Pathways of glycogen repletion.

P~IYSIOI,O~;I(.AI, REVIEWS Vol. ‘72, No. 4, October 1992

l’rit,tc7l

iu I ‘..S.:l.

Pathways GERALD Department

of Internal Departments

of Glycogen Repletion

I. SHULMAN

AND

BERNARD

R. LANDAU

Medicine, Yale University School of Medicine, New Haven, Connecticut; of Medicine and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio

I. Introduction .......................................................................................... II. Definition of Direct and Indirect Pathways ......................................................... ............................................................. III. Quantitation of Pathway Contributions A. Basis for concluding the indirect pathway predominates ........................................ B. Evaluation of the evidence ........................................................................ .............................................................................. IV. Regulation of Pathways A. Effect of fed versus fasted state .................................................................. B. Effect of dietary protein .......................................................................... C. Role of glucose versus insulin .................................................................... V. Source and Site of Three-Carbon Intermediates in Indirect Pathway .............................. VI. Is There Really a Glucose Paradox? ................................................................. ........................................................................................... VII. Conclusions

and

1019 1020 1021 1021 1023 1029 1029 1029 1030 1030 1032 1032

cose is built into glycogen molecules essentially as intact 6-carbon units.” Some 20 years later, Shikama and Ui glucose to fasted rats injected with In 1877 Claude Bernard declared (71, ll?), “the in- (99) administered [14C]bicarbonate and used the incorporation of 14C into disputable fact is that cane sugar administration considerably increases liver glycogen content, but how does blood glucose and liver glycogen as the measure of glucoor a substance neogenesis and glycogenesis. Increased incorporation of the sugar act- as a nutritive stimulator 14C into glycogen compared with glucose consequent to a directly converted to glycogen?” In 1923 intermediary glucose load led them to conclude that a glucose load metabolism of carbohydrates was reviewed in Physiologdirects the final product of hepatic gluconeogenesis icul Reviews (98). At that time, the initial step in glucose conversion to glycogen could only be ascribed to the from blood glucose to hepatic glycogen. transformation of glucose to a more reactive form that Although glycogen was readily formed in those in vivo glucose administrations, glucose proved a poor premight undergo condensation to glycogen. In 1931 Cori (18) described, again in Physiological Reviews, the cycle, cursor of glycogen in rat liver preparations in vitro (e.g., now bearing his name, in which glucose is converted to see Refs. 10, 26, 30, 42). That finding led to the suggesaccumulatlactic acid by muscle and the lactic acid reconverted to tion that much of hepatic glycogen initially glucose by liver. However, as Stetten and Stetten (109) ing in rat liver following refeeding after a period of wrote in 1960 in Physiological Reviews, measurement of starvation is derived from nonglucose precursors. That glucose administration readily replenished glycogen the actual pathway by which glycogen is formed from stores in vivo and yet glucose conversion to glycogen is glucose awaited the availability of isotopes. very limited in vitro was called by Katz and McGarry Hastings and colleagues (14, 112) in 1941-1942 and co-workers (47,88) the “glucose paradox.” They reshowed that “C from [llC]bicarbonate and [llC]lactate was incorporated into liver glycogen. Bollman (8) in examined the pathways by which hepatic glycogen stores, depleted on fasting, are replenished on glucose 1943 stated: “this may indicate that glucose is broken administration in vivo. From their quantitations they down to a three carbon intermediate before resynthesis to glycogen.” Boxer and Stetten (9) in 1944, from the concluded that the major pathway of glucose conversion to glycogen in vivo, after a fast, is by an indirect pathenrichment of deuterium in liver glycogen from fasted rats given glucose and lactate along with ‘H,O, con- way, i.e., glucose - glucose 6-phosphate - three-carbon compound(s) - glucose 6-phosphate - glycogen, and cluded that “glycogenesis was from fragments smaller not directly, i.e., glucose - glucose 6-phosphate - glythan hexose rather than glucose directly.” However, cogen (43, 47, 71). Cook and Lorber (17), eight years later, on injecting [lTheir experimental findings, which led to and were 14C]glucose into rats, found that the major portion of 14C in the glucosyl units of hepatic glycogen was retained in used to further support that conclusion, are contained in C-l. This finding was confirmed by others, i.e., 280% of five papers (46, 48, 51, 77, 78). These are summarized after first examining the definition of the pathways. the label from specifically labeled glucose was unrandomized in its conversion to glycogen. Hers (31) in 1955 Then that conclusion is evaluated in consideration of the findings by other investigators. We then examine data concluded: “this provides a strong indication that gluI. INTRODUCTION

0031~9333/92

$2.00 Copyright

0 1992 the American

Physiological

Society

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1020

GERALD

I. SHULMAN

AND BERNARD

on the regulation of the pathways, the site of formation of the three-carbon intermediate in the indirect pathway, and the validity of the conclusion that there is a glucose paradox. There have been other reviews on this subject (50, 60, 71). II. DEFINITION

OF DIRECT AND INDIRECT

PATHWAYS

The reactions in the direct pathway have then been taken as glucose - glucose 6-phosphate - glucose l-phosphate - UDP-glucose - glycogen. The carbon skeleton of glucose remains intact in that formation of glycogen. In contrast, in the indirect pathway, the carbon skeleton of glucose is cleaved with the glycogen formed from three-carbon intermediates. Distributions of label in glycogen on giving specifically labeled glucoses and the effect of an inhibitor of phosphoenolpyruvate carboxykinase, 3-mercaptopicolinate, on glycogen formation support lactate/pyruvate as the major intermediate (‘78,110). Therefore the reactions of the indirect pathway have been taken as glucose - lactate/pyruvate - glucose 6-phosphate - glycogen. Estimates of direct and indirect pathway contributions to glycogen formation from glucose have been made by tracing with radioactive and stable isotopes how much glycogen is formed from glucose with and without cleavage of the carbon skeleton of the glucose. In the circumstance depicted, one-half the glycogen is formed directly and one-half is formed indirectly (each pathway 10 units). If an approach is used that determines how much glycogen is formed from glucose with and without cleavage, the fraction via the direct pathway is then 0.5

glycogen glr;;e

~glucofZ-P

t

R. LANDAU

Volume

Therefore, by that definition, glucose 6-phosphate glucose is in the direct pathway. However, glucose-6phosphatase cannot differentiate between glucose 6-phosphate formed by the direct phosphorylation of glucose and that formed via lactate (56, 70). That is, of the 10 units of glucose 6-phosphate converted to glucose in the example, 5 units are formed directly and 5 units are formed indirectly. Thus glucose-6-phosphatase does not change, from the previous example, the proportion of glycogen formed from glucose that has been cleaved and uncleaved (it is still 0.5), but rather the total quantity of glycogen formed changes (10 rather than 20). Katz (41), in response to Rognstad’s definition, has in addition considered the consequence of the conversion of glucose to lactate. The example used to illustrate that view may be depicted as

glycogen 15

t 20 - glucose-6-P -

glucose \ 5

30

20 II lactate

By net balance, the contribution of each pathway is 10, i.e., a net uptake of 10 units of glucose (15 - 5) and 10 units of lactate in glucose equivalents (30 - 20). However, in terms of cleaved and uncleaved glucose, the glycogen would be formed one-third from intact glucose, i.e., 15/(30 + 15), where 30 is the number of molecules of glucose 6-phosphate formed cleaved via lactate and 15 is the number of molecules intact from glucose. This picture may be visualized as the sum of the individual fates of glucose and lactate. The one-third formed directly is then the 6.7 units of the 20 units of glycogen glycogen

Rognstad (89,90) concluded that because of hydrolysis of glucose 6-phosphate to glucose while glucose is being phosphorylated, i.e. “glucose cycling,” estimates using tracers can be in large error. Rognstad’s definition of the pathways is such that if the rate of glucose phosphorylation equals the rate of dephosphorylation, the direct pathway contribution is zero. That may be depicted as

glycogen 10 glucose .e

10

10

1

lactate

10 T glucose-6-P T I

72

glucose

t 6.7 glucose- 6-P + I 6.7 I lactate

glycogen glucose 1 33

t 13.3 glucose-6-P

There may be confusion in defining the pathways in other respects. First, in tracing the fate of a glucose load, the load has usually been labeled so that whether the glucose is cleaved in the formation of glycogen can be determined. However, the cleaved and uncleaved labeled fractions are not the measure of the pathway contributions to glycogen formation. This is because unlabeled substrates, i.e., not arising from the labeled gl ucose, can traverse the pathways during the tracing. The major contributors to that dilution of label are likely to be in the indirect pathway, i.e., lactate from muscle glycogen pyruvate from protein, and glycerol from triglyceride 9


October

PATHWAYS

1992

OF GLYCOGEN

glycogen galactose glucose

I t

lactate

4

r-4 I direct b glucose-6-P f w glycerol I\ indirect w pyruvate e 1 oxaloacetate

f prote/71-co* glycogen

+J

fatty acids Exchange of label with unlabeled carbons derived from acetyl-CoA formed from fatty acids can also occur via oxaloacetate in the tricarboxylic acid cycle (40). Galactose conversion to glycogen during the tracing would dilute label in the direct pathway. The conversion of mannose to glycogen is via fructose 6-phosphate, i.e., mannose 6-phosphate - fructose 6-phosphate - glucose 6-phosphate - glycogen. It is likely to make a small contribution to glycogen formation, but to the extent it did during tracing, since it would be uncleaved, it would dilute label in the direct pathway. Fructose, being cleaved (59), can be viewed as converted to glycogen via the indirect pathway. Second, glucose can be phosphorylated and then cleaved within the cell before its conversion to glycogen. Thus the sequences within the cell would be glucose glucose 6-phosphate - triose phosphate - glucose 6-phosphate - glycogen or glucose - glucose 6-phosphate - pyruvate -glucose 6-phosphate - glycogen. It seems best to define all cleavage of glucose before conversion to glycogen as occurring indirectly. Third, via the pentose cycle, glucose 6-phosphate via pentose 5-phosphate is converted to fructose 6-phosphate and glyceraldehyde 3-phosphate (5566). Because the carbons of glucose 6-phosphate are randomized and triose phosphate is formed, glycogen formed via the cycle is also considered indirect. The contribution of the cycle is also likely to be small (78, 97, 100). The indirect pathway then comprises several pathways by which glucose is cleaved before its conversion to glycogen. Which pathway is followed can determine physiological significance. Thus if glucose 6-phosphate is rapidly converted to pyruvate in the liver cell before its conversion to glycogen, as in

glycogen A 10

IO

glucose -glucose-6-P

essentially all the glycogen will have been formed after cleavage, i.e., formed indirectly. However, the physiological implications of this futile cycling, i.e., glucose

1021

REPLETION

6-phosphate conversion to pyruvate and its reversal in the same cellular environment, are quite different than the conversion of glucose to pyruvate in one cellular site, e.g., muscle or red blood cells, and then the conversion of that pyruvate to liver glycogen. In hepatocytes such cycling appears to occur (see sect. V). In conclusion, during glycogen formation from glucose, glucose cycling, glucose conversion to lactate, and the conversion of lactate to glucose, to the extent that these occur, will be determinants in the changes that occur in glucose, lactate, and glycogen content. Such changes may have profound physiological significance. However, those changes are not a measure of the pathways followed in the formation of the glycogen. The pathways of glycogen formation are defined as two, either with the carbon skeleton of the glucose intact, the direct pathway, or via three-carbon intermediates, the indirect pathway. This is the definition that has been used and is used in the presentation to follow. III.

