Cr supplementation decreases tyrosine phosphorylation of the CreaT in skeletal muscle during sepsis

Weiyang Wang, Michael A. Jobst, Brian Bell, Chun-Rui Zhao, Li-Hong Shang, and Danny O. Jacobs

Department of Surgery, Creighton University Medical Center, Omaha, Nebraska 68131


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myocellular creatine (Cr) uptake is predominantly governed by a sodium-dependent Cr transporter (CreaT) and plays a pivotal role in skeletal muscle energy metabolism. The CreaT belongs to a neurotransmitter transporter family that can be functionally regulated by protein tyrosine kinase-induced tyrosine phosphorylation. The association between myocellular Cr and c-Src-related tyrosine phosphorylation of the CreaT and the influence of oral Cr supplementation on this association were investigated during sepsis. Animals were randomized to receive standard rat chow or standard rat chow with oral Cr supplementation for 4 days followed by cecal ligation and puncture (CLP) or sham operation. Fast-twitch gastrocnemius muscles were harvested 24 h after operation. Myocellular free Cr levels were 70% higher after CLP. Western blotting of the immunoprecipitated CreaT with an anti-phosphotyrosine or anti-phospho-c-Src (Y-416) antibody revealed that tyrosine phosphorylation of the CreaT and tyrosine-phosphorylated c-Src (Tyr416) expression in the CreaT-c-Src complex were significantly increased after CLP compared with sham operation. These changes were observed in homogenates and plasma membrane fractions of gastrocnemius muscles. Although oral Cr supplementation increased myocellular free Cr levels equivalently in CLP and sham-operated animals, c-Src-related tyrosine phosphorylation of the CreaT in homogenates and plasma membrane fractions of gastrocnemius muscles was, however, downregulated in Cr-supplemented CLP animals compared with Cr-supplemented sham-operated rats. During sepsis, increased myocellular free Cr levels are associated with enhanced tyrosine phosphorylation of the CreaT, which is likely induced by active c-Src. Oral Cr supplementation downregulates c-Src-related tyrosine phosphorylation of the CreaT. The data suggest that myocellular Cr homeostasis and CreaT activity are tightly regulated and closely related during sepsis.

creatine transporter; phosphorylation; tyrosine; Src family kinase; sarcolemma; skeletal muscle; septic shock


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCELLULAR CREATINE (Cr) and its phosphorylated form, phosphocreatine (PCr), play a pivotal role in skeletal muscle energy metabolism. Intracellular PCr acts as a store of high-energy phosphate and can be converted into adenosine 5'-triphosphate (ATP) via the reversible reaction catalyzed by creatine kinase (CK): PCr + ADP + H+ left-right-arrow Cr + ATP. In turn, Cr is the precursor for PCr synthesis by CK and involves ATP production through its involvement in the PCr energy system. When the Cr/PCr/CK system is present and the CK reaction is near equilibrium with its substrates, the free energy available from ATP hydrolysis (Delta GATP) will be buffered at the expense of the PCr (10). Thus myocellular Cr is of great importance for cellular energy metabolism. Evidence suggests that dietary Cr supplementation can improve skeletal muscle energy metabolism and performance (6). For example, short-term oral Cr supplementation at a dose of 20 g/day for 5-7 days in humans increases myocellular Cr and PCr concentrations (5, 17) and improves muscle performance (5, 13). Long-term Cr supplementation at a dose of 7 g/day for 21 days also improves muscle performance (4). The improvements in muscle performance observed during exercise and recovery appear to parallel enhanced ATP synthesis, which occurs as a consequence of the higher myocellular PCr contents induced by dietary Cr supplementation (5, 12). Although some studies have not demonstrated any improvement in muscle performance induced by short-term Cr supplementation (11, 33), the total muscle Cr concentrations in these studies increased by far less than the amount observed in studies that demonstrated positive effects (5, 12). The explanation for this discrepancy is not known but is likely to be related to changes in the mechanisms that control influx or efflux of Cr across the cell membrane. Because the greatest increases in PCr availability and in muscle performance occur in humans that experience the greatest increases in muscle Cr concentration, the effects of Cr supplementation on muscle metabolism and performance must depend fundamentally on the extent of Cr uptake. Myocellular Cr transport is predominantly governed by a specific, Na+-dependent, plasma membrane creatine transporter (CreaT) (34, 42).

