title: A role for thioredoxin-interacting protein (Txnip) in cellular creatine homeostasis.
abstract: 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.
authors.affiliation: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Headington, Oxford, United Kingdom. szervou@well.ox.ac.uk
authors.forename: Sevasti
authors.initials: S
authors.lastname: Zervou
authors.forename: Tanmoy
authors.initials: T
authors.lastname: Ray
authors.forename: Natasha
authors.initials: N
authors.lastname: Sahgal
authors.forename: Liam
authors.initials: L
authors.lastname: Sebag-Montefiore
authors.forename: Rebecca
authors.initials: R
authors.lastname: Cross
authors.forename: Debra J
authors.initials: DJ
authors.lastname: Medway
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Ostrowski
authors.forename: Stefan
authors.initials: S
authors.lastname: Neubauer
authors.forename: Craig A
authors.initials: CA
authors.lastname: Lygate
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D003401:Creatine / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006706:Homeostasis / Q000502:physiology*; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D046228:Microarray Analysis; D009206:Myocardium / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013879:Thioredoxins / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C445672:Txnip protein, mouse; C080679:creatine transporter; D013879:Thioredoxins; D003513:Cycloheximide; D003401:Creatine
keywords: cardiomyocytes; redox