Comparative study of the usefulness of a novel insulin therapy in Japanese patients with Type 2 diabetes for concomitant use of an oral antidiabetic agent with twice-daily dosing either of insulin aspart, biphasic insulin aspart-30, or insulin detemir: Two times Insulin injection Combined with oral therapy Efficacy Study (TWICE study).

OBJECTIVES: It is common to treat type 2 diabetes by regular injections of insulin. We compared the efficacy and safety of twice-daily administration of short-acting, premixed, and long-acting insulins. METHODS: This was a multi-center, randomized, open-label, 52-week study. Patients were randomized to administer twice daily short-acting analog insulin (Aspart) plus a sulfonylurea (SU), premixed 70/30 analog insulin (Mix), or long-acting insulin (Detemir) plus a glinide derivative. RESULTS: Twelve (mean baseline HbA1c 9.86±1.71%), eight (9.24±1.14%), and eight (11.26±1.81%) patients were treated with Aspart, Mix, or Detemir, respectively, for 52 weeks. After 12 weeks, the reductions in HbA1c were similar in the groups. A further significant reduction in HbA1c occurred between weeks 12 and 52 in the Detemir, but not the Aspart or Mix groups. After 52 weeks, the target of an HbA1c <7.4% was achieved in 16.7% of the Aspart group, 37.5% of the Mix group, and 12.5% of the Detemir group (no significant differences among the three groups by χ2 analysis). The mean changes from baseline in blood glucose concentration measured after breakfast, and before and after dinner, were also similar in each group. CONCLUSIONS: Early and meaningful reductions in HbA1c were achieved by twice-daily administration of a premix, aspart plus an SU, and detemir plus a glinide, without severe hypoglycemia or an increase in body mass. However, the target HbA1c was achieved in relatively few participants, perhaps due to an insufficient dose of insulin or the small study size.

Level of urinary liver-type fatty acid-binding protein is associated with cardiac markers and electrocardiographic abnormalities in type-2 diabetes with chronic kidney disease stage G1 and G2.

Urinary liver-type fatty acid-binding protein (L-FABP) reflects the degree of stress in proximal tubules of the kidney. We examined the level of L-FABP in type-2 diabetes mellitus (T2DM) patients with chronic kidney disease (CKD) stage G1 and G2, and its relationship with cardiac markers and electrocardiographic (ECG) abnormalities. T2DM patients whose estimated glomerular filtration rate (eGFR) was ≥60 mL/min/1.73 m(2) were recruited [n = 276 (165 males), mean age 64 years]. The median level of urinary L-FABP was 6.6 µg/gCr. Urinary L-FABP showed significant correlation with urinary albumin-to-creatinine ratio (ACR) (r = 0.51, p < 0.0001). Median (25th-75th percentile) eGFR was 82 (72-95) mL/min/1.73 m2. We divided patients into four subgroups (group 1, L-FABP ≤8.4 µg/gCr and ACR ≤30 mg/gCr; group 2, L-FABP ≤8.4 µg/gCr and ACR >30 mg/gCr; group 3, L-FABP >8.4 µg/gCr and ACR ≤30 mg/gCr; group 4, L-FABP >8.4 µg/gCr and ACR >30 mg/gCr). Compared with group 1, group 4 was significantly higher in systolic blood pressure, and eGFR using standardized serum cystatin C, high-sensitivity troponin T, and N-terminal pro-brain natriuretic peptide (NT-proBNP). Group 4 had significantly higher level of NT-proBNP than group 3. Groups 2, 3 and 4 showed more ECG abnormalities than group 1. These findings suggest that simultaneous measurement of urinary L-FABP and ACR should be useful to assess cardiovascular damage reflecting on the elevation of cardiac markers and ECG abnormalities in T2DM with CKD G1 and G2.

[Assessment of the treatment of glucocorticoid-induced osteoporosis].

