Mitsuyasu Itoh

Post-transplantation bone diseases negatively affect the quality of life of solid organ recipients. Secondary or tertiary hyperparathyroidism is a frequent complication in kidney transplantation (KTx) recipients. Treatment with immunosuppressive agents including glucocorticoids can lead to deterioration in bone metabolism in these patients. In the present study, we explored the effects of a three-year treatment period with oral alendronate (ALN) in long-term KTx recipients. Post-KTx recipients were recruited (n = 24, M/F = 12/12, mean age 52.0 +/- A 7.8 years) into this study. All patients were prescribed methylprednisolone (4.07 +/- A 0.86 mg/day) with various immunosuppressive agents. Before treatment with oral ALN (35 mg/week), the mean concentrations of intact parathyroid hormone (iPTH) and 25-hydroxyvitamin D were 139.2 +/- A 71.4 pg/mL and 20.8 +/- A 4.1 ng/mL, respectively. After 36 months of ALN treatment, mean iPTH levels increased slightly (+20.9 %). Treatment with ALN reduced bone-specific alkaline phosphatase (-35.4 %), serum type I collagen N-terminal telopeptide (-31.2 %) and osteocalcin (-55.6 %) levels. ALN did not increase bone mass after 24 months. Four patients with the highest baseline iPTH levels suffered a clinical osteoporotic fracture during the 36-month ALN treatment period. Higher iPTH levels with chronic kidney disease (CKD) at baseline were associated with the incidence of new clinical fractures during ALN treatment. In conclusion, anti-resorptive therapy with ALN can suppress bone turnover even when iPTH concentration is elevated in long-term KTx recipients. However, hyperparathyroidism with CKD seems to be associated with new clinical fractures during ALN treatment.

Aripiprazole is the only atypical antipsychotic drug known to cause the phosphorylation of AMP-activated protein kinase (AMPK) in PC12 cells. However, the molecular mechanisms underlying this phosphorylation in aripiprazole-treated PC12 cells have not yet been clarified. Here, using PC12 cells, we show that these cells incubated for 24 h with aripiprazole at 50 mu M and 25 mM glucose underwent a decrease in their NAD(+)/NADH ratio. Aripiprazole suppressed cytochrome c oxidase (COX) activity but enhanced the activities of pyruvate dehydrogenase (PDH), citrate synthase and Complex I. The changes in enzyme activities coincided well with those in NADH, NAD(+), and NAD(+)/NADH ratio. However, the bioenergetic peril judged by the lowered COX activity might not be accompanied by excessive occurrence of apoptotic cell death in aripiprazole-treated cells, because the mitochondrial membrane potential was not decreased, but rather increased. On the other hand, when PC12 cells were incubated for 24 h with clozapine at 50 mu M and 25 mM glucose, the NAD(+)/NADH ratio did not change. Also, the COX activity was decreased; and the PDH activity was enhanced. These results suggest that aripiprazole-treated PC12 cells responded to the bioenergetic peril more effectively than the clozapine-treated ones to return the ATP biosynthesis back toward its ordinary level. This finding might be related to the fact that aripiprazole alone causes phosphorylation of AMPK in PC12 cells.

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc (1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc (1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc (1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc (1638T/1638T) and Apc (+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc (1638T/1638T) mice were significantly different whereas the Apc (+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc (1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc (+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc (1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc (+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc (1638T/1638T) mice and suggest suppressed secretory activities of these cells.

