Phosphoribosyl Pyrophosphate Formation in the Rat Adrenal Gland in Relation to Adrenal Growth in Experimental Diabetes SIRILAKSANA KUNJARA, MILENA SOCHOR, SALAH AHMED, A. LESLIE GREENBAUM, AND PATRICIA MCLEAN
Adrenal growth occurs in experimental diabetes, and evidence exists for increased adrenal function. The concentration of PPRibP has been examined in the rat adrenal gland at various times after induction of diabetes with STZ, in view of the key role it plays in the synthesis of Purs and Pyrs. The PPRibP level was exceptionally high in the adrenal gland and increased faster than the rate of growth during the initial rapid growth phase—the first 7 days after STZ was given; PPRibP synthetase showed a parallel increase. Formation of R5P via the oxidative and nonoxidative segments of the PPP also was measured. The oxidative enzymes, G-6-PD and 6-PGD, increased in parallel with growth during the early phase, but showed a more marked rise during the secondary, slower, growth phase seen 6 wk after STZ was given, when this may be associated with the known sustained rise in plasma corticosteroids. The nonoxidative enzymes of the PPP, an alternate route for the production of R5P, showed smaller changes. The specifically high adrenal concentration of PPRibP may be related to the high Km for PPRibP (250 |iM) of the first enzyme of the de novo pathway of Pur synthesis, as such synthesis may be required in the rat to replace the net loss of ATP associated with catecholamine secretion. Factors controlling PPRibP synthetase and their potential relative importance in the adrenal gland have been considered. Diabetes 41:1429-35,1992
From the Department of Biochemistry, University College and Middlesex School of Medicine, London, UK; and the Faculty of Medicine, University of Khartoum, Khartoum, Sudan. Address correspondence and reprint requests to Dr. S. Kunjara, Department of Biochemistry, University College and Middlesex School of Medicine, Cleveland Street, London, W1P 6DB, UK. Received for publication 17 December 1991 and accepted in revised form 23 April 1992. PPRibP, phosphoribosyl pyrophosphate; STZ, streptozocin; R5P, ribose 5 phosphate; PPP, pentose phosphate pathway; G-6-PD, glucose-6-phosphate dehydrogenase; 6-PGD, 6-phosphogluconate dehydrogenase; ALX, alloxan; CS, corticosterone; IGF-I, insulinlike growth factor I.
DIABETES, VOL. 41, NOVEMBER 1992
A
n increase in adrenal gland weight, with evidence for increased adrenai function and raised plasma corticoids, has been reported in experimental diabetes induced by subtotal pancreatectomy and with ALX or STZ (1-5). Rhees et al. (5) have shown that the increase in adrenal weight is related to hyperglycemia and/or lack of insulin because insulin therapy in STZ-induced diabetic rats reduced the elevated adrenal weight and plasma CS to normal values. Further, rats given STZ, but that did not develop diabetes, retained normal adrenal gland weights and levels of plasma CS (5). De Nicola et al. (4) have studied adrenal function at various time intervals after induction of diabetes with STZ, from short-term (5 days) to longer-term, chronic diabetes (2-3 mo). The most marked changes in adrenal weight and plasma CS were observed 5 days after administration of STZ, effects which could be related to the dual effect of diabetes and ACTH release; a less marked, but persistent, adrenal hyperactivity followed, which could be due to ACTH itself or to the action of growth factors such as IGF-I (3,4,6). The changing nature of the adrenal gland response to experimental diabetes, with an early rapid growth and corticosteroid production, has prompted studies of two key systems involved in growth and corticosteroid synthesis, namely, PPRibP formation and the activity of the PPP. PPRibP is essential to increased biosynthetic activity of the adrenal gland because it serves as a primary substrate in both the de novo and salvage pathways of nucleotide synthesis as well as being an activator of the first steps in the de novo routes of Pur and Pyr synthesis (Becker et al., 7). Thus, it is a key element in the provision of precursors for RNA and DNA synthesis. The PPP supplies two essential components for adrenal growth and hyperfunction: R5P, a precursor and known regulatory factor in PPRibP formation (7,8); and NADPH, re-
