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Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25-hydroxyvitamin D 3 1a-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 1a-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 1a,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. Administration of FGF-23 (5 mg i.p. at 12-hour intervals for 2 days) to ob/ob mice suppressed renal 1a-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 1a-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 1a-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. ß

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The intact Adda was isolated from microcystin-LR by a microbial degradation using an isolated Sphingomonas strain, B-9. The reaction of microcystin-LR with cell extract of this strain proceeded smoothly to give the final degradation product by way of two intermediates, linearized microcystin-LR and a tetrapeptide. The purified Adda that was structurally characterized using various spectral data did not show the toxicity to mice or inhibition to protein phosphatase activity in contrast to the native toxin.

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Microcystins and nodularins produced by cyanobacteria are potent hepatotoxins and tumor promoters. They are, respectively, cyclic heptapeptides and cyclic pentapeptides containing a characteristic beta-amino acid residue, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E ),6(E)-dienoic acid (Adda). Strain B-9 isolated from Lake Tsukui, Japan, degrades microcystin-LR, which is the most toxic among the microcystins, to nontoxic Adda as an end product. In the present study, we characterized the bacterial degradation process of the cyclic peptide hepatotoxins by liquid chromatography/ion trap tandem mass spectrometry. The use of protease inhibitors with a B-9 cell extract indicated that the degradation process of microcystin-LR consists of sequential enzymatic hydrolyses of Arg-Adda, Ala-Leu, and then Adda-Glu peptide bonds into two known nontoxic intermediate degradation products and then Adda, respectively. Subsequently, additional microcystins and nodularin were compared with microcystin-LR on substrate specificity. The cyclic peptides containing the Arg-Adda peptide bond were almost completely degraded to Adda as well as microcystin-LR, whereas microcystin-LF containing the Phe-Adda peptide bond instead of Arg-Adda peptide bond and 6(Z)-Adda-microcystin-LR and -RR which are geometrical isomers of the Adda residue were barely degraded. These results indicated that the degrading enzymes selectively hydrolyzed the Arg-Adda peptide bond as the initial ring opening of the cyclic peptide hepatotoxins, microcystins and nodularin.

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