QUANTITATION

OF PATHWAY

CONTRIBUTIONS

A. Basis for Concluding the Indirect Pathway Predominates

To quantify the extent exogenous glucose is converted directly and indirectly into hepatic glycogen, Newgard et al. (77) gave rats, weighing loo-160 g, fed a high-sucrose, low-fat diet, and then fasted 20 h, [U14C,3-3H]glucose, 167 and 334 mg . 100 g body wt-’ h-l ig, iv, and as a component of a solid diet eaten ad libitum. Glycogen was deposited linearly over a 3-h period, and portal plasma glucose concentrations seldom exceeded 8 mM. At a time when the specific activities of 3H and 14C in circulating glucose were about the same as in the administered glucose, the 3H specific activity of the glycogen was only 12-28% and the 14C specific activity was only 3250% as much as in the administered glucose. If it is accepted that no label is lost in the conversion of [U-14C]glucose and [3-3H]glucose to glycogen by the direct pathway and with negligible liver glycogen present in the fasting state, the specific activity of the glycogen formed would be expected to be that in the circulating glucose (74). Because, via the indirect pathway, all the 3H from [3-3H]glucose is removed, if conversion to glycogen were solely via the indirect pathway, the 3H specific activity of the glycogen would be zer0.l Newgard et al. (77) therefore concluded from the results with 3H that under their conditions only 1228% of the glycogen deposited in liver was deposited via the direct pathway. Via the indirect pathway, [U-14C]lactate, l

’ Tritium of [3-3H]glucose will be completely removed in the conversion of the glucose to lactate, i.e., the 3H is bound to the carbon that becomes the carboxyl group of the lactate. Tritium from [3-3H]glucose is also removed in the pentose cycle, i.e., 3 [3-3H]glucose 6-phosphate - 3C02 + 3 ribulose 5-phosphate - 2 glucose 6-phosphate + glyceraldehyde 3-phosphate, and in fructose cycling, i.e., [33H]fructose 6-phosphate - [3-3H]fructose-1,6-bisphosphate - 2 triose phosphate - fructose 1,6-bisphosphate - fructose 6-phosphate. Contributions of these two pathways are likely to be relatively small.


1022

GERALD

I. SHULMAN

AND BERNARD

formed from the [U-14C]glucose, would be converted to glycogen with a lower specific activity than that in the [U-14C]glucose to the extent 14C was diluted with 12C in the indirect pathway (60). Because 14C is then incorporated into glycogen by both the direct and indirect pathways, the results with 14C were concluded to indicate that at most 3250% of glycogen was formed directly. To provide more direct evidence for carbon flow from three-carbon compounds to glucose 6-phosphate continuing in the face of glucose load and active glycogen deposition, Newgard et al. (77) gave rats an intragastric load of glucose and infusions of [14C]lactate, [14C]alanine, and [14C]glutamine. The glucose load stimulated the incorporation of the 14C into glycogen. In a second study, Newgard et al. (78) infused rats with [l-14C]glucose under the same conditions as in their first study, except glucose was given at 40 mg. 100 g body wt-l h-l. Of the 14C in glucose from the glycogen synthesized, 38.8585% was in C-l. The [l-14C]glucose infused had 93-95% of its 14C in C-l by their assay. Plasma glucose concentrations ranged from 5.2 to 7.1 mM. When they gave rats [l-14C]glucose intragastrically at a dose of 84 mg 100 g body wt-’ . h-l, 54.7% was in C-l, and at 126 mg 100 g body wt-l h-l, 54.6% was in C-l. Samples of glucose from the glycogen were completely degraded, and the distribution of 14C in C-Z through C-6 was in accord with incorporations into C-Z through C-6 occurring via [3-14C]lactate, i.e., the indirect pathway.2 Calculating from these distributions the 14C that was incorporated into C-l of glucose via lactate, Newgard et al. (78) concluded that as a minimum two-thirds of the glucose converted to glycogen was via the indirect pathway. They also infused rats with [l-14C]glucose intragastrically at 84 and 126 mg. 100 g body wt-l. h-l, with and without mercaptopicolinate. In the presence of the inhibitor, in accord with the randomization occurring via lactate in the indirect pathway, the percentage of 14C in C-l increased from 54 to 84% at the lower glucose load and from 54 to 91% at the higher load. To provide evidence mercaptopicolinate was inhibiting the indirect pathway, presumably at the level of phosphoenolpyruvate formation, they showed that in its presence, glycogen was formed from glycerol and lactate accumulated. These findings were in accord with those of Sugden et al. (110). In a third study, Kuwajima et al. (52) allowed 18-h fasted rats, weighing -200 g, to refeed ad libitum either on a chow diet or the sucrose diet or the rats were given glucose intragastrically and intravenously, 126 and 167 mg 100 g body wt-’ h-l, respectively. Fifty minutes l

l

l

l

l

l

2 When [3-14C]lactate, which would be formed by glycolysis from either [1-‘4C]glucose or [6-14C]glucose, is converted to glycogen, the relative specific activities of the six carbons of the glucosyl unit of the glycogen, with C-l set to 1.0, are about C-Z = 0.8, C-3 = 0.15, C-4 = 0.15, C-5 = 0.8, and C-6 = 1.0 (33,60). However, in the fed state there may be incomplete isotopic equilibration of the triose phosphates, resulting in a difference in the specific activities of C-l, C-2, and C-3 compared with C-4, C-5, and C-6 (69).

R. LANDAU

Volume

72

after refeeding or beginning the glucose administration, 3H20 was infused. Twenty minutes later, livers were removed for determination of the 3H bound to C-2 and C-6 of the glucose from hepatic glycogen. Via the indirect pathway, the hydrogen bound to C-2 of glucose 6-phosphate becomes labeled during its formation from fructose 6-phosphate. In the direct pathway, glucose 6-phosphate, formed on phosphorylation of glucose, also becomes extensively labeled with 3H at C-2 because of the rapid equilibration of glucose 6-phosphate with fructose 6-phosphate. The bonding of 3H to C-6 is assumed to occur only in the indirect pathway at the level of pyruvate.3 Thus 3H bound to C-2 compared with that bound to C-6 provides a measure of the percentage of glucose 6-phosphate and hence glycogen formed via the indirect pathway. Their findings were as follows: chow ad libiturn, 75 and 77% formed through the indirect pathway; sucrose ad libitum, 43 and 55%; intragastric glucose, 61% ; and intravenous glucose plus mercaptopicolinate, 2.9-13.8%. The somewhat lower indirect pathway contribution in the sucrose-fed rats was presumed to be due to the conversion of fructose from sucrose to glycogen, since in that conversion labeling will be at C-2 and thus will appear as glucose converted to glycogen via the direct pathway. The decrease in the percentage on giving mercaptopicolinate is again in keeping with the incorporation at C-6 occurring via the indirect pathway before the conversion of oxaloacetate to phosphoenolpyruvate. In two studies, Katz and co-workers (46,48) infused chronically cannulated rats, fasted for 24 h, with [U13C]glucose at 180 mg 100 g body wt-’ h-l after giving a bolus of 90 mg/lOO g body wt. Portal blood glucose concentrations reached 8.0-10.2 mM. The enrichment in mass 186 of the glucosyl units of liver glycogen, calculated using an approach introduced by Kalderon et al. (39), was -50% of that of portal glucose, giving a direct pathway contribution estimate of 50%. Thus, by three independent methods (specific activity or enrichment in glycogen compared with that in glucose from which the glycogen was formed, randomization of specifically labeled glucose in its conversion to glycogen, and incorporation of 3H from 3H20 into glycogen formed from glucose), McGarry, Katz, and coll

l

3 Via the indirect pathway, 3H is bound to C-3 of pyruvate in the interconversion of pyruvate with alanine. Tritium is bound to C-3 of oxaloacetate in its equilibration with fumarate, and [3-3H]phosphoenolpyruvate will then be formed from the oxaloacetate. Carbon-3 of pyruvate becomes C-6 of the glucosyl units of glycogen. However, 3H from 3H20 can also be incorporated into C-6 of glycogen via 1) exchange in the interconversion of UDP-glucose with UDP-glucuronate and 2) the formation of [3-3H]dihydroxyacetone 3-phosphate from [1-3H]fructose 6-phosphate formed in exchanges catalyzed by phosphoglucoisomerase and phosphomannoisomerase; the [3-3H]dihydroxyacetone 3-phosphate is isomerized to [3-3H]glyceraldehyde 3-phosphate, with [1,6-3H]fructose 6-phosphate then formed by the condensation of the triose phosphates, i.e., fructose cycling, or by transaldolase exchange, i.e., [l-3H]fructose 6-phosphate + [3-3H]glyceraldehyde 3-phosphate - [l,6-3H]fructose 6-phosphate + glyceraldehyde 3-phosphate (55). How significant these conversions are in practice in a given circumstance is uncertain (44, 57).