1429
1 ADRENAL GROWTH AND PPRibP SYNTHESIS
TABLE 1 PPRibP content of the adrenal gland—comparison with other tissues and the effect of anesthesia and a p-blocker
Comparison with a range of normal rat tissues Intact adrenal gland Liver Kidney Lactating mammary gland (10 days) Testis Cerebral cortex Effect of anesthesia and propranolol on adrenal PPRibP Adrenal gland None Na pentobarbital, 30 min Na pentobarbital, 60 min Propranoiol Distribution of PPRibP in rabbit adrenal gland Intact gland Cortex Medulla
PPRibP concentration (nmol/g tissue)
PPRibP synthetase (U/g)
168 ± 10(30) 10.5 ±0.6 (17) 9.5 ± 0.5 (8) 9.7 ± 1.0(9) 5.0 ± 0.3 (20) 5.5 ± 0.4
12.0 ±0.6 (7) 22.7 ± 1.3(6) 25.3 ±0.6 (12) 63.2 ± 6.7 (4) 19.9 ± 1.2(13) 21.0 ± 1.0(4)
Reference
8 14 10 33
165 ± 1 4 (12) 196 ± 2 3 (5) 137 ± 9 (5) 156 ± 12(5) 255, 303 285, 280 276, 294
Values are means ± SE. (n), number of separate experiments. Each value for the.rat.adrenai cortex and medulla was obtained from the pooied samples from adult male rats. Values for the PPRibP conteht of the range of tissues come from published data (except those for testis, which are unpublished observations).
quired for the reductive synthetic steps of corticbsteroid synthesis. In this study, we measured the activity of these two systems in the rat adrenal gland during short-term (3-7 days) and chronic (6 wk) STZ-induced diabetes. RESEARCH DESIGN AND METHODS
Subjects were adult male Wistar rats, initial body weight 210-230 g. Diabetes was induced either by i.v. injection of STZ (60 mg/kg body weight) or i.p. injection of ALX monohydrate (200 mg/kg body weight) as described previously (8). Rats were grouped as follows: STZ-induced diabetic rats; 1) given no further treatment and then killed by cervical dislocation 3 or 7 days after diabetes induction; 2) pretreated with nicotinamide (500 mg/kg body weight) 10 min before STZ injection: this treatment has been shown to counteract the diabetogenic effects of STZ (9); 3) given insulin (4 U Ultratard human insulin and 2 U Actrapid insulin (Novo Nordisk, Bagsvaerd, Denmark) 24 and 48 h after STZ injection and 2 U Actrapid insulin after 72 h:.the animals were killed 1 h after the last injection. ALX-induced diabetic rats; 1) given no further treatment and then killed 3 days after diabetes induction; 2) given insulin (4 U Ultratard human insulin and 2 U Actrapid insulin (Novo Nordisk, Bagsvaerd, Denmark) 24 and 48 h after ALX injection and 2 U of Actrapid insulin aione 72 h after diabetes induction: the rats were killed 1 h after the last injection. Enzyme measurements. For PPRibP synthetase (EC 2.7.6.1.), preparation of the tissue extract and measurement of enzyme activity was as described previously (10). A unit of enzyme activity is 1 ixmole/14CO2 formed from (carboxyl labeled-14C) orotic acid • g~ 1 • h" 1 at 37°C. G-6-PD (EC 1.1.1.49.), 6-PGD (EC i.1.1.44.), transketolase (EC 2.2.1.1.), and transaldolase (EC 2.2.1.2.) were measured by the method of Novello & McLean (11) and Sochor et al. (12) in the high speed supernatant of a 1:5 tissue homogenate prepared in a
1430
