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J. Biol. Chem., Vol. 278, Issue 32, 30015-30021, August 8, 2003
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From the
Pacific Northwest Research Institute,
Seattle, Washington 98122 and the Departments of
Pharmacology and
¶Medicine, University of Washington, Seattle,
Washington 98195
Received for publication, March 12, 2003 , and in revised form, May 15, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The adverse effects of chronic fatty acids on beta cell function are dependent upon the presence of elevated glucose levels (reviewed in Ref. 3). Our working hypothesis, initially proposed by Prentki and Corkey (8), is that in the presence of physiological concentrations of glucose, fatty acids are readily oxidized in the mitochondria. In contrast, when glucose and fatty acid levels are simultaneously elevated, glucose inhibits fatty acid oxidation, which results in cytosolic accumulation of long-chain fatty-acyl coenzyme A (LC-CoA) and complex lipid synthesis (5, 9, 10). Indeed, cytosolic LC-CoA can be esterified into phospholipids and triglycerides (TG), or, in the case of palmitate, serve as precursor for ceramide synthesis. The lipid species responsible for the deleterious effects of fatty acids on beta cell function are unknown. It has been initially proposed that intracellular accumulation of TG might play an important role, because the occurrence of diabetes in the Zucker diabetic fatty (ZDF) rat is associated with a dramatic increase in islet TG content (11, 12). Recently, we have shown that forcing TG accumulation in isolated islets by overexpressing the TG synthesizing-enzyme diacylglycerol-acyltransferase-1 impairs insulin secretion, but does not affect preproinsulin mRNA levels (13), suggesting that TG accumulation is associated with, but not causally related to, decreased insulin gene expression. On the other hand, intracellular ceramide accumulation has been implicated in fatty acid-induced apoptosis in ZDF rat islets (14, 15). Thus, de novo ceramide formation is increased in islets from ZDF rats (14), and fatty acid-induced apoptosis is blocked by inhibitors of ceramide synthesis (15). However, the potential role of ceramide synthesis in the effects of palmitate on the insulin gene has not yet been examined.
This study was therefore aimed to determine, in primary rat islets, 1) whether palmitate affects preproinsulin mRNA stability and/or insulin promoter activity, and 2) whether ceramide synthesis is implicated in palmitate inhibition of insulin gene expression.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-32P]UTP and
[
-32P]ATP were from Amersham Biosciences. Propidium iodide
(PI) was from Molecular Probes (Eugene, OR). Interleukin-1
(IL-1) was
from R&D Systems (Minneapolis, MN). C18-ceramide was from Avanti Polar
Lipids (Alabaster, AL). C2-ceramide and fumonisin B1 were from Biomol
(Plymouth Meeting, PA). Collagenase, actinomycin D, diacylglycerol (DAG)
kinase, palmitic acid (sodium salt), fatty-acid-free bovine serum albumin
(BSA), L-cycloserine, myriocin, and all other reagents (analytical
grade) were from Sigma. Silica gel 60 thin-layer chromatography (TLC) plates
were from Whatman (Clifton, NJ). Animals—Six-week-old male Wistar rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Animals were housed on a 12-h light/dark cycle with free access to water and standard laboratory chow. All procedures using animals were approved by the Pacific Northwest Research Institute Animal Care and Use Committee.
Rat Islet Isolation and Culture—Rat islets were isolated by collagenase digestion as described (5). After an overnight culture in RPMI 1640 containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 11.1 mM glucose, islets were resuspended in fresh media and incubated in various experimental conditions as described under "Results." Preparation of culture media containing palmitate was as described (5). The total palmitate concentration ranged from 0.1 to 0.5 mM, and the final fatty acid-free BSA concentration was 0.1 mM. Therefore, the molar ratio of palmitate/BSA ranged from 1:1 to 5:1. All control conditions contained the same amount of BSA and vehicle (EtOH/H2O, 1:1, v/v) as those with palmitate.
