BIOCHEMICAL
Vol. 159, No. 2, 1989 March 15, 1989
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USEOFDEIJTERIUMLABELLEDGLUCOSEINEVALUATINGTBEPATHVAYOF BEPATICGLYCOGENSYBTBESIS Michael N. Goodman, Lorianne K. Masuoka, Jeffrey 5. deRopp, and A. Daniel Jones Department of Uedicine. Division of Endocrinology. Nuclear Magnetic Resonance Facility, Facility for Advanced Instrumentation, The University of California at Davis, School of Medicine, Sacramento, CA95517 Received
January
24,
1989
Summary: Deuterium iabelled glucose has been used to study the pathway of hepatic glycogen synthesis during the fasted-refed transition in rats. Deuterium enrichment of liver glycogen was determined using nuclear magnetic resonance as well as mass spectroscopy. Sixty minutes after oral administration of deuterated glucose to fasted rats , the portal vein blood was fully enriched with deuterated glucose. Despite this, less than half of the glucose molecules incorporated into liver glycogen contained deuterium. The loss of deuterium label from glucose is consistent with hepatic glycogen synthesis by an indirect pathway requiring prior metabolism of glucose. The use of deuterium iabelled glucose may prove to be a useful probe to study hepatic glycogen metabolism. Its use may also find application in the study of liver glycogen metabolism in humans by a noninvasive means. 0 1989 Academic Press, Inc.
It is now accepted that under certain conditions glycogen synthesis from glucose in liver occurs by both adirect as well as an indirect pathway (l-3 1. During the fasted-refed transition, it has been reported in animals that as much as 5065% of the glycogen synthesized in liver following oral glucose is derived from an indirect pathway (2,31. Studies in humans have also demonstrated a significant contribution of the indirect pathway in hepatic glycogen synthesis (4.51. In contrast to liver, glycogen synthesis in muscle for the most part occurs predominantly by the direct pathway (21. The indirect route for hepatic glycogen resynthesis presumably occurs via the gluconeogenic pathwayt23 l., making pyruvate. lactate, alanine, glycerol as well as other gluconeogenic amino acids likely candidates as presursors. Indeed it has been reported that after glucose ingestion in fasted rats, lactate and alanine contributed about 50% to hepatic glycogen formation (6.71. Previous studies have attempted to quantitate the contribution of the indirect pathway to hepatic glycogen synthesis by using radioactive isotopes of glucose (is.; 3 I-I and ‘?C) or %20 (2,3). More recently13 C-glucose has been used for this purpose (6,7).. An advantage of using ‘%-glucose is that it is a stable isotope and has applicability to human studies as the use of a radioactive isotope can be avoided. In addition, the use of13C-glucose coupled to detection by nuclear magnetic resonance shows promise as a noninvasive method to study glycogen metabolism in humansrS1. A disadvantage in using 13C-glucose is its high cost. Another stable isotope, much less expensive than l%-glucose, that has potential as a probe to study glycogen metabolism in humans is deuterium labelled glucose. This compound can also be deteckd over the liver noninvasively using nuclear magnetic resonance and 0006-291X/89 Copyright All rights
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0 1989 by Academic Press, Inc. of reproduction in any form reserved.