medium composed of 0.25 M sucrose • 0.02 M triethanolamine • 0.12 mM dithiothreitol, pH 7.4. Results are given as ixmoles substrate converted/min at 25°C. Flux studies. An estimate of the PPP activity was obtained by measuring the difference in 14CO2 yield from [1-14C]glucdse and [6-14C]glucose as described by Sochor et al. (12) and Larrabee (13). For this study, one adrenal sliced in half and weighing —30 mg, was incubated in 5 ml of Krebs-Ringer bicarbonate, pH 7.4, containing either 5 mM (control rats) or 20 mM (diabetic rats) glucose + 0.5 |xC radioactive glucose. Incubation was for 1 h, gas phase O2/CO2 (95/5) at 37°C. The reaction was stopped with 0.5 ml,5N HCI and the 14CO2 collected in 0.6 ml Scintran (BDH, Poole, Dorset, UK). The Scintran was transferred to vials containing 4.5 nil Optiphase (LKB, Leicester, UK) with the aid of 3 x 0.5 ml methanol and the radioactivity counted in a Beckman LS 7000 scintillation counter. Because the difference in 14 CO2 yield from [1-14G]glucose and [6-14C]glucose is the basis for the measurement of PPP activity, the left and right adrenals from a single rat were used. Chemical procedures. PPRibP was measured in adrenals by the method of Kunjara et al. (14). Because the values obtained in preliminary studies were some 15- to 30-fold greater than had been found in this laboratory in a wide range of normal tissues (Tabie 1), it became necessary to establish that the elevated levels fblind in the adrenal gland were hot artifactual, arising from alarm signals as a result of handling the intact animal. Therefore, parallel measurements were made in animals a) anesthetized for 30 or 60 min with Na pentobarbital (60 mg/kg body weight) before estimation of PPRibP or b) given a p-blocker, propranolol (30 mg/kg body weight) at 24 and 1 h before removal of the adrenal glands to estimate the tissue content of PPRibP. No differences could be detected between the PPRibP content of adrenals taken from animals with no pretreatment (165 ± 14
DIABETES, VOL. 41, NOVEMBER 1992
S. KUNJARA AND ASSOCIATES
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D)
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Adrenal growth occurs in experimental diabetes, and evidence exists for increased adrenal function. The concentration of PPRibP has been examined in the rat adrenal gland at various times after induction of diabetes with STZ, in view of the key role it plays in the synthesis of Purs and Pyrs. The PPRibP level was exceptionally high in the adrenal gland and increased faster than the rate of growth during the initial rapid growth phase—the first 7 days after STZ was given; PPRibP synthetase showed a parallel increase. Formation of R5P via the oxidative and nonoxidative segments of the PPP also was measured. The oxidative enzymes, G-6-PD and 6-PGD, increased in parallel with growth during the early phase, but showed a more marked rise during the secondary, slower, growth phase seen 6 wk after STZ was given, when this may be associated with the known sustained rise in plasma corticosteroids. The nonoxidative enzymes of the PPP, an alternate route for the production of R5P, showed smaller changes. The specifically high adrenal concentration of PPRibP may be related to the high Km for PPRibP (250 |iM) of the first enzyme of the de novo pathway of Pur synthesis, as such synthesis may be required in the rat to replace the net loss of ATP associated with catecholamine secretion. Factors controlling PPRibP synthetase and their potential relative importance in the adrenal gland have been considered. Diabetes 41:1429-35,1992
From the Department of Biochemistry, University College and Middlesex School of Medicine, London, UK; and the Faculty of Medicine, University of Khartoum, Khartoum, Sudan. Address correspondence and reprint requests to Dr. S. Kunjara, Department of Biochemistry, University College and Middlesex School of Medicine, Cleveland Street, London, W1P 6DB, UK. Received for publication 17 December 1991 and accepted in revised form 23 April 1992. PPRibP, phosphoribosyl pyrophosphate; STZ, streptozocin; R5P, ribose 5 phosphate; PPP, pentose phosphate pathway; G-6-PD, glucose-6-phosphate dehydrogenase; 6-PGD, 6-phosphogluconate dehydrogenase; ALX, alloxan; CS, corticosterone; IGF-I, insulinlike growth factor I.
