1 Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-0033; 2 Discovery Research Laboratory, Tanabe Seiyaku, Saitama 335-8505; and 3 Institute for Adult Disease, Asahi Life Foundation, Tokyo 160-0023, Japan
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ABSTRACT |
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To determine the contribution of hyperglycemia to the insulin resistance in various insulin-sensitive tissues of Zucker diabetic fatty (ZDF) rats, T-1095, an oral sodium-dependent glucose transporter (SGLT) inhibitor, was administered by being mixed into food. Long-term treatment with T-1095 lowered both fed and fasting blood glucose levels to near normal ranges. A hyperinsulinemic euglycemic clamp study that was performed after 4 wk of T-1095 treatment demonstrated partial recovery of the reduced glucose infusion rate (GIR) in the T-1095-treated group. In the livers of T-1095-treated ZDF rats, hepatic glucose production rate (HGP) and glucose utilization rate (GUR) showed marked recovery, with almost complete normalization of reduced glucokinase/glucose-6-phosphatase (G-6-Pase) activities ratio. In adipose tissues, decreased GUR was also shown to be significantly improved with a normalization of insulin-induced GLUT-4 translocation. In contrast, in skeletal muscles, the reduced GUR was not significantly improved in response to amelioration of hyperglycemia by T-1095 treatment. These results suggest that the contribution of hyperglycemia to insulin resistance in ZDF rats is very high in the liver and considerably elevated in adipose tissues, although it is very low in skeletal muscle.
glucose toxicity; Zucker diabetic fatty rat; sodium-dependent glucose transporter
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INTRODUCTION |
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IN NON-INSULIN-DEPENDENT diabetes mellitus (NIDDM),
insulin resistance is an initiating pathogenic mechanism, and when
pancreatic -cells fail to secrete enough insulin to overcome insulin
resistance, hyperglycemia becomes overt (38, 39, 40). However,
hyperglycemia is not only a consequence of, but also an important
factor in worsening both insulin resistance and insulin deficiency (4, 16, 38, 39, 40). Thus, in NIDDM patients whose glycemic control is
poor, not only initiating factors such as obesity, high-fat diet, and
insufficient exercise (23, 49), but also glucotoxicity, are regarded as
factors contributing to the development of insulin resistance (12, 17).
This study was designed to assess the contribution of hyperglycemia to
insulin resistance in the liver, adipose tissue, and skeletal muscle in
the Zucker diabetic fatty (ZDF) rat model.
ZDF rats are obese diabetic model animals that are polyphagic and hyperinsulinemic starting in the prehyperglycemic stage and then becoming hypoinsulinemic after establishment of hyperglycemia (42-44). This shift in disease phases is very similar to that seen in human NIDDM (2, 36). In this study, to remove the effect of hyperglycemia, a novel oral phlorizin derivative, T-1095, was administered to ZDF rats. An almost complete normalization of hyperglycemia was obtained at a high dosage of T-1095 without apparent changes in other factors, including overeating and obesity. By use of these T-1095-treated model animals, we investigated the extent to which hyperglycemia contributes to insulin resistance in the liver, adipose tissue, and skeletal muscle of ZDF rats. This is the first study to show clearly that the magnitude of the contribution of hyperglycemia to the development of insulin resistance differs among tissue types.
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METHODS |
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Chemicals and analytical methods. T-1095 was synthesized at the Discovery Research Laboratory (Tanabe Seiyaku, Saitama, Japan). Plasma insulin levels were assayed by means of an ELISA kit (Seikagaku, Tokyo, Japan) with rat insulin as a standard. An RIA kit (insulin kit "Eiken," Eiken Chemical, Tokyo, Japan) with human insulin serving as the standard was used for the assay in the hyperinsulinemic euglycemic clamp study. Blood glucose levels were determined by means of commercially available kits based on the glucose oxidase method (New Blood Sugar Test, Boehringer Mannheim, Germany). Plasma glucose levels in the euglycemic clamp study and urinary glucose content were measured by a glucose analyzer (Apec, Danvers, MA). The hemoglobin AIc (Hb AIc) level was determined by an affinity column method (Glyc-Affin, Seikagaku). All other chemicals were standard high-purity materials obtained from commercial sources.
