Role of Glucose Transporters in osteoblastic cells
Rais, Sana (2022-01-31)
Role of Glucose Transporters in osteoblastic cells
Rais, Sana
(31.01.2022)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
avoin
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2022030822404
https://urn.fi/URN:NBN:fi-fe2022030822404
Tiivistelmä
Human skeleton is undergoing a constant remodeling through coupled actions of bone resorbing osteoclasts and bone forming osteoblasts and this remodeling process requires a constant source of energy for the cells. Different energy substrates such as glucose or fatty acids are thought to be important for osteoblastic differentiation and act as a fuel source for bones. Glucose is a major source of energy for cells, and it is transported into the cells via glucose transporter proteins (GLUTs).
To date, it is unclear which GLUTs are essential for osteoblasts. Therefore, this study aims to optimize silencing of GLUTs using siRNA technology to find out which of the known class I GLUTs are most important for the osteoblasts and to determine how energy is utilized by the osteoblasts and their precursors, the bone marrow stromal cells (BMSCs).
Three main class I GLUTs (GLUT-1, GLUT-3, GLUT-4) were detected at mRNA level in rat osteosarcoma cell line UMR-106 by quantitative polymerase chain reaction (qPCR) and at proteins level by immunofluorescence. We silenced each of these GLUTs individually by siRNA in the cell line and observed an efficient silencing (>90%) for each of the GLUTs. Our results demonstrates that siRNA constructs work efficiently in GLUTs silencing. Similar silencing was also observed in primary osteoblasts differentiated from rat BMSCs. We have also optimized primers for GLUT-5-10 and -12 and checked their expression in control tissues with qPCR.
In summary, we have established siRNA methodology for silencing of GLUTs in UMR-106 cell line and in BMSCs. These methods allow us to study the role of each GLUT in skeletal glucose utilization and we can further gain knowledge about the specific roles of each GLUT in osteoblast homeostasis and energy metabolism.
To date, it is unclear which GLUTs are essential for osteoblasts. Therefore, this study aims to optimize silencing of GLUTs using siRNA technology to find out which of the known class I GLUTs are most important for the osteoblasts and to determine how energy is utilized by the osteoblasts and their precursors, the bone marrow stromal cells (BMSCs).
Three main class I GLUTs (GLUT-1, GLUT-3, GLUT-4) were detected at mRNA level in rat osteosarcoma cell line UMR-106 by quantitative polymerase chain reaction (qPCR) and at proteins level by immunofluorescence. We silenced each of these GLUTs individually by siRNA in the cell line and observed an efficient silencing (>90%) for each of the GLUTs. Our results demonstrates that siRNA constructs work efficiently in GLUTs silencing. Similar silencing was also observed in primary osteoblasts differentiated from rat BMSCs. We have also optimized primers for GLUT-5-10 and -12 and checked their expression in control tissues with qPCR.
In summary, we have established siRNA methodology for silencing of GLUTs in UMR-106 cell line and in BMSCs. These methods allow us to study the role of each GLUT in skeletal glucose utilization and we can further gain knowledge about the specific roles of each GLUT in osteoblast homeostasis and energy metabolism.