Glucocorticoid-induced osteoporosis (GIOP) is known as a serious adverse side effect during long-term glucocorticoid treatment. Several clinical guidelines are available to whom and how we should start to treat GIOP. However, the assessment of the treatment of GIOP is still controversial. Accumulating evidences suggest us that both bone mineral density and bone turn over markers could reflect the effect of some pharmacological agents on bone metabolism. However, further studies would be required for precise assessment of the efficacy of drugs on fracture prevention in GIOP.

Oral alendronate can suppress bone turnover but not fracture in kidney transplantation recipients with hyperparathyroidism and chronic kidney disease.

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 ± 7.8 years) into this study. All patients were prescribed methylprednisolone (4.07 ± 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 ± 71.4 pg/mL and 20.8 ± 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.

High prevalence of hypovitaminosis D in pregnant Japanese women with threatened premature delivery.

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.

Phosphate overload induces podocyte injury via type III Na-dependent phosphate transporter.

Uptake of P(i) 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 P(i) transporter Pit-1 to explore the role of extracellular P(i) 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 anti-connexin 43 and anti-desmin antibodies suggested the progressive injury of podocytes in TG rats. TG rats showed higher P(i) uptake in podocytes than wild-type rats, especially under low P(i) 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.

Local microbleeding facilitates IL-6- and IL-17-dependent arthritis in the absence of tissue antigen recognition by activated T cells.

Cognate antigen recognition by CD4(+) T cells is thought to contribute to the tissue specificity of various autoimmune diseases, particularly those associated with class II MHC alleles. However, we show that localized class II MHC-dependent arthritis in F759 mice depends on local events that result in the accumulation of activated CD4(+) T cells in the absence of cognate antigen recognition. In this model, transfer of in vitro polarized Th17 cells combined with the induction of experimental microbleeding resulted in CCL20 production, the accumulation of T cells in the joints, and local production of IL-6. Disease induction required IL-17A production by transferred T cells, IL-6 and CCL20 expression, and STAT3 signaling in type I collagen-expressing cells. Our data suggest a model in which the development of autoimmune disease in F759 mice depends on four events: CD4(+) T cell activation regardless of antigen specificity, local events that induce T cell accumulation, enhanced sensitivity to T cell-derived cytokines in the tissue, and activation of IL-6 signaling in the tissue. This model provides a possible explanation for why tissue-specific antigens recognized by activated CD4(+) T cells have not been identified in many autoimmune diseases, especially those associated with class II MHC molecules.

Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism.

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.

Sphingomyelins in four ascidians, Ciona intestinalis, Halocynthia roretzi, Halocynthia aurantium, and Styela clava.

Sphingomyelin is rarely found in lower animals, while sphingophospholipid is a characteristic of higher animals. In this study, sphingomyelin was first isolated and characterized from ascidian Ciona intestinalis. Ascidian sphingomyelin was prepared using ion exchange (QAE-Sphadex-A25) and silicic acid (Florisil and Iatrobeads) column chromatographies. The chemical structure was characterized by fatty acid analysis, sphingoid analysis, hydrogen fluoride degradation, acid hydrolysis, enzymatic hydrolysis, infrared analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ceramide moieties of C. intestinalis sphingomyelin consisted primarily of C16:0, C18:0, and C18:1 fatty acids and d18:2 sphingadiene. Furthermore, sphingomyelins were isolated and characterized from 3 other ascidians, Halocynthia roretzi, Halocynthia aurantium, and Styela clava using the same methods. Comparative analysis of the sphingomyelin structures in 4 ascidian species-C. intestinalis (Enterogona) and H. roretzi, H. aurantium, and S. clava (Pleurogona)-revealed that the major fatty acid composition of the ceramides was similar, and that they differed in minor components.

Effects of prostaglandin D2 on Na-dependent phosphate transport activity and its intracellular signaling mechanism in osteoblast-like cells.