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OF GLYCOGEN

1023

REPLETION

1. Percent label in speci$c carbon of liver glycogen on giving specifically labeled glucose with a glucose load

TABLE

Labeled Glucose

Species

Fast Period

Rat

24 h

1-14c

Rat

24 h

I-14C

Rat Rat Rat Rat Human Human Dog Rat

14-16 h Overnight 18 h 24 h Overnight Overnight 18 h 20 h

2J4c U-14C, 3-3H 1-14C, 6-14C 1-14c 6-14C 2-14c 1-14C 1-14c

Rat

20 h

Human Rat Human

lo-12 h 24 h 12 h

1J4C, 5J4C 6-14C 1-13C &14C 1-‘3c1 2J4c

Glucose Load

Mode of Administration

0.6 g/l00 g 0.05 g 14C/h 350 mg/lOO g 20 mg 14C 30 mg 100 g-’ h-l 243 mg 100 g-’ h-l 167 mg . 100 g-l. h-l 90 mg prime, 180 mg 100 g-’ h-’ 1 g/k 1 g/k 1.4 g/kg 40-126 mg 100 g-’ . h-l 400 mg/lOO g 84-126 mg* 100 g-l h-l 167-334 mg -100 g-’ h-l 540 mg/lOO g 165-170 mg/dl 100-600 mg/lOO g 2 mg kg-’ mine1 l

l

l

l

l

l

l

l

l

l

l

Stomach Intraperitoneal Stomach Intracardiac Intravenous Suprahepatic Duodenal Mouth Mouth Mouth Intravenous, Intragastric Intravenous Intravenous Intragastric Intravenous Intragastric Intravenous

intragastric

Time of Analysis

B. Evaluation of the Evidence 1. Randomization of specifically labeled glucose

Reference

4h

84

17

1, 3 h

86, 68

31

l-3 h 3h 2h 160 min 3-5 h 3-5 h 3.5 h 2, 3 h lh 2h 3h 3h 3.5-4.5 h 2-3 h 2-3.5 h

85-87” 86-87 60b 83-85 72” 80” 66 39-5gd 72 84-91d” 73 74 87” 82-87 79”

32 34 43 48 65 66 72 78

a Rats anesthetized. b Estimated from fractions of 14C in C-l-C-3 and C-4-C-6 of glucose from glycogen. d Rats restrained. e Rats given mercaptopicolinate. glycogen.

leagues arrived at the conclusion that one-half or more of liver glycogen formed from glucose after a fast is formed indirectly. They presented data to support a lack of phosphorylating capacity by liver as the explanation for the failure of glucose to be taken up in quantity directly by liver (52,71). Newgard et al. (76) also provided evidence in support of the hypothesis (24) that glucose administration resulted in a decrease in glucose-6-phosphatase activity, thus favoring the conversion to glycogen of glucose 6-phosphate formed by the indirect pathway.

% in Specific Carbon

’ 14Cin glucuronide

97 100 105 114 not

Katz et al. (48) gave [l-14C]glucose along with [U13C]glucose to rats. About 84% of the 14C in glycogen remained in C-l. This assumes about three times as much 14C was in C-2 through C-6 as was in C-6, i.e., was incorporated via pyruvate/lactate. Incorporation via transaldolase exchange and the pentose cycle to the extent, if any, that occurred would increase the percentage that remained in C-1.l~~ Newgard et al. (78) gave an intragastric bolus of [l-14C]glucose, 400 mg/lOO g body wt, to fasted rats, and 72% of the 14C in glucose from glycogen was in C-l after 1 h and 61% was in C-l after 2 h. Therefore they concluded that the degree of randomization was reduced with the large bolus. They suggested that the difference between the extent of randomization they had observed at 40-126 mg. 100 g body wt-’ h-l and that observed by Hers (31) was due to the fact that Hers gave a load of -350 mg/lOO g body wt, resulting in a higher blood glucose concentration. However, although portal glucose concentrations ranged between 6.7 and 16.4 mM in the study of Scofield et al. (97), there was no correlation between portal glucose concentration and the extent of randomization. Nor was there a difference discernible in the extent of randomization whether the glucose was given at a dose of 167 or 334 mg, just as there was no difference in the extent of randomization in the study of Newgard et al. (78) whether the dose was 40, 84, or 126 mg 100 g body wt-‘. h-l. Furthermore, Shulman et al. (105) gave 24-h fasted rats [1-13C]glucose by gavage in doses of 100 and 600 mg/ 100 g body wt. The percent 13C excess in C-l of glucose from the hepatic glycogen that formed ranged from 81.5 to 87.4%. There was no difference in the percent of 13Cin C-l at the two doses. Plasma glucose reached a peak of 8.9 mM at the lower dose and 12.7 mM at the higher dose. To examine the pathway contributions in humans, l

Table 1 records the conditions and results of studies in which the randomization of label from specifically labeled glucose into glycogen has been traced on giving a glucose load. Scofield et al. (97) gave glucose loads labeled with 1-14C, 5-14C, and 6-14C. The conditions were the same as those of Newgard et al. (77), except that the average weight of the rats was 198 g rather than between 100 and 160 g and no rats were restrained. Glucose was given at doses of 167 and 334 mg 100 g body wt-l. h-1 rather than the 40-126 mg. 100 g body wt-l h-l Newgard et al. (78) gave in their study of randomization of specifically labeled glucose. However, at the higher loads, Newgard et al. (77) also concluded that the indirect pathway contributed 270% to glycogen formation. Katz et al. (43) also found extensive randomization of 14C in glycogen on infusing [l-14C]glucose and [614C]glucose, 167 mg. 100 g body wt-l. h-l, into rats fasted 18 h. In contrast, Scofield et al. (97) found 73% of the 14C in glucose from the glycogen formed from the specifically labeled glucoses remained unrandomized. l

l

l


1024

GERALD

I. SHULMAN

AND

acetaminophen and diflunisal have been used to noninvasively sample intrahepatic UDP-glucose. Each is excreted in urine as a glucuronide conjugate. The glucuronide is formed in liver from UDP-glucose via UDP-glucuronic acid, and UDP-glucose is the immediate precursor of the glucosyl units of glycogen. There is no randomization of carbons in these conversions nor in the formation of UDP-glucose from glucose 6-phosphate. Magnusson et al. (65) gave healthy volunteers, after an overnight fast, diflunisal and [6-14C]glucose, 1 g/kg body wt. The distribution of 14C in the glucuronic acid moiety of the glucuronide excreted then presumably reflected the distribution of 14C from the [614C]glucose in the glucosyl units of glycogen (65). Of the 14C in the glucuronic acid, 71.9% was in C-6. Magnusson et al. (66) also gave subjects [2-14C]glucose in the same manner as [6-14C]glucose. Although 14C from [Z14C]glucose is randomized to some extent into C-l and C-3 of glucose 6-phosphate via the pentose cycle, 80.4% of the 14C in the glucuronic acid from of the excreted glucuronide was retained in C-Z. Wajngot et al. (114) infused [2-14C]glucose, 2 mg. kg body wt-’ min-‘, into each of two healthy subjects who had been fasted overnight and had ingested acetaminophen. Plasma glucose concentrations were 5.2 and 5.4 mM before infusion and rose to 6.3 and 6.7 mM, respectively, during infusion. Of the 14C in the glucuronic acid moiety, 76.6% in the first and 81.7% in the second subject were in C-Z. The reason Newgard et al. (78) observed extensive randomization of label of specifically labeled glucose in its conversion to glycogen while other investigators have not is unclear. It is probably related to differing conditions, since Landau and colleagues degraded samples provided by Newgard and co-workers and obtained results similar to theirs (78). Apparently it is not due to differing blood glucose concentrations nor to the mode of glucose administration. The [l-14C]glucose used by Newgard et al. (78) on their analysis had 93-95% of 14C in C-l, whereas Scofield et al. (97) used specifically labeled glucoses that on analysis had 297% of the 14C localized to the specific carbons, and Shulman et al. (105) used [l-13C]glucose that had 99% excess 13C in C-l. That would only minimally decrease the extents of randomization reported by Newgard et al. compared with those reported by the other investigators. Recycling of the circulating glucose with time, i.e., glucose - lactate - glucose, will also decrease the specificity of the position of the label in the glucose presented to the liver (46, 65). Newgard et al. (77) implanted intragastric, intravenous, and arterial catheters in their rats at the time fasting was begun. Huang and Veech (34) suggested that this “acute” rather than chronic implantation of catheters could have been responsible for at least some of the findings of Newgard et al. Newgard et al. (77, 78) restrained the rats given glucose intravenously and intragastrically for the 20 h of fast following catheterization but did not restrain the rats fed glucose ad libitum. Newgard et al. (78) noted that Hostetler and Landau (32) had infused [2-14C]glucose at only 30 mg 100 g body wt-l h-l into fasted rats under pentobarbital anesthel

