Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca²⁺ and cPKC

¹ Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8; ² Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8; and ³ Division of Molecular Genetics, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan

	ABSTRACT

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2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative chain from ATP production, preventing oxidative metabolism. The consequent increase in energy demand is, however, contested by cells increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal muscle cells, DNP rapidly doubles glucose transport, reminiscent of the effect of insulin. However, glucose transport stimulation by DNP does not require insulin receptor substrate-1 phosphorylation and is wortmannin insensitive. We report here that, unlike insulin, DNP does not activate phosphatidylinositol 3-kinase, protein kinase B/Akt, or p70 S6 kinase. However, chelation of intra- and extracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose uptake by 78.9 ± 3.5%. Because Ca²⁺-sensitive, conventional protein kinase C (cPKC) can activate glucose transport in L6 muscle cells, we examined whether cPKC may be translocated and activated in response to DNP in L6 myotubes. Acute DNP treatment led to translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from plasma membranes exhibited a twofold increase in kinase activity in response to DNP. Overnight treatment with 4-phorbol 12-myristate 13-acetate downregulated cPKC isoforms , , and  and partially inhibited (45.0 ± 3.6%) DNP- but not insulin-stimulated glucose uptake. Consistent with this, the PKC inhibitor bisindolylmaleimide I blocked PKC enzyme activity at the plasma membrane (100%) and inhibited DNP-stimulated 2-[³H]deoxyglucose uptake (61.2 ± 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC- inhibitor LY-379196 partially inhibited DNP effects on glucose uptake (66.7 ± 1.6%). The results suggest interfering with mitochondrial ATP production acts on a signal transduction pathway independent from that of insulin and partly mediated by Ca²⁺ and cPKCs, of which PKC- likely plays a significant role.

2,4-dinitrophenol; insulin; glucose uptake; glucose transporter-4 translocation; conventional protein kinase C

	INTRODUCTION

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IN MAMMALS, SKELETAL MUSCLE is the primary target tissue for insulin stimulation of glucose transport, a regulatory mechanism vital for glucose homeostasis. Insulin achieves this regulation by signaling the translocation of preformed glucose transporters from intracellular stores to the plasma membrane. The L6 muscle cell line has been used extensively to characterize physiological responses in muscle such as glucose transport, since it retains many morphological and metabolic properties of skeletal muscle (32, 61). Three glucose transporter (GLUT) isoforms are expressed in differentiated L6 myotubes, GLUT-1, GLUT-3, and GLUT-4 (5). There is mounting evidence that muscle cells respond to a variety of stimuli by rapidly elevating their rate of glucose uptake (reviewed in Ref. 23). These include, on one hand, the anabolic hormone insulin and, on the other hand, stimuli that increase energy demand such as exercise (23), hypoxia (8), environmental stress (11), and metabolic challenges to the oxidative chain. The mitochondrial uncoupler 2,4-dinitrophenol (DNP), a weak base that dissipates the H⁺ gradient of mitochondria, uncouples the oxidative chain from ATP production, thus compromising energy production (4). Previous work has shown that L6 muscle cells react to this metabolic challenge by increasing glucose transport to boost glycolytic ATP production, reminiscent of the response to hypoxia in vivo (4).

There are numerous contrasts between insulin and energy stressors in their mechanisms of glucose transport activation in skeletal muscle. Insulin and exercise recruit distinct intracellular pools of glucose transporters in skeletal muscle (13, 14), and the maximal effects of insulin and contraction or insulin and hypoxia on glucose uptake are additive (48, 62). Activation of phosphatidylinositol 3-kinase (PI3K) is utilized by insulin to induce glucose transporter translocation but does not participate in the responses to exercise or hypoxia (40, 42, 57). Moreover, insulin, but not contraction, causes a redistribution of Rab4 (a Ras-related GTP-binding protein) from internal compartments in skeletal muscle (53). In L6 muscle cells, insulin causes translocation to the cell membrane of GLUT-1, GLUT-3, and GLUT-4, whereas DNP mobilizes only GLUT-1 and GLUT-4 (57). Unlike insulin, DNP does not require PI3K activity and an intact actin cytoskeletal network (57) to mediate these effects. Collectively, these findings suggest that energy stressors utilize mechanisms other than insulin to increase muscle glucose influx; however, little is known about the mechanism by which these factors elicit this response. The purpose of this study was to use DNP as a model of exercise or hypoxia to investigate possible mediators of this alternative signaling pathway. Our findings provide evidence for the existence of a signaling system activated by metabolic challenge that regulates glucose transport in muscle cells by a mechanism distinct from that used by insulin.

