AMERICAN JOURNAL OF PHYSIOLOGY Vol. 230, No. 1, January 1976. Printed
Glucose
in U.S.A.
utilization
and recycling
M. S. ANWER, T. E, CHAPMAN, AND R. GRQNWALL Department of Physiological Sciences, College of Veterinary Kansas State University, Manhattan, Kansas 66506
ANWER, M.S.,T.E. CHAPMAN,AND R. GRONWALLX~UCOS~ utiliza;tion and recycling in ponies. Am. J. Physiol. 230(l): 138-142. 1976. - Variables of glucose metabolism determined by the use of [U-14C]glucose simultaneously with [6Tlglucose, [S-Tlglucose, or [2-T]glucose were compared in fed and fasted ponies. Relative recycling of glucose carbon with respect to tritium in fed animals was negligible for 6-T and 3-T and 16% for 2-T studies; in fasted animals relative recycling was 12 and 14% for 6-T and 3-T studies, respectively. Minimal mass of total-body glucose decreased significantly in the fasted ponies. I3ased on relative recycling of carbon to tritium, a negligible fraction of plasma glucose was produced via the Cori cycle or from glycerol in fed ponies; recycled tricarbon units contributed 12% of glucose production in ponies fasted 72 h. In fed ponies, 16% of plasma glucose carbon was recycled via a futile cycle at the glucose 6-phosphate stage. Glucose utilization was best estimated with the use of [6-Tlglucose (or 3-T). irreversible loss ; relative recycling; single injection; futile cycle
fasting;
labeled
MATERIALS
glucose;
TURNOVER in both monogastric and ruminant animals has been investigated extensively with the use of single injection and primed infusion of glucose labeled with 14C. Recycling of glucose carbon has been a problem in interpreting results of those investigations. Studies using glucose variously labeled with tritium along with [14C]glucose have shown that tritium disappeared from circulating plasma more rapidly than 14C(8, 17). Thus, glucose turnover, estimated from glucose variously labeled with tritium, exceeded that obtained simultaneously from [14C]glucose in rats (7), dogs (14), and sheep (16). Those differences are believed to result from less recycling of tritium than 14C (12, 16, 17). Formation of water appears to be the predominant early fate of hydrogen from metabolized glucose (16, 17). A pony can digest and absorb soluble carbohydrate and absorb volatile fatty acids produced in the large intestine by microbial cellulose digestion (1). [‘“Cl glucose (9) and [Z-Tlglucose (11) have been used in studying glucose turnover in horses and ponies, respectively. Studies of glucose recycling in ponies simultaneously using glucose labeled with 14C and variously labeled with tritium have not been reported. This study was designed a) to evaluate the use of glucose labeled variously with tritium for the determination of glucose utilization; and b) to estimate the extent of glucose resynthesis and its precursors. GLUCOSE
in ponies Medicine,
AND METHODS
Both jugular veins of ponies weighing lOGZOO kg were cannulated with polyethylene tubing (PE-190, Clay-Adams, Inc., Parsippany, N. J.) at least 32 h before each experiment, to minimize excitation and thus its effect on plasma glucose metabolism during experiments. One cannula was used to inject or infuse tracers; the other, to withdraw blood. [U-14C]glucose (3 Ci/mol), [6-Tlglucose (500 Ci/mol), and [3-Tlglucose (500 Ci/mol) were obtained from Amersham/Searle Corp., Arlington Heights, Ill.; [Z-Tlglucose (200 Cilmol) was from New England Nuclear Corp., Boston, Mass. Water and hay were available during all experiments except fasting trials, when only water was available. Timed blood samples (10 ml) were collected periodically in tubes containing 1 mg of a mixture of EDTA and NaF (9:l) and kept on ice. The plasma was removed after centrifugation at the end of each trial and frozen until subsequent analysis. Each experiment was started between 9:00 A.M. and 10:00 A.M. Blood samples were taken every 2 min for the first 20 min of each trial, when greatest changes in plasma isotope concentration occurred, to provide sufficient data for accurate analyses. Three fed ponies were each given single injections of a mixture of [U-14C]glucose (200 @i) and [6-Tlglucose (600 &i). Two months later the ponies were fasted 72 h and given the same injections of tracer glucose. After another 2 mo, the same three fed ponies were injected with a mixture of [U-14C]glucose (200 &i) and [3Tlglucose (600 &i). One of those ponies was later studied identically after a 72-h fast. Three other fed ponies received single intravenous injections of a mixture of [U-14C]glucose (100 &i) and [2-Tlglucose (300 &i). Blood samples were taken for 7 h after injection in all these experiments. Assay procedures. Plasma glucose concentrations were determined enzymatically (21) Plasma and dosage glucose were isolated and assayed for specific activity as glucose penta-acetate (15). Quenching was determined from changes in channels ratio in a liquid scintillation counter (4). Tritium and 14Cwere counted with efflcienties of 25 and 45%, respectively; approximately 0.3% of 14Cappeared in the tritium channel. Data analysis. The variables of glucose metabolism were defined and calculated essentially as described by Katz et al. (18, 19). Briefly the variables are 1) MS- the mass of glucose in the rapidly mixing pool (sampled
138
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GLUCOSE
UTILIZATION
AND
139
RECYCLING
pool), 21 R,,-- the rate of outflow from M, by catabolism and/or exchange, 3) M,i,- the limiting minimal mass of total-body glucose, 4) M,,,the limiting maximal mass of total-body glucose, 5) M,- total-body glucose, if uniform catabolic rate is assumed, and 6) R,- irreversible loss of glucose, which is the rate of glucose utilization or synthesis. The variables R,, M,, Rll, M,, and M,,, were calculated from expressions Za, 3a, 4a, 7b, and 8b of Katz et al. (18). The areas under the plasma specific radioactivity curves were measured by extrapolation and counting of squares on millimeter paper. The exponential components of the plasma specific radioactivity curves were obtained by fitting of data ti an exponential curve with the use of a digital computer. These exponents were used to calculate M,,, and M,. The limiting minimal mass (M,in) was calculated by multiplying R, by minimal transit time which was determined from the area under the cumulative curve for plasma specific radioactivity, normalized by dividing by the total area. Relative recycling, in this study, represents the relative recycling of 14C with respect to tritium and is calculated as the difference in irreversible loss between tritium and 14C (T - 14C) divided by the irreversible loss of tritium and expressed as percent (Table 1). StatisticaL anaZyses. The data were compared by analysis of variance; when the analysis indicated a significant difference, the means were compared by Fischer’s LSD test (10) unless otherwise mentioned. Significance in all cases was P < .05,
[UJ4C]glucose and [6-T]glucose or [3-Tlglucose injections (Fig. 2). Typical plasma glucose specific activities after single injections of [UJ4C]glucose and [6-Tlglucose in fasted ponies are shown in Fig. 1D. Unlike in fed ponies, tritium specific activity declined more rapidly than 14C specific activity in fasted ponies, and the ratio of 14Cto tritium increased after single injections of the same tracers (Fig. W). The outflow from the sampled pool (R,,) and irreversible loss (Rd for fasted ponies were relatively less, but did not differ significantly from those of fed ponies. Both R,, and R, obtained with [Z-T]glucose were significantly higher than those obtained with [UJ4C]glucose injected simultaneously. Irreversible losses of glucose did not differ significantly between 14Cand tritium after single injections of [UJ4C]glucose and [6-Tlglucose or [3Tlglucose.
