ANALYTICAL BIOCHEMISTRY 176,228-233 (1989)

An NADH-Linked Luciferase Assay for Glycogen: The Preparation of Glycogen-Free, Viable Human Eccrine Sweat Glands

Christine Welch, Arthur Bryant, and Terence Kealeyl Departments of Clinical Biochemistry, University of Newcastle upon Tyne and University of Oxford, Oxford OX3 9DU. United Kingdom

Received June 23,1988

A glycogen assay based on bacterial NADH luciferase is described. It is free of tissue interference. The detec- tion limit is 0.12 nmol glycogen, and the coefficient of variation is 5.5%. A method of depleting human eccrine sweat glands while retaining their viability is de- scribed. This depends on their incubation in 10m5 M ace- tylcholine and 1 mM pyruvate. This method may be ap- plicable to other tissues. The evidence for the viability of glycogen-depleted human eccrine sweat glands is re- ported and includes tissue contents of ATP and the rates of oxidation of glucose, pyruvate, B-hydroxybutyrate, and palmitate. 0 1989 Academic Press. Inc.

Metabolic studies that depend upon monitoring the rates of metabolism of exogenous radioactive carbohy- drate substrates by tissues in vitro may be invalidated by endogenous glycogen metabolism. So, for example, we have studied pyruvate dehydrogenase in isolated, intact human eccrine sweat glands by following [l-14C]- pyruvate oxidation. However, the extracellular specific activity of [l-14C]pyruvate cannot be extrapolated into the intracellular specific activity, because of the un- known amount of pyruvate that might be produced from glycogen metabolism. We have, therefore, developed a technique for preparing glycogen-free human eccrine sweat glands, and we show that such glands retain their viability.

Because sweat glands are small (wet weight approxi- mately 10 pg) we have needed a sensitive assay to moni- tor glycogen depletion. The most sensitive assays cur- rently available for glycogen depend upon the oxidation

’ To whom correspondence should be addressed at: Nuffield Depart- ment of Clinical Biochemistry, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, UK.

228

of glycogen-derived glucose by glucose oxidase. The hy- drogen peroxide thus produced may be measured by vari- ous chromophores. Unfortunately, tissues contain agents that interfere with such methods which necessi- tates the destruction of those agents prior to assay (1). We have, therefore, developed a sensitive but specific NADH-linked bacterial luciferase assay which does not suffer from tissue interference.

The principle of the coupled enzyme assay is given by the equation

glUCOSe

amyloglucosidase dehydrogenase

i I Glycogen -j Glucose \ Gluconolactone

NAD

bacterial

luciferase

MATERIALS AND METHODS

Materials

Bacterial NADH and firefly ATP luciferase monitor- ing kits were obtained from LKB Wallac, Finland. Glu- cose dehydrogenase and amyloglucosidase were from Sigma (Poole, UK). NAD and ATP were from BCL (Lewes, East Sussex, UK). Radiochemicals were from New England Nuclear (Boston, MA). All other chemi-

0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc.

All rights of reproduction in any form reserved.

GLYCOGEN DEPLETION MONITORED BY LUCIFERASE 229

cals were from BDH (Enfield, Middlesex, UK) and of the highest purity available.

Methods

Hydrolysis of glycogen. An adaptation of the method of Johnson and Fusaro (2) was employed. Glycogen stan- dards were incubated in 200 ~10.2 M sodium acetate, pH 4.8, containing 0.3 units amyloglucosidase for 90 min at 40°C. Control experiments showed the reaction to be complete by 90 min. Because glycogen is of indetermi- nate molecular weight, it was reported in glucose units.

Glucose Assam. Glucose was measured by glucose dehy- drogenase. Aliquots (20 ~1) were made up to final volume of 400 ~1 with 0.1 M potassium phosphate, 100 PM NAD+, 0.4 units glucose dehydrogenase, pH 7.5, and incubated for 30 min at 37”C, by which time control experiments showed the reaction to be complete. Aliquots (200 ~1) were then removed for NADH determination by bacte- rial luciferase following the manufacturers’ adaptation of Stanley’s method (3).

