Departments of Medicine, Biochemistry and Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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Simultaneous synthesis and breakdown of glycogen is called glycogen cycling. The extent of hyperglycemia and decreased glycogen stores in diabetes mellitus may relate in part to the extent cycling occurs. Four methods have been introduced to estimate its extent in liver in humans. 1) In the fasted state, the rate of net hepatic glycogenolysis, i.e., glycogen breakdown minus synthesis, is estimated using NMR, and the rate of glycogenolysis is estimated from deuterium labeling of blood glucose on 2H2O ingestion. 2) The rate of glycogen synthesis is estimated from the rate of labeling of carbon 1 of glycogen on [1-13C]glucose infusion, monitored by NMR, and the rate of breakdown from the rate of disappearance of that labeling on unlabeled glucose infusion. 3) The rate of synthesis from glucose-1-P, formed by glycogenolysis, is measured by the decrease in the 3H/14C ratio in acetaminophen glucuronide on acetaminophen and [2-3H,6-14C]galactose administration. 4) The rate of synthesis is estimated from the dilution of label from labeled galactose in its conversion to the acetaminophen glucuronide, and the rate of glycogenolysis is estimated from the amount of label in blood glucose. In the first method, the fate of glucose-6-P is assumed to be only to glycogen and glucose. In the second, only glucose-6-P molecules formed by breakdown that are not cycled back to glycogen are measured. In the third, 3H is assumed to be removed completely during cycling, and only the molecules cycled back to glycogen are measured. In the fourth, galactose conversion to glucose is assumed to be via glycogen. Quantitations in all four methods depend on assuming the order in which the molecules deposited in glycogen are released.
liver; glycogenolysis; gluconeogenesis; glycogenesis
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INTRODUCTION |
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SIMULTANEOUS SYNTHESIS AND BREAKDOWN of glycogen has been called glycogen cycling. Recently, four methods have been introduced to quantitate hepatic glycogen cycling in humans. The first method to be examined combines rates of glycogenolysis and gluconeogenesis determined by use of 2H2O with rates of net glycogenolysis determined by 13C NMR. Analysis of that method will serve as a basis for analyzing the other three methods and their applications. First, the definitions to be used of the pathways by which glucose and glycogen are produced and utilized are given.
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DEFINITIONS |
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Glucose-6-P is assumed to completely equilibrate with
glucose-1-P (Fig. 1). The rate
of gluconeogenesis is the rate of glucose synthesis via
glucose-6-P from noncarbohydrate precursors, e.g., lactate,
alanine, pyruvate, and glycerol (40). The rate of
glucose-6-phosphoneogenesis is then the rate of synthesis of
glucose-6-P from those precursors. The rate of
glyconeogenesis is the rate of glycogen synthesis via
glucose-6-P from those same precursors. The rate of
glycogenolysis is the rate at which glucose is formed from glycogen.
The rate of glycogen breakdown is the rate of conversion of glycogen to glucose-6-P1. The
rate of glycogenesis is the sum of the rates of glyconeogenesis and of
glycogen formed from glycogen via glucose-6-P, i.e.,
glycogen glucose-6-P
glycogen. Net glycogenolysis is
the rate of glycogen breakdown minus the rate of glycogenesis. The rate
of glucose production is the sum of the rates of glycogenolysis and
gluconeogenesis. Of note, the rate of glucose-6-phosphoneogenesis is
called the rate of gluconeogenesis by Shulman and associates (see
Rothman et al., Ref. 37). Thus they use the definition of
gluconeogenesis that was proposed by Krebs (22) and is
generally accepted: the formation of carbohydrate, i.e., both glucose
and glycogen, from noncarbohydrate precursors.
