Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho's known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant beta-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by beta-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.

Fibroblast growth factor (FGF) 23 inhibits renal phosphate reabsorption by activating FGF receptor (FGFR) 1c in a Klotho-dependent fashion. The phosphaturic activity of FGF23 is abrogated by proteolytic cleavage at the RXXR motif that lies at the boundary between the FGF core homology domain and the 72-residue-long C-terminal tail of FGF23. Here, we show that the soluble ectodomains of FGFR1c and Klotho are sufficient to form a ternary complex with FGF23 in vitro. The C-terminal tail of FGF23 mediates binding of FGF23 to a de novo site generated at the composite FGFR1c-Klotho interface. Consistent with this finding, the isolated 72-residue-long C-terminal tail of FGF23 impairs FGF23 signaling by competing with full-length ligand for binding to the binary FGFR-Klotho complex. Injection of the FGF23 C-terminal tail peptide into healthy rats inhibits renal phosphate excretion and induces hyperphosphatemia. In a mouse model of renal phosphate wasting attributable to high FGF23, the FGF23 C-terminal peptide reduces phosphate excretion, leading to an increase in serum phosphate concentration. Our data indicate that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a dual mechanism: by removing the binding site for the binary FGFRKlotho complex that resides in the C-terminal region of FGF23, and by generating an endogenous inhibitor of FGF23. We propose that peptides derived from the C-terminal tail of FGF23 or peptidomimetics and small-molecule organomimetics of the C-terminal tail can be used as therapeutics to treat renal phosphate wasting.

A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double-knockout (Fgf23(-/-)/klotho(-/-)) mice to test the hypothesis whether Fgf23 has a klotho-independent function. Fgf23(-/-)/klotho(-/-) mice are viable and have high serum phosphate levels, similar to Fgf23(-/-) and klotho(-/-) single-knockout mice. In addition, the Fgf23(-/-)/klotho(-/-) mice have increased renal expression of the sodium/phosphate cotransporter NaP(i)2a and of 1- alpha-hydroxylase concomitant with increased serum levels of 1,25-dihydroxyvitamin-D, as also observed in the Fgf23(-/-) and klotho(-/-) mice. Moreover, Fgf23(-/-)/klotho(-/-) mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23(-/-) and klotho(-/-) mice. Notably, injection of bioactive FGF23 protein into Fgf23(-/-)/klotho(-/-) and klotho(-/-) mice does not lower serum phosphate, whereas in wild-type and Fgf23(-/-) mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho-independent role in the regulation of systemic phosphate and vitamin D homeostasis.

Rodents fed fish oil showed less obesity with a reduction of triglyceride synthesis in liver, relative to other dietary oils, along with a decrease of mature form of sterol regulatory element binding protein-1 (SREBP-1) and activation of peroxisome proliferator-activated receptor ␣ (PPAR ␣ ). Decrease of mature SREBP-1 protein by fish oil feeding was due to either inhibition of SREBP-1 proteolytic cascade or to decrease of its mRNA. To clarify its mechanism and relation to antiobesity effect, mice were fed fish oil in a range from 10 to 60 energy percent (en%). Fish oil feeding decreased body weight and fat mass in a dosedependent manner, in parallel with PPAR ␣ activation and a decrease of SREBP-1 mRNA. However, compared with 0 en% fish oil feeding, 10 en% fish oil feeding decreased mature SREBP-1 protein by 50% with concomitant decreases of lipogenic genes, while precursor SREBP-1 protein rather increased by 1.3-fold. These data suggest that physiological doses of fish oil feeding effectively decrease expression of liver lipogenic enzymes by inhibiting SREBP-1 proteolytic cascade, while substantial decrease of SREBP-1 expression is observed in its pharmacological doses, and that activation of PPAR ␣ rather than SREBP-1 decrease might be related to the antiobesity effect of fish oil feeding. -Nakatani, T., H-J. Kim, Y. Kaburagi, K. Yasuda, and O. Ezaki. A low fish oil feeding inhibits SREBP-1 proteolytic cascade, while a high fish oil feeding decreases SREBP-1 mRNA in mice liver: its relationship to anti-obesity effect. J. Lipid Res.

Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice.

Hyp mice possess a mutation that inactivates the phosphate-regulating gene, which is homologous to the endopeptidases of the X-chromosome (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of fibroblast growth factor (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hypophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a cofactor of FGF23 signaling). We generated Hyp and klotho double-mutant mice (Hyp/klotho(-/-)). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho(-/-) double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho(-/-) mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1-34 down-regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho(-/-) mice. Moreover, in contrast to the Hyp mice, the Hyp/klotho(-/-) mice showed significantly higher serum levels of 1,25-dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho(-/-) mice were smaller in size, showed features of generalized tissue atrophy, and generally died by 15-20 wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in diseases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeutically modulated by manipulating the effects of klotho.