l

l

BERNARD

R. LANDAU

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sia and found little randomization of label in glycogen. When Newgard et al. (78) gave [l-14C]glucose intravenously to rats anesthetized with pentobarbital, the percentage 14C in C-l of glycogen increased from 55 to 70%, and when i ntragastrically given, it i ncreased from .49 to 64% . They interpreted this to mean pentobar bital anesthesia decreased the indirect pathway contribution. Pentobarbital anesthesia has been reported to alter glucose kinetics (63). Alternatively, the anesthetic decreased stress and thus decreased the indirect pathway contribution. Shalwitz et al. (98a) gave [l-13C]glucose by introd uodenal infusion to 1 8-h fasted rats und .er halothane anesthesia and meas ured glycogen form ati .on in situ by 13C-nuclear magnetic resonance. From the distribution of 13C in glycogen they estimate a 70% direct contribution. They gave evidence that halothane did not alter the formation of glycogen. Retention of the position of a specific carbon label of 270% in the conversion of glucose to glycogen does not mean the direct pathway contribution to glycogen formation is 270%. Some 14C retained has been converted to [14C]triose via the indirect pathway and then returned to the specific carbon in glycogen. If [l14C]glucose is given and the distribution in glycogen is in percent, C-l = 77%, C-Z = 6%, C-3 = 2%, C-4 = 2%, C-5 = 6%) C-6 = 7%; 7% of the 14C has been incorporated into C-l via the indirect pathway (see footnote 2) and incorpor ation via the direct pathway is maximally 70 % (C-l-C-6). To the extent there is dilution of label in the conversion of glucose carbon to glycogen via the indirect pathway, the direct pathway contribution will be overestimated. Dilution can occur in three segments: conversion of glucose to the three-carbon compound, presumably lactate; conversion of the lactate to phosphoenolpyruvate; and conversion of the phosphoenolpyruvate to glycogen (60). Shulman et al. (103) estimated the extent of dilution of glucose to glycogen via the indirect pathway. They gave [l-13C]glucose by intraduodenal infusion to 24-h fasted rats to achieve a steady-state plasma glucose concentration of 160 mg/dl. The enrichment of lactate in the portal vein was about one-third that of glucose. If there had been no dilution, it would have been one-half, so the dilution was 1.5-fold. Furthermore, the enrichment of lactate in the liver was two-thirds that in the portal vein, giving an additional dilution of 1.5-fold for a total dilution between glucose and liver lactate of 2.25. The enrichment of 13C in lactate was 2.3-2.5 times as much as in C-Z and C-3 of glutamate. If it is assumed that C-Z and C-3 of glutamate give the measure of the enrichment in C-Z and C-3 of oxaloacetate and hence C-Z and C-3 of phosphoenolpyruvate, this gives an overall dilution of ~5.3. Further dilution could occur through entrance of unlabeled carbon in the pathway between phosphoenolpyruvate and glucose 6-phosphate, for example, from glycerol. A dilution factor of 5 with 70% of label in C-l of glycogen would mean a direct pathway contribution of 100[70/(70 + 30 x 5)] = 32%. Probably the major assumption in the above estimate of Shulman et al. (103) is that the glutamate iso-


Ocfotxr /

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l!I!Id

OF

GLYCOGEN

lated was formed solely from hepatic a-ketoglutarate. However, evidence that the enrichments in the C-3 and C-Z positions of intrahepatic glutamate reflect the enrichments in the C-Z and C-3 positions of oxaloacetate is suggested by the fact that the enrichments in the C-Z and C-3 positions of aspartate in their hepatic extracts were similar to those in the C-3 and C-Z positions of glutamate. Magnusson et al. (69) estimated that in humans fasted overnight and given a glucose load, 1 g/kg body wt, and trace [3-14C]lactate there was essentially no dilution of the 14C from the [3-14C]lactate in its conversion to phosphoenoLpyruvate via the tricarboxylic acid cycle. The dilution was estimated from the distribution of 14C in the carbons of glutamine from the glutamine conjugate of phenylacetate. The subjects were given phenylacetate along with the [3-14C]lactate, and the distribution in glutamine was assumed to reflect that in hepatic cu-ketoglutarate. Katz et al. (46) by dividing the enrichment in liver glucose by that in liver lactate arrived at a dilution factor of 1.3. The enrichment in blood lactate was the same as in liver lactate, in contrast to the finding of Shulman et al. (105) but under different conditions. More recently, Katz et al. (48), from mass isotopomer analyses, have estimated under their conditions dilutions in the tricarboxylic acid cycle of 1.4- to 1.5-fold. Dilution of lactate carbon from other than glucose, e.g., muscle glycogen, from their data was --2.5-fold, giving an overall dilution of -3.5. Knowing the direct pathway contribution, the overall dilution in the indirect pathway can be estimated using data from 14C randomization or 3H/14C ratios (see next section). Thus, if the direct pathway is shown to be 50% and there is 80% of 14Cin C-l of glycogen and 6% in C-6, the overall dilution is 2.8, i.e., 0.5 = 0.74/(0.74 + 0.26~); thus x = 2.8 (60). The dilution of 2.8 is calculated knowing the direct pathway contribution. Dilution of the specific activity or enrichment of a labeled carbon, as in lactate, may only reflect an exchange of 12C with the labeled carbon and not a real metabolic change (28, 61). Thus a dilution of 3 in itself does not mean the amount of glycogen synthesized from lactate would be three times the amount that would be formed if there were no dilution. 2. Tritium-to-carbon-14

ratios

Table 2 records the 3H/14C ratios in glycogen relative to those in glucose on giving [14C,3H]glucose with glucose loads. The ratios of 3H and 14C in hepatic glycogen, relative to the ratios in [U-14C,3-3H]glucose administered by Newgard et al. (‘77), ranged from 0.35 to 0.55. They assumed that if the glucose had been directly converted to glycogen, the 3H/14C ratio would have been the same as in the administered glucose, i.e., the relative ratio should have been 1.0. However, when [3-3H]glucose is converted to [3-3H]glucose 6-phosphate and before its conversion to glycogen, 3H can be removed in the pentose cycle and in cycling between fructose 6-phosphate

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and fructose 1,6-bisphosphate.’ Scofield et al. (97), when they administered [6-14C,3-3H]glucose and [U-14C,3“HIglucose under the conditions of Newgard et al. (77), obtained relative ratios averaging 0.44. The ratio was increased to 0.65 when corrected for estimated pentose cycle activity and cycling between the fructose phosphates. In contrast, Katz et al. (48), in their recent study, administered [3-3H]glucose as well as [l-14C]glucose to rats and reported 3H/14C ratios of 0.84 and 0.94. When Scofield et al. (97) administered [6-14C,63H]glucose, the ratios in glycogen relative to those in the administered glucose averaged 0.88. Correction for the estimated retention of 3H in the conversion of [63H]glucose to glycogen via the indirect pathway reduced the ratio to 0.85. Moriwaki and Landau (74) reported that when [U-14C,6-3H]glucose was administered to rats, the relative ratio was near 1.0. Baker (3) gave [63H]glucose and [U-14C]glucose to mice trained to eat for 2 h each day. The 3H/14C ratio in liver glycogen was 0.8. Hellerstein et al. (29) administered [U-14C,l-3H]glucose to rats. Although 3H at C-l can be removed in the pentose cycle and between fructose 6-phosphate and mannose 6-phosphate to the extent mannose phosphoisomerase is active (57), they observed 3H/14C ratios of ~0.8. Lang et al. (62) infused [U-14C,6-3H]glucose at rates of 22-248 mg ~100 g body wt-’ h-l in rats. They found no difference in the rate of glycogen deposition over that range of glucose administered despite plasma glucose concentrations ranging from -9 to 23 mM. The 3H/14C ratios ranged between 0.8 and 0.9. Huang and Veech (34) on administering [U-14C,3-3H]glucose found ratios of 0.83-0.97. In contrast, Katz, McGarry, and colleagues (43, 71) reported that when they administered [14C,3-3H]glucose and [14C,6-3H]glucose to rats, ratios in glycogen were 0.5-0.6. The explanation for these differing results is again probably differing conditions. Possibly, these include diet, the light cycle to which the rats were exposed, and the length of fasting. Stress of the rats may have played a role in the differences, as emphasized by Huang and Veech (34). As noted, Newgard et al. (77,78) cannulated their rats just before beginning a fast for 20 h, and the rats were restrained during that period, except when fed the labeled glucoses ad libitum. Scofield et al. (97) did not restrain their rats. Huang and Veech (34) concluded from ratios of 0.83-0.93 they observed on giving [U14C,3-3H]glucose by portal vein that the majority of the glucose deposited in glycogen was via the direct pathway. Cannulation was done 8 days before administering the labeled glucose. The glucose was given by portal vein immediately after the rats, trained to eat their daily rations in 2 h, had eaten. The ratios were at 6 min after giving the labeled glucose. Ratios of the other investigators were obtained at rl h after labeled glucose administration. Because 14C of the [14C]glucose, to be expresssed in glycogen via the indirect pathway, has to proceed through many intermediates, including lactate, steady state should not have been achieved in 6 min. Ratios near 1.0 would then be expected even if the indirect pathway predominated. However, when Huang and l


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TABLE 2. “HP-% ratio in liver glycogen relative to that in glucose on administering [‘T, ‘H]glucose with a glucose load Species

Diet

Fast Period

Labeled Glucose

Mouse Rat Rat

58% Glucose Chow Chow

22 h 20-24 h Overnight

u-14c, 6-3H U-14C, 1-3H U-14C, 3-“H

Rat Rat

Chow Chow

Overnight 18 h

Rat

Chow

24 h

U-14C, 3-3H 14C, 3-3H 14c, 6-3H 1 -14C, 3-3H

Rat

Chow High low High low

* Rats

sucrose, fat sucrose, fat

48 h 20 h 20 h

Time of Analysis

120-240 mg/ZO g 90-300 mg 100 g-’ h 50% Glucose 3H, 14C 243 mg 100 g-’ . h-l 167 mg 100 g-’ h-l

Meal fed Intravenous Meal fed Intravenous Suprahepatic

30-150 2-8 h 6 min

90 mg prime, 180 mg 100 g-’ h-’ 22-248 mg 167,334 mg 100 g-’ h-l 40% Glucose diet 167,334 mg 100 g-l h-’ 270-540 g/100 g 40% Glucose diet

l

l

l

l

min

3H/‘4C Ratio

Glucose for Ratio

Reference

0.8 0.8-0.9 0.8-0.9

Diet Portal Liver

3 29 34

3 h 2h

0.97 0.5-0.6

Portal Blood

34 43

Duodenal

160 min

0.84, 0.94

Blood

48

Intravenous Intravenous Ad libitum Intravenous Stomach

3 h 1-3 h l-3 h 3 h

0.8-0.9 0 35-o 55* 0:64-0:71 0.44 0.88

Arterial Portal

62 77

Load

97

l

l

Rat Rat

Mode of Administration

Glucose Load

l

l

l

l

l

restrained.

