Rate of Incorporation of CO, Carbon into Glucose and Other Body Constituents in Vivo Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Entomology on 08/09/12 For personal use only.

R. A . SHIPLEY AND .4. P. GIBBONS Veterans Administration Hospital und Department of Medicine, Cuse- Western Reserve University, Clevelund, Ohio 44105 Received April 28, 1975

SHIPLEY, R. A . , and GIBBONS, A. P. 1975. Kate of incorporation of CO, carbon into glucose and other body constituents in viva. Can. J. Physiol. Pharmacol. 53,895-902. Specific activity curves of respired CO, and of body glucose after intravenous NaHI4CO, as tracer and, in separate experiments, after [U-14C]glucoseas tracer were employed to assess rate of interchange of carbon between HCO, and glucose, and to calculate other rates of input and output for each of these substances. Solution for six rates attending the model was by integrals rather than by curve analysis. Fasting caused a twofold increase in rate of transport of CO, carbon to glucose. Whereas in fed animals this rate was only 7% of the fonvard flow from glucose to CO, , it rose to 41% during fasting. Glucose carbon derived from CO, rose from 3.7 to 20%. As expected. the rates of entry of new glucose to blood. and the conversion rate of glr~coseto products in body depots and to CO, were reduced by fasting, whereas, the non-glucose input to CO, was increased. Fast~ngwas attended by a 20-fold increase in rate of conversion of C02-derivedcarbon to hepatic glycogen and a fourfold increase to non-hepatic glycogen. Protein exceeded all whole-body depots for rate of acceptance of such carbon, and total lipids received an appreciable amount, but fasting caused no overall increase for either. SHIPLEY. R. A., et GIBBONS, A . P. 1975. Rate of incorporation of CO, carbon into glucose and other body constituents in vivo. Can. J. Physiol. Pharmacol. 53,895-902. On utilise les courbes d'activite specifique du CO, expire et du glucose sanguin apres injection de NaH14C0,, 011 de [U-14Cjglucose pour determiner la vitesse d'echange du carbone entre HCO, et le glucose, et calculer les autres vitesses d'entree ou de sortie de chacune de ces substances. Les solutions des six parametres composant le modele peuvent 2tre calculees sous forme d'integrales plutbt que par analyse de courbes. I,e jeGne double la vitesse de transport du carbone provenant du CO, vers le glucose. Chez l'animal nourri. cette vitesse est 7% du flux du glucose vers le CO, mais elle monte h 31% pendant le jeiine. Le glucose contient du carbone provenant de CO, dans la proportion de 3.7% chez I'animal nourri, et 20% chez l'animal hjeun. Comme prevu. la vitesse de neoformation du glucose sanguin et la vitesse de transformation du glucose en dep6ts organiques et en CO, sont rkduits par le jefine, tandis que le C 0 2 d'origine proteique ou lipidique est augment&.Le jeClne est suivi d'une augmentation de 20 fois dans la conversion du carbone venant du CO, en glycogene hepatique et de quadruple en glycogene non hepatique. I x s proteines constituent de loin les dep6ts organiques qui acceptent le plus de carbone en provenance du C 0 2 ,et les lipides totaux en resoivent une quantite appreciable, mais le jeilne ne cause aucune augmentation globale de ces reactions. [ Traduit par le journalj

Models devised to represent glucose kinetics in vivo frequently assume non-reversibility in the pathway from glucose to CQ2 (e.g. Baker et al. 1961 ) . Yet evidence exists for the reverse reaction via C 0 2 fixation in intermediates such as rndate and oxalacetate. Reverse conversion is easily shown by recovering tracer in b l o d glucose after giving kJaW1CCO3 to rats (Ashmore 1959; Ashmore et a&.1961; Landau et al. 1962; Wagle and Ashmore 19631, or human subjects (Adlung et al. 197 1) . The reverse pathway in a working model may bc neglected if the rate is insignificant in 'Veterans Administpa tiara Project No. 7869-0 1.

relation to other rates in the overall system. This can be decided only by measuring the actual conversion rate of C 0 2 carbon to @ucose along with other attendant rates. In the present report these rates were calculated from specific activity (sa) curves of C 0 2 and glucose with NaH1*C03 as tracer, and again with [U-14&l]glucoseas tracer. The computation was based on integrals (subtended area) of the curves rather than on curve analysis in which slopes and intercepts are interpreted in terms of a complex pool system. It was possible to calculate all six rates attending a simple reversible gluc~se-HCO:~ system centered in blood.