Background: Interactions between CD40 and its ligand (CD40L) have important roles in T-cell-dependent activation of B cells, which may be related to the thyrotoxic activity of Graves' disease (GD). Soluble forms of CD40 ligand (sCD40L) are released from activated T cells and platelets, and several types of inflammatory cytokines are increased in patients with hyperthyroid GD. The aim of this study was to assess sCD40L and other cytokines as clinical indicators of disease activity or as possible markers of remission in GD. Methods: Serum levels of sCD40L, interleukin 18 (IL-18), tumor necrosis factor-alpha (TNF alpha), and TNF alpha receptors 1 and 2 (TNFR1 and TNFR2) were investigated in patients with active GD (GD-A), intractable GD (GD-IT), inactive GD (GD-IA), GD in remission (GD-R), and Hashimoto's thyroiditis (HT), and in control subjects (CON). Results: Serum concentrations of sCD40L were higher in the GD-A and GD-IT groups than in the HT and CON groups. Similarly, serum concentrations of IL-18, which induces Th1 cytokines, such as interferon-gamma, were higher in the GD-A and GD-IT groups than in all other groups. Serum levels of TNFR1 and TNFR2 were also significantly higher in the GD-A than in all other groups. The mean serum concentration of TNF alpha was higher in the GD-R compared with the GD-A and GD-IT groups, although the difference was not significant. Serum sCD40L concentrations in the GD-R group were lower than in the GD-A and GD-IT groups. Finally, the ratio of serum TNF alpha to sCD40L was higher in the GD-R group than in the GD-A and GD-IT groups. This is the first report that serum sCD40L is increased in active GD, and that the serum TNF alpha:sCD40L ratio is a marker for remission in GD. Conclusions: Our results suggest that not only thyrotoxicosis, but also the activity of the immunoreaction presenting as anti-thyrotropin receptor antibodies (TRAb) titer in GD, affects inflammatory cytokine serum profiles. Serum profiles of cytokines vary in patients with GD depending on disease activity. An elevated serum TNF alpha:sCD40L ratio indicates declining disease activity and reflects a shift from Th2 to Th1 dominance, suggesting that suppression of sCD40L or increased production of TNF alpha is required to initiate or maintain remission of GD.

Background: Chronic elevation of cardiac troponin T (TnT) levels as measured by conventional assays is strongly associated with structural heart disease and cardiovascular events. A new high-sensitivity assay for TnT makes it possible to measure concentrations more than a factor of 5 lower than the limits of detection of conventional assays. We evaluated the utility of serum TnT as a risk marker of cardiovascular disease in 409 outpatients with type-2 diabetes mellitus (T2DM). Results: TnT was detectable (&gt 0. 002 ng/mL) in 80% of patients, and elevation in TnT levels (&gt 0. 014 ng/mL) was found in 19. 3%, suggesting a higher prevalence of structural heart diseases in T2DM patients. A history of cardiovascular disease was noted in 89 (22%) patients. Patients with diabetic microvascular complications and those with abnormal electrocardiogram including left ventricular hypertrophy had higher TnT levels. Patients with increased levels of TnT (&gt 0. 014 ng/mL) were older, had higher values of N-terminal pro-B-type natriuretic peptide (NT-proBNP), C-reactive protein, cystatin C, and urinary albumin/creatinine ratio, had lower values of hemoglobin and estimated glomerular filtration rate, and had a higher prevalence of cardiovascular disease compared with those without increased TnT levels. In stepwise logistic analysis, NT-proBNP (odds ratio 7. 40 per 10-fold increase, P &lt 0. 0001) and cystatin C (18. 0 per 1. 0 mg/L, P &lt 0. 0001) were independently associated with elevation of TnT levels. HsTnT level, cystatin C, and HDL-cholesterol were also independent risk factors for history of major cardiovascular diseases in T2DM patients. Conclusion: This new high-sensitivity TnT assay may be useful for stratifying cardiovascular risk in outpatients with T2DM. © 2011 The Japan Diabetes Society.

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc (1638T/1638T) mice, which express a truncated Apc that lacks the C-terminal domain. Apc (1638T/1638T) mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc (1638T/1638T) mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc (1638T/1638T) and Apc (+/+) mice. Thyroid follicle size was significantly larger in Apc (1638T/1638T) mice than in Apc (+/+) mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc (1638T/1638T) mice than in Apc (+/+) mice. TSH also induced a greater reduction in thyroid follicle size in Apc (1638T/1638T) mice than in Apc (+/+) mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc (1638T/1638T) mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.

The present study was undertaken to compare the efficacy of pitavastatin and colestimide in patients with diabetes mellitus complicated by hyperlipidemia and metabolic syndrome. 48 diabetic patients with metabolic syndrome were randomly assigned to a pitavastatin group or colestimide group. The clinical parameters, serum lipids, fasting (FPG) and postprandial plasma glucose(PPG), HOMA-IR, hemoglobin A1c(HbA1c), hs-CRP and urinary albumin were measured before/after 24-week administration. Treatment with pitavastatin reduced LDL-C and TG, while that with colestimide significantly reduced waist circumference, BMI, LDL-C, HbA1c, FPG, PPG, HOMA-R, hs-CRP and urinary albumin. Percent improvement in LDL-C was greater in the pitavastatin group than in the colestimide group. Colestimide appeared to be useful in the management of Japanese patients with diabetes mellitus complicated by metabolic syndrome, since it alleviates obesity and insulin resistance in addition to exhibiting lipid profile-improving effects, and can thus improve markers of atherosclerosis.