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METHODS |
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Combining 2H2O with 13C NMR. Rates of gluconeogenesis and glycogenolysis in the fasted state are estimated from the enrichment of 2H in the hydrogens bound to carbons 5 and 2 of blood glucose on 2H2O ingestion (the 5/2 ratio) (26). The rate of net glycogenolysis is estimated from the decline in liver glycogen content measured by 13C NMR (37). Because net glycogenolysis is glycogen breakdown minus glycogenesis, by measuring glycogenolysis and net glycogenolysis, glycogenesis can be estimated.
Figure 2 depicts rates calculated from results of 2H2O-NMR measurements in normal subjects and in type 2 diabetics with fasting plasma glucose concentration ~15 mM before and after their treatment with metformin (18). If we focus first on results before treatment, glucose production measured from dilution of a labeled glucose, i.e., [6,6-2H2]glucose, was 0.70 mmol · m body surface area
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Pulse-chase of glycogen monitored by 13C NMR. In this method, [1-13C]glucose and then unlabeled glucose are continuously infused, and the intensity of the C1 resonance of the glucosyl units of glycogen is monitored (28). The rate of glycogen synthesis is estimated from the increase in that intensity during the [1-13C]glucose infusion and the rate of breakdown from its decline during the unlabeled glucose infusion.
When normal subjects were fasted for 5-10 h and maintained at a plasma glucose concentration of ~9.5 mM, the rate of breakdown was estimated to be 57% of the rate of glycogen synthesis and 31% when the subjects were fasted for 12-14 h (28). Although the estimate of 31% has been referred to as an estimate in the fasted state, the determinations were then made when the subjects were hyperglycemic. Hepatic glycogen concentration in the subjects after the 5-10 h of fasting was estimated to be ~400 mmol/l. It seems unlikely that those concentrations were significantly underestimated because of incomplete visualization by NMR (31), because higher concentrations would approach those found in glycogen storage disease. Again, these estimates depend on an assumption of the extent to which the last deposited glucosyl units of glycogen are first released. If the extent of these units was absolute, only unlabeled glucosyl units from the unlabeled glucose infused would be released, and there would be no decline in the intensity of the C1 resonance. To the extent those unlabeled units were released, the rate of glycogen breakdown was therefore underestimated. Furthermore, it was underestimated to the extent that [1-13C]glucosyl units released from glycogen were reincorporated into glycogen. That is, what may be called true cycling, i.e., [1-13C]glycogen[2-3H,6-14C]galactose cycling.
[2-3H,6-14C]galactose is continuously
infused, and acetaminophen is given (42). The
3H/14C ratio in the excreted glucuronide
compared with the ratio in the galactose is the measure of UDPglucose
glycogen
glucose-1-P
UDPglucose. Thus the
galactose is converted to UDPglucose, the immediate precursor of the
glucosyl unit of glycogen and of the glucuronide moiety of the
glucuronide, without loss or change in the position of the labels. With
no glycogen cycling, the ratio in UDPglucose and hence in the
glucuronide should then be the same as in the galactose.
[2-3H,6-14C]glucose-1-P will be
formed if there is glycogenolysis. Assuming extensive equilibrium
between glucose-1-P and fructose-6-P, i.e., glucose-1-P
glucose-6-P
fructose-6-P, the 3H, but not the
14C, is removed. The conversion of that
[6-14C]glucose-1-P to UDPglucose will then
lower the 3H/14C ratio in the glucuronide. Thus
the method measures only what may be called true cycling. That is, it
measures glucose-1-P that is reconverted to UDPglucose, and
not glucose-1-P that is metabolized via
glucose-6-P, as measured by the previous method.
[1-2H]galactose and glucuronide formation. A trace amount of [1-2H]galactose, along with acetaminophen, is infused (16). The turnover of UDPglucose, equated with the rate of glycogen synthesis, is calculated from the enrichment of the [1-2H]galactose, the amount infused, and the enrichment in the excreted glucuronide, assumed to reflect the enrichment in hepatic UDPglucose. The rate of release of [1-2H]glucose into the systemic circulation is equated with the rate of glycogenolysis.
