULTS: The levels of Angptl4 mRNA expression in liver, white adipose tissue (WAT), and brown adipose tissue (BAT) were found to increase in the STZ diabetic mice relative to control mice. This effect was attenuated by insulin administration. In the HFD diabetic mice, the Angptl4 mRNA expression levels were increased in liver, WAT, and BAT. Treatment with metformin for 4 weeks attenuated the increased levels of Angptl4 mRNA. Fatty acids (FAs) such as palmitate and linoleate induced Angptl4 mRNA expression in H4IIE hepatoma cells and 3T3-L1 adipocytes. Treatment with insulin but not metformin attenuated FA-induced Angptl4 mRNA expression in H4IIE. Both insulin and metformin did not influence the effect of FAs in 3T3-L1 cells. CONCLUSION: These observations demonstrated that Angptl4 mRNA expression was increased through the elevated free FAs in diabetic mice.

Palmitate induces apoptosis in Schwann cells via both ceramide-dependent and independent pathways.

Peripheral neuropathy has been reported to prevail in obese or pre-diabetic individuals, yet its etiology remains unknown. Palmitate, a saturated fatty acid increased in obesity and diabetes, is known to induce apoptosis in multiple types of cells and this effect may be mediated by ceramide, a member of the sphingolipid family. To clarify whether de novo ceramide synthesis from palmitate contributes to apoptosis of Schwann cells, we cultured immortalized mouse Schwann cells (IMS) and rat primary Schwann cells with palmitate, a ceramide analogue C2-ceramide as well as inhibitors of the de novo ceramide synthesis (myriocin and fumonisin B1). Apoptosis of IMS detected by nuclear staining and cell membrane inversion was significantly increased by incubation with palmitate for 48 h in a dose-dependent fashion. This enhanced apoptosis was partially but significantly suppressed by myriocin and fumonisin B1. Western blot analysis and immunostaining revealed that palmitate clearly activated caspase-3 in IMS. Unexpectedly, the ceramide synthesis inhibitors failed to suppress the palmitate-induced caspase-3 activation in spite of complete restoration in ceramide accumulation. The results seemed relevant to the observations that C2-ceramide did not activate caspase-3 while provoking apoptosis with a clear dose-dependency. In agreement, the pro-apoptotic action of C2-ceramide was not attenuated by caspase inhibitors that partially suppressed palmitate-induced apoptosis. These results in IMS were well reproducible in rat primary Schwann cells, indicating that the observed phenomena are not specific to the cell line. Collectively, we have reached a conclusion that palmitate induces apoptosis in Schwann cells via both a ceramide-mediated, caspase-3-independent pathway and ceramide-independent, caspase-3-dependent pathways. Given the fact that palmitate and ceramide are increased in obese or pre-diabetic subjects, these lipids may be implicated in the pathogenesis of peripheral neuropathy observed in these disorders.

Angiopoietin-like protein 2 sensitively responds to weight reduction induced by lifestyle intervention on overweight Japanese men.

OBJECTIVE: Overexpression of Angiopoietin-like protein 2 (Angptl2) in obese adipose tissues promotes adipose tissue inflammation and its-related metabolic abnormalities. In a comparative study with adiponectin, we investigated whether alterations in serum Angptl2 concentrations reflect the effect of lifestyle intervention on weight loss and improved metabolic parameters in overweight subjects. METHODS: A total of 154 Japanese men (age, 40.9±5.1 years; body mass index, 26.9±3.6 kg m(-2); abdominal circumference, 94.1±8.9 cm) underwent a 3-month lifestyle intervention and underwent follow-up for 3 months thereafter. RESULTS: Decreased serum Angptl2 levels, but not increased serum adiponectin levels, were immediately apparent at the end of 3-month lifestyle intervention. Angptl2 levels continued to decrease for 3 months in parallel with body weight loss and improvement in metabolic indicators. In subjects showing 6% weight reduction, markedly reduced Angptl2 levels were detected at the end of 3-month intervention, whereas increased adiponectin levels were detected 3 months after the end of intervention. Multivariate analysis revealed changes in serum Angptl2 levels associated with changes in triglycerides (TGs), aspartate aminotransferase and alanine aminotransferase. In contrast, changes in serum adiponectin levels were associated with altered high-density lipoprotein cholesterol (HDL-C) and fasting plasma glucose levels. CONCLUSION: A 3-month lifestyle intervention promoted weight reduction and improved glucose and lipid metabolism, an effect maintained 3 months later. Notably, our findings indicate that decreased Angptl2 levels are a good indicator of reduced visceral fat and metabolic improvement at early stages of lifestyle intervention. Thus, Angptl2 reflects adiposity and might be a key protein to regulate inflammation and TG metabolism, whereas adiponectin levels could reflect improved glucose and HDL-C metabolism.

