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Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet
ARCHIVES
OF BIOCHEMISTRY
Vol. 244, No. 2, February
AND
BIOPHYSICS
1, pp. ‘787-‘794,1986
Precocious Induction of Hepatic Glucokinase and Malic Enzyme in Artificially Reared Rat Pups Fed a High-Carbohydrate Diet PETER
M. HANEY,’
CINDY RAEFSKY AND MULCHAND
ESTRIN, ANGELA S. PATEL’
CALIENDO,
Departments of Biochemistry and Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 4.4106 Received
July
22,1985,
and in revised
form
October
‘7,1985
Glucokinase and NADP:malate dehydrogenase (malic enzyme) first appear in liver when rat pups are weaned from milk which is high in fat to lab chow which is high in carbohydrate. To examine the influence of diet during the early neonatal period, before developmental changes in the circulating concentrations of thyroid and adrenocortical hormones occur, high-carbohydrate formula (56% of calories from carbohydrate), isocaloric and isonitrogenous with rat milk, was intermittently infused via gastrostomy starting on the second day of life. Pups had no further access to their dams. Body weights attained by these pups were at least 90% of those attained by mother-fed pups, which served as controls. In artificially reared rats fed the high-carbohydrate formula, on Day 4, glucokinase and malic enzyme were 30 and 18% of adult activity, respectively; on Day 10, glucokinase and malic enzyme were 71 and 96% of adult activity, respectively. On Days 4 and 10 glucose-6-phosphate dehydrogenase was elevated four- to fivefold in pups fed the high-carbohydrate formula compared to mother-fed pups. A second isocaloric formula, with 22% of calories from carbohydrate but low in protein, resulted in intermediate levels of all three enzymes on Day 10. Pups fed the high-carbohydrate formula has plasma insulin concentrations four- to fivefold greater than mother-fed pups on both Days 4 and 10. Triiodothyronine administration (1 pg/g body wt) on Day 1 enhanced the induction of malic enzyme but not glucokinase on Day 4 in pups fed the high-carbohydrate formula. The results demonstrate that neonatal rat liver is competent to respond to high carbohydrate intake by induction of glucokinase and malic enzyme. 0 1986 Academic
Press. Inc.
The timing of the initial appearance of several key enzymes of carbohydrate and lipid metabolism in developing rat liver allows their individual classification into one of three enzyme “clusters,” late fetal, neonatal, or late suckling (1). Two members of the late suckling cluster, glucokinase (EC 2.7.1.2) and NADP:malate dehydrogenase (malic enzyme) (EC 1.1.1.40), appear in rat liver for the first time during the third postnatal week and attain adult activities
within days. Effects of the transition from rat milk (high in fat) to lab chow (high in carbohydrate) on the development of enzymes of the late suckling cluster have been examined by weaning rats prematurely as early as the sixteenth postnatal day (2) and by weaning rats to a high-fat diet (3). However, these approaches provide little insight into the metabolic potential of younger animals, and interpretation of these studies is complicated by changes in the hormonal milieu which also take place during the third postnatal week, including increases in serum thyroid hormones (4) and corticosterone (5). The influence of
’ Predoetoral Fellow supported by NIH Metabolism Training Grant AM-07319. ’ To whom correspondence should be addressed. 787
0003-9861/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
788
HANEY
these hormonal changes on glucokinase and malic enzyme has been studied by injection of hormones into neonatal rats; appearance of even marginal activity has been interpreted as evidence for an important regulatory role of the hormone in the normal emergence of enzyme activity. However, inherent limitations imposed by the need of suckling rat pups to consume rat milk have restricted examination of nutritional influences on the development of enzyme activity to studies of the shortterm effect of oral or intraperitoneal administration of glucose to suckling pups. The advent of techniques for the artificial rearing of neonatal rats (6) made possible our examination of the capability of neonatal rat liver to induce glucokinase and malic enzyme in response to metabolic conditions normally encountered at the time of weaning. EXPERIMENTAL
PROCEDURES
Materials. ATP (disodium salt), NADP, triiodothyronine (sodium salt), and 6-phosphogluconate dehydrogenase were from Sigma Chemical Company, St. Louis, Missouri. Glucose-g-phosphate dehydrogenase was purchased from Calbiochem, Los Angeles, California. All other reagents were of the highest purity commercially available. Animals. Pregnant Sprague-Dawley rats (Zivic Miller Laboratories, Pittsburgh, Pa.) were fed Purina rat chow and water ad libitum. Pups were born naturally and remained with their dams until the time of cannulation. Pups from litters of less than 9 or greater than 12 pups were not studied. Animals were killed at essentially the same time of day. Rats killed on Day 31 were males which had been weaned to lab chow on Day 21. Artijcial rearing. Rat pups, either 1 or 4 days old, received intragastric cannulas under light ether anesthesia and were raised from that point on in isolation from their dams using an artificial rearing system (6). Modified milk formulas were prepared using a Waring blender and frozen, then thawed as needed and refrigerated for up to 2 days before use. Formulas were delivered for 21- to 29-min periods every 2 h at a rate of 0.45 kcal/g body wt per day. Pups were housed in Styrofoam cups floating on a temperature-regulated water bath. The internal temperature of the cups were maintained at about 32°C. Twice daily, pups were stroked to promote urination and defecation, cleaned, and weighed, and the rate of formula delivery was adjusted. When indicated, pups received intraperitoneal injections of triiodothyronine (1 cg/g body wt
ET
AL.
