CORRESPONDENCE

FUTILE CYCLING AND PURITY REVISITED To the Editor:

The purpose of this letter is to examine statements made by Rizza and his associates, in recent publications,‘-’ bearing significantly on measurements of futile cycling and the use of labeled substrates. I believe the statements require further definition to avoid possible misinterpretation. Both [3-jH] and [C’Hlglucose are stated to “have the disadvantage of retaining their label during incorporation and release from glycogen” and cycling of glycogen is said “to likely be a particular problem if the most recently formed outer layers of glycogen are first to be degraded during glycolysis.” It is further stated that “unless synthesis is negligible or glucose release is completely suppressed, the difference between turnover measured with [2-‘H] and either [3-‘HIglucose or [6-‘HIglucose cannot be equated with the activity of the glucose/glucose-6-P cycle.“’ Consider that there is only glucose cycling and glycogen is deposited without (1A) or with glycogen cycling (1 B): IO 5 1A: G ; G6P - glycogen 10 10 1B: G .- G6P - glywgen

5

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ie, with concurrent glycogen synthesis and breakdown. The net flux of glucose is toward glycogen formation and so, as an example, I have set G-+G6P to 10 pmol/min and G6P-+G to 5 pmollmin. With [2-‘HIglucose the ‘H would be completely removed at G6P, assuming complete detritiation,4 so the specific activity of the G6P and glycogen would be 0 in IA and 1B. With [3-3H]glucose (or 6-3H glucose in this and the remainder of this letter), the specific activity of the glucose-6-P and glycogen would be that of the [3-‘HIglucose, also in both 1A and 1B. Thus, glycogen cycling is without effect. The change in specific activity of the circulating glucose, the measure of the glucose/glucose-6-P cycling would be determined in both circumstances by the difference between 5 pmol of unlabeled glucose-6-P from [2-3H]glucose and 5 rmol of [3-‘HIglucose-6-P from [3‘HIglucose hydrolyzed to glucose. It would be a disadvantage if the label of [3-‘HIglucose was not retained during incorporation and release from glycogen for then, if glycogen cycling occurred, the turnover of [2-‘HIglucose and [3-‘HIglucose could not be equated with the activity of the glucose/glucose-6-P cycle. The same conclusion is apparent if there is indirect as well as direct formation of glycogen5 The rates of G+G6P and G6P-G in 2A and 2B are the same as in the first example and glycogen is formed at 5 ~mol/rnin via the direct pathway and also 5 qmol/min via the indirect pathway. There is no cycling of glycogen in 2A, and one-third of the glycogen deposited is cycled in 2B. By both the direct and indirect pathways ‘H will be lost from [2-‘HIglucose, so the specific activity of the glucose-6-P will again be 0 in both 2A and 2B. By the indirect, but not direct pathway, ‘H from [3-3H]glucose will be lost, so with [3-‘HIglucose, the specific activity of the glucose-6-P will be greater than 0, but less than that of the circulating glucose. The specific activity of glucose-6-P will again be the same in both 2A and 2B. Again, glycogen cycling plays no role in the amount of ‘H flowing from G+G6P and G6P-+G.

Metabolism, Vol39,

No

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(March), 1990: pp 327-333

In these examples, the system is at steady-state and it is assumed that there is no glycogen present initially or the “recently formed layers are the first to be degraded,” ie, those layers that are formed from the detritiated [2-‘HIglucose and the undetritiated [3-‘HIglucose are first degraded. To the extent with [3-3H]glucose, it is unlabeled glycogen present initially that is degraded, the difference in turnover between [2-‘HIglucose and [3-3H]glucose will decrease and hence lead to a falsely low estimate of the extent of cycling between glucose and glucose-6-P. Efendic et al6 reported an increase in glucose cycling measured with [2-‘HIglucose and [3-‘HIglucose in patients with glucose intolerance during an intravenous infusion of glucose. Bell et al’ found no such difference. McMahon et al3 state that increased glycogen turnover could account for the increased futile cycling observed by Efendi6 et al6 As just shown, if increased turnover of glycogen occurred and was from the layers of glycogen formed from the administered glucoses, the estimates of glucose cycling would be unaltered. If from preformed glycogen the futile cycling estimates would be less than the actual extent of cycling and possibly different for one condition than another. Bell et al,’ demonstrate that if you use a labeled glucose to follow glucose kinetics while glucose is being deposited as glycogen, and the label is retained in the glycogen, you cannot use that labeled glucose to study kinetics while the glycogen is being released. Spccificially, you cannot detect glycogen breakdown when the label glucose administered into the circulation is the same as that in the glycogen being broken down, as occurs when [3-‘HIglucose is administered and as one proceeds from the fed to the fasted state. Efendic et al* administered a glucose load intravenously and presumably a net deposition of glycogen continued during the course of the infusion. In 1986, Bell et al reported glucose turnover measurements using tritiated glucoses whose purity was reported by the manufacturer to be 97% to 99%’ 98.5%: or was unstated.’ In a January 1989 report in Diobetes, referring to the data obtained with [6-‘HIglucose, previously reported in abstract form,” McMahon et al’ state, “At first it seems incongrous that the presence of a 1.5% contaminant in a

