Uncarboxylated osteocalcin reverses the high glucose‑induced inhibition of the osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway

  • Authors:
    • Luyao Ma
    • Fangzi Gong
    • Jiaojiao Xu
    • Jianhong Yang
  • View Affiliations

  • Published online on: March 29, 2021     https://doi.org/10.3892/ijmm.2021.4924
  • Article Number: 91
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Abstract

Diabetic osteoporosis is a serious complication of diabetes affecting human bones. Uncarboxylated osteocalcin (GluOC), a small molecular protein specifically synthesized and secreted from osteoblasts, is of importance in regulating energy metabolism. In previous studies, the authors demonstrated that high glucose inhibited osteoblastic differentiation, but promoted adipocytic differentiation. GluOC promoted osteogenic and inhibited adipogenic differentiation under high glucose conditions. However, the corresponding receptors and signaling pathways through which GluOC exerts its effects on MC3T3E1 cells remain elusive. Thus, in the present study, Cell Counting kit‑8 assays and western blot analysis were performed to assess the proliferation of MC3T3E1 cells. Alizarin Red S or Oil Red O staining, as well as reverse transcription‑quantitative PCR analysis were performed to examine osteogenic and adipogenic differentiation. The cells were transfected with short interfering RNA or inhibitors to investigate the possible signaling pathways involved. The results revealed that G‑protein coupled receptor, class C, group 6, subtype A (GPRC6A) receptor expression was markedly increased following the addition of GluOC to the MC3T3E1 cells. GPRC6A silencing decreased osteogenic gene expression, while it increased adipogenic gene expression. Furthermore, GluOC promoted osteoblast differentiation via the subsequent activation of the cyclic AMP (cAMP)/protein kinase A(PKA)/AMP‑activated protein kinase (AMPK) signaling pathway in MC3T3E1 cells. On the whole, the results of the present study suggest that GluOC reverses the high glucose‑induced inhibition of osteogenic differentiation via the GPRC6A/cAMP/PKA/AMPK signaling pathway in MC3T3E1 cells, and thus may prove to be beneficial in the treatment of diabetic osteoporosis.

Introduction

Osteocalcin (OC) is a multipurpose bone-derived hormone (1), which can be γ-carboxylated at one or more of its glutamic acid residues (2). Therefore, OC can be present as one of 2 forms, carboxylated and undercarboxylated OC. Uncarboxylated osteocalcin (GluOC) is the form with no γ-carboxylated glutamic acid residu (3). GluOC is not only an index used to assess the health of bones, but also a biologically active molecule directly secreted into the blood to mediate glucose and energy metabolism (4,5). GluOC can affect the role of glucose by promoting the proliferation of β cells, increasing the secretion of insulin and improving the sensitivity of insulin target tissues, such as muscle and adipogenic tissue (6,7). However, the precise mechanisms through which GluOC improves the effects of glucose on osteoblasts have not yet been fully elucidated, at least to the best of our knowledge.

Osteoporosis is considered a serious skeletal disease, characterized by abnormal bone structure and low bone mineral density (8,9). The imbalance between osteoblast and adipocyte differentiation of bone marrow-derived stromal cells (BMSCs) is a major cause of osteoporosis (10-12). Diabetic osteoporosis is a severe diabetic complication affecting the bones. The number of patients with diabetic osteoporosis is increasing, and the disease is thus gaining increasing attention (13-15). Previous studies have suggested that diabetic patients have a lower bone quality primarily due to hyperglycemia (16,17). In a previous study on primary rat osteoblasts, the authors demonstrated that high glucose induced oxidative stress and resulted in the activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway to induce adipogenic differentiation (18). Moreover, it was demonstrated that high glucose conditions activated the cyclic AMP (cAMP)/protein kinase A(PKA) pathway to stimulate the adipocytic differentiation of MG63 cells (19). GluOC has been shown to inhibit the high glucose-induced inhibition of cellular proliferation, as well as the osteogenic differentiation of MC3T3E1 cells (20). However, the corresponding receptors and signaling pathways through which GluOC exerted its effects on osteoblasts have not yet been fully elucidated.