Animals. ZDF ( fa/fa) and lean control (fa/+) rats were purchased from Charles River Japan at 6 wk of age, housed in stainless wire cages, and given normal laboratory chow (CE-2, Clea Japan, Tokyo, Japan) and water ad libitum. The experiments were started at 9 wk of age. The animals were divided into experimental groups matched for both body wt and blood glucose level. All experiments that used animals were approved by the Animal Care and Use Committee at Tanabe Seiyaku.
For the single oral administration experiment, T-1095 was suspended in 0.1% hydrogenated caster oil polyethyleneglycol ether solution (Nikkol HCO-60, Nikko Chemical, Japan) and administered orally via stomach tube at a volume of 5 ml/kg. Blood samples were taken from the tail vein before, and 1, 2, 3, 5, 8, and 24 h after, drug administration to determine glucose levels. Urine samples were collected to measure glucose content by means of metabolic cages. To achieve continuous administration, T-1095 was given as food admixtures. Both lean control and ZDF rats (9 wk old) were kept on a CE-2 diet containing 0.03 or 0.1% (wt/wt) T-1095. Doses were estimated from food intake and body wt. Blood and urine samples were collected as described above.Hyperinsulinemic euglycemic clamp study.
A hyperinsulinemic euglycemic clamp study was performed after 4 wk of
continuous T-1095 administration. The animals were fasted for 16 h
before this experiment to assure that the drug had been entirely
eliminated, and insulin sensitivity was then investigated by use of the
hyperinsulinemic euglycemic clamp technique (20, 25). In brief, rats
were anesthetized with pentobarbital sodium (60 mg/kg ip), and
catheters were inserted into both the right and left femoral veins for
insulin and glucose infusion, respectively. A catheter in the left
jugular vein was used for blood sampling and injection of
[14C]glucose and
deoxy-[2-3H]glucose. Regular human insulin (1.8 U · kg1 · h
1,
Humulin R, Eli Lilly, Indianapolis, IN) was infused
intravenously, and whole blood glucose concentrations were determined
at 5-min intervals. A 10% glucose solution was infused to maintain the blood glucose concentration at 120 mg/dl. When the blood glucose level
reached a steady-state level,
D-[U-14C] glucose (Amersham Life
Science, Buckinghamshire, UK) was administered as an initial
intravenous priming dose (4 µCi), and immediately after by continuous
infusion at a rate of 0.2 µCi/min. After a stabilization period of
30 min, the bladder was emptied and urine sampling was started (i.e.,
time = 0 min). The steady-state glucose infusion rate (SSGIR)
was determined as the mean of values obtained during a 0- to 60-min
period. At 15 min, 2-deoxy-[1-3H]glucose (50 µCi, Amersham Life Science) was intravenously administered as a bolus
injection. Additional blood samples were collected for the
determination of plasma tracer concentrations and insulin levels at 17, 20, 25, 30, 35, 45, and 60 min, and at 0, 20, 40, and 60 min, respectively.
Glucokinase activity assay. Hepatic glucokinase (GK) activity was measured by the spectrophotometric continuous assay method (10). Liver homogenates were prepared in ice-cold buffer (pH 7.5) containing 50 mM HEPES, 250 mM sucrose, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5 mM dithioerythritol and centrifuged at 105,000 g for 60 min to sediment the microsomal fraction which was used for measuring glucose-6-phosphatase (G-6-Pase) activity. The enzyme activity of the supernatant (soluble fraction) was assayed in a buffer (pH 7.4) containing 50 mM HEPES, 7.5 mM MgCl2, 100 mM KCl, 5 mM ATP, 2.5 mM dithioerythritol, 10 mg/ml BSA, 0.5 mM NAD+, 4 U/ml glucose-6-phosphate dehydrogenase (L. mesenteroides) and 0.5 mM for hexokinase or 50, 25, 12.5, and 6.25 mM glucose for total phosphorylating activity. The hexokinase reaction was initiated by adding ATP, and the rate of NAD+ reduction was recorded at 340 nm. Total phosphorylating activity was determined as the difference of absorbance change in the presence and absence of ATP. GK activity was calculated as the difference between the total phosphorylating activity and hexokinase activities.