	MATERIALS AND METHODS

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Materials. Tissue culture medium, serum, and other tissue culture reagents were obtained from Life Technologies (Burlington, ON, Canada). Human insulin was a kind gift from Eli Lilly Canada (Toronto, ON, Canada). DNP, 4-phorbol-12-myristate-13-acetate (PMA), and cytochalasin B were obtained from Sigma Chemical (St. Louis, MO). Bisindolylmaleimide I (BIM) was from Calbiochem (La Jolla, CA). The protein kinase C (PKC)- inhibitor LY-379196 was a kind gift from Eli Lilly (Indianapolis, IN). Protein A- and protein G-Sepharose were from Pharmacia Biotechnology (Uppsala, Sweden). [-³²P]ATP and enhanced chemiluminescence reagents were purchased from Amersham (Oakville, ON, Canada). 2-[³H]deoxyglucose and 3-O-[methyl-³H]methylglucose were obtained from DuPont NEN (Boston, MA). Monoclonal antibody against PKC-, -, and - used for immunoprecipitation and polyclonal antibody to PKC-_II were kind gifts from Kinetek Pharmaceuticals (Vancouver, BC, Canada). Polyclonal antibodies to PKC-, PKC-_I, and PKC- used for immunoblotting were purchased from Signal Transduction Laboratories (Lexington, KY). Crosstide peptide, protein kinase A (PKA) and PKC inhibitor peptides, and polyclonal antibodies to PKC-, p70 S6 kinase (p70^S6K), and protein kinase B/Akt (PKB/Akt) were obtained from Santa Cruz (Santa Cruz, CA). PKC kinase assay kit and monoclonal anti-phosphotyrosine antibody (used for PI3K activity assay) were purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibody McK1 to the ₁-subunit of the Na⁺-K⁺-ATPase was a kind gift from Dr. K. Sweadner (Massachusetts General Hospital, Boston, MA).

Cell culture. L6 muscle cells were maintained in myoblast monolayer culture in -MEM containing 10% vol/vol fetal bovine serum (FBS) and 1% vol/vol antibiotic-antimycotic solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin, and 25 mg/ml amphotericin B) in an atmosphere of 5% CO₂ at 37°C as described previously (15). Cells were maintained in continuous passages by trypsinization of subconfluent cultures using 0.25% trypsin. Myoblasts were seeded in medium containing 2% vol/vol FBS at ~4 × 10⁴ cells/ml in 10-cm-diameter dishes and used 6-8 days postseeding for plasma membrane preparations and kinase activity assays. L6 cells were seeded in 12-well or 6-well plates for glucose uptake experiments. Cells were fed fresh medium every 48 h and used at the stage of myotubes.

L6 muscle cells expressing c-myc epitope-tagged GLUT-4 (GLUT-4-myc) were constructed as described (29). The human c-myc epitope (14 amino acids) was introduced into the first ectodomain of GLUT-4, and the epitope does not affect GLUT-4 activity (29, 59). GLUT-4-myc cDNA was subcloned into the mammalian expression vector pCXN (pCXN-GLUT-4-myc). L6 myoblasts were transfected with pCXN-GLUT-4-myc and pSV2-bsr, a blasticidin S deaminase expression plasmid, and selected with blasticidin S hydrochloride (Funakoshi, Tokyo, Japan). Cell surface GLUT-4-myc was detected by a colorimetric assay as described previously (59).

Hexose transport determinations. Measurements of 2-[³H]deoxyglucose and 3-O-[methyl-³H]methylglucose uptake were carried out as previously described (34, 38). Briefly, differentiated L6 myotube monolayers grown in 12-well plates (used for 2-deoxyglucose uptake) or 6-well plates (used for 3-O-methylglucose uptake) were rinsed twice with HEPES-buffered saline (HBS; in mM: 140 NaCl, 20 NaHEPES, 2.5 MgSO₄, 1 CaCl₂, and 5 KCl, pH 7.4). Glucose uptake was quantitated by exposing the cells to 10 µM 2-[³H]deoxyglucose (1 µCi/ml) for 5 min or 10 µM 3-O-[methyl-³H]methylglucose (2 µCi/ml) for 2 min. Nonspecific uptake was determined by quantitating cell-associated radioactivity in the presence of 10 µM cytochalasin B, which blocks transporter-mediated uptake. At the end of the 5-min period, the uptake buffer was aspirated rapidly and the cells were washed three times with ice-cold isotonic saline (0.9% wt/vol NaCl, containing 1 mM HgCl₂ in the case of 3-O-[methyl-³H]methylglucose uptake assays). The cells were lysed in 0.05 N NaOH, and the associated radioactivity was determined by liquid scintillation counting. Each condition was assayed in triplicate for 2-[³H]deoxyglucose assays and in duplicate for 3-O-[methyl-³H]methylglucose uptake experiments.

PI3K, Akt/PKB, and p70^S6K activity assays. PI3K activity and p70^S6K activity were assayed exactly as described previously (17, 56).

Immunoprecipitation of Akt1 and kinase assay were performed as described (37) with modifications. Cells were lysed with lysis buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10% vol/vol glycerol, 1% vol/vol Triton X-100, 30 mM sodium pyrophosphate, 10 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 1 mM Na₃VO₄, 1 mM dithiothreitol (DTT), and 100 nM okadaic acid. Anti-Akt1 antibody was precoupled to a mixture of protein A- and protein G-Sepharose beads by incubating 2 µg of antibody per condition with 20 µl of the protein A- and protein G-Sepharose beads (100 mg/ml) for a minimum of 2 h. These anti-Akt1 beads were washed twice with ice-cold PBS and once with ice-cold lysis buffer. Akt1 was immunoprecipitated by incubating 200 µg of total cellular protein with the anti-Akt1-bead complex for 2-3 h under constant rotation (4°C). Akt1 immunocomplexes were isolated and washed four times with 1 ml of wash buffer [25 mM HEPES (pH 7.8), 10% vol/vol glycerol, 1% vol/vol Triton X-100, 0.1% wt/vol BSA, 1 M NaCl, 1 mM DTT, 1 mM PMSF, 1 µM microcystin, and 100 nM okadaic acid] and twice with 1 ml of kinase buffer [50 mM Tris · HCl (pH 7.5), 10 mM MgCl₂, and 1 mM DTT]. This was then incubated under constant agitation for 30 min at 30°C with 30 µl of reaction mixture (kinase buffer containing 5 µM ATP, 2 µCi [-³²P]ATP, and 100 µM Crosstide). After the reaction, 30 µl of the supernatant were transferred onto Whatman p81 filter paper and washed with 3 ml of 175 mM phosphoric acid four times for 10 min and once with distilled water for 5 min. Filters were air dried and then subjected to liquid scintillation counting.

