Assessing Gluten-Free Soy Bread Quality and Amino Acid Content.

The nutritional and palatability relevance of bread prepared with soy flour was examined. There are a few effective nutritional measures that combine palatability, convenience, and functionality in the suppression of muscle loss (contributing to the improvement and prevention of sarcopenia). Therefore, in the present study, we attempted to produce bread using soybeans, which are rich in amino acids involved in the synthesis and degradation of skeletal muscle proteins. Rice flour was also used to avoid gluten intolerance. The bread was baked in an automatic bread maker, and the rheological properties of its breadcrumbs were determined using a creep meter. We found that a 70 g slice of soy bread satisfied approximately one-fifth of the daily nutritional requirement for leucine. Although soy decreased the specific volume of bread by preventing starch construction, the use of preprocessed rice flour recovered the volume, and corn starch improved the taste. We propose that the addition of soy bread to the daily diet may be an effective protein source.

The Critical Role of MMP13 in Regulating Tooth Development and Reactionary Dentinogenesis Repair Through the Wnt Signaling Pathway.

Matrix-metalloproteinase-13 (MMP13) is important for bone formation and remodeling; however, its role in tooth development remains unknown. To investigate this, MMP13-knockout (Mmp13 -/- ) mice were used to analyze phenotypic changes in the dentin-pulp complex, mineralization-associated marker-expression, and mechanistic interactions. Immunohistochemistry demonstrated high MMP13-expression in pulp-tissue, ameloblasts, odontoblasts, and dentin in developing WT-molars, which reduced in adults, with human-DPC cultures demonstrating a >2000-fold increase in Mmp13-expression during mineralization. Morphologically, Mmp13 -/- molars displayed critical alterations in the dentin-phenotype, affecting dentin-tubule regularity, the odontoblast-palisade and predentin-definition with significantly reduced dentin volume (∼30% incisor; 13% molar), and enamel and dentin mineral-density. Reactionary-tertiary-dentin in response to injury was reduced at Mmp13 -/- molar cusp-tips but with significantly more dystrophic pulpal mineralization in MMP13-null samples. Odontoblast differentiation-markers, nestin and DSP, reduced in expression after MMP13-loss in vivo, with reduced calcium deposition in MMP13-null DPC cultures. RNA-sequencing analysis of WT and Mmp13 -/- pulp highlighted 5,020 transcripts to have significantly >2.0-fold change, with pathway-analysis indicating downregulation of the Wnt-signaling pathway, supported by reduced in vivo expression of the Wnt-responsive gene Axin2. Mmp13 interaction with Axin2 could be partly responsible for the loss of odontoblastic activity and alteration to the tooth phenotype and volume which is evident in this study. Overall, our novel findings indicate MMP13 as critical for tooth development and mineralization processes, highlighting mechanistic interaction with the Wnt-signaling pathway.

The Deletion of Hdac4 in Mouse Osteoblasts Influences Both Catabolic and Anabolic Effects in Bone.

Histone deacetylase 4 (Hdac4) is known to control chondrocyte hypertrophy and bone formation. We have previously shown that parathyroid hormone (PTH) regulates many aspects of Hdac4 function in osteoblastic cells in vitro; however, in vivo confirmation was previously precluded by preweaning lethality of the Hdac4-deficient mice. To analyze the function of Hdac4 in bone in mature animals, we generated mice with osteoblast lineage-specific knockout of Hdac4 (Hdac4ob-/- ) by crossing transgenic mice expressing Cre recombinase under the control of a 2.3-kb fragment of the Col1a1 promoter with mice bearing loxP-Hdac4. The Hdac4ob-/- mice survive to adulthood and developed a mild skeletal phenotype. At age 12 weeks, they had short, irregularly shaped and stiff tails due to smaller tail vertebrae, with almost no growth plates. The tibial growth plate zone was also thinned, and Mmp13 and Sost mRNAs were increased in the distal femurs of Hdac4ob-/- mice. Immunohistochemistry showed that sclerostin was elevated in Hdac4ob-/- mice, suggesting that Hdac4 inhibits its gene and protein expression. To determine the effect of PTH in these mice, hPTH (1-34) or saline were delivered for 14 days with subcutaneously implanted devices in 8-week-old female Hdac4ob-/- and wild-type (Hdac4fl/fl ) mice. Serum CTX, a marker of bone resorption, was increased in Hdac4ob-/- mice with or without PTH treatment. Tibial cortical bone volume/total volume (BV/TV), cortical thickness (Ct.Th), and relative cortical area (RCA) were decreased in Hdac4ob-/- mice, but PTH caused no further decrease in Hdac4ob-/- mice. Tibial trabecular BV/TV and thickness were not changed significantly in Hdac4ob-/- mice but decreased with PTH treatment. These results indicate that Hdac4 inhibits bone resorption and has anabolic effects via inhibiting Mmp13 and Sost/sclerostin expression. Hdac4 influences cortical bone mass and thickness and knockout of Hdac4 prevents the catabolic effect of PTH in cortical bone. © 2018 American Society for Bone and Mineral Research.