% I B
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10
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AbA
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c RESULTS
Plasma glucose concentrations were essentially constant through all experimental periods in all studies. Fasted ponies maintained a lower average plasma glucose concentration that was not significantly different from that of fed ponies (Table 1). The rate of decline of plasma glucose specific activities was essentially the same for both tritium and 14C after single injections of mixtures of [UJ*C]glucose and [6-Tlglucose or 3-T, whereas the specific activity of tritium declined more rapidly than that of 14C when a mixture of [UJ4C]glucose and [Z-Tlglucose was injected (Fig. 1). The ratio of 14C to tritium specific activity in plasma glucose increased with time after a single injection of [Z-Tlglucose and [U-14C]glucose but did not after TABLE
1
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x*00 *X0
AQ
x
0 x O 00
QQRQ Q’?o Kg0
xoo
2
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1 0
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x KX
--
3
5 tlourr
after
x
6
7
injection
FIG. 1. Specific activities of plasma glucose after a single injection of a mixture of [UJ4C]glucose and [G-Tlglucose (A), [3-Tlglucose (B] , or 1%Tlglucose (C) in fed pony or [6-Tlglucose (DI in a fasted pony. [14C Jglucose (0) and [Tlglucose (X > specific activities are adjusted to 1 mCi of injected tracer. Where they differ by less than 5%, a common symbol (n> is used.
1. Variables of glucose metabolism in fed and fasted ponies
Isotopes in Glucose
U-14C 6-T UJ4C 6-T UJ4C 3-T u- 14C 3-T UJ4C 2-T Values recycling
10
% % 0. A
Body Weight,
145 (109-172) 148 (llal70)
kg
Plasma
glucose concn, mg/lOO ml
112 (lO&lZl) 95
(84-102)
Fed (3) Fasted
177 (163-195)
123 (105164)
Fed 13)
162
101
Fasted
174 (165-193)
101
are means and range. = 100 x (irreversible
(94-107)
Significant difference (P < 0.05) loss of T minus U- 14C)/(irreversible
Outflow From Sampled Pool, mglmin per kg
Expt Types
Fed (3)
(3) 1
(1)
5.7 5.1 3.4 3.6 5.7 5.6
Irreversible Loss, mgl min per kg
(W-5.9) (4.S6.3) (2.0-4.3) (1.S4.8) (4.C8.9) (3. S9.1)
1.3 1.2 0.8 1.0 1.4 1.4 1.0 1.2 1.5 1.8
i’: 4:6 (4.5-4.7>n 4.9 (4.7-5.ly
- b > a, and d > c. Numbers loss of T).
of animals
(1.21.4) (1.1-1.4) (0.~1.1) (O.M.3) (1.1-1.7) (1.1-1.9)
Relative
-7
Recycling, %8
(-16-S)
12 (10-16) -3
(-148)
14 (l.H.6)c (1.7-1.9)”
are in parentheses.
16 (14-22) *Relative
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140
ANWER,
Relative recycling was -7, -3, and 16% for 6-T, 3-T, and 2-T, respectively, in fed ponies, and 12 and 14% for 6-T and 3-T, respectively, in fasted pomes (Table 1). The ratio of 14C/tritium in plasma glucose indicated the relative recycling of 14Cwith respect to tritium (20). Since that ratio did not increase for 3-T and 6-T, the relative recycling values of -7 and -3% for 6-T and 3-T, respectively, were considered negligible. Relative recycling of 14% for 3-T in a fasted pony was within the 95% cotidence interval for 6-T in fasted ponies. Glucose mass of the sampled pool (MJ and total-body pool differed little between isotopes for a particular trial. Therefore, the masses calculated from 14C and tritium data are combine,d together (Table 2). Minimal mass of total-body glucose decreased significantly in fasted ponies compared to fed ponies. The sampled pool varied from 50 to 66% of the minimal mass in fed Denies and from 61 to 67% in the fasted ponies. The miximal mass (Mmax), which was relatively higher than total2T
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Isotopes
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injection
2. Ratio of 14C to tritium in plasma injections of a mixture of [UJ4C]glucose and TJglucose (B) , or [2-Tlglucose (CJ in fed ponies a faskd pony. Values normalized to a ratio of FIG.
TABLE
6
glucose after single [6-TJglucose CA), [3or [6-TJglucose (II] in 1 for dose.