Inorganic phosphate (Pi) is required in many biological processes, including signaling cascades, skeletal development, tooth mineralization, and nucleic acid synthesis. Recently, we showed that Pi transport in osteoblasts, mediated by Slc20a1, a member of the type III sodium-dependent phosphate transporter family, is indispensable for osteoid mineralization in rapidly growing rat bone. In addition, we found that bone mineral density decreased slightly with dysfunction of Pi homeostasis in aged transgenic rats overexpressing mouse Slc20a1 (Slc20a1-Tg). Bone and tooth share certain common molecular features, and thus, we focused on tooth development in Slc20a1-Tg mandibular incisors in order to determine the role of Slc20a1 in tooth mineralization. Around the time of weaning, there were no significant differences in serologic parameters between wild-type and Slc20a1-Tg rats. However, histological analysis showed that Slc20a1-Tg ameloblasts formed clusters in the papillary layer during the maturation stage as early as 4 weeks of age. These pathologies became more severe with age and included the formation of cyst-like or multilayer ameloblast structures, accompanied by a chalky white appearance with abnormal attrition and fracture. Hyperphosphatemia was also observed in aging Slc20a1-Tg rats. Micro-computed tomography and electron probe microanalysis revealed impairments in enamel, such as delayed mineralization and hypomineralization. Our results suggest that enamel formation is sensitive to imbalances in Pit1-mediated cellular function as seen in bone, although these processes are under the control of systemic Pi homeostasis.

Serum 25-hydroxyvitamin D (25-OHD) concentrations are thought to accurately reflect vitamin D stores, and vitamin D deficiency causes secondary hyperparathyroidism, irreversible bone loss, and increased risk of fracture. Recent studies suggest that decrease of serum 25-OHD level in mothers could increase the risk of preeclampsia, cesarean section, and craniotabes. Furthermore, this deficiency may affect bone mass and the incidence of neuromuscular diseases of their children in the future. In the present study, the serum concentration of 25-OHD in 93 pregnant women after the 30th week of their gestation was determined by direct radioimmunoassay. Mean 25-OHD levels in spring, summer, fall, and winter were 14.3 +/- 5.1, 15.7 +/- 6.4, 13.7 +/- 5.1, and 13.9 +/- 4.2 ng/ml, respectively. Severe vitamin D deficiency (25-OHD < 10 ng/ml) was found in 10 of these 93 women. Overall, hypovitaminosis D, which was defined as serum 25-OHD concentration equal to or less than 20 ng/ml, was revealed in 85 mothers (89.5%). Serum 25-OHD levels were not associated with either intact parathyroid hormone or corrected calcium concentrations, but were negatively associated with serum type I collagen N-terminal telopeptide and bone-specific alkaline phosphatase in these subjects. Mothers with threatened premature delivery had significantly lower 25-OHD levels (11.2 +/- 3.2 ng/ml) than those in mothers with normal delivery (15.6 +/- 5.1 ng/ml). In conclusion, the present data suggest a high prevalence of hypovitaminosis D in perinatal pregnant Japanese women throughout the year, which seems to affect bone metabolism and to be associated with threatened premature delivery.

Sekiguchi S, Suzuki A, Asano S, Nishiwaki-Yasuda K, Shibata M, Nagao S, Yamamoto N, Matsuyama M, Sato Y, Yan K, Yaoita E, Itoh M. Phosphate overload induces podocyte injury via type III Na-dependent phosphate transporter. Am J Physiol Renal Physiol 300: F848-F856, 2011. First published February 9, 2011; doi:10.1152/ajprenal.00334.2010.-Uptake of Pi at the cellular membrane is essential for the maintenance of cell viability. However, phosphate overload is also stressful for cells and can result in cellular damage. In the present study, we investigated the effects of the transgenic overexpression of type III Pi transporter Pit-1 to explore the role of extracellular Pi in glomerular sclerosis during chronic renal disease. Pit-1 transgenic (TG) rats showed progressive proteinuria associated with hypoalbuminemia and dyslipidemia. Ultrastructural analysis of TG rat kidney by transmission electron microscopy showed a diffuse effacement of the foot processes of podocytes and a thickening of the glomerular basement membrane, which were progressively exhibited since 8 wk after birth. TG rats died at 32 wk of age due to cachexia. At this time, more thickening of the glomerular basement membrane and segmental sclerosis were observed in glomeruli of the TG rats. Immunohistochemical examination using anticonnexin 43 and anti-desmin antibodies suggested the progressive injury of podocytes in TG rats. TG rats showed higher Pi uptake in podocytes than wild-type rats, especially under low Pi concentration. When 8-wk-old wild-type and TG rats were fed a 0.6% normal phosphate (NP) or 1.2% phosphate (HP) diet for 12 wk, HP diet-treated TG rats showed more progressive proteinuria and higher serum creatinine levels than NP diet-treated TG rats. In conclusion, our findings suggest that overexpression of Pit-1 in rats induces phosphate-dependent podocyte injury and damage to the glomerular barrier, which result in the progression of glomerular sclerosis in the kidney.