Plasma membrane-enriched fraction and immunoblotting. Myotube monolayers grown on 10-cm-diameter dishes were gently scraped with a rubber policeman in 5 ml of ice-cold homogenization buffer (in mM: 250 sucrose, 20 HEPES, 2 EGTA, and 3 NaN₃, pH 7.4) containing freshly added protease inhibitors (in µM: 200 PMSF, 1 leupeptin, and 1 pepstatin A) and homogenized in a 40-ml Dounce type A homogenizer on ice (20 strokes). The homogenate was centrifuged at 760 g for 5 min at 4°C, and the resultant supernatant was centrifuged at 31,000 g for 20 min to separate a plasma membrane-enriched pellet from an intracellular microsome supernatant. The plasma membrane fraction was resuspended in homogenization buffer. Membrane protein content was determined by the bicinchoninic acid method (Pierce, Rockford, IL). Fifty micrograms of protein were separated by 7.5% SDS-PAGE, electrotransferred onto polyvinylidene difluoride membrane, and immunoblotted for various PKC isoforms or for the ₁-subunit of the Na⁺-K⁺-ATPase. For monoclonal and polyclonal antibody detection, horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were used, respectively, followed by enhanced chemiluminescence.

PKC activity assay. Plasma membranes were resuspended in 0.5 ml of immunoprecipitation buffer [50 mM HEPES (pH 7.8), 1% vol/vol Triton X-100, 2.5 mM EDTA, 200 µM PMSF, 1 µM leupeptin, and 1 µM pepstatin A] and lysed by passing through a 27-gauge syringe five times. The homogenate was centrifuged at 12,000 g for 5 min, and the supernatant was incubated overnight with 20 µl of anti-PKC-,, monoclonal antibody at 4°C with rotary shaking. To this mixture was added 50 µl of 50% wt/vol protein A-Sepharose beads for 1 h. The Sepharose beads and attached proteins were pelleted by centrifugation and washed three times with PBS plus 0.1% vol/vol Triton X-100. The phosphotransferase activity of PKC in immunoprecipitates from plasma membranes was measured using a PKC assay kit. The assay is based on phosphorylation of a specific substrate peptide (Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu) using the transfer of the -phosphate of [-³²P]ATP by PKC kinase. The immunoprecipitated protein was diluted in 20 mM MOPS (pH 7.2) containing 25 mM -glycerol phosphate, 1 mM sodium vanadate, 1 mM DTT, and 1 mM CaCl₂. To the enzyme preparation were added 100 µM peptide substrate, lipid activators (0.1 mg/ml phosphatidylserine and 0.01 mg/ml diglyceride), and kinase inhibitors (100 nM PKA inhibitor peptide and 4 µM R-24571). The kinase reaction was started by adding Mg²⁺/ATP reaction buffer containing 15 mM MgCl₂ and 100 µM ATP (1.5 µCi [-³²P]ATP) in assay dilution buffer. The mixture was incubated at 30°C for 10 min. The phosphorylated substrate was then separated from the residual [-³²P]ATP using Whatman p81 filter paper, washed in 175 mM phosphoric acid, air dried, and then quantitated using a liquid scintillation counter.

Statistical analysis. X-ray films were quantified in the linear range by densitometry using National Institutes of Health Image software. The detection and quantitation of [³²P]phosphatidylinositol 3-phosphate (PI3P) on TLC plates were performed with a Molecular Dynamics PhosphorImager system (Sunnyvale, CA). Statistical analysis was performed using the ANOVA test (Fisher, multiple comparisons).

	RESULTS

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DNP does not activate the insulin signal transduction pathway. The time course in Fig. 1 demonstrates that the maximal effects of DNP and insulin on glucose transport were additive over a 60-min period after addition, reminiscent of previous observations of additivity between insulin and hypoxia or insulin and exercise in skeletal muscle (48, 62). This suggests that different signals may participate in relaying the signal from insulin and from DNP to the glucose transporters.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   2,4-Dinitrophenol (DNP)-stimulated glucose transport is additive to insulin. L6 myotubes were serum deprived for 5 h and then incubated for 0-60 min in -MEM with 0.5 mM DNP, 100 nM insulin (INS), or both at 37°C. Immediately after these incubations, cells were washed twice with HEPES-buffered saline (HBS) and 2-[3H]deoxyglucose uptake was assayed as described in MATERIALS AND METHODS. Transport rates were normalized with respect to basal rate of untreated control, which was assigned a value of 1. Shown is a representative of 3 independently performed experiments. Results are expressed as means ± SE of 3 replicates.