MEF2C Interacts With c-FOS in PTH-Stimulated Mmp13 Gene Expression in Osteoblastic Cells.

Parathyroid hormone (PTH) regulates the transcription of many genes in the osteoblast. One of these genes is Mmp13, which is involved in bone remodeling and early stages of endochondral bone formation. Previously, we reported that PTH induces Mmp13 transcription by regulating the dissociation of histone deacetylase 4 (HDAC4) from runt-related transcription factor 2 (Runx2), and the association of the HATs, p300, and p300/CREB binding protein (CBP)-associated factor. It is known that, in addition to Runx2, HDAC4 binds to the transcription factor, myocyte-specific enhancer factor 2c (MEF2C), and represses its activity. In this work, we investigated whether MEF2C participates in PTH-stimulated Mmp13 gene expression in osteoblastic cells and how it does so. Knockdown of Mef2c in UMR 106-01 cells repressed Mmp13 messenger RNA expression and promoter activity with or without PTH treatment. Chromatin immunoprecipitation (ChIP) assays showed that MEF2C associated with the Mmp13 promoter; this increased after 4 hours of PTH treatment. ChIP-reChIP results indicate that endogenous MEF2C associates with HDAC4 on the Mmp13 promoter; after PTH treatment, this association decreased. From gel shift, ChIP, and promoter-reporter assays, MEF2C was found to associate with the activator protein-1 (AP-1) site without directly binding to DNA and had its stimulatory effect through interaction with c-FOS. In conclusion, MEF2C is necessary for Mmp13 gene expression at the transcriptional level and participates in PTH-stimulated Mmp13 gene expression by increased binding to c-FOS at the AP-1 site in the Mmp13 promoter. The observation of MEF2C interacting with a member of the AP-1 transcription factor family provides knowledge of the functions of HDAC4, c-FOS, and MEF2C.

MMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton.

Histone deacetylase 4 (Hdac4) regulates chondrocyte hypertrophy. Hdac4(-/-) mice are runted in size and do not survive to weaning. This phenotype is primarily due to the acceleration of onset of chondrocyte hypertrophy and, as a consequence, inappropriate endochondral mineralization. Previously, we reported that Hdac4 is a repressor of matrix metalloproteinase-13 (Mmp13) transcription, and the absence of Hdac4 leads to increased expression of MMP-13 both in vitro (osteoblastic cells) and in vivo (hypertrophic chondrocytes and trabecular osteoblasts). MMP-13 is thought to be involved in endochondral ossification and bone remodeling. To identify whether the phenotype of Hdac4(-/-) mice is due to up-regulation of MMP-13, we generated Hdac4/Mmp13 double knockout mice and determined the ability of deletion of MMP-13 to rescue the Hdac4(-/-) mouse phenotype. Mmp13(-/-) mice have normal body size. Hdac4(-/-)/Mmp13(-/-) double knockout mice are significantly heavier and larger than Hdac4(-/-) mice, they survive longer, and they recover the thickness of their growth plate zones. In Hdac4(-/-)/Mmp13(-/-) double knockout mice, alkaline phosphatase (ALP) expression and TRAP-positive osteoclasts were restored (together with an increase in Mmp9 expression) but osteocalcin (OCN) was not. Micro-CT analysis of the tibiae revealed that Hdac4(-/-) mice have significantly decreased cortical bone area compared with the wild type mice. In addition, the bone architectural parameter, bone porosity, was significantly decreased in Hdac4(-/-) mice. Hdac4(-/-)/Mmp13(-/-) double knockout mice recover these cortical parameters. Likewise, Hdac4(-/-) mice exhibit significantly increased Tb.Th and bone mineral density (BMD) while the Hdac4(-/-)/Mmp13(-/-) mice significantly recovered these parameters toward normal for this age. Taken together, our findings indicate that the phenotype seen in the Hdac4(-/-) mice is partially derived from elevation in MMP-13 and may be due to a bone remodeling disorder caused by overexpression of this enzyme.

PTH Signaling and Epigenetic Control of Bone Remodeling.