the specific activity of the glucosyl units of glycogen is that of the specific carbon of glucose administered, corrected for label incorporated via the indirect pathway. Thus, with specifically [14C]glucose administration, both the specific activities of glucose and glycogen and the distribution of label in the glucosyl units of glycogen must be determined. Importantly, estimates of pathway contributions made in this way are independent of dilution of label in the indirect pathway. In the fasted state when there is no glycogen in liver, the specific activity (or enrichment) of the glycogen present with glucose administration is the specific activity of the glycogen deposited during that administration. Otherwise correction must be made for the unlabeled glycogen present before glucose administration. In all but one of the animal studies in Table 3 (97), glycogen content in animals at the end of the fast period was determined. Content was small, or presumed so (97), relative to the quantity of glycogen formed in response to the glucose load. An exception is in the overnight fasted rats studied by Huang and Veech (34). In some studies glycogen specific activity was calculated, correcting for the small quantity of glycogen present before administering the glucose load (62, 73,74,105). In others (3,23, 3. Glycogen to glucose speci& activities and enrichments 46,48,77), correcting the direct pathway contribution in Table 3 would increase the estimates by ~0.05. Only diTable 3 records studies reporting specific activity or lution of the labeled glucose in its conversion to glycoenrichment of 13C in glycogen relative to that in admingen via the direct pathway could otherwise result in an istered or circulating glucose when [3H]-, [‘“Cl-, or underestimation of the direct pathway. This could occur [13C]glucose was given in a glucose load. If only the direct from sources of glucosyl units of glycogen, such as manpathway is operative, hepatic glycogen formed from spe- nose, galactose, oligosaccharides, hexoses from glycocifically [3H]- and [14C]glucose should have the same spe- protein degradadation, and protein-bound primers of cific activity as that of the glucose (74). Substituting 13C glycogen, present in or presented to the liver. Their contributions seem likely to be small to insignificant for 14C, it would be excess isotope rather than radioactiv(11, 106). ity that would be the determinant in the discussion that follows. To the extent the indirect pathway contributes, In the mice trained to eat in 2 h that Baker (3) fed [UJ4C]glucose in small meals, liver glycogen specific acthe specific activity of glycogen should be less than that tivity was one-half that of glucose. Moriwaki and Lanof glucose. The ratio of glycogen to glucose specific activity then equals the fraction of glycogen formed via the dau (74) gave rats fasted for 48 h [U-14C]glucose by stomach tube (540 mg/lOO g body wt). The specific activity of direct pathway. With the use of specifically [14C]glucose, Veech (34) gave labeled glucose and then determined the ratio in glycogen at 3 h it was 0.97. The labeled glucose was then given through a suprahepatic catheter, a catheter placed just below the diaphragm of the rat. Ratios cannot be taken as a measure of the pathway contributions unless the extent of dilution of 14Cwith 12C in the indirect pathway is known, the same limitation as in the case of randomization from specifically [14C]glucose. Thus, if there was extensive dilution so that 14C, just as 3H, was not incorporated into glycogen via the indirect pathway, ratios of -1.0 would be obtained despite a predominance of the indirect pathway. However, if, for example, each pathway contributed 50% to glycogen formation and a ratio of 0.9 was found, dilution in the indirect pathway would have to be about ninefold, i.e., 0.9 = 50/(50 + 50/x); x = 9. A ratio of 0.8 would only mean a dilution in the indirect pathway of fourfold. Furthermore, a fourfold dilution with a ratio of 0.9 would mean a 70% direct pathway; a ratio of 0.8, as just noted, a 50% direct pathway; and a ratio of 0.7 a 37% direct pathway. Thus ratios are relatively insensitive to pathway contributions.


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3. Ratio of specific activity or 13Cexcess in glycogen to glucose on administering specifically labeled ofr [13C]glucose /with a glucose load

TABLE

Species

Mouse

Labeled Glucose

Fast Period

Diet

58% Glucose in meal NIH-31 50% Glucose

22 h

u-14C,

6-3H

22 h Overnight 18 h 24 h

3-3H U-14C,

3-3H

Glucose

120-240

Mode Administration

Load

mg/ZO

g

Meal

48 h 42 h

6-3H 1-13C

Stock High sucroselow fat

48 h 20 h

u-14C 3-3H u-14C

6.5 g Carbohydrate 243 mg 100 g-’ h-’ mg 100 g-’ h-l 90 mg prime, 180 mg 100 g-’ h-l 90 mg prime, 180 mg 100 g-l h-l 22-248 mg 150 mg/kg bolus, 480 mg kg-l h-l 540 mg/lOO g 167-334 mg 100 g-l h-l 40% Glucose diet

20 h

6-14C

167-334

Human Rat

High sucroselow fat 200 g Carbohydrate Chow

lo-12 24 h

1-13C l-13C

Rat

Chow

24 h

165-170 mg/dl 0.7 g prime, 210 mg 100 8-l. h-’ 100-600 mg/lOO g

Rat Rat Rat Rat

1-‘4C6-14C 167 u-136

24 h

Rat Rat Dog

Chow Meat,

Rat Rat

Rat

chow

1-14C , 3-3H

l

l

l

l

l

l

l

l

l

h

Glucose for Ratio

Ratio

Reference

150 min

0.5

Diet

Duodenal

2h 3h 2h 140-160

min

0.73 0.86-0.87 0.5-0.6 o-44-0.54*

Portal vein Portal Blood Liver/blood

Duodenal

140-160

min

0.50-0.57

Liver/blood

48

Intravenous Duodenal

3h 4h

0.13-0.74 0.49

Arterial Portal

62 73

Stomach tube Intravenous, intragastric Ad libitum Intravenous

3h 3h

0.76 0.12-0.28 0.32-0.50

Blood Loadt

74 77

3h

0.42

Load

97

Intravenous Duodenal

3.5-4.5 3h

0.49$

0.34

Vein Portal

100 103

Intragastric

2-3 h

0.31-0.36

Portal

105

Meal fed Suprahepatic

3 23 34 43 46,48

l

l

mg

fed

Time of Analysis

l

l

100 g-l

l

h-’

h

l

* Concentration glucuronide to glucose.

TYL

+ 6 isotopomer

l-13C in glycogen

to glucose.

t Specific

activity

liver glycogen deposited over a 3-h period was 0.76 that of the blood glucose, leading to the conclusion that a majority of the glycogen was formed directly from glucose. Liver glycogen in adrenalectomized rats given 360 mg/lOO g body wt of [U-14C]glucose via stomach tubes and injected with cortisol had -0.8 the specific activity of blood glucose. When only trace amounts of labeled glucose were injected, the specific activity of glycogen deposited in response to the administration of cortisol was ~0.1 that of blood glucose.4 In more recent investigations, except those of Lang et al. (62), Huang and Veech (34), and Dobson et al. (23), specific activities of or carbon excess in glucosyl units of glycogen on the administration of labeled glucoses have been reported to be one-half or less than those of the circulating glucose, indicating that one-half or less of the glycogen was formed via the direct pathway. Thus Newgard et al. (77) observed 14C specific activities in glycogen of 32-50%, and Scofield et al. (97) observed activities averaging 42% that of administered [‘“Clglucose. Katz and co-workers (45, 46, 48) observed enrichments in glycogen and specific activities of -50% of circulating glucose.5 Shulman et al. (105) found 13C en4 Trace-labeled glucose along with labeled glycerol and alanine were administered. Cortisol administration increased glycogen content but not its relative sources, i.e., the same percentages of label in glycogen were from glucose, glycerol, and alanine, suggesting that cortisol increased the conversion of glucose 6-phosphate to glycogen without altering the relative pathways of its formation (74). 5 Katz et al. (48) state and Katz and Lee restate (45) that Des Rosiers et al. (21) claimed a direct pathway of 65-71% on recalculating isotopomer data reported by Katz et al. (46). Des Rosiers et al. (21) showed the need for the factor 0.5 in the equation used by Katz et al. (46) and that their estimates of direct pathway contributions using that equation then increased from -50 to 65-71%. They indicated, as Katz et al. (46) had, that the estimates using that equation were over-

of portal

glucose

88-10070

of administered

glucose.

$ 13C excess

in C-l

richments in glycogen 31-36Y0 of that in glucose in portal blood on giving [l-13C]glucose to rats in doses of 100 and 600 mg/lOO g body wt. In humans fasted overnight, given acetaminophen, and infused with [1-13C,6-14C]glucose to achieve a plasma glucose concentration of -170 mg/dl, the specific activity and enrichment in the glucuronide they excreted was -0.49 of that in their circulating glucose (100). Lang et al. (62) found ratios of glycogen to glucose specific activities ranging from 0.13 when glucose was given at the rate of 22 mg 100 g body wt-’ h-l to 0.74 at 248 mg 100 g body wt-’ . h-l, supporting an increase in direct pathway contribution with increasing amounts of glucose administered. However, they found no increase in the rate of glycogen formation with increasing doses of glucose. They noted that at the higher doses of glucose, plasma glucose concentrations were above those encountered under physiological circumstances. Huang and Veech (34), giving [U-14C,3-3H]glucose via suprahepatic catheter, 243 mg. 100 g body wt-’ h-l, for 3 h to rats fasted overnight, found a 3H specific activity in glyl

l

l

l

estimations because of dilution. They indicated the need to introduce a dilution factor and showed that with a dilution factor of 2-3, the estimates of the indirect pathway, under the conditions of Katz et al. (48), would be -50%. Glucosyl units in glycogen of mass we + 6 to those in circulating glucose on giving a load of [U-13C]glucose are then taken as the measure of the direct pathway (45, 46, 48). During direct conversion [U-‘3C]fructose 6-phosphate will be formed from [U-‘3C]glucose 6-phosphate. Unlabeled glyceraldehyde 3-phosphate formed from unlabeled glucose in the load and other sources can exchange with the fructose 6-phosphate, catalyzed by transaldolase (see footnote 3, i.e., [U-13C]fructose 6-phosphate + glyceraldehyde 3-phosphate - [1,2,313C]fructose 6-phosphate + [U-13C]glyceraldehyde 3-phosphate). To the extent that occurs, glucosyl units of mass yn + 3 can result and hence lead to an underestimation of the direct pathway, the indirect pathway then being defined as glycogen formed via trioses (45,46,48).