G-protein coupled receptor, class C, group 6, subtype A (GPRC6A), a relatively recently discovered G-protein-coupled receptor, has been identified in several tissues and organs in humans, mice and rats, including skeletal muscle, pancreas, kidneys, heart, liver, lungs and brain (21). It has been reported that GPRC6A can bind to a variety of ligands, such as L-α amino acids, calcium, magnesium, testosterone and osteocalcin (22,23). Pi et al (24) demonstrated that 293 cells transfected with full-length GPRC6A cDNA exhibited a dose-dependent response to treatment with GluOC; GluOC activated the extracellular signal-regulated kinase (ERK) pathway via the GPRC6A/phospholipase C (PLC)/protein kinase C (PKC) signaling pathway. Otani et al (25) found that in 3T3L1 cells, GluOC was assumed to bind to GPRC6A, and this resulted in the increased intracellular accumulation of cAMP, and thus, in the subsequent activation of PKA to promote the synthesis and secretion of adiponectin. Karsenty and Oury (26) also found that GluOC upregulated cAMP levels via GPRC6A to promote the synthesis and secretion of testosterone in testicular mesenchymal cells. In another study, in TC-6 cells, GluOC stimulated PKD1 in a dose-dependent manner via GPRC6A, suggesting the active participation of GPRC6A in the maintenance of glucose homeostasis (27). However, whether GPRC6A acts as a GluOC receptor in MC3T3E1 has not yet been confirmed, at least to the best of our knowledge.

The present study assessed whether GPRC6A functions as a receptor of GluOC in the high glucose-induced inhibition of the osteogenic differentiation of MC3T3E1 cells. To the best of our knowledge, the present study is the first to demonstrate that GluOC activates the GPRC6A/cAMP/PKA/AMP-activated protein kinase (AMPK) signaling pathway to reverse the high glucose-induced inhibition of the osteogenic differentiation of MC3T3E1 cells. Thus, the results of the present study add to a growing body of evidence indicating that in MC3T3E1 cells, GPRC6A plays a significant role in osteogenic differentiation promoted by GluOC. Additionally, these results also highlight a potentially novel means for the prevention or treatment of diabetic osteoporosis.

Materials and methods

Materials

Alizarin Red S and Oil Red O were obtained from Sigma-Aldrich; Merck KGaA. Small interfering (si) RNAs were synthesized by Shanghai Gima Corp. Inhibitors, including SQ22536 [adenylate cyclase (AC) inhibitor], U73122 (PLC inhibitor), H-89 (PKA inhibitor) and BML-275 (AMPK inhibitor) were obtained from TargetMol. Antibodies against PKA (cat. no. 4782), AMPKα (cat. no. 5831), phospho-PKA (cat. no. 5661) and phospho-AMPKα (cat. no. 2535) were purchased from Cell Signaling Technology, Inc. Antibodies against cyclin D1 (cat. no. ab134175), proliferating cell nuclear antigen (PCNA; cat. no. ab29) and β-actin (cat. no. ab8226) were purchased from Abcam.

Cells and cell culture

MC3T3E1 cells (cat. no. GNM15) were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Initially, the cells were grown in α-MEM (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) with 5% CO2 at 37°C. The concentration of FBS in α-MEM was lowered to 4% when treating the cells, and the medium was replaced every day during the treatments. The control group was exposed to a glucose concentration of 5.5 mM.

Cell proliferation assay

A Cell Counting kit-8 (cat. no. CK04; Dojindo Molecular Technologies, Inc.) was used to detect the proliferation of the MC3T3E1 cells. MC3T3E1 cells were plated in 96-well plates (3×103 cells/well) for 24 h, after which the cells were cultured in 4% FBS α-MEM with various concentrations of GluOC (0, 0.1, 1, 3 or 10 ng/ml) and high glucose (25.5 mM) for 1, 2 or 3 days, as previously described (20). Subsequently, 100 μl fresh medium and 10 μl CCK-solution 8 were added to each well, and the cells were cultured for 30 min in a humidified incubator with 5% CO2 at 37°C. An automated microplate reader (Synergy H1; Biotek Instruments, Inc.) at 450 nm was used to measure the absorbance.

Mineralization assay

The MC3T3E1 cells were grown overnight at a density of 1×106 cells/well in 6-well plates, and then cultured in osteoblastic differentiation medium (HyClone; Cytiva) (α-MEM, 4% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate disodium, 50 mg/l vitamin C) containing high glucose (25.5 mM) or GluOC (3 ng/ml) for 28 days. The cells were fixed at room temperature with 10% formaldehyde for 30 min and calcium nodules were stained using Alizarin Red S. The staining was imaged using a microscope (magnification, ×100, DM750; Leica Microsystems, Inc.) and the red areas represented calcified nodules. For quantitative detection, 0.1 M cetylpyridinium chloride was used to dissolve the calcium nodules for 15 min. Following a 20-fold dilution, the absorbance value was measured at 570 nm by an automated microplate reader (Synergy H1; Biotek Instruments, Inc.).