CreaT-encoding cDNAs have recently been cloned from a wide variety of tissues (15, 27, 42). Two human CreaT genes are identified on chromosomes Xq28 and 16p11.1 (20, 30). The mRNA of the CreaT coded by the gene at chromosome Xq28 is widely expressed in human brain, heart, skeletal muscle, kidney, and testis (15, 27); however, the mRNA of the CreaT coded by the gene at chromosome 16p11.1 is present only in the testis (20). These two CreaT proteins share 98% homology. Analysis of the encoded protein sequence has shown that the CreaT has 12 putative transmembrane domains and probably displays both NH2 and COOH termini directed toward the cytosol (15, 27). The CreaT belongs to the Na+/Cl--dependent neurotransmitter transporter family and shares significant homology with neurotransmitter and amino acid transporter subfamilies (i.e., 46-53% with GABA/taurine transporters and 38-44% with glycine/proline transporters) (27, 42). In rat skeletal muscle, the CreaT has two isoforms, CreaT-70 and CreaT-55; CreaT-70 is believed to be a glycosylated version of CreaT-55 (14, 26, 38).

A high-affinity, Na+-dependent, saturable uptake mechanism for Cr has been identified in cultured skeletal muscle cell lines (28, 37) and isolated soleus muscle strips (40), which suggests that Cr uptake via the CreaT is closely regulated and that changes in Cr uptake affect myocellular Cr content. An important property of the neurotransmitter transporter family in general is their ability to be functionally regulated by intracellular kinases (2). Protein kinases can directly phosphorylate transporters or influence the interaction of transporters with other proteins (2). Tyrosine phosphorylation by protein tyrosine kinase is one of the primary mechanisms by which neurotransmitter transporter function is modulated (2). For example, when the GABA transporters (GAT1) are expressed in hippocampal neurons or in Chinese hamster ovary cells, a decrease in GABA uptake is correlated with a decrease in tyrosine phosphorylation of the GAT1 (23). Tyrosine phosphorylation is also known to functionally regulate Na+-K+-ATPase activity (16), which directly influences myocellular Cr transport (28).

The purpose of this study, therefore, was to determine 1) whether changes in myocellular free Cr are associated with tyrosine phosphorylation of the CreaT, which is related to active c-Src kinase, and 2) whether oral Cr supplementation modulates tyrosine phosphorylation of the CreaT during sepsis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN), weighing 250-300 g, were acclimatized to 12:12-h light-dark cycles for 3 days and housed in a temperature-controlled room (22°C) with 50-70% humidity before any experiments were initiated. During this time, all animals were fed standard rat chow and water ad libitum. After acclimatization, the rats were randomized to receive cecal ligation and puncture (CLP) or sham operation. The experiments described herein adhered to the National Institutes of Health guidelines for the use of experimental animals and were approved by the Institutional Animal Care and Use Committee of Creighton University.

Oral Cr supplementation. Animals were randomized to receive standard rat chow or standard rat chow with oral supplementation of 1% Cr in drinking water for 4 days followed by CLP or sham operation. Cr solutions (1%) were used because we found that concentrations >1% were not stable at room temperature (i.e., Cr precipitation occurs). Preliminary experiments also showed that normal adult rats weighing ~250 g drink 40-50 ml of 1% Cr-containing water per day during 4 days of Cr supplementation, which indicates that ~1.6-2.0 g of Cr are ingested per kilogram of body weight. Although the amount of Cr consumed orally by animals cannot be measured exactly, we have found that intracellular Cr concentrations in skeletal muscle are significantly increased after 4 days of oral 1% Cr supplementation (17.7 ± 1.1 vs. 9.0 ± 0.8 µmol/g wet wt, Cr-supplemented vs. non-Cr-supplemented normal rats, respectively).

CLP. Sepsis was produced by CLP. Briefly, the rats were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). After preparation of the abdominal skin by shaving and scrubbing with povidone-iodine solution (Clinipad, Guilford, CT), the peritoneal cavity was entered aseptically through a small incision. Stool was milked into the cecum from the ascending colon, and the base of the cecum was ligated in a nonobstructing fashion just distal to the ileocecal valve. The ligated cecum was punctured once along the antimesenteric border with an 18-gauge needle and then returned to the abdominal cavity. In the sham-operated rats, the cecum was manipulated but was neither ligated nor punctured. The abdominal incision was then closed in two layers with ethilon suture (Ethicon, Somerville, NJ), and the animals were given 6 ml of normal saline per 100 g body wt by intraperitoneal injection to compensate for volume depletion. After operation, the rats were caged individually and were allowed water ad libitum. Twenty-four hours after operation, the animals were used for experimentation.

Experimental groups. Four groups of animals were studied: CLP (n = 6), sham operation (SHAM, n = 6), CLP plus Cr (Cr+CLP, n = 6), and sham operation plus Cr (Cr+SHAM, n = 6). In addition, baseline myocellular free Cr levels were measured in eight normal, well nourished, nonoperated rats.