as 0.01% in 0.9% NaCl, pH 10) on Day 1 just after cannulation; these animals were killed on Day 4. In pilot experiments, pups cannulated on Day 1 tended to pull their cannulas out before Day 17. Therefore, animals killed on Day 1’7 were cannulated on Day 4, when a more secure cannula could be safely introduced. This cannula differed from those used in animals cannulated on Day 1 by having a wider flange (up to 1.1 mm) and a thicker polyethylene disk (1.75 mil). In all groups, survival to the designated time point was about 50-70%; deaths usually occurred within 3 days of cannulation and were associated either with trauma or, more commonly, with a syndrome which included impaired gastric emptying, abdominal distention, and diarrhea. Formulas. Naturally reared animals consumed rat milk, which consists of 68% fat, 24% protein, and 8% carbohydrate on an energy basis (7). Preliminary experiments revealed that a fat content of less than 20% of calories was poorly tolerated. Therefore, the highcarbohydrate formula (8) consisted of 20% fat, 24% protein, and 56% carbohydrate. The high-carbohydrate formula contained skimmed evaporated cow milk (375 ml, Carnation, Los Angeles, Calif.), hydrolyzed casein (21.2 g), L-methionine (500 mg), corn oil (17 g, Best Foods, Englewood Cliffs, N. J.), sodium deoxycholate (85 mg), vitamin mix (10 ml, Munchables Formula 78, Plus Products, Irvine, Calif.), FeSOI* 7HzO (13.5 mg), CuSO,. 5HzO (7.5 mg), ZnSO, * 7HsO (8.0 mg), linolenic acid (750 /.d), Polycose (67.3 g, Ross Laboratories, Columbus, Ohio), and distilled water to a final volume of 500 ml. Preliminary trials of high-carbohydrate formulas containing dextrose and lactose were unsuccessful because of osmotic diarrhea; inclusion of Polycose, a mixture of glucose oligomers, allowed substantial changes in the carbohydrate content of the formula without causing a dangerously high osmotic pressure. A second formula, devised by Messer et al. (9), contained 68% of calories from fat, 10% from protein, and 22% from carbohydrate, was designated as high-fat, and was used to control for dietary and environmental influences not specific to the high-carbohydrate formula. The highcarbohydrate formula was isonitrogenous with rat milk but the high-fat formula was not. Both formulas were isocaloric with rat milk. Enzyme assays. Livers were removed from decapitated rats and homogenized in 5 vol of ice-cold buffer (250 IIIM sucrose, 10 mM Tris, 1 mM mercaptoethanol, pH 7.4). Portions of the homogenates were saved for protein and DNA determination and the remainder was centrifuged at 100,OOOg for 1 h at 4°C. The clear supernatant of this centrifugation was assayed for enzyme activity at 37°C. Hexokinase (EC 2.7.1.1) and glucokinase were assayed as described by Partridge et al. (10). Addition of purified 6-phosphogluconate dehydrogenase to the assay did not affect the rate of formation of NADPH, suggesting that endogeneous
PRECOCIOUS
INDUCTION
OF
GLUCOKINASE
6-phosphogluconate dehydrogenase activity was sufficient to reduce all the 6-phosphogluconate formed. Therefore, the rates of NADPH formation were divided by two in the calculation of hexokinase and glucokinase activity. Glucosed-phosphate dehydrogenase (EC 1.1.1.49) was assayed as described by Langdon (11). Malic enzyme was assayed by the method of Hsu and Lardy (12). Absorbance changes in the blank, usually very small, were subtracted. Passage of cytosol over a Sephadex G-50 column (0.8 X 5.0 cm) reduced absorbance changes in the blank of the malic enzyme assay. One unit of enzyme activity catalyzes the formation of 1 pmol of product per minute. Other detemtinationa Plasma insulin was determined by radioimmunoassay using ‘%I-labeled rat insulin as standard (13). Plasma glucose (14), acetoacetate, and n-3-hydroxybutyrate (15) were determined enzymatically. Liver protein was quantitated with crystalline bovine serum albumin as standard (16). DNA was assayed with calf thymus DNA as standard (17). Glycogen was determined by the method of Van Handel (18). Statistical analysis. Results are presented as means + SEM for groups of four to seven animals. Significance of differences between groups was determined by Student’s t test.
RESULTS
AND
DISCUSSION
Artificially reared rats were fed either high-fat or high-carbohydrate formulas. The high-fat formula contained 68% of calories as fat, the same proportion as in rat milk, while the high-carbohydrate formula contained only 20% of calories as fat. The high-carbohydrate formula contained 56% of calories from carbohydrate, compared to rat milk, which contains only 8% of its calories as carbohydrate. Body weights of pups fed the high-carbohydrate formula approximated those of mother-fed pups at all ages (Table I). Pups fed the high-fat formula had body weights only 76% of control values on Day 10, although the delivery rate of 0.45 kcal/g body wt per day was the same as for the pups fed the high-carbohydrate formula. This difference may be related to the low protein content of the high-fat formula. In contrast to other studies of artificially reared rats (19), no tremors or cataracts were observed. Hepatic glycogen normally accumulates prenatally, but large amounts present at birth are utilized during the perinatal period to maintain the concentration of glu-
AND
MALIC
ENZYME
789
cose in the blood. Thereafter, hepatic glycogen concentration remains at low levels during the suckling period and rises to adult levels after weaning. In contrast, pups fed the high-carbohydrate formula maintained hepatic glycogen concentration at adult levels even as 2-day-olds (Table I). Hepatic glycogen content was elevated between 3- and 5-fold at all ages in pups fed the high-carbohydrate formula compared to mother-fed pups. Pups fed the high-fat formula exhibited hepatic glycogen content intermediate between the other two groups. Differences in liver weight between mother-fed pups and pups fed the highcarbohydrate formula could be attributed to glycogen content; there were no significant differences in liver DNA between agematched pups, and a significant increase in liver protein content was found only on Day 17 in pups fed the high-carbohydrate formula. The high fat content of rat milk causes hyperketonemia in suckling pups. Serum acetoacetate and D-&hydroxybutyrate concentrations normally return