Recent studies have identified three kinases rapidly activated by insulin in muscle cells: PI3K (56), PKB (Akt/PKB) (37), and p70^S6K (17). We have shown that the selective PI3K inhibitor wortmannin does not affect the activation of glucose transport by DNP in L6 muscle cells but abolishes the insulin response (57). However, PI3K activity was not directly measured in that study. Because arsenite-induced activation of glucose transport and vanadate-mediated antilipolytic actions are wortmannin insensitive although PI3K is activated by both agents (41, 46), it was important to test whether DNP also activates PI3K. Unlike the hormonal response of L6 muscle cells, DNP did not activate PI3K (Fig. 2A). It is widely held that insulin-mediated Akt/PKB and p70^S6K activation occurs downstream of PI3K and is dependent on the lipid products of PI3K (1, 9), yet recent reports have uncovered stress-induced activation of Akt/PKB in COS-7 cells (39) and, in cardiomyocytes, arsenite-induced activation of p70^S6K (60), which are both PI3K independent. We therefore tested whether DNP activated either Akt/PKB or p70^S6K directly. As shown in Fig. 2, DNP did not activate either Akt/PKB (Fig. 2B) or p70^S6K (Fig. 2C) in L6 muscle cells. Furthermore, selective inhibition of the p70^S6K pathway by rapamycin in L6 muscle cells failed to prevent the DNP-induced activation of glucose transport (Khayat and Klip, unpublished observations).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   DNP does not activate lipid and protein kinases activated by insulin. Serum-depleted L6 myotubes (5 h) were treated with 0.5 mM DNP or 100 nM insulin for up to 30 min. Phosphatidylinositol 3-kinase (PI3K; A), Akt/protein kinase B (PKB; B), or p70 S6 kinase (p70S6K; C) kinase activity was assayed in cell lysates as described in MATERIALS AND METHODS. Observed kinase activities were normalized relative to basal activities in untreated cells, which were assigned a value of 1. Results are expressed as means ± SE of 3 independent experiments.

Role of intracellular Ca²⁺ in DNP-stimulated glucose uptake. There is evidence that mitochondrial uncoupling provokes a rapid rise in intracellular Ca²⁺ that coincides with an acceleration of glucose flux in muscle and liver cells (11, 44). To ascertain the demand for Ca²⁺ in the activation of glucose transport by DNP, L6 muscle cells were loaded with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM incubated simultaneously with EGTA (to buffer extracellular Ca²⁺) before challenge with DNP or insulin (as in Ref. 33) followed by 2-[³H]deoxyglucose uptake measurements. The Ca²⁺ chelators inhibited DNP-stimulated glucose uptake by 78.9 ± 3.5% (P < 0.01), without affecting insulin-stimulated glucose uptake (Fig. 3). Buffering extracellular Ca²⁺ with 2.5 mM EGTA alone or 15 µM BAPTA-AM alone did not significantly affect the DNP response (results not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Role of Ca2+ in response to DNP. Serum-depleted L6 myotubes (5 h) were pretreated with or without 15 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM and 2.5 mM EGTA in Ca2+-free HBS supplemented with 10 mM D-glucose for 10 min, followed by stimulation for 30 min with 0.5 mM DNP or 100 nM insulin in HBS. Cells were washed twice with HBS, and 2-[3H]deoxyglucose uptake was measured as described in MATERIALS AND METHODS. Transport rates were normalized with respect to basal rate of untreated control (CON), which was assigned a value of 1. Results are expressed as means ± SE of 5 independent experiments. * P < 0.01 vs. control DNP.

Role of Ca²⁺-sensitive PKC in DNP action. A rise in intracellular Ca²⁺ triggers the activation of a variety of cellular proteins, including Ca²⁺-sensitive, conventional PKC (cPKC) (reviewed in Ref. 49). To assess the involvement of cPKC in the glucose transport response, we utilized the potent PKC inhibitor BIM, which inhibits Ca²⁺-dependent cPKC isoforms at lower doses (<1 µM) than those affecting novel or atypical isoforms, which do not require Ca²⁺ for activation (45). At 1 µM, BIM caused a 61.2 ± 2.4% (P < 0.05) reduction in the stimulation of glucose transport by DNP (Fig. 4A), but it did not affect the response to insulin. Furthermore, pretreatment with 1 µM BIM did not further reduce DNP-stimulated glucose transport beyond the 80% inhibition observed with BAPTA-EGTA pretreatment (Table 1). At a higher dose of BIM (10 µM), which is known to inhibit novel and atypical PKC isoforms, no additional inhibition of DNP-stimulated glucose uptake was observed; however, insulin-stimulated glucose transport was inhibited by 50%. The latter observation is in agreement with recent evidence supporting the involvement of atypical PKC- in the insulin-dependent glucose transport pathway (2).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   DNP-stimulated glucose transport is reduced by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM). A: serum-depleted L6 myotubes (5 h) were preincubated for 15 min in -MEM in absence or presence of indicated doses of BIM. Cells were then stimulated for 30 min at 37°C with 0.5 mM DNP or 100 nM insulin in continuous presence of BIM. At end of this period, cells were washed twice with HBS and 2-[3H]deoxyglucose uptake was measured. Transport rates were normalized with respect to basal rate of untreated control (UNT), which was assigned a value of 1. B: effect of 1 µM BIM on DNP-, insulin-, and 4-phorbol-12-myristate-13-acetate (PMA; 1 µM, 30 min)-stimulated glucose uptake. Results are expressed as means ± SE of 6 independent experiments. * P < 0.05 vs. respective control.