1028

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cogen 85% that of the liver glucose and a 14C specific activity 87% that of the glucose. Dobson et al. (23) meal fed [3-3H]glucose in ground chow to rats chronically cannulated and fasted for 22 h. The glycogen content of liver increased from 5 to 101 pmol/g, and the specific activity of the glycogen relative to that of the portal glucose was 0.73. Therefore they concluded that a minimum of 73% of the newly synthesized glycogen was formed directly. Portal glucose concentrations were between 10 and 12 mM. Whether the apparently large direct pathway contributions observed by Veech and colleagues (23,34) are related to the rats being meal fed or to administration via suprahepatic catheter is unclear. In meal-fed rats there is the suggestion that three-carbon compounds are preferentially converted to lipid and glucose to glycogen (80). Huang and Veech (34) noted that Newgard et al. (77) used the specific activity of the administered glucose rather than that of the circulating glucose for their estimates. They believed this might explain why their estimates of direct pathway contributions were more than those of Newgard et al. (77). However, Newgard et al. (77) reported specific activities of circulating glucose to be 88-100% of those of the administered glucose so this could explain little of the difference. Shulman et al. (103) used a time-integrated plasma glucose enrichment that was 80-85% that of the administered glucose. Most of the ratios in Table 3 were also obtained using a timeintegrated specific activity or enrichment for blood glucose (23, 34, 62, 73, 100, 105). Katz et al. (46) found that with infusing [l-13C]glucose intraduodenally with a priming dose, enrichment of arterial glucose was 7080% of the infusate at 30 min and became virtually constant at 60 min after beginning infusion. In another study (48), they reported that the enrichment and specific activity of portal blood glucose and glucose in liver exceeded that in peripheral blood until steady state was achieved so that correction for the period before peripheral glucose reached steady state was not indicated. In some studies where a relatively large number of administrations were performed under a given condition, estimates cover a broad range (e.g., see Refs. 73, 97). Thus, in Moore et al. (73), estimates of direct pathway contributions range from 0.21 to 0.91. Mongrel dogs were studied in which obtaining uniformity of animal breed was not possible. The observations, however, emphasize the possible uncertain factors that may influence estimates. Interestingly, in the dogs exhibiting the greatest net glycogen synthesis, the indirect pathway contributions were the largest. 4. Incorporation

of tritium from tritiated water

The assumption, as previously noted, is that for every glucose unit of glycogen formed via the indirect pathway, two hydrogens from water are incorporated at C-6 and for every glucose unit formed via either pathway a hydrogen from water is found at C-Z. When mea-

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sured, rather than 2 hydrogens binding to C-6, 1.5-1.8 hydrogens were bound (91), and incorporation into C-Z was between 0.77 and 0.92 hydrogens rather than 1 (51, 83).

Kuwajima et al. (51) injected 3H,0 into their rats 50 min after beginning refeeding and killed them 20 min later. Thus their estimates are for a short time after breaking the fast, and direct pathway contributions may increase with the duration of feeding. In mice injected with “H,O and fed chow and sucrose diets, glycogen analyzed at 3 h gave estimates of 72 and 50% indirect pathway contributions (71). Postle and Bloxham (83) injected 3H,0 into rats fed a standard diet and a high-glucose-containing diet. From the incorporations of 3H observed at C-Z and C-6 of glucose from liver glycogen, the indirect pathway contribution can be calculated to be 22 and 41%, respectively, for the two diets (60). The difference between the estimates from the data of Postle and Bloxham and of Kuwajima et al. may be due to Postle and Bloxham, as Huang and Veech (34), meal feeding their rats over a Z-h period daily (80) and injecting 3H,0 at the beginning of a meal and killing the rats 4 h later. 5. Incorporation of carbon-14 from carbon-l&labeled carbon dioxide into glycogen

Radziuk (84,86) estimated the sources of carbon for glycogen synthesis by tracing with [l-14C]glucose the total amount of glycogen formed, using glucagon to release glycogen and measuring synthesis from gluconeogenie precursors from the uptake of 14C from [14C]bicarbonate into glycogen. Radziuk concluded that of a 100-g oral glucose load given to 12-h fasted humans, no more than IO g is converted directly to liver glycogen, whereas at least 15 g is formed by synthesis from gluconeogenic precursors. For that estimate, 14Cfrom [14C]bicarbonate fixed to pyruvate to form [4-14C]oxaloacetate in liver was assumed, relative to its other fates, to completely equilibrate with fumarate, so that equal carboxyl-labeled [1,4-14C]oxaloacetate was formed. There is evidence for extensive, but not complete, equilibration (69). The specific activity of circulating 14C0, was assumed to be that fixed by the pyruvate. There is also evidence that equilibration between 14C0, from bicarbonate in the circulation with CO2 in liver is extensive (25, 27). Incomplete equilibrations would result in an underestimation of glycogen synthesis from gluconeogenic precursors. The estimate was also made assuming the [1,4-14C]oxaloacetate was diluted threefold with unlabeled oxaloacetate formed in the tricarboxylic acid cycle, i.e., [1,414C]oxaloacetate + acetyl-CoA - [1,6-14C]citrate zZ~~CO,+ oxaloacetate. Without dilution only ~5 g glycogen would have resulted from precursors. The threefold dilution was based on an estimate, from the tracing of 14C from [Z-14C]acetate into glucose, that the rate of tricarboxylic acid cycle flux was two to


Octobur

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I!)!)2

OF

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three times the rate of the conversion of oxaloacetate to glucose and glycogen. That rate was likely lower (69,96), resulting in less dilution. IV.

REGULATION

A. Eflect ofFed

OF PATHWAYS

Versus Fasted

State

Stetten and Boxer (108) fed rats a diet containing 60% glucose along with 2H20. From the enrichment of deuterium in the hepatic glycogen to that in body water, they concluded that glycogen was formed about equally from dietary glucose directly and “from fragments smaller than hexose.” They reported that when the rats were fasted 24 h and then given glucose and 2H20, glycogenesis was preferentially from fragments smaller than hexose (9). Radziuk (85) concluded that in humans the amount of hepatic glycogen formed directly is not altered by extending fasting from 12 to 24 h. To examine the effect of the fed and fasted states on the pathways of liver glycogen repletion in humans, Magnusson et al. (67) gave acetaminophen to humans along with glucose loads labeled with [6-14C]glucose. After an overnight fast a maximum of 65% of glycogen was formed from glucose via the direct pathway, and a maximum of 77% of glycogen was formed via the direct pathway when a glucose load was ingested at lunch time. Those estimates were based on the extent of randomization of 14C in the carbons of the glucuronic acid moiety of urinary glucuronide. The glucuronide has the advantage that it reflects the labeling pattern in UDPglucose from which glycogen is being formed irrespective of the quantity of glycogen present before the administration of label except for glycogen cycling. The estimates were considered maximal because dilution of 14C with 12C in the formation of the glycogen via the indirect pathway was not determined. To overcome the uncertainty of dilution in the indirect pathway, Shulman et al. (100) gave acetaminophen and infused [l-13C,6-14C] glucose for 4 h into four subjects after an overnight fast. This was repeated with the same subjects but 4 h after a breakfast of 600 cal. Plasma glucose concentrations were ~165-170 mg/dl for the last 2 h of infusion. With plasma glucose concentrations +5% higher than blood glucose concentrations, these seem to be reasonable physiological glucose concentrations in the fed state. Estimates of the pathway contributions were similar when made from 1) the 13C enrichments in C-l and C-6 of glucose from the urinary acetaminophen glucuronide compared with enrichments in plasma glucose and 2) the 14C specific activities of C-l and C-6 of glucose from the glucuronide compared with the specific activities in the plasma glucose. During the last hour of infusion after the overnight fast, 49% of the glycogen was estimated to have been formed via the direct pathway (Table 3), increasing to 69% after breakfast. The specific activities of the glucuronide had not yet reached steady state so that the percentages are at

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least to a degree underestimates. This suggests that under normal conditions, when individuals consume multiple meals daily, most hepatic glycogen repletion occurs by the direct pathway. The estimates of 49 and 69% may also be minimal because of dilution of label with 12C in the direct pathway due to glycogen cycling. Glycogen cycling is the simultaneous synthesis and breakdown of glycogen. If unlabeled glycogen is broken down while the glucuronide is being formed from labeled glucose, the specific activity of the glucuronide will be reduced. Glycogen cycling has been reported to occur in humans (68,113), as well as in rats (24,104), and to be greater in the fed state (20,68). Recently, Kunnecke and Seelig (49) estimated pathway contributions in fed and 48-h fasted rats. The rats were given a single intraperitoneal injection of [1,213C2]glucose, loo-125 mg/lOO g body wt. They were anesthetized with Nembutal, so along with pentobarbital they also received -20 mg/lOO g body wt of propylene glycol and 4 mg/lOO g body wt of ethanol. The number of the glucosyl units of liver glycogen formed 90 min after injection in which the two 13Cnuclei remained bonded in their positions was determined, which is the measure of the direct pathway. Estimates of the indirect pathway contribution depended on the presence of [5,6-13C2]glucosyl units in the glycogen. In the fed rats the direct pathway contribut ‘ion was estimated to be at least 67%, with no detectable indirect pathway con tri bution. In the fasted rats, the indirect pathway contribution was estimated to be at least 9% and not more than 30%. The indirect pathway contributions are underestimates because the extent of dilution in the pathway was not determined. Assuming a two- to threefold dilution gave indirect pathway estimates of 18 and 27%. B. Eflect of Dietary

Protein

Diet can profoundly affect the uptake of glucose by liver (79). Thus in conscious dogs, fasted for 14-16 h and then infused with glucose, when the dogs had been maintained on a high-carbohydrate diet a net uptake of glucose began at a plasma glucose concentration of -120 mg/dl and when maintained on a high-protein diet net uptake began at -220 mg/dl (58). To examine the influence of dietary manipulation on the pathways of liver glycogen repletion rats, Rossetti et al. (94) pair fed rats a euc aloric die tb”f either high-protein, lowcarboh .ydrate or low-pro tein, high-carbohydrate chow for 10 days. A constant intraduodenal infusion of [l13C]glucose was given, and the direct pathway contribution was calculated from the enrichment of 13C in liver glycogen to that in portal vein glucose. Rats eating the high-protein diet had only 20% of their glycogen synthesized by the direct pathway, whereas rats receiving the high-carbohydrate diet had 52% repleted by the direct pathway. This occurred despite no change in the overall rates of net hepatic glycogen synthesis. The increased flux of gluconeogenic precursors to glycogen via the indi-


