INTRODUCTION

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THE FOUR MAMMALIAN HEXOKINASE (HK) family members catalyze the phosphorylation of glucose to glucose 6-phosphate. HK I is relatively ubiquitous, whereas HK II, III, and IV display more limited tissue distribution. HK II is primarily expressed in insulin-sensitive tissues (skeletal and cardiac muscle, adipose tissue), HK III is expressed in the liver and vascular endothelial cells, and HK IV (glucokinase) is expressed primarily in the liver and pancreatic  cells (30). Whereas both HK I and HK II are expressed in skeletal muscle, HK II constitutes the vast majority of total HK activity in this tissue (30). Glucose phosphorylation plays an essential role in skeletal muscle glucose uptake. Because muscle glucose transport is mediated by facilitated transporters that are able to move glucose both into and out of cells, the rapid phosphorylation of glucose is critical for maintaining net inward transport of glucose. As alterations in HK II activity are able to influence the rate of exercise- and insulin-stimulated muscle glucose uptake in vivo (9), understanding the regulation of HK II expression is important.

The skeletal muscle HK II gene is highly sensitive to alterations in contractile activity. As little as 30-60 min of moderate-intensity exercise results in an increase in HK II mRNA and protein in both rat (15) and human (12) skeletal muscle. An increased rate of gene transcription after exercise accounts for the increased HK II mRNA in rats (14). The molecular link between muscle contraction and increased expression of the HK II gene has not been determined. Experiments in which muscle contractions are generated by electrical stimulation suggest that the signal leading to the increase in HK II is intrinsic to the contracting muscle, as these interventions do not generally result in major hormonal alterations, and the increases are specific to contracting muscle (10, 27, 28). Because alterations in intracellular Ca²⁺ homeostasis may regulate the expression of certain genes in contracting skeletal muscle (4, 11, 24), the ability of Ca²⁺ to increase HK II mRNA was investigated.

Transient increases in cytosolic Ca²⁺ occur in muscle in response to sarcolemmal depolarization, resulting in interaction between myosin and actin filaments and muscle contraction. The cytosolic Ca²⁺ concentration varies from <100 nM in resting muscle to ~1-2 µM in contracting cells (1, 29). The primary source of the increased cytosolic Ca²⁺ is the sarcoplasmic reticulum (SR), a tubular membranous network containing high concentrations of Ca²⁺ that surround the myofibrils (21). After each contraction, active reuptake of Ca²⁺ back into the SR occurs through a Ca²⁺-ATPase pump located in the SR membrane. Although the role of Ca²⁺ in mediating contraction has been well characterized, the potential role of Ca²⁺ in regulating skeletal muscle gene expression has received less attention (11, 24). Fluctuations of cytosolic Ca²⁺ have been implicated in the alteration of gene expression in a number of cell types. Several mechanisms appear to be involved in this process (7).

One difficulty encountered in attempting to study the role of Ca²⁺ in skeletal muscle gene regulation is the inability to manipulate intramuscular Ca²⁺ concentrations in vivo independently of muscle contraction. For this reason, these experiments were performed on a cultured muscle cell line, L6 myotubes (17). The purpose of this project was to determine whether alterations of Ca²⁺ homeostasis in L6 cells increase HK II mRNA and, if so, to determine the mechanism by which this increase occurs.

	MATERIALS AND METHODS

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L6 cell maintenance and differentiation. L6 myoblasts (obtained from Robert J. Smith, Joslin Diabetes Center, Boston, MA) were grown in 100-mm dishes to near confluency in DMEM supplemented with 10% fetal calf serum. To induce differentiation into myotubes, cells were maintained for 3-4 days in DMEM supplemented with 2% horse serum, 2 nM triiodothyronine, and 20 nM insulin, as described previously (20). Cells were placed in serum-free DMEM 24 h before the experiments. DMEM contains 1.8 mM Ca²⁺.