The method depends on the assumption that galactose has to be converted to glycogen before its conversion to glucose. That requires that the conversion of glucose-1-P to UDPglucose in the reaction glucose-1-P + UTP
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CONCLUSIONS |
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During fasting, simultaneous glycogenesis and glycogenolysis occur in type 2 diabetics and liver cirrhotics. Evidence for that cycling includes measurements made of those rates by combining estimates of net glycogenolysis by the 13C NMR method with estimates of the contribution of gluconeogenesis to glucose production by the 2H2O method. During fasting in normal subjects, glycogenesis is not significant. In normal subjects given glucose loads, glycogenesis and glycogen breakdown simultaneously occur.
The significance of glycogen cycling in normal individuals in the fed
state is uncertain. In the conversion of a molecule of
glucose-1-P glycogen
glucose-1-P, the
equivalent of one high-energy bond is expended, but the complete
oxidation of one molecule of glucose will generate 36-38
high-energy bonds. Only high rates of cycling would then impact
significantly on energy expenditure. Also, that cycling was
demonstrated at relatively high glycogen concentrations. Glycogen may
regulate its concentration, such that, at high concentrations,
glycogenolysis may be a physiological response not occurring at lower
concentrations. Also, hydrolysis of glycogen to glucose, catalyzed by
lysosomal acid
-glucosidase, may be a means of protecting the liver
from high concentrations of glycogen.
The minimum rate of cycling in the diabetic subject after an overnight fast is estimated to be 40% of the rate of glucose production (Fig. 2). Such a rate, if it occurred in the diabetics in the fed state, as suggested by the pulse-chase studies (34), could explain the lower than normal hepatic glycogen stores found in the diabetics (29). The hyperglycemia could then be attributable, at least in part, to decreased hepatic glycogen deposition. Therapy to inhibit cycling, e.g., the use of a phosphorylase inhibitor (4), would then be expected to decrease the hyperglycemia and increase the glycogen stores. As suggested by the results of administering metformin, inhibition of glucose-6-phosphoneogenesis could result not only in decreased glucose production but also a decrease in the rate of glyconeogenesis and, hence, cycling.
It should be emphasized that the rates of cycling depicted in Figs. 2 and 3 are calculated by assuming that the differences between estimates of gluconeogenesis defined using the NMR and 2H2O methods are due to glycogen cycling. Support for that is obtained from the significant difference also found in cirrhotics, but not normal subjects, as well as the other evidence that cycling does not occur in normal subjects. However, other explanations for at least some of those differences cannot be dismissed.
The quantitations of cycling by all four methods rely on the validity of the assumptions made. A major assumption in all four methods is the order in which glucosyl units in glycogen are removed. Experimental evidence supporting glucosyl units last incorporated being first removed rests on studies on the breakdown of labeled glycogen. Thus radioactive galactose has been infused to add labeled glucosyl units to unlabeled glycogen in livers of rats and dogs. When the glycogen was isolated from rat liver and degraded, the radioactive units incorporated last were liberated first (11). When glucagon was infused into dogs, the glucose first appearing in the circulation was also labeled (25).
However, there is evidence that last-deposited-first- removed can be far from absolute. Otherwise, after deposition of [1-13C]glucosyl units, the infusion of unlabeled glucose with continued synthesis of glycogen could not be accompanied by a large disappearance of the [1-13C]glucosyl unit of glycogen (28). Differential labeling of the glucosyl units at sites on the glycogen granules, and then their breakdown, remain a possible explanation for last-deposited not being first removed4. Synthesis and breakdown in different areas of the liver lobule could also be a possible explanation (1, 19). Thus the absolute order of release of the glucosyl units of glycogen in relationship to the time of their deposition remains uncertain. Hence, rates of cycling measured by any of the methods are uncertain.
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ACKNOWLEDGEMENTS |
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This review was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-14507.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. R. Landau, Dept. of Medicine, Case Western Reserve Univ. School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4951 (E-mail: brl{at}po.cwru.edu).
