Lactate from glucose metabolism regulates osteogenic differentiation of human periodontal ligament stem cells

doi:10.21203/rs.3.rs-1553439/v1

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Research Article

Lactate from glucose metabolism regulates osteogenic differentiation of human periodontal ligament stem cells

https://doi.org/10.21203/rs.3.rs-1553439/v1

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Objectives: Periodontal ligament stem cells (PDLSCs), as one of mesenchymal stem cells in the oral cavity, are closely associated with periodontal hard tissue regeneration. However, the effect of local tissue glucose deficiency on periodontal tissue regeneration, such as immediately after surgical operation, is still unknown. Therefore, this study sought to investigate the effect of a low-glucose environment on the proliferation and osteogenic differentiation of PDLSCs.

Material and methods: This study used medium at five glucose concentrations (100, 75, 50, 25 and 0 mg/dL) and focused on the effects on PDLSCs of changes in lactate in the low-glucose environment. Lactate uptake by PDLSCs in a standard glucose environment was suppressed by the addition of AZD3965, a monocarboxylate transporter-1 inhibitor.

Results: We investigated cell proliferation, migration and assessed osteogenic differentiation factors. Furthermore, PDLSC lactate production was suppressed under low-glucose conditions. We compared that condition to the low-glucose environment to assess lactate's effect on the proliferation and osteogenic differentiation of PDLSCs. The low-glucose environment decreased PDLSC lactate production and inhibited PDLSC osteogenic differentiation. In low glucose environment, the phosphorylation of extracellular signal-regulated kinase, protein kinase B, and the activation of hypoxia-inducible factor 1α signaling pathways were inhibited, also, AZD3965 inhibited the activation of these signaling pathways.

Conclusion: The results of this study suggested that lactate production by glucose metabolism is involved in periodontal tissue regeneration.

Clinical relevance: The findings of close relationship about lactate metabolism in periodontal tissue may contribute new insights into future periodontal regenerative therapies. Lactate may be a new treatment option for periodontal regenerative therapy in the future.

Lactate

periodontal ligament stem cells

glucose metabolism

osteogenic differentiation

Glucose is a major energy source for most mammalian cell types and is metabolized to synthesize ATP. Homeostasis is maintained in various cells and tissues by the use of this ATP as an energy source. Just as various tissues metabolize and utilize glucose, bone tissue also uses glucose to maintain homeostasis[1–4]. Glycolysis, a central biochemical reaction pathway[5], describes the metabolic process whereby glucose is broken down into organic acids such as pyruvate and lactate, and converted to the major energy source ATP.

Among various cellular functions, bone metabolism and glucose metabolism, and the connection between these processes, have been the focus of much interest. Lactate, the end product of anaerobic glycolysis, long has been considered to be harmful. However, it has become clear that lactate plays an important role in various cellular functions. Particularly, it has been suggested that uptake of secreted lactate may promote osteogenic differentiation, possibly by activating the hypoxia-inducible factor 1α (HIF-1α) signaling pathway[6]. HIF-1α is known to induce transcription of multiple glycolytic genes, thereby enhancing bone formation in vivo; this protein also has an important role in maintaining cellular homeostasis[7]. Although the relationship between bone formation and glucose metabolism is becoming more apparent, the relationship between periodontal hard tissue regeneration and glucose metabolism remains poorly understood.

The periodontal ligament (PDL) is a connective tissue consisting of fibroblasts, osteoblasts, cementoblasts, and mesenchymal stem cells; the PDL exists between the cementum and alveolar bone[8, 9]. Among the cells of the PDL, periodontal ligament stem cells (PDLSCs) play a central role in regenerating periodontal tissues consisting of cementum, periodontal ligament, and alveolar bone. Therefore, PDLSCs play a critical role in the regeneration of periodontal tissues[10].