G-6-Pase activity assay. The microsomal fraction was resuspended in the homogenization buffer and diluted with ice-cold buffer (pH 6.5) containing 100 mM HEPES and 0.1 mM EDTA. The G-6-Pase reaction was initiated by adding 10, 5, 2.5, 1.25, and 0.625 mM glucose 6-phosphate at 30°C and stopped after 10 min with a 2.2-fold volume of the stop solution containing 3.7 mM ammonium molybdate and 240 mM SDS in 270 mM H2SO4. The absorption at 750 nm was monitored after color development by adding a 1/5 volume of 1.2 M ascorbic acid. Pi was used as a standard (26).
Preparation of isolated adipocytes from ZDF rats. ZDF and T-1095-treated ZDF rats were killed by decapitation after 5 wk of treatment. Epididymal fat pads were excised and digested with collagenase (1 mg/ml, Sigma, St. Louis, MO), and the isolated adipocytes were suspended in Krebs-Ringer bicarbonate (KRB) buffer supplemented with 30 mM HEPES, 1% BSA fraction V, 3 mM glucose, and 200 mM adenosine at pH 7.4 as described (9, 21). Adipocytes were prepared with a 25% adipocrit.
Subcellular fractionation and Western blotting. Isolated adipocytes were suspended in KRB buffer containing BSA (10 mg/ml, pH 7.4) and preincubated with or without insulin for 30 min at 37°C. After incubation, subcellular membrane fractions of adipocytes were prepared by differential centrifugation (9, 21). The homogenates were centrifuged at 3,000 g for 15 min to sediment the crude membrane fraction. The fat cakes were discarded, and the supernatant was centrifuged at 12,000 g for 15 min to sediment the plasma membrane (PM) fraction. The supernatant was centrifuged at 28,000 g for 15 min, and the resulting supernatant was centrifuged at 146,000 g for 75 min, yielding a particulate fraction termed low-density microsomes (LDM). Proteins (0.05 mg) in PM and LDM fractions were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 3% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T, vol/vol), and then incubated for 2 h at room temperature with antiserum to the COOH terminus of GLUT-4. After being washed with TBS-T, the bound immunoglobulins were probed with anti-rabbit IgG antibody. The membranes were immediately reacted with an enhanced chemiluminescence (ECL) reagent (Amersham Life Science), and the chemiluminescence level was quantified with a molecular imager GS-525 by use of Screen-HC.
Data analyses. The data are presented as means ± SE. Statistical analyses were performed by closed testing procedures. In brief, the untreated diabetic control group was initially compared with the lean group by unpaired Student's t-test. When the difference between these two groups was significant, a multiple comparison was performed with Dunnett's test to compare each T-1095-treated group with the diabetic control group. P values <0.05 were considered to be statistically significant.
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RESULTS |
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Effect of a single administration of T-1095 on blood glucose and
urinary glucose excretion.
When administered orally, T-1095 is absorbed from the gut into the
bloodstream and metabolized to an active form termed T-1095A. T-1095A
suppresses the activity of SGLTs in the kidney, thereby inhibiting
reabsorption of glucose (34, 46). Thus a dose-dependent increase in
urinary glucose excretion was observed with the administration of
T-1095 to ZDF rats (at 3 and 8 h after administration, as shown in Fig.
1A). Concurrently, T-1095 dose
dependently lowered blood glucose levels in ZDF rats (Fig. 1B).