Values
AND
GRONWALL
body glucose (M,) calculated by assuming uniform catabolic rate, ranged from 147 to 353 mg/kg body wt. DISCUSSION
The precise estimation of sampled pool and outflow from this pool depends on the estimation of zero-time intercept and the initial slope, which in turn depend on the rapidity of sampling. Any problem with sampling, and thereby the estimation of zero-time intercept and initial slope, would result in large variations in the estimates of those variables. In the present study, with the exception of one pony, the estimates of sampled pool ranged from 70 to 95 mg/kg (Table 2>, and those of the outflow from that pool ranged from 3.9 to 6.3 mglmin per kg (Table 1) in fed ponies. This small range suggested that the sampling was rapid enough for relatively precise estimations of those variables. Production of new plasma glucose from tricarbon units which originated from plasma glucose (Cori cycle) can be estimated from the relative recycling of carbon obtained by using [6-Tlglucose simultaneously with [U14C]glucose (7). If recycled tricarbon units, like lactate and pyruvate, contribute significantly to glucose production, a significant portion of tritium from position 6 of glucose would be exchanged with protons of the medium during conversion of lactate or pyruvate to phosphoenol pyruvate via oxaloacetate (16). That would result in a higher irreversible loss of glucose when estimated from [6-Tlglucose than from [UJ4C]glucose. In our fed ponies, when [6-Tlglucose was injected simultaneously with [ UJ4C]glucose, a negligible relative recycling of carbon suggested that the Cori cycle contributed little, if at all, to production of glucose. Glucose production via the Cori cycle has been reported as 4 and 23% in sheep (16) and postabsorptive rats (7), respectively. The negligible relative recycling of carbon in fed ponies given [6-Tlglucose or [S-T]glucose indicated that resynthesis of glucose carbon from glycerol also was negligible. Resynthesis of glucose from glycerol would return tritium to plasma glucose when [6-Tlglucose is used but not when [3-Tlglucose is used, because a significant portion of tritium from position 3 of glucose would be lost during the conversion of glycerol, which enters the gluconeogenic pathway distal to oxaloacetate, to glyceraldehyde 3-phosphate via dihydroxyacetone 3-phosphate (16). Therefore, if glucose were resynthesized extensively via glycerol, estimates of irreversible glucose loss obtained with [ST]glucose would be significantly higher than with [G-Tlglucose. That would result in higher relative recycling of carbon to 3-T than to 6-T.
2. Glucose masses in fed and fasted ponies in Glucose
Total-Body Expt
Types
Sampled
Pool,
+ + + + +
6-T 6-T 3-T 3-T 2-T
are means
Fed (3) Fasted (3) Fed (3) Fasted (1) Fed (3) and range.
Significant
86 86 106 95 92 difference
Mass,
“g/kg
mglkg M ml”
UJ4C u-14C UJ4C UJ4C UJ4C
CHAPMAN,
(70-95) (63-99) (81-144) (8&95) dp < 0.05)
153 131 172 151 167
M”
(124167)” (97-148)b (12%147) (14%X4) (160-171)
- b < a. Numbers
166 168 211 193 227 of animals
(13&200) (157-179) (143-330) (17%207) (19%252)
k&Y
178 179 225 202 239
(147-213) (165-191) (15&353) (185-218) (21@266)
are in parentheses.
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GLUCOSE
UTILIZATION
AND
141
RECYCLING
Glycerol provided about 5 and 11% of glucose carbon, respectively, in sheep (3) and postabsorptive rats (6). The difference in estimates of irreversible loss of glucose, determined simultaneously with [Z-Tlglucose and [UJ4C]glucose, indicated a selective loss of tritium in position 2 relative to 14C, which is also evident from Fig. 1C. The loss of tritium inposition. 2 most likely occurs at the level of hexose 6-phosphate isomerase, and a single cycle between glucose 6-phosphate and fructose 6-phosphate would remove 75% of tritium from position 2 of glucose (14, 17). The selective loss of tritium in position 2 relative to 14C was first interpreted as the result of recycling of plasma glucose via ihe Cori cycle and the degradation of labeled glycogen (17). It is now believed to be due to the recycling of glucose via the Cori cycle and to futile cycle of glucose into glucose 6-phosphate and fructose 6-phosphate (13, 23). Futile cycle at glucose 6-phosphate stage would cause detritiation of [2Tlglucose and futile cycle at the triose phosphate stage would cause detritiation from positions 3, 4, and 5 (5). Since the Cori cycle did not contribute significantly to glucose resynthesis and no detritiation was evident with 13-T]glucose in the present study, in fed ponies the selective detritiation of [2-Tlglucose is due to the futile cycle at the glucose 6-phosphate stage, and there is no significant futile cycle at the triose phosphate stage. Thus, the relative recycling of carbon with respect to [ZTlglucose (Table 1) indicated that 16% of plasma glucose carbon was recycled via the futile cycle at glucose 6phosphate stage. The presence of such a futile cycle has been demonstrated in isolated hepatocytes (5) and in other species (19), but its physiological significance, if any, is not understood. Irreversible loss of glucose did not decrease significantly during fasting. A significant decrease in irreversible loss has been reported for fasting dogs (22), rats (2) and horses (9), after use of [ 14C]glucose, and in ponies (I 1) after use of [2-Tlglucose. The relatively small number of animals in this study may be the reason for the lack of a significant difference. A significant difference between the sampled pool of fed and fasted ponies was not expected, since plasma glucose concentration did not decrease significantly during fasting. The relative recycling of 14C with respect to [6-Tlglucose in fasting ponies suggested that about l&16% of glucose carbon was derived via recycled tricarbon units. This is consistent with the idea that recycling is highest in animals with a high rate of glucose turnover. In one study where [3-Tlglucose was simultaneously injected with [UJ4C]glucose into fasted ponies, the calculated relative recycling was not different from that estimated from [ 6-T]glucose and [ UJ4C]glucose. That result suggested that glycerol was not a source of recycled glucose carbon in fasting ponies. However, more studies are needed before a definite conclusion may be drawn. Ponies derive volatile fatty acids from cellulose diges-