The osteogenic differentiation of PDLSCs is key to successful periodontal tissue regeneration. Although many studies have examined the relationship between osteogenesis and glucose metabolism, the molecular biological mechanisms of this connection remain poorly defined. Notably, in a recent study, we clarified that a low-glucose environment could affect gingival fibroblast proliferation, glucose metabolism and induce autophagy to maintain homeostasis[11]. However, the mechanism whereby glucose metabolism, including the key metabolite lactate, regulates the proliferation and osteogenic differentiation of PDLSCs remains (to our knowledge) uninvestigated. Furthermore, the effect of local tissue glucose deficiency on periodontal tissue regeneration, such as immediately after surgical operation, is not clear.

The purpose of this study was to examine the effects of glucose on the proliferation and osteogenic differentiation of human PDLSCs. Moreover, we investigated whether lactate is involved in these processes by employing a selective lactate transport inhibitor.

Isolation and culture of PDLSCs

PDLSCs were cultured from three females (aged 20 to 28 years), as described in previous studies[8]. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Teaque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Marlborough, MA, USA) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B; Nacalai Teaque, Inc.) at 37 °C under 5% CO₂. Passage-zero PDLSCs were seeded and cultured until Passage 4 to 7 for use in experiments. Characterization of PDLSCs was performed as previously described, including analysis by flow cytometry using a FACSVerse (BD Pharmingen, NY, USA) with antibodies (obtained from BioLegend, Inc. San Diego, CA, USA) against human cluster of differentiation 34 (CD34), CD45, CD73, CD90, CD105, and stromal cell surface marker 1 (STRO-1).

Culture medium

For growth at various glucose concentrations, PDLSCs were cultured in DMEM supplemented with 10% FBS and glucose at one of the five following concentrations: 100 mg/dL (physiological control), 75 mg/dL, 50 mg/dL, 25 mg/dL and 0 mg/dL.

Immunofluorescent staining

PDLSCs were cultured with antibodies against human CD34, CD45, CD73, CD90, CD105, and STRO-1 (Santa Cruz Biotechnology, Inc. Dallas, Texas, USA). Fluorescent immunostaining was performed using Alexa Fluor 488^® (Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the secondary antibody, and the nuclei were counterstained using 4’,6-diamidino-2-phenylindole (DAPI) (Dojindo Molecular Technologies, Inc. Kumamoto, Japan). Images of stained cells were obtained using an all-in-one fluorescence microscope (BZ-9000, Keyence Corp., Osaka, Japan).

Cell proliferation assay

PDLSCs were cultured in 96-well plates at 2×10⁴ cells/mL in glucose-supplemented culture medium (at 100, 75, 50, 25, or 0 mg/dL) for 1, 3, and 7 days. The number of viable cells at each timepoint was determined using a cell proliferation assay kit (Cell Count Reagent SF, Nacalai Teaque, Inc.) according to the manufacturer’s protocol; results were determined by measuring absorbance at 450 nm with a spectrophotometer (SpectraMax iD3, Molecular Devices Inc., San Jose, CA, USA).

Staining of live cells

PDLSCs were cultured in 24-well plates at 2×10³ cells/mL in glucose-supplemented culture medium (at 100, 75, 50, 25, or 0 mg/dL) for 1, 3, and 7 days. At each time point, live cells were stained for 15 min at 37 °C with 4 μM Calcein-AM solution (Dojindo Molecular Technologies, Inc.). Images were obtained with an all-in-one fluorescence microscope (BZ-9000, Keyence Corp.) and the percentage of positive staining area in images was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell migration assay

Cell migration was assessed using a wound-repair assay kit (Ibidi GmbH, Inc., Martinsried, Germany). Briefly, PDLSCs were cultured in a cell culture insert at 5×10⁵ cells/mL. The culture insert was removed after the cells reached confluence, leaving a 500-μm wide “wound” in the confluent monolayer, and the medium was replaced with serum-free low-glucose medium. The images of each wound at 0, 12 and 24 h (as the cells migrated to fill in the intervening space) were photographed with a digital microscope camera (Olympus LS, Olympus Corp., Tokyo, Japan). The percentage of healed-wound area was determined by using ImageJ software.

