—NOTE— Glycolytic Activities in Leukocytes of Thoroughbreds Undergoing Training Exercise

Toshiro ARAI1*, Takumi KAWAUE1, Maki ABE1, Etsuko KURAMOTO1, Ryuma NURUKI3 and Toshinori SAKO2 Divisions of 1Veterinary Biochemistry and 2Veterinary Internal Medicine, Nippon Veterinary and Animal Science University, 7-1 Kyonancho 1 chome, Musashino, Tokyo 180-8602 and 3YCL Horse Clinic, 9-6 Kaminoda 6 chome, Urawa, Saitama 337-0977, Japan

Glycolytic enzyme activities and expression of (GK) were investigated in leukocytes J. Equine Sci. (WBC) from Thoroughbreds, racehorses undergoing continuous training exercise and riding Vol. 8, No. 4 horses. Glycolytic enzyme activities in WBC from racehorses were higher than those from pp. 113–116, 1997 riding horses. Especially activities of GK, rate-limiting enzyme of glycolysis, were significantly higher in WBC from racehorses than in those from riding horses. Analysis of reverse transcription- chain reaction (RT-PCR) with specific primer for GK showed that expression of mRNA of GK was remarkably increased in WBC from racehorses. This suggested that activities and gene expression of some kinds of glycolytic including GK in WBC are possibly a good indicator for assessing glucose metabolism in Thoroughbreds undergoing training exercise. Key words: glycolytic enzyme, leukocytes, RT-PCR, Thoroughbreds

Since glucose is an important energy source for all isolation of samples than assay of enzyme activity and mammalian cells, blood glucose concentrations should can obtain primary information on the regulative be controlled strictly in animals. In racehorses, mechanism of the gene expression. In the present study, concentrations of blood glucose and plasma insulin were we measured the activities of cytosolic enzymes and maintained constantly within the day [6]. On the other detected the conserved region of mRNA of glucokinase hand, the activities of D-glucose transport and glycolytic ( IV), the rate-limiting enzyme of glycolysis, enzymes varied in the erythrocytes from various animals by using the sensitive reverse transcription-polymerase with different concentrations of blood glucose [5]. chain reaction (RT-PCR) in leukocytes (WBC) of Continuous training increases glucose utilization in tissues Thoroughbreds. of animals [10]. In the racehorse erythrocytes (RBC), All the horses examined were Thoroughbreds. Twelve the higher glucose transport and glycolytic activities racehorses (4 to 8 years old, 5 female and 7 male) were compared with those in untrained horses are observed kept and trained at Kaminoda Training Center of Urawa under continuous training exercise [4]. It is very difficult Race Course, National Association of Racing. Each to obtain tissue samples, e.g. skeletal muscle and liver, racehorse was usually exercised for 9 of every 10 days. from racehorses. If it may be presumed that glycolytic Their 9 days work in every 10 days included 3 days of fast enzyme activities in blood cells reflect those in other galloping at 13 to 17 m/sec for 1000 to 1200 m and 6 days tissues, they would be of value in assessing the metabolic of slow work at 6 to 8 m/sec for 1500 to 2000 m, including status of glucose in racehorses undergoing continuous warming up and cooling down for 2 hr. Ten riding horses training. Measuring the mRNA amount of enzyme (5 to 16 years old, 2 female and 8 gelding) kept at the requires simpler treatments for the preservation or Equestrian Club of St. Paul’s University were examined as comparable references. All the horses were maintained on grass supplemented with good-quality hay and This article was submitted July 17, 1997 and was accepted concentrate. Blood was withdrawn from the jugular veins November 6, 1997 *corresponding author. of the animals into heparinized tubes between 1400 and 114 T. ARAI, T. KAWAUE, M. ABE ET AL.