DIABETES, VOL. 41, NOVEMBER 1992
A
n increase in adrenal gland weight, with evidence for increased adrenai function and raised plasma corticoids, has been reported in experimental diabetes induced by subtotal pancreatectomy and with ALX or STZ (1-5). Rhees et al. (5) have shown that the increase in adrenal weight is related to hyperglycemia and/or lack of insulin because insulin therapy in STZ-induced diabetic rats reduced the elevated adrenal weight and plasma CS to normal values. Further, rats given STZ, but that did not develop diabetes, retained normal adrenal gland weights and levels of plasma CS (5). De Nicola et al. (4) have studied adrenal function at various time intervals after induction of diabetes with STZ, from short-term (5 days) to longer-term, chronic diabetes (2-3 mo). The most marked changes in adrenal weight and plasma CS were observed 5 days after administration of STZ, effects which could be related to the dual effect of diabetes and ACTH release; a less marked, but persistent, adrenal hyperactivity followed, which could be due to ACTH itself or to the action of growth factors such as IGF-I (3,4,6). The changing nature of the adrenal gland response to experimental diabetes, with an early rapid growth and corticosteroid production, has prompted studies of two key systems involved in growth and corticosteroid synthesis, namely, PPRibP formation and the activity of the PPP. PPRibP is essential to increased biosynthetic activity of the adrenal gland because it serves as a primary substrate in both the de novo and salvage pathways of nucleotide synthesis as well as being an activator of the first steps in the de novo routes of Pur and Pyr synthesis (Becker et al., 7). Thus, it is a key element in the provision of precursors for RNA and DNA synthesis. The PPP supplies two essential components for adrenal growth and hyperfunction: R5P, a precursor and known regulatory factor in PPRibP formation (7,8); and NADPH, re-
1429
1 ADRENAL GROWTH AND PPRibP SYNTHESIS
TABLE 1 PPRibP content of the adrenal gland—comparison with other tissues and the effect of anesthesia and a p-blocker
Comparison with a range of normal rat tissues Intact adrenal gland Liver Kidney Lactating mammary gland (10 days) Testis Cerebral cortex Effect of anesthesia and propranolol on adrenal PPRibP Adrenal gland None Na pentobarbital, 30 min Na pentobarbital, 60 min Propranoiol Distribution of PPRibP in rabbit adrenal gland Intact gland Cortex Medulla
PPRibP concentration (nmol/g tissue)
PPRibP synthetase (U/g)
168 ± 10(30) 10.5 ±0.6 (17) 9.5 ± 0.5 (8) 9.7 ± 1.0(9) 5.0 ± 0.3 (20) 5.5 ± 0.4
12.0 ±0.6 (7) 22.7 ± 1.3(6) 25.3 ±0.6 (12) 63.2 ± 6.7 (4) 19.9 ± 1.2(13) 21.0 ± 1.0(4)
Reference
8 14 10 33
165 ± 1 4 (12) 196 ± 2 3 (5) 137 ± 9 (5) 156 ± 12(5) 255, 303 285, 280 276, 294
Values are means ± SE. (n), number of separate experiments. Each value for the.rat.adrenai cortex and medulla was obtained from the pooied samples from adult male rats. Values for the PPRibP conteht of the range of tissues come from published data (except those for testis, which are unpublished observations).