Protective effect of hedgehog signaling on cytokine-induced cytotoxicity in pancreatic beta-cells.

BACKGROUND: Hedgehog (Hh) signaling plays an important role in pancreas development. However, its role in the developed endocrine pancreas remains to be elucidated. To clarify whether Hh signaling participates in beta-cell survival, we investigated the role of Hh signaling in cytokine-induced apoptosis in pancreatic beta-cells. METHODS: Insulin-producing INS-1E cells were transfected with Sonic Hh (Shh) expression vector or siRNA against Indian Hh (siIhh). The Hh signal inhibitor cyclopamine were pretreated in INS-1E cells and rat islets. The cells were exposed to 200 U/ml IL-1ß and 200 U/ml IFN-γ for 48 h. Apoptosis was estimated by flow cytometory and immunofluorescence staining for cleaved caspase-3. Nitric oxide generation was measured by Griess reaction. RESULTS: We found that exposure to proinflammatory cytokines increased Ihh expression in rat islets and INS-1E cells. Overexpression of Shh reduced cytokine-induced apoptosis. By contrast, treatment with cyclopamine increased cytokine-induced apoptosis in INS-1E cells and rat islets. Treatment with the siIhh showed same results in INS-1E cells. Forced expression of Shh suppressed cytokine-induced nuclear factor-κB promoter activity, leading to attenuation of nitric oxide synthase 2 expression and nitric oxide production, while Ihh knockdown enhanced this pathway in INS-1E cells. CONCLUSION: Our findings suggest that Hh signaling is implicated in protecting beta-cells from cytokine-induced cytotoxicity.

Mechanisms underlying progressive polyuria in familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). The analyses of knock-in mice expressing a mutant NPII that causes FNDI in humans demonstrated that polyuria progressed substantially in the absence of loss of AVP neurones. Morphological analyses revealed that inclusion bodies were present in the AVP neurones in the supraoptic nucleus and that the size and numbers of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) of AVP neurones. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregate formation in the ER is likely to be related to the pathogenesis of the progressive polyuria.

Prevention of neural tube defects by loss of function of inducible nitric oxide synthase in fetuses of a mouse model of streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: Maternal diabetes during pregnancy increases the risk of congenital malformations such as neural tube defects (NTDs). Although the mechanism of this effect is uncertain, it is known that levels of nitric oxide synthase (NOS) and nitric oxide are elevated in embryos of a mouse model of diabetes. We postulated that overproduction of nitric oxide causes diabetes-induced congenital malformations and that inhibition of inducible NOS (iNOS) might prevent diabetic embryopathy. METHODS: Mice were rendered hyperglycaemic by intraperitoneal injection of streptozotocin. The incidence of congenital malformations including NTDs was evaluated on gestational day 18.5. We assessed the involvement of iNOS in diabetes-induced malformation by administering ONO-1714, a specific inhibitor of iNOS, to pregnant mice with streptozotocin-induced diabetic mice and by screening mice with iNOS deficiency due to genetic knockout (iNos(-/-)). RESULTS: ONO-1714 markedly reduced the incidence of congenital anomalies, including NTDs, in fetuses of a mouse model of diabetes. It also prevented apoptosis in the head region of fetuses, indicating that iNOS is involved in diabetes-related congenital malformations. Indeed, no NTDs were observed in fetuses of diabetic iNos(-/-) mice and the incidence of other malformations was also markedly reduced. CONCLUSIONS/INTERPRETATION: We conclude that increased iNOS activity during organogenesis plays a crucial role in the pathogenesis of diabetes-induced malformations and suggest that inhibitors of iNOS might help prevent malformations, especially NTDs, in diabetic pregnancy.

Alpha-lipoic acid reduces congenital malformations in the offspring of diabetic mice.