Free Cr assay. Myocellular free Cr levels were measured using high-performance liquid chromatography (HPLC) according to the method described by Wiseman et al. (41). Briefly, freeze-clamped gastrocnemius muscles (mixed red and white gastrocnemius muscle tissues), harvested from normal or experimental animals, were weighed and pulverized to fine powders in liquid nitrogen. A portion of each gastrocnemius muscle powder was mixed with 1.0 ml of 0.02 N HCl. Next, 112.5 µl of 3.0 N perchloric acid were added, and extraction was performed under gentle agitation for 20 min at 4°C followed by vortex mixing. The supernatants were collected by centrifugation, neutralized with 225 µl of 2.5 M KHCO3, and then centrifuged to remove neutralized KClO4. The resulting supernatant was subjected to HPLC. A reversed-phase HPLC Binary Gradient System (model 338; Beckman Instruments, San Ramon, CA) consisting of two model 110B solvent delivery modules, a 330 organizer with 210A sample injection valve and dynamic mixer, a model 167 programmable scanning detector, a model 406 analog interface, and an IBM PC equipped with Beckman System Gold Software, was used to measure myocellular free Cr contents. Separation was performed on a reversed-phase C2/C18 silica column, Mino RPC S5/20 (5 µm, 4.6 × 200 mm) at 25°C. The free Cr concentrations were normalized to gram wet weight of muscle. The values for each were determined by averaging triplicate samples.

Plasma membrane isolation. The CreaT is present not only on plasma membranes (26, 43) but also in the intracellular locations (such as mitochondria) (34). Because the protein expression and tyrosine phosphorylation status of the CreaT in muscle homogenate samples may be influenced by changes at both sites, we isolated muscle plasma membrane fractions to determine whether 1) changes in the CreaT occurred on the plasma membrane and 2) changes in tyrosine-phosphorylated CreaT were related to the plasma membrane-associated c-Src protein tyrosine kinase, which would suggest that the CreaT could be a targeted substrate for c-Src. Myocellular plasma membrane fractions were isolated according to the methods described by Deems et al. (8) and modified by Shimoda et al. (32). Three gastrocnemius muscles (~3.5-4.5 g) harvested from CLP or sham-operated animals were pooled, pulverized to a fine powder in liquid nitrogen, diluted 10-fold, and homogenized in buffer A (10 mM NaHCO3, 0.25 M sucrose, and 5 mM NaN3, pH 7.4) at 4°C by sonication. Protease inhibitors (a mixture of aprotinin, pepstatin, and leupeptin from Sigma Chemical) and 1 mM phenylmethylsulfonyl fluoride (PMSF) were added. The homogenate was centrifuged at 1,200 g for 10 min to remove cellular debris. This step was repeated once. The supernatant was then centrifuged at 9,000 g for 10 min to remove mitochondria and nuclei. Next, the resulting supernatant was centrifuged at 190,000 g for 60 min. The resulting pellet containing crude membranes was recovered, applied to a discontinuous sucrose density gradient (25, 30, and 35% sucrose), and centrifuged at 150,000 g for 16 h with an SW411 bucket (Optima LE-80K ultracentrifuge, Beckman Coulter, Fullerton, CA). A portion of each crude membrane fraction was also extracted and used for marker enzyme and protein assays. The plasma membrane fractions were collected from the top 25% layer. This suspension of plasma membranes was pelleted by ultracentrifugation (150,000 g for 1 h) and recovered in an appropriate amount of buffer A. The purity of the plasma membrane fraction was assessed by determining the enrichment of specific enzyme activity (5'-nucleotidase) relative to the crude membrane portion. The 5'-nucleotidase activity was 6 times higher in the plasma membrane fraction (156 ± 20 vs. 25 ± 5 µmol · min-1 · mg protein-1, plasma vs. crude membrane, respectively). Protein concentrations were measured using a protein assay kit (Bio-Rad, Hercules, CA) and were adjusted to a concentration appropriate for immunoprecipitation and Western blotting, as described in the following section.