Glucose values were found to be 76% of the values for corresponding NADH standards, but the addition of known amounts of NADH to the glucose incubation showed that this was due to quenching, not incomplete conversion. Glycogen standards were shown to be 75% of the values obtained for corresponding NADH standards, which confirmed no additional quenching.

Standard curves for glucose were linear for up to 900 nmol of glucose.

Measurement of the glycogen content of sweat glands. Sweat glands were isolated by shearing (4), our adapta- tion of the collagenase method (5). Shearing is per- formed by the repeated dissection of skin biopsies wit,h scissors. This separates the basal lamina of the glands from the surrounding dermis, and yields glands that are free of adipocytes or connective tissue as determined by binocular light microscopy (see review in Ref. (6)). Sliv- ers of skin, 10 X 0.5 cm, were obtained from the incisions of elective chest operations on adult male patients at the Freeman Hospital, Newcastle upon Tyne. Ethical Com- mittee permission has been received for this.

For assays of glycogen, a modification of the method of Keppler and Decker was employed (7). Glands were rinsed in glucose-free bicarbonate-buffered medium (8) and single glands were then placed into 20 ~1 of 0.6 M ice

cold perchloric acid. Thirteen microliters of 1~ potas- sium hydrogen carbonate and 67 ~1 of amyloglucosidase (14 units ml- ‘) in 0.2 M sodium acetate, pH 4.8, were added, and the aliquots were then incubated at 40°C for 2 h. Following centrifugation (10,OOOg for 15 min), 20 ~1 of supernatant was removed for glucose assay as de- scribed above. Control experiments without amyloglu- cosidase showed that the glandular content of glucose was undetectable.

Control experiments showed that perchloric acid did

not, affect the time of completion of the amyloglucosi- dase or glucose dehydrogenase reactions, but it was noted that glycogen standards were only 60% of the val- ues for equivalent NADH standards. Control experi- ments showed this was due to quenching which was at- tributable to the perchloric acid, which therefore added to the quenching caused by the glucose dehydrogenase buffer.

Control experiments showed that sonication, in addi- tion to exposure to perchloric acid, gave no increase in glycogen content, which confirmed perchloric acid as an effective chaotropic reagent.

Glycogen breakdown. Glycogen depletion experiments were carried out to investigate the rate at which sweat glands would utilize their glycogen when incubated in glucose-free medium. Isolated glands were incubated in glucose-free bicarbonate-buffered medium at 37°C equil- ibrated with O,:CO, (19:l). At l-h intervals five glands were removed and their glycogen contents estimated and compared with those of glands incubated in the presence of 5 mM glucose. Identical experiments were then per- formed in the presence of 20 pM acetylcholine, the major sweat gland secretagogue, and 5 mM glucose.

Glycogen repletion. To examine the viability of sweat glands after glycogen depletion, refeeding experiments were performed. Following depletion, glands were trans- ferred to bicarbonate-buffered medium equilibrated with O,:CO, (19:l) containing either 5 mM glucose or 1 mM

pyruvate and incubated at 37°C for 2 h, after which gly- cogen contents were estimated. The ATP contents of these glands were determined.

ATP assay. This was performed using LKB ATP mon- itoring kits, based on firefly luciferin/luciferase (9). Indi- vidual glands were added to 10 ~1 of 4% HCLO, and 50 ~1 of 0.12 M KOH, and made up to a total volume of 80 ~1 with distilled water. Following centrifugation, 10 ~1 of the supernatant was transferred to 0.1 M Tris/EDTA buffer, pH 7.5, and the peak light emission was measured following addition of the ATP monitoring reagent. Con- trol experiments with ATP in HCL04 showed that stan- dard curves were linear over the range O-200 pmol and that ATP was stable in HCLO, for up to 6 h, within which time it was assayed.