BACKGROUND: The mechanism of diabetes-induced congenital malformation remains to be elucidated. It has been reported that alpha-lipoic acid (LA) prevents neural tube defects (NTDs) in offsprings of rats with streptozotocin-induced diabetes. Here, we evaluate the protective effect of LA against diabetic embryopathy, including NTDs, cardiovascular malformations (CVMs), and skeletal malformations, in mice. METHODS: Female mice were rendered hyperglycemic using streptozotocin and then mated with normal male mouse. Pregnant diabetic or non-diabetic mice were treated daily with either LA (100 mg/kg body weight) or saline between gestational days 0 and 18. On day 18, fetuses were examined for congenital malformations. RESULTS: Plasma glucose levels on day 18 were not affected by LA treatment. No congenital malformations were observed either in the saline-treated or LA-treated non-diabetic group. In the saline-treated diabetic group, 39% of fetuses had external malformations and 30% had NTDs. In the LA-treated diabetic group, the corresponding proportions were 11 and 8%, respectively. LA treatment also decreased the incidence of CVMs from 30-3% and of skeletal malformations from 29-6%. CONCLUSIONS: We conclude that LA can reduce NTDs, CVMs and skeletal malformations in the offspring of diabetic mice at term delivery.

Tranilast inhibits glucose-induced insulin secretion from pancreatic beta-cells.

Tranilast, N-(3,4-demethoxycinnamoyl)-anthranilic acid, is an anti-allergic agent identified as an inhibitor of mast cell degranulation. Recently, tranilast was shown to decrease albuminuria in a rat model of diabetic nephropathy and to ameliorate vascular hypertrophy in diabetic rats, suggesting that it may be clinically useful in the treatment of diabetic complications. However, the effects of tranilast on glucose tolerance have not been elucidated. Thus, the aim of this study is to investigate the effect of tranilast on insulin secretion in pancreatic beta-cells. Treatment with tranilast significantly suppressed insulin secretion in INS-1E cells and rat islets induced by 16.7 mmol/l glucose. Furthermore, tranilast inhibited tolbutamide-induced insulin secretion. Treatment with tranilast increased (86)Rb (+) efflux from COS-1 cells in which pancreatic beta-cell-type ATP-sensitive K (+) (K (ATP)) channels were reconstructed and suppressed the cytosolic ATP/ADP ratio in INS-1E cells. Interestingly, treatment with tranilast enhanced glucose uptake in INS-1E cells. In the present study, we demonstrated that tranilast inhibited glucose- and tolbutamide-induced insulin secretion through the activation of K (ATP) channels in pancreatic beta-cells.

Impaired parathyroid hormone response to hypocalcemic stimuli in a patient with hypomagnesemic hypocalcemia.

Magnesium (Mg) deficiency sometimes causes hypocalcemia with impaired PTH secretion although the precise mechanism remains unclear. We examined the PTH secretion in response to physiological hypocalcemic stimuli in a patient with hypomagnesemic hypocalcemia. We adopted sodium bicarbonate infusion test, which we recently developed, to evaluate the PTH response to acute decrease in plasma ionized Ca. The results showed that, before Mg replacement and when the patient was mildly hypocalcemic, absolutely no PTH release to hypocalcemic stimuli was observed. In contrast, the plasma Ca was promptly normalized following the start of Mg replacement, and brisk PTH response to hypocalcemic stimuli was obtained during the same test carried out a week after the Mg replacement. The data in this case thus suggest that: a) the acute regulation of PTH release by plasma ionized Ca is lost in the patient with hypomagnesemic hypocalcemia, and b) Mg deficiency itself is likely to be a primary cause of this disorder because the hormone response was clearly restored after shortterm Mg replacement alone.

Aldose reductase gene is associated with diabetic macroangiopathy in Japanese Type 2 diabetic patients.

AIMS: The aldose reductase (AR) gene, a rate-limiting enzyme of the polyol pathway, has been investigated as a candidate gene in determining susceptibility to diabetic microangiopathy. However, the association of the AR gene with diabetic macroangiopathy has not been investigated. Therefore, the present study was conducted to determine whether genetic variations of AR may determine susceptibility to diabetic macroangiopathy. METHODS: There were 378 Type 2 diabetic patients enrolled in this study. A single nucleotide polymorphism in the promoter region (C-106T) was genotyped and the AR protein content of erythrocytes measured by ELISA. RESULTS: There were no significant differences in genotypic or allelic distribution in patients with or without ischaemic heart diseases, but there was a significant increase in the frequency of the CT + TT genotype and T allele in patients with stroke (P = 0.019 and P = 0.012). The erythrocyte AR protein content was increased in patients with the CT and TT genotype compared with those with the CC genotype. After adjustment for age, duration of diabetes, body mass index, systolic blood pressure, HbA1c, and serum creatinine, triglycerides, and total cholesterol in multivariate logistic-regression models, the association between this AR genotype and stroke remained significant. CONCLUSIONS: Our results suggest that the CT or TT genotype of the AR gene might be a genetic marker of susceptibility to stroke in Type 2 diabetic patients. This observation might contribute to the development of strategies for the prevention of stroke in Type 2 diabetic patients.