Immunoprecipitation and Western blotting. Gastrocnemius muscles were homogenized and then lysed in lysis buffer containing 0.5% Triton X-100, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 100 mM sodium fluoride, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin and aprotinin, and 50 mM HEPES, pH 7.5. The lysates were centrifuged to remove cellular debris. Total protein concentration of the resulting supernatants was quantified and adjusted to a final protein concentration of 0.2 mg/ml using Bio-Rad protein assay kits (Bio-Rad). Next, ~2 µg of a polyclonal rabbit anti-rat CreaT (affinity pure) antibody (Alpha Diagnostic, San Antonio, TX) was added to 500 µl of each gastrocnemius muscle lysate or plasma membrane fraction (~100 µg of homogenate or plasma membrane proteins). The reaction mixture was incubated overnight with gentle agitation at 4°C. Protein A-Sepharose 4B (20 µl/tube, Zymed Laboratories, South San Francisco, CA) was added, and then incubation and agitation continued for another hour at room temperature. The reaction tubes were then centrifuged, and the supernatants were discarded. Each Sepharose pellet was washed three times with lysis buffer. The pellets were resuspended in reducing sample buffer, boiled for 5 min, and centrifuged. Appropriate amounts of the supernatants containing ~20 µg of homogenate proteins or 40 µg of plasma membrane proteins (with the assumption that no proteins were lost during immunoprecipitation) were then applied to 12% SDS-PAGE gels and electrophoretically transferred to a hydrophobic polyvinylidene difluoride (PVDF) membrane.

Western immunoblotting was performed using monoclonal mouse anti-phosphotyrosine (Zymed Laboratories) or polyclonal rabbit anti-phospho-c-Src (Y-416; Upstate Biotechnology, Lake Placid, NY) as a primary antibody. The membrane was reacted with an alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG as a secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ). Immunoreactive bands on the membrane were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) and scanned using a Storm 860 Gel and Blot Imaging System (Molecular Dynamics, Sunnyvale, CA). The corresponding protein expressions were quantified using ImageQuant (Molecular Dynamics). Values are expressed in arbitrary densitometry units (ADU).

The same membranes were stripped and reprobed with the corresponding antibody for immunoprecipitation (i.e., a polyclonal rabbit anti-rat CreaT antibody) to confirm that equivalent amounts of immunoprecipitated proteins were loaded. In addition, we have previously found that protein expression of the CreaT in gastrocnemius muscle homogenates and plasma membrane fractions is equivalent 24 h after CLP compared with sham operation (data not shown). This suggests that sepsis does not alter protein expression of the CreaT under our experimental conditions.

Statistics. All results are expressed as means ± SE. A Student's t-test was used to compare tyrosine phosphorylation of the CreaT and tyrosine-phosphorylated c-Src (Tyr416) expression in the CreaT-c-Src complex between corresponding groups, i.e., CLP and SHAM or Cr+CLP and Cr+SHAM. One-way analysis of variance (ANOVA) was used to compare myocellular free Cr levels in four groups, i.e., CLP, SHAM, Cr+CLP, and Cr+SHAM. For ANOVA, post hoc pairwise comparisons were performed using Fisher's least significant difference test if necessary. Probability values <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of sepsis and oral Cr supplementation on myocellular free Cr levels. Polymicrobial sepsis was produced by CLP. All CLP, but no sham-operated, animals had positive blood cultures 24 h after operation. This indicates that sepsis had developed as expected. Myocellular free Cr concentrations were measured using HPLC and are expressed as micromoles per gram wet weight in Fig. 1. Intracellular free Cr levels in fast-twitch gastrocnemius muscles were 9.0 ± 0.8 in normal, well nourished, non-operated animals. Myocellular free Cr levels increased significantly by 70% in fast-twitch gastrocnemius muscles harvested from animals 24 h after CLP compared with sham-operated rats (13.9 ± 1.2 in CLP vs. 8.2 ± 1.0 in SHAM, P < 0.05), which demonstrates that sepsis increases myocellular free Cr contents. Four days of oral 1% Cr supplementation before operation increased myocellular Cr levels equivalently in animals subjected to CLP or sham operation relative to normal control rats (19.8 ± 1.9 in Cr+CLP or 18.0 ± 3.4 in Cr+SHAM vs. 8.2 ± 1.0 in SHAM, P < 0.05). The myocellular free Cr concentrations of the Cr+CLP, Cr+SHAM, and CLP groups were not significantly different.


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Fig. 1.   Myocellular free creatine (Cr) levels were significantly increased in fast-twitch gastrocnemius muscles harvested 24 h after cecal ligation and puncture (CLP, n = 6) relative to sham operation (SHAM, n = 6). Four days of oral 1% Cr supplementation before operation increased myocellular Cr levels equivalently in animals subjected to CLP (Cr+CLP, n = 6) or sham operation (Cr+SHAM, n = 6). Values are expressed as means ± SE. * P < 0.05 vs. SHAM; not significant (NS) vs. CLP by one-way ANOVA with post hoc pairwise comparisons by Fisher's least significant difference test.