tion in the large intestine (1), which may not be completely empty ifter 72 h of fasting, so propionic acid from cellulose digestion could sti .ll contribute to glucose production during fasting. Liver g plycogen stores should be exhausted by 72 h of fasting. Thus, gluconeogenesis from amino acids probably played a major role in glucase nroduction in our fasted ponies. Th; minimal glucose m&s calculated from the plasma specific radioactivity curve would represent total-body glucose if the major site of catabolism is in or near the rapidly mixing pool (sampled pool). On the other hand, the maximal mass would represent totalbody glucose if catabolism occurred at a site as far as possible from the sampled pool (18). The total-body glucose for fed ponies could be between 124 and 353 mg/kg, since the site of catabolism is not known. If the maximal mass represents total-body glucose, then fasting did not have any effect on total-body glucose in ponies. However, if the minimal mass represents total-body glucose, as it is shown for rats and rabbits (19), then total-body glucose decreased during fasting in ponies. In that case it would appear that mass of glucose some place other than the sampled pool is decreased, since there was no change in the sampled pool during fasting. No definite conclusions regarding the effect of fasting on the variables of glucose metabolism in ponies could be drawn because of the limited number of animals. However, it appears that ponies adjusted to fasting by lowering outflow from the sampled pool and irreversible loss, and possibly by decreasing total-body glucose. Relative recycling of l&16% in fasted ponies suggested increased gluconeogenesis. The use of [UJ4C]glucose, [6-Tlglucose and [3Tlglucose resulted in similar estimates of irreversible loss of glucose in fed ponies, indicating negligible glucase resynthesis via the Cori cycle and glycerol. Studies with [2-Tlglucose estimated the recycling of plasma glucase carbon which occurred via the futile cycle at the glucose 6-phosphate stage. This futile cycle would overestimate glucose resynthesis and hence the irreversible loss, since detritiation of labeled glucose would occur without any net synthesis of glucose. Thus, estimates of irreversible loss of glucose from [6-Tlglucose and [3Tlglucose would better estimate glucose utilization, Use of [UJ4C]glucose along with glucose variously labeled with tritium should be useful in quantitative estimation of precursors of glucose resynthesis in various physiological and nutritional conditions. This
study
was supported
in part
AM11384 and RR()5619.
This work is contribution Sciences, Kansas Agricultural
tan*
Present Universitat Present University Received
no.
by Public
Health
146, Department Experimentation
Service
Grants
of Physiological Station, Manhat-
address of M. S. Anwer: Institut fur Pharmakologie, Munchen, West Germany. address of R. Gronwall: College of Veterinary Medicine, of Florida, Gainesville, Fla. for publication
7 November
1974.
REFERENCES 1. ARGENZIO,
of volatile J. Anim.
R. A., AND H. F. HINTZ. Glucose tolerance fatty acid on plasma glucose concentrations Sci. 30: 514-518, 1970.
and effect in ponies.
2, BAKER, studies turnover
N., R. A. SHIPLEY, R. E. CLARK, AND G. E. INCEFY. 14C in carbohydrate metabolism: glucose pool size and rate of in the normal rat. Am. J. Physiol. 196: 24%252,1959.
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ANWER,
142 3. BERGMAN, E. N., D. J. STARR, AND S. S. REULEIN, JR. Glycerol metabolism and gluconeogeneris in the normal and hypoglycemic ketotic sheep. Am. J. Physiol. 215: 847-880, 1968. 4. BRUNO, G. A., AND J. E. CHRISTIAN. Correction for quenching associated with liquid scintillation counting. Anal. Chem. 33: 650--651, 1961. 5. CLARK, D. G., R. RONGSTAD, AND 5. KATZ. Isotope evidence for futile cycles in liver cells. Biochem. Biophys. Res. Commun. 54: 1141-1148, 1973. 6. DE FREITAS, A. S. W., AND F. DEPOCAS. Glyceride-glycerol release and the interconversion of glucose and glycerol in normal and fasted rats. Can. J. Physiol. Pharmacol. 48: 561-568, 1970. 7. DUNN, A., M. CHENOWETH, AND L. D. SCHAEFFER. Estimation of glucose turnover and Cori cycle using glucose-6-T-14C. Biochemistry 6: 6-11, 1967. 8. DUNN, A., AND S. STRAHS. A comparison of 3H and 14C glucose metabolism in the intact rat. Nature 205: 705-706, 1965. 9. EVANS, J. W. Effect of fasting, gestation, lactation and exercise on glucose turnover in horses. J. Animal. Sci. 33: 1001-1004, 1971. 10. FRYER, H. C. Concepts and Methods of Experimental Statistics. Boston: Allyn and Bacon, 1966, p. 260-261. 11. GINOCHIO, R. J., AND J. W. EVANS. Glucose-2-T turnover in Shetland ponies. J. Anim. Sci. 37: 484-487, 1973. 12. HETENYI, G., JR., AND D. MAK. “H-2-glucose as tracer in turnover studies. Can. J. Physiol. Pharmacol. 48: 732-734, 1970. 13. HUE, L., AND H. G. HERS. On the use of c3H, 14C) labeled glucose in the study of the so-called “futile cycles” in liver and muscle. Biochem. Biophys. Res. Commun. 58: 532-539, 1974. 14. ISSEKUTZ, B., JR., M. ALLEN, AND I. BORKOW. Estimation of
15.
16.
17. 18.
19.
20. 21.
22.
23.