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CAN. J. PHYSIOL. PHARMACOL. VOL. 53, 1975

Hours

FIG.1. Specific activity (sa) curves for COz and for glucose. The uppermost curves are for COa when H1*COsis given as tracer. At bottom are subordinate curves for glucose in these same animals. Curves at the top in B are for glucose when [U-'*C]glucose is given as tracer, and those below are for the attendant sa of COr. Specific activity is expressed as percentage dose per milligram C . Each data point is the mean for 3-16 rats, with the number per group averaging seven for COBvalues and six for glucose. The vertical bars define standard errors of the means. When not seen they are within the solid circles. The initial values for nominal zero time in the case of COP after giving NaH1'C03 are those for the first minute of collection. For glucose after giving [U-l'C]glucose, the zero-time value is taken as 100 divided by the total body glucose in the homogenized whole rat (Shipley et al. 1967).

Rates of movement of C 0 2 carbon to protein, lipids, and glycogen also were estimated by a calculation which employed the sa curves of expired CO., after giving NaN14CO:3,and the values for tracer uptake in these recipient materials at 6 h after injection.

Experimental Procedures Male rats of Sprague-Bawley strain weighing between 170 and 260 g maintained on Purina laboratory chow were given NaH14C03into a tail vein after being lightly anesthetized with sodium pentobarbital, 4 mg/100 g, given intraperitoneally. Animals being fasted were deprived of food overnight (17-21 h ) . The dose of labeled bicarbonate varied from 20 to 44 pCi in 0.3 ml of a solution of 0.017 M NaHC03 and 0.0003 M NaOH. The rats were retained in a glass jar through which COz-free air was drawn, then delivered to a C02-absorbing system containing 2 N NaOH (Skipley et al. 1967). Occasional animals, despite confidencz that tracer was cleanly injected into the tail vein, showed evidence of extravasation as indicated by a slow increase in expired 14Cduring the first 5-30 min following the immediate sharp rise attending the injection. Such animals were discarded because direct venous delivery of all tracer is followed by prompt progressive decline in output after the first minute. Serial samples of expired air

provided data points for time-curves of the specific activity (sa) of COa carbon (Fig. 1A). At progressive intervals after delivery s f tracer, separate series of rats were homogenized irt toto to provide attending sa values of body glucose. Specific activity curves for the C 0 2 of breath and of glucose also were constructed for experiments in which [U-l'C]glucose was given as tracer (Fig. 1R). Data points were mean values previously obtained in normal rats (Shipley et al. 1967, 1970, 1974). Previous publication~describe methods for homogenizing the whole rat (Shipley et (11. 1947), and for homogenizing liver separately (Shipley et al. 1974). Also described previously are all methods for chemical and physical processing of body constituents and for estimation of sa (Shipley ct al. 1967).