Sepsis increases tyrosine phosphorylation of the CreaT in skeletal muscle. To determine whether sepsis induces tyrosine phosphorylation of the CreaT, immunoprecipitation-Western blotting experiments were performed. Immunoblotting of the immunoprecipitated CreaT with an anti-phosphotyrosine antibody showed that tyrosine phosphorylation of the CreaT was significantly increased in gastrocnemius muscle homogenates (Fig. 2A) and plasma membrane fractions (Fig. 2B) from rats 24 h after CLP relative to sham operation. We also observed that tyrosine-phosphorylated CreaT-55, but not the CreaT-70 isoform, was predominantly detected in all studied groups, i.e., CLP, SHAM, Cr+CLP, and Cr+SHAM (data not shown).


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Fig. 2.   Creatine transporter (CreaT) in fast-twitch gastrocnemius muscle homogenates and plasma membrane fractions prepared 24 h after CLP (CLP, n = 6) or sham operation (SHAM, n = 6) was immunoprecipitated with anti-CreaT antibody and then analyzed by Western blotting with anti-phosphotyrosine antibody under reducing conditions. Sepsis significantly increased tyrosine phosphorylation of the CreaT in homogenates (A) and plasma membrane fractions (B) in CLP relative to SHAM. Data are shown by representative blots and values expressed in arbitrary densitometry units (ADU). Values of plasma membrane fractions from pooled gastrocnemius muscles were averaged from 3 measurements. A molecular mass marker in kDa is shown on left. * P < 0.05 vs. SHAM by Student's t-test.

c-Src is physically associated with the CreaT in skeletal muscle during sepsis. Src family kinase is a soluble nonreceptor protein tyrosine kinase that is closely associated with the plasma membrane (3, 19, 35, 36). Tyrosine (Tyr416) phosphorylation of Src protein completely activates Src family kinase and provides a binding site for the SH2 or SH3 domain of its downstream proteins (3, 19, 35, 36). Therefore, we investigated whether tyrosine-phosphorylated c-Src (Tyr416) is involved in enhanced tyrosine phosphorylation of the CreaT in homogenates or plasma membrane fractions of gastrocnemius muscles during sepsis. Immunoblotting of the immunoprecipitated CreaT with anti-phospho-c-Src (Y-416) antibody showed that protein expression of tyrosine-phosphorylated c-Src (Tyr416) in the CreaT-c-Src complex was significantly increased in gastrocnemius muscle homogenates (Fig. 3A) and plasma membrane fractions (Fig. 3B) harvested from rats 24 h after CLP compared with sham-operated animals. These data indicate that activated c-Src is physically associated with the CreaT and strongly suggest that tyrosine phosphorylation of the CreaT is related to active c-Src.


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Fig. 3.   CreaT in fast-twitch gastrocnemius muscle homogenates and plasma membrane fractions prepared 24 h after CLP (CLP, n = 6) or sham operation (SHAM, n = 6) was immunoprecipitated with anti-CreaT antibody and then analyzed by Western blotting with anti-phospho-c-Src (Y-416) antibody under reducing conditions. Sepsis significantly increased protein expression of tyrosine-phosphorylated c-Src (Tyr416) in a CreaT-c-Src complex in homogenates (A) and plasma membrane fractions (B) in CLP relative to SHAM. Data are shown by representative blots and values expressed in ADU. Values of plasma membrane fractions from pooled gastrocnemius muscles were averaged from 3 measurements. A molecular mass marker in kDa is shown on left. * P < 0.05 vs. SHAM by Student's t-test.

Oral Cr supplementation downregulates tyrosine phosphorylation of the CreaT in skeletal muscle during sepsis. Animals received 1% Cr supplementation orally for 4 days before CLP or sham operation. Immunoblotting of immunoprecipitated CreaT with an anti-phosphotyrosine antibody showed that tyrosine phosphorylation of the CreaT was decreased after CLP in gastrocnemius muscle homogenates (Fig. 4A) and plasma membrane fractions (Fig. 4B). This finding suggests that oral Cr supplementation, which increases myocellular Cr levels, downregulates tyrosine phosphorylation of the CreaT in skeletal muscle during sepsis.


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Fig. 4.   Cr was orally supplemented 4 days before CLP or sham operation. CreaT in fast-twitch gastrocnemius muscle homogenates and plasma membrane fractions, prepared 24 h after CLP (Cr+CLP, n = 6) or sham operation (Cr+SHAM, n = 6), was immunoprecipitated with anti-CreaT antibody and then analyzed by Western blotting with anti-phosphotyrosine antibody under reducing conditions. Tyrosine phosphorylation of the CreaT in homogenates (A) and plasma membrane fractions (B) was significantly reduced in Cr-supplemented CLP relative to Cr-supplemented SHAM. Data are shown by representative blots and values expressed in ADU. Values of plasma membrane fractions from pooled gastrocnemius muscles were averaged from 3 measurements. A molecular mass marker in kDa is shown on left. * P < 0.05 vs. Cr+SHAM by Student's t-test.

