Shigehisa Taketomi

In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats.

Intravenous administration of human basic fibroblast growth factor (bFGF) for 2 weeks stimulated osteoblast proliferation and new bone formation in various skeletal bones in young and aged rats at dosage levels of 0.1 mg/kg/day and greater. Morphometry of the soft X-ray radiograms of cross sections of the tibia indicated about a 20% increase in the calcified bone area of the diaphysis at 0.1 mg/kg/day. The Ca and hydroxyproline contents showed statistically significant increases at this dosage. The new bone formation was found only on the endosteal side, and no periosteal bone formation was found. Similar systemic osteogenic potential was seen after intravenous administration of other growth factors of the FGF family, human acidic FGF and human heparin-binding secretory transforming protein-1. The above results suggest a potential therapeutic role for these growth factors in bone-loss diseases such as osteoporosis.

Reduction of Insulin Resistance in Obese and/or Diabetic Animals by 5-[4-(1-Methylcyclohexylmethoxy)benzyl]-thiazolidine-2,4-dione (ADD-3878, U-63,287, Ciglitazone), a New Antidiabetic Agent

Effects of 5-[4-(1-methylcyclohexylmethoxy)benzyl]-thiazolidine-2,4-dione (ADD-3878, U-63,287, Ciglitazone) on glucose and lipid metabolism were examined in various animal models. ADD-3878, administered as a dietary admixture (30–186 mg/kg/day) to obese-diabetic yellow KK (KK-Ay) mice, markedly suppressed the diabetic syndromes (hyperglycemie, hypertriglyceride-mia, and hyperinsulinemia), accompanied by the reduction of insulin resistance as manifested by improvement of overall insulin sensitivity in either the insulin tolerance test òr the steady-state blood glucose test. Chronic administration of ADD-3878 for as long as 12 wk to young yellow KK mice, which were in the early stage of diabetes and obesity, depressed age-dependent rises in blood glucose, plasma triglyceride, and insulin without exerting any effect on obesity. When orally administered to obese Zucker-fatty rats, ADD-3878 decreased plasma insulin and triglyceride in a dose-dependent manner (5–100 mg/kg/day). The treated rats showed increased tolerance and decreased insulin secretion in response to oral glucose. The glycemie response to insulin and the steady-state plasma glucose were also normalized in the treated rats. Chronic administration of ADD-3878 to young fatty rats for as long as 12 wk decreased the dose-dependent rises in blood glucose, plasma triglyceride, and insulin without exerting any effect on body weight. ADD-3878 had no effect on glucose and lipid metabolism of young Sprague-Dawley rats and mild strepto-zotocin-diabetic rats. However, in old Sprague-Dawley rats that were moderately insulin resistant and hyperli-pidemic compared with young ones, ADD-3878 decreased plasma triglyceride and insulin and improved insulin sensitivity. Five-day administration of ADD-3878 to beagle dogs with slightly impaired glucose tolerance increased glucose tolerance and suppressed postprandial rises in plasma glucose, insulin, and triglyceride. Based on these results, ADD-3878 is effective on abnormal glucose and lipid metabolism associated with insulin resistance or obesity through reduction of peripheral insulin resistance. Therefore, ADD-3878 is expected to be useful in the treatment of hyperglycemie, hyperinsulinemia, and hyperlipemia in obese type II diabetes and Obesity.

Overexpression of GPR40 in Pancreatic β-Cells Augments Glucose-Stimulated Insulin Secretion and Improves Glucose Tolerance in Normal and Diabetic Mice

OBJECTIVE GPR40 is a G protein–coupled receptor regulating free fatty acid–induced insulin secretion. We generated transgenic mice overexpressing the hGPR40 gene under control of the mouse insulin II promoter and used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis. RESEARCH DESIGN AND METHODS Normal (C57BL/6J) and diabetic (KK) mice overexpressing the hGPR40 gene under control of the insulin II promoter were generated, and their glucose metabolism and islet function were analyzed. RESULTS In comparison with nontransgenic littermates, hGPR40 transgenic mice exhibited improved oral glucose tolerance with an increase in insulin secretion. Although islet morphologic analysis showed no obvious differences between hGPR40 transgenic and nontransgenic mice, isolated islets from hGPR40 transgenic mice had enhanced insulin secretion in response to high glucose (16 mmol/l) compared with those from nontransgenic mice, and they both had similar low glucose (3 mmol/l)-stimulated insulin secretion. In addition, hGPR40 transgenic islets significantly increased insulin secretion against a naturally occurring agonist palmitate in the presence of 11 mmol/l glucose. hGPR40 transgenic mice were also found to be resistant to high-fat diet–induced glucose intolerance, and hGPR40 transgenic mice harboring KK background showed augmented insulin secretion and improved oral glucose tolerance compared with nontransgenic littermates. CONCLUSIONS Our results suggest that GPR40 may have a role in regulating glucose-stimulated insulin secretion and plasma glucose levels in vivo and that pharmacological activation of GPR40 may provide a novel insulin secretagogue beneficial for the treatment of type 2 diabetes.

