Development and Control of Intestinal and Hepatic Fructokinase
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INTESTINAL AND HEPATIC FRUCTOKINASE 765 expansion on intrarenal distribution of plasma flow in the dog. 42. The authors acknowledge the expert assistance of Mrs. Ann- Amer. J. Physiol., 223: 125 (1972). Christine Eklof and Mr. Gothe Carlebjork. 37. West, G. R., Smith, H. W., and Chasis, H.: Glomerular filtration 43. This research was supported by Grant no. B74-19X-3644-03Bfrom rate, effective renal blood flow and maximal tubular excretory the Swedish Medical Research Council and by a grant from the capacity in infancy. J. Pediat., 32: 10 (1948). Prenatal Research Funds of Expressen. 38. Laevosan Gesellschaft, Linz, Austria. 44. Requests for reprints should be addressed to: P. Herin, M.D., 39. 3M Company, St. Paul, Minn. Karolinski Institutet, Pediatriska Kliniken, St: Goran's Barn- 40. Sage Instruments, Inc., White Plains, N. Y. liniker, Box 12500, 112 81 Stockholm, Sweden. 41. Gilford Instrument Labs., Inc., Oberlin, Ohio. 45. Accepted for publication April 4, 1974. Copyright O 1974 International Pediatric Research Foundation, Inc. Printed in U.S.A. Pediat. Res. 8: 765-770 (1974) Developmental biochemistry intestine fetus liver fruct okinase Development and Control of Intestinal and Hepatic Fructokinase RICHARD J. GRAND,(^') MARIA I. SCHAY, AND STEPHANIE JAKSINA Department of Pediatrics, Harvard Medical School, and the Department of Medicine (Division of Clinical Nutrition), the Children's Hospital Medical Center, Boston, Massachusetts, USA Extract experimental animals (1, 2, 20, 26) and in man (I 5), although the mechanism of this regulation has not yet been elucidated. The patterns of development of intestinal and hepatic In the rat, adrenalectomy prevents the rise in the activity of fructokinase have been studied in the rat, in the rabbit, and in the hepatic enzyme expected after fructose feeding; hydro- man, and information regarding the mechanism of control of cortisone administered to the intact animal greatly enhances this enzyme in mature organs has been obtained. Fructokinase activity. The intestinal enzyme has not been so studied. activity was detectable 4 days before term in fetal rat jejunum (1.0 nmol/min/mg protein) and rose progressively to a plateau During gestation and postnatal development in many species, fetal hepatic fructokinase activity is low (3, 21, 23), 20-fold greater by the 20th day of life (20 nmol/min/mg protein). The enzyme in rat ileum and liver and in rabbit liver even when fructose levels in fetal blood are appreciable (23), and the developmental pattern of fructokinase activity in the behaved similarly (rising from 0.5 to 4.0 nmol/min/mg protein liver has been shown to correlate with the utilization of (ileum) and from 0.5 to 8.5 nmol/min/mg protein (liver)). In exogenously administered (' 4C)fructose (3). human fetal liver, there was a threefold rise in fructokinase Because no data are available regarding the fetal levels or activity from approximately the 10th to 24th week of gestation (increasing from 2.1 to 7.8 nmol/min/mg protein); postnatal development of fructokinase in the intestine, the jejunal activity rose slightly, but not significantly, during this present studies were undertaken to compare such patterns in period. As expected, feeding of mature rats with sucrose after the rat, in the rabbit, and in the human fetus. In addition, the availability of a highly specific assay technique for the enzyme prolonged carbohydrate starvation led to a twofold increase in jejunal (from 6.05 to 13.0 nmol/min/mg protein) and hepatic (25) has made it worthwhile to re-examine similar patterns in the liver and to study further possible mechanisms of control (from 14.1 to 24.2 nmol/min/mg protein) fructokinase of the activity of this enzyme. The data show a progressive rise activity. The administration of actinomycin before the in the specific activity of fmctokinase during development and introduction of sucrose inhibited the carbohydrate-stimulated rise in fructokinase activity in both organs. Similarly, injection suggest that its activity in the mature animal is regulated at the of actinomycin during sucrose feeding produced a significant level of transcription. fall in enzyme activity which greatly exceeded that produced by fasting alone. The data suggest that the substrate-dependent METHODS activity of this enzyme may be regulated at the level of transcription. ANIMALS AND TISSUE PREPARATION Fetuses were obtained from timed pregnancies in New Speculation Zealand white rabbits and in albino Charles Riverr (Sprague- These studies support the hypothesis that the genetic Dawley derived) rats. For rabbits, only the second through control of certain enzymes involved in carbohydrate metabo- fifth pregnancies in a given doe were utilized; in rats, any but lism can be influenced by exogenous factors, among them the first pregnancy was acceptable. Mature tissues were dietary carbohydrate. obtained from adult animals of the same species. Human fetal liver and intestine were obtained from nonviable fetuses during therapeutic abortion, according to practices in operation at the Substrate-dependent control of the activity of hepatic and time of the study and approved by the Committee on Human intestinal fructokinase has been demonstrated in mature Investigation, Children's Hospital Medical Center. 