Calculations The Glucose-C02 System Calculations were based solely on the subtended areas to infinity of the four curves of Fig. 1. Areas to 6 h were measured by planirnetry after plotting the curves on rectilinear coordinates. Residual area to infinity under the tail of each curve was estimated by extrapolating the segment between 5 and 6 h toward infinity and calculating the integral to infinity

$97

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SHIPLEY AND GIBBONS: INCORPORATION OF CO, CARBON

FIG.2. Model for central blood pools of glucose carbon and bicarbonate carbon. Sources and destinations of moving carbon, along with calculated rates of movement ( R ) are given in Table 1.

for this terminal slope with onset at an intercept adjusted to 6 h. i.e., sa value at 6 h / (0.693/half time) = residual area to infinity. This additional area ranged from less than 1 % of total to a maximum of 6 % . The model employed for rate calculations is illustrated in Fig. 2. Compartments shown represent glucose carbon (pool a ) and HCO:! carbon (pool b ) , both of which are centered in the mixed central blood of heart and lungs. The sa of whole-body glucose is a close approximation of that in this blood (Shipley et al. 1967). Likewise the sa of C 0 2 in the breath reflects that of HCO,< in the central blood. It is assumed that essentially all glucose arising from gut or via gluconeogenesis will traverse this pool, as will all C 0 2 derived from oxidation within the body. Exceptions are the fraction of new glucose in portal blood intercepted by liver during the fed statc, and the fraction of synthesized glucose retained by liver during gluconeogenesis. Also excluded is that portion of CO, generated in sifu by liver, and fixed therein without reaching the central blood pool. These hepatic fractions are assumed to represent only a small portion of total body movement of glucose and WCO::. A compartment of Fig. 2 need not be construed as a pool in the sense of being delimited by discrete anatomic boundaries. Each simply represents a compound in blood which receives essentially all tracer and its kinetically active tracee mixed together for purposes of sampling. The pattern of intermediate products between the two is not relevant to calculations. With [U-lT1gIucose as tracer, the overall disposal rate of glucose carbon is a weighted mean of rates for separate carbons at all 6-positions, but the rclative contribution of carbon atoms from various sites is not necessarily the same in R2 as in R,,. From the area under the sa curve of glucose after giving tracer as bicarbonate, coupled with

the area under the concomitant curve for expired C 0 2 , the fraction of incorporated glucose carbon arising from COZ carbon is calculable, thus: [I] Fraction, glucose carbon from CO, = sa glucose d t

To calculate the actual rate of conversion of CQ2 carbon to glucose carbon along with other rates centered in the glucose-COz system the integrals for the two sa curves, after giving glucose as tracer, also must be employed. Although several different equations are applicable for solution of the model via integrals of the four observed sa curves, expressions [2] through [5] were chosen for this purpose:

[3] R2

sa," dt

+ R,

Somsa," dt

=

100

Specific activity is expressed as percentage of dose per milligram C corrected to a rat weight of 100 g. The superscripts represent the pool labcled and the subscripts the pool sampled, e.g., saila means inject labeled glucose and sample C 0 2 for specific activity. Equation 2 says simply that when pool a is labeled the ultimate loss of tracer is 100?41, that it leaves the system via both pool a ( R 2 ) and pool b ( R e ) , and that the amount lost via each route is equal to the integral to infinity of the satime curve at each point of exit inultiplied by the corresponding rate of carbon loss. Equation 3 is a similar equation when bicarbonate (pool b ) is labeled. Expressions [2] and [3] are simultaneous equations that may be solved for R2 and Ro. An expression for fraction of glucose derived from CQ2, i.e., a from b is R 4 / ( R 4 -t R 1 ) . Or, because of assumed steady state about each pool, the denominator also can be written R2 R3. Thus, [ l ] becomes:

+

iw

sa,b dt

L-43

R,/(R, 6 K3)

=

s a t dr

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C.4N. J . PHYSIOL. PtI.4RM.4C(lL. VOL. 53, 1975

Another expression gives fraction of CQ2 carbon derived from glucose carbon, i.e., b from a:

im Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Entomology on 08/09/12 For personal use only.

sacdt

1151 R , / ( R , + R , )

= K,/484

Rd = sa; dt

With R p and R6 now being known, the simultaneous 141 and [ 5 ] are solved for Rs and R4. Then, from assumed steady state: R1 == R2 R3 - Rq and R, = Rq R(;- R3. This completes the solution. Other qualities are: Fraction of C 0 2 to glucose ( b to a ) =

bonate carbon (from 191) gives separate rates of transport (Shipley et aE. 1967).

