Determination of Intrinsic Creatine Transporter (Slc6a8) Activity and Creatine Transport Function of Leukocytes in Rats

Abstract The objective of this study was to evaluate the effects of dietary creatine and SID methionine+cysteine (Met+Cys) levels on the performance, blood parameters and gene expression of the creatine transporter (SLC6A8) in finishing gilts. Forty gilts, averaging 75.26 ± 0.87 kg of initial weight, were distributed in a randomized blocks design in a 2x2 factorial scheme, consisting of two creatine monohydrate (CMH) supplementation levels (0.00 and 0.10%) and two levels of SID Met+Cys (0.40 and 0.44%, considering 0.44% as the requirement), with 10 replicates. DL methionine was used to ensure the dietary SID Met+Cys levels. Upon reaching a mean weight of 100± 5.85, blood was collected for the determination of urea, creatinine, lactate, glucose and homocysteine plasma concentrations. Afterwards, the gilts were slaughtered for the collection of Longissimus dorsi muscle samples, for further determination of the gene expression of the creatine transporter (SLC6A8). No interactions (P > 0.05) we observed between the CMH and SID Met+Cys on the performance and gene expression of the SLC6A8 transporter, and also were not affected (P > 0.05) by the dietary levels of CMH or SID Met+Cys, individually. However, there was an interaction (P = 0.03) between SID Met+Cys and CMH levels on the plasma creatinine concentration, showing a lower (P = 0.018) concentration (6.40 mg/dL) supplementing 0.10% CMH than not supplementing (8.96 mg/dL), only at 0.44% of SID Met+Cys. There were no interactions (P > 0.05) between SID Met+Cys and CMH on the other blood parameters, and also no individual effects were observed for the studied factors. It is concluded that supplementing 0.10% CMH reduced plasma creatinine concentration only at conventionally dietary SID Met+Cys level (0.44%), not affecting other blood parameters, growth performance and the gene expression of the creatine transporter SLC6A8 of finishing gilts.

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Down-Regulation of the Na+,Cl- Coupled Creatine Transporter CreaT (SLC6A8) by Glycogen Synthase Kinase GSK3ß

Cellular Physiology and Biochemistry ◽

10.1159/000453177 ◽

2016 ◽

Vol 40 (5) ◽

pp. 1231-1238 ◽

Cited By ~ 2

Author(s):

Myriam Fezai ◽

Mohamed Jemaà ◽

Hajar Fakhri ◽

Hong Chen ◽

Bhaeldin Elsir ◽

...

Keyword(s):

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Xenopus Laevis Oocytes ◽

Induced Current ◽

Wild Type ◽

Creatine Transporter ◽

Electrode Voltage ◽

Creatine Transport ◽

The Brain ◽

Current Kinetic

Background: The Na+,Cl- coupled creatine transporter CreaT (SLC6A8) is expressed in a variety of tissues including the brain. Genetic defects of CreaT lead to mental retardation with seizures. The present study explored the regulation of CreaT by the ubiquitously expressed glycogen synthase kinase GSK3ß, which contributes to the regulation of neuroexcitation. GSK3ß is phosphorylated and thus inhibited by PKB/Akt. Moreover, GSK3ß is inhibited by the antidepressant lithium. The present study thus further tested for the effects of PKB/Akt and of lithium. Methods: CreaT was expressed in Xenopus laevis oocytes with or without wild-type GSK3ß or inactive K85RGSK3ß. CreaT and GSK3ß were further expressed without and with additional expression of wild type PKB/Akt. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp. Results: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes, co-expression of GSK3ß but not of K85RGSK3ß, resulted in a significant decrease of creatine induced current. Kinetic analysis revealed that GSK3ß significantly decreased the maximal creatine transport rate. Exposure of CreaT and GSK3ß expressing oocytes for 24 hours to Lithium was followed by a significant increase of the creatine induced current. The effect of GSK3ß on CreaT was abolished by co-expression of PKB/Akt. Conclusion: GSK3ß down-regulates the creatine transporter CreaT, an effect reversed by treatment with the antidepressant Lithium and by co-expression of PKB/Akt.