Assay of lipid droplets

The MC3T3E1 cells were grown overnight at a density of 1×106 cells/well in 6-well plates and then cultured in adipocytic differentiation medium (α-MEM, 4% FBS, 1 mM dexamethasone, 10 mg/l insulin) containing high glucose (25.5 mM) or GluOC (3 ng/ml) for 16 days. The cells were fixed at room temperature with 10% formaldehyde for 30 min and Oil Red O was used to stain the lipid droplets. The staining was imaged using a microscope (magnification, ×500, DM750; Leica Microsystems, Inc.), and the red areas represented lipid droplets. For quantitative detection, isopropanol was used to dissolve lipid droplets for 10 min and the absorbance value was measured at 510 nm by an automated microplate reader (Synergy H1; Biotek Instruments, Inc.).

Alkaline phosphatase (ALP) assay

The MC3T3E1 cells were seeded into 6-well plates (1×106 cells/well) and cultured in 4% FBS α-MEM containing high glucose (25.5 mM), siRNAs (100 pmol), inhibitors (10 μM) or GluOC (3 ng/ml) for 7 days. A total of 200 μl pre-cooled PBS was added to collect the cells, and the supernatant was retained to measure ALP activity, using an Alkaline Phosphatase assay kit (cat. no. A059-2; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

Type I collagen (COLI) assay

The secretion of COLI was performed as described above for the measurement of ALP activity. The secretion of COLI was quantified using a specific ELISA kit (cat. no. SEA571Mu; Cloud-Clone Corp.), according to the manufacturer's protocol.

Reverse transcription-quantitative PCR (RT-qPCR)

The MC3T3E1 cells in 6-well plates (1×106 cells/well) were treated with or without high glucose (25.5 mM), siRNAs (100 pmol), inhibitors (10 μM) or GluOC (3 ng/ml). Total RNA extraction from the MC3T3E1 cells was performed using a Total RNA kit (cat. no. DP419; Tiangen Biotech, Co., Ltd.). A total of 2 μg RNA was reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (cat. no. A T311-03; TransGen Biotech.) and quantitative PCR (qPCR) was performed in a 20 μl reaction volume with the TransStart Top Green qPCR SuperMix (+Dye II) (cat. no. AQ132-24; TransGen Biotech.). The following thermocycling conditions were used: 40 cycles at 94°C for 5 sec, 60°C for 15 sec and 72°C for 10 sec. To quantify GPRC6A expression, qPCR was performed after 24 h. The expression of the adipogenic genes, peroxisome proliferator-activated receptor γ (PPARγ) and FAS, was assessed after 3 days. The expression of the osteogenic genes, Runt-related transcription factor 2 (Runx2) and osterix (Osx), was assessed after 5 days. The sequences of the primers used are listed in Table I. Expression data were normalized to β-actin, which was used as the internal standard.

Table I

Primer sequences designed for RT-qPCR.

Table I

Primer sequences designed for RT-qPCR.

GeneForward primers (5′→3′)Reverse primers (5′→3′)
Runx2GCCTTCAAGGTGGTAGCCC C GTTACCCGCCATGACAGTA
Osx ACTGGCTAGGTGGTGGTCAG GGTAGGGAGCTGGGTTAAGG
PPARγ GCATGGTGCCTTCGCTGA TGGCATCTCTGTGTCAACCATG
FAS GGCTGCAGTGAATGAATTTG TTCGTACCTCCTTGGCAAAC
GPRC6A TCCGGAGTCAAGCTGGGATA CCCTTGGCATGTAGCTGGAA
β-actin GCTCTTTTCCAGCCTTCCTT AGGTCTTTACGGATGTCAACG

[i] Runx2, runt-related transcription factor 2; Osx, osterix; PPARγ, peroxisome proliferator-activated receptor γ; FAS, fatty acid synthase; GPRC6A, GPCR class C group 6 subtype A receptor.