766 GRAND, SCHAY, AND JAKSINA Animals were anesthetized either by rapid cooling or with Intestinal disaccharidases were assayed by a modification (5) ether, and exsanguinated bx severing the aorta. Livers were of the method of Messer and Dahlqvist (13). Results were rapidly excised and kept at 4 until the intestine was removed, expressed as nanomoles of glucose liberated per minute per stripped of its mesentery, flushed with iced saline solution (pH milligram of protein in the EDTA homogenate. 7.4), and the mucosa scraped with glass slides on an iced glass plate. Fetal intestine was not scraped, but was used as FEEDING STUDIES AND ACTINOMYCIN D stripped. The first third of the intestine measured from the ligament of Treitz was termed jejunum and the last third, In all experiments, adult rats (120-150 g) were fed a ileum. Histologic study of the fetal intestine showed that the carbohydrate-free diet (86% protein, 10% fat, 4% salt mixture stripped preparation represented intact mucosa and approxi- (30)) for 5-7 days before each study. They were then fasted mately one-half the thickness of the muscularis. for 48 hr, and subsequently re-fed a solid sucrose-rich ,test diet Tissues (liver or intestine) were then weighed, minced and, (68% sucrose, 18% protein, 8% fat, 4% salt mixture, 2% yeast when fructokinase was to be assayed, homogenized in 10 (30)) with additional sucrose (30% w/v) in the drinking water volumes of 25 mM potassium phosphate buffer, pH 7.4, for various periods of time. Whenever the effect of actino- containing 5 mM EDTA and 0.5 mM dithiothreitol, using a mycin D was studied, the drug was dissolved in distilled water glass homogenizer with 10 passes of a Teflon pestle (25). A (250 pg/ml) and administered intraperitoneally according to high speed supernatant was prepared by centrifugation at the schedules shown in the legends to the figures and tables; 20,000 X g for 30 min. (Sorvall model RC-2B). Preliminary control animals received sterile 0.9% NaCl solution. Previous studies revealed that the fructokinase preparation from this studies (5) have demonstrated that the additives in the material had activity identical with that in the 105,000 X g actinomycin, as prepared by the manufacturer, have no effect supernatant in the original method (25). When intestinal on intestinal enzyme levels. disaccharidases were to be measured, an additional homoge- nate was prepared in 100 volumes of 5 mM EDTA, pH 7.4 (5). ESTIMATION OF RNA SYNTHESIS Previous studies from this (5, 6) and other laboratories (9) ENZYME ASSAYS have characterized the effects of actinomycin on total jejunal Fructokinase activity was measured as described by Weiser RNA synthesis, and many data are also available regarding the and Quill (25) with modifications; in this assay, fructose effects of the drug on hepatic RNA synthesis (14). In the 1-phosphate is the only reaction product. The high speed present study, hepatic RNA synthesis was measured only as an supernatants were acidified to pH 6.1 with 0.05 N HC1, indication of the activity of the actinomycin preparation. centrifuged at 20,000 X g for 20 min, and then returned to pH Eight and 20 hr after the injection of actinomycin D, 7.4 with 0.05 N KOH. This acidified supernatant usually (14C)orotic acid (0.1 pCi/g, body wt) was administered contained 2.0-3.0 mg protein/ml (intestine) or 3.5-4.5 intravenously. After 45 min, rats were killed, the livers excised mg/ml (liver) as determined by the method of, Lowry et al. and homogenized, and incorporation of radioactivity into cold (12). The incubation mixture contained 50 mM Tris-HC1 trichloroacetic acid (TCA)-precipitable material was measured buffer, pH 7.4, 10 mM MgCl,, 10 mM ATP, 50 mM as described previously (5). Results were expressed as counts N-acetylglucosamine, 10 mM D-fructose. The fructose for the per minute per milligram of protein in the alkaline digest. incubation mixture was prepared by diluting 100 mM D-(' C-Wfructose with enough unlabeled 100 mM D-fructose MATERIALS to give a solution which contained 750-900 cpm/nmol. The Disaccharides and actinomycin D were obtained as described final volume of the incubation mixture was 0.2 ml. The usual previously (5): (I4 C)Orotic acid (57.7 mCi/mmol) and concentration of enzyme was 0.2-0.3 mg/assay. Incubation D-(' C)fructose (5.1 -9.8 mCi/mmol) were products of New (performed in duplicate) was begun by adding 0.1 ml enzyme England Nuclear Corporation (31). ATP and N-acetylglucosa- suspension and was continued for 30 min at 37'.