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AMPK and substrate availability regulate creatine transport in cultured cardiomyocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00554.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. E870-E876 ◽

Cited By ~ 21

Author(s):

Marcus D. Darrabie ◽

Antonio Jose Luis Arciniegas ◽

Rajashree Mishra ◽

Dawn E. Bowles ◽

Danny O. Jacobs ◽

...

Keyword(s):

Heart Failure ◽

Energy Homeostasis ◽

Cardiac Cells ◽

Substrate Availability ◽

Human Heart Failure ◽

Positive Role ◽

Creatine Transporter ◽

Neonatal Cardiomyocytes ◽

Transporter Protein ◽

Creatine Transport

Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in Vmax that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.

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Creatine transport and pathological changes in creatine transporter deficient mice

Journal of Inherited Metabolic Disease ◽

10.1002/jimd.12358 ◽

2021 ◽

Author(s):

Adam M. Wawro ◽

Chandresh R. Gajera ◽

Steven A. Baker ◽

Jeffrey J. Nirschl ◽

Hannes Vogel ◽

...

Keyword(s):

Pathological Changes ◽

Creatine Transporter ◽

Creatine Transport ◽

Deficient Mice

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Removal of Potential Phosphorylation Sites does not Alter Creatine Transporter Response to PKC or Substrate Availability

Cellular Physiology and Biochemistry ◽

10.1159/000430359 ◽

2015 ◽

Vol 37 (1) ◽

pp. 353-360 ◽

Cited By ~ 1

Author(s):

Lucia Santacruz ◽

Marcus D. Darrabie ◽

Rajashree Mishra ◽

Danny O. Jacobs

Keyword(s):

Intracellular Signaling ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Structural Elements ◽

Substrate Availability ◽

Bioinformatics Analyses ◽

Creatine Transporter ◽

Transporter Protein ◽

Creatine Transport ◽

Pkc Activation

Background: Creatine, Phosphocreatine, and creatine kinases, constitute an energy shuttle that links ATP production in mitochondria with cellular consumption sites. Myocytes and neurons cannot synthesize creatine and depend on uptake across the cell membrane by a specialized transporter to maintain intracellular creatine levels. Although recent studies have improved our understanding of creatine transport in cardiomyocytes, the structural elements underlying the creatine transporter protein regulation and the relevant intracellular signaling processes are unknown. Methods: The effects of pharmacological activation of kinases or phosphatases on creatine transport in cardiomyocytes in culture were evaluated. Putative phosphorylation sites in the creatine transporter protein were identified by bioinformatics analyses, and ablated using site-directed mutagenesis. Mutant transporter function and their responses to pharmacological PKC activation or changes in creatine availability in the extracellular environment, were evaluated. Results: PKC activation decreases creatine transport in cardiomyocytes in culture. Elimination of high probability potential phosphorylation sites did not abrogate responses to PKC activation or substrate availability. Conclusion: Modulation of creatine transport in cardiomyocytes is a complex process where phosphorylation at predicted sites in the creatine transporter protein does not significantly alter activity. Instead, non-classical structural elements in the creatine transporter and/or interactions with regulatory subunits may modulate its activity.

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Determination of Transcoronary Circulatory Transport Function

The Tohoku Journal of Experimental Medicine ◽

10.1620/tjem.122.43 ◽

1977 ◽

Vol 122 (1) ◽

pp. 43-50

Author(s):

KAI TSUIKI ◽

JUNICHIRO NAGASAWA ◽

TAKASHI NAKAMURA

Keyword(s):

Transport Function

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Electrolysis stimulates creatine transport and transporter cell surface expression in incubated mouse skeletal muscle: potential role of ROS

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00060.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. E1250-E1257 ◽

Cited By ~ 7

Author(s):

Wim Derave ◽

Nadine Straumann ◽

Robert A. Olek ◽

Peter Hespel

Keyword(s):