to low levels at the time of weaning in naturally reared pups. Pups fed the high-carbohydrate diet had low levels of both ketone bodies as early as Day 4 (Table I). Pups fed the highfat formula exhibited ketone body levels intermediate between the other two groups. Both the relative deficiency in medium-chain triglycerides in the high-fat formula and the high carbohydrate content (22% of calories) may be responsible for this. However, transient hyperglycemia and marked hyperinsulinemia observed in pups fed the high-carbohydrate diet are not observed on weaning of naturally reared pups. Plasma insulin concentration was 93 pU/ml in 2-day-old pups fed the high-carbohydrate diet, compared to only 17 pU/ ml in age-matched mother-fed pups. Strikingly high plasma insulin concentrations were observed in pups fed the high-carbohydrate formula at all ages (Fig. 1). Only transient hyperinsulinemia was observed in pups fed the high-fat formula. Glucose-6-phosphate dehydrogenase activity first appears in rat liver during the prenatal period, but declines after birth in mother-fed pups. The activity of the en-
,,
24.9 + 0.4 19.0 + 0.2* 22.4 + 0.5*
44.8 + 0.5 43.5 + 1.2
117
10
17
Day
Day l-Day MF HF HC
Day I-Day MF HC
Mother-fed, MF
1540 2 20
1290 + 20 1830 k 10*
633 f 15 636 f 22 999 f 24*
374 + 18 341 + 11 470 k 25*
368 2 17 388 + 24 389 + 16
Weight (mg)
ND
135.2 ? 3.6 178.5 + 5.5*
101.9 + 4.0 97.3 t 4.1 106.8 + 7.7
75.2 + 1.7 72.0 + 2.1 83.4 + 4.8
ND ND ND
(mg)
ND
2.94 f 0.12 2.81 & 0.17
2.90 + 0.03 3.01 + 0.25 2.78 + 0.10
2.46 t 0.10 2.31 f 0.10 2.46 f 0.07
ND ND ND 1.5 6.2* 5.9*
73.5 f
25.9 + 63.0 +
9.2
1.3 4.8*
23.2 k 1.1 65.0 f 3.9* 78.0 k 15.5*
19.2 + 38.8 t 79.8 f
ND 46.9 f 11.3 66.0 k 16.8
mg/g of liver
ON LIVER
Protein DNA bg)
I
FORMULAS
TABLE
Liver
MILK
Liver
AND HIGH-FAT
1.0 1.s* 2.6*
5.1 5.8
5.2 +
0.3
33.5 + 1.9 116.1 + 12.7*
14.7 + 0.9 40.5 + 3.1* 77.1 + 14.s*
7.3 + 19.1 * 36.0 f
ND 18.8 f 24.9 +
mg/liver
glycogen
CONSTITUENTS
354
0.3 0.1*
0.3 0.3* 1.1
0.4 0.9* 1.6
0.2 1.3 0.3*
k 54
7.2 IL 5.9 +
7.8 -t 6.1 k 6.2 +
7.5 * 9.1 + 8.9 +
6.5 + 7.6 f 8.6 +
67f
7
406 + 11 51 f 4*
362 + 13 275 f 21* 88 f lO*
531 f 38 372 f 15* 41 + 4*
660 + 63 ND ND
AcAc (PM)
Serum
METABOLITES
Glucose (m@
AND SERUM
81 9*
58 62* 15*
131 zk 12
830 + 772
603 ?I 440 f 136 f
1045 f 111 596 + lOO* 242 + 51*
775 + 144 662 + 158 289 + 21*
BOHB (PM)
“. * .,
._-
,-
I-
_- ,-
-
-- t-
-
._- ,-.- “-
--
-^- --
“-- “_”
_..-
“-
-
,.- _-- --
--__ “-- .-‘ .,.^_ - ~.- --
--
-,
-
Note. Rat pups were artifically reared as described under Experimental Procedures. Mother-fed (MF) pups served as controls. The contribution of calories from carbohydrate was 56% in the high-carbohydrate formula (HC) and 8% in rat milk. High-fat formula (HF) contained 22% of calories from carbohydrate. Rats killed on Day 1’7 were cannulated on Day 4; all other artifically reared rats were cannulated on Day 1. Assays of protein, DNA, glycogen, glucose, acetoacetate (AcAc), and D-3-hydroxybutyrate (BOHB) were performed as described under Experimental Procedures. Results are means + SEM of four to seven pups per group. Body weights and serum metabolite concentrations in 10 and 1’7-day-old pups have been reported previously (8). N.D., not determined. * Significant difference (P < 0.05) between naturally and artificially reared pups.
+ 3.7
12.4 ? 0.1 11.7 f 0.2* 11.2 + 0.1*
4
Day l-Day MF HF HC
31
10.0 + 0.1 9.8 t 0.1 9.8 f 0.2
2
wt W
OF HIGH-CARBOHYDRATE
Day l-Day MF HF HC
Body
EFFECT
-_. -
b
z
z
PRECOCIOUS
I OO
INDUCTION
I
I
5
IO Postnotol
OF
GLUCOKINASE
I 15 cgs (days.1
/+e
FIG. 1. Plasma insulin concentration in pups artificially reared on high-carbohydrate (A) and high-fat (Cl) milk formulas and in naturally reared (0) pups. Results are means + SEM for four to seven animals per experimental point.
zyme was elevated 3- to &fold at all ages in pups fed the high-carbohydrate formula (Fig. Z), in keeping with the high concentrations of plasma insulin. Induction of glucose-6-phosphate dehydrogenase activity not only serves to enhance catabolism of glucose by the pentose phosphate pathway, but also provides NADPH required for lipogenesis. Glucose-&phosphate dehydrogenase activity in livers of pups fed the high-fat formula was significantly increased compared to livers of mother-fed pups, in contrast to a previous report (20). Hexokinase activity peaks in rat liver during the fetal period and then gradually declines during the suckling period to the low levels characteristic of adulthood. Marked increases in hexokinase activity compared to age-matched mother-fed pups were observed on Day 4 in pups fed the high-carbohydrate formula and in pups fed the high-fat formula, but on Day 10 there were no significant differences among the groups (Fig. 3). This is the first report of a dietary effect on the activity of hexokinase. The question of which of the three isoenzymes of hexokinase described by Ureta et
AND
MALIC
791
ENZYME
al. (21) is affected remains to be investigated. Several factors are implicated in the regulation of glucokinase in developing rat liver. Walker and Eaton (2) found that 16day-old rats, but not younger rats, respond to a high-carbohydrate diet by induction of glucokinase, and that weaning animals to a high-fat diet delays glucokinase induction. The reported increase in hepatic glucokinase activity in g-day-old rats after injection of hydrocortisone and intraperitoneal glucose (22) has not been confirmed in subsequent reports (10,23). The possible importance of triiodothyronine was suggested by the correlation between circulating triiodothyronine levels and glucokinase activity and by the induction of marginal glucokinase activity in livers of 2- to 16-day-old rats after combined administration of triiodothyronine and glucose (10). However, hypothyroid animals respond to oral glucose administration at the time of weaning with glucokinase ac-
I OO
I 5
I IO Postnatal
I 09s
I5 (days)
‘31
FIG. 2. Hepatic glucose-6-phosphate dehydrogenase activity in pups artificially reared on high-carbohydrate (A) and high-fat (Cl) milk formulas and in naturally reared (0) pups. Enzyme activity on Day 31 is equivalent to 3.1 U/g liver. Enzyme activity in liver of pups injected with 1 pg/g body wt triiodothyronine is also shown (A, 0). Results are means + SEM for four to seven animals per experimental point.