1500 hr under unfasting conditions at rest. In general, the volume of 100 µl. The mixture was overlaid with mineral horses showed no excitement or fear and they were not oil and amplified by 30 cycles of denaturaion at 94°C for forcibly restained. The plasma was recovered by 30 sec (the first cycle was for 2 min), primer annealing at centrifugation at 4°C and stored at － 20°C. WBC 60°C for 30 sec and extension at 72°C for 90 sec, by using containing mainly lymphocytes were collected by gradient a Perkin Elmer DNA thermal cycler (Nowalk, CT, USA). centrifugation with lymphocyte isolating solution, Ten µl of PCR product was electrophoresed in 2% agarose SEPARATE-L (Muto Pure Chemicals, Tokyo, Japan), gels and visualized by ethidium bromide staining. The followed by washing twice with phosphate buffered saline glucose concentrations in whole blood were measured by (pH 7.2). For the assay of enzyme activities, the cytosol a glucose-oxidase method [12]. The plasma insulin fraction from washed WBC was prepared [3], and stored concentrations were determined by a micro ELISA at －80°C until used. Enzyme activities were measured sandwich method [2]. Each value was expressed as the in the cytosol fraction by the methods cited in the mean ± SD and the differences between the means were following references: hexokinase I, II and III (HK) and analysed by Student’s t-test. glucokinase (GK) [18], pyruvate (PK) [11], glucose- Table 1 shows blood glucose and plasma insulin 6-phosphate dehydrogenase(G6PD) [7] and lactate concentrations and cytosolic enzyme activities in WBC dehydrogenase (LDH) [13]. All enzyme assays were from race and riding horses. The mean blood glucose conducted at 25°C and the activities were expressed as and plasma insulin concentrations from racehorses were nmol of substrate degraded per min per mg of protein. almost the same as those from riding horses. Activities Protein concentrations in cytosol were measured by the of all cytosolic enzymes examined in WBC from method of Bradford [8]. Total RNAs were isolated by racehorses were higher than those from riding horses. using a commercial RNA isolation kit, RNeasy Total Especially GK activities from racehorses were RNA Kit (Qiagen, Hilden, Germany). RT-PCR was used singnificantly higher than those from riding horses. for detecting the region containing glucose- and ATP- We applied the RT-PCR method for detection of binding domains of glucokinase mRNA which were glucokinase mRNA in WBC from horses (Fig. 1). The positioned at 162 to 736 of the cDNA sequence of rat fragment with a predicted size of 574 bp was detected in liver glucokinase [1]. The sense and antisense primers PCR products from both race and riding horse WBC. were 5’-GTCGAGCAGATCCTGGCAG-3’ and 5’- In the WBC from racehorse with high GK activities (10.8 ACTGTGTCGTTCACCATTGCC-3’, respectively. The nmol/min/mg), the fragment was stained strongly with RT-PCR was carried out according to the protocol of a ethidium bromide (Fig. 1, lane 2) but the fragment was commercial kit, TaKaRa RNA PCR Kit AMV (Takara obscure in the WBC from a riding horse with low Shuzo, Ohtu, Japan). A 20 µl reverse transcription reaction activities of 2.8 nmol/min/mg (Fig. 1, lane 1). mixture containing 1 µg of total RNA, 1x PCR buffer (10 Blood glucose and plasma insulin concentrations of mM Tris-HCl, 50 mM KCl, pH 8.3), 5 mM MgCl2, 1 mM horses in the present sutdy were considerably lower than dNTPs, 1 U/ml RNAase inhibitor, 2.5 µM random those reported previously [4]. This finding was hexamers and 0.25 U/µl Avian myeloblastosis virus considered to be owing to the sampling time, around , was preincubated at 30°C for 10 min, one hour before feeding in the afternoon but there were then incubated at 55°C for 30 min, heated to 99°C for 5 no significant differences between race and riding horses min and maintained at 5°C for 5 min. The PCR was in blood glucose and plasma insulin concentrations at carried out at a final concentration of 1x PCR buffer, 2.5 any time. Glucose utilization was increased in the RBC mM MgCl2, 0.2 mM dNTPs, 0.16 µM of each sense and from the racehorses owing to an increased basal antisense primer and 2.5 U of Taq polymerase in a total metabolic energy associated with continuous exercise

Table 1. Blood glucose and plasma insulin concentrations and cytosolic enzyme activities in white blood cells from race and riding horses