PI Staining and Fluorescence-activated Cell Sorter Analysis— Batches of 100 islets each were washed with phosphate-buffered saline (PBS) and incubated in 300 µl of 0.25% trypsin/1 mM EDTA for 5 min at 37 °C followed by gentle mechanical dispersion through a 1-ml pipette tip. The reaction was stopped by addition 700 µl of cold PBS, and islet cells were then collected by centrifugation and resuspended in 500 µl of PBS. PI was added to a final concentration of 6 µg/ml, and the cells were incubated in the dark at 4 °C for 30 min. Single-cell populations, defined based on size on a forward versus size scatter, were analyzed for PI exclusion using a Coulter EpicsXL flow cytometer (Coulter Corp., Miami, FL).
Ribonuclease Protection Assay (RPA)—RPAs were carried out
using the Direct Protect Lysate RPA kit (Ambion, Austin, TX). Briefly, batches
of 100 cultured islets were washed twice in Hank's balanced salt solution,
resuspended in 150 µl of lysis buffer (provided in the kit), and sonicated
4 x 1 s at low power. [-32P]UTP-labeled antisense
probes were transcribed from a T7 promoter to a 360-bp sequence for the rat II
preproinsulin gene cloned into a pBLUEscript plasmid (Stratagene Inc., La
Jolla, CA). Control probes were made to a 316-bp conserved sequence of mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion) and a 245-bp sequence
of mouse -actin (Ambion). Probes were hybridized directly in the lysate
overnight at 42 °C. The lysate was then treated as directed by the
manufacturer, and protected fragments were resolved by 5% denaturing gel
electrophoresis and analyzed by autoradiography and phosphorimaging. The
probes were determined to be in excess for each experiment.
Generation of Recombinant Adenoviruses and Transient Transfections—A 298-bp fragment of the rat I insulin promoter (corresponding to bases –310 to –12 relative to the transcription start site) was used to drive transcription of an RNA carrying the untranslated regions of the rat preproinsulin II mRNA and encoding firefly luciferase. This construct was subcloned into pShuttle (Qbiogene, Carlsbad, CA) and used to construct a recombinant adenovirus (Ad-RIP1-Luc) as described (13). A control adenovirus encoding firefly luciferase under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Luc) was used as a control (16). Duplicate batches of 100 islets each were infected overnight with 107 plaque-forming units per islet of Ad-RIP1-Luc or Ad-CMV-Luc in RPMI 1640 containing 10% fetal bovine serum and 11.1 mM glucose. Islets were then cultured in RPMI 1640 containing 10% fetal bovine serum and 2.8 mM glucose for 8 h, to lower RIP1 activity to basal values. Islets were then transferred to fresh RPMI 1640 containing 0.1% BSA and various glucose and palmitate concentrations for 24 h. Luciferase activity was measured in islet extracts using the Luciferase Assay kit (Promega, Madison, WI) according to the manufacturer's instructions.
Immunostaining and Confocal Microscopy—Islets were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.1% saponin in PBS for 30 min at room temperature, blocked with 10% donkey serum in PBS for 30 min at room temperature, and incubated with a rabbit anti-firefly luciferase antibody (Cortex Biochem, San Leandro, CA) and a guinea pig anti-bovine insulin antibody (Sigma; 1:100 dilution) overnight at 4 °C in 5% BSA in PBS. After washing in PBS, islets were incubated with a Cy2-conjugated donkey anti-rabbit immunoglobulin and a Cy5-conjugated donkey anti-guinea pig immunoglobulin (both from Jackson Immunoresearch, West Grove, PA; 1:300 dilution) for1hat room temperature in 5% BSA in PBS. After washing, islets were mounted on slides and examined with a Fluoview 500 Olympus confocal microscope.
Determination of Ceramide Content—Batches of 100 islets each
were washed in ice-cold PBS and lipids were extracted by chloroform/methanol
(1:2, v/v). Extracted lipids were dried under N2, and ceramide
content was measured by the DAG kinase assay and TLC analysis as originally
described by Preiss et al.