CHAPMAN,
AND
GRONWALL
glucose turnover in the dog with glucose-2-T and glucose-U-14C. Am. J. Physiol. 222: 710-712, 1972. JONES, G. D. Determination of the specific activity of labeled blood glucose by liquid scintillation using glucose penta-acetate. Anal. Biochem. 12: 249-253, 1965. JUDSON, G. J., AND R. A. LENG. Estimation of total entry rate and resynthesis of glucose in sheep using glucose uniformly labeled with 14C and variously labeled with :
Glucose
in U.S.A.
utilization
and recycling
M. S. ANWER, T. E, CHAPMAN, AND R. GRQNWALL Department of Physiological Sciences, College of Veterinary Kansas State University, Manhattan, Kansas 66506
ANWER, M.S.,T.E. CHAPMAN,AND R. GRONWALLX~UCOS~ utiliza;tion and recycling in ponies. Am. J. Physiol. 230(l): 138-142. 1976. - Variables of glucose metabolism determined by the use of [U-14C]glucose simultaneously with [6Tlglucose, [S-Tlglucose, or [2-T]glucose were compared in fed and fasted ponies. Relative recycling of glucose carbon with respect to tritium in fed animals was negligible for 6-T and 3-T and 16% for 2-T studies; in fasted animals relative recycling was 12 and 14% for 6-T and 3-T studies, respectively. Minimal mass of total-body glucose decreased significantly in the fasted ponies. I3ased on relative recycling of carbon to tritium, a negligible fraction of plasma glucose was produced via the Cori cycle or from glycerol in fed ponies; recycled tricarbon units contributed 12% of glucose production in ponies fasted 72 h. In fed ponies, 16% of plasma glucose carbon was recycled via a futile cycle at the glucose 6-phosphate stage. Glucose utilization was best estimated with the use of [6-Tlglucose (or 3-T). irreversible loss ; relative recycling; single injection; futile cycle
fasting;
labeled
MATERIALS
glucose;
TURNOVER in both monogastric and ruminant animals has been investigated extensively with the use of single injection and primed infusion of glucose labeled with 14C. Recycling of glucose carbon has been a problem in interpreting results of those investigations. Studies using glucose variously labeled with tritium along with [14C]glucose have shown that tritium disappeared from circulating plasma more rapidly than 14C(8, 17). Thus, glucose turnover, estimated from glucose variously labeled with tritium, exceeded that obtained simultaneously from [14C]glucose in rats (7), dogs (14), and sheep (16). Those differences are believed to result from less recycling of tritium than 14C (12, 16, 17). Formation of water appears to be the predominant early fate of hydrogen from metabolized glucose (16, 17). A pony can digest and absorb soluble carbohydrate and absorb volatile fatty acids produced in the large intestine by microbial cellulose digestion (1). [‘“Cl glucose (9) and [Z-Tlglucose (11) have been used in studying glucose turnover in horses and ponies, respectively. Studies of glucose recycling in ponies simultaneously using glucose labeled with 14C and variously labeled with tritium have not been reported. This study was designed a) to evaluate the use of glucose labeled variously with tritium for the determination of glucose utilization; and b) to estimate the extent of glucose resynthesis and its precursors. GLUCOSE
in ponies Medicine,
AND METHODS
Both jugular veins of ponies weighing lOGZOO kg were cannulated with polyethylene tubing (PE-190, Clay-Adams, Inc., Parsippany, N. J.) at least 32 h before each experiment, to minimize excitation and thus its effect on plasma glucose metabolism during experiments. One cannula was used to inject or infuse tracers; the other, to withdraw blood. [U-14C]glucose (3 Ci/mol), [6-Tlglucose (500 Ci/mol), and [3-Tlglucose (500 Ci/mol) were obtained from Amersham/Searle Corp., Arlington Heights, Ill.; [Z-Tlglucose (200 Cilmol) was from New England Nuclear Corp., Boston, Mass. Water and hay were available during all experiments except fasting trials, when only water was available. Timed blood samples (10 ml) were collected periodically in tubes containing 1 mg of a mixture of EDTA and NaF (9:l) and kept on ice. The plasma was removed after centrifugation at the end of each trial and frozen until subsequent analysis. Each experiment was started between 9:00 A.M. and 10:00 A.M. Blood samples were taken every 2 min for the first 20 min of each trial, when greatest changes in plasma isotope concentration occurred, to provide sufficient data for accurate analyses. Three fed ponies were each given single injections of a mixture of [U-14C]glucose (200 @i) and [6-Tlglucose (600 &i). Two months later the ponies were fasted 72 h and given the same injections of tracer glucose. After another 2 mo, the same three fed ponies were injected with a mixture of [U-14C]glucose (200 &i) and [3Tlglucose (600 &i). One of those ponies was later studied identically after a 72-h fast. Three other fed ponies received single intravenous injections of a mixture of [U-14C]glucose (100 &i) and [2-Tlglucose (300 &i). Blood samples were taken for 7 h after injection in all these experiments. Assay procedures. Plasma glucose concentrations were determined enzymatically (21) Plasma and dosage glucose were isolated and assayed for specific activity as glucose penta-acetate (15). Quenching was determined from changes in channels ratio in a liquid scintillation counter (4). Tritium and 14Cwere counted with efflcienties of 25 and 45%, respectively; approximately 0.3% of 14Cappeared in the tritium channel. Data analysis. The variables of glucose metabolism were defined and calculated essentially as described by Katz et al. (18, 19). Briefly the variables are 1) MS- the mass of glucose in the rapidly mixing pool (sampled
138
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GLUCOSE
UTILIZATION
AND
139
RECYCLING