Alkaline phosphatase (ALP) staining and activity

PDLSCs were cultured in 24-well plates at 4×10⁴ cells/mL in glucose-supplemented osteogenic medium (at 100, 75, 50, 25, 0 mg/dL) and incubated for 7 and 14 days. At each time point, PDLSCs were stained using an ALP staining kit (Sigma-Aldrich, Inc. St. Louis, MO) in accordance with the manufacturer’s protocol. At the same time points, PDLSCs were treated with 0.2% Triton X-100 (Sigma-Aldrich, Inc.) and ALP activity was measured following addition of 1-step PNPP (Pierce Biotechnology, Inc., Rockford, IL, USA.); product formation data were determined by measuring absorbance at 405 nm with a spectrophotometer (SpectraMax iD3, Molecular Devices Inc.), and the values were normalized to the DNA content measured by Pico Green dsDNA Assay Kit (Invitrogen, Inc. Paisley, UK.) in the respective sample.

Extracellular calcium deposition and Alizarin red S (ARS) staining

PDLSCs were cultured in 24-well plates at 4×10⁴ cells/mL as for the ALP staining assay. After the osteogenic medium was removed, cells were washed with PBS and extracellular calcium was dissolved with 10% formic acid. The deposition of extracellular calcium was quantified using the Calcium E-test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s protocol. For the ARS assay, PDLSCs were cultured for 7 and 14 days with low-glucose osteogenic medium, then stained for 3 minutes at room temperature with a solution of 1% ARS (Wako Pure Chemical Industries, Ltd.) and washed three times with PBS.

Real-time polymerase chain reaction (Real-time PCR)

Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Inc., Venlo, the Netherlands) according to the manufacturer’s instructions, and 10 μL total RNA from each sample was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (TAKARA Bio, Inc., Shiga, Japan) on the StepOne^Plus Real-time PCR System (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. PCR was performed to determine the expression level of Runt-related transcription factor 2 (Runx2), and gene expression was normalized to that of the housekeeping gene encoding glyceraldehyde phosphate dehydrogenase (GAPDH).

Measurement of osteocalcin (OCN)

PDLSCs were cultured in low-glucose osteogenic medium for 14 days. The spent culture medium then was collected and the OCN was quantified using an OCN detection kit (TAKARA Bio, Inc.) according to the manufacturer’s protocol.

Western blot analysis

Total protein was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and a phosphatase cocktail (Nacalai Teaque, Inc.). Protein samples were separated on a 12.5% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked for 60 min at room temperature using Blocking One (Nacalai Teaque, Inc.) and then incubated overnight at 4 °C with primary antibodies against Akt, p-Akt (S473), HIF-1α, ERK1/2, p-ERK1/2, or β-actin (Cell Signaling Technology, Inc., Danvers, Massachusetts, USA). Detection of primary antibodies was performed by incubation for 1 hour at room temperature with horseradish peroxidase (HRP) -conjugated secondary antibodies (Invitrogen, Inc.). Immunoreactive bands were visualized with a chemiluminescence kit (Nacalai Teaque, Inc.) and signals were detected with the ChemiDoc MP system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed using ImageJ software.

Lactate deposition

After achieving confluence, PDLSCs were stimulated by osteogenic medium for 7 and 14 days, and the supernatants at each time point were collected for analysis. Lactate secretion into the medium was measured using a Lactate Assay Kit-WST (Dojindo Molecular Technologies, Inc.) according to the manufacturer’s instructions.

Statistical Analysis

Data are presented as mean ± SD. Parametric data from three or more groups were analyzed using a two-tailed One-way Analysis of Variance (ANOVA) with post hoc Tukey’s test. Values of p <0.05 were considered significant.

Characterization of PDLSCs

PDLSCs were characterized at Passage 4 by immunostaining and flow cytometric analysis (Fig. 1). Staining showed that the cells were positive for the mesenchymal stem cell markers CD73, CD90, CD105, and STRO-1, and were negative for primitive hematopoietic progenitors and endothelial cells marker CD34 and pan-leukocyte marker CD45.