1030

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I. SHULMAN

AND

rect pathway in rats fed a high-protein diet could be consequent to an increase in plasma concentrations of gluconeogenic substrates. These could augment gluconeogenesis by a mass action effect. Also, a high-protein diet would be expected to enhance the secretion of glucagon, a stimulator of gluconeogenesis. However, Katz et al. (48), giving glucagon acutely to rats along with [U-13C]glucose, reported an increase in the direct pathway contribution to glycogen formation from an average of 49 to 65%, with a decrease in overall dilution in the indirect pathway. C. Role qf’Glucose Versus Insulin

The studies by Newgard et al. (78) suggested that glucose uptake by the liver and its direct incorporation into glycogen is influenced by the prevailing plasma glucose concentration. Lang et al. (62) observed a positive relationship between plasma glucose concentration and the amount of glycogen repleted by the direct pathway during progressively increasing rates of glucose infusion (Table 3). At the highest dose of glucose infused, 248 mg 100 g body wt-’ h-‘, plasma glucose concentration reached 23 mM. In both of these studies the plasma insulin concentration presumably also increased with increasing plasma glucose levels. Shulman et al. (105) observed no change in the pathway contributions when 100 and 600 mg/lOO g body wt doses of glucose were given (Table 3), but plasma glucose concentration only ranged from 8.9 to 12.7 mM. Variation in diet, use of restraint, mode of administration, rat age, and/or weight of the rats may explain why at similar doses of glucoses there are such significant differences in glucose concentrations in the several reports. To delineate the roles of hyperglycemia and insulin on the direct versus indirect pathways of liver glycogen synthesis, Shulman et al. (101) performed euglycemic and hyperglycemic clamps in chronically catheterized rats under similar conditions of hyperinsulinemia. They found that under conditions of hyperinsulinemia, hyperglycemia per se markedly increased the percentage of hepatic glycogen synthesized by the direct pathway and played a major role in augmenting hepatic glycogen synthesis. Shulman and Rossetti (102) gave [l-13C]glucose intravenously or intraduodenally to chronically catheterized 24-h fasted rats and estimated direct pathway contributions from the enrichment of 13C in C-l of glycogen compared with the enrichment of 13C in C-l of glucose from portal vein. The percent direct pathway contribution was the same, 43-46%, by both routes of administration, despite differing insulin concentrations and rates of glycogen formation but in the face of similar portal vein plasma glucose concentrations. Rossetti and Giaccari (93) gave rats [3-3H]glucose and from the specific activity of hepatic UDP-glucose, relative to that in portal vein glucose, estimated the pathway contributions. The rats were fasted 6 and 24 h and were studied under hyperglycemia clamped at 300 mg/dl. Insulin and somatostatin were given to achieve l

l

BERNARD

Vohme 72

R. LANDAU

plasma insulin concentrations of -5 and 500 pU/ml. The estimated direct pathway contributions were similar -6O%, in the 6- and 24-h fasted rats whether the insulin was at the low or high concentrations. Similarly, in diabetic rats, the direct pathway contribution, -35%, was unaltered by the differing insulin concentrations. V.

SOURCE

AND

INTERMEDIATES

SITE

OF THREE-CARBON IN INDIRECT

PATHWAY

The source of the precursors for indirect synthesis has been in doubt (e.g., see Refs. 60, 71, 115, 116). Gluconeogenic precursors, particularly lactate produced in the periphery, have been proposed to be the primary substrates for indirectly synthesized glycogen (15, 19). Others have proposed the intestine or the liver, rather than the periphery, as the major site of formation of lactate (37, 8.2). Measurements of splanchnic uptake of gluconeogenie substrates after a glucose load to humans allow estimates of the amount of hepatic glycogen that can be synthesized from substrates from extrahepatic tissues. Although glucose ingestion by humans is accompanied by a rise in arterial lactate concentrations, release of lactate from the periphery is unchanged or diminished, and net splanchnic uptake in the basal state disappears in response to a glucose load (60). Similar findings exist for pyruvate, glycerol, and alanine (60). This suggests either the indirect pathway makes a minimal contribution under the conditions of these studies in humans or extrasplanchnic tissues cannot be a significant source of carbons for glycogen synthesis via the indirect pathway. Consoli et al. (16) infused [14C]lactate into subjects and determined net balance and specific activities of arterial and venous lactate across muscle. They concluded from the changes in specific activities that muscle produces more lactate than determined by net balance and thus plays a larger role than previously recognized in the gluconeogenic process. However, net balance provides the real measure of how much lactate is produced. Changes in specific activities can be attributed to exchange between the lactate administered and unlabeled pyruvate formed in the muscle and thus cannot be taken as a measure of real metabolic change (28, 61).

The possibility exists that the glucose conversion to lactate occurs in the intestine, since that conversion would be undetected from measurements across the splanchnic bed. However, in 4- to 11-day postoperative patients with portal vein catheters, the amount of lactate released from the gut after oral glucose ingestion could only account for 4% of the glucose absorbed (7). Balance studies in rats agree with that finding in humans. Rich-Denson and Kimura (87) chronically catheterized the stomach, portal vein, and aorta of rats. They found that of the glucose absorbed by the intestine, a minimal amount was converted to lactate. Dobson et al. (23) also determined portal-hepatic vein differences in


October

1992

PATHWAYS

OF

GLYCOGEN

plasma concentrations of glucose, lactate, and alanine. Of those three, glucose accounted for 82% of the carbon taken up by the liver at 20 min into the meal feeding of the rats. The possibility also exists that the formation of lactate in the indirect pathway actually occurs within the liver. Perivenous hepatocytes have been suggested to metabolize glucose to lactate, and after recirculation the lactate has been proposed to be converted to glycogen in periportal hepatocytes (4,36,37,82). However, Chen and Katz (12) found no heterogeneity of lactate formation in preparations from the different zones of the liver, and studies by Agius et al. (2) agree with that finding. Furthermore, Cline and Shulman (13) determined the distributions of 13C in glucosyl units of glycogen from the periportal and perivenous zones of livers from rats given [l-13C]glucose. Distributions were similar, indicating that in both zones glycogen was synthesized via the direct and indirect pathways to similar extents. Moore et al. (73) infused [l-13C]glucose duodenally into 42-h fasted dogs. Net hepatic balance of glucose, gluconeogenic amino acids, lactate, and glycerol were measured, as well as the amount of liver glycogen formed. The contribution of the direct pathway to net glycogen synthesis was 57%, measured by the enrichment of 13C in C-l of glycogen compared with that in glucose from portal vein blood. Net hepatic glucose uptake was sufficient to account for all the glycogen formed by the direct and indirect pathways. Net glucose uptake of the amino acids, glycerol, and lactate accounted for only 20% of the glycogen synthesized by the indirect pathway. Therefore these data support an intrahepatic origin for most of the three-carbon precursors of glycogen. Mitrakou et al. (72) estimated in dogs a direct pathway contribution to glycogen formation of 50% but, in contrast to the finding of Moore et al. (73), found the uptake of gluconeogenic substrates by the livers of the dogs could account for the amount of glycogen made via the indirect pathway. However, they estimated the direct pathway from the percentage of 14C in C-l minus that in C-6 of the glucosyl units of the glycogen when the glucose load given was labeled with [l-14C]glucose. Because they did not take into account dilution in the indirect pathway, their estimate of a 50% direct pathway is presumably an overestimate. Wajngot et al. (114) found minimal incorporation of 14C into C-5 of circulating glucose on giving humans [2-14C]galactose with glucose, 2 mg . kg-’ min? There was much more 14Cin C-5 of glucuranic acid from the acetaminophen glucuronide excreted on giving [2-14C]glucose with acetaminophen to humans under the same conditions (114). This means the glucose and galactose were cleaved in different cellular environments. Because galactose is utilized primarily in the liver, this suggests cleavage of glucose occurred primarily outside the liver. However, the two hexoses might be metabolized in different cellular sites within the liver. Possible heterogeneity of galactose metabolism within liver has not been examined (38). Youn and Bergman (116) gave dogs [U-14C]glucose l

REPLETION

1031

and measured changes in arterial blood lactate specific activity with time and the amount of [14C]lactate released from the splanchnic bed into the circulation. They then infused unlabeled glucose with [U-14C]lactate at a rate matching that release. They reasoned that if the splanchnic bed was the major site of glucose conversion to lactate, the concentration of arterial [14C]lactate on infusing the [14C]lactate should be similar to that when [U-14C]glucose was infused. It was markedly less, so they concluded the splanchnic bed is not the major site of glucose conversion to lactate after glucose ingestion. However, the incorporation of 14C into and dilution of 14C in lactate cannot be equated with the production of lactate (61). Thus nonsplanchnic bed tissues could have metabolized the [U-14C]glucose to [14C]pyruvate, with exchange with circulating unlabeled lactate then forming labeled lactate in the arterial circulation without a net production of lactate. When the labeled lactate was infused, it would then have exchanged with the unlabeled pyruvate now formed in those tissues from unlabeled glucose, thus resulting in a decrease in the specific activity of lactate in the arterial circulation. Cleavage of glucose to pyruvate within the liver cell could proceed simultaneously with glycogen formation from pyruvate. Spence and Koudelka (107) incubated primary cultures of hepatocytes from 24-h fasted rats with 10 mM [U-14C,3-3H]glucose and [6-14C,6-3H]glucose and found 3H/14C ratios in glycogen -0.5-0.6 of those in glucose in the incubation medium. Salhanick et al. (95) obtained similar results on incubating primary cultures of hepatocytes from rats fasted 24 h with [U-14C,3“HIglucose. Lahtela et al. (54) incubated hepatocytes from 24-h fasted mice with [3-3H]glucose, [6-3H]glucose, and [U-14C]glucose at 20-120 mM and found 3H/14C ratios in glycogen 0.6-0.7 times those in the medium glucose. Although 3H from [3-3H]glucose can be lost at the triose phosphate level in the conversion of glucose to glycogen, accepting that 3H of [6-3H]glucose is essentially lost at the pyruvate level (see footnote 3), these ratios indicate glucose proceeds to pyruvate in hepatocytes in significant measure before its conversion to glycogen. When Spence and Koudelka (107) incubated [U-14C,3-3H]glucose with hepatocytes from fed rats, the ratio of 3H/14C in glycogen to that in the medium glucose increased to -0.9. Similarly, Parniak and Kalant (81) incubated hepatocytes in a primary culture with 8 mM [U-14C,3-3H]glucose and observed relative ratios of 0.7-0.8. Lahtela et al. (54) estimated that of the glucose utilized in their study, 35% recycled between glucose 6-phosphate and pyruvate. A reservation in that estimate is that when they incubated hepatocytes with [l14C]glucose only 7-10% of the 14C in glycogen was in its C-6. With 35% cycling more randomization into C-6 might have been expected unless there was intrahepatic dilution. Also, the extent of incorporation into C-2 and C-5 of the glycogen was not measured. If the incorporation into C-6 occurred through recycling to pyruvate and not to the triose phosphate level, incorporation would be expected in C-2 and C-5 (57). Chen and Katz (12) did not