Experimental methods. Total cellular RNA was isolated from L6 cells by using Tri Reagent (Molecular Research Center, Cincinnati, OH). RNA was quantified by measuring the absorbance at 260 nm, and integrity was assessed by visual inspection of the 18S and 28S RNA bands on an ethidium bromide-stained formaldehyde-agarose gel. RNase protection assays (RPAs) were performed by using the RPA I kit (Ambion, Austin, TX) on 10 µg of total cellular RNA. Advantages of the RPA include high sensitivity and the ability to quantify more than one mRNA simultaneously, allowing for an internal control for each sample. Antisense probes labeled with [-³²P]UTP (Amersham Pharmacia Biotech, Piscataway, NJ) were generated from linearized plasmids containing fragments of HK I cDNA or HK II cDNA downstream from the T7 promoter and transcribed with the Maxiscript T7 kit (Ambion). As described previously, the protected fragment lengths for HK I and HK II are 396 and 247 nucleotides, respectively (15, 20). Protected fragments were electrophoresed through a denaturing gel, dried, and exposed to film at 70°C. Autoradiographs were scanned, and the density of bands was determined by using Collage (Fotodyne).

	RESULTS

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HK I mRNA is unaltered by treatment with A-23187, EGTA, and BAPTA-AM. HK I and HK II mRNA were measured in all experiments. None of the interventions used in these experiments altered the expression of HK I mRNA. Shown in Fig. 1 is a representative autoradiograph showing the lack of an effect of 500 nM A-23187 on HK I mRNA expression. This same intervention resulted in an increase in HK II mRNA, as discussed below. The fact that HK I mRNA is not altered by these interventions is important for two reasons. First, it is evident that the effects described in subsequent sections are not simply global effects on mRNA transcription or stability. Second, this allowed us to use HK I mRNA as an internal control for each sample in each experiment and to normalize for differences in the RPA between samples.

View larger version (66K): [in this window] [in a new window]   Fig. 1.   Representative autoradiograph showing that treatment of L6 cells with A-23187 increases hexokinase (HK) II mRNA without altering HK I mRNA expression. Cells were incubated with 500 nM A-23187 for the length of time indicated before isolation of RNA. mRNA was quantitated by the RNase protection assay (RPA), as described in MATERIALS AND METHODS. HK II mRNA was increased by A-23187, as described later in RESULTS. Protected HK I and HK II mRNA fragments are indicated by arrows.

A-23187 increases HK II mRNA in a dose- and time-dependent manner. L6 myotubes were incubated with A-23187 to investigate the role that alterations in Ca²⁺ homeostasis play in the regulation of the HK II gene. The divalent cation ionophore A-23187 increases cytosolic Ca²⁺ and decreases the concentration of Ca²⁺ in intracellular stores (5). Whereas cytosolic Ca²⁺ concentration was not measured in these experiments, it is predicted from previous studies that treatment of cells with the highest A-23187 concentrations used in the present study results in a transient increase in intracellular Ca²⁺ concentration [to ~2 µM (16)], followed by a steady-state increase of ~50% over basal concentrations (5). Shown in Fig. 2 are the results of 6 h of incubation of L6 cells in media containing 0, 50, 100, 500, and 1,000 nM A-23187 on HK II mRNA. The increases in HK II mRNA above the control value were 1.8 ± 0.2-, 2.1 ± 0.2-, 3.7 ± 0.7-, and 3.5 ± 0.8-fold with increasing concentrations of A-23187 [all significantly increased above basal (P < 0.05)].

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of increasing concentrations of A-23187 on HK II mRNA in L6 cells. Cells were incubated with A-23187 for 6 h before isolation of RNA. The mRNA was quantitated by the RPA as described in MATERIALS AND METHODS. Data are presented as means ± SE of 3 separate treatments at each ionophore concentration. Significant increase in HK II mRNA compared with the untreated control, * P < 0.05.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effect of increasing duration of A-23187 exposure on HK II mRNA content in L6 cells. Cells were incubated with 500 nM A-23187 for length of time indicated before isolation of RNA. mRNA was quantitated by RPA as described in MATERIALS AND METHODS. Data are presented as means ± SE of 3 separate treatments at each duration. Significant increase in HK II mRNA compared with the untreated control, * P < 0.05.