Cell Surface ◽

Electrical Field Stimulation ◽

Surface Expression ◽

Cell Surface Expression ◽

Field Stimulation ◽

Electrical Field ◽

Creatine Transporter ◽

Creatine Uptake ◽

Creatine Transport ◽

Stimulation Of

Electrical field stimulation of isolated, incubated rodent skeletal muscles is a frequently used model to study the effects of contractions on muscle metabolism. In this study, this model was used to investigate the effects of electrically stimulated contractions on creatine transport. Soleus and extensor digitorum longus muscles of male NMRI mice (35–50 g) were incubated in an oxygenated Krebs buffer between platinum electrodes. Muscles were exposed to [14C]creatine for 30 min after either 12 min of repeated tetanic isometric contractions (contractions) or electrical stimulation of only the buffer before incubation of the muscle (electrolysis). Electrolysis was also investigated in the presence of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and catalase. Both contractions and (to a lesser degree) electrolysis stimulated creatine transport severalfold over basal. The amount of electrolysis, but not contractile activity, induced (determined) creatine transport stimulation. Incubation with SOD and catalase at 100 and 200 U/ml decreased electrolysis-induced creatine transport by ∼50 and ∼100%, respectively. The electrolysis effects on creatine uptake were completely inhibited by β-guanidino propionic acid, a competitive inhibitor of (creatine for) the creatine transporter (CRT), and were accompanied by increased cell surface expression of CRT. Muscle glucose transport was not affected by electrolysis. The present results indicate that electrical field stimulation of incubated mouse muscles, independently of contractions per se, stimulates creatine transport by a mechanism that depends on electrolysis-induced formation of ROS in the incubation buffer. The increased creatine uptake is paralleled by an increased cell surface expression of the creatine transporter.

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Exposing cardiomyocytes to subclinical concentrations of doxorubicin rapidly reduces their creatine transport

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00108.2012 ◽

2012 ◽

Vol 303 (5) ◽

pp. H539-H548 ◽

Cited By ~ 14

Author(s):

Marcus D. Darrabie ◽

Antonio Jose Luis Arciniegas ◽

Jose Gabriel Mantilla ◽

Rajashree Mishra ◽

Miguel Pinilla Vera ◽

...

Keyword(s):

Cell Surface ◽

Peak Plasma ◽

Plasma Concentrations ◽

Soft Tissue Sarcomas ◽

Cardiac Cells ◽

Early Event ◽

Creatine Transporter ◽

Transporter Protein ◽

Creatine Uptake ◽

Creatine Transport

Doxorubicin is commonly used to treat leukemia, lymphomas, and solid tumors, such as soft tissue sarcomas or breast cancer. A major side effect of doxorubicin therapy is dose-dependent cardiotoxicity. Doxorubicin's effects on cardiac energy metabolism are emerging as key elements mediating its toxicity. We evaluated the effect of doxorubicin on [14C]creatine uptake in rat neonatal cardiac myocytes and HL-1 murine cardiac cells expressing the human creatine transporter protein. A significant and irreversible decrease in creatine transport was detected after an incubation with 50–100 nmol/l doxorubicin. These concentrations are well below peak plasma levels (5 μmol/l) and within the ranges (25–250 nmol/l) for steady-state plasma concentrations reported after the administration of 15–90 mg/m2 doxorubicin for chemotherapy. The decrease in creatine transport was not solely because of increased cell death due to doxorubicin's cytotoxic effects. Kinetic analysis showed that doxorubicin decreased Vmax, Km, and creatine transporter protein content. Cell surface biotinylation experiments confirmed that the amount of creatine transporter protein present at the cell surface was reduced. Cardiomyocytes rely on uptake by a dedicated creatine transporter to meet their intracellular creatine needs. Our findings show that the cardiomyocellular transport capacity for creatine is substantially decreased by doxorubicin administration and suggest that this effect may be an important early event in the pathogenesis of doxorubicin-mediated cardiotoxicity.

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