792
HANEY
Postnotol
age
(days)
FIG. 3. Hepatic hexokinase activity in pups artificially reared on high-carbohydrate (A) and high-fat (0) milk formulas and in naturally reared (0) pups. Enzyme activity in liver of pups injected with 1 pg/g body wt triiodothyronine is also shown (A, 0). Enzyme activity on Day 31 is equivalent to 0.34 U/g liver. Results are means + SEM for four to seven animals per experimental point.
tivity equal to that of age-matched control animals (24). The relative amounts of glucose and insulin in the plasma were suggested as important determinants of hepatic glucokinase activity by Wakelam et al. (25), who observed that induction of glucokinase to about 30% of adult levels after oral glucose administration to 13-dayold rats was inhibited by mannoheptulose, which inhibited insulin secretion, and by galactose, which stimulated insulin secretion. The inability to experimentally induce appreciable glucokinase activity before the time at which it normally appears led to the conclusion that a sequence of differentiation events must occur to permit the establishment of competence to synthesize the enzyme (26). However, pups fed the high-carbohydrate formula had 30% of adult glucokinase activity by Day 4 and 71% of adult activity by Day 10 (Fig. 4), indicating that competence for glucokinase synthesis exists at a very early age but requires an effective stimulus for its dem-
ET
AL.
onstration. The suggestion that excessive insulin secretion prevents glucokinase induction (25) is inconsistent with the striking hyperinsulinemia observed in pups fed the high-carbohydrate formula. The high galactose content of the high-carbohydrate formula (approximately threefold elevated compared to rat milk) did not prevent glucokinase induction, indicating that the galactose component of rat milk is not a potent inhibitor of glucokinase induction. The results do not support a role for thyroid hormones in the development of glucokinase activity. Injection of triiodothyronine into pups fed the high-carbohydrate formula did not enhance glucokinase activity compared to control animals. The possibility exists that thyroid hormone metabolism is altered in pups fed the high-carbohydrate formula, since nutritional effects on the peripheral conversion of thyroxine to triiodothyronine are well documented in adult rats (27). The data indicate that nutritional changes normally en-
Postnatal
age (days)
FIG. 4. Hepatic glucokinase activity in pups artificially reared on high-carbohydrate (A) and high-fat (0) milk formulas and in naturally reared (0) pups. Enzyme activity in liver of pups injected with 1 pg/g body wt triiodothyronine is also shown (A, 0). Enzyme activity on Day 31 is equivalent to 0.92 U/g liver. Results are means f SEM for four to seven animals per experimental point.
PRECOCIOUS
INDUCTION
OF
GLUCOKINASE
countered during the third postnatal week which result in increased plasma insulin and, presumably, decreased plasma glycagon, are sufficient to cause induction of significant glucokinase activity even in neonates. The precocious induction of malic enzyme was observed in 16-day-old rats after weaning prematurely to a high-carbohydrate diet, but rats weaned to a carbohydrate-free diet eventually develop adult levels of malic enzyme activity (19). Acute intragastric administration of glucose to 14-day-old pups did not cause induction of malic enzyme (28), but injection of triiodothyronine resulted in adult activity of malic enzyme in g-day-old pups (28). Pups injected with IX-thyroxine and then killed on Day 2 had 22% of the malic enzyme activity observed in 35-day-old pups (29). In the results reported here, pups fed the high-carbohydrate formula had 18% of adult activity by Day 4 and 96% of adult activity by Day 10 (Fig. 5), indicating that
Postnatal
age (days)
FIG. 5. Hepatic malic enzyme activity in pups artificially reared on high-carbohydrate (A) and highfat (0) milk formulas and in naturally reared (0) pups. Enzyme activity in liver of pups injected with 1 pgg/g body wt triiodothyronine is also shown (A, 0). Enzyme activity on Day 31 is equivalent to 4.10 U/g liver. Results are means + SEM for four to seven animals per experimental point.
AND
MALIC
ENZYME
793
dietary manipulation alone can cause precocious induction of malic enzyme. Furthermore, a synergistic effect of dietary and hormonal stimulation of malic enzyme activity was observed. Pups injected with triiodothyronine (1 pg/g body wt) on Day 1 and fed the high-carbohydrate formula show an induction of malic enzyme activity to 2-fold above the adult level by postnatal Day 4. The injection of triiodothyronine into mother-fed pups on Day 1 resulted in malic enzyme levels on Day 4 that were approximately one-third of the adult activity. Thus, the results indicate the importance of both dietary carbohydrate and serum triiodothyronine in the regulation of malic enzyme. The levels of glucose-&phosphate dehydrogenase, hexokinase, glucokinase, and malic enzyme in pups fed the high-carbohydrate formula are the highest ever observed in rats at this stage of development. Intermediate activities of all enzymes were observed in animals fed the high-fat diet, which contained 22% of calories as carbohydrate, compared to 56% of calories as carbohydrate in the high-carbohydrate formula and only 8% of calories as carbohydrate in rat milk. Induction of glucose6-phosphate dehydrogenase was maximal by Day 4, but accumulation of glucokinase and malic enzyme in pups fed the highcarbohydrate diet followed a slower time course, since slowly degraded enzymes approach new steady-state levels more slowly than rapidly degraded enzyme (30). Substantial activities of both enzymes in loday-old rats fed the high-carbohydrate formula suggest that developmental events during the third postnatal week do not play a critical role. Even as neonates, rats can successfully adapt to the demands for enhanced carbohydrate metabolism and lipogenesis normally encountered at the time of weaning. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Service Grant HD-15778. We gratefully acknowledge the assistance of Veanne Anderson and Dr. Grant Smith of McMaster University, Hamilton, Ontario, Canada, and Christopher Fomon and Dr. Samuel Fomon of the University of Iowa, Iowa City, regarding
794
HANEY
artificial rearing of rat pups. We thank Dr. Alan Goodridge and Dr. Richard Miller for their careful reading of the manuscript and valuable suggestions.
