Date Published: May 28, 2013
Publisher: American Physiological Society
Author(s): Sevasti Zervou, Tanmoy Ray, Natasha Sahgal, Liam Sebag-Montefiore, Rebecca Cross, Debra J. Medway, Philip J. Ostrowski, Stefan Neubauer, Craig A. Lygate.
Creatine is important for energy metabolism, yet excitable cells such as cardiomyocytes do not synthesize creatine and rely on uptake via a specific membrane creatine transporter (CrT; SLC6A8). This process is tightly controlled with downregulation of CrT upon continued exposure to high creatine via mechanisms that are poorly understood. Our aim was to identify candidate endogenous CrT inhibitors. In 3T3 cells overexpressing the CrT, creatine uptake plateaued at 3 h in response to 5 mM creatine but peaked 33% higher (P < 0.01) in the presence of cycloheximide, suggesting CrT regulation depends on new protein synthesis. Global gene expression analysis identified thioredoxin-interacting protein (Txnip) as the only significantly upregulated gene (by 46%) under these conditions (P = 0.036), subsequently verified independently at mRNA and protein levels. There was no change in Txnip expression with exposure to 5 mM taurine, confirming a specific response to creatine rather than osmotic stress. Small-interfering RNA against Txnip prevented Txnip upregulation in response to high creatine, maintained normal levels of creatine uptake, and prevented downregulation of CrT mRNA. These findings were relevant to the in vivo heart since creatine-deficient mice showed 39.71% lower levels of Txnip mRNA, whereas mice overexpressing the CrT had 57.6% higher Txnip mRNA levels and 28.7% higher protein expression compared with wild types (mean myocardial creatine concentration 124 and 74 nmol/mg protein, respectively). In conclusion, we have identified Txnip as a novel negative regulator of creatine levels in vitro and in vivo, responsible for mediating substrate feedback inhibition and a potential target for modulating creatine homeostasis.
This study confirmed that exposure of cells to millimolar levels of Cr results in reduced Cr uptake (substrate inhibition) and that this is dependent on the synthesis of new protein (8, 19). However, the identities of these protein(s) were unknown. We therefore took a nonbiased gene array approach that identified Txnip as the only gene to be upregulated under these conditions, and this was confirmed at the protein level both in vitro and in vivo. In particular, in vitro knockdown of Txnip abolished the downregulation of Cr uptake and gene expression in response to high Cr levels, implicating Txnip as a mediator of substrate feedback inhibition.
This work was funded by the British Heart Foundation (BHF) Programme Grant RG/10/002/28187 and by the BHF Centre of Research Excellence, Oxford. N. Sahgal is supported by a Wellcome Trust Grant [090532/Z/09/Z].
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: S.Z., S.N., and C.A.L. conception and design of research; S.Z., T.R., L.S.-M., R.C., D.J.M., and P.J.O. performed experiments; S.Z., T.R., N.S., and R.C. analyzed data; S.Z. and C.A.L. interpreted results of experiments; S.Z. prepared figures; S.Z. drafted manuscript; S.Z., T.R., N.S., L.S.-M., S.N., and C.A.L. edited and revised manuscript; S.Z., T.R., N.S., L.S.-M., R.C., D.J.M., P.J.O., S.N., and C.A.L. approved final version of manuscript.