Results The Glucose-GO2 System The upper curves of Fig. BA are those for the specific activity (sa) of C 0 2 after giving WaHC03 to fed and fasted rats. The areas under these curves defined by the integral

+

+

are, respectively, 103 and 186 units whcn abscissa1 values are expressed in minutes. The lower companion curves are for the sa of @ucose with the samc bicarbonate tracer. Their subtcilded areas

Fraction of glucose to CQ, ( a to 6) =

Bu 'l

R,l(B,

f

R,?

are, respectively, 3.8 and 23.3 for fed and fasted rats. Figure 1B shows curves obtained when [U-14C]glucose was administered. The upper curves for glucose have areas

=

'Irreversible disposal' of glucose

C81

R2

+ K3LR61'(R6 +

=

=

100 sa," d t

'Irreversible disposal' of C 0 2 =

estimated to be 160 and 455, respectively, for fed and fasted rats. Attending curves for sa of C 0 2 in the Bower part of the figure have respective areas

Disposal t9J C 0 2 Carbon to Sites Other tkzan Gbuco.re Whereas the sojourn of tracer from labeled HCQI in recipient glucose is transient, that in large carbon reservoirs such as fat, protein, arnd the bulk of glycogen, is long-sustained. Thcse sites. acting as sinks, are considered to remove tracer from the syctem irreversibly during the 6-h period of observation. The combined rate of movement of natural carbon to such sinks plus breath a n d snrine is given by [9]. The fraction of total disposed tracer recovered at any site in 6 h is proportional to the rate of movement of natural carbon to each. Thus, suck fracti~~nal amounts of tracer multiplied by overall disposal rat% of bicar-

of 5 1 and 74. Table 1 includes rates ( R ) for the inodel of Fig. 2 as calculated by [2] through [ 5 ] . Also shown are fractional conversion values (see [6j and 171) expressed as percentage, and rates of irreversible disposal calculated by [8] and 191. The rate of new glucose carbon entry to blood (Rr) is 0.6 1 rng C per minute in the fcd state as compared to 0. B 8 during fasting when alimented glucose does not enter. Rate of conversion to products in body depots (&) drops from 0.30 to 0.882 as a result of fasting, and that to C 0 2 ( R 3 ) falls from 8.33 to 0.15. Of particular interest is the effect of fasting on the rate of conversion of C 0 2 carbon to glucose

SHIPEEY AN11 GIBBONS: INCORPORATION OF CO, CARBON

TABLE 1. Rates of transport of carbon (R), rng mins ' (100 g rat)-', and fractional conversions. See Fig. 2

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Fig. 2 symbol Non-CO, input to glucose (gut, hepatic synthesis) Glucose output to body depots (including some fixed C02) Glucose to CO2 (partly reversible) C 0 2 to glucose (largely reversible) C 0 2from non-glucose precursors Direct loss of COs to excreta, and nsn-glucose fixation 'Irreversible loss', glucose (breath and body depots) [8] 'Irreversible loss' 602(breath and body depots) [9] Percentage of glucose carbon from C 0 2(€41 x 100%) Percentage of C 0 2carbon from glucose ( [ 5 ] x 100%) Percentage of C 0 2converted to glucose ([6j x 1 0 % ) Percentage of glucose converted to C 0 2 ([7] x 100x)