The oxidation of [I - “‘C lpyruuate, D-[ U-“Cl glucose, D- [3- “C’]-&hydroxybutyrate, and [ U-‘4C]palmitate. So as to confirm the viability of glycogen-depleted glands, their metabolism was compared with that of freshly iso- lated glands. Pairs of glands were incubated for 3 h in 0.15 ml of bicarbonate-buffered medium at 37”C, equili- brated with 02:COZ (19:1), and made up to either 1 mM [l-l”C]pyruvate, 5 mMD-[U-l”C]glucose, 1 mMD-[3-14C]-

b-hydroxybutyrate, or 0.5 mM [U-14C]palmitate/0.75% defatted bovine serum albumin. All specific activities were 0.6 Ci/mol. Oxidation was then arrested by the in- jection of HClO, and the l”CO, trapped in Hyamine as previously described (5).

230 WELCH, BRYANT, AND KEALEY

3 -

nmols glgcogenl gland 1, a

FIG. 1. Rates of glycogen utilization in glucose-free medium by hu- man eccrine sweat glands. Human eccrine sweat glands from three adult subjects were incubated in glucose-free bicarbonate-buffered medium as described under Materials and Methods. At l-h intervals five glands from each subject were removed and glycogen contents esti- mated as described under Materials and Methods. Each point repre- sents the mean glycogen content of five such glands.

[2-3H]Glucose hydrolysis. Glycolysis was monitored by following the loss of tritium from carbon 2 during the conversion of [2-3H]glucose 6-phosphate to fructose 6- phosphate (10). Pairs of glands were incubated in 0.15 ml of bicarbonate-buffered medium as described above containing 5 mM [2-3H]glucose (S.A. 60 Ci/mol) for 3 h. 3Hz0 was then sep arated by ion exchange chromatogra- phy as described by Hammerstedt (10).

RESULTS

Glycogen Assay

The assay was shown to be linear for up to 20 nmol glycogen. The sensitivity of the glycogen assay was in- vestigated by determining the detection limit, which was performed by examining 12 separate values of the zero glycogen standard (between batch). The detection limit is a formal description of the lowest quantity of a mate- rial that can be confidently assayed. It is defined as twice the standard deviation of the figure an assay yields when it is used to measure control (non-material-containing) samples. The detection limit was shown to be 0.12 nmol glycogen/sample. Between batch assay precision, based on values for 2 nmol glycogen, revealed a coefficient of variation of 5.5%.

Glycogen Content of Human Eccrine Sweat Glands

Analysis of eight glands, each taken from five male adult patients (a total of 40), showed the glycogen con- tent to be 2.28 + 0.28 nmol/gland (mean f SE).

Rates of Glycogen Utilization

(a) In the absence of 5 mM glucose. Figure 1 illustrates the rates of glycogen utilization in sweat glands taken from three different patients, maintained in glucose- free, bicarbonate-buffered medium. In the absence of ex- ternal nutrients, the glands from three different subjects showed similar rates of glycogen depletion despite differences in initial glycogen content. The initial glyco- gen depletion rate measured over 2 h was found to be 0.497 ? 0.015 (mean + SE) nmol glycogen/gland/h. This compares very well with the rate of glucose metabolism of 0.48 nmol/gland/h which has been reported for iso- lated human eccrine sweat glands as determined by the rate of 14C02 evolution from exogenous [U-‘4C]glucose and by the rate of lactate production (5).

(b) In the presence of 5 mM glucose. Experiments in which sweat glands were incubated in bicarbonate- buffered medium containing 5 mM glucose at 37°C showed there was no change in the glycogen content dur- ing a 3-h incubation (Fig. 2).

(c) In the presence of 5 mM glucose and 20 yM acetyl- choline. Figure 2 demonstrates the effect of acetylcholine on glycogen depletion when glands were incubated in bi- carbonate-buffered medium containing 5 mM glucose. Glycogen depletion was complete within 60 min in all glands examined.

Glycogen Repletion

Following a l-h depletion in bicarbonate-buffered me- dium containing 20 yM acetylcholine and 5 mM glucose,

nmds glycogenl gland.

I \

FIG. 2. The effect of acetylcholine on the rate of glycogen depletion of human eccrine sweat glands. Sets of five glands each from three male patients were incubated in bicarbonate-buffered medium con- taining 5 mM glucose as described under Materials and Methods, ei- ther with (0) or without (0) 2 X 10m5 M acetylcholine. At each time point five glands were removed for each glycogen estimation as de- scribed under Materials and Methods. Each time point represents the mean + SE glycogen content (n = 15 glands).