quired for the reductive synthetic steps of corticbsteroid synthesis. In this study, we measured the activity of these two systems in the rat adrenal gland during short-term (3-7 days) and chronic (6 wk) STZ-induced diabetes. RESEARCH DESIGN AND METHODS
Subjects were adult male Wistar rats, initial body weight 210-230 g. Diabetes was induced either by i.v. injection of STZ (60 mg/kg body weight) or i.p. injection of ALX monohydrate (200 mg/kg body weight) as described previously (8). Rats were grouped as follows: STZ-induced diabetic rats; 1) given no further treatment and then killed by cervical dislocation 3 or 7 days after diabetes induction; 2) pretreated with nicotinamide (500 mg/kg body weight) 10 min before STZ injection: this treatment has been shown to counteract the diabetogenic effects of STZ (9); 3) given insulin (4 U Ultratard human insulin and 2 U Actrapid insulin (Novo Nordisk, Bagsvaerd, Denmark) 24 and 48 h after STZ injection and 2 U Actrapid insulin after 72 h:.the animals were killed 1 h after the last injection. ALX-induced diabetic rats; 1) given no further treatment and then killed 3 days after diabetes induction; 2) given insulin (4 U Ultratard human insulin and 2 U Actrapid insulin (Novo Nordisk, Bagsvaerd, Denmark) 24 and 48 h after ALX injection and 2 U of Actrapid insulin aione 72 h after diabetes induction: the rats were killed 1 h after the last injection. Enzyme measurements. For PPRibP synthetase (EC 2.7.6.1.), preparation of the tissue extract and measurement of enzyme activity was as described previously (10). A unit of enzyme activity is 1 ixmole/14CO2 formed from (carboxyl labeled-14C) orotic acid • g~ 1 • h" 1 at 37°C. G-6-PD (EC 1.1.1.49.), 6-PGD (EC i.1.1.44.), transketolase (EC 2.2.1.1.), and transaldolase (EC 2.2.1.2.) were measured by the method of Novello & McLean (11) and Sochor et al. (12) in the high speed supernatant of a 1:5 tissue homogenate prepared in a
1430
medium composed of 0.25 M sucrose • 0.02 M triethanolamine • 0.12 mM dithiothreitol, pH 7.4. Results are given as ixmoles substrate converted/min at 25°C. Flux studies. An estimate of the PPP activity was obtained by measuring the difference in 14CO2 yield from [1-14C]glucdse and [6-14C]glucose as described by Sochor et al. (12) and Larrabee (13). For this study, one adrenal sliced in half and weighing —30 mg, was incubated in 5 ml of Krebs-Ringer bicarbonate, pH 7.4, containing either 5 mM (control rats) or 20 mM (diabetic rats) glucose + 0.5 |xC radioactive glucose. Incubation was for 1 h, gas phase O2/CO2 (95/5) at 37°C. The reaction was stopped with 0.5 ml,5N HCI and the 14CO2 collected in 0.6 ml Scintran (BDH, Poole, Dorset, UK). The Scintran was transferred to vials containing 4.5 nil Optiphase (LKB, Leicester, UK) with the aid of 3 x 0.5 ml methanol and the radioactivity counted in a Beckman LS 7000 scintillation counter. Because the difference in 14 CO2 yield from [1-14G]glucose and [6-14C]glucose is the basis for the measurement of PPP activity, the left and right adrenals from a single rat were used. Chemical procedures. PPRibP was measured in adrenals by the method of Kunjara et al. (14). Because the values obtained in preliminary studies were some 15- to 30-fold greater than had been found in this laboratory in a wide range of normal tissues (Tabie 1), it became necessary to establish that the elevated levels fblind in the adrenal gland were hot artifactual, arising from alarm signals as a result of handling the intact animal. Therefore, parallel measurements were made in animals a) anesthetized for 30 or 60 min with Na pentobarbital (60 mg/kg body weight) before estimation of PPRibP or b) given a p-blocker, propranolol (30 mg/kg body weight) at 24 and 1 h before removal of the adrenal glands to estimate the tissue content of PPRibP. No differences could be detected between the PPRibP content of adrenals taken from animals with no pretreatment (165 ± 14
DIABETES, VOL. 41, NOVEMBER 1992
S. KUNJARA AND ASSOCIATES
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D)
a> r^ i n o CM T-
ppp odd
OO O O
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CM ^ O CO O O
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