Role of Pentose phosphate pathway during osteoblast differentiation
Document Type
Oral Presentation
Abstract
Glucose metabolism is crucial for osteoblast differentiation. Our previous studies have shown that differentiated osteoblasts generate a majority of ATP in response to glucose through glycolysis compared to oxidative phosphorylation. To understand the mechanisms that are active during energy generation we performed unbiased metabolomics to study metabolites that are significantly different comparing undifferentiated MC3T3E1 osteoblasts versus differentiated osteoblasts. Metaboanalyst 5.0 was used to identify significantly different pathways. A number of different pathways were significantly different between the groups. Metabolites involved in the Pentose phosphate pathway (PPP), purine and pyrimidine metabolism were the top three pathways significantly different in the differentiated group. PPP helps breakdown Glucose-6-Phosphate (G6P) generated during glycolysis to produce ribose-5-phosphate (R5P) and nicotinamide adenine dinucleotide phosphate (NADPH). To study PPP during osteoblast differentiation we used MC3T3E1 (pre-osteoblasts) and differentiated them in osteogenic media (Ascorbic acid and β-glycerophosphate) in the presence of glucose-6-phosphate dehydrogenase inhibitor 1 (G6PDi-1) inhibitor. G6PDi-1 inhibitor blocks the activity of the rate limiting enzyme glucose-6-phosphate dehydrogenase of the PPP pathway. We used different concentrations of G6PDi based on the published studies (0.5, 1.0 and 10µM) and studied cell viability and protein expression using western blotting. Higher concentrations (10µM) of the inhibitor proved toxic to the cells and we are currently in the process of optimizing the dose of G6PDi-1 for further studies.
Role of Pentose phosphate pathway during osteoblast differentiation
Glucose metabolism is crucial for osteoblast differentiation. Our previous studies have shown that differentiated osteoblasts generate a majority of ATP in response to glucose through glycolysis compared to oxidative phosphorylation. To understand the mechanisms that are active during energy generation we performed unbiased metabolomics to study metabolites that are significantly different comparing undifferentiated MC3T3E1 osteoblasts versus differentiated osteoblasts. Metaboanalyst 5.0 was used to identify significantly different pathways. A number of different pathways were significantly different between the groups. Metabolites involved in the Pentose phosphate pathway (PPP), purine and pyrimidine metabolism were the top three pathways significantly different in the differentiated group. PPP helps breakdown Glucose-6-Phosphate (G6P) generated during glycolysis to produce ribose-5-phosphate (R5P) and nicotinamide adenine dinucleotide phosphate (NADPH). To study PPP during osteoblast differentiation we used MC3T3E1 (pre-osteoblasts) and differentiated them in osteogenic media (Ascorbic acid and β-glycerophosphate) in the presence of glucose-6-phosphate dehydrogenase inhibitor 1 (G6PDi-1) inhibitor. G6PDi-1 inhibitor blocks the activity of the rate limiting enzyme glucose-6-phosphate dehydrogenase of the PPP pathway. We used different concentrations of G6PDi based on the published studies (0.5, 1.0 and 10µM) and studied cell viability and protein expression using western blotting. Higher concentrations (10µM) of the inhibitor proved toxic to the cells and we are currently in the process of optimizing the dose of G6PDi-1 for further studies.