GLYCOGEN DEPLETION MONITORED BY LUCIFERASE 231

TABLE 1

Glycogen and ATP Contents of Human Eccrine Sweat Glands Maintained under Different Conditions

Parameter (mean k SE) Freshly isolated glands

Following 1-h incubation in

20 fiM acetylcholine, 5 mM glucose

Table la

Following a further 2-h incubation in acetylcholine-free

medium containing 5 mM glucose

Following a further 2-h incubation in acetylcholine-free

medium containing 1 mM pyruvate

Glycogen (nmol/gland) 1.51 * 0.13 0.09 i 0.01*** 0.89 f 0.09***

Table lb

Glycogen (nmol/gland) ATP (pmol/gland)

1.25 k 0.05 0.03 -+ 0.01*** 0.02 f 0.01*** 47.5 + 2.4 44.3 * 4.9 52.7 k 3.1

Table la. Fifteen glands, each from three male adult patients (a total of 45 glands) were isolated as described under Materials and Methods. Each time point represents the mean zk SE of the glycogen content of 5 glands from each patient, a total of 15. The first time point represents the glycogen contents of the glands on isolation. The second time point represents their glycogen contents after an hour’s incubation in bicarbo- nate-buffered medium supplemented with 20 fiM acetylcholine and 5 mM glucose. The third time point represents their glycogen contents after a further 2 h incubation in acetylcholine-free bicarbonate-buffered medium supplemented with 5 mM glucose. *** P < 0.001 when values are compared by Student’s t test with the glycogen contents of freshly isolated glands.

Table lb. Thirty glands, each from three male adult patients (a total of 90 glands) were isolated by shearing. Each time point represents (a) the mean t SE of glycogen contents of 5 glands from each patient, a total of 15, and (b) the mean + SE of the ATP contents of a further 5 glands from each patient, a total of 15. The conditions of incubation were the same as for Table la, except that the final 2 h incubation was in acetylcholine-free medium containing 1 mM pyruvate. *** P < 0.001 when values are compared by Student’s t test with the glycogen contents of freshlv isolated glands. The ATP contents were not sienificantlv different under different conditions of incubation (P > 0.2 when compared with freshly isolated glands).

sweat glands were transferred into bicarbonate-buffered medium containing 5 mM glucose without acetylcholine and incubated for 2 h at 37°C. Table 1 shows that such glands repleted half of their original glycogen content within 2 h. However, glands incubated in bicarbonate- buffered medium containing 1 mM pyruvate for 2 h showed no increase in glycogen contents. They did, how- ever, retain their viability as determined by ATP con- tent.

Table 2 shows why 1 mM pyruvate maintains the via- bility of the glands, because the rates of pyruvate oxida- tion in glycogen-depleted glands were either five times (Table 2a) or two times (Table 2b) those of glucose oxi- dation. The predicted value would have been four times, for two reasons: (i) pyruvate oxidation yields approxi- mately half the energy of glucose oxidation, and (ii) the eccrine sweat gland engages in aerobic glycolysis. Krebs defined aerobic glycolysis as the conversion of glucose to lactate by tissues despite the presence of oxygen (ll), and we have previously shown that the human eccrine sweat gland engages in aerobic glycolysis (5). This ex- plains the high lactate content of sweat. We have pre- viously shown that the human eccrine sweat gland de- rives about half of its energy from glucose from aerobic glycolysis and about half from oxidation (5). The devia- tion reported here from the predicted value of pyruvate oxidation may be attributable to the variations that are seen between the activities of different sweat glands (12).

Intermediary Metabolism

Table 2 shows that acetylcholine stimulates glucose oxidation and glycolysis 2.5-fold in freshly isolated glands, while isoprenaline (isoproterenol), a P-adrener- gic agonist, stimulates these by 50%. These are similar both to our own previous findings (5) and to those of Sato and Dobson who worked on microdissected glands (13). Glycogen-depleted glands show a similar baseline rate of glucose oxidation, but in them acetylcholine stim- ulates glucose oxidation 3.75-fold. Although this differ- ence does not attain statistical significance, it might be attributable to a lack of endogenous glycogen breakdown which, as Fig. 2 shows, occurs in freshly isolated glands with acetylcholine.