Effect of incubation of L6 myotubes with EGTA, an extracellular Ca²⁺ chelator. The induction of HK II mRNA by A-23187 could be due to either the increase in cytosolic Ca²⁺ or the depletion of intracellular Ca²⁺ stores (i.e., SR). The combined effect of EGTA and A-23187 was investigated to determine whether a rise in intracellular Ca²⁺ was necessary to observe the A-23187 effect. HK II mRNA was unchanged by a 6-h incubation with EGTA, even at a 10:1 EGTA-to-media Ca²⁺ molar ratio, indicating that removal of extracellular Ca²⁺ did not affect HK II mRNA (Fig. 4). EGTA also did not affect the response of HK II mRNA to A-23187, as the increase in HK II mRNA was similar to that noted in the absence of EGTA (Fig. 4). A-23187 functions by permeabilizing membranes to Ca²⁺; therefore, when the medium Ca²⁺ concentration is lower than the Ca²⁺ concentration in the cytosol, due to its chelation by EGTA, Ca²⁺ moves down the concentration gradient and exits cells. Therefore, the combination of A-23187 and EGTA reduces the Ca²⁺ concentration in both the SR and the cytosol compared with that observed when cells are incubated in DMEM alone.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Effect of the extracellular Ca2+ chelator EGTA on HK II mRNA in L6 cells. Cells were incubated with 500 nM A-23187, 10 mM EGTA, or both for 6 h before isolation of RNA. mRNA was quantitated by RPA as described in MATERIALS AND METHODS. Data are presented as means ± SE of 3 separate samples for each treatment. Significant increase in HK II mRNA compared with the untreated control, * P < 0.05.

Effect of incubation of L6 myotubes with BAPTA-AM, an intracellular Ca²⁺ chelator. BAPTA-AM is a membrane-permeable Ca²⁺-specific chelator. The presence of four AM groups on the compound allows BAPTA-AM to cross cellular membranes. Esterases located in the cytosol then cleave the AM groups, serving to trap the active chelator inside the cell where it binds Ca²⁺ and effectively decreases the cytosolic-free Ca²⁺ concentration by ~50% (19). In addition, by binding to Ca²⁺ that escapes from the SR through a slow leak current and preventing its active reuptake, incubation with BAPTA-AM slowly depletes SR Ca²⁺ stores as well (5). Six hours after loading of BAPTA-AM into L6 myotubes, HK II mRNA was significantly increased compared with basal (3.7 ± 0.3-fold; Fig. 5). The combination of BAPTA-AM and A-23187 resulted in a similar increase in HK II mRNA (3.8 ± 0.4-fold; Fig. 5), suggesting that these two compounds operate to increase HK II mRNA through similar mechanisms. The time course of the BAPTA-AM effect on HK II mRNA was similar to that of A-23187, as increases in HK II mRNA were not observed until 4 h after BAPTA-AM loading (data not shown). The finding that incubation of cells with BAPTA-AM, either in the presence or absence of A-23187, increases HK II mRNA serves as evidence that a depletion of intracellular Ca²⁺ stores, and not an increase in cytosolic Ca²⁺, is involved in the induction of HK II mRNA.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) on HK II mRNA in L6 cells. Cells were incubated with 500 nM A-23187, 50 µM BAPTA-AM, or both for 6 h before isolation of RNA. mRNA was quantitated by RPA as described in MATERIALS AND METHODS. Data are presented as means ± SE of 3-4 separate samples for each treatment. Significant increase in HK II mRNA compared with the untreated control, * P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results of these experiments indicate that the Ca²⁺ ionophore A-23187 is able to increase expression of HK II mRNA in L6 myotubes. This is due to the ability of A-23187 to deplete intracellular stores of Ca²⁺ and not due to an increase in the cytosolic Ca²⁺ concentration. This is supported by the observation that treatment of cells with intracellular and extracellular Ca²⁺ chelators that decrease the SR Ca²⁺ concentration also results in an increase of HK II mRNA.