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glycogen t10 ’ G6P

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CORRESPONDENCE

[6-3H]glucose infusate could lead to such a serious error in measurement of glucose turnover. In the past, 98.5% purity of a tracer has been considered satisfactory.” They also report the presence of a contaminant in lots of [3-‘H]glucose and conclude “these experiments emphasize the need for specific methods of measuring the concentration of the tracer in both the infusate and plasma.” Despite these statements in the March 1989 report in Diabetes, McMahon et al’ state, “all glucose isotopes were greater than 98% pure.” In the report in the January 1989 Metabolism, Bell et al.] give no measure by them or from the manufacturer of the purity of the tritiated glucoses they used. There is no mention in either report of the possible role of a contaminant in the tritiated glucoses. I wonder whether a contaminant of a few percent, and perhaps more, if it accumulated, would affect the estimates in the reports, and particularly since [2-3H]glucose and [3-3H]glucose were administered together. The specific activities of plasma glucose attributable to [3-3H]glucose, as in the previous reports of the authors, is taken as the difference between the ‘H remaining after treatment with phosphohexose isomerase to remove ‘H from carbon 2 of glucose and the total amount of ‘H in a deproteinized-deionized sample. I believe

‘H from contaminants from both [2-‘HIglucose and [3-‘HIglucose administration would then be a part of the [3-3H]glucose specific activities they report. Did the authors demonstrate in their studies that they were specifically measuring the tracer in both the glucose administered and the plasma? In the Metabolism report,’ as noted, results for four of the diabetic subjects were reported in part in one of the 1986 reports.’ As a reviewer of the manuscript of one of those papers’ wrote to the editor of that journal at that time (personai communication): “If one is going to use different ‘H-labeled glucoses, one has to begin by making sure the glucoses purchased are really what they are stated to be and then document what you think you measure, you really measure. I believe that a few percent nonspecificity of label, residues not quite all glucose, will not alter the conclusions, but this should be. shown.” Bernard R. Landau. PhD, IUD

Departments of Medicine and Biochemistry Case Western Reserve University School of Medicine Cleveland, OH

REFERENCES

1. Bell PM, Firth RG, Rizza RA: Assessment of the postprandial pattern of glucose metabolism in nondiabetic subjects and patients with noninsulin-dependent diabetes mellitus using a simultaneous infusion of [2-‘H] and [3-‘HIglucose. Metabolism 38:38-45, 1989 2. McMahon MM, Schwenk WF, Haymann MW, et al: Underestimation of glucose turnover measure with [6-‘HI- and [6,6-2H2]But not [6-‘4C]glucose during hyperinsulinemia in humans. Diabetes 38:97-107, 1989 3. McMahon MM, Marsh HM, Rizza RA: Effects of basal insulin supplementation on disposition of mixed meal in obese patients with NIDDM. Diabetes 38:291-303, 1989 4. Wajngot A, Chandramouli V, Schumann WC, et al: Testing of the assumptions made in estimating the extent of futile cycling. Am J Physiol256:E668-E675,1989 5. Landau BR, Wahren J: Quantitation of the pathways followed in hepatic glycogen formation from glucose. FASEB J 2:2368-2375, 1988

6. EfendiC S, Wajngot A, VraniC M: Increased activity of glucose cycle in liver, early characteristics of type 2 diabetes. Prcc Nat1 Acad Sci USA 82:2965-69.1985 7. Bell PM, Firth RG, Rizza R: Assessment of insulin action in insulin-dependent diabetes mellitus using [6-‘4C]glucose, [3-‘HIglucose and [2-‘HIglucose: Differences in apparent pattern of insulin resistance depending on the isotope used. J Clin Invest 78:1479-1486, 1986 8. Bell PM, Firth RG, Rizza RA: Effects of hyperglycemia on glucose production and utilization in humans. Diabetes 35:642-648, 1986 9. Firth RG, Bell PM, Marsh HM, et al: Postprandial hyperglycemia in patients with non-insulin dependent diabetes mellitus. Role of hepatic and extrahepatic tissues. J Clin Invest 77:1525-1532, 1986 10. Schwenk WF, Butler P, McMahon M, et al: Accumulation in plasma of a radioactive contaminant present in 6-‘H but not 6-“C glucose leads to a systematic underestimation of glucose turnover. Clin Invest. 36:490A, 1988 (abstr)

REPLY To the Editor:

We appreciate Dr Landau’s efforts to define the effects of glucose/glucose-6-phosphate cycling and/or incorporation of isotope into and release of isotope from glycogen on the measurement of glucose turnover with [2-‘HIglucose and [3-3H]glucose (or [6-‘HIglucose). We agree that if glucose-6-phosphate cycling is defined as shown in either (a) or (b) then incorporation of glucose

Blood

Hepatocyte

Glucose -glucose-6-phosphate

~

(a) Glucose T

into and release from glycogen will not alter apparent glucose-6phosphate cycle activity, since both processes result in detritiation of [2-‘H]glucose, but not [3-‘H]glucose. However, we believe that for the sake of clarity and because they potentially have different physiologic implications, (a) should be referred to as glucose-6phosphate cycling, and (b) should not. The former consumes ATP (and therefore energy while shuttling back and forth between glucose and glucose-6-phosphate. It is for this reason it has pop-

glucose-6-phosphate

Fructose-6-phosphate

Futile cycling and purity revisited.

CORRESPONDENCE FUTILE CYCLING AND PURITY REVISITED To the Editor: The purpose of this letter is to examine statements made by Rizza and his associat...
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