siRNAs and transfection

siRNA-1 (siRNA.m1638, CCA ACA CAG CTG TTG CTA T) and siRNA-2 (siRNA.m2553, GCAGAAGACTAACACCAAA), which were specific for GPRC6A were used in the present study. GPRC6A (GenBank accession no. NM_153071) was used as a template for synthesizing the siRNAs. Cells at a density of 2×105 cells/well in 24-well plates were cultured overnight in 10% FBS α-MEM, and then transiently transfected with 100 pmol siRNA and 5 μl Lipofectamine® 2000 (cat. no. 11668-019, Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RT-qPCR analysis was used to quantify GPRC6A expression after 24 h. Subsequently, the medium was changed to 4% FBS α-MEM containing 25.5 mM glucose or 3 ng/ml GluOC. Scrambled siRNA (UUC UCC GAA CGU GUC ACG UTT) was used as a negative control.

Radioimmunoassay for cAMP

MC3T3E1 cells at a density of 2×105 cells/well in 24-well plates were cultured in 4% FBS α-MEM and divided into a control group, high glucose group, GluOC group and a SQ22536 group. The cAMP assay kit (cat. no. ab133051; Abcam) was used to measure the intracellular accumulation of cAMP after 24 h, according to the manufacturer's protocol.

Western blot analysis

MC3T3E1 cells in 6-well plates (1×106 cells/well) were treated either with or without glucose (25.5 mM), siRNAs (100 pmol), inhibitors (10 μM) and GluOC (3 ng/ml). The cold cell lysates were acquired by ultrasonic disruption with RIPA lysis buffer on ice after two ice-cold rinses with PBS. Cell lysates were centrifuged (10,000 × g) at 4°C for 20 min, and the protein concentrations of the extracted proteins were measured using a BCA Detection kit (cat. no. B5000; Beijing Lablead Biotech, Co., Ltd.). A total of 10 μg proteins (for each lane) in cell lysates were loaded and separated by 12% SDS-PAGE, and then transferred to PVDF membranes. The membranes were blocked in TBS-5% Tween-20 (TBST) containing 5% non-fat milk at room temperature for 2 h and incubated overnight at 4°C with rabbit-derived antibodies, including anti-cyclin D1 (1:10,000), anti-PCNA (1:2,000), anti-β-actin (1:1,000), anti-PKA (1:1,000), anti-phosphorylated (p-)PKA (1:1,000), anti-AMPKα (1:1,000), anti-p-AMPKα (1:1,000). The membranes were then washed 3 times with TBST (10 min each), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no. ab6721; Abcam) at 25°C for 2 h. The protein bands were visualized using an ECL kit (Biomiga). To quantify the expression of proteins, the intensity of bands was normalized to the respective β-actin control, and the bands of the phospho-proteins were normalized to the total levels of that specific protein. ImageJ version 6 (National Institutes of Health) was used for densitometric analysis.

Statistical analysis

Data are presented as the means ± standard deviation of at least 3 independent repeats. Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, Inc.). Differences between the groups were compared using a one-way ANOVA followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

GluOC promotes the proliferation of MC3T3E1 cells under high glucose conditions

First, the MC3T3E1 cells were treated with high glucose or various concentrations of GluOC (0.1, 1, 3, 10 and 30 ng/ml) to confirm whether GluOC promotes the proliferation of MC3T3E1 cells under high glucose conditions. High glucose inhibited cell proliferation, whereas treatment with 3 ng/ml GluOC significantly alleviated the inhibitory effects of high glucose on the proliferation of the MC3T3E1 cells (Fig. 1A). Thus, in all subsequent experiments, 3 ng/ml GluOC was used as the working dose to treat the MC3T3E1 cells. Similarly, the expression levels of cyclin D1 and PCNA were reduced by high glucose, whereas the addition of GluOC reversed the inhibitory effects of high glucose on cyclin D1 and PCNA expression (Fig. 1B-D). These results suggest that GluOC reversed the high glucose-induced inhibition of the proliferation of MC3T3E1 cells.

GluOC promotes the osteogenic differentiation and inhibits the adipogenic differentiation of MC3T3E1 cells under high glucose conditions GluOC promotes the mineralization and inhibits the high glucose-induced formation of lipid droplets in MC3T3E1 cells

It has been reported that MC3T3E1 cells generate calcium nodules during mineralization, and these calcium nodules are considered as an osteoblastic parameter (28). Alizarin Red S staining of calcium nodules indicated that cells treated with GluOC generated significantly more calcium nodules, whereas fewer calcium nodules were observed in the high glucose group (Fig. 2A). Fewer lipid droplets stained by Oil Red O were observed in the GluOC-treated cells (Fig. 2B). GluOC did not affect the cells in the normal glucose group. Both of the images and the results of quantitative analysis indicated that GluOC promoted the mineralization and inhibited the high glucose-induced formation of lipid droplets in MC3T3E1 cells.