Cell proliferation and cell migration

We first assessed the effects of five different glucose concentrations (100, 75, 50, 25, or 0 mg/dL) on PDLSC proliferation after culturing for 1, 3, and 7 days (Fig. 2 a-c). Low-glucose medium had no significant effect on cell viability at 24 h compared to the 100-mg/dL culture (physiological control). However, the cells grown in medium containing 25 and 0 mg/dL glucose exhibited significantly decreased cell viability after 72 h, and all of the tested low-glucose-medium groups showed significantly decreased cell viability at 120 h. Similarly, growth in low-glucose medium significantly inhibited the proportion of live cell staining. Furthermore, the percentage of healed-wound area also was decreased in low-glucose medium (Fig. 2 d and e).

Osteogenic differentiation

Then we assessed the expression of Runx2 mRNA expression and ALP activity in low glucose concentrations (Fig. 3 a-c). The expression of Runx2 mRNA was significantly enhanced after culturing for 3 days in low-glucose osteogenic medium. ALP activity was decreased after culturing in low-glucose medium for 7 and 14 days, consistent with the observation that ALP staining also appeared progressively weaker as the glucose concentration decreased.

Extracellular calcium deposition, ARS staining for calcium, and OCN production

The study also assessed the effects of glucose on the mineralization potential of PDLSCs (Fig. 3 d-f). Extracellular calcium deposition was significantly inhibited in low-glucose osteogenic medium. ARS staining revealed that decreased glucose concentrations significantly resulted in decreased formation of mineralized nodules. The study further demonstrated that OCN production was significantly decreased in the lower glucose concentration.

Lactate production and western blotting analysis

Lactate production in the culture medium supernatant was significantly decreased on both Day 7 and Day 14 of growth in low-glucose medium (Fig. 4 a). Low glucose concentrations also resulted in decreases in the protein levels of HIF-1α, ERK, and Akt; the greatest effects were seen, at both 6 and 24 h, for cultures grown in osteogenic medium containing 0 mg/dL glucose (Fig. 4 b-e).

AZD3965 prevents the effects on PDLSCs of the low-glucose environment

AZD3965, an inhibitor of monocarboxylate transporter-1 (MCT1), has been shown to exert concentration-dependent inhibition of the production of lactate[12]. Following the addition of the AZD3965 to PDLSC cultures growing in medium containing 100 mg/dL glucose, cell proliferation was significantly inhibited (compared to 0-μM AZD3965 control cultures) after 3 and 7 days (Fig. 5 b-d); similar inhibition was seen for the percentage of healed-wound areas (Fig. 5 e-f). Furthermore, the addition of AZD3965 to cultures growing in low-glucose medium resulted in significant changes (compared to 0-μM AZD3965 control cultures) in multiple parameters (Fig. 6 and 7), including the following: accumulation of Runx2 mRNA; decreased ALP activity on both Days 7 and 14, consistent with results observed by ALP staining; decreases in extracellular calcium deposition on both Days 7 and 14, consistent with the results of ARS staining; decreases in OCN levels; and decreases in the levels of HIF-1α and of activated p-ERK and p-Akt.

In this study, we investigated the effect of a low glucose environment on periodontal hard tissue regeneration using PDLSCs. Our results show that the low-glucose environment suppresses the cell proliferation and osteogenic differentiation of PDLSCs. Additionally, we found that lactate may be a key factor in periodontal tissue regeneration.

The cell proliferation of stem cells is considered an important factor in the regeneration of various tissues; particularly, several studies have reported the importance of PDLSCs in periodontal tissue regeneration[10]. In the present study, a low-glucose environment inhibited the proliferation of PDLSCs. On the other hand, we previously have reported that high-glucose conditions also inhibit the proliferation of PDLSCs[13]. Therefore, we suggest that the appropriate (physiologically relevant) glucose concentration (100 mg/dL) is important for maintaining the proliferative potential of PDLSCs.

Cell migration of stem cells contributes to tissue regeneration. Similarly, during the regeneration of periodontal tissue, the migration of PDLSCs to destroyed tissue plays an important role in the repair of tissue defects caused by periodontal disease[10]. The present study showed that low glucose concentrations inhibit cell migration by PDLSCs. Thus, one of glucose’s essential roles in this model of periodontal hard tissue regeneration is to promote cell migration by PDLSCs.