Table 2 shows that [l-14C]pyruvate oxidation in glyco- gen-free glands is not stimulated either by lob5 M acetyl- choline or by 1 mM dichloroacetic acid. This shows that acetylcholine does not activate eccrine sweat gland pyru- vate dehydrogenase directly. Glucose oxidation in glyco- gen-depleted glands, but not in freshly isolated glands, is stimulated by 1 mM dichloroacetic acid. The reason for this difference is not known, but dichloroacetic acid has a number of contradictory and poorly understood effects on carbohydrate metabolism in addition to those on pyruvate dehydrogenase, and these might vary with glycogen status (14).

Because exogenous lipids cannot sustain eccrine sweat gland fluid secretion in vitro, it has been suggested

232 WELCH, BRYANT, AND KEALEY

TABLE 2

Metabolic Studies on Freshly Isolated and Glycogen-Depleted Human Eccrine Sweat Glands

Conditions of incubation

Freshly isolated Glycogen depleted

5mM 5 mM 5mM 1mM 1 mM D-[3-“Cl-& 0.5 mM [U-‘4C]glucose [2-3H]glucose [U-‘~C]glucose [l-“C]pyruvate hydroxyhutyrate [U-“C]palmitate

(nmol of substrate oxidized or hydrolyzed/gland/h)

Table 2a

Control 10m5 M acetylcholine lo-’ M dichloroacetic acid

3 subjects 5 pairs of glands 0.037 k 0.009 0.102 * 0.028’

0.038 IO.012

-

-

3 subjects 3 pairs of glands 0.074 + 0.026 0.280 k 0.062*

0.120 k 0.013’

4 subjects 3 pairs of glands 0.39 A 0.06 0.37 kO.08

0.41 kO.09

Table 2b

Control 10e5 M isoprenaline 10e5 M acetylcholine

3 subjects 5 pairs of glands

0.062 s 0.009 0.097 + 0.023 0.147 zt 0.038*

3 subjects 5 pairs of glands

0.63 + 0.12 0.86 t 0.17 1.41 + 0.21**

No additions

Table 2c

3 subjects 3 pairs of glands 0.087 2 0.002

-

3 subjects 3 pairs of glands

0.045 + 0.017

3 subjects 3 pairs of glands

0.012 * 0.006

Table 2d

Control 1 mM @-hydroxyhutyrate 0.5 mM pahnitate

- -

3 subjects 3 subjects 3 pairs of glands 3 pairs of glands 0.120 -+ 0.030 0.230 + 0.160 -

0.105 f 0.020 0.140 + 0.010** - -

0.040 + 0.005 ** 0.255 k 0.020 -

IVote. Each subsection of the table (Za, Zb, 2c, and 2d) represents a different experiment, performed on different glands from different adult male patients. All experiments were performed on pairs of glands incubated for 3 h in 150 ~1 bicarbonate-buffered medium. The rates of substrate oxidation or hydrolysis were determined as described under materials and methods. As is indicated in the table title, glands were either studied on isolation or following glycogen depletion.

Table 2a. This represents three separate experiments. Freshly isolated glands were obtained from three subjects, and their rates of 5 mM [U-

“Clglucose oxidation determined. The glycogen-depleted glands were obtained from four different subjects, three of whom supplied enough glands to study both 5 mM [U-‘*C]glucose and 1 mM [1-*‘C]pyruvate oxidation. *P < 0.05 as compared to the control value for each substrate as determined by Student’s t test.

Table 2b. This represents experiments performed on the glands of three subjects (a total of 90 pairs of glands from three subjects). * P < 0.05, ** P < 0.01 as compared to the control value for each substrate as determined by Student’s t test.