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OF BIOCHEMISTRY
Vol. 244, No. 2, February
AND
BIOPHYSICS
1, pp. ‘787-‘794,1986
Precocious Induction of Hepatic Glucokinase and Malic Enzyme in Artificially Reared Rat Pups Fed a High-Carbohydrate Diet PETER
M. HANEY,’
CINDY RAEFSKY AND MULCHAND
ESTRIN, ANGELA S. PATEL’
CALIENDO,
Departments of Biochemistry and Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 4.4106 Received
July
22,1985,
and in revised
form
October
‘7,1985
Glucokinase and NADP:malate dehydrogenase (malic enzyme) first appear in liver when rat pups are weaned from milk which is high in fat to lab chow which is high in carbohydrate. To examine the influence of diet during the early neonatal period, before developmental changes in the circulating concentrations of thyroid and adrenocortical hormones occur, high-carbohydrate formula (56% of calories from carbohydrate), isocaloric and isonitrogenous with rat milk, was intermittently infused via gastrostomy starting on the second day of life. Pups had no further access to their dams. Body weights attained by these pups were at least 90% of those attained by mother-fed pups, which served as controls. In artificially reared rats fed the high-carbohydrate formula, on Day 4, glucokinase and malic enzyme were 30 and 18% of adult activity, respectively; on Day 10, glucokinase and malic enzyme were 71 and 96% of adult activity, respectively. On Days 4 and 10 glucose-6-phosphate dehydrogenase was elevated four- to fivefold in pups fed the high-carbohydrate formula compared to mother-fed pups. A second isocaloric formula, with 22% of calories from carbohydrate but low in protein, resulted in intermediate levels of all three enzymes on Day 10. Pups fed the high-carbohydrate formula has plasma insulin concentrations four- to fivefold greater than mother-fed pups on both Days 4 and 10. Triiodothyronine administration (1 pg/g body wt) on Day 1 enhanced the induction of malic enzyme but not glucokinase on Day 4 in pups fed the high-carbohydrate formula. The results demonstrate that neonatal rat liver is competent to respond to high carbohydrate intake by induction of glucokinase and malic enzyme. 0 1986 Academic
Press. Inc.
The timing of the initial appearance of several key enzymes of carbohydrate and lipid metabolism in developing rat liver allows their individual classification into one of three enzyme “clusters,” late fetal, neonatal, or late suckling (1). Two members of the late suckling cluster, glucokinase (EC 2.7.1.2) and NADP:malate dehydrogenase (malic enzyme) (EC 1.1.1.40), appear in rat liver for the first time during the third postnatal week and attain adult activities
within days. Effects of the transition from rat milk (high in fat) to lab chow (high in carbohydrate) on the development of enzymes of the late suckling cluster have been examined by weaning rats prematurely as early as the sixteenth postnatal day (2) and by weaning rats to a high-fat diet (3). However, these approaches provide little insight into the metabolic potential of younger animals, and interpretation of these studies is complicated by changes in the hormonal milieu which also take place during the third postnatal week, including increases in serum thyroid hormones (4) and corticosterone (5). The influence of
’ Predoetoral Fellow supported by NIH Metabolism Training Grant AM-07319. ’ To whom correspondence should be addressed. 787
0003-9861/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
788
HANEY
these hormonal changes on glucokinase and malic enzyme has been studied by injection of hormones into neonatal rats; appearance of even marginal activity has been interpreted as evidence for an important regulatory role of the hormone in the normal emergence of enzyme activity. However, inherent limitations imposed by the need of suckling rat pups to consume rat milk have restricted examination of nutritional influences on the development of enzyme activity to studies of the shortterm effect of oral or intraperitoneal administration of glucose to suckling pups. The advent of techniques for the artificial rearing of neonatal rats (6) made possible our examination of the capability of neonatal rat liver to induce glucokinase and malic enzyme in response to metabolic conditions normally encountered at the time of weaning. EXPERIMENTAL
PROCEDURES
Materials. ATP (disodium salt), NADP, triiodothyronine (sodium salt), and 6-phosphogluconate dehydrogenase were from Sigma Chemical Company, St. Louis, Missouri. Glucose-g-phosphate dehydrogenase was purchased from Calbiochem, Los Angeles, California. All other reagents were of the highest purity commercially available. Animals. Pregnant Sprague-Dawley rats (Zivic Miller Laboratories, Pittsburgh, Pa.) were fed Purina rat chow and water ad libitum. Pups were born naturally and remained with their dams until the time of cannulation. Pups from litters of less than 9 or greater than 12 pups were not studied. Animals were killed at essentially the same time of day. Rats killed on Day 31 were males which had been weaned to lab chow on Day 21. Artijcial rearing. Rat pups, either 1 or 4 days old, received intragastric cannulas under light ether anesthesia and were raised from that point on in isolation from their dams using an artificial rearing system (6). Modified milk formulas were prepared using a Waring blender and frozen, then thawed as needed and refrigerated for up to 2 days before use. Formulas were delivered for 21- to 29-min periods every 2 h at a rate of 0.45 kcal/g body wt per day. Pups were housed in Styrofoam cups floating on a temperature-regulated water bath. The internal temperature of the cups were maintained at about 32°C. Twice daily, pups were stroked to promote urination and defecation, cleaned, and weighed, and the rate of formula delivery was adjusted. When indicated, pups received intraperitoneal injections of triiodothyronine (1 cg/g body wt
ET
AL.