(17) and optimized for
ceramide determination (18,
19). Dried lipids were
solubilized in 20 µl of detergent solution (7.5%
n-octyl--glucopyranoside/5 mM cardiolipin in 1 mM
diethylenetriamine-penta-acetic acid (DETAPAC)). After adding 50 µl of
assay buffer (120 mM HEPES/100 mM LiCl/25 mM
MgCl2/2 mM EGTA), 10 µl of 20 mM
dithiothreitol and 10 µl of 1:1 diluted DAG kinase/enzyme diluent (1
mM DETAPAC/10 mM imidazole), the reaction was started by
adding 10 µl of 10 mM [-32P]ATP (specific
activity: 80 mCi/mmol) prepared in 100 mM imidazole/1 mM
DETAPAC. After mixing, the reaction was continued for 30 min at 30 °C.
Lipids were extracted again and dried under N2. Samples were
resuspended in 100 µl of chloroform, spotted onto TLC plates, and developed
with chloroform/methanol/acetone (65:15:5, v/v/v). The radioactive spot
corresponding to ceramide-1-phosphate was identified using autoradiography,
scraped, and quantified by scintillation counting. Ceramide standards were run
on each plate. Linear responses were obtained between 0 and 2500 pmol.
Expression of Data and Statistics—Data are expressed as mean ± S.E. Intergroup comparisons were performed by Student's paired t test or analysis of variance with post-hoc Dunnet t test, where appropriate. p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Palmitate Decreases Preproinsulin mRNA Levels—Previously (4), we have shown that a 72-h exposure of isolated rat islets to 0.5 mM palmitate does not affect preproinsulin mRNA levels in the presence of 2.8 mM glucose but significantly decreases it in the presence of 16.7 mM glucose. Batches of 100 isolated islets were cultured in RPMI 1640 with either 2.8 or 16.7 mM glucose plus 0, 0.3, or 0.5 mM palmitate (fatty-acid-to-BSA ratio of 0, 3:1, and 5:1, respectively) for 72 h. Steady-state levels of preproinsulin mRNA were measured by RPA (Fig. 2). GAPDH mRNA was measured as a control for loading variations between lanes, and the results are expressed as the ratio of preproinsulin/GAPDH mRNA. As expected, the presence of high glucose led to a significant increase in preproinsulin mRNA levels (3.6 ± 0.6-fold increase, n = 5, p < 0.05). At the concentration of 0.5 mM, palmitate decreased preproinsulin mRNA levels to 29.2 ± 5.8% (n = 5, p < 0.0001) of control values.
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Palmitate Does Not Affect Preproinsulin mRNA Stability— Because steady-state mRNA levels are the product of the rate of mRNA synthesis versus the rate of mRNA decay, palmitate inhibition of preproinsulin mRNA levels could be due to decreased transcription, decreased mRNA stability, or both. To determine the effects of palmitate on preproinsulin mRNA stability, we measured preproinsulin and GAPDH mRNA levels 0, 6, 12, 24, 30, and 36 h after addition of actinomycin D (5 µg/ml) in the absence or presence of 0.5 mM palmitate. The half-life of preproinsulin and GAPDH mRNA was estimated from the slope of the best-fit curve of mRNA level (expressed in percent of time 0) as a function of time (Fig. 3). As expected, glucose significantly increased the half-life of preproinsulin mRNA from 27.7 ± 1.5 h at 2.8 mM to 40.8 ± 3.0 h at 16.7 mM (n = 3, p < 0.05). In contrast, the presence of 0.5 mM palmitate did not affect preproinsulin mRNA stability, either at 2.8 (27.2 ± 3.1 versus 27.7 ± 1.5 h, n = 3, NS) or 16.7 (39.0 ± 2.0 versus 40.8 ± 3.0 h, n = 3, NS) mM glucose. Neither glucose nor palmitate had an effect on GAPDH mRNA half-life (not shown). These results suggested that fatty acid inhibition of preproinsulin mRNA is not due to changes in mRNA half-life, and prompted us to investigate insulin promoter activity.