Human proinsulin C-peptide prevents proliferation of rat aortic smooth muscle cells cultured in high-glucose conditions.

AIMS/HYPOTHESIS: Proinsulin C-peptide is involved in several biological activities. However, the role of C-peptide in vascular smooth muscle cells is unclear. We therefore investigated its effects, in vascular smooth muscle cells in high-glucose conditions. METHODS: Rat aortic smooth muscle cells were cultured with 5.5 or 20 mmol/l glucose with or without C-peptide (1 to 100 nmol/l) for 3 weeks. Proliferation activities, the protein expression of platelet-derived growth factor (PDGF)-beta receptor, the phosphorylation of p42/p44 mitogen-activated protein (MAP) kinases, and glucose uptake were measured. RESULTS: The proliferation activities increased approximately three-fold under high-glucose conditions (p<0.05). C-peptide suppressed hyperproliferation activities that were induced by high glucose. This happened in a dose-dependent manner from 1 to 100 nmol/l of C-peptide. C-peptide (10 and 100 nmol/l) inhibited the increased protein expression of PDGF-beta receptor and the phosphorylation of p42/p44 MAP kinases that had been induced by high glucose (p<0.05). Furthermore, 100 nmol/l of C-peptide augmented the impaired glucose uptake in the high-glucose conditions. CONCLUSIONS/INTERPRETATION: These observations suggest that C-peptide could prevent diabetic macroangiopathy by inhibiting smooth muscle cell growth and ameliorating glucose utilisation in smooth muscle cells. C-peptide may thus be a novel agent for treating diabetic macroangiopathy in patients with type 1 and type 2 diabetes.

Calcium/calmodulin kinase IV pathway is involved in the transcriptional regulation of the corticotropin-releasing hormone gene promoter in neuronal cells.

Although corticotropin-releasing hormone (CRH) plays a pivotal role in the regulation of the hypothalamo-pituitary-adrenal axis, the mechanism of CRH gene expression in the neuronal cell is not completely understood. In this study, we examined the transcriptional regulation of human CRH gene 5'-promoter, using a human BE(2)C neuroblastoma cell line expressing intrinsic CRH. In particular, we focused on the involvement of calmodulin kinases (CaMKs), which are known to play an important role in excitation-induced gene expression through the rise in intracellular calcium in the central nervous system. RT-PCR analysis confirmed the expression of CaMK as well as CRH mRNA in BE(2)C cells. When we introduced approximately 1.1 kb of the 5'-promoter region of the human CRH fused with luciferase reporter gene into the cells, a substantial transcriptional activity was observed, and this was further increased by the activation of the cAMP/PKA pathway. We then examined the effect of activation of CaMKs by introducing the expression vectors of each kinase, revealing a potent stimulatory effect of CaMKIV, but no effect of CaMKII. Depolarization of the cells caused an increase in CRH promoter activity, which was completely abolished by the treatment with the CaMK antagonist K252a. Interestingly, KCREB, a dominant negative form of CREB, antagonized the effect of the CaMKIV-mediated effect. Altogether, we conclude that not only the cAMP/PKA but also the calcium/CaMKIV signaling pathway is involved in the regulation of CRH gene expression. Furthermore, CREB is thought to be involved in CaMK- as well as cAMP/PKA-mediated CRH gene expression. Since the CRH gene is expressed in the neuronal cells of the hypothalamus, the calcium/CaMKIV signaling pathway may play an important role in the excitation-mediated regulation of CRH synthesis.

A novel deletion mutation in the arginine vasopressin receptor 2 gene and skewed X chromosome inactivation in a female patient with congenital nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare inherited disorder caused by mutations in the arginine vasopressin receptor 2 (V2R) gene. The clinical phenotype is fully expressed in hemizygous male patients and is usually asymptomatic in heterozygous females. In the present study, a 51-yr-old Japanese female with congenital NDI and her family members were examined. The patient developed severe hypernatremia accompanied by hypoosmotic polyuria after gynecological surgery, and was unable to concentrate urinary osmolality in response to exogenous vasopressin. Direct sequencing analysis of the propositus and her two affected sons revealed a two-nucleotide deletion change at codon 30 (g.452-453delAC) in the V2R gene, resulting in a frameshift and premature termination in translation at codon 190. The X chromosome inactivation pattern was investigated in the propositus using methylation analysis of the polymorphic CAG repeat in the androgen receptor gene, and the value for relative X chromosome inactivation of one allele was 70.2%. In conclusion, we identified a novel V2R gene mutation in a female patient and her sons with congenital NDI, and her phenotype may be caused by skewed X chromosome inactivation.