View this table: [in this window] [in a new window]   Table 1.   Combination of PKC inhibition and Ca2+ chelation does not further inhibit DNP response

The PKC-stimulating phorbol ester PMA is also able to stimulate glucose transporter translocation (30), albeit to a lesser extent than insulin or other stimuli. To confirm that the action of 1 µM BIM is on cPKC, we tested the effect of BIM (1 µM) on PMA-stimulated glucose transport in L6 muscle cells. Figure 4B demonstrates that PMA increased glucose transport by 30% and 1 µM BIM inhibited this stimulation by 100%, whereas DNP-dependent glucose uptake was partially inhibited (60%) by the same treatment. BIM had no effect on the basal value of glucose transport (Fig. 4, A and B).

To further clarify the dependence of cPKC in DNP-dependent glucose transport activation, cPKCs were depleted from L6 cells by overnight PMA treatment. PMA treatment (cPKC downregulation) eliminated all cPKC isoforms but not the atypical PKC- (Fig. 5A). Furthermore, cPKC downregulation partially inhibited the stimulation of glucose transport by DNP by 45.0 ± 3.6%, whereas it fully blocked PMA-stimulated glucose uptake. PKC downregulation did not affect the stimulation of glucose transport by insulin (Fig. 5B). The findings for insulin and PMA were consistent with previous observations reported by Bandyopadhyay et al. (2) in L6 muscle cells. As with 1 µM BIM, the inhibition of DNP action by PKC depletion in combination with Ca²⁺ chelation was no greater than the effect of Ca²⁺ buffering alone (Table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Conventional, Ca2+-sensitive PKC (cPKC) downregulation reduces DNP-stimulated glucose transport. L6 myotubes were incubated for 16 h in absence or presence of 100 nM PMA (downregulated). A: cells were lysed, and various PKC isoforms were detected in 15 µg of cell lysates by SDS-PAGE followed by immunoblotting. B: cells were stimulated for 30 min with 0.5 mM DNP, 100 nM insulin, or 1 µM PMA. At end of this period, cells were washed and 2-[3H]deoxyglucose transport was assayed as described in MATERIALS AND METHODS. Transport rates were normalized with respect to basal rate of untreated control, which was assigned a value of 1. Results are expressed as means ± SE of 5 independent experiments. * P < 0.05 vs. respective control.

The extent of cPKC activation by DNP was ascertained by two methods. The first approach involved purification of fractions enriched in plasma membranes derived from DNP-treated L6 cells, followed by immunoblotting for cPKC isoforms with an antibody that recognized PKC-, -, and - isoforms. DNP generated a 2.6-fold increase in PKC-, -, and - levels in the plasma membrane compared with unstimulated cells (Fig. 6, A and B). PMA, on the other hand, provoked a marked redistribution of cPKC to the plasma membrane (ninefold), whereas insulin treatment resulted in only a modest increase in the plasma membrane levels of cPKCs. As expected, cPKC could not be detected in plasma membrane fractions isolated from cells pretreated with PMA overnight that were not stimulated or were stimulated with insulin, DNP, or PMA for 30 min (Fig. 6A). The second approach involved measuring in vitro cPKC activity directly in plasma membrane fractions derived from unstimulated or from DNP-, insulin-, or PMA-stimulated cells. PKC-, -, and - activity in cPKC immunoprecipitates from plasma membranes was elevated by 200% by DNP (Fig. 7A). This activation was completely blocked by pretreatment of cells with 1 µM BIM. Comparable with its higher stimulation of cPKC translocation to the plasma membrane, PMA also induced a much greater activation of cPKC activity (sevenfold) than DNP, whereas insulin elevated cPKC activity by only 30%.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   DNP increases translocation of cPKC to plasma membrane. Serum-depleted L6 myotubes (5 h) were stimulated with 0.5 mM DNP, 100 nM insulin, or 1 µM PMA in -MEM at 37°C for 30 min. Subcellular fractionation was performed to obtain a plasma membrane-enriched fraction. Equal amounts of this fraction (50 µg) were separated by 7.5% SDS-PAGE, electrotransferred onto polyvinylidene difluoride membrane, and immunoblotted for PKC-, -, and -. To ensure equality of protein loading, blots were simultaneously probed with monoclonal antibody against 1-subunit of Na+-K+-ATPase (results not shown). A: representative immunoblot. B: quantitation of immunoblots (n = 4) expressed as means ± SE relative to basal levels of PKC in plasma membrane from untreated cells. * P < 0.05, ** P < 0.001 vs. untreated control.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   DNP induces a cPKC activity that is inhibitable by BIM. Serum-depleted L6 myotubes (5 h) were treated with or without 1 µM BIM in -MEM for 15 min before stimulation with 0.5 mM DNP or 100 nM insulin (A) or with 1 µM PMA (B) for 10 min at 37°C. Cells were fractionated to obtain a plasma-membrane enriched fraction. cPKC was immunoprecipitated from this fraction using a monoclonal antibody against PKC-, -, and -. A nonspecific rabbit antibody (IgG) was used as a background control. Kinase activity of immunoprecipitated PKC on an exogenous substrate was measured. Observed PKC activity was normalized relative to basal activity in untreated cells. Results are expressed as means ± SE of 4-6 independent experiments. * P < 0.05, ** P < 0.001 vs. untreated control.