Blood glucose Plasma insulin Cytosolic enzyme activity (nmol/min/mg) (mg/dl) (µU/ml) HK GK PK G6PD LDH Race (n=12) 64 ± 11 16 ± 6 1.6 ± 0.6 8.4 ± 2.4* 53 ± 16 11 ± 4 196 ± 46 Riding (n=10) 62 ± 12 14 ± 7 0.9 ± 0.3 4.1 ± 1.2 34 ± 12 10 ± 3 136 ± 34 Values represent the means ± S.D. *Significantly different from the riding horses (p<0.05). GLYCOLYTIC ENZYME IN LEUKOCYTES OF THOROUGHBREDS 115

showed GK activities. GK is expressed in the liver and pancreatic B cells and plays a key role in the regulation of glucose homeostasis and its expression is changed in various metabolic conditions [14, 15]. In the present study, a significant increase in the activities and gene expression of GK were observed in WBC from racehorses undergoing continuous exercise training. This result suggests that continuous training plays important roles in increasing glucose utilization even in the WBC of Thoroughbreds, so activities or gene expression of some kinds of enzymes including GK are possibly a good indicator to assess the metabolic condition of glucose which is a major energy source in Thoroughbreds also. But the relationship between glycolytic enzyme in tissues and racing performance is not known. Further studies on the regulative mechanism of glycolytic enzyme Fig. 1. Analysis of glucokinase mRNA with activities and gene expression are necessary to determine RT-PCR in WBC from race and riding horses. PCR products were stained whether they are effective indicators for assessing the with ethidium bromide after agarose gel racing performance of racehorses. electrophoresis. Lane 1, riding horse; lane 2, race horse; lane M, size marker containing 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 base pairs. References 1. Andreone, T.L., Printz, R.L., Pilkis, S.J., Magnuson, M.A. and Granner, D.K. 1989. The amino acid training [4]. The WBC from racehorses also showed sequence of rat liver glucokinase deduced from higher activities of cytosolic enzymes than those from cloned cDNA. J. Biol. Chem. 264: 363–369. riding horses. Especially GK activities were significantly 2. Arai, T., Machida, Y., Sasaki, M. and Oki, Y. 1989. high in the WBC from racehorses. This corresponds to Hepatic enzyme activities and plasma insulin the result of RT-PCR analysis of GK mRNA. The WBC concentrations in diabeteic herbivorous voles. Vet. from a racehorse with high activities of GK had a Res. Commun. 13: 421–426. fragment strongly stained with 574 bp. The staining 3. Arai, T., Ageto, H., Shinofuji, T. and Sugawara, density of the fragment appeared to be accompanied with M. 1992. Activities of certain cytoplasmic enzymes GK activities. The fragment with a size of 574 bp contains in bovine T lymphoma. J. Vet. Med. Sci. 54: 1067– putative amino acid residues involved in the binding of 1068. glucose and of ATP, and this region is highly conserved 4. Arai, T., Washizu, T., Hamada, S., Sako, T., Takagi, in the hexokinase family of man and rat [9, 17]. S., Yashiki, K. and Motoyoshi, S. 1994. Glucose Comparison of the structures of the HK II and GK genes transport and glycolytic enzyme activities in supports the hypothesis that the 100-kDa HK arose by erythrocytes of two-year-old thoroughbreds gene duplication and tandem ligation of a 50-kDa GK- undergoing training exercise. Vet. Res. Commun. 18: like ancestral gene [16]. The primer set used in this study 417–422. were specific to the region containing glucose- and ATP- 5. Arai, T., Washizu, T., Sagara, M., Sako, T., Nigi, binding domain of the HK family. The fragments with H., Matsumoto, H., Sasaki, M. and Tomoda, I. different sizes except 574 bp may be PCR products of a 1995. D-glucose transport and glycolytic enzyme region of a gene of the HK family. To clarify this activities in erythrocytes of dogs, pigs, cats, horses, supposition sequencing of the PCR products cattle and sheep. Res. Vet. Sci. 58: 195–196. should be performed. The fragment with 574 bp was 6. Arai, T., Sugawara, M., Sako, T. and Motoyoshi, also detected in liver of dog and mouse, but was absent S. 1996. Within day changes in blood glucose and from the liver of cattle when the same RT-PCR method plasma insulin concentrations and erythrocytes as in our preliminary experiment was used (unpublished enzyme activities in race horses at rest. data). This fragment was detected in tissues which Pferdeheilkunde 12: 485–487. 116 T. ARAI, T. KAWAUE, M. ABE ET AL.

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