The potential of PDLSCs to differentiate into osteoblasts is one of the key factors for the success of periodontal regeneration therapy. Glucose starvation suppresses osteogenic differentiation, and another study has shown that bone formation is inhibited in Glucose transporter 1 (GLUT1) knockout mice (in which the intracellular glucose concentration would be decreased)[3]. However, little is known about effect of low glucose on osteogenic differentiation of PDLSCs. Thus, the present study is the first (to our knowledge) to examine various osteogenic markers in a low-glucose environment, assessing the effect of glucose deprivation on the osteogenic differentiation of PDLSCs.

We first examined Runx2 mRNA expression, which encodes a transcription factor essential for osteoblast differentiation. The Runx2 mRNA transcript accumulates early in osteoblast differentiation and subsequently is depleted. In other words, Runx2 is a gene that maintains osteoblasts in an immature stage after mesenchymal stem cells have been induced to differentiate into osteoblasts[14]. Although our initial expectation was that the expression of Runx2 would be suppressed under glucose starvation, we found that the low-glucose environment promotes Runx2 mRNA expression in PDLSCs. This result suggests that the PDLSCs may be maintained in an undifferentiated state (precluded from osteogenic differentiation) in a low-glucose environment[13, 15]. That is, mature osteoblast differentiation of PDLSCs may not be induced during glucose starvation, thereby inhibiting periodontal hard tissue regeneration under such conditions.

To identify other factors affecting osteoblast differentiation, we examined the effects of a low-glucose environment on ALP activity, OCN production, and mineralization in PDLSCs. ALP is a marker of intermediate osteoblast differentiation[16, 17], OCN is a marker of late osteoblast differentiation[18], and extracellular calcium deposition is an important indicator for assessing the degree of osteoblast differentiation[8, 19]. In the present work study, growth of PDLSCs in a low-glucose environment resulted in decreases in ALP activity, OCN production, and the amounts of mineralized nodules. These results suggest that a low-glucose environment impedes mid to late-stage osteoblast differentiation of PDLSCs. Therefore, it appears that an adequate glucose concentration is essential for the osteogenic differentiation of PDLSCs to form hard tissues.

Next, we focused on glucose metabolites, to investigate the mechanism whereby a low-glucose environment suppresses osteogenic differentiation. As is seen for the Warburg effect in cancer cells, some studies have reported that ATP production by glucose metabolism in osteoblasts employs anaerobic glycolysis rather than the tricarboxylic acid cycle[5]. Anaerobic glycolysis is a complex biochemical process mediated by multiple glycolytic genes and pathways, such that lactate, an intermediate metabolite, is produced in the cytoplasm by pyruvate reduction. Another study reported that lactate stimulates osteogenic differentiation and mineralization[6]. These facts imply a mechanism whereby osteoblasts regulate their differentiation by reuptake of lactate that the cells have themselves secreted. Therefore, we measured the production of lactate that was secreted into the culture supernatant by PDLSCs. This analysis showed that lactate secretion was decreased in a low-glucose environment. This result suggested that lactate may regulate the differentiation of PDLSCs.

MCT1 is a cellular membrane protein that transports monocarboxylic acids such as lactate and pyruvate, as well as ketones, in and out of cells[20]. Recent research has shown that osteogenic differentiation is regulated by MCT1-mediated lactate reuptake[6, 21, 22]. Therefore, we additionally investigated the effects of AZD3965 (an MCT1-specific inhibitor) on the cell proliferation and osteogenic differentiation of PDLSCs.

Specifically, we tested the effect of adding AZD3965 to cultures growing in medium containing a normal (physiologically relevant) concentration of glucose (100 mg/dL). Under these conditions, AZD3965 inhibited lactate secretion and inhibited cell proliferation, although the effect on cell proliferation appeared to be of a smaller magnitude than that seen under glucose starvation. Cells use ATP derived from glucose metabolism to proliferate. This ATP is generated from lactate metabolism in the mitochondrion, where most of the ATP is produced via the citric acid cycle[23]. In earlier work, we initially supposed that the effect of MCT1 inhibitors on cell proliferation might be insignificant. However, in contrast to our expectation, proliferation by small cell lung cancer cells was partially inhibited by the MCT1 inhibitor[24]. In the current study, we extended that work by showing that AZD3965 inhibits the cell proliferation and osteogenic differentiation potential of PDLSCs. Thus, osteogenic differentiation was suppressed by an MCT1 inhibitor in a manner similar to that seen in a low-glucose environment. These results suggest that the depletion of lactate may be the primary mechanism for inhibiting osteogenic differentiation of PDLSCs in a low-glucose environment.