pool), 21 R,,-- the rate of outflow from M, by catabolism and/or exchange, 3) M,i,- the limiting minimal mass of total-body glucose, 4) M,,,the limiting maximal mass of total-body glucose, 5) M,- total-body glucose, if uniform catabolic rate is assumed, and 6) R,- irreversible loss of glucose, which is the rate of glucose utilization or synthesis. The variables R,, M,, Rll, M,, and M,,, were calculated from expressions Za, 3a, 4a, 7b, and 8b of Katz et al. (18). The areas under the plasma specific radioactivity curves were measured by extrapolation and counting of squares on millimeter paper. The exponential components of the plasma specific radioactivity curves were obtained by fitting of data ti an exponential curve with the use of a digital computer. These exponents were used to calculate M,,, and M,. The limiting minimal mass (M,in) was calculated by multiplying R, by minimal transit time which was determined from the area under the cumulative curve for plasma specific radioactivity, normalized by dividing by the total area. Relative recycling, in this study, represents the relative recycling of 14C with respect to tritium and is calculated as the difference in irreversible loss between tritium and 14C (T - 14C) divided by the irreversible loss of tritium and expressed as percent (Table 1). StatisticaL anaZyses. The data were compared by analysis of variance; when the analysis indicated a significant difference, the means were compared by Fischer’s LSD test (10) unless otherwise mentioned. Significance in all cases was P < .05,
[UJ4C]glucose and [6-T]glucose or [3-Tlglucose injections (Fig. 2). Typical plasma glucose specific activities after single injections of [UJ4C]glucose and [6-Tlglucose in fasted ponies are shown in Fig. 1D. Unlike in fed ponies, tritium specific activity declined more rapidly than 14C specific activity in fasted ponies, and the ratio of 14Cto tritium increased after single injections of the same tracers (Fig. W). The outflow from the sampled pool (R,,) and irreversible loss (Rd for fasted ponies were relatively less, but did not differ significantly from those of fed ponies. Both R,, and R, obtained with [Z-T]glucose were significantly higher than those obtained with [UJ4C]glucose injected simultaneously. Irreversible losses of glucose did not differ significantly between 14Cand tritium after single injections of [UJ4C]glucose and [6-Tlglucose or [3Tlglucose.
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Plasma glucose concentrations were essentially constant through all experimental periods in all studies. Fasted ponies maintained a lower average plasma glucose concentration that was not significantly different from that of fed ponies (Table 1). The rate of decline of plasma glucose specific activities was essentially the same for both tritium and 14C after single injections of mixtures of [UJ*C]glucose and [6-Tlglucose or 3-T, whereas the specific activity of tritium declined more rapidly than that of 14C when a mixture of [UJ4C]glucose and [Z-Tlglucose was injected (Fig. 1). The ratio of 14C to tritium specific activity in plasma glucose increased with time after a single injection of [Z-Tlglucose and [U-14C]glucose but did not after TABLE
1
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FIG. 1. Specific activities of plasma glucose after a single injection of a mixture of [UJ4C]glucose and [G-Tlglucose (A), [3-Tlglucose (B] , or 1%Tlglucose (C) in fed pony or [6-Tlglucose (DI in a fasted pony. [14C Jglucose (0) and [Tlglucose (X > specific activities are adjusted to 1 mCi of injected tracer. Where they differ by less than 5%, a common symbol (n> is used.
1. Variables of glucose metabolism in fed and fasted ponies
Isotopes in Glucose
U-14C 6-T UJ4C 6-T UJ4C 3-T u- 14C 3-T UJ4C 2-T Values recycling
10
% % 0. A
Body Weight,
145 (109-172) 148 (llal70)
kg
Plasma
glucose concn, mg/lOO ml
112 (lO&lZl) 95
(84-102)
Fed (3) Fasted
177 (163-195)
123 (105164)
Fed 13)
162
101
Fasted
174 (165-193)
101
are means and range. = 100 x (irreversible
(94-107)
Significant difference (P < 0.05) loss of T minus U- 14C)/(irreversible
Outflow From Sampled Pool, mglmin per kg
Expt Types
Fed (3)
(3) 1
(1)
5.7 5.1 3.4 3.6 5.7 5.6
Irreversible Loss, mgl min per kg
(W-5.9) (4.S6.3) (2.0-4.3) (1.S4.8) (4.C8.9) (3. S9.1)
1.3 1.2 0.8 1.0 1.4 1.4 1.0 1.2 1.5 1.8
i’: 4:6 (4.5-4.7>n 4.9 (4.7-5.ly
- b > a, and d > c. Numbers loss of T).
of animals
(1.21.4) (1.1-1.4) (0.~1.1) (O.M.3) (1.1-1.7) (1.1-1.9)
Relative
-7
Recycling, %8
(-16-S)
12 (10-16) -3
(-148)
14 (l.H.6)c (1.7-1.9)”
are in parentheses.
16 (14-22) *Relative
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140
ANWER,
Relative recycling was -7, -3, and 16% for 6-T, 3-T, and 2-T, respectively, in fed ponies, and 12 and 14% for 6-T and 3-T, respectively, in fasted pomes (Table 1). The ratio of 14C/tritium in plasma glucose indicated the relative recycling of 14Cwith respect to tritium (20). Since that ratio did not increase for 3-T and 6-T, the relative recycling values of -7 and -3% for 6-T and 3-T, respectively, were considered negligible. Relative recycling of 14% for 3-T in a fasted pony was within the 95% cotidence interval for 6-T in fasted ponies. Glucose mass of the sampled pool (MJ and total-body pool differed little between isotopes for a particular trial. Therefore, the masses calculated from 14C and tritium data are combine,d together (Table 2). Minimal mass of total-body glucose decreased significantly in fasted ponies compared to fed ponies. The sampled pool varied from 50 to 66% of the minimal mass in fed Denies and from 61 to 67% in the fasted ponies. The miximal mass (Mmax), which was relatively higher than total2T
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2. Ratio of 14C to tritium in plasma injections of a mixture of [UJ4C]glucose and TJglucose (B) , or [2-Tlglucose (CJ in fed ponies a faskd pony. Values normalized to a ratio of FIG.
TABLE
6
glucose after single [6-TJglucose CA), [3or [6-TJglucose (II] in 1 for dose.