GluOC promotes the expression of COLI and inhibits high glucose-induced ALP activity in MC3T3E1 cells

Osteoblasts express various phenotypic markers including ALP and collagenous bone matrix protein-COLI (29,30). Thus, ALP activity and the COLI secretion of MC3T3E1 cells cultured with or without GluOC for 7 days under high glucose conditions were examined in the present study. As shown in Fig. 2C, high glucose upregulated ALP activity, whereas treatment with GluOC reversed this effect. Conversely, an increased COLI production was observed in the cells treated with GluOC (Fig. 2D). GluOC did not affect the cells in the normal glucose group. These findings suggested that GluOC increased COLI expression and decreased the high glucose-induced ALP activity in MC3T3E1 cells.

GluOC promotes the expression of osteogenic genes and inhibits the high glucose-induced expression of adipogenic genes in MC3T3E1 cells

RT-qPCR was used to further confirm the effects of GluOC on the expression of relevant genes. As shown in Fig. 2E, the gene expression levels of the osteogenic markers (Runx2 and Osx) in the high glucose group were significantly decreased, whereas the expression of adipogenic genes (PPARγ and FAS) was increased. The effects of high glucose were reversed by GluOC; however, GluOC did not affect the cells in the normal glucose group. These results demonstrated that GluOC significantly affected the upregulation of osteogenic gene expression and downregulated adipogenic gene expression in MC3T3E1 cells. Thus, GluOC reversed the inhibitory effects of high glucose on the osteogenic differentiation of MC3T3E1 cells.

GluOC reverses the high glucose-induced inhibition of osteogenic differentiation via GPRC6A in MC3T3E1 cells GluOC increases the expression of GPRC6A in MC3T3E1 cells

The expression of GPRC6A, which was hypothesized to be a GluOC receptor in MC3T3E1 cells, was detected to determine whether GPRC6A is the receptor of GluOC. Confluent cells were cultured in 6-well plates with 4% FBS and treated with high glucose or GluOC for 6, 12 or 24 h, respectively. RT-qPCR analysis indicated that treatment with GluOC significantly increased the expression of GPRC6A in MC3T3E1 cells (Fig. 3A).

GPRC6A functions as a receptor of GluOC in MC3T3E1 cells

To further confirm whether GluOC stimulates the osteogenic differentiation of MC3T3E1 cells under high glucose conditions via GPRC6A, siRNAs were used to knockdown GPRC6A expression. RT-qPCR analysis indicated that GPR6CA siRNA-2 significantly reduced the expression of GPRC6A by 90% (Fig. 3B). Based on these results, GPR6CA siRNA-2 was selected to knockdown GPRC6A expression in all subsequent experiments. To determine the effects of GPRC6A on the osteoblast differentiation of MC3T3E1 cells, the present study examined the activity of ALP and the expression of COLI, as well as the expression of the aforementioned genes (Runx2, Osx, PPARγ and FAS) following GPRC6A knockdown. The promoting effects of GluOC on the osteogenic differentiation of MC3T3E1 cells were reversed by the knockdown of GPRC6A (the knockdown of GPRC6A increased ALP activity, decreased COLI production, decreased Runx2 and Osx expression, and increased PPARγ and FAS expression), and the results were similar to those in the HG group (Fig. 3C-E). Taken together, these results suggest that GPRC6A functions as a receptor of GluOC in MC3T3E1 cells.

GluOC reverses the high glucose-induced inhibition of osteogenic differentiation via the GPRC6A/cAMP/PKA/AMPK signaling pathway in MC3T3E1 cells GluOC promotes the osteogenic differentiation of MC3T3E1 cells by activating AC through GPRC6A

