Nutrient-Dependent Mitochondrial Fission Enhances Osteoblast Function

Abstract

Background: The bone synthesizing function of osteoblasts (OBs) is a highly demanding energy process that requires nutrients. However, how nutrient availability affects OBs behavior and bone mineralization remain to be fully understood.

Methods: MC3T3-E1 cell line and primary OBs (OBs) cultures were treated with physiological levels of glucose (G; 5.5 mM) alone or with the addition of palmitic acid (G+PA) at different concentrations. Mitochondria morphology and activity were evaluated by fluorescence microscopy, qPCR, and oxygen consumption rate (OCR) measurement, and OBs function was assessed by mineralization assay.

Results: The addition of non-lipotoxic levels of 25 μM PA to G increased mineralization in OBs. G+25 μM PA exposure reduced mitochondria size in OBs, which was associated with increased activation of dynamin-related protein 1, a mitochondrial fission protein, enhanced mitochondria OCR and ATP production, and increased expression of oxidative phosphorylation genes. Treatment with Mdivi-1, a putative inhibitor of mitochondrial fission, reduced osteogenesis and mitochondrial respiration in OBs.

Conclusions: Our results revealed that OBs function was enhanced in the presence of glucose and PA at 25 μM. This was associated with increased OBs mitochondrial respiration and dynamics. These results suggest a role for nutrient availability in bone physiology and pathophysiology.

Keywords: bioenergetics; glucose; metabolism; mitochondrial fission; osteoblasts; palmitate.

Figure 1

Effect of PA on MC3T3-E1 cell proliferation, metabolic activity, and osteogenesis. MC3T3-E1 cells were treated for 4 days with Glucose 5.5 mM (G) plus different PA concentrations ranging from 12.5 μM to 100 μM. (A) CFSE representative fluorescence histograms and (B) mean fluorescence intensity (MFI) at 96 h after treatments (n = 3 per group). (C) MTT assay metabolic activity relative fold to t0 at 96 h after treatments (n = 6 per group). Osteogenic-induced cells treated with G or G+PA25. (D) Representative images for ALP staining (Scale bar 400 μm) and (E) ALP enzyme activity (n = 6 per group). (F) Representative images for ARS staining (Scale bar 400 μm) and (G) relative ARS quantization (n = 10 per group). (H–K) qPCR analysis for osteogenic marker genes (n = 4 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. G 5.5 mM; # p < 0.05, ### p < 0.001 vs. G+PA12.5 μM; °° p < 0.01 vs. G+PA25 μM.

Figure 2

PA 25 μM dose effect on mitochondria morphology in MC3T3-E1 cells. MC3T3-E1 cells undergoing osteogenesis and treated with G or G+PA25 were labeled with Mitotracker Red CMXRos probe. (A) Representative images for Mitotracker Red CMXRos staining (scale bar: 25 μm for lower magnification, 40 μm for higher magnification). (B–D) Morphological parameters evaluation and mitochondrial number/mitochondrial area ratio analysis. (E–G) Mitochondrial network analysis (three samples per group; n = 30 cells for G and n = 24 cells for G+PA. (H) Δψm (fold) analysis calculated as probe fluorescence intensity/cell area (three samples per group; n = 75 cells for G and n = 70 cells for G+PA25). (I) Δψm analysis represented as Mean Fluorescence Intensity obtained by flow cytometry (n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Mitochondrial activity profile upon PA 25 μM administration in MC3T3-E1 cells. (A) Seahorse analysis on osteogenic-induced MC3T3-E1 cells undergoing G and G+PA25 treatments, and (B–D) evaluation of respiratory parameters expressed as OCR, pmol/min/cells (n = 3 per group). (E–K) qPCR analysis of gene expression related to fatty acid metabolism (n = 4 per group). (L) mtROS production analysis calculated as MitoSox RED probe fold fluorescence intensity/cell area (three samples per group; n = 29 cells for G and n = 38 cells for G+PA25). * p < 0.05, ** p < 0.01.

Figure 4

PA 25 μM dose effect on mineralization in pOBs. pOBs in osteogenic conditions treated with G or G+PA25. (A) Representative images for ALP staining (Scale bar 400 μm) and (B) ALP enzyme activity (n = 4 per group). (C) Representative images for ARS staining (Scale bar 400 μm) and (D) relative ARS quantization (n = 4 per group). (E–H) qPCR analysis for osteogenic marker genes (n = 4 per group). * p < 0.05.

Figure 5

Mitochondrial fission induced by PA25 dose in pOBs. Osteogenic-induced pOBs treated with G or G+PA25 were labeled with a Mitotracker Red CMXRos probe. (A) Representative images for Mitotracker Red CMXRos staining (scale bar: 25 μm for lower magnification, 40 μm for higher magnification). (B–D) Morphological parameters evaluation and mitochondrial number/mitochondrial area ratio analysis. (E–G) Mitochondrial network analysis (four mice per group; n = 25 cells for G and n = 20 cells for G+PA. (H) Δψm analysis calculated as probe fold fluorescence intensity/cell area (four mice per group; n = 20 cells for G and n = 12 cells for G+PA25). (I) Δψm analysis represented as Mean Fluorescence Intensity obtained by flow cytometry (n = 4 per group). (J) Representative images for pDRP1[Ser616] immunofluorescence staining (scale bar: 25 μm for lower magnification, 40 μm for higher magnification) and (K) relative quantification (four mice per group; n = 39 cells for G and n = 35 cells for G+PA25) expressed as fluorescence intensity/cell area. (L) Representative western blot image and analysis of DRP1 expression in its phosphorylated form related to total DRP1 protein (normalized on actin) (n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

PA 25 μM dose influence mitochondrial metabolic profile in pOBs. Mitochondrial activity profile upon low PA levels administration to G in pOB cells. (A) Seahorse analysis on osteogenic-induced pOBs undergoing G and G+PA25 treatments, and (B–D) evaluation of respiratory parameters expressed as OCR, pmol/min/cells (n = 3 per group). (E–K) qPCR analysis of gene expression related to fatty acid metabolism (n = 4 per group). (L) mtROS production analysis calculated as MitoSox RED probe fold fluorescence intensity/cell area (four mice per group; n = 39 cells for G and n = 27 cells for G+PA25). * p < 0.05, ** p < 0.01.

Figure 7

Mitochondrial fission inhibition effect on pOBs mineralization and metabolism in PA conditions. pOBs in osteogenic conditions treated with G or G+PA25 or treated with G+PA25+Mdivi-1 compound. (A) Representative images for ALP staining (Scale bar 400 μm) and (B) ALP enzyme activity (n = 4 per group). (C) Representative images for ARS staining (Scale bar 400 μm) and (D) relative ARS quantization (n = 3 for G, n = 3 for G+PA25, n = 5 for G+PA25+Mdivi-1). (E–H) qPCR analysis for osteogenic marker genes (n = 4 per group). Mitochondrial activity profile upon Mdivi-1 administration to G+PA25 in pOB. (I) Seahorse analysis on osteogenic-induced pOBs undergoing G, G+PA25, and G+PA25+Mdivi-1 treatments, and (J–L) evaluation of respiratory parameters expressed as OCR, pmol/min/cells (n = 4 per group). * p < 0.05, ** p < 0.01, *** p < 0.001.