The type III inorganic phosphate (Pi) transporter Pit-1 was previously found to be preferentially expressed in developing long bones. Several studies also described a regulation of its expression in cultured bone cells by osteotropic factors, suggesting a role of this transporter in bone metabolism. In the present study, we investigated the effects of the transgenic overexpression of Pit-1 in Wistar male rats on calcium phosphate and bone metabolism. A threefold increase and doubling of Pi transport activity were recorded in primary cultured osteoblastic cells derived from calvaria of two transgenic (Tg) lines compared with wild-type littermates (WT), respectively. Skeletal development was not affected by the transgene, and bone mass, analyzed by DXA, was slightly decreased in Tg compared with WT. Enhanced Pi uptake in calvaria-derived osteoblasts from Pit-1 Tg was associated with a significantly decreased expression of alkaline phosphatase activity and a normal deposition and calcification of the collagenous matrix. In 4-month-old adult Tg rats, serum Pi and renal Pi transport were increased compared with WT. The decrease of serum Ca concentration was associated with increased serum parathyroid hormone levels. Variations in serum Pi in Pit-1 Tg rats were negatively correlated with serum fibroblast growth factor-23, whereas 1,25-dihydroxyvitamin D(3) was not affected by Pit-1 overexpression. In conclusion, transgenic Pit-1 overexpression in rats affected bone and calcium phosphate metabolism. It also decreased alkaline phosphatase activity in osteoblasts without influencing bone matrix mineralization as well as skeletal development.

The aim of this study was to determine whether a relatively low dose of pioglitazone or metformin was effective in diabetic patients with metabolic syndrome. Fifty diabetic patients with metabolic syndrome were randomly assigned to a low-dose pioglitazone (15mg/day) treatment group or a low-dose metformin (500mg/day) treatment group. Drugs were administered for 12 weeks. Systolic and diastolic blood pressure, heart rate, body mass index, triglyceride (TG), HDL and LDL-cholesterol, fasting plasma glucose (FPG), fasting plasma insulin (IRI), postprandial glucose, and HOMA-IR in the 75gOGTT, HbA1c, high-sensitivity CRP (hs-CRP) determined by cervical artery echography, and pulse wave velocity (PWV) were measured before/after 12-week drug administration. Significant decreases in HbA1c and HOMA-IR were noted in the pioglitazone group, along with significant decreases in TG, AST, ALT, blood pressure, hs-CRP and PWV. Significant decreases in HbA1c, HOMA-IR, BMI and waist circumference were noted in the metformin group. The pioglitazone group significantly improved the values for ALT, systolic blood pressure, hs-CRP and PWV compared to the metformin group. However, the metformin group demonstrated significant improvement in BMI compared with the pioglitazone group. Using a low dose regimen, pioglitazone significantly improved blood pressure and hepatic function and may be more effective than metformin to reduce risk factors in Japanese diabetic patients with metabolic syndrome at preventing atherosclerosis.

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D-2 (PGD(2)) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD(2) stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD(2) on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD(2)-induced Pi uptake. The inhibitors of phosphaticlylinositol (PI) 3-kinase and S-6 kinase reduced this effect of PGD(2), while Akt kinase inhibitor did not. These results suggest that PGD(2) stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S-6 kinase. (C) 2009 Elsevier Ltd. All rights reserved.

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (-T3 and -T4) and human thyroglobulin (hTg) contained in the thyroid follicles, -T3, -T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, -T3 and -T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC50 of hTg: ca 0.45M: inhibiting concentration of hTg was from 0.15M to 3.0M). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.