The effects of GH-releasing hormone/somatostatin on the 5'-promoter activity of the GH gene in vitro.

The two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF), are known to regulate GH secretion. However, the effects of these hormones on GH gene expression are not completely clear, partly because of the lack of appropriate host cells maintaining the original characteristics of the somatotroph. Since MtT/S, a pure somatotroph cell line, has become available, the effects of GHRH and SRIF on GH gene transcription have been studied using a subclone of MtT/S (MtT/SGL), in which the GH gene 5'-promoter-luciferase fusion gene was stably incorporated. The expression of GHRH receptor and SRIF receptor subtypes was also studied by RT-PCR. The results showed that MtT/SGL cells intrinsically expressed the functional GHRH receptor and all of the SRIF receptor subtypes. The expression of GHRH receptor was markedly enhanced by glucocorticoid pretreatment and, in the presence of corticosterone and 3-isobutyl-1-methylxanthine, GHRH (at or above 100 pM) stimulated GH gene 5'-promoter activity in a dose-dependent manner. On the other hand, SRIF (100 nM) significantly antagonized the effect of GHRH, which was completely reversed by pretreatment with pertussis toxin (50 ng/ml). Taken together, the present data indicated that both GHRH and SRIF are involved in the transcriptional regulation of the GH gene, and that the effect of SRIF is mediated through pertussis toxin-sensitive G protein. The MtT/SGL cell line is a good in vitro model for studying the molecular mechanisms of GH gene transcription by GHRH and/or SRIF.

Involvement of stress-activated protein kinase/c-Jun N-terminal kinase in endothelin-1-induced heat shock protein 27 in osteoblasts.

OBJECTIVE: We have reported that endothelin-1 (ET-1) activates p38 mitogen-activated protein (MAP) kinase through protein kinase C in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase plays a role in the ET-1-induced heat shock protein 27 (HSP27). Recently, we found that stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is activated by ET-1 in these cells. In the present study, we have investigated the involvement of SAPK/JNK in ET-1-induced HSP27 in MC3T3-E1 cells. METHODS: The concentration of HSP27 in soluble extracts of the cells, the expression of mRNA for HSP27, and the phosphorylation of SAPK/JNK were determined by an enzyme immunoassay, Northern blot analysis, and Western blot analysis respectively. RESULTS: SP600125, a specific inhibitor of SAPK/JNK, markedly reduced ET-1-stimulated HSP27 accumulation. The inhibitory effect of SP600125 was dose dependent in the range between 1 and 50 microM. SP600125 reduced the ET-1-increased level of HSP27 mRNA. Calphostin C and Go 6976, inhibitors of protein kinase C, reduced the ET-1-induced phosphorylation of SAPK/JNK. 12-O-Tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C, induced SAPK/JNK phosphorylation, which was suppressed by SP600125. A combination of SP600125 and p38 MAP kinase inhibitor such as SB203580 and PD169316 additively reduced the ET-1-stimulated accumulation of HSP27. CONCLUSIONS: These results strongly suggest that JNK plays a part in ET-1-induced HSP27 in addition to p38 MAP kinase in osteoblasts.

Suppressive effects of a selective inducible nitric oxide synthase (iNOS) inhibitor on pancreatic beta-cell dysfunction.