The antihyperglycemic effect was apparent at a dose of 30 mg/kg, and
the maximal dose of 100 mg/kg induced a sustained decrease in blood
glucose levels for 24 h. In contrast, there was only a marginal effect
on blood glucose levels in lean rats, even at a dosage of 100 mg/kg
(Fig. 1C).
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Effect of continuous treatment with T-1095 on hyperglycemia and
physiological parameters.
Continuous administration of T-1095 was shown to lower blood glucose
levels significantly in ZDF rats at both low (0.03%) and high (0.1%)
doses (Fig. 2A). In contrast, no
such hypoglycemic effect of T-1095 was observed in lean rats (control
lean rats, 86.3 ± 2.9 mg/dl, T-1095-treated lean rats, 86.1 ± 2.7 mg/dl). ZDF rats exhibited apparent hyperphagia and continued to weigh more than lean rats (Fig. 2B). T-1095 treatment did not affect the body wt increase of ZDF rats, despite slightly suppressing hyperphagia at 1 wk after the beginning of T-1095 administration (Fig.
2C).
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Effect of continuous T-1095 treatment on insulin sensitivity in
hyperinsulinemic euglycemic clamp study.
In vivo insulin sensitivity was quantitatively determined by the
hyperinsulinemic euglycemic clamp study. During the clamp study, there
were no significant differences in steady-state plasma glucose (SSPG),
steady-state plasma insulin (SSPI), or urinary glucose loss (UGL) among
lean, ZDF, and T-1095-treated ZDF rats (Table
2). When compared with lean control rats,
there were decreases in steady-state GIR and GUR, and an increase in
HGP in ZDF rats (Fig. 4). T-1095 treatment
normalized GIR, GUR, and HGP in ZDF rats (Fig. 4).
Rg' in skeletal muscle (quadriceps femoris), liver,
white adipose tissue (epididymal fat pad), and brown adipose tissue
were decreased in ZDF rats. T-1095 at a high dose elevated the
Rg' levels significantly in liver and both types of adipose tissue (Fig. 5, A,
C, and D). T-1095 produced no significant change in the
Rg' of skeletal muscles (Fig. 5B).
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Effects of continuous T-1095 treatment on hepatic GK and G-6-Pase
activities.
To study changes in the key steps of hepatic glucose metabolism in ZDF
rats, we measured the activities of both GK (Fig.
6A) and G-6-Pase (Fig. 6B).
GK activity was decreased significantly to 46.0% of the control value,
whereas G-6-Pase activity was increased to 152.3% in ZDF rats (Fig.
6). T-1095 treatment restored both GK and G-6-Pase activities in ZDF
rats to those in lean control rats. There were no differences in
Michaelis constant (Km) values for GK and G-6-Pase
activities among groups.
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Effect of continuous T-1095 treatment on insulin-induced GLUT-4
translocation in isolated adipocytes.
Insulin stimulation of isolated adipocytes induced dramatic recruitment
of GLUT-4 protein from LDM to PM. The GLUT-4 protein increase in the PM
with insulin stimulation was markedly blunted in ZDF rats, and the
T-1095 treatment apparently normalized this impairment (Fig.
7, A and B). Similarly,
impaired insulin-induced reduction of GLUT-4 in LDM of ZDF rats was
partially normalized with T-1095 treatment (Fig. 7, C and
D). These results indicate that T-1095 treatment ameliorates
impaired GLUT-4 translocation, which is in good agreement with
increased Rg' levels in the hyperinsulinemic
euglycemic clamp study.
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DISCUSSION |
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T-1095 is a derivative of phlorizin but, unlike phlorizin, it is absorbed efficiently from the gut into the bloodstream when administered orally. After entering the circulation, T-1095 is metabolized to an active form, termed T-1095A (33, 45), that suppresses the activity of renal SGLTs involved in glucose reabsorption in proximal tubules (22, 33, 34, 45). Thus orally administered, T-1095 exerts essentially the same effect as intravenously injected phlorizin, which cannnot be absorbed from the gut (8). However, taken as a food admixture, T-1095 efficiently suppresses postprandial blood glucose elevation, which results in a more efficient antihyperglycemic effect than phlorizin when given as a continuous venous infusion (45).