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene.

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.

Inhibitory effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation in long-term cultures of mouse unfractionated bone cells

SummaryTo study the effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation, we used an unfractionated bone cell culture system containing mature osteoclasts from femur and tibia of newborn mice. Ipriflavone (10−5 M) inhibited pit formation on dentin slices and caused a decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive (+) multinucleate cells (MNCs) in a 4-day culture period in which no increase in the number of TRAP(+)-MNCs was observed in the presence of 5% fetal bovine serum (FBS) and 10−8 M 1α,25-dihydroxy-vitamin D3 (1α,25(OH)2D3). During the following 12 days, both the total area of the pits and the number of TRAP(+)-MNCs increased in the control. Continuous treatment with ipriflavone also inhibited the increase in pit area during this period. These effects of ipriflavone were reversible. Furthermore, the differentiation of osteoclasts was examined when preexisting TRAP(+)-MNCs were removed by incubation in the absence of 1α,25(OH)2D3 for the initial 4 days in culture dishes without dentin slices. When 1α,25(OH)2D3 and ipriflavone were added to the medium on the 4th day, ipriflavone inhibited new TRAP(+)-MNC formation stimulated by 1α,25(OH)2D3 in a dose-dependent manner. However, pretreatment of the cells with ipriflavone before the addition of 1α,25(OH)2D3 did not inhibit TRAP(+)-MNC formation. These results indicate that ipriflavone inhibits both the activation of mature osteoclasts and the formation of new osteoclasts without affecting growth of TRAP-negative progenitor cells.

Inhibitory effect of ipriflavone on pit formation in mouse unfractionated bone cells

SummaryEffects of ipriflavone (7-isopropoxyisoflavone) on osteoclast-induced bone resorption were evaluated using an unfractionated bone cell culture system containing mature osteoclasts from the femur and tibia of newborn mice. When cells were cultured for 4 days on dentin slices in the presence of 5% fetal bovine serum and 10−8 M 1α,25(OH)2D3, ipriflavone (3 x 10−7-3 x 10−5 M) inhibited pit formation and caused a decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). The lowest significant effect was observed at a concentration of 10−6 M. Unlike ipriflavone, calcitonin inhibited pit formation 4 days after the culture was started without affecting the number of TRAP-positive MNCs. Ipriflavone still inhibited pit formation when the culture period was 13 days, when new osteoclasts were expected to be formed. These findings suggest that ipriflavone inhibits new osteoclast formation and bone resorption at the cellular level.

Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture

SummaryThe effects of ipriflavone (IP) (10−5 M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of β-glycerophosphate and dexamethasone. Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time. The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days. Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells. After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells. In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days. These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP.

Similarities and differences between the effects of ipriflavone and vitamin K on bone resorption and formation in vitro

The effects of ipriflavone and vitamin K on bone metabolism were examined using a culture system. Vitamin K1 and vitamin K2 (10(-7)M-10(-5)M) inhibited both the activation of mature osteoclasts and the formation of new osteoclasts without affecting the growth of progenitor cells in cultures of mouse unfractionated bone cells. The inhibitory effects of vitamin K on bone resorption were similar to those of ipriflavone and were not affected by the vitamin K antagonist warfarin. When ipriflavone was added to the culture medium in combination with vitamin K2, an additive inhibitory effect on bone resorption was observed. An additive effect was also observed in organ cultures of mouse calvaria. On the other hand, ipriflavone, but neither vitamin K1 nor vitamin K2, stimulated cellular alkaline phosphatase (ALP) activity on rat bone marrow stromal cells under culture conditions in which cells subsequently form mineralized bone-like tissue. Vitamin K1 and vitamin K2 also did not modulate the stimulatory effect of ipriflavone on the ALP activity of the cells. These results suggest that the inhibitory effects of vitamin K on bone resorption are similar to those of ipriflavone through mechanisms that may be independent of the gamma-carboxylation system, while the effects of vitamin K on osteoblast phenotype expression are different from those of ipriflavone.