The availability of a selective PKC- inhibitor, 379196 (27), allowed us to test the participation of this isoform in DNP-stimulated glucose transport. As shown in Fig. 8, pretreatment of L6 cells with 379196 inhibited DNP-stimulated glucose uptake in a dose-dependent manner. At a concentration of 100 nM 379196, which will effectively inhibit PKC- (IC₅₀ 150 nM), the stimulation of glucose uptake by DNP was reduced by 66.7% (P < 0.01). Insulin-stimulated glucose uptake was not affected by the inhibitor (results not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Inhibition of PKC- reduces DNP-mediated glucose transport. Serum-depleted L6 myotubes were preincubated for 15 min in -MEM containing indicated doses of the PKC--specific inhibitor 379196. Cells were then stimulated for 30 min with 0.5 mM DNP in continuous presence of inhibitor. At end of this period, cells were washed and 2-[3H]deoxyglucose uptake was measured. Transport rates were normalized with respect to basal rate of untreated control, which was assigned a value of 1. Results are expressed as means ± SE of 3 independent experiments. * P < 0.01 vs. control DNP.

3-O-methylglucose uptake and GLUT-4 translocation also depend on Ca²⁺ mobilization and cPKC. The uptake of 2-deoxyglucose is the sum of transmembrane transport and phosphorylation. We and others previously measured the transport rate and found that changes in 2-deoxyglucose uptake reflect changes in transport under the assay conditions used. Nonetheless, the effect of ATP depletion by DNP on hexose uptake may have either triggered the stimulation of hexose transport or modulation of the activity of hexokinase, the enzyme that phosphorylates 2-deoxyglucose to form 2-deoxyglucose-6-phosphate. To ensure that incubation with DNP brings about a response of hexose transport specifically, the uptake of 3-O-methylglucose (a nonphosphorylatable analog of glucose) was also measured. Table 2 shows that DNP increased 3-O-methylglucose uptake by about twofold relative to control cells. Furthermore, pretreatment with BAPTA-EGTA, BIM, or cPKC downregulation partially inhibited DNP-stimulated 3-O-methylglucose uptake (Table 2). The extent of reduction in DNP-stimulated glucose uptake closely paralleled the results using 2-deoxyglucose as the transported sugar (Figs. 3, 4, and 5B).

View this table: [in this window] [in a new window]   Table 2.   DNP also stimulates 3-O-methylglucose uptake in a Ca2+- and PKC-dependent manner

To confirm that the observed effects of Ca²⁺ chelation and cPKC inhibition of DNP-stimulated glucose uptake resulted from impaired glucose transporter translocation, we utilized L6 cells stably transfected with a GLUT-4 protein containing an exofacial myc epitope tag (L6 GLUT-4-myc) (29). These cells were treated with or without DNP along with various manipulations of Ca²⁺ or cPKC, and myc-tagged GLUT-4 was detected on the surface of intact cells, as described in MATERIALS AND METHODS. Pretreatment with Ca²⁺ chelation, BIM (1 µM), or cPKC downregulation decreased the GLUT-4-myc at the cell surface to 37, 47, and 46% of the DNP response, respectively (Fig. 9A). As in wild-type L6 muscle cells, DNP-stimulated glucose uptake was also partially inhibited in L6 GLUT-4-myc cells by pretreatment with BAPTA-EGTA, 1 µM BIM, or cPKC downregulation (Fig. 9B). The inhibitory effects of Ca²⁺ buffering and interfering with cPKC activity on DNP-mediated GLUT-4 translocation in L6 GLUT-4-myc cells were qualitatively comparable to the observed inhibition of DNP-stimulated 2-deoxyglucose uptake.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   DNP-stimulated glucose transporter-4 (GLUT-4)-myc translocation depends on Ca2+ and cPKC. L6 GLUT-4-myc cells were grown to stage of myotubes and were pretreated with BAPTA-AM and EGTA (BAP/EGTA), 1 µM BIM (BIM), or were depleted of cPKC [downregulated (DR)], as described for Figs. 3-5, respectively, before treatment with 0.5 mM DNP for 30 min. DNP-stimulated increase in GLUT-4-myc translocation (A) or 2-[3H]deoxyglucose uptake (B) was assigned a value of 100%. Inhibition of this value toward basal (0%) was calculated after inhibition with BAPTA-EGTA, BIM, or downregulation.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Distinct pathways for glucose transport stimulation. Several lines of evidence suggest that a pathway exists for the stimulation of glucose transport into skeletal muscle by insulin that differs from that for stimulation of energy demand (contraction or hypoxia). For example, a combination of the two stimuli produces an additive stimulation of glucose uptake. Furthermore, insulin signaling requires activation of PI3K and Akt/PKB, but hypoxia and contraction do not (40, 43, 57). Here we show that the stimulation of glucose uptake by DNP in L6 muscle cells is additive to that induced by insulin. Also, we demonstrate that, unlike insulin, DNP does not activate PI3K, Akt/PKB, or p70^S6K. The lack of activation of Akt/PKB by DNP is in agreement with the recent study by Lund et al. (43) that showed muscle contraction had no effect Akt/PKB activity. These findings support the notion that at least two distinct pathways leading to the stimulation of glucose uptake also exist in L6 muscle cells. The insulin-independent pathway will hence be termed "the alternative pathway" for the purpose of this discussion.

