Glutamine Regulates Cardiac Progenitor Cell Metabolism and Proliferation

Abstract

Autologous transplantation of cardiac progenitor cells (CPCs) alleviates myocardial dysfunction in the damaged heart; however, the mechanisms that contribute to their reparative qualities remain poorly understood. In this study, we examined CPC metabolism to elucidate the metabolic pathways that regulate their proliferative capacity. In complete growth medium, undifferentiated CPCs isolated from adult mouse heart proliferated rapidly (Td = 13.8 h). CPCs expressed the Glut1 transporter and their glycolytic rate was increased by high extracellular glucose concentration, in the absence of insulin. Although high glucose concentrations did not stimulate proliferation, glutamine increased CPC doubling time and promoted survival under conditions of oxidative stress. In comparison with glucose, pyruvate or BSA-palmitate, glutamine, when provided as the sole metabolic substrate, increased ATP-linked and uncoupled respiration. Although fatty acids were not used as respiratory substrates when present as a sole carbon source, glutamine-induced respiration was doubled in the presence of BSA-palmitate, suggesting that glutamine stimulates fatty acid oxidation. Additionally, glutamine promoted rapid phosphorylation of the mTORC1 substrate, p70S6k, as well as retinoblastoma protein, followed by induction of cyclin D1 and cdk4. Inhibition of either mTORC1 or glutaminolysis was sufficient to diminish CPC proliferation, and provision of cell permeable α-ketoglutarate in the absence of glutamine increased both respiration and cell proliferation, indicating a key role of glutamine anaplerosis in cell growth. These findings suggest that glutamine, by enhancing mitochondrial function and stimulating mTORC1, increases CPC proliferation, and that interventions to increase glutamine uptake or oxidation may improve CPC therapy.

Type 2 Diabetes Dysregulates Glucose Metabolism in Cardiac Progenitor Cells

“…After outgrowth, the CPCs were subjected to sequential sorting for c-kit ϩ /lin Ϫ markers using magnetic immunobeads (29,30). As described previously (30,31), the CPCs were cultured in growth medium. Passages 3-9 were used for these studies.…”

“…Proliferation, Viability, and Differentiation MeasurementsCell proliferation was assessed by counting cells using a hemocytometer or a BD Accuri C6 flow cytometer, as described previously (31). Cell viability was assessed by lactate dehydrogenase assay, as described previously (31).…”

“…Cell viability was assessed by lactate dehydrogenase assay, as described previously (31). Differentiation of CPCs was assessed by quantitative RT-PCR after incubating cells in DMEM/F-12 medium containing 2% FBS, 1% penicillin/ streptomycin, and 10 nM dexamethasone (32)(33)(34).…”

“…For Western blotting, 0.5-50 g of protein from crude CPC lysates were applied to each lane of a 12.5 or 10.5-14% Bis-Tris-HCl gel and electrophoresed. The separated proteins were then electroblotted onto a PVDF membrane, and immunoblotting was performed as described (31). Immunoreactive bands were detected using a Typhoon variable mode imager (GE Healthcare) or a Fuji LAS-3000 Bio Imaging analyzer after exposure to ECL detection reagent.…”

“…Extracellular Flux Analysis-Mitochondrial activity and surrogate measures of glycolytic flux were measured in WT and db/db CPCs, similar to that described previously (31,35).…”