Abstract
Osteocytes are the main cells of bone tissue and play a crucial role in bone formation and resorption. Recent studies have indicated that Diabetes Mellitus (DM) affects bone mass and potentially causes higher bone fracture risk. Previous work on osteocyte cell cultures has demonstrated that mechanotransduction is impaired after culture under diabetic pre-conditioning with high glucose (HG), specifically osteoclast recruitment and differentiation. The aim of this study was to analyze the extracellular metabolic changes of osteocytes regarding two conditions: pre-conditioning to either basal levels of glucose (B), mannitol (M) or HG cell media, and mechanical stimulation by fluid flow (FF) in contrast to static condition (SC). Secretomes were analyzed using Liquid Chromatography and Capillary Electrophoresis both coupled to Mass Spectrometry (LC-MS and CE-MS, respectively). Results showed the osteocyte profile was very similar under SC, regardless of their pre-conditioning treatment, while, after FF stimulation, secretomes followed different metabolic signatures depending on the pre-conditioning treatment. An important increment of citrate pointed out that osteocytes release citrate outside of the cell to induce osteoblast activation, while HG environment impaired FF effect. This study demonstrates for the first time that osteocytes increase citrate excretion under mechanical stimulation, and that HG environment impaired this effect.
Highlights
Mechanical loading is an important regulator of bone mass, by which our skeleton adapts to the changes in load by altering its mass, shape and microstructure[1]
The signal intensity of each cell medium (CM) profile reflected by their total useful signal (TUS) along the worklist showed that quality control (QC) injections were all in the same level for the three techniques (Fig. S1B)
A second principal components analysis (PCA) model was performed after removing Quality control (QC) (Fig. 1), in which we observed a separation of samples treated with fluid flow (FF) (B-FF, high glucose (HG)-FF and M-FF) in respect to the clustering of static condition (SC) (B-SC, HG-SC and M-SC) samples
Summary
Mechanical loading is an important regulator of bone mass, by which our skeleton adapts to the changes in load by altering its mass, shape and microstructure[1]. Our group has shown that HG alters the secretome of mechanically stimulated osteocyte cells, affecting the manner these cells communicate with osteoclasts[14]. To investigate the effect of HG on mechanically stimulated osteocytes, we performed metabolomics analysis on cell culture media using a multiplatform approach including liquid chromatography and capillary electrophoresis, both coupled to mass spectrometry (LC-MS and CE-MS, respectively). With this approach, we managed to investigate the metabolic background of osteocyte communication and the effect of HG environment on osteocyte metabolic activity after mechanical stimulation. Our findings highlight citrate as a key metabolite in mechanically stimulated osteocytes, and that this process is impaired by the HG environment
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