Oral Cr supplementation decreases protein expression of tyrosine-phosphorylated c-Src (Tyr416) in the CreaT-c-Src complex. The CreaT proteins in gastrocnemius muscle homogenates or plasma membrane fractions were immunoprecipitated and then analyzed by Western blotting with an anti-phospho-c-Src (Y-416) antibody. Protein expression of tyrosine-phosphorylated c-Src (Tyr416) in the CreaT-c-Src complex was significantly decreased in gastrocnemius muscle homogenates (Fig. 5A) and plasma membrane fractions (Fig. 5B) harvested from rats 24 h after CLP relative to sham operation, which suggests that oral Cr supplementation reduces the amount of tyrosine-phosphorylated c-Src (Tyr416) coimmunoprecipitated with CreaT during sepsis.


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Fig. 5.   Cr was orally supplemented 4 days before CLP or sham operation. CreaT in fast-twitch gastrocnemius muscle homogenates and plasma membrane fractions, prepared 24 h after CLP (Cr+CLP, n = 6) or sham operation (Cr+SHAM, n = 6), was immunoprecipitated with anti-CreaT antibody and then analyzed by Western blotting with anti-phospho-c-Src (Y-416) antibody under reducing conditions. Protein expression of tyrosine-phosphorylated c-Src (Tyr416) in CreaT-c-Src complex in homogenates (A) and plasma membrane fractions (B) was significantly decreased in Cr-supplemented CLP relative to Cr-supplemented SHAM. Data are shown by representative blots and values expressed in ADU. Values of plasma membrane fractions from pooled gastrocnemius muscles were averaged from 3 measurements. A molecular mass marker in kDa is shown on left. * P < 0.05 vs. Cr+SHAM by Student's t-test.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that myocellular free Cr levels increase significantly in fast-twitch skeletal muscle after CLP. Although intracellular PCr levels do not directly affect Cr transport (24), sepsis significantly alters skeletal muscle energy metabolism in association with an increase in PCr breakdown and a decrease in PCr stores, which suggests that PCr stores can be used to buffer the Delta GATP (21, 22). Thus it is possible that the elevated PCr breakdown rate observed during sepsis could increase intracellular free Cr level via the reversible, CK-catalyzed reaction. However, observed increase in myocellular free Cr does not appear to be secondary simply to a decrease in PCr, because total CK activity does not change significantly and because the percentage decrease in PCr cannot alone account for the increase in myocellular free Cr as has been reported previously in a similar model (21). Therefore, myocellular Cr transport, which is predominantly governed by a specific, Na+-dependent, plasma membrane CreaT, may be enhanced, and, thereby, is likely responsible for the observed increase in myocellular Cr content.

The CreaT in skeletal muscle is primarily located on the plasma membrane (sarcolemma), where regulation of Cr uptake via the CreaT occurs (26, 43). We have shown that increased myocellular free Cr levels are associated with enhanced tyrosine phosphorylation of the CreaT in gastrocnemius muscles, which suggests that tyrosine phosphorylation of the CreaT may mediate, at least in part, changes in myocellular free Cr levels during sepsis by altering Cr transport. Our findings are similar to other studies showing that decreased GABA transport by the GAT1, endogenously expressed in hippocampal neurons or heterologously expressed in Chinese hamster ovary cells, is correlated with a decrease in tyrosine phosphorylation of the GAT1 (23). The CreaT is similar to the GAT1, and its amino acid sequence contains consensus tyrosine phosphorylation sites (such as Tyr11) (27). It is, therefore, logical to hypothesize that tyrosine phosphorylation of the CreaT by protein tyrosine kinases could functionally modulate CreaT function and its expression. Tyrosine phosphorylation accomplished by protein tyrosine kinase may be one of the primary mechanisms by which modulation of the function of the neurotransmitter transporter family, including GAT1 and CreaT, occurs.