A depletion of intracellular Ca²⁺ stores also has been demonstrated to increase the expression of two molecular chaperone proteins, glucose regulated protein (grp) 78 and grp94 (5, 13), that reside in the endoplasmic reticulum (ER). The link between intracellular Ca²⁺ depletion and increased grp78 and grp94 expression has been established in fibroblasts by using interventions similar to the ones described in this work in conjunction with measurements of intracellular Ca²⁺ (5). In addition, upregulation of grp78 and grp94 mRNA is observed after treatment of fibroblasts with the ER Ca²⁺-ATPase inhibitor thapsigargin. Thapsigargin prevents the reuptake of Ca²⁺ that leaks out of the ER by directly inhibiting the ER Ca²-ATPase (25) and effectively decreases ER Ca²⁺ content (13). The promoter of the grp78 gene has been characterized, and the putative Ca²⁺ depletion response elements were identified (5, 22). The CCAAT-binding factor [CBF, also called NF-Y or CP-1 (18)], binding to a CCAAT element located ~100 bases upstream of the grp78 transcription start site, is the primary mediator of the Ca²⁺ depletion-induced upregulation of grp78 (22). Interestingly, the HK II promoter also contains a CBF-binding CCAAT element located ~100 bases upstream of the transcription start site (18). Taken together, these findings suggest that a similar molecular mechanism may regulate the grp78 and grp94 genes in fibroblasts, and the HK II gene in L6 myotubes, in response to alterations of Ca²⁺ homeostasis.

In contrast to the apparent mechanism involved in the upregulation of the HK II gene by Ca²⁺, increases in intracellular Ca²⁺ concentration may also participate in the regulation of genes by contractile activity in muscle. Subunits of the acetylcholine receptor are rapidly downregulated in mouse and chicken skeletal muscle in response to contraction or an elevation in cytosolic Ca²⁺ concentration (11, 26). For this response to occur, the Ca²⁺ must enter cells from the extracellular medium and not from the SR (11). An elevation in intracellular Ca²⁺ has also been implicated in the increase in inwardly rectifying K⁺ channel 1 (IRK1) mRNA observed with contraction. This response is also observed when cultured muscle cells are incubated with ionophore or depolarizing concentrations of KCl, responses that are prevented when EGTA was present in the media (24). Interestingly, the IRK1 effects are not due to alterations in the rate of IRK1 transcription, but, instead, the Ca²⁺ ionophore increases the stability of the IRK1 transcripts (24). A third mechanism of Ca²⁺-dependent muscle gene regulation may be through activation of the Ca²⁺-sensitive serine/threonine phosphatase calcineurin (4). In this system, small, chronic elevations in intracellular Ca²⁺ (as exist in slow, oxidative muscle) signal through the calcineurin pathway, resulting in the upregulation of a number of slow-twitch muscle-enriched genes (4). The expression of many genes is either increased or decreased in response to acute or chronic exercise (for review, see Ref. 2). Interestingly, many different signal transduction pathways, most likely acting on the various processes of gene expression (ranging from gene transcription to protein stability), are used to regulate these muscle genes. This diversity would allow specific genes to be regulated with different time courses and varying magnitudes of change and could accommodate exercise-specific adaptations.

The demonstration that HK II mRNA is regulated by depletion of intracellular Ca²⁺ stores in cultured L6 myotubes does not necessarily mean that this is the mechanism by which the contraction-induced alterations occur. The degree of SR Ca²⁺ depletion in L6 myotubes necessary to invoke the increase in HK II mRNA cannot be determined from these experiments. It has been estimated that ~30% of SR Ca²⁺ is released during contractions of the perfused heart (3). Whether there is a decrease in SR Ca²⁺ content in fatigued muscle is controversial. It has been demonstrated that a decrease in the force of muscle contraction in electrical stimulation protocols correlates with decreased Ca²⁺ release from the SR (6, 29), and a decrease in the SR Ca²⁺ content has been reported (23). However, a decrease in the SR Ca²⁺ with fatigue has not been a universal finding (8). Whether a lower time-averaged SR Ca²⁺ concentration exists during or after prolonged exercise in vivo and whether this degree of Ca²⁺ depletion is sufficient to invoke alterations in HK II mRNA are questions that remain to be addressed.

In conclusion, the results reported here support the idea that depletion of intracellular Ca²⁺ stores in cultured L6 myotubes results in a specific increase of HK II mRNA. The same manipulations of Ca²⁺ homeostasis that increase HK II mRNA expression also increase expression of the grp78 and grp94 genes, suggesting that similar mechanisms may be involved in the regulation of these three genes. We speculate that depletion of intracellular Ca²⁺ stores is one mechanism by which muscle contraction increases HK II mRNA.