Leptin corrects increased gene expression of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase and -24-hydroxylase in leptin-deficient, ob/ob mice.

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We investigated leptin effects on bone metabolism using male leptin-deficient obese (ob/ob) mice, which had lower bone mineral density (BMD) and shorter femurs than lean (?/+) controls. Serum concentrations of calcium, phosphate, tartrate-resistant acid phosphatase (a bone resorption marker) and alkaline phosphatase, and urinary calcium and phosphate excretion were significantly elevated in ob/ob mice, whereas urinary concentrations of deoxypyridinoline did not differed between ob/ob and control mice. Because ob/ob mice develop severe hypogonadism, testosterone was administered to these mice for 10 wk (5 mg/kg, sc, twice weekly); this did not affect femoral BMD. Control and ob/ob mice showed similar vitamin D-receptor densities in bone and kidney; the obese mice had marked increases in serum 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] and in mRNA expression and activities of renal 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (CYP27B1) and -24-hydroxylase (CYP24) compared with control mice. Leptin administration to ob/ob mice (4 mg/kg body weight, ip, every 12 h for 2 d) greatly reduced mRNAs and activities of 1 alpha-hydroxylase and 24-hydroxylase. Elevated concentrations of serum calcium, phosphate, and 1,25-(OH)(2)D(3) were normalized by leptin treatment. Thus, leptin suppresses renal gene overexpression for 1 alpha-hydroxylase and 24-hydroxylase and corrects increased serum concentrations of calcium and phosphate in ob/ob mice. Therefore, low BMD in leptin-deficient mice appears to be related to stimulation of bone resorption by 1,25-(OH)(2)D(3).

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1alpha,25-dihydroxyvitamin D3 synthesis in leptin-deficient mice.

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone-derived fibroblast growth factor 23 (FGF-23) as a mediator of the influence of leptin on renal 1alpha-hydroxylase mRNA expression in leptin-deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF-23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12-hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF-23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Administration of FGF-23 (5 microg i.p. at 12-hour intervals for 2 days) to ob/ob mice suppressed renal 1alpha-hydroxylase mRNA expression. The main site of FGF-23 mRNA expression was the bone, and leptin markedly increased the FGF-23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1alpha-hydroxylase and sodium-phosphate cotransporters (NaP(i)-IIa and NaP(i)-IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF-23 level and renal expression of 1alpha-hydroxylase mRNA were not influenced by administration of leptin to leptin receptor-deficient (db/db) mice. These results indicate that leptin directly stimulates FGF-23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)(2)D(3) synthesis in these mice is at least partly due to elevated bone production of FGF-23.

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor.

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells.

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1alpha,25-dihydroxyvitamin D3.

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK1 kidney cells. To induce 24-hydroxylase expression, 1,25(OH)2D3 (10(-7)M) and dexamethasone were added simultaneously to the medium of LLC-PK1 cells, and 24 h before dexamethasone treatment, 1,25(OH)2D3 was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells. Maximal stimulation was observed with 10(-6) M dexamethasone in both cell lines. The addition of 10(-6) M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10(-6) M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)2D3-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)2D3-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)2D3 enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.