1 Glycogenolysis in liver is defined as the formation of glucose from glycogen. However, the breakdown of glycogen to glucose-6-P in tissue where the glucose-6-P is not converted to glucose is also called glycogenolysis. Therefore, that rate of conversion of glycogen to glucose-6-P in liver is here called the rate of glycogen breakdown.
2 The rate of glycogen cycling in the fasted state, because glycogen breakdown exceeds synthesis, is therefore the rate of synthesis, i.e., the rate of glycogenesis. In the fed state, since synthesis exceeds breakdown, the rate of cycling is the rate of breakdown.
3 By the NMR method, Petersen et al. (35) estimated a contribution of gluconeogenesis of 55 ± 6% in normal subjects fasted for 12 h, Rothman et al. (37) 64 ± 5% in subjects fasted for 22 h, and Magnusson et al. (29) 70 ± 6% in subjects fasted for 23 h.
4 Enzymes catalyzing the synthesis and degradation of glycogen are bound to glycogen, although not in stoichiometric ratios (40). Thus synthesis could be occurring in portions of glycogen molecules (labeled units deposited), and in other portions, degradation could be occurring simultaneously (unlabeled units released).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Babcock MB and Cardell RR Jr. Hepatic glycogen patterns in fasted
and fed rats. J Anat 299-338, 1974.
2.
Barrett, EJ,
and
Liu Z.
Hepatic glucose metabolism and insulin resistance in NIDDM and obesity.
Bailliere's Clin Endocrinol Metab
7:
875-901,
1993[ISI][Medline].
3.
Basu, A,
Basu L,
Shah P,
Vella A,
Johnson CM,
Nair KS,
Jensen MD,
Schwenk WF,
and
Rizza RA.
Effects of type 2 diabetes on the ability of insulin and glucagon to regulate splanchnic and muscle glucose metabolism: evidence for a defect in hepatic glucokinase activity.
Diabetes
49:
272-283,
2000[Abstract].
4.
Bergans, N,
Stalmans W,
Goldmann S,
and
Vanstapel F.
Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.
Diabetes
49:
1419-1426,
2000[Abstract].
5.
Biava, C,
Grossman A,
and
West M.
Ultrastructural observations on renal glycogen in normal and pathologic human kidneys.
Lab Invest
15:
330-356,
1966[ISI][Medline].
6.
Boden, G,
Chen X,
and
Stein TP.
Gluconeogenesis in moderately and severely hyperglycemic patients with type 2 diabetes mellitus.
Am J Physiol Endocrinol Metab
280:
E23-E30,
2001
7.
Chandramouli, V,
Ekberg K,
Schumann WC,
Kalhan SC,
Wahren J,
and
Landau BR.
Quantifying gluconeogenesis during fasting.
Am J Physiol Endocrinol Metab
273:
E1209-E1215,
1997[ISI][Medline].
8.
Chandramouli, V,
Ekberg K,
Schumann WC,
Wahren J,
and
Landau BR.
Origins of the hydrogen bound to carbon 1 of glucose in fasting: significance in gluconeogenesis quantitation.
Am J Physiol Endocrinol Metab
277:
E717-E723,
1999
9.
Christiansen, MP,
Linfoot PA,
Neese RA,
and
Hellerstein M.
Metformin: effects upon postabsorptive intrahepatic carbohydrate fluxes (Abstract).
Diabetes
46, Suppl1:
244,
1997[Abstract].
10.
Cusi, K,
Consoli A,
and
DeFronzo RA.
Metabolic effects of metformin on glucose and lactate metabolism in non-insulin dependent diabetes mellitus.
J Clin Endo Metab
81:
4059-4067,
1996[Abstract].
11.
Devos, P,
and
Hers HG.
A molecular order in the synthesis and degradation of glycogen in the liver.