Finally, we investigated the molecular biological pathways by which lactate derived from glucose metabolism affects PDLSCs. Previous work has shown that ERK promotes cell proliferation and osteoblast differentiation[25–27], and that Akt controls an important signaling pathway that promotes osteogenic differentiation by regulating cellular glucose metabolism[28, 29]. In addition, HIF-1α has been reported to promote bone formation by osteoblasts, and accumulation of HIF-1α in osteoblasts is known to enhance the glycolytic pathway, promote osteogenic differentiation, and increase bone formation[30–34]. In the present study, we found that activation of p-ERK and p-Akt and the accumulation of HIF-1α were attenuated in PDLSCs in a low-glucose environment and upon inhibition of lactate production by MCT1 inhibition. Decreases in the activation of the ERK, Akt, and HIF-1α pathways, would in turn inhibit osteoblast differentiation. These results suggest a role for glucose metabolism in periodontal hard tissue regeneration, and suggest that we have, for the first time, elucidated a potential mechanism of these effects.

This study focused on in vitro experiments, and did not employ animal models. Although few lactate-related in vivo studies have been reported (to our knowledge), the previous literature has shown that lactate can promote hard tissue formation in vitro[6, 21, 22, 35, 36]. Animal experiments will be necessary to determine whether the hard tissues of periodontal regions depend on lactate to regenerate; for instance, lactate could be injected into alveolar bone defects. Moreover, various factors other than the signals examined in this study are known to be involved in bone formation. We are planning to address these issues by conducting further basic in vitro and in vivo research. We expect that such experiments will lead, in the future, to new periodontal hard tissue regeneration therapies using lactate.

In clinical, the success or failure of periodontal regenerative therapy depends on blood supply in the operation area. We found that the local low glucose environment inhibits PDLSCs proliferation and osteoblast differentiation, and the production of lactate is one of the key factors involved in this mechanism. Although the biological mechanisms of lactate have been well studied, much remains unknown about the potential of lactate in periodontal regenerative therapy. Lactic acid has been considered a waste product, but this finding suggests that lactate may be a new treatment option for periodontal regenerative therapy in the future.

In summary, the findings from this study suggest that lactate production by glucose metabolism regulates PDLSC osteogenic differentiation, thereby altering ERK, Akt, and HIF-1α signaling pathways. Furthermore, the involvement of lactate, a product of glycolytic metabolism, reveals a lactate-mediated mechanism in periodontal hard tissue regeneration. Therefore, these findings will contribute to a better understanding of the involvement of energy metabolism in periodontal hard tissue regeneration and the development for future periodontal regenerative therapies.

Acknowledgements

This study was supported partially by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (C) (21K09946, 22K17084, 22K09993) and Osaka Dental University Research Promotion Grant (22-03).

Author contributions

X.D. contributed to conception, design, data acquisition and interpretation, performed all statistical analyses, drafted and critically revised the manuscript; H.K. contributed to conception, design, data acquisition and inter-pretation, drafted and critically revised the manuscript; Y.T. contributed to the conception, design, and critically revised the manuscript; T.N. contributed to the conception and data acquisition; M.U. contributed to the project administration and supervision. All authors gave final approval and agreed to be accountable for all aspects of the work.

Funding

This work was supported by the Japan Society for the Promotion of Science (C) (21K09946, 22K17084, 22K09993) and Osaka Dental University Research Promotion Grant (22-03).

Compliance with ethical standards

Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval: PDLSCs were obtained in accordance with the guidelines of the Osaka Dental University for Medical Ethics, and all experiments were approved by the Osaka Dental University Medical Ethics Committee (approval no. 111132). This study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013.