As our understanding of the mechanisms that govern bone development advance, the role of epigenetic modifications in these processes become increasingly evident. Interestingly, in parathyroid hormone (PTH)-induced bone metabolism and remodeling, recent evidence shows that PTH signaling employs a particular facet of the epigenetic machinery to elicit its desired effects. In this review, we briefly discuss the known epigenetic events occurring in cells of the osteoblast lineage. More specifically, we elaborate on current findings that reveal the utilization of histone deacetylating enzymes (HDACs) in PTH-regulated modulation of gene expression in bone.

Parathyroid hormone regulates histone deacetylase (HDAC) 4 through protein kinase A-mediated phosphorylation and dephosphorylation in osteoblastic cells.

Histone deacetylases (HDACs) are crucial regulators of gene expression in transcriptional co-repressor complexes. Previously, we reported that HDAC4 was a basal repressor of matrix metalloproteinase-13 (MMP-13) transcription and parathyroid hormone (PTH) regulates HDAC4 to control MMP-13 promoter activity through dissociation from Runx2. Here, we show that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of the rat osteoblastic cell line, UMR 106-01. We demonstrate that PKA-dependent phosphorylated HDAC4 is released from Runx2 bound to the MMP-13 promoter in these cells. Point mutation of Ser-740 in rHDAC4 prevents the release of HDAC4 from Runx2 on the MMP-13 promoter and also prevents the PTH stimulation of MMP-13 transcription. Thus, PTH-induced phosphorylation of rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcription in osteoblasts. PTH causes degradation of HDAC4, and this product appears in the cytoplasm. The cytoplasmic degradation of HDAC4 is blocked by PKA and lysosomal inhibitors, but is not affected by proteasome, caspase-3, or serine and aspartic protease inhibitors. In addition, the phosphatase inhibitor, okadaic acid, prevents degradation indicating that dephosphorylation is associated with degradation. These mechanisms regulating HDAC4 and their roles in such processes are crucial for bone and chondrocyte development. Our data support a link between PTH regulating HDAC4 phosphorylation by PKA, trafficking, partial degradation, and the control of MMP-13 transcription through association with Runx2.

FGF23-induced hypophosphatemia persists in Hyp mice deficient in the WNT coreceptor Lrp6.

Deregulated phosphate homeostasis can lead to a wide range of disorders, including myopathy, cardiac dysfunction, and skeletal abnormalities. Therefore, characterization of the molecular regulation of phosphate metabolism is of pathophysiological and clinical significance. Hyp mouse is the model for human X-linked hypophosphatemia which is due to mutations that inactivate the endopeptidases of the X chromosome (PHEX). PHEX inactivation leads to increased serum levels of fibroblast growth factor 23 (FGF23), a phosphaturic hormone that induces excessive renal phosphate excretion and severe hypophosphatemia. The expression of WNT signaling components is increased in Hyp mice. To determine the potential role of WNT signaling in FGF23-mediated hypophosphatemia, we cross-bred Hyp mice with mice deficient in the WNT coreceptor low-density lipoprotein receptor-related protein 6 (Lrp6) to generate Hyp and Lrp6 double mutant mice (Hyp/Lrp6). Like Hyp mice, Hyp/Lrp6 double mutants maintained high serum levels of FGF23, and accordingly exhibited hypophosphatemia to the same degree as the Hyp mice did, indicating that genetically reducing WNT signaling does not impact FGF23-induced phosphaturia. Moreover, similar to Hyp mice, the Hyp/Lrp6 double mutants also exhibited reduced mineralization of the bone, further supporting that reduced WNT signaling does not affect the chronic phosphate wasting caused by excess FGF23 in these mice. In further support of our finding, injection of bioactive FGF23 protein into Lrp6 mutant mice reduced serum phosphate levels to a similar degree as FGF23 injection into wild-type mice. Our in vivo studies provide genetic and pharmacological evidence for a WNT-independent function of FGF23 in the regulation of phosphate homeostasis.

Triclosan blocks MMP-13 expression in hormone-stimulated osteoblasts.

BACKGROUND: Matrix metalloproteinase-13 (MMP-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of MMP-13 are associated with alveolar bone resorption, periodontal ligament breakdown, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Evidence continues to suggest that periodontal disease contributes to oral tissue breakdown and is linked to numerous systemic conditions. Triclosan (TCN) is a long-standing, proven antibacterial and anti-inflammatory agent found in the only Food and Drug Administration-approved dentifrice for the treatment of plaque and gingivitis. METHODS: This study examines the inhibitory effects of TCN on lipopolysaccharide-, parathyroid hormone (PTH)-, and prostaglandin E2 (PGE2)-induced expression of MMP-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE2 to induce MMP-13 mRNA expression, and real-time reverse transcription-polymerase chain reaction was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. MMP-13 promoter reporter assay was used to explore possible direct effects of TCN on the MMP-13 promoter. RESULTS: TCN significantly reduced PTH or PGE2 elevated expression of MMP-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, TCN enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated that TCN directly inhibits the activation of the PTH-responsive minimal promoter of MMP-13. CONCLUSION: The present study appears to have identified a nuclear mechanism of action of TCN that accounts for the ability of TCN to inhibit PTH- or PGE2-induced MMP-13 expression in osteoblastic cells.