Context and objective: Arterial stimulation and venous sampling (ASVS) is an important technique for localizing insulinoma. The principle behind ASVS is that insulin secretion is promoted from insulinoma cells by the injection of calcium into the insulinoma-feeding artery. However, the mechanism for ASVS-induced insulin secretion remains unclear. Both insulnoma and familial hypocalciuric hypercalcemia (FHH) are rare diseases. This study reports on a case in which both of these diseases occur concurrently. Design and patient: The patient with FHH also suffered from insulinoma. We reasoned that insulin secretion for ASVS is dependent on the calcium-sensing receptor (CaSR). ASVS was performed on this patient. The expression of the CaSR protein and corresponding mRNA were confirmed. Results: No significant changes in the plasma levels of insulin and C-peptide were observed during ASVS. The patient was clinically diagnosed as having FHH. We confirmed that a mutation in the CaSR gene was present in the genomic DNA of this patient and that there were no mutations in the multiple endocrine neoplasia type I gene. In addition, expression of both CaSR mRNA and CaSR protein was confirmed in the insulinoma samples. Conclusion: These results suggest that the CaSR gene is involved in ASVS-induced insulin secretion.

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH4 biosynthesis and is widely used in order to elucidate the possible biological function of BH4 in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH4) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH4 might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH4 and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH4 level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH4 level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH4 production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH4-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappa B (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappa B level in the nucleus of HUVEC. (C) 2008 Elsevier B.V. All rights reserved.

Aims A rare case of the insulin autoimmune syndrome (IAS) accompanied by insulin receptor anomaly is reported. Methods Antibodies to insulin and insulin receptor were determined in the patient with severe hypoglycaemia before and after the treatment with prednisolone. Results Titers of antibody to insulin and insulin receptors were 73.0% and 41.5%, respectively. Drug-induced lymphocyte stimulation tests were all negative for the suspicious drugs. Her HLA-DR was DRB1*0403/04051. Following steroid therapy, the formation of antibodies was suppressed and alleviated her symptoms. Scatchard analysis yielded findings specific to polyclonal antibodies. Conclusions The changes in autoantibodies resulted in alleviation of the hypoglycemic symptoms as a result of steroid therapy.

Introduction: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. Materials and Methods: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). Results: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V-max of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1. but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. Conclusions: Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1. cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.

Aldolase, a glycolytic enzyme, consists of 3 immunologically different subunits as the tetrameric form and may correlate with cell carcinogenesis. In the present study, we investigated the role of aldolase isozymes in the thyroid tissue of various thyroid disorders using immunohistochemical staining and highly sensitive enzyme immunoassay for quantitative analysis of the isozymes. Aldolase A had low tissue specificity but correlated with tumor proliferation. The relation to tumor proliferation, however, was found only in a few cases of the thyroid tumors. Aldolase B was barely detectable in these thyroid tissues. Aldolase C, however, had high activities suggesting the high thyroid tissue specificity, particularly for parafollicular cells. It was considered to be a useful marker of tumor cell differentiation. The analysis of these isozymes may be helpful for the diagnosis of medullary thyroid carcinoma and neuroendocrine tumors.

We conducted an observational study in order to assess the prevalence of hypovitaminosis D and its seasonal changes, in the Tokai area (N35.3 E137.0), in 197 normal subjects in Japan. The mean serum 25-hydroxyvitamin D (25-OHD) level measured by direct radioimmunoassay (RIA) was lowest at the end of winter, and highest at the end of summer (15.1 +/- 7.1 ng/ml in March; 21.5 +/- 5.5 ng/ml in June; 31.6 +/- 5.6 ng/ml in September; 23.1 +/- 5.3 ng/ml in December; mean +/- SD). The prevalence of hypovitaminosis D ( < 20 ng/ml) was 86.7%, 33.4%, 1.0%, and 26.0% in March, June, September, and December, respectively. Mean plasma intact parathyroid hormone (iPTH) concentration was lowest at the end of summer and highest at the end of winter (28.2 +/- 9.3 pg/ml in March; 21.7 +/- 7.0 pg/ml in June; 19.8 +/- 6.9 pg/ml in September; and 25.7 +/- 9.2 pg/ml in December; mean +/- SD). Serum 25-OHD was inversely associated with iPTH ( coefficient, - 0.223; r = 0.251; P < 0.001). Serum 25-OHD levels were higher in men than in women. The serum 25-OHD level was positively associated with age, body weight, and body mass index, but not with body fat content. These results suggest a high prevalence of hypovitaminosis D associated with elevation of iPTH in Japan, in winter, even in a sunny area.