as 0.01% in 0.9% NaCl, pH 10) on Day 1 just after cannulation; these animals were killed on Day 4. In pilot experiments, pups cannulated on Day 1 tended to pull their cannulas out before Day 17. Therefore, animals killed on Day 1’7 were cannulated on Day 4, when a more secure cannula could be safely introduced. This cannula differed from those used in animals cannulated on Day 1 by having a wider flange (up to 1.1 mm) and a thicker polyethylene disk (1.75 mil). In all groups, survival to the designated time point was about 50-70%; deaths usually occurred within 3 days of cannulation and were associated either with trauma or, more commonly, with a syndrome which included impaired gastric emptying, abdominal distention, and diarrhea. Formulas. Naturally reared animals consumed rat milk, which consists of 68% fat, 24% protein, and 8% carbohydrate on an energy basis (7). Preliminary experiments revealed that a fat content of less than 20% of calories was poorly tolerated. Therefore, the highcarbohydrate formula (8) consisted of 20% fat, 24% protein, and 56% carbohydrate. The high-carbohydrate formula contained skimmed evaporated cow milk (375 ml, Carnation, Los Angeles, Calif.), hydrolyzed casein (21.2 g), L-methionine (500 mg), corn oil (17 g, Best Foods, Englewood Cliffs, N. J.), sodium deoxycholate (85 mg), vitamin mix (10 ml, Munchables Formula 78, Plus Products, Irvine, Calif.), FeSOI* 7HzO (13.5 mg), CuSO,. 5HzO (7.5 mg), ZnSO, * 7HsO (8.0 mg), linolenic acid (750 /.d), Polycose (67.3 g, Ross Laboratories, Columbus, Ohio), and distilled water to a final volume of 500 ml. Preliminary trials of high-carbohydrate formulas containing dextrose and lactose were unsuccessful because of osmotic diarrhea; inclusion of Polycose, a mixture of glucose oligomers, allowed substantial changes in the carbohydrate content of the formula without causing a dangerously high osmotic pressure. A second formula, devised by Messer et al. (9), contained 68% of calories from fat, 10% from protein, and 22% from carbohydrate, was designated as high-fat, and was used to control for dietary and environmental influences not specific to the high-carbohydrate formula. The highcarbohydrate formula was isonitrogenous with rat milk but the high-fat formula was not. Both formulas were isocaloric with rat milk. Enzyme assays. Livers were removed from decapitated rats and homogenized in 5 vol of ice-cold buffer (250 IIIM sucrose, 10 mM Tris, 1 mM mercaptoethanol, pH 7.4). Portions of the homogenates were saved for protein and DNA determination and the remainder was centrifuged at 100,OOOg for 1 h at 4°C. The clear supernatant of this centrifugation was assayed for enzyme activity at 37°C. Hexokinase (EC 2.7.1.1) and glucokinase were assayed as described by Partridge et al. (10). Addition of purified 6-phosphogluconate dehydrogenase to the assay did not affect the rate of formation of NADPH, suggesting that endogeneous
PRECOCIOUS
INDUCTION
OF
GLUCOKINASE
6-phosphogluconate dehydrogenase activity was sufficient to reduce all the 6-phosphogluconate formed. Therefore, the rates of NADPH formation were divided by two in the calculation of hexokinase and glucokinase activity. Glucosed-phosphate dehydrogenase (EC 1.1.1.49) was assayed as described by Langdon (11). Malic enzyme was assayed by the method of Hsu and Lardy (12). Absorbance changes in the blank, usually very small, were subtracted. Passage of cytosol over a Sephadex G-50 column (0.8 X 5.0 cm) reduced absorbance changes in the blank of the malic enzyme assay. One unit of enzyme activity catalyzes the formation of 1 pmol of product per minute. Other detemtinationa Plasma insulin was determined by radioimmunoassay using ‘%I-labeled rat insulin as standard (13). Plasma glucose (14), acetoacetate, and n-3-hydroxybutyrate (15) were determined enzymatically. Liver protein was quantitated with crystalline bovine serum albumin as standard (16). DNA was assayed with calf thymus DNA as standard (17). Glycogen was determined by the method of Van Handel (18). Statistical analysis. Results are presented as means + SEM for groups of four to seven animals. Significance of differences between groups was determined by Student’s t test.
RESULTS
AND
DISCUSSION
Artificially reared rats were fed either high-fat or high-carbohydrate formulas. The high-fat formula contained 68% of calories as fat, the same proportion as in rat milk, while the high-carbohydrate formula contained only 20% of calories as fat. The high-carbohydrate formula contained 56% of calories from carbohydrate, compared to rat milk, which contains only 8% of its calories as carbohydrate. Body weights of pups fed the high-carbohydrate formula approximated those of mother-fed pups at all ages (Table I). Pups fed the high-fat formula had body weights only 76% of control values on Day 10, although the delivery rate of 0.45 kcal/g body wt per day was the same as for the pups fed the high-carbohydrate formula. This difference may be related to the low protein content of the high-fat formula. In contrast to other studies of artificially reared rats (19), no tremors or cataracts were observed. Hepatic glycogen normally accumulates prenatally, but large amounts present at birth are utilized during the perinatal period to maintain the concentration of glu-
AND
MALIC
ENZYME
789
cose in the blood. Thereafter, hepatic glycogen concentration remains at low levels during the suckling period and rises to adult levels after weaning. In contrast, pups fed the high-carbohydrate formula maintained hepatic glycogen concentration at adult levels even as 2-day-olds (Table I). Hepatic glycogen content was elevated between 3- and 5-fold at all ages in pups fed the high-carbohydrate formula compared to mother-fed pups. Pups fed the high-fat formula exhibited hepatic glycogen content intermediate between the other two groups. Differences in liver weight between mother-fed pups and pups fed the highcarbohydrate formula could be attributed to glycogen content; there were no significant differences in liver DNA between agematched pups, and a significant increase in liver protein content was found only on Day 17 in pups fed the high-carbohydrate formula. The high fat content of rat milk causes hyperketonemia in suckling pups. Serum acetoacetate and D-&hydroxybutyrate concentrations normally return