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Palmitate Decreases Glucose-stimulated Rat Insulin Promoter Activity—As shown in Fig. 4, luciferase immunoreactivity was detected in the vast majority of islet cells after infection with Ad-CMV-Luc (panels A–C) or Ad-RIP1-Luc (panels D–F). Glucose increased RIP1-Luc activity in a dose-dependent manner (ANOVA, n = 2–7 replicates for each glucose concentration, p < 0.0005; Fig. 5A). RIP1-Luc activity was 7.8 ± 1.4-fold higher at 16.7 mM glucose than at 2.8 mM glucose (n = 7, p < 0.0001; Fig. 5A). CMV-Luc activity was also slightly augmented by glucose, although this effect was not statistically significant (ANOVA, n = 2–7 replicates for each glucose concentration, NS; Fig. 5A). The ratio of RIP1-Luc over CMV-Luc was 3.8 ± 0.5-fold higher at 16.7 mM glucose than at 2.8 mM glucose (n = 7, p < 0.0001). The stimulation of RIP1-Luc activity by glucose was not due to a nonspecific osmolar effect, because an equivalent concentration of mannitol was ineffective (not shown). The response of the RIP1-Luc reporter to glucose was then tested in the absence or presence of 0.5 mM palmitate (Fig. 5B). The presence of palmitate did not affect basal RIP1-Luc activity at 2.8 mM glucose (94.3 ± 16.7% of the control without palmitate, n = 3, NS), but markedly blunted the response to glucose, which was no longer statistically significant (ANOVA, n = 3, NS). Palmitate decreased RIP1-Luc activity stimulated by 16.7 mM glucose in a dose-dependent manner (ANOVA, n = 4, p < 0.005; Fig. 5C).
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Palmitate Increases Ceramide Content in Isolated Islets— Palmitate is a substrate for de novo ceramide synthesis, which involves condensation of palmitoyl-CoA with L-serine by the enzyme serine palmitoyltransferase (20). Culture of ZDF rat islets in the presence of fatty acids results in increased ceramide generation via de novo synthesis (14, 15). Whether prolonged exposure of islets from normal animals to palmitate is associated with increased ceramide content has not been directly assessed. Here we show that the presence of 16.7 mM glucose without palmitate did not increase ceramide content (0.89 ± 0.26 versus 0.89 ± 0.30 pmol/islet, n = 6, NS; Fig. 6A), but that palmitate increased ceramide levels (ANOVA, p < 0.05; Fig. 6, A and B). At the fatty-acid concentration of 0.5 mM, ceramide content was increased to 417 ± 117% of control values by palmitate (n = 7, p < 0.005; Fig. 6B). Addition of the inhibitors of de novo ceramide synthesis fumonisin B1 (which inhibits ceramide synthase; 50 µM) and myriocin (which inhibits serinepalmitoyl transferase; 50 nM) (20) to the culture medium significantly inhibited ceramide generation induced by palmitate (both n = 7, NS from control in the absence of palmitate, Fig. 6, C and D). This suggests that palmitate induces ceramide generation at least in part via de novo synthesis.
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Inhibition of de Novo Ceramide Synthesis Prevents Palmitate Impairment of Insulin Gene Expression—First, we observed that exposure of islets for 72 h to the cell-permeable analogue C2-ceramide decreased preproinsulin mRNA levels (Fig. 7A). We then sought to ascertain whether inhibition of de novo ceramide formation could prevent the decrease in preproinsulin mRNA levels observed after culture with palmitate. Preproinsulin mRNA levels were measured after culture for 72 h in the presence of 16.7 mM glucose and 0.5 mM palmitate with or without 50 nM myriocin, 50 µM fumonisin B1, or 2 mM Lcycloserine (an serine-palmitoyl transferase inhibitor). Fig. 7, B and C, show representative experiments with the three inhibitors. Both fumonisin B1 (Fig. 7B) and L-cycloserine (Fig. 7C) partially prevented palmitate inhibition of preproinsulin mRNA levels. Myriocin was the most potent inhibitor and completely prevented the decrease in preproinsulin mRNA levels induced by palmitate (Fig. 7, B and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Importantly, palmitate inhibition of insulin gene expression (Fig. 2) is not merely due to fatty acid-induced cell death, because the number of PI-positive cells after 72 h of exposure was unchanged (Fig. 1). The lack of effect of palmitate on cell viability appears in conflict with previous studies in rat islet cells (21, 22). However, the study by Cnop et al. (21) used purified rat beta cells, which might be more susceptible to the effects of palmitate, and Maedler et al. (22) investigated the effect of palmitate after 4 days of exposure. In our study, the rationale for assessing the effects of palmitate on PI staining was not to fully investigate palmitate-induced apoptosis, but only to exclude, under our culture conditions, effects on cell viability that could have accounted for the observed decrease in preproinsulin mRNA levels.