1032

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I. SHULMAN

AND BERNARD

find much glycolytic activity in hepatocytes incubated at physiological concentrations of glucose rather than at 20-120 mM glucose. Bismut and Plas (5) made similar observations to those of Spence and Kouldelka (107), Salhanick et al. (95), and Lahtela et al. (54) when they incubated [U14C,3-3H]glucose, 4 mM, with cultured fetal hepatocytes? From the 3H/14C ratio in glycogen to that in the glucose they estimated that 40% of the glucose reached the triose phosphate level before incorporation into glycogen. REALLY

A GLUCOSE

Volume

7.2

glucose being presented to the liver. Shulman et al. (102) administered [l-13C]glucose either by intravenous or duodenal infusion to chronically catheterized 24-h fasted rats. Despite similar or lower portal vein insulin and glucose concentrations, the duodenal route of glucose administration compared with the intravenous route markedly increased the total amount of liver glycogen synthesized without altering the percent of the direct versus indirect pathways by which liver glycogen was repleted. VII.

VI. IS THERE

R. LANDAU

CONCLUSIONS

PARADOX?

The basis of the glucose paradox was that at physiological glucose concentrations in vivo, glycogen was deposited in liver, whereas with in vitro liver preparations glucose concentrations ~20 mM were necessary for net glycogen formation. With regard to other metabolic processes, similar findings in vivo and with hepatocytes were used to support hepatocytes also reflecting adequately glycogen repletion in vivo (71). The paradox was explained by glycogen not being formed by the phosphorylation of glucose in liver. Rather lactate, formed from glucose in other tissues, was converted to glycogen in liver (71). In support of this explanation were assays of glucokinase interpreted to indicate phosphorylating capacity in liver is inadequate to account for glycogen formation in liver (52). Other enzymatic analyses indicate that there is sufficient phosphorylating capacity to account for glycogen formation (50, 82). The possibility exists that glucokinase is under metabolic control such that its activity measured in liver homogenates does not reflect its activity in situ (22, 111). However, the observation that in conscious dogs, given an intraduodenal infusion of glucose (73), hepatic glucose uptake was sufficient to account for all the glycogen formed by the pathways provides further evidence that the phosphorylation of glucose is not rate limiting for synthesis by the direct pathway. The paradox may then rest with hepatocytes and perfused liver preparations not taking up significant amounts of glucose at physiological glucose concentrations. Thus the paradox may rest with the failure to reproduce in vitro the in vivo conditions necessary to achieve glycogen formation at physiological glucose concentrations. This could perhaps be due to the lack of a portal-arterial gradient for glucose in vitro. A gradient has been reported to be needed for normal glucose uptake in vivo (1,35,75,102). Adkins et al. (1) and Myers et al. (75) demonstrated an enhanced uptake of glucose by dog liver when glucose was infused intraportally versus peripherally, despite identical loads of insulin and

6 However, in fetal liver, gluconeogenesis from lactate/pyruvate is minimal because of negligible phosphoenolpyruvate carboxykinase activity. Bismut and Plas (6) have identified serine as a possible source of carbon for glycogen synthesis in fetal rat hepatocytes.

There is rationale for the functioning of the indirect pathway in glycogen formation, particularly in the time immediately following a fast. There cannot be assurance that a fast is at an end and gluconeogenesis should be discontinued with the initial entrance of exogenous glucose into the liver. To turn on and off the gluconeogenic process over brief periods could be energetically expensive, and a mechanism could be expected to exist to avoid that. Ingestion of food includes ingestion of glycogenic substrates such as fructose, pentose, glycerol, and amino acids. With meal feeding then the enzymes associated with gluconeogenesis would be reasonably expected to remain active. Percentage contributions by the direct and indirect pathways must be considered in terms of the conditions of the administration of glucose as well as the method used to estimate those contributions. Estimates of direct pathway contributions range from ~0 to ~100%. In part, this range reflects limitations in the methods of estimation but appears more importantly to reflect varying experimental conditions, i.e., species studied, dietary state, size of glucose load, mode of administration, time of measurement, and approach to steady state. The largest contributions of the indirect pathway are reported by McGarry, Katz, and colleagues, whose observations stimulated the recent interest in this area. Other investigators have generally not observed as much randomization of specifically labeled glucoses in conversion to glycogen and as low 3H/14C ratios in glycogen on giving [3H,14C]glucoses. Furthermore, whether the glucose loads used by several of the investigators can be considered physiological may be questioned. A load of 1 g/kg body wt to humans is routinely used to assess tolerance to glucose, but that is not the quantity of glucose usually ingested in a meal as free glucose. The direct pathway may predominate in the meal-fed rat, but the rat left to its own devices does not eat its daily requirement within 2 h. Estimates of pathway contributions in rats have generally been made 220 h into a fast, not a rat’s usual eating pattern. In such circumstances, fixation on any particular percentage contribution of a pathway or which pathway contributes more may well be counterproductive. Nevertheless, on the average and under most of the conditions studied, following a fasting period about one-half of he-


October

PATHWAYS

1992

OF GLYCOGEN

patic glycogen formation was indirect on giving glucose. The direct pathway appears to dominate in the fed state in humans. If that is so, then in the course of feeding over a 24-h period, i.e., multiple meals a day for humans, most glycogen deposited is probably deposited by the direct pathway. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40936 to G. I. Shulman and DK-14507 to B. R. Landau. REFERENCES

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DOBSON, G. P., R. L. VEECH, J. V. PASSONNEAU, AND M. T. HUANG. In vivo portal-hepatic venous gradients of glycogenic precursors and incorporation of D-[3-3H]glucose into liver glycogen in the awake rat. J. Biol. Chem. 265: 16350-16357,199O. 24. EL-REFAI, M., AND R. N. BERGMAN. Simulation study of control of hepatic glycogen synthesis by glucose and insulin. Am. J, Physiob 231: 1608-1619, 1976. 25. ESENMO, E., V. CHANDRAMOULI, W. C. SCHUMANN, K. KUMARAN, J. WAHREN, AND B. R. LANDAU. Use of 14C0, in estimating rates of hepatic gluconeogenesis. Am. J. Physiol. 263 (Endocrinoh Metab. 26): E36-E41, 1992. 26. GEELEN, M. J. H., E. L. PRUDEN, AND D. M. GIBSON. Restoration of glycogenesis in hepatocytes from starved rats. Life Sci. 20: 23.

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HOSTETLER, K. Y., H. R. WILLIAMS, W. W. SHREEVE, AND B. R. LANDAU. Conversion of specifically 14C-labeled lactate and pyruvate to glucose in man. J. BioZ. Chem. 244: 2075-2077,1969. 34. HUANG, M. T., AND R. L. VEECH. Role of the direct and indirect pathways for glycogen synthesis in rat liver in the postprandial state. J Clin. Invest. 81: 872-878, 1988. 35. ISHIDA, T., Z. CHAP, J. CHOW, R. LEWIS, C. HARTLEY, M. ENTMAN, AND J. B. FIELD. Differential effects of oral, peripheral, intravenous and intraportal glucose on hepatic glucose uptake and insulin and glucagon extraction in conscious dogs. J. 33.

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Differential effect of hyperglycemia and hyperinsulinemia on pathways of hepatic glycogen repletion.

Muscle glycogen repletion after high-intensity intermittent exercise.

Post-exercise muscle glycogen repletion in the extreme: effect of food absence and active recovery.

Rat skeletal muscle glycogen degradation pathways reveal differential association of glycogen-related proteins with glycogen granules.

Regulation of muscle glycogen repletion, muscle protein synthesis and repair following exercise.

Glycogen Repletion in Brown Adipose Tissue upon Refeeding Is Primarily Driven by Phosphorylation-Independent Mechanisms.

Predominant role of gluconeogenesis in the hepatic glycogen repletion of diabetic rats.

Glycogen repletion and exercise endurance in rats adapted to a high fat diet.

Relation between plasma K+ and ventilation during incremental exercise after glycogen depletion and repletion in man.

On the measurement of pathways of glycogen synthesis.

Glycogen pathways in disease: new developments in a classical field of medical genetics.

Abnormal erythrocyte metabolism in hepatic disease: effect of NADP repletion.

Postnatal selenium repletion protects lungs of neonatal rats from hyperoxia.

Schistosoma haematobium: histochemistry of glycogen, glycogen phosphorylase a and glycogen branching enzyme in niridazole-treated females.

Incorporation of phosphate into glycogen by glycogen synthase.

Ascorbic acid repletion: A possible therapy for diabetic macular edema?

Determination of pathways of glycogen synthesis and the dilution of the three-carbon pool with [U-13C]glucose.

Retinoid repletion of vitamin A-deficient mice restores IgG responses.

Neuroendocrine factors affecting the glycogen metabolism of purified Mytilus edulis glycogen cells: partial characterization of the putative glycogen mobilization hormone--demonstration of a factor that stimulates glycogen synthesis.

Disorders of glycogen metabolism.

Biosynthesis of glycogen in Neurospora crassa. Chemical composition and binding to glycogen of UDP-glucose: glycogen 4-alpha-glucosyltransferase.

Hepatic glucose-6-phosphate cycling has no bearing on recently used isotopic procedures to investigate the pathways of glycogen synthesis.

Vitamin D Repletion in Korean Postmenopausal Women with Osteoporosis.

The structure of liver glycogen.