Eur J Biochem
99:
161-167,
1979[ISI][Medline].
12.
Ekberg, K,
Landau BR,
Wajngot A,
Chandramouli V,
Efendic S,
Brunengraber H,
and
Wahren J.
Contributions by kidney and liver to glucose production in the post-absorptive state and after 60 h of fasting.
Diabetes
48:
292-298,
1999
13.
Froesch, ER,
Ashmore J,
and
Renold AE.
Comparison of renal and hepatic effects of fasting, cortisone administration and glucose infusion in normal and adrenalectomized rats.
Endocrinology
62:
614-620,
1958[ISI].
14.
Guynn, RW,
Veloso D,
Randolph Lawson JW,
and
Beech RL.
The concentration and control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo.
Biochem J
140:
369-375,
1974[Medline].
15.
Hellerstein, M.
Detection of secreted glucuronide from labeled galactose does measure hepatic UDP-glucose turnover and (with proper correction) can be useful for estimating hepatic glycogen synthesis.
Metabolism
49:
1375-1378,
2000[ISI].
16.
Hellerstein, MK,
Neese RA,
Linfoot P,
Christiansen M,
Turner S,
and
Letscher A.
Hepatic glyconeogenic fluxes and glycogen turnover during fasting in humans.
J Clin Invest
100:
1305-1319,
1997
17.
Hers, HG,
Van Hoof F,
and
de Barsy T.
Glycogen storage diseases.
In: The Metabolic Basis of Inherited Disease (6th ed.), edited by Scriver CR,
Beaudet AL,
Sly WS,
and Valle D. New York: McGraw-Hill, 1989, vol. I, p. 425-452.
18.
Hundal, RS,
Krssak M,
Dufour S,
Laurent D,
Lebon V,
Chandramouli V,
Inzucchi SE,
Schumann WC,
Petersen K,
Landau BR,
and
Shulman GI.
Mechanism by which metformin reduces glucose production in type 2 diabetes.
Diabetes
49:
2063-2069,
2000[Abstract].
19.
Jungermann, K,
and
Katz N.
Functional specialization of different hepatocyte populations.
Physiol Rev
69:
708-764,
1989
20.
Kark, RM,
and
Gellman DD.
Renal disease in diabetes.
In: Diabetes, edited by Williams RH. New York: Harper and Row, 1960, p. 563-581.
21.
Khandelwal, RL,
Zinman SM,
and
Knull HR.
The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.
Arch Biochem Biophys
197:
310-316,
1979[ISI][Medline].
22.
Krebs, HA.
Renal gluconeogenesis.
Adv Enzyme Regul
1:
385-400,
1963[ISI].
23.
Landau, BR.
Quantifying the contribution of gluconeogenesis to glucose production in fasted human subjects using stable isotopes.
Proc Nutr Soc
58:
963-973,
1999[ISI][Medline].
24.
Landau, BR.
Problems with the assumed biochemical basis for estimating hepatic glycogen turnover using glucuronide formation and labeled galactose.
Metabolism
49:
1374-1375,
2000[ISI][Medline].
25.
Landau, BR,
Leonard JR,
and
Barry FM.
A quantitative study of glucagon-induced hepatic glycogenolysis.
Am J Physiol
199:
231-234,
1960[ISI].
26.
Landau, BR,
Wahren J,
Chandramouli V,
Schumann WC,
Ekberg K,
and
Kalhan SC.
Contributions of gluconeogenesis to glucose production in the fasted state.
J Clin Invest
98:
378-385,
1996
27.
Ljungdahl, L,
Wood HG,
and
Racker E.
Formation of unequally labeled fructose-6-phosphate by an exchange reaction catalyzed by transaldolase.
J Biol Chem
236:
1622-1625,
1961[ISI][Medline].
28.
Magnusson, I,
Rothman DL,
Jucker B,
Cline CW,
Shulman RG,
and
Shulman GI.