Cyclic nucleotides (cAMP and cGMP) phosphodiesterase (PDE) activities and expression are altered in the cardiac muscle of cardiomyopathic heart failure, and PDE inhibitors improve the abnormal muscle condition through changing the cyclic nucleotide concentration. These observations prompted us to investigate the role of calmodulin (CaM) in the regulation of cyclic nucleotide PDE activities, and moreover to study the modulation of the PDE isozymes in heart failure, using cardiac muscles of cardiomyopathic hamster. The CaM concentrations in the heart muscle of the normal control and cardiomyopathic hamsters (each of three to four hamsters) varied with cell fraction and with the age of the animal. The CaM concentrations in the soluble fraction obtained from cardiomyopathic hamster tissue were significantly increased at 25 and 32 weeks of age (2.02 +/- 0.62 mug/mg protein (mean S.E.), and 3.21 +/- 0.95) compared with that obtained from the control (0.60 +/- 0.04) or cardiomyopathic (0.95 +/- 0.12) hamsters at 8 weeks of age. The solubilized PDE isolated from the hamster heart muscle (three or four hamsters in each age) by column chromatography on diethylaminoethyl (DEAE)-cellulose revealed three peaks of activity, which may correspond to the isozymes of PDE classified recently, namely PDE I, II, and III. These three peaks of activity, particularly peak 111, seen in the soluble fraction of cardiomyopathic hamster heart declined in proportion to the age of the animal compared with that of the control hamster heart. In the cGMP-PDE assay system, the concentration of CaM inhibitor W-7 required for 50% inhibition (IC50) of PDE 1, 11, and III peak activities was 140, 29, and 46 muM respectively, suggesting that PDE 11 is more sensitive to W-7. These results suggest that alteration in these isozyme activities accompanied with changes of CaM concentration may influence the cardiac muscle contractility in cardiomyopathic hamster via changes of cyclic nucleotide concentration. (C) 2004 Elsevier Ltd. All rights reserved.

ErbB family members, such as epidermal growth factor receptor 1 (erbB1), erbB2, erbB3 and erbB4, are widely distributed in organ tissues, and these receptors are suspected tumorigenesis factors. We measured erbB mRNA in thyroid tissues of benign and malignant thyroid tumors or Graves' disease using Genescan. ErbB2 is associated with aggressive cancers and is used as a biological marker for the disease; Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses have shown it to be increased in Graves' disease. Additional studies indicated a similar result in papillary carcinoma cells; mRNAs of crbB2 and erbB3 were increased but erbB4 mRNA was decreased, suggesting distorted erbB expression may be associated with tumorigenesis. However, only erbB2 overexpression is associated with Graves' disease. These data further implicate transmembrane-type receptors in tumorigenesis in the thyroid. (C) 2004 Elsevier B.V. All rights reserved.

Aims: To confirm whether a prostacyclin (prostaglandin 12) affects the increased TNF-alpha concentration in sera of diabetic patients, we measured serum TNF-alpha concentration and treated these patients with oral administration of the stable prostacyclin analogue (Beraprost). Twelve of 20 type 11 diabetic patients were investigated for follow up-study and 6 of those patients were for therapy with Beraprost for diabetic neuropathy. Subjects and Methods: Serum TNF-alpha concentration was quantified by EASIA using monoclonal antibodies directed against distinct epitopes of TNF-alpha. Results: In diabetic patients, serum TNF-alpha concentration was significantly increased compared with that of healthy subjects. The augmented TNF-alpha concentration in these patients was not decreased by diabetic control using antihyperglycemic agents for 8 weeks but was reduced with oral administration of a stable prostacyclin (prostaglandin 12) analogue for 5 weeks without any changes of blood glucose levels. Conclusions: Stable prostacyclin analogue administration for a short term period reduced increased TNF-alpha levels in diabetic patients, not through the improved hyperglycemic condition but another pathway, probably a cAMP system. These results imply that treatment with the prostacyclin analogue may contribute to the prevention of progression in diabetic complications.

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E-1 (PGE(1)) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE(1) in MC3T3-E1 osteoblast-like cells. PGE(1) dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 muM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE(1). Further analysis with western blotting suggested that PGE(1) induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt(2)cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE(1)-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE(1) stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity. (C) 2003 Elsevier Ltd. All rights reserved.