Values
AND
GRONWALL
body glucose (M,) calculated by assuming uniform catabolic rate, ranged from 147 to 353 mg/kg body wt. DISCUSSION
The precise estimation of sampled pool and outflow from this pool depends on the estimation of zero-time intercept and the initial slope, which in turn depend on the rapidity of sampling. Any problem with sampling, and thereby the estimation of zero-time intercept and initial slope, would result in large variations in the estimates of those variables. In the present study, with the exception of one pony, the estimates of sampled pool ranged from 70 to 95 mg/kg (Table 2>, and those of the outflow from that pool ranged from 3.9 to 6.3 mglmin per kg (Table 1) in fed ponies. This small range suggested that the sampling was rapid enough for relatively precise estimations of those variables. Production of new plasma glucose from tricarbon units which originated from plasma glucose (Cori cycle) can be estimated from the relative recycling of carbon obtained by using [6-Tlglucose simultaneously with [U14C]glucose (7). If recycled tricarbon units, like lactate and pyruvate, contribute significantly to glucose production, a significant portion of tritium from position 6 of glucose would be exchanged with protons of the medium during conversion of lactate or pyruvate to phosphoenol pyruvate via oxaloacetate (16). That would result in a higher irreversible loss of glucose when estimated from [6-Tlglucose than from [UJ4C]glucose. In our fed ponies, when [6-Tlglucose was injected simultaneously with [ UJ4C]glucose, a negligible relative recycling of carbon suggested that the Cori cycle contributed little, if at all, to production of glucose. Glucose production via the Cori cycle has been reported as 4 and 23% in sheep (16) and postabsorptive rats (7), respectively. The negligible relative recycling of carbon in fed ponies given [6-Tlglucose or [S-T]glucose indicated that resynthesis of glucose carbon from glycerol also was negligible. Resynthesis of glucose from glycerol would return tritium to plasma glucose when [6-Tlglucose is used but not when [3-Tlglucose is used, because a significant portion of tritium from position 3 of glucose would be lost during the conversion of glycerol, which enters the gluconeogenic pathway distal to oxaloacetate, to glyceraldehyde 3-phosphate via dihydroxyacetone 3-phosphate (16). Therefore, if glucose were resynthesized extensively via glycerol, estimates of irreversible glucose loss obtained with [ST]glucose would be significantly higher than with [G-Tlglucose. That would result in higher relative recycling of carbon to 3-T than to 6-T.
2. Glucose masses in fed and fasted ponies in Glucose
Total-Body Expt
Types
Sampled
Pool,
+ + + + +
6-T 6-T 3-T 3-T 2-T
are means
Fed (3) Fasted (3) Fed (3) Fasted (1) Fed (3) and range.
Significant
86 86 106 95 92 difference
Mass,
“g/kg
mglkg M ml”
UJ4C u-14C UJ4C UJ4C UJ4C
CHAPMAN,
(70-95) (63-99) (81-144) (8&95) dp < 0.05)
153 131 172 151 167
M”
(124167)” (97-148)b (12%147) (14%X4) (160-171)
- b < a. Numbers
166 168 211 193 227 of animals
(13&200) (157-179) (143-330) (17%207) (19%252)
k&Y
178 179 225 202 239
(147-213) (165-191) (15&353) (185-218) (21@266)
are in parentheses.
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GLUCOSE
UTILIZATION
AND
141
RECYCLING
Glycerol provided about 5 and 11% of glucose carbon, respectively, in sheep (3) and postabsorptive rats (6). The difference in estimates of irreversible loss of glucose, determined simultaneously with [Z-Tlglucose and [UJ4C]glucose, indicated a selective loss of tritium in position 2 relative to 14C, which is also evident from Fig. 1C. The loss of tritium inposition. 2 most likely occurs at the level of hexose 6-phosphate isomerase, and a single cycle between glucose 6-phosphate and fructose 6-phosphate would remove 75% of tritium from position 2 of glucose (14, 17). The selective loss of tritium in position 2 relative to 14C was first interpreted as the result of recycling of plasma glucose via ihe Cori cycle and the degradation of labeled glycogen (17). It is now believed to be due to the recycling of glucose via the Cori cycle and to futile cycle of glucose into glucose 6-phosphate and fructose 6-phosphate (13, 23). Futile cycle at glucose 6-phosphate stage would cause detritiation of [2Tlglucose and futile cycle at the triose phosphate stage would cause detritiation from positions 3, 4, and 5 (5). Since the Cori cycle did not contribute significantly to glucose resynthesis and no detritiation was evident with 13-T]glucose in the present study, in fed ponies the selective detritiation of [2-Tlglucose is due to the futile cycle at the glucose 6-phosphate stage, and there is no significant futile cycle at the triose phosphate stage. Thus, the relative recycling of carbon with respect to [ZTlglucose (Table 1) indicated that 16% of plasma glucose carbon was recycled via the futile cycle at glucose 6phosphate stage. The presence of such a futile cycle has been demonstrated in isolated hepatocytes (5) and in other species (19), but its physiological significance, if any, is not understood. Irreversible loss of glucose did not decrease significantly during fasting. A significant decrease in irreversible loss has been reported for fasting dogs (22), rats (2) and horses (9), after use of [ 14C]glucose, and in ponies (I 1) after use of [2-Tlglucose. The relatively small number of animals in this study may be the reason for the lack of a significant difference. A significant difference between the sampled pool of fed and fasted ponies was not expected, since plasma glucose concentration did not decrease significantly during fasting. The relative recycling of 14C with respect to [6-Tlglucose in fasting ponies suggested that about l&16% of glucose carbon was derived via recycled tricarbon units. This is consistent with the idea that recycling is highest in animals with a high rate of glucose turnover. In one study where [3-Tlglucose was simultaneously injected with [UJ4C]glucose into fasted ponies, the calculated relative recycling was not different from that estimated from [ 6-T]glucose and [ UJ4C]glucose. That result suggested that glycerol was not a source of recycled glucose carbon in fasting ponies. However, more studies are needed before a definite conclusion may be drawn. Ponies derive volatile fatty acids from cellulose diges-