The ZDF rat is regarded as an insulin-resistant diabetic model exhibiting obesity and overeating because of an abnormality of the leptin system (18, 32, 35). In our experiments, the nonfasting blood glucose levels of ZDF rats averaged 250 ± 10 mg/dl at 9 wk of age, and a defect in insulin secretion had already been indicated in an oral glucose tolerance test at 11 wk (data not shown). Although the molecular mechanisms underlying insulin resistance remain unclear, the major cause of insulin resistance in ZDF rats has been considered to be obesity related (3, 18, 32, 35, 41-44, 48). Although the blood glucose level was normalized, the fasting insulin level was not reduced by T-1095 treatment. The ZDF strain of rat is a substrain of the Zucker fatty strain that is obese, hyperinsulinemic, and normoglycemic. Thus we speculate that these Zucker-strain rats are genetically hyperinsulinemic irrespective of blood glucose levels, and that correction of hyperglycemia does not, therefore, reduce hyperinsulinemia in ZDF rats.
In the present study, despite the degree of obesity being unchanged, the normalization of hyperglycemia with T-1095 treatment ameliorated insulin resistance significantly, as shown by a reduction in GIR in the hyperinsulinemic euglycemic clamp study. In fact, T-1095 treatment slightly improved total GUR. On the other hand, the elevated triglyceride (TG), free fatty acid (FFA), and total cholesterol (TCHO) values in ZDF rats were not altered by T-1095 treatment. We speculate that these levels are not related to abnormal leptin metabolism, because there is a functional leptin receptor defect in ZDF rats. In fact, plasma leptin levels in T-1095-treated ZDF rats were similar to those in untreated ZDF rats (data not shown). Thus it is very likely that the improved insulin sensitivity achieved with T-1095 treatment is attributable to removal of the effect of hyperglycemia (so-called glucotoxicity).
Of great interest in this study is the observation that the degree of insulin sensitivity improvement differs among tissues after blood glucose normalization. Insulin resistance in the liver was markedly suppressed by normalizing blood glucose levels. In contrast, although insulin resistance in adipose tissues was also improved significantly, this normalization was partial. Finally, insulin resistance in muscle was apparently not improved. These results show clearly that the contribution of hyperglycemia to insulin resistance differs in these three insulin-sensitive tissues.
First, T-1095 treatment profoundly reversed both reduced glucose uptake and elevated glucose production in the liver, as demonstrated by the hyperinsulinemic euglycemic clamp study. Although abrupt correction of hyperglycemia might have counterregulated HGP in ZDF rats during the glucose clamp study, elevations in fasting glucose level and the ratio of GK to G-6-Pase activities agree well with the result of the glucose clamp study. GK and G-6-Pase play important roles in blood glucose regulation. GK and G-6-Pase, both of which are known to be regulated by insulin, convert glucose to glucose 6-phosphate, and glucose 6-phosphate to glucose through glucose production, respectively (19, 30, 46, 47). In our experiment, the reduced GK/G-6-Pase activity ratio in ZDF rats was corrected by continuous T-1095 treatment. Hepatic GK activity and GK expression levels are reportedly increased in streptozotocin-treated (STZ) rats, and phlorizin treatment ameliorates the impaired GK activity (6, 27); therefore, it is likely that hyperglycemia altered GK and G-6-Pase activities, and that T-1095 treatment corrected the enzyme activities through amelioration of hyperglycemia.
