A role for sirtuin 1 in FGF23 activation following ß-glycerophosphate treatment.

Phosphate homeostasis is vital for many biological processes and disruptions in circulating levels can be detrimental. While the mechanisms behind FGF23 regulation have been regularly studied, the role of extracellular phosphate sensing and its impact on fibroblast growth factor 23 (FGF23) expression remains unclear. This study aimed to investigate the involvement of reactive oxygen species (ROS), silent information regulator 1 (SIRT1), and Hairy and Enhancer of Split-1 (HES1) in regulating FGF23 in FGF23 expressing MC3T3-E1 cells. MC3T3-E1 cells treated with ß-glycerophosphate (BGP) resulted in increased Fgf23 expression. Inhibition of ROS formation by inhibition of NADPH oxidase, which is essential for ROS production, did not affect this response to BGP, suggesting ROS is not involved in this process. Moreover, treatment with tert-butyl hydroperoxide (TBHP), a ROS-inducing agent, did not increase Fgf23 expression. This suggests that ROS machinery is not involved in FGF23 stimulation as previously suggested. Nonetheless, inhibition of SIRT1 using Ex527 eliminated the Fgf23 response to BGP, indicating its involvement in FGF23 regulation after BGP treatment. Indeed, activation of SIRT1 using SRT1720 increased Fgf23 expression. Moreover, transcription factor Hes1 was upregulated by BGP treatment, which was diminished when cells were treated with Ex527 implying it is also regulated through SIRT1. These findings suggest the existence of an upstream SIRT1-HES1 axis in the regulation of FGF23 by phosphate, though we were unable to find a role for ROS in this process. Further research should provide insights into phosphate homeostasis and potential therapeutic targets for phosphate-related disorders.

Mir-195-5p targets Smad7 regulation of the Wnt/ß-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells.

In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium ß-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/ß-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/ß-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by ß-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/ß-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.

Defining the Most Potent Osteoinductive Culture Conditions for MC3T3-E1 Cells Reveals No Implication of Oxidative Stress or Energy Metabolism.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and ß-glycerophosphate (ßGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 µg.mL-1/2 mM Asc/ßGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes.

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and âˆ¼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 µM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 µM) and mGPDH (Ki 54.70 µM) activities. siRNAs transfection showed ∼50 % and âˆ¼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Enhanced anti-biofilm and anti-caries potential of arginine combined with calcium glycerophosphate and fluoride.

OBJECTIVE: The aim of this work was to evaluate the antibiofilm and anticaries properties of the association of arginine (Arg) with calcium glycerophosphate (CaGP) and fluoride (F). METHODS: An active attachment, polymicrobial biofilm model obtained from saliva and bovine teeth discs were used. After the initial biofilm growth period, the enamel discs were transferred to culture medium. The treatment solutions were added to the culture media to achieve the desired final concentration. The following groups were used: negative control (Control); F (110 ppm F); CaGP (0.05 %); Arg (0.8 %) and their associations (F + CaGP; Arg + F; Arg + CaGP; Arg +F + CaGP). The following analyses were carried out: bacterial viability (total bacteria, aciduric bacteria and mutans streptococci), pH assessment of the spent culture medium, dry weight quantification, evaluation of surface hardness loss (%SH) and subsurface mineral content. Normality and homoscedasticity were tested (Shapiro-Wilk and Levene's test) and the following tests were applied: two-way ANOVA (acidogenicity), Kruskall-Wallis (microbial viability) and one way ANOVA (dry weight, %SH, mineral content). RESULTS: The association Arg + F + CaGP resulted in the lowest surface hardness loss in tooth enamel (-10.9 ± 2.3 %; p < 0.05). Arg +F + CaGP exhibited highest values of subsurface mineral content (10.1 ± 2.9 gHAP/cm3) in comparison to Control and F (p < 0.05). In comparison to Control and F, Arg +F + CaGP promoted the highest reduction in aciduric bacteria and mutans streptococci (5.7 ± 0.4; 4.4 ± 0.5 logCFU/mL, p < 0.05). CONCLUSIONS: The Arg-F-Ca association demonstrated to be the most effective combination in protecting the loss of surface hardness and subsurface mineral content, in addition to controlling important virulence factors of the cariogenic biofilm. CLINICAL SIGNIFICANCE: Our findings provide evidence that the Arg-F-Ca association showed an additive effect, particularly concerning protection against enamel demineralization. The combination of these compounds may be a strategy for patients at high risk of caries.

Calcium glycerophosphate supplemented to soft drinks reduces bovine enamel erosion

OBJECTIVE: This in vitro study evaluated the effect of calcium glycerophosphate (CaGP) supplemented to soft drinks on bovine enamel erosion. MATERIAL AND METHODS: Four pH-cycles were performed, alternating demineralization by the beverage and remineralization in artificial saliva. RESULTS: Mean wear (±SD, µm) was 7.91±1.13, 7.39±1.01, 7.50±0.91 and 5.21±1.08 for Coca-Cola® without CaGP or containing CaGP at 0.1, 1.0 or 2.0 mM, respectively, while no wear was detected for CaGP at 5.0 and 10.0 mM. Corresponding figures for Sprite Zero® without CaGP or containing CaGP at 0.1, 1.0, 2.0, 5.0 or 10.0 mM were 8.04±1.30, 7.84±0.71, 7.47±0.80, 4.96±0.81, 3.99±0.10 and 1.87±0.12, respectively. CONCLUSION: Supplementation of both beverages with CaGP seems to be an alternative to reduce their erosive potential.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Efectividad del glicerofosfato de calcio como agente reparador de caries dentales / Effectiveness of calcium glycerophosphate as healing agent of dental caries

El presente informe responde a una solicitud del Departamento de Salud Bucal de la División de Rectoría y Regulación Sanitaria, para evaluar la evidencia científica disponible sobre la efectividad del glicerofosfato de calcio presente en pastas dentífricas, en el tratamiento de las caries dentales. Lo anterior, en el contexto de una campaña publicitaria que afirma que este agente tendría capacidad "reparadora" de las caries, lo que ha desatado polémica en cuanto al significado y los fundamentos técnicos de esa afirmación. En rigor, la campaña señala que el producto "repara y protege los dientes de tu familia día a día", que el calcio activo "entrega Calcio activo, el cual trabaja directamente en estos daños incipientes del esmalte dental, reponiendo el Calcio perdido", que "trabaja sobre los daños microscópicos producidos por las bacterias en los dientes" , que el resultado es "esmalte dental mucho más fuerte, enriquecido con Calcio, y más protegido contra las caries", y que " logra con sus ingredientes retardar el aparecimiento de caries". La página web de la empresa (Unilever) señala que el ingrediente activo al que se atribuye este efecto reparador es el Glicerofosfato de Calcio.