Congenitally impaired hormone sensitivity of the adipose tissue of spontaneously diabetic mice, KK. Validity of thrifty genotype in KK mice.

SummaryAdipose tissue of KK mice was less sensitive to insulin in its stimulatory action on glucose oxidation or its inhibitory action on lipolysis, and to epinephrine in its stimulatory action on lipolysis as compared to that of C57BL mice. A lack of appreciable increase in plasma NEFA in response to fasting of the KK mice might be caused by the impaired response of lipolysis to the hormones. — Adipose tissue of KK mice was also less sensitive to insulin-like action of concanavalin A, ouabaine or omission of K+ in the medium on glucose oxidation or lipolysis. Lipolysis in response to theophylline and/or DcAMP was less marked in the tissue of KK mice. The mean diameter of the adipocytes was larger in KK than in C57BL mice. In the experiments using adipocytes with the same diameter, however, the cells of KK mice were less sensitive to the hormones than those of C57BL. — With respect to tissue sensitivity to the hormones, the mean diameter of adipocytes and the response of plasma NEFA to fasting, the F1-hybrid mice of KK and C57BL showed values between those of the parental strains. Yellow hybrid (genetically obese F1-hybrid) mice showed higher sensitivity to insulin in the adipose tissue, more remarkable response of plasma NEFA to fasting as compared with KK mice, although hypertrophy of adipocytes was more pronounced in the yellow hybrid mice. — These findings suggest that insulin insensitivity is associated with epinephrine insensitivity in adipocytes of KK mice; both appear to be subjected to genetic factor(s)per se rather than hypertrophy of the adipocytes. Furthermore, insensitivity to both hormones in the KK mice appears to be caused by a common defect in cellular process other than the hormone receptor systems. These genetically determined abnormalities of adipocytes may result in fat-storage metabolism through hyperinsulinemia; this is very similar to the metabolic profile due to thrifty genotype in human diabetes as proposed by Neel.

Increase in femoral bone mass by ipriflavone alone and in combination with 1α-hydroxyvitamin D3 in growing rats with skeletal unloading

We assessed the possibility that ipriflavone treatment might result in bone restoration in immobilized rats. We also investigated the effect of combined treatment with ipriflavone and vitamin D3 on the bone. Male Sprague-Dawley rats, 6 weeks of age, were subjected to unilateral sciatic neurectomy. Three weeks after the operation, ipriflavone (100 mg/kg), 1α-hydroxyvitamin D3 [1α(OH)D3, 25 ng/kg], or both ipriflavone and 1α(OH)D3 were orally administered every day for 12 or 24 weeks. After 12 weeks of treatment, only the group receiving combined treatment with ipriflavone and 1α(OH)D3 showed increases in total femur calcium content (+16.4%, compared with the control). After 24 weeks, both animals treated with ipriflavone alone and those that had received the combination of ipriflavone and 1α(OH)D3 showed significant increases in femur calcium content (+18.0% and +23.8%, respectively). In these treatment groups, X-ray analysis revealed an increase in bone mineral density over the entire length of the femur, and an increase in cortical diameter at the midshaft without affecting medullary width. Administration of 1α(OH)D3 (25 ng/kg) alone had no effect. Body weight, femur length, and serum markers of calcium and bone metabolism were not affected in any group. We evaluated the relationship between ipriflavone and vitamin D3 in bone cells in a culture system using rat bone marrow stromal cells in which the cells subsequently form mineralized bone-like tissue. Continuous treatment with ipriflavone (10−5 M) for 21 days resulted in an increase in osteocalcin secretion, and enhanced its response to 1α,25-dihydroxyvitamin D3 (10−11 M-10−8 M). These findings indicate that ipriflavone treatment increases the femoral bone mass in immobilized rats. In addition, a low dose of 1α(OH)D3, which did not induce hypercalcemia, in combination with ipriflavone, augmented the stimulatory effect of ipriflavone alone on the bone mass, possibly due to a direct effect of each agent on osteoblastic cells.