Informed consent: All participants provided written informed consent to participate in this study, and the study design was approved by the appropriate ethics review boards.

Karner CM, Long F (2018) Glucose metabolism in bone. Bone 115:2–7. https://doi.org/10.1016/j.bone.2017.08.008
Lee W-C, Guntur AR, Long F, Rosen CJ (2017) Energy Metabolism of the Osteoblast: Implications for Osteoporosis. Endocr Rev 38:255–266. https://doi.org/10.1210/er.2017-00064
Wei J, Shimazu J, Makinistoglu MP, et al (2015) Glucose Uptake and Runx2 Synergize to Orchestrate Osteoblast Differentiation and Bone Formation. Cell 161:1576–1591. https://doi.org/10.1016/j.cell.2015.05.029
Zoch ML, Abou DS, Clemens TL, et al (2016) In vivo radiometric analysis of glucose uptake and distribution in mouse bone. Bone Research 4:16004. https://doi.org/10.1038/boneres.2016.4
Lee W-C, Ji X, Nissim I, Long F (2020) Malic Enzyme Couples Mitochondria with Aerobic Glycolysis in Osteoblasts. Cell Reports 32:108108. https://doi.org/10.1016/j.celrep.2020.108108
Wu Y, Wang M, Feng H, et al (2017) Lactate induces osteoblast differentiation by stabilization of HIF1α. Mol Cell Endocrinol 452:84–92. https://doi.org/10.1016/j.mce.2017.05.017
Doherty JR, Cleveland JL (2013) Targeting lactate metabolism for cancer therapeutics. Journal of Clinical Investigation 123:3685–3692. https://doi.org/10.1172/JCI69741
Seo B-M, Miura M, Gronthos S, et al Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364:149–55. https://doi.org/10.1016/S0140-6736(04)16627-0
Nagatomo K, Komaki M, Sekiya I, et al (2006) Stem cell properties of human periodontal ligament cells. J Periodontal Res 41:303–10. https://doi.org/10.1111/j.1600-0765.2006.00870.x
Gronthos S, Mrozik K, Shi S, Bartold PM (2006) Ovine periodontal ligament stem cells: isolation, characterization, and differentiation potential. Calcif Tissue Int 79:310–7. https://doi.org/10.1007/s00223-006-0040-4
Li R, Kato H, Taguchi Y, Umeda M (2022) Intracellular glucose starvation affects gingival homeostasis and autophagy. Sci Rep 12:1230. https://doi.org/10.1038/s41598-022-05398-2
Bola BM, Chadwick AL, Michopoulos F, et al (2014) Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport. Mol Cancer Ther 13:2805–16. https://doi.org/10.1158/1535-7163.MCT-13-1091
Kato H, Taguchi Y, Tominaga K, et al (2016) High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts. Journal of Periodontology 87:e44–e51. https://doi.org/10.1902/jop.2015.150474
Komori T (2006) Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 99:1233–9. https://doi.org/10.1002/jcb.20958
García-Hernández A, Arzate H, Gil-Chavarría I, et al (2012) High glucose concentrations alter the biomineralization process in human osteoblastic cells. Bone 50:276–88. https://doi.org/10.1016/j.bone.2011.10.032
Weinreb M, Shinar D, Rodan GA (1990) Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization. J Bone Miner Res 5:831–42. https://doi.org/10.1002/jbmr.5650050806
Hwang SK, Park JS (1995) Cementless total hip arthroplasty with AML, PCA and HGP prostheses. Int Orthop 19:77–83. https://doi.org/10.1007/BF00179964
Ikeda T, Nomura S, Yamaguchi A, et al (1992) In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections. J Histochem Cytochem 40:1079–88. https://doi.org/10.1177/40.8.1619274
Park J-C, Kim J-M, Jung I-H, et al (2011) Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations. J Clin Periodontol 38:721–31. https://doi.org/10.1111/j.1600-051X.2011.01716.x
Halestrap AP (2013) Monocarboxylic acid transport. Compr Physiol 3:1611–43. https://doi.org/10.1002/cphy.c130008