Second, in adipose tissues, the normalization of insulin resistance with T-1095 treatment was demonstrated by the increased Rg' values in the glucose clamp study. It is well known that the rate-limiting step for glucose uptake into adipocytes is governed by the amount of GLUT-4 on the cell surface (11, 24, 28, 37). In fact, the translocation of GLUT-4 protein from LDM to PM was markedly impaired in adipose tissues of ZDF rats, and this impairment was shown to be ameliorated by T-1095 treatment. However, the degree of normalization was partial, such that factors worsening insulin sensitivity in ZDF fat tissues apparently persist. We speculate that the enlargement of adipocytes is likely to be one such factor, because it has been reported that larger adipocytes possess lower insulin sensitivity in glucose transport activity (29).
Finally, the reduced skeletal muscle Rg' of ZDF rats
was not restored significantly by T-1095 treatment. A previous study
demonstrated that impaired insulin sensitivity in STZ diabetic rats was
improved by long-term correction of hyperglycemia with phlorizin (5).
Thus, although in most nongenetic diabetic models (e.g., STZ rats)
hyperglycemia is likely to be a major cause of the development of
insulin resistance in peripheral tissues (5-7, 27, 30, 31, 38),
glucotoxicity is not involved in the mechanism underlying insulin
resistance in ZDF rat muscles (1, 51). Moreover, it was also reported
that insulin resistance was not improved in muscle of ZDF rats treated
with pioglitazone, an insulin sensitizer of the peroxisome
proliferator-activated receptor- (PPAR-
) agonist
(13). On the contrary, it has been reported that the PPAR
-agonist
improves muscle insulin resistance (51) and
-glucosidase inhibitor
recovers muscle GLUT-4 contents in ZDF rats (15). In the present study,
T-1095 reduced hyperglycemia without affecting plasma TG, FFA, and
total cholesterol. These results suggest that insulin resistance in
skeletal muscle is already developed by other mechanisms including
lipotoxicity when hyperglycemia is established, and that hyperglycemia
does not further increase insulin resistance, in ZDF rats. Hawkins et
al. (16) have suggested that the increased availability of glucose and
FFA affects insulin-induced glucose uptake via a common mechanism. In
the present study, FFA levels were elevated in ZDF rats, whereas muscle
TG contents were not determined. Therefore, although we speculate that
the impaired insulin sensitivity in ZDF rat muscle may be related to an
abnormality in lipid metabolism as well as the leptin system, further
study is necessary to clarify this issue.
In summary, insulin resistance in hepatic and adipose tissues is induced by glucotoxicity resulting from long-term hyperglycemia in ZDF rats. In contrast, insulin resistance in skeletal muscles is not due to glucotoxicity. Taken together, these raise the possibility of the tissue dependence of glucotoxicity not only in other animal models but also in individual diabetic patients (14). However, although hyperglycemia is a consequence of insufficient insulin secretion and insulin resistance, these results suggest that normalization of hyperglycemia leads to an apparent improvement in insulin resistance. Thus hyperglycemia should be considered not only a consequence of but also a major factor in worsening insulin sensitivity. We now consider T-1095 to be a potentially novel therapeutic strategy for diabetes management, because it evidently reduces hyperglycemia-induced insulin resistance in liver and adipose tissues. Further study is required to elucidate the molecular mechanisms underlying insulin resistance in the skeletal muscle of ZDF rats.
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ACKNOWLEDGEMENTS |
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We thank Drs. Mamoru Matsumoto, Kenji Tsujihara, Takeshi Matsumoto (Tanabe Seiyaku, Japan) for reviewing the manuscript, Mitsuya Hongu for supplying T-1095, and Yasuo Kuronuma (Tanabe Seiyaku, Japan) for technical support.
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FOOTNOTES |
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This work was supported by a grant from Tanabe Medical Frontier Conference (TMFC, Tomoichiro Asano, Japan).
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 and other correspondence: A. Saito, Discovery Research Laboratory, Tanabe Seiyaku, 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan (E-mail: a-saito{at}tanabe.co.jp).
Received 24 June 1999; accepted in final form 15 October 1999.
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