The effect of palmitate on preproinsulin mRNA is consistent with previous studies by us (4, 5) and others (6, 7). Our experiments using actinomycin D confirmed in rat islets that glucose increases preproinsulin mRNA half-life, as originally shown by Welsh et al. (23) in RINm5f cells. Palmitate, however, did not affect preproinsulin mRNA decay at either low or high glucose (Fig. 3). These results contrast with those of Ritz-Laser et al. (7), who observed that 2 mM palmitate decreased preproinsulin mRNA half-life in MIN6 cells. This discrepancy might be due to differences between cell lines and primary islets, or to the fact that the concentration of palmitate in our experiments was much lower (0.5 mM). Transcriptional regulation of insulin promoter activity was assessed in primary islets using an adenoviral-mediated reporter gene assay. Glucose specifically and dose-dependently increased insulin promoter activity (Fig. 5), consistent with previous studies in fetal islet cells (24) and intact adult islets (25). Palmitate did not affect basal insulin promoter activity, but strongly inhibited its stimulation by glucose (Fig. 5). These results establish for the first time that palmitate inhibits insulin promoter activity in primary islets. They are consistent with our previous observations in HIT-T15 cells (4) and those of Ritz-Laser et al. (7) in MIN6 cells. They are also consistent with the finding that palmitate inhibits glucose-stimulated, but not basal, preproinsulin mRNA levels (4, 5, 7), and support our hypothesis that fatty acids affect beta cell function only in the context of chronic hyperglycemia (2). There are several examples in mammalian cells of transcriptional regulation by fatty acids (26). In the pancreatic beta cell, prolonged fatty acids increase expression of the carnitine palmitoyltransferase 1 gene (27) and inhibit expression of the genes encoding acetyl-CoA carboxylase (28) and PDX-1 (6). In addition, expression profiling studies using microarrays have identified a number of genes modulated by fatty acids in beta cells, with an overall up-regulation of genes of fatty-acid oxidation and down-regulation of lipogenic genes (29, 30). Negative regulation of PDX-1 (6) is probably a mechanism underlying the fatty-acid inhibition of insulin gene transcription observed in our study. Whether this also involves alterations in the expression or binding activity of other glucose-responsive transactivating factors remains to be determined.