Liver glycogen turnover in fed and fasted humans.
Am J Physiol Endocrinol Metab
266:
E796-E803,
1994
29.
Magnusson, I,
Rothman DL,
Katz LD,
Cline GW,
Shulman RG,
and
Shulman GI.
Increased rate of gluconeogenesis in type II diabetes mellitus. A 13C nuclear magnetic resonance study.
J Clin Invest
90:
1323-1327,
1992[ISI][Medline].
30.
Meyer, C,
Stumvoll M,
Nadkarni V,
Dostou J,
Mitrakou A,
and
Gerich J.
Abnormal renal and hepatic glucose metabolism in type 2 diabetes mellitus.
J Clin Invest
102:
619-624,
1998
31.
Murphy, E,
and
Hellerstein M.
Is in vivo nuclear magnetic resonance spectroscopy currently a quantitative method for whole-body carbohydrate metabolism?
Nutr Rev
58:
304-314,
2000[ISI][Medline].
32.
Newsholme, E,
and
Leech A.
Biochemistry for the Medical Sciences. New York: Wiley, 1985, p. 585.
33.
Petersen, KF,
Krssak M,
Navarro V,
Chandramouli V,
Hundal R,
Schumann WC,
Landau BR,
and
Shulman GI.
Contributions of net glycogenolysis and gluconeogenesis to glucose production in cirrhosis.
Am J Physiol Endocrinol Metab
276:
E529-E535,
1999
34.
Petersen, KF,
Laurent D,
Rothman DL,
Cline GW,
and
Shulman GI.
Mechanisms by which glucose and insulin inhibit net hepatic glycogenolysis in humans.
J Clin Invest
101:
1203-1209,
1998
35.
Petersen, KF,
Price T,
Cline GW,
Rothman DL,
and
Shulman GI.
Contribution of net hepatic glycogenolysis to glucose production during the early postprandial period.
Am J Physiol Endocrinol Metab
270:
E186-E191,
1996
36.
Robbins, SL.
The reversibility of glycogen nephrosis in alloxan treated diabetic rats.
Am J Med Sci
216:
376-381,
1950.
37.
Rothman, DL,
Magnusson I,
Katz LD,
Shulman RG,
and
Shulman GI.
Quantitation of hepatic glycogenolysis and gluconeogenesis in fasting humans with 13C NMR.
Science
254:
573-576,
1991[ISI][Medline].
38.
Schneiter, P,
Gillet M,
Chiolero R,
Jequier E,
and
Tappy L.
Hepatic nonoxidative disposal of an oral glucose meal in patients with liver cirrhosis.
Metabolism
48:
1260-1266,
1999[ISI][Medline].
39.
Segal, S,
and
Berry GT.
Disorders of galactose metabolism.
In: The Metabolic and Molecular Bases of Inherited Disease (7th ed.), edited by Scriver CR,
Beaudet AL,
Sly WS,
and Valle D. New York: McGraw-Hill, 1995, vol. I, p. 967-1000.
40.
Stryer, LL.
Biochemistry (3rd ed.). New York: Freeman, 1988, p. 438, 450.
41.
Stumvoll, M,
Nurjhan N,
Perriello G,
Dailey G,
and
Gerich JE.
Metabolic effects of metformin in non-insulin dependent diabetes mellitus.
N Engl J Med
333:
550-554,
1995
42.
Wajngot, A,
Chandramouli V,
Schumann WC,
Efendic S,
and
Landau BR.
Quantitation of glycogen/glucose-1-P cycling in liver.
Metabolism
40:
877-881,
1991[ISI][Medline].
43.
Wajngot, A,
Chandramouli V,
Schumann WC,
Ekberg K,
Jones PK,
Efendic S,
and
Landau BR.
Quantitative contributions of gluconeogenesis to glucose production during fasting in type 2 diabetes mellitus.
Metabolism
50:
47-52,
2001[ISI][Medline].