The Ca²⁺-PKC hypothesis. It has long been considered that the rise in intracellular Ca²⁺ is a critical mediator of increased glucose transport during skeletal muscle contraction and hypoxia (7, 25). This has been proposed mostly on the basis of inhibition of the stimulation of glucose transport during hypoxia and contraction by agents that are thought to block Ca²⁺ channels [e.g., verapamil (7)] or lower Ca²⁺ efflux from the sarcoplasmic reticulum [e.g., dantrolene (25)]. Additionally, several studies have shown that rates of glucose transport can be increased in mammalian muscle when cytoplasmic Ca²⁺ concentrations are raised using agents such as W-7, caffeine, and Ca²⁺ ionophores (24, 25, 47). In contrast, insulin does not significantly affect cytosolic Ca²⁺ levels (33, 35). Ca²⁺ is, however, released from mitochondria as a result of DNP dissipation of the H⁺ gradient (44). We therefore reasoned that Ca²⁺ may be a trigger in the insulin-independent mechanism of glucose transport activation. Our findings with the buffering of intra- and extracellular Ca²⁺ provide more direct evidence that Ca²⁺ plays a significant role in the stimulation of glucose transport induced by DNP but not in stimulation induced by insulin.

A rise in cytoplasmic Ca²⁺ levels may facilitate the activation of key intracellular signaling molecules that lead to increased muscle glucose transport. PKC is a Ca²⁺-dependent signaling intermediary that can be activated by increases in cellular Ca²⁺. Because Ca²⁺ can activate cPKCs and PMA (a known activator of cPKC) can increase glucose by a transport mechanism distinct from insulin (2, 18, 36, 58), we explored the potential role of cPKC in DNP-stimulated glucose transport. On the basis of four lines of evidence, we propose that DNP, acting through Ca²⁺-sensitive PKC, can modify L6 muscle cell glucose transport. 1) The downregulation of cPKC, but not of atypical PKC protein isoforms, decreased DNP-stimulated glucose transport by 45%, with no effect on insulin-induced glucose uptake. 2) The DNP-induced rise in glucose transport was lowered by 60% with a low dose of BIM (1 µM) that is known to effectively inhibit cPKC, whereas the insulin response was only affected at a far greater BIM concentration. 3) DNP caused a rapid translocation of PKC-, -, and - to the cell surface and brought about their activation. It is conceivable that, in addition to Ca²⁺ activation, the kinase molecules experienced covalent modifications that contributed to this activation. 4) Using 379196 to selectively inhibit PKC-, we observed a partial decrease (67%) in the stimulation of glucose transport by DNP, which closely approximates the inhibition observed with BIM treatment (60%). Therefore, we propose that PKC- may account for the cPKC isoform participating in glucose transporter mobilization during metabolic challenge. Previous reports have revealed PKC activation during muscle contraction (12, 50). However, which of the 12 different PKC isoforms was responsible for this effect was not determined. In light of our findings with 379196, it is plausible that PKC- may also relay the signal to glucose transporters in the exercising muscle. If specific antagonists for the other Ca²⁺-sensitive PKC isotypes become available, it will be possible to verify the specific cPKC mediators of the alternative mechanism of glucose transport activation.

Because PMA-stimulated glucose transport was completely inhibited by 1 µM BIM and PMA downregulation of cPKC and yet no more than 60% of the stimulation by DNP was inhibited by these manipulations, we postulate that there may be a PKC-independent component to the stimulation of glucose uptake by DNP. Conversely, PMA stimulates cPKC activity by eightfold but is only able to induce a 50% rise in glucose transport. Therefore, robust activation of PKC alone is not sufficient to increase glucose transport to levels comparable to those induced by DNP or insulin. Discrepant effects of phorbol esters, insulin, and hypoxia on glucose transport have been noted previously (18, 20, 58). Ca²⁺ chelation was more effective than cPKC inhibition in reducing the DNP stimulation of glucose uptake. However, even this treatment left a residual increase in glucose uptake. Also, the effect of Ca²⁺ buffering on DNP action was not enhanced by simultaneous cPKC inhibition or cPKC deletion (Table 1). Assuming that all treatments were fully effective on their targets (i.e., they fully inhibited cPKC and prevented rises in cytoplasmic Ca²⁺, as appropriate), then it is possible that three types of signals cooperate to bring about the DNP effect on glucose uptake: cPKC activation, a secondary effect of Ca²⁺, and a Ca²⁺-independent signal. This concept is illustrated in Fig. 10.

View larger version (51K): [in this window] [in a new window]   Fig. 10.   Ca2+- and cPKC-dependent components of DNP-stimulated glucose transport. Whole pie graph represents 100% of glucose transport stimulated by DNP. Hatched and stippled regions represent Ca2+-dependent component (80%) of this activation. Within this Ca2+-dependent element, we postulate that 60% of DNP response (stippled region) is mediated by cPKCs that are sensitive to intracellular Ca2+ levels (likely PKC-). Remaining Ca2+-independent component (~20%, open region) involves unknown mediators.

Using L6 GLUT-4-myc cells, we were able to show that the inhibition of DNP-stimulated 2-[³H]deoxyglucose uptake caused by Ca²⁺ chelation or by interference with cPKC activation is reflected by a decrease in the mobilization of GLUT-4-myc to the cell surface. GLUT-4-myc translocation, assessed by a colorimetric detection assay, was impaired by these manipulations to nearly the same extent as glucose transport in wild-type L6 or L6 GLUT-4-myc muscle cells. It was shown previously that the GLUT-4-myc expressed in L6 myotubes is functional for glucose uptake and behaves like endogenous GLUT-4 (31, 59). Therefore, the inhibitory effect of cPKC inhibition or downregulation and Ca²⁺ buffering on DNP-stimulated glucose transport occurred at a signaling step proximal to GLUT-4 translocation rather than at the level of GLUT-4 vesicle docking and fusion or by a direct effect on glucose transporter activity. Consistent with a role for PKC stimulation of GLUT-4 vesicle translocation, numerous early studies report that agents that activate PKC can stimulate exocytosis in a variety of cell types (26, 28). Billiard et al. (6) observed that the exocytosis of secretory vesicles in rat pituitary gonadotropes could be stimulated independently by either Ca²⁺ elevations or PKC activation with PMA. Because the stimulation of glucose uptake by DNP involves incorporation of GLUT-containing vesicles into the cell surface (57), the participation of cPKC in this step is a distinct possibility.