The nature of the downstream effectors that mediate fatty acid inhibition of insulin gene expression in the presence of high glucose are unknown. The possibility has been raised that TG accumulation might indeed be the mechanism underlying these effects, because TG accumulation precedes the development of diabetes in islets from ZDF rats (11, 12), and TG depletion reverses the diabetic phenotype (31–33). In isolated rat islets, we found an inverse correlation between the levels of preproinsulin mRNA and TG content (5). To directly test the role of TG accumulation in fatty-acid impairment of beta cell function, we overexpressed the last and only dedicated enzyme of triglyceride synthesis, diacylglycerol-acyltransferase-1, in isolated islets by adenoviral-mediated transduction (13). Diacylglycerol-acyltransferase-1 overexpression inhibited glucose-induced insulin secretion after prolonged culture in elevated glucose, but preproinsulin mRNA levels were unaffected (13). These results led us to hypothesize that the mechanisms underlying fatty-acid impairment of gene expression are distinct from those responsible for the decrease in insulin secretion and do not directly involve TG accumulation. Here we investigated the alternative possibility that negative regulation of insulin gene expression by fatty acids might involve ceramide synthesis. This hypothesis is supported by observations by the group of Unger in the ZDF rat, in which fatty acid induced beta cell dysfunction and death is associated with intracellular generation of ceramide (14, 15), and by the fact that inhibitors of de novo ceramide synthesis prevent, at least in part, the pro-apoptotic effects of fatty acids in beta cells (22, 34). The observation that ceramide content is increased by palmitate and blocked by inhibitors of de novo synthesis (Fig. 6) is consistent with our hypothesis. Ceramide has been implicated as a potential mediator of cytokine-induced beta cell death (35–37), although some reports have been controversial (19, 38). However, the functional role of ceramide in beta cells has, to our knowledge, not been investigated, and our results represent the first demonstration of a role for ceramide as a mediator of palmitate inhibition of insulin gene expression (Fig. 7), in the absence of any noticeable cell death. Although a potential nonspecific effect of the inhibitors of ceramide synthesis used in this study cannot be completely ruled out, our conclusion is supported by the facts that the analogue C2-ceramide decreases insulin mRNA levels, and that three structurally different inhibitors, which act on two different enzymes of ceramide synthesis, prevented palmitate inhibition of insulin gene expression. The time course of ceramide increase in the presence of palmitate was not investigated in these studies because of the limited sensitivity of the ceramide measurement from primary islets. However, de novo generation of detectable ceramide levels is thought to require several hours after exposure to the stimulus (39), consistent with the long-term effects of palmitate on the insulin gene.
In addition to its well defined role as a mediator of apoptosis, ceramide modulates a number of signaling pathways and has a variety of functional effects in mammalian cells (39). Several mechanisms can be postulated to explain ceramide inhibition of insulin gene transcription. One of the possible candidates is the stress-activated protein kinase c-jun N-terminal kinase (JNK), which is activated by ceramide (39) and inhibits insulin gene transcription both via c-jun-dependent (40, 41) and -independent (42) pathways. The possible involvement of JNK is currently under investigation in our laboratory. Ceramide has also been shown to activate the extracellular signal-regulated kinase pathway (43), which, on the other hand, might play a role in glucose-regulation of insulin gene transcription (44). The atypical isoform of protein kinase C zeta is also a target for ceramide (45) and has been implicated in transcriptional regulation of the insulin gene (46). Finally, de novo ceramide synthesis mediates palmitate inhibition of protein kinase B in muscle cells (47). Therefore, our results suggest that ceramide modulates one or several signaling pathways implicated in the transcriptional regulation of the insulin gene by glucose. Identification of the targets of ceramide in the beta cell and of the glucose-responsive transactivating factors whose binding activity is impaired by ceramide synthesis requires further investigation.
In conclusion, our results uniquely demonstrate that fatty acids impair insulin gene expression by inhibiting insulin promoter activity in primary rat islets, without affecting preproinsulin mRNA stability. Further, our data establish that palmitate inhibition of insulin gene expression is mediated by de novo ceramide generation in the beta cell. The mechanisms whereby ceramide affects insulin gene transcription remain to be determined, but our results raise the intriguing possibility that interfering with the ceramide pathway, which is being considered as a promising option for the treatment of certain cancers, coronary disease, and immune disorders (48), may also be a strategy to prevent the deterioration of beta cell function in type 2 diabetes.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122. Tel.: 206-860-6755; Fax: 206-726-1217; E-mail: vpoitout{at}pnri.org.
1 The abbreviations used are: PDX-1, pancreatic and duodenal homeobox-1; BSA,
bovine serum albumin; CMV, cytomegalovirus; DAG, diacylglycerol; DETAPAC,
diethylenetriamine-penta-acetic acid; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase, JNK, c-jun N-terminal kinase; IL-1, interleukin-1;
LC-CoA, long-chain fatty-acyl coenzyme A; NS, not significant; PBS,
phosphate-buffered saline; PI, propidium iodide; RPA, ribonuclease protection
assay; TLC, thin-layer chromatography; TG, triglycerides; ZDF, Zucker diabetic
fatty; Luc, luciferase; Ad, adenovirus; ANOVA, analysis of variance.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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