Thyroid hormone and COUP-TF1 regulate kallikrein-binding protein (KBP) gene expression.

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T(3) and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5' flanking region (-53 to -29) and nTRE2, located in the first intron (104-132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T(3). COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T(3) repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T(3). Nuclear corepressor knockdown resulted in loss of T(3) repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T(3) induction of positive thyroid hormone response elements, reverses T(3) repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T(3) and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T(3).

Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

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.

Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation.

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 FGFR-Klotho 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.

In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin d levels.

BACKGROUND: Klotho-knockout mice (klotho(-/-)) have increased renal expression of sodium/phosphate cotransporters (NaPi2a), associated with severe hyperphosphatemia. Such serum biochemical changes in klotho(-/-) mice lead to extensive soft-tissue anomalies and vascular calcification. To determine the significance of increased renal expression of the NaPi2a protein and concomitant hyperphosphatemia and vascular calcification in klotho(-/-) mice, we generated klotho and NaPi2a double-knockout (klotho(-/-)/NaPi2a(-/-)) mice. METHODS AND RESULTS: Genetic inactivation of NaPi2a activity from klotho(-/-) mice reversed the severe hyperphosphatemia to mild hypophosphatemia or normophosphatemia. Importantly, despite significantly higher serum calcium and 1,25-dihydroxyvitamin D levels in klotho(-/-)/NaPi2a(-/-) mice, the vascular and soft-tissue calcifications were reduced. Extensive soft-tissue anomalies and cardiovascular calcification were consistently noted in klotho(-/-) mice by 6 weeks of age; however, these vascular and soft-tissue abnormalities were absent even in 12-week-old double-knockout mice. Klotho(-/-)/NaPi2a(-/-) mice also regained body weight and did not develop the generalized tissue atrophy often noted in klotho(-/-) single-knockout mice. CONCLUSIONS: Our in vivo genetic manipulation studies have provided compelling evidence for a pathological role of increased NaPi2a activities in regulating abnormal mineral ion metabolism and soft-tissue anomalies in klotho(-/-) mice. Notably, our results suggest that serum phosphate levels are the important in vivo determinant of calcification and that lowering serum phosphate levels can reduce or eliminate soft-tissue and vascular calcification, even in presence of extremely high serum calcium and 1,25-dihydroxyvitamin D levels. These in vivo observations have significant clinical importance and therapeutic implications for patients with chronic kidney disease with cardiovascular calcification.

Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic (Hyp) mouse model.

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.

Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1alpha-hydroxylase.

Changes in the expression of klotho, a beta-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 1alpha-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 1alpha-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.

In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23) -mediated regulation of systemic phosphate homeostasis.

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.

Does Fgf23-klotho activity influence vascular and soft tissue calcification through regulating mineral ion metabolism?

Recent studies describe a novel role of fibroblast growth factor-23 (Fgf23)-klotho activity in the systemic regulation of calcium and phosphate homeostasis. Both Fgf23 and klotho ablated mice develop extensive vascular and soft tissue calcification. Inability to clear the required amount of phosphate by the kidney, due to the absence of Fgf23-klotho activity, leads to increased accumulation of serum phosphate in these genetically modified mice, causing extensive calcification. Serum calcium and 1,25 hydroxyvitamin D levels are also elevated in both Fgf23 and klotho ablated mice. Moreover, increased sodium phosphate co-transporter activity in both Fgf23 and klotho ablated mice increases renal phosphate reabsorption which in turn can facilitate calcification. Collectively, these observations bring new insights into our understanding of the roles of the Fgf23-klotho axis in the development of vascular and soft tissue calcification.

Effects of fish oil feeding and fasting on LXRalpha/RXRalpha binding to LXRE in the SREBP-1c promoter in mouse liver.