One family of cytoplasmic nonreceptor protein tyrosine kinases that are capable of communicating with a large number of different channels, receptors, and transporters on the plasma membrane is the Src family kinases (3, 19, 35, 36). Src family kinases contain a myristoylated amino terminus that interacts with phospholipid head groups, a short region with low sequence homology, an SH3 domain (a binding site for proline-containing sequences), an SH2 domain (a binding site for phosphotyrosine-containing sequences), a tyrosine kinase domain, and a short carboxy-terminal tail (19, 35, 36). The Src protein possesses two important regulatory tyrosine phosphorylation sites: phosphorylation of Tyr527 in the carboxy-terminal tail inhibits Src kinase activity, whereas phosphorylation of Tyr416 in the activation loop maximally stimulates its activity (19, 35, 36). In the present study, Western blotting with an anti-phospho-c-Src (Y-416) antibody against immunoprecipitated CreaT revealed evidence of increased tyrosine-phosphorylated c-Src (Tyr416) expression in the CreaT-c-Src complex in homogenates and plasma membrane fractions of gastrocnemius muscles after CLP, which strongly suggests that activated c-Src (Tyr416) is physically associated with the CreaT and that enhanced tyrosine phosphorylation of the CreaT is related to active c-Src. Furthermore, our data suggest that regulation of the CreaT by active c-Src occurs, at least, on the plasma membrane. Previous investigations have revealed that Src family kinases mediate some of the biological effects of gram-negative bacterial lipopolysaccharide (LPS) on monocyte or macrophage activation via tyrosine phosphorylation of Src downstream proteins (1, 18, 31, 39). A significant reduction in liver and kidney injury and in neutrophil migration after LPS injection was also found in the Src family kinase, Hck and Fgr knockout mice (25). Thus an increase in Src family kinase activity resulting from an enhanced tyrosine phosphorylation of Src on its Tyr416 site may regulate the CreaT function during sepsis.

Several consensus protein phosphorylation sites for protein kinase C (such as Thr257, Ser625, and Ser626) are also present in the protein sequence of the mammalian CreaT (27), which suggests that threonine/serine phosphorylation of the CreaT by protein kinase C (PKC) may also modulate transporter function and its expression. Several lines of evidence have demonstrated that activation of PKC by phorbol 12-myristate 13-acetate, or PMA, inhibits Cr uptake in the Xenopus oocytes expressing human CreaT (7, 27). In addition, activation of PKC and inhibition of tyrosine kinase comparably decreases GABA uptake by the GAT1 endogenously expressed in hippocampal neurons or heterologously expressed in Chinese hamster ovary cells through a redistribution of the transporter from the cell surface to intracellular locations (23). In our previous experiments, when the CreaT was immunoprecipitated and then immunoblotted with an anti-phosphoserine or anti-phosphothreonine antibody, threonine or serine phosphorylation of the CreaT was equivalent in gastrocnemius muscle homogenates from CLP and sham-operated rats (data not shown). Thus PKC-mediated phosphorylation of the CreaT most likely does not play a major role in regulation of the CreaT observed during sepsis. However, we cannot rule out the possibility that PKC activation may affect Cr uptake in other models through its interaction with tyrosine phosphatase, because evidence indicates that stimulation of GABA transport by a tyrosine phosphatase inhibitor can be blocked by PKC activation (23).

The influence of external Cr or dietary Cr supplementation on the Cr transport and the CreaT has been investigated. Loike et al. (24) showed that extracellular Cr induces the expression of a protein that functionally inactivates the CreaT and attenuates Cr transport in cultured human and rat myoblasts and myotubes. Consistent with this observation, Dodd et al. (9) demonstrated that the accumulation of intracellular Cr decreases Cr uptake in CreaT-expressing HEK293 cell lines. It is not clear how extracellular Cr generates an intracellular signal to modulate CreaT activity. Guerrero-Ontiveros and Wallimann (14) also reported that long-term, high-dose Cr supplementation decreases protein expression of the CreaT in rat skeletal muscle in vivo. However, to our knowledge, the effects of oral Cr supplementation on phosphorylation of the CreaT have not been reported. In the present study, we have shown that oral 1% Cr supplementation increased free Cr levels equivalently in gastrocnemius muscles harvested from animals 24 h after CLP or sham operation. Furthermore, tyrosine phosphorylation of the CreaT was markedly decreased in homogenates or plasma membrane fractions of gastrocnemius muscles after oral Cr supplementation in CLP rats relative to sham-operated animals and was associated with decreased protein expression of tyrosine-phosphorylated c-Src (Tyr416) coimmunoprecipitated with the CreaT (Figs. 4 and 5). Because myocellular free Cr levels in CLP or sham-operated animals after 4 days of oral 1% Cr supplementation were equivalently increased compared with those in non-Cr-supplemented sham-operated rats, the effect of sepsis to increase myocellular Cr levels was abrogated, and sepsis-induced c-Src-related tyrosine phosphorylation of the CreaT to enhance Cr transport did not occur. This apparently differs from the alterations observed in non-Cr-supplemented CLP and sham-operated animals, where sepsis significantly increased myocellular free Cr levels, tyrosine phosphorylation of the CreaT, and tyrosine-phosphorylated c-Src (Tyr416) expression in the CreaT-c-Src complex. Thus, on the basis of the results presented in the current study and the data available in the literature, it is reasonable to hypothesize that extracellular Cr must be transported into the cells where accumulated Cr feedback inhibits c-Src-related tyrosine phosphorylation of the CreaT. Although the mechanism(s) by which the downregulatory effects on c-Src-related tyrosine phosphorylation of the CreaT after dietary Cr supplementation occur is unknown, we speculate that preaccumulation of intracellular Cr in muscle after oral Cr supplementation may cause acquired intrinsic alteration(s) in the control of myocellular energy metabolism that may trigger changes in intracellular signaling pathways by which the downregulatory effects on c-Src-related tyrosine phosphorylation of the CreaT after Cr supplementation occurs in skeletal muscle during sepsis. These remain to be defined and are the subjects of future investigations. Also, acquired intrinsic alteration(s) after Cr supplementation may be responsible for the observed differential tyrosine phosphorylation between the CreaT and tyrosine-phosphorylated c-Src (Tyr416) expression in the CreaT-c-Src complex after CLP.