tion in the large intestine (1), which may not be completely empty ifter 72 h of fasting, so propionic acid from cellulose digestion could sti .ll contribute to glucose production during fasting. Liver g plycogen stores should be exhausted by 72 h of fasting. Thus, gluconeogenesis from amino acids probably played a major role in glucase nroduction in our fasted ponies. Th; minimal glucose m&s calculated from the plasma specific radioactivity curve would represent total-body glucose if the major site of catabolism is in or near the rapidly mixing pool (sampled pool). On the other hand, the maximal mass would represent totalbody glucose if catabolism occurred at a site as far as possible from the sampled pool (18). The total-body glucose for fed ponies could be between 124 and 353 mg/kg, since the site of catabolism is not known. If the maximal mass represents total-body glucose, then fasting did not have any effect on total-body glucose in ponies. However, if the minimal mass represents total-body glucose, as it is shown for rats and rabbits (19), then total-body glucose decreased during fasting in ponies. In that case it would appear that mass of glucose some place other than the sampled pool is decreased, since there was no change in the sampled pool during fasting. No definite conclusions regarding the effect of fasting on the variables of glucose metabolism in ponies could be drawn because of the limited number of animals. However, it appears that ponies adjusted to fasting by lowering outflow from the sampled pool and irreversible loss, and possibly by decreasing total-body glucose. Relative recycling of l&16% in fasted ponies suggested increased gluconeogenesis. The use of [UJ4C]glucose, [6-Tlglucose and [3Tlglucose resulted in similar estimates of irreversible loss of glucose in fed ponies, indicating negligible glucase resynthesis via the Cori cycle and glycerol. Studies with [2-Tlglucose estimated the recycling of plasma glucase carbon which occurred via the futile cycle at the glucose 6-phosphate stage. This futile cycle would overestimate glucose resynthesis and hence the irreversible loss, since detritiation of labeled glucose would occur without any net synthesis of glucose. Thus, estimates of irreversible loss of glucose from [6-Tlglucose and [3Tlglucose would better estimate glucose utilization, Use of [UJ4C]glucose along with glucose variously labeled with tritium should be useful in quantitative estimation of precursors of glucose resynthesis in various physiological and nutritional conditions. This
study
was supported
in part
AM11384 and RR()5619.
This work is contribution Sciences, Kansas Agricultural
tan*
Present Universitat Present University Received
no.
by Public
Health
146, Department Experimentation
Service
Grants
of Physiological Station, Manhat-
address of M. S. Anwer: Institut fur Pharmakologie, Munchen, West Germany. address of R. Gronwall: College of Veterinary Medicine, of Florida, Gainesville, Fla. for publication
7 November
1974.
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R. A., AND H. F. HINTZ. Glucose tolerance fatty acid on plasma glucose concentrations Sci. 30: 514-518, 1970.
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N., R. A. SHIPLEY, R. E. CLARK, AND G. E. INCEFY. 14C in carbohydrate metabolism: glucose pool size and rate of in the normal rat. Am. J. Physiol. 196: 24%252,1959.
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ANWER,
142 3. BERGMAN, E. N., D. J. STARR, AND S. S. REULEIN, JR. Glycerol metabolism and gluconeogeneris in the normal and hypoglycemic ketotic sheep. Am. J. Physiol. 215: 847-880, 1968. 4. BRUNO, G. A., AND J. E. CHRISTIAN. Correction for quenching associated with liquid scintillation counting. Anal. Chem. 33: 650--651, 1961. 5. CLARK, D. G., R. RONGSTAD, AND 5. KATZ. Isotope evidence for futile cycles in liver cells. Biochem. Biophys. Res. Commun. 54: 1141-1148, 1973. 6. DE FREITAS, A. S. W., AND F. DEPOCAS. Glyceride-glycerol release and the interconversion of glucose and glycerol in normal and fasted rats. Can. J. Physiol. Pharmacol. 48: 561-568, 1970. 7. DUNN, A., M. CHENOWETH, AND L. D. SCHAEFFER. Estimation of glucose turnover and Cori cycle using glucose-6-T-14C. Biochemistry 6: 6-11, 1967. 8. DUNN, A., AND S. STRAHS. A comparison of 3H and 14C glucose metabolism in the intact rat. Nature 205: 705-706, 1965. 9. EVANS, J. W. Effect of fasting, gestation, lactation and exercise on glucose turnover in horses. J. Animal. Sci. 33: 1001-1004, 1971. 10. FRYER, H. C. Concepts and Methods of Experimental Statistics. Boston: Allyn and Bacon, 1966, p. 260-261. 11. GINOCHIO, R. J., AND J. W. EVANS. Glucose-2-T turnover in Shetland ponies. J. Anim. Sci. 37: 484-487, 1973. 12. HETENYI, G., JR., AND D. MAK. “H-2-glucose as tracer in turnover studies. Can. J. Physiol. Pharmacol. 48: 732-734, 1970. 13. HUE, L., AND H. G. HERS. On the use of c3H, 14C) labeled glucose in the study of the so-called “futile cycles” in liver and muscle. Biochem. Biophys. Res. Commun. 58: 532-539, 1974. 14. ISSEKUTZ, B., JR., M. ALLEN, AND I. BORKOW. Estimation of
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glucose turnover in the dog with glucose-2-T and glucose-U-14C. Am. J. Physiol. 222: 710-712, 1972. JONES, G. D. Determination of the specific activity of labeled blood glucose by liquid scintillation using glucose penta-acetate. Anal. Biochem. 12: 249-253, 1965. JUDSON, G. J., AND R. A. LENG. Estimation of total entry rate and resynthesis of glucose in sheep using glucose uniformly labeled with 14C and variously labeled with :