High-fish oil feeding and fasting down-regulate sterol regulatory element-binding protein-1c (SREBP-1c) mRNA level and suppress lipogenesis in mouse liver. Previous promoter analysis revealed that liver X receptor alpha (LXRalpha)/retinoid X receptor alpha (RXRalpha) complex was required for SREBP-1c gene expression in cell culture. In in vitro studies, polyunsaturated fatty acids (PUFAs, n-6, n-3) inhibited binding of LXRalpha/RXRalpha heterodimer to LXR responsive elements (LXREs) in the SREBP-1c promoter. To examine whether fish oil feeding and fasting would also inhibit its binding to LXREs in mouse liver, active liver nuclear extracts were prepared by percoll gradient centrifugation, and gel mobility shift assay was conducted. Although 1- to 5-day fish oil feeding and 2-day fasting decreased SREBP-1c mRNA by 45-68% and 65%, respectively, fish oil feeding decreased binding of LXR/RXR heterodimer to LXREs by 0-26%, while 2-day fasting decreased their binding by 40-56%. Luciferase assay using mutation of LXREs in mouse primary hepatocytes revealed that the LXR ligand, T0901317, induced increased transcription of SREBP-1c mRNA was mediated by LXREs, but it is unknown whether fish oil/eicosapentaenoic acid (EPA)-induced down-regulation of SREBP-1c mRNA was mediated by LXREs. These data indicate that high-fish oil feeding might decrease SREBP-1c mRNA partly by decreased transcription of SREBP-1c, but if so, the binding inhibition of LXRalpha to LXREs might not be a major cause, while fasting decreased SREBP-1c mRNA, mainly by its binding inhibition of LXRalpha to LXREs in the SREBP-1c promoter.

Combined intervention of soy isoflavone and moderate exercise prevents body fat elevation and bone loss in ovariectomized mice.

Body fat accumulation and bone loss are both often associated with estrogen deficiency following menopause. In this study, we examined whether soy isoflavone, one of the phytoestrogens, and moderate exercise interventions exhibit cooperative effects on body composition and bone mass in ovariectomized (OVX) mice. Eight-week-old female mice were assigned to 6 groups: (1) sham-operated (sham); (2) OVX; (3) OVX with received a soy isoflavone diet (OVX+ISO); (4) OVX with exercised on a treadmill (OVX+EX); (5) OVX with given both isoflavone and exercise (OVX+ISO&EX ); and (6) OVX with treated with 17 beta-estradiol subcutaneously (OVX+E2). Body composition and bone mineral density (BMD) were estimated by dual-energy x-ray absorptiometry (DXA). After the 6-week intervention, whole body fat (%) in the OVX group showed significantly higher than that in the sham group. Intervention of exercise and isoflavone alone partially inhibited OVX-induced body fat gain, and the combined intervention as well as E2 treatment completely restored fat mass to the sham level. Lean body mass in the whole body was not different in OVX group compared with that in OVX+ISO, OVX+EX, and OVX+E2 groups, but it was significantly higher in OVX+ISO&EX than in other groups. BMD of the whole body, lumbar spine, or femur showed significantly reduced by OVX, and the bone loss was partially inhibited by intervention of exercise or isoflavone alone. However, the combined intervention completely restored the bone mass to the level of sham, as did E2. Serum total cholesterol was significantly increased by OVX, which was normalized by the combined intervention or E2 treatment. These results demonstrate that combined intervention of soybean isoflavone and exercise prevented body fat accumulation in the whole body with an increase in lean body mass and restoration of bone mass, and reduced high serum cholesterol in OVX mice.

Involvement of intracellular glutathione in zinc deficiency-induced activation of hepatic stellate cells.

Hepatic stellate cells (HSC) play an important role in the development of liver cirrhosis. They are a major source of extracellular matrix and during fibrogenesis undergo an activation process characterized by increased proliferation and collagen synthesis. In this study, we investigated the anti-fibrogenic effect of zinc supplementation on zinc deficiency induced HSC activation. Isolated HSC were incubated with or without zinc chelator, diethylenetriamine penta-acetic acid (DTPA). Type I collagen expression in HSC was detected by immunohistochemistry. The involvement of glutathione (GSH) homeostasis in the anti-fibrogenic action of zinc was also investigated, as GSH is implicated in many cellular events, such as regulation of cell proliferation, remodeling of extracellular matrix and oxidative stress. Intracellular GSH was measured by HPLC. Enhanced type I collagen expression, apoptosis and cell cycle arrest were found in HSC when DTPA was added, but they were inhibited with supplementation with zinc. Zinc deficiency caused a reduction in intracellular GSH 8 h after the addition of DTPA compared with control levels. The results of this study show that in HSC, the chelation of zinc triggers a progression of collagen synthesis and this involves the depletion of intracellular GSH levels after the addition of DTPA.