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its detection is slightly more sensitive thad3 C-glucose. For thesereasons,we have begun to evaluate the useof deuterium labelled glucose to study hepatic glycogen metabolism in humans. This brief communicationdescribesinitial studiesusing a rat model. Materials and Methods
Amimfs sod tie& Male Sprague-Dawley rats (190-200 g) were obtained from Charles River Breeding Laborstories (Wilmington. MA) and were kept in wire-bottomed cages in animal quarters maintained at 72 F with a light-dark cycle of 12h (6 am- 6 pm). Ratswere fed ad libitum with Purina lab chow and given free accessto water. After one week on this diet. rats were divided into various groups. One group was fasted for one day beginning at 9 am: a secondgroup was fasted for one day and given 1.0 gram of glucose via stomach tube and sacrificed after 30, 60 or 180minutes; a third group wasfasted for one day and then given 1.Ogram of deuterium-labelled glucosevia stomach tube and sacrificed after 30,60 or 1.30minutes; and a fourth group was treated as in group three except that 90 minutes prior to oral dueterated glucose administration animals were treated with mercaptopicolinic acid (25 mg/kg, orally). All rats were studiedin the awake condition. Mareriafs Chemicalwere reagent grade and were obtained from Sigma ChemicalCo. (St. Louis, MO) or Fisher Scientific Co. (Santa Clara, CA). Enzymes and cofactors were obtained from BoehringerMannheim (Indianapolis, IN). Deuterium labelled D-glucose(6.6-d ; 95-999, purity) was obtained from Tracer Technologies, Inc. (Somerville , I-$A). 3-mercaptopicolinic acid was a gift of Smith, Kline and French Laboratories (Philadelphia, PA). H-2,3, alanine (Specific activity 60 Ci/mmole) was purchad from ICNRadiochemicals(Irvine, CA), 10 viva s&fu!& At appropriate times, control and treated rats were anesthetized with sodium pentobarbital (5 mg/lOO g body wt). The abdomenwas then opened and blaodwas removed from the aorta and portal vein. Bloodwasallowedto clot and then centrifuged for 20 minutes at 10,000g (4-C). Immediately after bloodwithdrawal a lobe of the liver was freeze clampedwith clampspre-cooled in liquid nitrogen. Tissuesampleswere stored in liquid nitrogen and serum was stored at - 2O’Cuntil analyzed. An&-ti&fp/oc&ures. Frozen liver was pulverized to a fine powder under liquid nitrogen. A weighed samplewas placedinto tubescontaining 2 ml of hot 30% KOHand digestedin a water bath at 100 C for 20 minutes for glycogen isolation. Glycogen was isolated and hydrolyzed to glucose as described previously ( 9 ). Glucose was measured as described previously (9 ). 3 H-alanine radioactivity in seurm and in glucoseisolatedfrom glycogen was determined by liquid scintillation spectroscopy. The amount of deuterated glucosein glycogen and in serum was determined by nuclear magnetic resonance and also by gas chromatography-mass spectroscopy. Hepatic glycogen was isolated and hydrolyzed to free glucose by standard procedures i9).. A sample of this hydrolysate or serum was analyzed by nuclear magnetic resonance and massspectoscopy.For massspectroscopy analyses, a sampleof the hydrolysate or serumwaslyophilized and to 10ug of solid material was added 50 ul each of pyridine and N,O-bis(trimethyls$yl) trifluoracetamide containing 1% trimethylchlarosilane. The samplewas heated for 1 hour at 60 C and an aliquot was analyzed on a Trio-2 gas chromatography massspectrometer (VG Masslab:Manchester, UK) by selectedion monitoring at 435,436 and 437 (m/z). A 30 m ED-1 capillary gas chromatographic column 1J and V Scientific; Folsom,CA) was used.For nuclear magnetic resonance analyses, the concentration of deuterated glucose in the glycoqen hydrolysate or serum was determined using a Nicolet NM 500 11.75T (500 MHz) vertical bore, h
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4
3
2
PPM
Fi ure 1 Nuclearmagneticresonancespectrumof glucoseisolatedfrom liver glycogenof a fasted rat--J-F given 50mgof deuteriumlabelledglucose.A, deuteratedwater.B,deuteratedglucose.