We investigated the effect of platelet-derived growth factor B homodimer (PDGF-BB) on inorganic phosphate (Pi) transport activity, which has been reported to be involved in the mechanism of atherosclerosis, in A-10 rat aortic vascular smooth muscle cells (VSMCs). PDGF-BB time- and dose-dependently stimulated Pi transport in A-10 cells. Using northern blot analysis, the PDGF-BB-enhanced Pi transporter (PiT) in A-10 cells was identified as Pit-1 (Glvr-1), a member of the type III Na-dependent PiT. An inhibitor of PDGF P-receptor tyrosine kinase suppressed PDGF-BB-induced Pi transport. Both a protein kinase C (PKC) inhibitor calphostin C and PKC down regulation suppressed the stimulatory effect of PDGF-BB on Pi transport. On the other hand, inhibition of mitogen-activated protein (MAP) kinases by selective inhibitors did Dot affect Pi transport. Ly294002, a phosphatidylinositol (PI) 3-kinase inhibitor, partially attenuated PDGF-BB-induced Pi transport. A selective inhibitor Of S-6 kinase, rapamycin, reduced this effect of PDGF-BB, while Akt kinase inhibitor did not. In summary, these results indicated that PDGF-BB is a potent and selective stimulator of Pi transport in VSMCs. The mechanism responsible for this effect is not mediated by MAP kinase, but involves activation of PKC, PI 3-kinase and S-6 kinase. (C) 2004 Published by Elsevier Ireland Ltd.

Understanding the causes of diabetic vascular complications has become an increasingly important issue because of the rapidly rising prevalence of diabetes. Recently discovered vasoconstrictors and angiogenesis regulators, such as endothelin (ET) and vascular endothelial growth factor (VEGF), have been intensely studied for possible pathogenic roles in diabetic vascular complications. The present study was undertaken to clarify the effect of glycemic control on serum VEGF and plasma ET-1 concentrations in diabetic patients, and to identify other factors that may cause fluctuations of these substances. Plasma VEGF and ET-1 concentrations of 45 hospitalized diabetic patients and 54 control subjects were measured by enzyme immunoassay (EIA) and radioimmunoassay (RIA), respectively. Plasma VEGF was elevated in poorly controlled diabetic patients compared with healthy subjects and plasma VEGF concentrations declined after hospitalized treatment with either insulin or oral hypoglycemic agents in combination with diet. There was a significant correlation between plasma VEGF concentration and both fasting plasma glucose (FPG) and hemoglobin A(1c) (HbA(1c)). Plasma ET-1 in poorly controlled diabetic patients was higher than in healthy controls, but improved glycemic control did not affect plasma ET-1 concentrations. Thus, poor glycemic control causes increased levels of plasma VEGF, which may result in hypertension and vascular complications in diabetes. Short-term treatment resulting in good glycemic control can improve levels of VEGF and may provide beneficial effects on diabetic vascular complications. (C) 2004 Elsevier Inc. All rights reserved.

Tetrahydrobiopterin is an essential cofactor for nitric oxide synthase (NOS). This study was undertaken to examine the effects of intraperitoneally injected lipopolysaccharide on tetrahydrobiopterin biosynthesis in murine white and brown adipose tissues. Tetrahydrobiopterin content, catalytic activity and mRNA expression level of GTP cyclohydrolase I (GCH), rate-controlling enzyme in de novo biosynthesis of tetrahydrobiopterin, in both adipose tissues were up-regulated by 500-gg lipopolysaccharide at 6 h after the injection. On the contrary, treatment of 3T3-L1 adipocytes with lipopolysaccharide alone did not affect GCH mRNA expression level, whereas the combination of lipopolysaccharide, tumor necrosis factor (TNF)-alpha, and interferon gamma induced the increase in expression levels of GCH mRNA and CD14 mRNA. Collectively, our results showed that tetrahydrobiopterin biosynthesis can be augmented by increased GCH activity caused by a synergistic effect of lipopolysaccharide and cytokines in white and brown adipose tissues. These observations support the view that tetrahydrobiopterin biosynthesis in the adipose tissues is a target of inflammatory events triggered by peripheral LPS injection. (C) 2004 Elsevier B.V All rights reserved.

The amounts of sorbitol (SOR) excreted in 24-h urine were determined on two groups, i.e., diabetic and nondiabetic patients, using an improved method in which ion exchange resin column processing was applied, and these levels were compared with SOR levels in whole blood. Urinary SOR concentration was also determined in diabetic and normal rats in the same manner and its relationship to aldose reductase (AR) activity in whole blood was investigated. Changes in SOR levels in urine and whole blood were compared in diabetic rats after administration of an AR inhibitor (ARI). Whole blood SOR levels and urinary SOR excretion were significantly higher in diabetic patients than in nondiabetic patients. The same results were obtained in the animal models. In diabetic rats, the urinary SOR excretion was about five times higher than that in control rats, and the AR activity in whole blood was also significantly higher. The increase in urinary SOR excretion and whole blood SOR levels, as well as AR activity, in blood in the diabetic state was inhibited by ARI administration. The influence of the diabetic state and the efficacy of the ARI were more marked in urinary SOR excretion than in whole blood SOR levels. These data indicate that determinations of urinary SOR excretion and AR activity are easily measurable and of benefit to assessing the diabetic condition. (C) 2003 Elsevier Inc. All rights reserved.