to low levels at the time of weaning in naturally reared pups. Pups fed the high-carbohydrate diet had low levels of both ketone bodies as early as Day 4 (Table I). Pups fed the highfat formula exhibited ketone body levels intermediate between the other two groups. Both the relative deficiency in medium-chain triglycerides in the high-fat formula and the high carbohydrate content (22% of calories) may be responsible for this. However, transient hyperglycemia and marked hyperinsulinemia observed in pups fed the high-carbohydrate diet are not observed on weaning of naturally reared pups. Plasma insulin concentration was 93 pU/ml in 2-day-old pups fed the high-carbohydrate diet, compared to only 17 pU/ ml in age-matched mother-fed pups. Strikingly high plasma insulin concentrations were observed in pups fed the high-carbohydrate formula at all ages (Fig. 1). Only transient hyperinsulinemia was observed in pups fed the high-fat formula. Glucose-6-phosphate dehydrogenase activity first appears in rat liver during the prenatal period, but declines after birth in mother-fed pups. The activity of the en-
,,
24.9 + 0.4 19.0 + 0.2* 22.4 + 0.5*
44.8 + 0.5 43.5 + 1.2
117
10
17
Day
Day l-Day MF HF HC
Day I-Day MF HC
Mother-fed, MF
1540 2 20
1290 + 20 1830 k 10*
633 f 15 636 f 22 999 f 24*
374 + 18 341 + 11 470 k 25*
368 2 17 388 + 24 389 + 16
Weight (mg)
ND
135.2 ? 3.6 178.5 + 5.5*
101.9 + 4.0 97.3 t 4.1 106.8 + 7.7
75.2 + 1.7 72.0 + 2.1 83.4 + 4.8
ND ND ND
(mg)
ND
2.94 f 0.12 2.81 & 0.17
2.90 + 0.03 3.01 + 0.25 2.78 + 0.10
2.46 t 0.10 2.31 f 0.10 2.46 f 0.07
ND ND ND 1.5 6.2* 5.9*
73.5 f
25.9 + 63.0 +
9.2
1.3 4.8*
23.2 k 1.1 65.0 f 3.9* 78.0 k 15.5*
19.2 + 38.8 t 79.8 f
ND 46.9 f 11.3 66.0 k 16.8
mg/g of liver
ON LIVER
Protein DNA bg)
I
FORMULAS
TABLE
Liver
MILK
Liver
AND HIGH-FAT
1.0 1.s* 2.6*
5.1 5.8
5.2 +
0.3
33.5 + 1.9 116.1 + 12.7*
14.7 + 0.9 40.5 + 3.1* 77.1 + 14.s*
7.3 + 19.1 * 36.0 f
ND 18.8 f 24.9 +
mg/liver
glycogen
CONSTITUENTS
354
0.3 0.1*
0.3 0.3* 1.1
0.4 0.9* 1.6
0.2 1.3 0.3*
k 54
7.2 IL 5.9 +
7.8 -t 6.1 k 6.2 +
7.5 * 9.1 + 8.9 +
6.5 + 7.6 f 8.6 +
67f
7
406 + 11 51 f 4*
362 + 13 275 f 21* 88 f lO*
531 f 38 372 f 15* 41 + 4*
660 + 63 ND ND
AcAc (PM)
Serum
METABOLITES
Glucose (m@
AND SERUM
81 9*
58 62* 15*
131 zk 12
830 + 772
603 ?I 440 f 136 f
1045 f 111 596 + lOO* 242 + 51*
775 + 144 662 + 158 289 + 21*
BOHB (PM)
“. * .,
._-
,-
I-
_- ,-
-
-- t-
-
._- ,-.- “-
--
-^- --
“-- “_”
_..-
“-
-
,.- _-- --
--__ “-- .-‘ .,.^_ - ~.- --
--
-,
-
Note. Rat pups were artifically reared as described under Experimental Procedures. Mother-fed (MF) pups served as controls. The contribution of calories from carbohydrate was 56% in the high-carbohydrate formula (HC) and 8% in rat milk. High-fat formula (HF) contained 22% of calories from carbohydrate. Rats killed on Day 1’7 were cannulated on Day 4; all other artifically reared rats were cannulated on Day 1. Assays of protein, DNA, glycogen, glucose, acetoacetate (AcAc), and D-3-hydroxybutyrate (BOHB) were performed as described under Experimental Procedures. Results are means + SEM of four to seven pups per group. Body weights and serum metabolite concentrations in 10 and 1’7-day-old pups have been reported previously (8). N.D., not determined. * Significant difference (P < 0.05) between naturally and artificially reared pups.
+ 3.7
12.4 ? 0.1 11.7 f 0.2* 11.2 + 0.1*
4
Day l-Day MF HF HC
31
10.0 + 0.1 9.8 t 0.1 9.8 f 0.2
2
wt W
OF HIGH-CARBOHYDRATE
Day l-Day MF HF HC
Body
EFFECT
-_. -
b
z
z
PRECOCIOUS
I OO
INDUCTION
I
I
5
IO Postnotol
OF
GLUCOKINASE
I 15 cgs (days.1
/+e
FIG. 1. Plasma insulin concentration in pups artificially reared on high-carbohydrate (A) and high-fat (Cl) milk formulas and in naturally reared (0) pups. Results are means + SEM for four to seven animals per experimental point.
zyme was elevated 3- to &fold at all ages in pups fed the high-carbohydrate formula (Fig. Z), in keeping with the high concentrations of plasma insulin. Induction of glucose-6-phosphate dehydrogenase activity not only serves to enhance catabolism of glucose by the pentose phosphate pathway, but also provides NADPH required for lipogenesis. Glucose-&phosphate dehydrogenase activity in livers of pups fed the high-fat formula was significantly increased compared to livers of mother-fed pups, in contrast to a previous report (20). Hexokinase activity peaks in rat liver during the fetal period and then gradually declines during the suckling period to the low levels characteristic of adulthood. Marked increases in hexokinase activity compared to age-matched mother-fed pups were observed on Day 4 in pups fed the high-carbohydrate formula and in pups fed the high-fat formula, but on Day 10 there were no significant differences among the groups (Fig. 3). This is the first report of a dietary effect on the activity of hexokinase. The question of which of the three isoenzymes of hexokinase described by Ureta et
AND
MALIC
791
ENZYME
al. (21) is affected remains to be investigated. Several factors are implicated in the regulation of glucokinase in developing rat liver. Walker and Eaton (2) found that 16day-old rats, but not younger rats, respond to a high-carbohydrate diet by induction of glucokinase, and that weaning animals to a high-fat diet delays glucokinase induction. The reported increase in hepatic glucokinase activity in g-day-old rats after injection of hydrocortisone and intraperitoneal glucose (22) has not been confirmed in subsequent reports (10,23). The possible importance of triiodothyronine was suggested by the correlation between circulating triiodothyronine levels and glucokinase activity and by the induction of marginal glucokinase activity in livers of 2- to 16-day-old rats after combined administration of triiodothyronine and glucose (10). However, hypothyroid animals respond to oral glucose administration at the time of weaning with glucokinase ac-
I OO
I 5
I IO Postnatal
I 09s
I5 (days)
‘31
FIG. 2. Hepatic glucose-6-phosphate dehydrogenase activity in pups artificially reared on high-carbohydrate (A) and high-fat (Cl) milk formulas and in naturally reared (0) pups. Enzyme activity on Day 31 is equivalent to 3.1 U/g liver. Enzyme activity in liver of pups injected with 1 pg/g body wt triiodothyronine is also shown (A, 0). Results are means + SEM for four to seven animals per experimental point.