glucose output ( 21. Additionally, hepatic glucose extraction may be augmented. As shown in Table 1. a one day fast depleted the liver of almost all glycogen. Liver glycogen in well fed rats averages 250*13 umol/gram t 2 1. Administration of either D-glucose or deuterium labelled glucose repleted liver glycogen to similar extents and by 180 minutes liver glycogen was about 32-39 7, of fed levels. The deuterium enrichment of serum glucose and liver glycogen is presented in Table 2. Deuterium enrichment was quantitated by two procedures. Using nuclear magnetic resonance, we obtained the molar concentration of deuterium relative to glucose in both portal vein serum and in glucose isolated from liver glycogen. The parent deuterated glucose compound given orally was 95-99% pure and should result in a molar deuterium:glucose concentration ratio of 1.90-1.98 A second procedure used to measure deuterium enrichment was gas chromatography mass spectrometry by which we obtained the percentage of glucose molecules labelled with deuterium. As shown in Table 2. at 60 minutes following deuterated glucose administration, serum glucose in the portal vein was almost totally enriched with deuterium. Despite this, the deuterium labelling pattern in liver glycogen was quite different. At 30, 60 as well as 180 minutes after deuterium labelled glucose administration. the deuterium enrichment of liver glycogen was always less ,than SO%.In other words, less than 50% of the glucose molecules in liver glycogen contained the deuterium label. This suggests significant loss
Table 1. Effect of oral glucose administration to one day fasted rats on arterial andportalveinglucose and hepatic glycogen content Group
Time after
glucose OoiIlUteS) D-glucose
0 2: ia0
Deuterium labelled glucose
!iO 60 ia0
Arterial qlucose bw 7.7io.4 8.850.4 10.7*0.5* 9.2*0.5*
6.6k0.3 7.3AO.4 9.3*0.5* 7.9*0.4*
Fortalvein glucose
Hepatic a-3-
WI)
bml/g)
6.4i0.3 12.0*0.6*
12.8*0.6*
lO*l 39*2* 59*3*
80&4* 6.5k0.3 9.0*0.5* 10.1*0.5*
15*1 25&l* 52*3* 97*5*
Results are means*SENwith 4 rats perpoint.Cmeday fasted ratsweregiven either 1.0 gram of D-glucose or 1.0 gram of deuterium labelled glucose via stanach tube and sacrificed at appropriate times. Liver and blood was collected as described in Methods. * p < 0.05 vs time 0 minutes in each group. 524
labelled
2. Deuterium
Results are glucose via topicolinic spsctroscopy
glycogen and portal administration toone
9.0*0.5 1o.1io.5
30 60 180
70*4 83Lk5
(%) 1.46kO.07 1.84iO.09 04*4
25*1 52k3 97i5
(umol/g)
labelled
glucose
7%7
l&2 24*2 45*3
(%I
1.8OkO.09
0.72kO.04 0.78+0.04 0.89kO.06
Hepatic glyccgen deuterium Deuterium:glucose labelby ratio by NMR* GOIS*
deuterirnn
4 rats p=r point. One day fasted rats were given 1.0 gram of deuterium labelled One group of rats was pretreated with niercap sacrificed at the times indicated. orally) 90 minutes prior to glucose administration. * CCklS-gas chraatography mass magnetic resonance.
180
OH
oral
content
vein glucose following day fasted rats
Portal vein glucose concentration deuterium Deuteriumglucose label by ratio by WFP GCltS*
of hepatic
bsinutes)
Time after glucose
enrichment
mean f SE24with stomach tuba ard acid (25 mg/kg, : Mft- nuclear
Deuteriumlaixlled glucose after pretreatment with mrcaptopicolinic acid
Deuterim glucose
GYP
Table
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of deterium label prior to glycogen synthesis consistent with the indirect pathway of glycogen synthesis (2.3). Alternatively, glycogen may have been formed via a direct pathway, followed by degradation and metabolismof phosphorylated glucoseprior to reincorporation back into glycogen. However, this type of cyclic mechanismwould not account for extensive glycogen repletion. The finding of extensive lossof deuterium label from glucosein glycogen was not surprising as it is now acceptedthat during the fasted-refed transition in rats. hepatic glycogen repletion occurs by an indirect pathway (2.3 I. It has been proposedthat prior to glycogen synthesis glucoseis first metabolised to 3-carbon intermediates (ie., lactate, pyruvate.