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 ostcoblast-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 MC3T3E- E1 osteoblast-like cells. PGF(2alpha) stimulated Na-dependent Pi transport dose dependently in the range between 1 nM and 10 muM 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. (C) 2003 Elsevier Science Ltd. All rights reserved.

The 112/121 haplotype combination defined by the UCSNP-43, -19, and -63 alleles in the calpain-10 gene is associated with type 2 diabetes in Mexican Americans. To determine whether this genetic variation constitutes risk of type 2 diabetes in Japanese, we investigated its frequency in 177 patients with type 2 diabetes and 172 controls. Though this variation occurs in Japanese more frequently than in Mexican Americans, there is no significant difference in frequency between diabetic (29.9%) and control (31.9%) subjects. We also screened all exons and the putative promoter of the calpain-10 gene for mutations in 96 of the genotyped patients, resulting in the identification of 7 coding variants, including 3 missense mutations and 5 nucleotide alterations in the promoter. However, their frequencies all are similar in patients and controls, suggesting that these genetic variations are not a major factor in the occurrence of type 2 diabetes in Japanese, although they could yet. be associated with various phenotypes of the disease.

In view of the fact that a deficient calcium (Ca) intake results in osteoporosis in elderly males, we conducted an animal experiment on aged male Wistar rats given a Ca-deficient diet. The rats were divided into 2 groups according to diet: a Ca-deficient diet group (Ca content, 0.08% to 0.1%) and a regular diet group (Ca content, 0.8% to 1.2%). The Ca-deficient diet reduced bone mineral density (BMD) by approximately 12%. Administration of menatetrenone or elcatonin was able to reverse the reduction in BMD induced by Ca deficiency. The mean estradiol level in sera of rats fed the Ca-deficient diet was significantly increased to 4.3 times that in the regular diet group. However, the increased estradiol concentration was reduced after the administration of menatetrenone or elcatonin. The estrone concentrations in sera of menatetrenone- or elcatonin-treated rats fed the Ca-deficient diet decreased to a level lower than that of animals fed the regular diet. Testicular aromatase cytochrome P450 (P450(arom); estrogen synthetase) activity was significantly increased by 2.4-fold in the Ca-deficient diet group compared to that in the regular diet group, and the aromatase mRNA level was also significantly increased 1.45-fold. Testicular aromatase activity was strongly correlated with aromatase mRNA level and serum estradiol level. These data suggest that the change in testicular aromatase expression might be, in part, a compensatory mechanism for the bone mineral deficiency induced by the Ca-deficient diet in aged male rats. Copyright 2002, Elsevier Science (USA). All rights reserved.

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8+ cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th-1/Th-2 balance to Th-2 dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th2 cells. (C) 2002 Elsevier Science Ltd. All rights reserved.

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8+ cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th-1/Th-2 balance to Th-2 dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th2 cells. (C) 2002 Elsevier Science Ltd. All rights reserved.

In general, many cases of malignancy-associated hypercalcemia are due to HHM. In patients with humoral hypercalcemia of malignancy (HHM), it has been reported that plasma parathyroid hormone-related protein (PTHrP) and cyclic adenosine monophosphate (cAMP) levels were elevated, while plasma PTH and active vitamin D-3 levels were suppressed. Our patient showed hypercalcemia with a concurrent increase in plasma and tumor tissue PTHrP and PTH concentrations and also high cAMP and low 1-25(OH)(2)VD3 levels in the plasma. These data suggest that the hypercalcemia exhibited by our patient was consistent with HHM due to lung cancer and its liver metastasis. Moreover, diagnostic imaging and autopsy findings showed no appreciable lesions of the parathyroid gland. In addition, histopathologic examination of the primary and metastatic tumors revealed the existence of PTH immunohistochemically stained with anti-PTH antibodies, suggesting an ectopic-PTH-producing lung tumor. From these data, our patient was diagnosed with a rare case of lung cancer, which produced both ectopic PTH and PTHrP. Copyright 2002, Elsevier Science (USA). All rights reserved.