Table 2c. This represents experiments performed on the glands of three subjects, a total of 27 pairs of glands from three subjects. Table 2d. This represents experiments performed on the glands of three subjects, a total of 54 pairs of glands from three subjects. ** P < 0.01

as compared to the control value for each substrate by Student’s t test.

that this tissue cannot metabolize lipids (15), but Table 2 shows that both a-hydroxybutyrate and palmitate are oxidized by the glands. However, it can be calculated that they yield only I.125 and 1.548 pmol ATP/gland/h, respectively, which is only a fifth of that obtainable from glucose metabolism at 6.6 pmol ATP/gland/h (5), which may account for their inability to sustain fluid secretion. So as to test the glucose fatty cycle (16) in this tissue, it was noted that palmitate inhibits glucose oxidation in glycogen-depleted glands, while P-hydroxybutyrate in- hibits pyruvate oxidation.

DISCUSSION Bioluminescence provides a rapid and accurate glyco-

gen assay which is linear for up to 20 nmol of glycogen. The recovery of glycogen may be assumed to be complete for two reasons: (i) there is no separation step, and (ii) the technique of trichloroacetic acid disruption followed by 2 h of incubation in amyloglucosidase, pH 4.8, has already been shown by Becker (17) to result in the com- plete hydrolysis of yeast cell glycogen. When eccrine sweat glands were glycogen depleted, their glycogen con- tent fell to 0.095 f 0.014 nmol/gland which is below the

GLYCOGEN DEPLETION MONITORED BY LIJCIFERASE 233

detection limit of the assay at 0.12 nmol glycogen/sam- ple. This shows that eccrine sweat glands do not contain agents that interfere with the assay.

Early attempts at glycogen depletion of eccrine sweat glands by incubating them in glucose-free medium (Fig. 1) failed because this invariably killed them. The method we now describe leaves them viable. The acetyl- choline-stimulated breakdown of glycogen in vitro is fur- ther evidence of the viability of sheared sweat glands, because acetylcholine has been reported histochemically to deplete glands of glycogen in situ (18). Whether the acetylcholine acts via the calcium activation of phos- phorylase, or by increasing the energetic requirements of the gland by stimulating secretion, remains to be de- termined.

The method of glycogen depletion we have described was developed so as to exploit two properties of pyru- vate: (i) pyruvate is oxidized by pyruvate dehydrogenase, and so it should maintain tissue viability; (ii) mamma- lian gluconeogenesis, and therefore glycogenesis from pyruvate, is restricted to the liver and kidney cortex (19). This technique of glycogen depletion should, therefore, be applicable to all other tissues.

The metabolic studies reported here show that glyco- gen-depleted glands demonstrate rates of glucose oxida- tion similar to those of freshly isolated human eccrine sweat glands and will, furthermore, metabolize fats.

Dichloroacetate is an activator of pyruvate dehydro- genase (20), but it failed to stimulate pyruvate oxidation in the eccrine sweat gland. However, pyruvate dehydro- genase resistance to dichloroacetic acid appears to be a feature of other tissues such as the gut that engage in aerobic glycolysis (2 1).

Acetylcholine, the major secretagogue of the sweat gland, stimulates both glucose oxidation and glycolysis equally (Table 2), but it does not stimulate [ l-‘“Cl- pyruvate oxidation. This indicates that in this tissue secretagogue stimulation of glucose metabolism occurs upstream of pyruvate production. The @-adrenergic stimulation of glycolysis suggests that the cyclic AMP- mediated inhibition of phosphofructokinase 2 (22) is not a feature of the human eccrine sweat gland. The variable inhibition of glucose and pyruvate oxidation by exoge- nous fats is hard to explain, but it should be noted that the absence of a glucose-fatty acid cycle effect is a fea- ture of other aerobic tissues such as the brain whose ma- jor fuel is glucose rather than fats (23).

In summary, we have described a specific, sensitive

glycogen assay which has enabled us to devise a method of glycogen-depleting human eccrine sweat glands which retain their viability as determined by a number of meta- bolic criteria. This glycogen assay may be valuable not only in metabolic research, but also in the clinical deter- mination of the glycogen content of small biopsies of liver, muscle, or other tissues obtained from patients.

ACKNOWLEDGMENTS

We thank Dr. L. Agius, Mrs. G. Bates, Dr. A. L. Kerbey, and Dr. A. Skillen for their help and advice. This work was supported by the Cys- tic Fibrosis Research Trust and the Northern Regional Health Au- thority. T.K. is a Wellcome Senior Clinical Research Fellow.

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