792
HANEY
Postnotol
age
(days)
FIG. 3. Hepatic hexokinase activity in pups artificially reared on high-carbohydrate (A) and high-fat (0) milk formulas and in naturally reared (0) pups. Enzyme activity in liver of pups injected with 1 pg/g body wt triiodothyronine is also shown (A, 0). Enzyme activity on Day 31 is equivalent to 0.34 U/g liver. Results are means + SEM for four to seven animals per experimental point.
tivity equal to that of age-matched control animals (24). The relative amounts of glucose and insulin in the plasma were suggested as important determinants of hepatic glucokinase activity by Wakelam et al. (25), who observed that induction of glucokinase to about 30% of adult levels after oral glucose administration to 13-dayold rats was inhibited by mannoheptulose, which inhibited insulin secretion, and by galactose, which stimulated insulin secretion. The inability to experimentally induce appreciable glucokinase activity before the time at which it normally appears led to the conclusion that a sequence of differentiation events must occur to permit the establishment of competence to synthesize the enzyme (26). However, pups fed the high-carbohydrate formula had 30% of adult glucokinase activity by Day 4 and 71% of adult activity by Day 10 (Fig. 4), indicating that competence for glucokinase synthesis exists at a very early age but requires an effective stimulus for its dem-
ET
AL.
onstration. The suggestion that excessive insulin secretion prevents glucokinase induction (25) is inconsistent with the striking hyperinsulinemia observed in pups fed the high-carbohydrate formula. The high galactose content of the high-carbohydrate formula (approximately threefold elevated compared to rat milk) did not prevent glucokinase induction, indicating that the galactose component of rat milk is not a potent inhibitor of glucokinase induction. The results do not support a role for thyroid hormones in the development of glucokinase activity. Injection of triiodothyronine into pups fed the high-carbohydrate formula did not enhance glucokinase activity compared to control animals. The possibility exists that thyroid hormone metabolism is altered in pups fed the high-carbohydrate formula, since nutritional effects on the peripheral conversion of thyroxine to triiodothyronine are well documented in adult rats (27). The data indicate that nutritional changes normally en-
Postnatal
age (days)
FIG. 4. Hepatic glucokinase activity in pups artificially reared on high-carbohydrate (A) and high-fat (0) milk formulas and in naturally reared (0) pups. Enzyme activity in liver of pups injected with 1 pg/g body wt triiodothyronine is also shown (A, 0). Enzyme activity on Day 31 is equivalent to 0.92 U/g liver. Results are means f SEM for four to seven animals per experimental point.
PRECOCIOUS
INDUCTION
OF
GLUCOKINASE
countered during the third postnatal week which result in increased plasma insulin and, presumably, decreased plasma glycagon, are sufficient to cause induction of significant glucokinase activity even in neonates. The precocious induction of malic enzyme was observed in 16-day-old rats after weaning prematurely to a high-carbohydrate diet, but rats weaned to a carbohydrate-free diet eventually develop adult levels of malic enzyme activity (19). Acute intragastric administration of glucose to 14-day-old pups did not cause induction of malic enzyme (28), but injection of triiodothyronine resulted in adult activity of malic enzyme in g-day-old pups (28). Pups injected with IX-thyroxine and then killed on Day 2 had 22% of the malic enzyme activity observed in 35-day-old pups (29). In the results reported here, pups fed the high-carbohydrate formula had 18% of adult activity by Day 4 and 96% of adult activity by Day 10 (Fig. 5), indicating that
Postnatal
age (days)
FIG. 5. Hepatic malic enzyme activity in pups artificially reared on high-carbohydrate (A) and highfat (0) milk formulas and in naturally reared (0) pups. Enzyme activity in liver of pups injected with 1 pgg/g body wt triiodothyronine is also shown (A, 0). Enzyme activity on Day 31 is equivalent to 4.10 U/g liver. Results are means + SEM for four to seven animals per experimental point.
AND
MALIC
ENZYME
793
dietary manipulation alone can cause precocious induction of malic enzyme. Furthermore, a synergistic effect of dietary and hormonal stimulation of malic enzyme activity was observed. Pups injected with triiodothyronine (1 pg/g body wt) on Day 1 and fed the high-carbohydrate formula show an induction of malic enzyme activity to 2-fold above the adult level by postnatal Day 4. The injection of triiodothyronine into mother-fed pups on Day 1 resulted in malic enzyme levels on Day 4 that were approximately one-third of the adult activity. Thus, the results indicate the importance of both dietary carbohydrate and serum triiodothyronine in the regulation of malic enzyme. The levels of glucose-&phosphate dehydrogenase, hexokinase, glucokinase, and malic enzyme in pups fed the high-carbohydrate formula are the highest ever observed in rats at this stage of development. Intermediate activities of all enzymes were observed in animals fed the high-fat diet, which contained 22% of calories as carbohydrate, compared to 56% of calories as carbohydrate in the high-carbohydrate formula and only 8% of calories as carbohydrate in rat milk. Induction of glucose6-phosphate dehydrogenase was maximal by Day 4, but accumulation of glucokinase and malic enzyme in pups fed the highcarbohydrate diet followed a slower time course, since slowly degraded enzymes approach new steady-state levels more slowly than rapidly degraded enzyme (30). Substantial activities of both enzymes in loday-old rats fed the high-carbohydrate formula suggest that developmental events during the third postnatal week do not play a critical role. Even as neonates, rats can successfully adapt to the demands for enhanced carbohydrate metabolism and lipogenesis normally encountered at the time of weaning. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Service Grant HD-15778. We gratefully acknowledge the assistance of Veanne Anderson and Dr. Grant Smith of McMaster University, Hamilton, Ontario, Canada, and Christopher Fomon and Dr. Samuel Fomon of the University of Iowa, Iowa City, regarding
794
HANEY
artificial rearing of rat pups. We thank Dr. Alan Goodridge and Dr. Richard Miller for their careful reading of the manuscript and valuable suggestions.
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