alanine). In the liver,
these
intermediatesare incorporated into glycogen via the gluconeogenic pathway which is still active in liver of a fasted rat several hours after refeeding. The site of formation of these prcursors is not exactly known but they may be producedin the liver. cells of the gastrointestinal tract and/or muscle. The indirect pathway of hepatic glycogen repletion requires an active gluconeogenic pathway (2,3). Becauseof this, we next studiedthe deuterium labelling pattern of liver glycogen in animals pre-treated with an inhibitor of hepatic gluconeogenesis.One day fasted rats were pre-lreated with mercaptopicolinic acid 90 minutes prior to oral glucoseadministration. Mercaptopicolinic acid is an effective inhibitor of phosphoenolpyruvate carboxykinase (10). Ninety minutes after treating rats with mercaptopicolinic acid, they were hypoglycemic (serum glucose 0.55 *0.03 mM) and the concentration of lactate and alanine in serumwaselevated indicating inhibition of gluconeogenesis. In addition, when these animals were given 3H-alanine. its incorporation into hepatic glycogen was diminished by SO 7.. Despite inhibition of gluconeogenesis, fasted rats pre-treated with mercaptopicolinic acid formed similar amounts of liver glycogen as rats not treated with the drug (Table2). However, the labelling pattern of deuterium in glycogen was quite different. In rats given only deuterated glucose, less than fifty
percent of the glucose molecules in liver glycogen were
labelledwith deuterium, whereas in rats pre-treated with mercaptopicolinic acid, most of the glucose molecules in glycogen were labelled with deuterium (Table 2). This suggests that when gluconeogenesisis inhibited, hepatic glycogen synthesis occurred by the direct pathway. A previous study using mercaptopicolinic acid reported that the quantity of hepatic glycogen formed in the fasted-refed rat wasmarkedly blunted (11 I. This result differs from ours for unknown reasons,but it could be due to differences in the amount of glucose administered to fasted rats and time course of mercaptopicolinic acid treatment. The results presented in this report indicate significant loss of deterium from glucose prior to hepatic glycogen synthesis. Glucoseused in this study was labelled with two deuteriums at carbon-& Metabolismof glucosethrough glycolysis in peripheral tissuesor the liver would yield pyruvate, lactate and alanine both unlabelled or labelled with deuterium at carbon-3. In the liver, one proton is from carbon-3 of pyruvate as it is converted to odoac&te during gluconeogenesis.Ad&tiond,
10~1
even extensive further lossof protons from carbon-3 of pyruvate can occur if phosphoenolpyruvate recycles through pyruvate and oxaloacetate.Recycling between phosphoenolpyruvate and pyruvate can be quite extensive and has been found to be on the order of 30-50%of gluconeogenic flux (7). Although we did not test this. a major portion of the lossof deuterium may have occurred in this futile cycle. On the other hand. due to the kinetic isotopic effect a carbon-deuterium bond may be hydrolyted in vivo at a rate slower than a carbon-hydrogen bond (12). Becauseof this, the extent of 526
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loss of a deuterium vs a proton from cwhon-3 of pyruvate as it is converted to oxaloacetate in vivo is unknown. Left unresolved then is the precise mechanism and pathway&i by which a major loss of deuterium occurs. In conclusion, these initial studies using deuterium Iahelled glucose suggest it may prove to be a novel probe for evaluating the pathway of hepatic glycogen synthesis. Also deuterium is a stable isotope and has applicability to use in human studies. Coupled to nuclear magnetic resonance imaging over the liver, the use of deuterium labelled glucose may prove to be a useful marker to study liver glycogen metabolism noninvasively. Acknovledgment
This work was supported in part by U.S. Public Health Service grant DK 39740. References
1. 2. 3. 4.
Foster, D. W. (19S4) Diabetes. 33, 1188-1199. McGarry, J. D., Kuwajima, M., Newgard. C. B., and Foster, D. W. (19$7) Ann. Rev. Nutr. ,7,51-73, Landau, B. R.. and Wahren, J. (198,881 FASEBJ,, 2,2365-2375. Magnusson. I., Chandramouli. V., Schumann, W. C., Kumaran, g,, Wahren, J., and La&m, B, R, (19871 J. Clin. Invest., 80,174~-1754. 5. Radziuk, J. (1982)Fed. Proc., 41, 110-116. 6. Kalderon, B. , Gopher, A., and iapidot, A. (1986) FEBSLebrs, 204,29-32. 7. Shulman, G. I.. Rosseui. L., Rothman, D. L. , Blair. J. B. , and Smith, D. (19S757) J. Clin. Invest., 80,387-393 8. Jue, T.. Lehman, J. A. B. , Ordidge, R. J. , and Shulman, R. G. (1987) Msg. Res. Med. ,5.377-379. 9. Goodman, M. N., Berger, M., and Ruderman, N. B. (1974) Diabetes, 23,851~858. 10. Goodman, M. N. (1975) Biochem. J., 150.137-139. 11. Newgard. C. B.. Moore, S. V., Foster. D. W., and McGarry, J. D. (1984) J. Biol. Chem. ,259,6958-6963. 12. Atkins, P. W. (19783 In, Physical Chemistry, Oxford University Press, Great Britain, pp. 912-913,
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