Sasa K, Yoshimura K, Yamada A, et al (2018) Monocarboxylate transporter-1 promotes osteoblast differentiation via suppression of p53, a negative regulator of osteoblast differentiation. Scientific Reports 8:10579. https://doi.org/10.1038/s41598-018-28605-5
Wu Y, Wang M, Zhang K, et al (2018) Lactate enhanced the effect of parathyroid hormone on osteoblast differentiation via GPR81-PKC-Akt signaling. Biochemical and Biophysical Research Communications 503:737–743. https://doi.org/10.1016/j.bbrc.2018.06.069
Wellen KE, Hatzivassiliou G, Sachdeva UM, et al (2009) ATP-Citrate Lyase Links Cellular Metabolism to Histone Acetylation. Science (1979) 324:1076–1080. https://doi.org/10.1126/science.1164097
Polański R, Hodgkinson CL, Fusi A, et al (2014) Activity of the Monocarboxylate Transporter 1 Inhibitor AZD3965 in Small Cell Lung Cancer. Clinical Cancer Research 20:926–937. https://doi.org/10.1158/1078-0432.CCR-13-2270
Ge C, Xiao G, Jiang D, Franceschi RT (2007) Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development. Journal of Cell Biology 176:709–718. https://doi.org/10.1083/jcb.200610046
Ge C, Xiao G, Jiang D, et al (2009) Identification and Functional Characterization of ERK/MAPK Phosphorylation Sites in the Runx2 Transcription Factor. Journal of Biological Chemistry 284:32533–32543. https://doi.org/10.1074/jbc.M109.040980
Matsushita T, Chan YY, Kawanami A, et al (2009) Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Play Essential Roles in Osteoblast Differentiation and in Supporting Osteoclastogenesis. Molecular and Cellular Biology 29:5843–5857. https://doi.org/10.1128/MCB.01549-08
Mukherjee A, Rotwein P (2009) Akt promotes BMP2-mediated osteoblast differentiation and bone development. Journal of Cell Science 122:716–726. https://doi.org/10.1242/jcs.042770
Robey RB, Hay N (2009) Is Akt the “Warburg kinase”?-Akt-energy metabolism interactions and oncogenesis. Seminars in Cancer Biology 19:25–31
Tomlinson RE, Silva MJ (2015) HIF-1α regulates bone formation after osteogenic mechanical loading. Bone 73:98–104. https://doi.org/10.1016/j.bone.2014.12.015
Chen D, Li Y, Zhou Z, et al (2012) Synergistic Inhibition of Wnt Pathway by HIF-1α and Osteoblast-Specific Transcription Factor Osterix (Osx) in Osteoblasts. PLoS ONE 7:e52948. https://doi.org/10.1371/journal.pone.0052948
Chen D, Tian W, Li Y, et al (2012) Osteoblast-specific transcription factor Osterix (Osx) and HIF-1α cooperatively regulate gene expression of vascular endothelial growth factor (VEGF). Biochemical and Biophysical Research Communications 424:176–181. https://doi.org/10.1016/j.bbrc.2012.06.104
Johnson RW, Schipani E, Giaccia AJ (2015) HIF targets in bone remodeling and metastatic disease. Pharmacology & Therapeutics 150:169–177. https://doi.org/10.1016/j.pharmthera.2015.02.002
Rankin EB, Wu C, Khatri R, et al (2012) The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO. Cell 149:63–74. https://doi.org/10.1016/j.cell.2012.01.051
Esen E, Chen J, Karner CM, et al (2013) WNT-LRP5 Signaling Induces Warburg Effect through mTORC2 Activation during Osteoblast Differentiation. Cell Metabolism 17:745–755. https://doi.org/10.1016/j.cmet.2013.03.017
Hong M, Zhang X-B, Xiang F, et al (2020) MiR-34a suppresses osteoblast differentiation through glycolysis inhibition by targeting lactate dehydrogenase-A (LDHA). In Vitro Cellular & Developmental Biology - Animal 56:480–487. https://doi.org/10.1007/s11626-020-00467-0

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Lactate from glucose metabolism regulates osteogenic differentiation of human periodontal ligament stem cells

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