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 D2 (PGD2) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD2 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 PGD2 on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD2-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S6 kinase reduced this effect of PGD2, while Akt kinase inhibitor did not. These results suggest that PGD2 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 S6 kinase.

[Biochemical markers of bone turnover. New aspect. Biochemical markers of bone turnover in long term treatment with bisphosphonate].

Bisphosphonates (BPs) are now widely used for the prevention of osteoporotic fracture. The elevation of bone turnover markers (BTM) is an independent risk factor for the bone fracture, and their significant decrease with BPs treatment is considered to reflect BPs' efficacy to "adjust" bone turnover. A large-scale study so far showed that BPs continuously suppress both BTM levels and the incidence of fracture as long as 7 to 10 years, though possible deterioration of bone strength with long-term severe suppression of BTM has been pointed out.

[Etidronate for treatment of osteoporosis].

The treatment with anti-resorptive agents including bisphosphonates and selective estrogen receptor modulators (SERM) has clinical relevance to reduce osteoporotic fractures in postmenopausal women. Among them, etidronate has a unique mechanism to suppress osteoclastic activity, and could be used to treat long-term immobilized patients, to whom other bisphosphonates or SERM are not available because of their adverse effects. In addition, intermittent therapy regimen (two weeks every three months) and less gastroesophageal reflux disorder may be suitable for some patients. Etidronate has less and lower clinical evidences than other bisphosphonates and SERM, but still has clinical efficacy and advantage to treat osteoporotic patients.

Pharmacological topics of bone metabolism: recent advances in pharmacological management of osteoporosis.

The prevention of osteoporotic fracture is an essential socioeconomical priority, especially in the developed countries including Japan. Estrogen, selective estrogen-receptor modulators (SERMs), and bisphosphonate are potent inhibitors of bone resorption; and they have clinical relevance to reduce osteoporotic fractures in postmenopausal women. However, we can prevent at most 50% of vertebral fractures with these agents. For the better compliance of aminobisphosphonate, the use of a daily bisphosphonate regimen is moving to a weekly or monthly bisphosphonate regimen. Both cathepsin K inhibitors and modulators of the RANK-RANKL system, which can reduce bone resorption, are the candidates for the future treatment of osteoporosis. As well as bone resorption, we need to increase bone formation to prevent osteoporotic fractures, particularly in elderly patients with low bone turnover. In the U.S., Europe, and Australia, they have already started intermittent parathyroid hormone injection and/or oral strontium ranelate to stimulate bone formation. We still need to discover new agents to reduce osteoporotic fractures for the better quality of life without fractures.

Vasopressin stimulates Na-dependent phosphate transport and calcification in rat aortic smooth muscle cells.

We investigated the effect of arginine vasopressin (AVP) on inorganic phosphate (Pi) transport in A-10 rat aortic vascular smooth muscle cells (VSMCs). AVP time- and dose-dependently stimulated Na-dependent Pi transport in A-10 cells. This stimulatory effect of AVP on Pi transport was markedly suppressed by V1 receptor antagonist. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of AVP. The selective inhibitors of c-Jun-NH2-terminal mitogen-activated protein (MAP) kinase (Jun kinase) attenuated AVP-induced Pi transport, but Erk kinase or p38 MAP kinase inhibitors did not. Wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, suppressed AVP-induced Pi transport. Rapamycin, a selective inhibitor of S6 kinase, reduced this effect of AVP, while Akt kinase inhibitor did not. The combination of inhibitors for PKC, Jun kinase and PI 3-kinase completely suppressed the AVP-enhanced Pi transport. Furthermore, AVP rescued the VSMC from high phosphate-induced cell death and enhanced mineralization of these cells. In summary, these results suggest that AVP stimulates both Na-dependent Pi transport and mineralization in VSMCs. The mechanism is mediated by the activation of multiple signaling pathways including PKC, PI 3-kinase, S6 kinase and Jun kinase.