The intracellular signaling pathways activated by the GluOC-mediated activation of GPRC6A in MC3T3E1 cells were determined. The results of western blot analysis revealed that GluOC reversed the high glucose-induced decrease in the phosphorylation of PKA and AMPK, and that this effect was abrogated by the knockdown of GPR6CA or SQ22536, an AC inhibitor. Additionally, it was confirmed that the addition of U73122, a PLC inhibitor, did not affect the effects of GluOC on PKA and AMPK phosphorylation under high glucose conditions (Fig. 4A-F). These findings suggest that GluOC activated downstream PKA and AMPK through GPRC6A-AC rather than GPRC6A-PLC. As shown in Fig. 4G, GluOC increased the intracellular cAMP concentrations, reversing the decrease observed in the HG group; the addition of SQ22536 attenuated this effect, suggesting that GluOC promoted the production of cAMP through AC. Additionally, SQ22536 reversed the increase in the expression of COLI, Runx2 and Osx induced by GluOC, whereas ALP activity, and the expression of PPARγ and FAS were significantly increased under high glucose conditions. U73122, a PLC inhibitor, did not affect the effects of GluOC under high glucose conditions (Fig. 4H-J). These findings suggest that the activation of AC rather than PLC through GPRC6A is the mechanism through which GluOC reverses the inhibitory effects of high glucose on the osteogenic differentiation of MC3T3E1 cells.

GluOC phosphorylates PKA and AMPK to promote the osteogenic differentiation of MC3T3E1 cells

Finally, the crucial functions of PKA and AMPK on the GluOC-mediated promotion of the osteogenic differentiation of MC3T3E1 cells were determined. The addition of H-89 inhibited the phosphorylation of PKA and AMPK, and treatment with BML-275 did not affect the phosphorylation of PKA, but only inhibited the phosphorylation of AMPK, suggesting that AMPK was a downstream factor of GPRC6A/cAMP/PKA (Fig. 5A-C). As shown in Fig. 5D-F, the expression of COLI, Runx2 and Osx induced by GluOC was suppressed by H-89 and BML-275, whereas ALP activity, and the expression of PPARγ and FAS were significantly increased similar to the results in the HG group. These results demonstrated that the sequential activation of PKA and AMPK was the mechanism through which GluOC reversed the inhibitory effects of high glucose on the osteogenic differentiation of MC3T3E1 cells. Taken together, GluOC activated AC and PKA through GPRC6A, and the effects of GluOC were AMPK phosphorylation-dependent, which further promoted the osteogenic differentiation and inhibited the adipogenic differentiation of MC3T3E1 cells under high glucose conditions (Fig. 6).

Discussion

In the present study, it was found that GluOC reversed high glucose-induced inhibition of osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway, manifested by an increase in COLI protein expression and a decrease in ALP activity, as well as the increased expression of osteoblast-specific genes (Runx2 and Osx), and the decreased expression of adipogenic genes (PPARγ and FAS). The results of the present study provide novel insight into the role of GluOC and the molecular mechanisms underlying the inhibition of the high glucose-induced inhibition of osteogenic differentiation of MC3T3E1 cells.

Diabetic osteoporosis is a severe complication of bones in patients with diabetes, and is becoming an ever-increasing public health concern (31). In a previous study by the authors, it was demonstrated that high glucose, a major cause of diabetic osteoporosis, resulted in abnormal structural changes and a decrease in bone density, specifically of the bone trabecula in KK-Ay mice (32). Additionally, in the present study, it was confirmed that high glucose exerted an inhibitory effect on the osteoblastic differentiation of MC3T3E1 cells. Thus, the identifying factors that inhibit the adipogenic differentiation of osteoblasts induced by high glucose has potentially wide prospects for the prevention of diabetic osteoporosis.

In recent years, bone has been considered as not only a structural organ, but also as an endocrine organ that can secrete protein factors into the circulation (33). GluOC, the active form of OC, is crucial for the regulation of energy metabolism and glucose metabolism (34,35). GluOC relieves diabetes by increasing insulin synthesis and secretion. GluOC can also act on adipocytes and Leydig cells to promote the production of adiponectin and testosterone, respectively (36,37). Studies have demonstrated that GluOC functions by binding to its putative receptor, GPRC6A. GPRC6A is a relatively recently discovered GluOC receptor. A growing number of studies have confirmed that GPRC6A is expressed by β cells, and GluOC directly activates GPRC6A to regulate β-cell proliferation and insulin secretion (38,39). GluOC activates GPRC6A to stimulate mammalian target of rapamycin (mTOR) phosphorylation in myocytes (40) and stimulates Leydig cells to produce vitamin D via the activation of GPRC6A (41). In a previous study, the authors demonstrated that GluOC ameliorated hepatic glucose and lipid metabolism in KK-Ay mice (42). Moreover, GluOC has been shown to exert a positive effect on the osteogenic differentiation of BMSCs (43). The conclusion that GluOC promotes the osteogenic differentiation of MC3T3E1 cells was confirmed in the present study. However, the receptor of GluOC in osteoblasts has not yet been identified. To the best of our knowledge, the present study demonstrated, for the first time, that GPRC6A functioned as a receptor of GluOC in MC3T3E1 cells. GluOC promoted the expression of GPRC6A, irrespective of the glucose concentration. Following transfection with GPR6CA siRNA, the inhibitory effects of GluOC on ALP activity and adipogenic gene expression were abrogated, as well as the effect on the increase in COLI and osteogenic gene expression. These results demonstrated that GPRC6A functioned as a receptor of GluOC in MC3T3E1 cells.