Other potential mediators. The alternative pathway appears to involve Ca²⁺-dependent and Ca²⁺-independent signals, since Ca²⁺ chelation could not fully inhibit DNP-stimulated glucose transport. From our studies, the origin and nature of the Ca²⁺-independent pathway is not evident, but it does not include the type 1A PI3K-Akt axis or other wortmannin-sensitive PI3Ks. This pathway probably also does not include PKC-, since inhibition of all known subfamilies of PKC with 10 µM BIM did not further inhibit DNP-stimulated glucose transport even though it reduced insulin-stimulated glucose transport, which has been linked in part to a requirement for PKC- (2, 3) (Fig. 4A). It is noteworthy that activation of PKC- by insulin probably occurs via PI3K lipid products (2, 54).

A major unresolved issue is whether other signaling molecules, in addition to cPKC, are responsible for mediating the effect of DNP on glucose transport. Recently, it was proposed that 5'-AMP-activated protein kinase (AMPK) may be involved in hypoxia- or exercise-stimulated glucose transport but not in the insulin-dependent pathway (22, 52). Given that metabolic stressors such as DNP are known activators of AMPK, we tested this hypothesis by chemically activating AMPK with the AMP analog 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Maximal AICAR-stimulated glucose transport was not additive to insulin- or DNP-dependent glucose uptake in L6 muscle cells (Khayat and Klip, unpublished observations). Therefore, the participation of AMPK in the action of DNP on glucose transport is not likely. Other signals proposed to lead to glucose transport stimulation during muscle contraction are nitric oxide (NO) (51) and bradykinin, acting through the trimeric G protein G_q (31). Whether these signals are implicated in DNP action is not presently known. Goodyear et al. (19) have shown that exercise, a physiological stressor, can activate the stress-activated mitogen-activated protein kinase (MAPK) p38^MAPK in rat skeletal muscle. Similarly, we have shown rapid phosphorylation of p38^MAPK by DNP in L6 muscle cells (55). However, DNP-stimulated glucose uptake is likely not dependent on p38^MAPK activity, since the selective p38^MAPK inhibitor SB-203580 failed to prevent the DNP response of glucose transport (Khayat and Klip, unpublished observations).

Three recent studies have used two chemical conditions that stimulate glucose transport independently of insulin signals to elucidate the molecular mechanisms underlying these distinct pathways. These are hyperosmolarity and guanosine 5'-O-(3-thiotriphosphate) (GTPS). In 3T3-L1 adipocytes, osmotic shock- and GTPS-mediated elevations in GLUT-4 translocation are PI3K independent but are prevented by inhibitors of tyrosine kinases (10) or by microinjection of anti-phosphotyrosine antibodies (16, 21), suggesting that as yet unidentified tyrosine kinases may be activated by these stimuli and participate in glucose transport stimulation. In a previous study, we reported that treatment of L6 cells with DNP does not alter the pattern of tyrosine-phosphorylated proteins of myotube lysates assayed by immunoblotting with phosphotyrosine-specific antibodies (57). Indeed, we have tested three structurally unrelated tyrosine kinase inhibitors, erbstatin (30 µg/ml), genistein (50 µM), and herbimycin A (50 µM), for inhibitory effects on DNP-stimulated glucose transport. None were able to reduce the DNP stimulation of 2-deoxyglucose uptake (DNP, 100%; DNP + erbstatin pretreatment, 93.4%; DNP + herbimycin A pretreatment, 89.2%; DNP + genistein pretreatment, 89.5%), whereas insulin-dependent glucose uptake was blocked by all three agents (Khayat and Klip, unpublished results). Therefore, it is not likely that mitochondrial uncoupling engages tyrosine kinase signaling pathway(s) similar to those of these other activators of glucose transport.

In summary, the findings presented suggest that DNP may employ Ca²⁺ as a secondary messenger to activate cPKCs, forming part of the alternative signaling system leading to the regulation of glucose transport by energy demand in L6 muscle cells. This alternative pathway functions independently of the PI3K signaling pathway utilized by insulin to increase muscle cell glucose influx.

	ACKNOWLEDGEMENTS

We thank Dr. Philip J. Bilan for useful discussions.

	FOOTNOTES

This work was supported by grants from the Canadian Diabetes Association and the Eli Lilly/Banting and Best Diabetes Centre (to A. Klip). Z. Khayat was supported by a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship. T. Tsakiridis was supported by a fellowship from the Medical Research Council of Canada. A. Ueyama received financial support from the Otsuka Pharmaceutical Co., Ltd.

Current address of T. Tsakiridis: Department of Medicine, University of Toronto, Toronto, ON, M5S 1A8, Canada.

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. §1734 solely to indicate this fact.

Address for reprint requests: A. Klip, Programme in Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8.

Received 15 June 1998; accepted in final form September 1 1998.

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