In the current study, we focused on four experimental groups, i.e., CLP, SHAM, Cr+CLP, and Cr+SHAM. Tyrosine phosphorylation of the CreaT and tyrosine-phosphorylated c-Src (Tyr416) expression in the CreaT-c-Src complex were compared only between corresponding groups, i.e., CLP and SHAM or Cr+CLP and Cr+SHAM, whereas myocellular free Cr levels were compared among four groups, i.e., CLP, SHAM, Cr+CLP, and Cr+SHAM. Because sham operation is the most appropriate control group for CLP, we did not study tyrosine phosphorylation of the CreaT or the effects of Cr supplementation in normal, nonoperated animals. Preliminary experiments showed that protein expression and tyrosine phosphorylation of the CreaT, as well as its association with tyrosine-phosphorylated c-Src (Tyr416) in gastrocnemius muscles, were equivalent in normal, nonoperated and sham-operated animals (data not shown). We assume, therefore, that the effects of oral 1% Cr supplementation for 4 days on the CreaT would be similar between the two groups. The equivalency of myocellular free Cr levels in normal, nonoperated and sham-operated gastrocnemius muscles is consistent with this assumption (Fig. 1).

Murphy et al. (26) have demonstrated that protein expression of the CreaT is greater in fast-oxidative, red gastrocnemius muscles than in fast-glycolytic, white gastrocnemius muscles (26). In the current study, myocellular free Cr levels and tyrosine phosphorylation of the CreaT were increased in mixed red and white gastrocnemius muscles. For this reason, we cannot isolate our findings to a specific fiber type. Furthermore, because the CreaT is also present in skeletal and cardiac muscle mitochondria (34), mitochondrial contamination of our plasma membrane fractions cannot be completely excluded. However, because the purity of our plasma membrane fractions as assessed by the enrichment of plasma membrane marker enzyme (5'-nucleotidase) activity was sufficiently high (8, 32), and because the CreaT is primarily located on the plasma membrane (26, 43), the amount of mitochondrial contamination that occurred, if any, was likely negligible.

In summary, sepsis increases myocellular free Cr levels, which are associated with enhanced tyrosine phosphorylation of the CreaT. Because the CreaT is physically associated with an active form of c-Src, the CreaT may be a targeted substrate for c-Src during sepsis. c-Src-related tyrosine phosphorylation of the CreaT in skeletal muscle is, however, downregulated in Cr-supplemented septic animals compared with Cr-supplemented sham-operated rats. Although the physiological relevance of Cr supplementation's downregulatory effects on tyrosine phosphorylation of the CreaT during sepsis is not yet clear, it is obvious that myocellular CreaT function is tightly regulated and involves c-Src-related tyrosine phosphorylation during sepsis. Whether or not oral Cr supplementation improves skeletal muscle energetics or performance during sepsis is not yet determined. Given substantial evidence that dietary Cr supplementation not only improves skeletal muscle energy metabolism and performance in healthy men and women (6) but is also therapeutic for patients with certain diseases, e.g., Huntington's disease, Parkinson's disease, Duchenne muscular dystrophy, amyotrophic lateral sclerosis, and congestive heart failure (29), it is important to understand how the CreaT is modulated. The current study provides new insights into this process.


    ACKNOWLEDGEMENTS

This study was supported by the Creighton University Health Future Foundation and, in part, by the National Institute of General Medical Sciences Grant P50 GM-52585.


    FOOTNOTES

Address for reprint requests and other correspondence: D. O. Jacobs, Dept. of Surgery, Creighton Univ. School of Medicine, 601 N 30th St., Suite 3520, Omaha, NE 68131 (E-mail: djacobs{at}creighton.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 15, 2002;10.1152/ajpendo.00506.2001

Received 8 November 2001; accepted in final form 9 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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