The signaling pathways that are mediated via GPRC6A vary. It has been shown that GluOC activates GPCR6A, thereby increasing the expression of testosterone via the cAMP/CREB pathway (44). GluOC binding to GPRC6A also increases the cAMP concentrations and consequently, PKA phosphorylation in 3T3L1 adipocytes, thereby promoting the production of adiponectin (5). In the present study, it was found that GluOC subsequently activated the GPRC6A/cAMP/PKA/AMPK signaling pathway to reverse the high glucose-induced inhibition of osteoblastic differentiation of MC3T3E1 cells. As shown in Fig. 4, the addition of SQ22536, an inhibitor of AC, reduced the phosphorylation levels of PKA and AMPK compared with the GluOC group, intracellular CAMP accumulation and the expression of osteogenic-specific markers decreased and the expression of adipogenic-specific markers increased. However, the addition of the PLC inhibitor, U73122, did not affect the results, indicating that GluOC activated AC, rather than PLC after binding to GPRC6A, and that AC mediated the high glucose-induced inhibition of osteoblastic differentiation of MC3T3E1 cells. Blocking the cAMP/PKA/AMPK signal transduction pathway with H-89 or BML-275 also abrogated the effects of GluOC on osteoblastic differentiation, as evidenced by the increased expression of adipogenic gene expression and the increased activity of ALP, as well as the reduced production of COLI and reduced osteogenic gene expression. The above-mentioned results suggest that the GPRC6A/cAMP/PKA/AMPK signaling pathway was responsible for GluOC mediated inhibition of the high glucose-induced inhibition of osteoblastic differentiation of MC3T3E1 cells.

In conclusion, to the best of our knowledge, the present study is the first to demonstrate that GluOC reverses high glucose-induced inhibition of osteogenic differentiation by activating the GPRC6A/cAMP/PKA/AMPK signaling pathway in MC3T3E1 cells (Fig. 6). These findings further clarify the pathogenesis of diabetic osteoporosis at the cellular level, indicating that the GluOC/GPRC6A signaling pathway may potentially serve as a therapeutic target for the prevention or treatment of diabetic osteoporosis.

Availability of data and materials

All analyses and results obtained from the current experiments are included in the present study.

Authors' contributions

All authors (LM, FG, JX and JY) contributed to the conception and design of the entire study. FG and JX performed an abundant search of relevant literature on osteocalcin. LM performed the experiments and wrote the manuscript. LM and JY were responsible for modifying the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they no competing interests.

Acknowledgements

Not applicable.

Funding

No funding was received.

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May-2021
Volume 47 Issue 5

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Online ISSN:1791-244X

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Spandidos Publications style
Ma L, Gong F, Xu J and Yang J: Uncarboxylated osteocalcin reverses the high glucose‑induced inhibition of the osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway. Int J Mol Med 47: 91, 2021
APA
Ma, L., Gong, F., Xu, J., & Yang, J. (2021). Uncarboxylated osteocalcin reverses the high glucose‑induced inhibition of the osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway. International Journal of Molecular Medicine, 47, 91. https://doi.org/10.3892/ijmm.2021.4924
MLA
Ma, L., Gong, F., Xu, J., Yang, J."Uncarboxylated osteocalcin reverses the high glucose‑induced inhibition of the osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway". International Journal of Molecular Medicine 47.5 (2021): 91.
Chicago
Ma, L., Gong, F., Xu, J., Yang, J."Uncarboxylated osteocalcin reverses the high glucose‑induced inhibition of the osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway". International Journal of Molecular Medicine 47, no. 5 (2021): 91. https://doi.org/10.3892/ijmm.2021.4924