AIMS/HYPOTHESIS: Type 1 diabetes mellitus is an autoimmune disease characterized by dysfunction and destruction of the pancreatic beta cells. Interleukin-1beta (IL-1beta) has been reported to cause suppression of insulin secretion from pancreatic islets via induction of inducible nitric oxide synthase (iNOS) followed by nitric oxide (NO) production. In this study, we investigated the effects of inhibition of iNOS on pancreatic beta-cell dysfunction in non-obese diabetic (NOD) mice and IL-1beta-treated isolated rat pancreatic islets using a novel specific inhibitor, ONO-1714. METHODS: Female NOD mice which received subcutaneous infusion of ONO-1714 (4 microg/kg/day or 40 microg/kg/day) from 10 to 14 weeks after birth were compared with untreated NOD mice. In addition, pancreatic islets were isolated from Sprague-Dawley rats and cultured for 24 h with IL-1beta (100 U/mL) with or without ONO-1714 or the non-selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). We measured insulin secretion and insulin content of the islets by ELISA, iNOS mRNA expression by reverse transcription-polymerase chain reaction, and NO generation by Griess Reagent System. RESULTS: Hyperglycaemia was observed in NOD mice. ONO-1714 treatment blunted this increase and tended to preserve insulin secretion, although body weight increase did not differ between the groups. Insulitis was also attenuated in the ONO-1714-administered group compared to the control group. Furthermore, in isolated rat pancreatic islets ONO-1714 prevented IL-1beta-induced inhibition of insulin secretion, this protection being evident in much lower concentrations than with L-NMMA. While ONO-1714 completely inhibited IL-1beta-induced NO production, it did not reduce expression of islet iNOS mRNA. CONCLUSION/INTERPRETATION: These findings indicate that ONO-1714 is promising as a therapeutic agent for autoimmune diabetes.

Involvement of SAPK/JNK in basic fibroblast growth factor-induced vascular endothelial growth factor release in osteoblasts.

We previously reported that basic fibroblast growth factor (FGF-2) activates p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in FGF-2-induced VEGF release in these cells. FGF-2 markedly induced the phosphorylation of SAPK/JNK. SP600125, an inhibitor of SAPK/JNK, markedly reduced the FGF-2-induced VEGF release. SP600125 suppressed the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase induced by FGF-2. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase, failed to affect the FGF-2-induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the FGF-2-stimulated VEGF release in an additive manner. These results strongly suggest that FGF-2 activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in FGF-2-induced VEGF release.

Contrasting effects of triiodothyronine on heat shock protein 27 induction and vascular endothelial growth factor synthesis stimulated by TGF-beta in osteoblasts.

In osteoblast-like MC3T3-E1 cells, we recently reported that transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein 27 (HSP27). In the present study, we investigated the effects of triiodothyronine (T(3)) on the TGF-beta-stimulated induction of HSP27 and synthesis of vascular endothelial growth factor (VEGF) in these cells. T(3) by itself had little effect on the level of HSP27, however, it significantly reduced the TGF-beta-stimulated HSP27 accumulation in a dose-dependent manner in the range between 1 pM and 100 nM. The TGF-beta-stimulated increase in the level of mRNA for HSP27 was also attenuated by T(3). On the other hand, T(3), which alone stimulated the release of VEGF, more than additively stimulated the TGF-beta-induced VEGF release. T(3) enhanced the TGF-beta-induced increase in the levels of mRNA for VEGF. These results strongly suggest that T(3) has contrasting effects on HSP27 induction and VEGF synthesis induced by TGF-beta in osteoblasts.

Stimulatory effect of prostaglandin F(2alpha) on Na-dependent phosphate transport in osteoblast-like cells.

Prostaglandin F(2alpha) (PGF(2alpha)) has been reported to activate protein kinase C (PKC) through both phospholipase (PL) C and D, resulting in the proliferation of osteoblast-like cells. In addition, it has also been reported that Erk mitogen-activated protein kinase is also involved in the mechanism of PGF(2alpha)-induced proliferation of these cells. Recently, we have reported that several growth factors stimulate Na-dependent phosphate transport (Pi transport) activity of osteoblast-like cells, which has been recognized to play an important role in their mineralization. In the present study, we investigated the effect of PGF(2alpha) on Pi transport in MC3T3-E1 osteoblast-like cells. PGF(2alpha) stimulated Na-dependent Pi transport dose dependently in the range between 1nM and 10 micro M in MC3T3-E1 cells. The effect was time dependent up to 24h. Kinetic analysis revealed that PGF(2alpha) induces newly synthesized Pi transporter. Pretreatment with actinomycin D and cycloheximide suppressed PGF(2alpha)-induced enhancement of Pi transport. Combined effect of PMA and PGF(2alpha) was not additive in Pi transport. Calphostin C, a PKC inhibitor, dose-dependently suppressed Pi transport induced by PGF(2alpha). On the contrary, U0126, which inhibits an upstream kinase of Erk (MEK), did not affect PGF(2alpha)-induced enhancement of Pi transport. In conclusion, PGF(2alpha) stimulates Pi transport through activation of PKC in osteoblast-like cells.