Fish oil rich in n-3 polyunsaturated fatty acids has been shown to reduce the risk of cardiovascular diseases partly by reduction of blood triglyceride concentration. This favorable effect mainly results from the combined effects of inhibition of lipogenesis by decrease of SREBP-1 and stimulation of fatty acid oxidation by activation of peroxisome proliferator-activated receptor-alpha (PPARalpha) in liver. However, because fish oil is easily peroxidized to form hydroperoxides and increases oxidative stress, some defense mechanism(s) against oxidative stress might occur. To understand these complex effects of fish oil diet, the gene expression profile of mice liver was analyzed using high-density oligonucleotide arrays. High-fat diet (60% of total energy intake) as either safflower oil or fish oil (tuna) was given to mice. After 6 mo of feeding, expression levels of a total of 6,521 genes were analyzed. In fish oil diet compared with safflower oil diet, immune reaction-related genes, antioxidant genes (several glutathione transferases, uncoupling protein 2, and Mn-superoxide dismutase), and lipid catabolism-related genes upregulated, whereas cholesterol and fatty acid synthesis-related genes and 17-alpha hydroxylase/C17-20 lyase and sulfotransferases related to production of endogenous PPARalpha ligands and reactive oxygen species (ROS) downregulated markedly. Because upregulation of these antioxidant genes and downregulation of sulfotransferases were also observed in mice administered fenofibrate, altered gene expression related to antioxidant system observed in fish oil feeding was mediated directly and indirectly by PPARalpha activation. However, downregulation of 17-alpha hydroxylase/C17-20 lyase was not due to PPARalpha activation. These data indicate that fish oil feeding downregulated the endogenous PPARalpha-activation system and increased antioxidant gene expressions to protect against ROS excess.

Peroxisome proliferator-activated receptor-␣ (PPAR␣) activators, fish oil feeding, or fibrate administration upregulated mitochondrial uncoupling protein (UCP2) mRNA expression in mouse liver by 5-9-fold, whereas tumor necrosis factor-␣ (TNF␣) also up-regulated UCP2 in liver. In this study, the mechanisms for PPAR␣ activators-induced up-regulation of UCP2 mRNA, related to TNF␣ and reactive oxygen species (ROS), were investigated. PPAR␣ activators-induced UCP2 up-regulation in mouse/rat liver tissues was due to their increases in hepatocytes but not in non-parenchymal cells. Addition of PPAR␣ activators, WY14,643 or fenofibrate, to cultured hepatocytes up-regulated UCP2 mRNA by 5-10-fold. PPAR␣ activators-induced up-regulation of UCP2 mRNA was not due to increased mRNA stability and required cycloheximide-sensitive short term turnover protein(s). However, expression of PPAR␣/retinoid X receptor-␣ and PGC-1 was not rate-limiting for WY14,643-induced UCP2 up-regulation. In primary hepatocytes, an exogenous oxidant, tert-butyl-hydroperoxide (TBHP), which increased ROS production, up-regulated UCP2 mRNA, whereas WY14,643 treatment did not produce detectable ROS under the condition that fibrate markedly up-regulated UCP2. In in vivo studies, PPAR␣ activators moderately up-regulated TNF␣ mRNA expression in mouse liver. An anti-oxidant pyrrolidine dithiocarbamate ammonium salt injection completely prevented their TNF␣ mRNA increases but did not prevent most of their UCP2 mRNA increases. These data indicate that PPAR␣ activators upregulate UCP2 expression in hepatocytes through unknown proteins by increased transcription, and neither ROS nor TNF␣ production are the major causes for PPAR␣ activators-induced UCP2 up-regulation.UCPs 1 (UCP1, UCP2, and UCP3) are mitochondrial transporters that are capable of dissipating the proton gradient and increasing thermogenesis while reducing the efficiency of ATP synthesis (1). In addition, there are several other hypotheses concerning the physiological roles of UCPs, regulation of fatty acid and glucose oxidation, and reduction of mitochondrial reactive oxygen species (ROS) generation (2). UCP2 was expressed in liver tissues, but hepatocytes of the adult rat liver did not express UCP2, although non-parenchymal cells especially Kupffer cells did (3). However, UCP2 in hepatocytes will be up-regulated under some metabolic conditions. Bacterial lipopolysaccharide (LPS) stimulation or lipid emulsions (linoleic or oleic acid) treatment induced UCP2 mRNA accumulation in rat hepatocytes (4, 5). Furthermore, in in vivo mice studies, fatty liver increased UCP2 expression in hepatocytes (6). We also have shown that compared with safflower oil feeding, fish oil feeding up-regulated UCP2 by 5-fold and fenofibrate administration also induced UCP2 expression by 9-fold in mouse liver tissues (7). Because n-3 fatty acids rich in fish oil and fibrate compounds are peroxisome proliferator-activated receptor-␣ (PPAR␣) activators that stimulate ␤-oxidation of fatty acids in hepatocytes, and because ther...