RI 2

R3

R4 R5

R6

Fed 0.61 0.30 0.33 0.024 0.65 0.96 0.62 0.97 3.7z

32% 2.4% 52%

Fasted 0.18 0.082 0.15 0.047 0.75 0.85 0.22 0.86 20% 16% 5.2% 64%

multiplied by the overall disposal rate csf C 0 2 carbon (last column) gives the separate transport rates in the bottom two rows. The rate of conversion of C02-derived carbon to whole body protein exceeded that to any other internal product, and was four o r five times greater than the rate to lipids (Table 2 ) . In fed rats the tracer uptake in these two products was measured separately for liver Fixation of C 0 2 in Body Depots and non-hepatic tissue. The calculated rate to As noted in the calculations section, the cal- hepatic protein was 0.0020 mg C per minute caalatiora of rate of transport to body depots is as compared to 0.01 85 for non-hepatic promade under the assumption that no significant tein. For lipids these respective rates were amount of tracer is lost from tlaese large pools 0.00069 and 0.0025, Thus, absolute values during the 6-h period of observation. Such a sink were greater for non-hepatic tissue, but hepatic effect should be evidenced by a progressive rise conversion would be greater in relation to the of accumulated tracer toward a sustained pla- sizc of recipicnt mass of material. teau. This behavior has been demonstrated for Because previous studies (Shipley et al. whole body lipids and protein with labeled 1974) have shown that glycogen is an unglucose as tracer (Shipley et al. 1967, 1974). depeindable true sink for tracer, the uptake in It was verified in the present series of fed both hepatic and non-hepatic glycogen was rats after NaM1*@03by following the uptake measured serially in fed and fasted rats given at 4, 1, 4, and 6 h in those two products. The NaH1-iCO:+.Departure from an ideal ccsntour problem of glycogen will be discussed s~abse- is seen in the curvcs for fed rats (Fig. 3) in y uently. that tracer content falls off somewhat after the In Table 2 are values for perccntagc of dose initial uupslope. I n any case a well-marked of tracer recovered in breath and body depots effect of fasting is apparent. For hepatic glycoof rats receiving NaH14C03. Recovery in urine gen all data points show a difference which (not shown), approximated 1.,5 76,and that in exceeds the 99% confideilce limits. For nonbody bicarbonate (largely lost as C 0 2 during hepatic glycogen the same holds true at 2, 4, homogenization) would probably amount to and 6 h. Based on the uptake at 6 h the rates about L % (Shipley et a/. 1967). Thus, the of transport of C 0 2 carbon to glycogen were total recovery at 6 h approximated 100%. The calculated in the same manner as for lipids and fraction recovered in a product (expressed as protein. As shown in Table 2 this rate is over percentage in the first two rows of the table) 20-fold greater for hepatic glycogen during

(It4). It rises from 0.024 to 0.047 as a consequence of fasting. In terms of percentage glucose carbon derived from C 0 2 the increase is from 3.7% in the fed state to 20%, during fasting. Norm-glucose-derived COz formation ( H i 5 ) rises from 0.65 to 0.75 mg C per minute, and C 0 2 output rate to breath ( R 6 ) falls from 0.96 to 0.85.

1.4540.044 0.0133 0.0125

0 . 2 8 4 0.015 0.0029 0.0024

92.4+ 1.6 0.93

0.79

Fasted Fed

Fasted

1.37k0.067

0.30+0.028

96.0+ 1.5

Fed

*Fraction of dose recovered times overall disposal rate. ?Equation 9.

Percentage of dose Recovered Rate of disposal, rng c I&(100 g rat)-'*

Total body protein

Total body lipids

Expired air

0.0089

1.03k0.14 0.00042

0.043f 0.014

Liver glycogen

0.0030

0.35k0.068 0.00066

0.068f 0.023

Non-hepatic glycogen

TABLE2. Percentage of dose recovered at 6 h (+_SE of mean), rates of loss in breath, and rates of disposal of CO, carbon to depot materials after giving NaH14C0,

96.8

1.2540.083 0.00107 0.0108

97.9

0.86t

0.97t

Total Overall activity disposal recovered rate 0.11 k0.033

Total glycogen

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Rate of incorporation of CO2 carbon into glucose and other body constituents in vivo.

Specific activity curves of respired CO2 and of body glucose after intravenous NaH14CO3 as tracer and, in separate experiments, after [U-14C]glucose a...
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