Hexokinase Isozymes of Normal Human Subcutaneous Adipose Tissue Victor

The

isozymes

specimens tissue

of

were

7.4.

1 eluted

0.19M

KCI.

glucose 2. 1.5

with

Two

Peak

peaks

linear

adipose

were

more

1 to inactivation

2 at

(K,)

x lo-“M;

2 was

at

found:

KCI, and Peak

1, 6.5

Peak

from

KCI gradients

constants

Peak

Peak

surgical

elution

of activity

Michaelis

were:

ble than

by

at 0.05M

x 1 0e4M.

in

subcutaneous

separated

DEAE-cellulose pH

hexokinase

of human

R. Lavls

0.1 mg/ml,

and was

cose.

peaks

Both

for

(45”

comprised

66

hexokinase

activity.

tected

Peak

tics,

suscepti-

f

the

5

percent No

isozymes

from

glucose. of

of human

the

was

de-

characteris-

adipose

I

hexokinases

2

total

with

III

In all these

heat

Peak

the

activity

hexokinase

fat.

resemble

by 0.1 M glu-

protected

by O.lM

of

in human

closely

by trypsin,

)

inactivation

characteristics

protected

were

and

tissue II from

rats.

T

HERE ARE four principal forms of hexokinase (ATP:D-hexose 6-phosphotransferase, E.C. 2.7.1.1) in mammalian tissues, distinguishable by electrophoretic and ion-exchange behavior,‘-5 kinetic constants,‘,‘j.4.” stability to heat and proteases,‘.‘S,i,X and antigenic determinants.“-“i Rat adipose tissue contains primarily hexokinase I and II, the relative proportions of which vary with age, nutritional and hormonal status.‘,i,‘4-“Z However, the number and identity of the hexokinases of human adipose tissue remain in dispute. Published studies, employing electrophoretic separation of isozymes, have variously shown one,“’ two”.J.i three,“l or moreZ5 bands of hexokinase activity from human fat. In particular, the published results have been conflicting with regard to the question of whether or not human fat contains an isozyme corresponding to hexokinase II of rats. Hexokinase II is of particular interest, because it is dependent on insulin, in muscle and adipose tissue of rats:. i ,li,1x--““.L’?.‘lfi.~~ As discussed below, hexokinase II may be an index of the protein-synthetic action of insulin, which is worthy of further study in human tissues. Because of the potential importance of hexokinase II, I chose to reexamine the hexokinases of human adipose tissue. In this work, special care was taken to avoid loss of labile isozymes. DEAE-cellulose chromatography was employed for separation and recovery of isozymes, in order to permit quantification of activity and for partial characterization. The results indicate that human and rat adipose hexokinases are highly similar. MATERIALS

AND

METHODS

Tissue Preparation Abdominal

subcutaneous

nephrolithotomy,

From

Supported search

AM

January

reprint

requests

P.O. Box 20708.

c 1978 hy Grune

Diabetes

of TexasMedicalSchool.

& Stratton,

Houston.

Te.yas.

the National

Institutes

of

Health

(NIAMDD].

and hi, a Re-

Foundation.

io Victor R. Lavis, Department

Houston,

cholecystectomy.

consent. None of the donors were

27, 1978.

02456 from

Grant from the Juvenile

Address School.

publication by Grant

on patients who gave informed

of’ Medicine, University

the Department

Receivedfor

fat was obtained during elective surgery (hysterectomy,

or herniorrhaphy)

of Medicine. l_‘ni\‘ersity of

Te.yas Medical

Te.uas 77025.

inc. 0026 -O495/7~/27O9-OOllSOl,~~J~

Metabolism. Vol. 27. No. 9 (September).

1978

1101

1102

VICTOR R. LAWS

diabetic or acutely ill. All operations were done under general anesthesia, after an overnight fast. Specimens of fat weighing l-4 g were either homogenized within 20 min of excision, or stored at -80” for future study. The fat specimens were minced and homogenized in 2 vol of the following buffer: IOmM Tris-Cl, pH 7.4; 5 mM Na,EDTA, 5 mM mercaptoethanol, 5% (v/v) glycerol. Homogenization was with a PotterElvehjem homogenizer with Teflon pestle, at 23”. All succeeding steps were at 4”. Homogenates were centrifuged at 30,000 g for 30 min, the fat cakes discarded, the supernatants desalted on columns of Sephadex G-25, and then applied to columns of DEAE-cellulose equilibrated with homogenization buffer. The columns were developed with linear gradients of 0 to 0.6M KCI in the same buffer. Fractions were assayed for hexokinase activity at 0. I mM or I9 mM glucose, and peaks pooled for further study. In the fractions from DEAE-cellulose chromatography. hexokinase activities were stable at 4” for at least 48 hr.

Hexokinase Assay Hexokinase activity was assayed at 30” by an enzymatic fluorometric method similar to that that 0.1 ml of sodium dodecyl sulfate, IO described by Bernstein and Kipnis.’ with the modification mg/ml in ethanol/H,0 (l/l, v/v), was added at the end of incubations to stop the progress of the reactions. Routine assay mixtures contained (final concentrations, in a volume of 1.05 ml): 75 mM Tris-Cl, pH, 7.4; 2.5 mM MgCI,; 6 mM mercaptoethanol; 0.1 mM or I9 mM glucose; 140 mU/ml glucose-6phosphate dehydrogenase (type XI, Sigma); 0.33 mM NADP+ (Sigma), I.1 mM disodium ATP (Calbiochem), and 80 bg/ml bovine serum albumin (Armour). By definition, I U of hexokinase activity catalyzes phosphorylation of I pmol glucose/min, at 30” There was negligible reduction of NADP+ by DEAE-cellulose chromatographic fractions in the absence of glucose or ATP, confirming the specificity of the assay for hexokinase activity. RESULTS

The fat specimens studied were from donors ranging in age from ll:s~-69 yr, and in body mass index (wt/ht2)28 from 18.0 to 44.7 kg/m’. All specimens yielded peaks of hexokinase activity which eluted from DEAE cellulose at 0.05M KC1 (peak 1) and at 0.19M KC1 (peak 2). A typical elution pattern is shown in Fig. 1. On rechromatography of peak 2, there was no change in elution behavior (data not shown). When assayed at various concentrations of glucose, both peaks yielded linear Lineweaver-Burk plotszg as shown in Fig. 2. With peak 1, K, for glucose was 6.5 x 10-5M (range, 4.7 to 8.0 x lo-“M; n = 4). With peak 2, K, for glucose was 1.5 x 10maM (range, 1.2-1.8 x 10mlM; n = 5). Under identical conditions, hexokinases I and II from rat epididymal fat pads eluted at 0.04M and 0.18&I L

’

"1 3-

10

15 FRACTION

20 25 NUMBER

Fig. 1. Hexokinase isozymes in 3.3 g of adipose tissue from a 46.yr old man. Tissue preparation and DEAE-cellulose chromatography were as described under “Methods.” Each point represents the mean of duplicate hexokinase assays.

HEXOKINASE

Fig. Peaks point

ISOZYMES

2.

Lineweaver-Burk

1 and

2 were

represents

(Milliunits

pooled

a single

hexokinase

OF

SUBCUTANEOUS

plot from

of

hexokinase

DEAE-cellulose

hexokinase

assay.

TISSUE

activities

in adipose

chromatography, Units

1103

ADIPOSE

of absicssa:

tissue

as described l/[glucose](M).

from

a 41 -yr

under Units

old

woman.

“Methods.” of ordinate:

Each l/V

activity/tube)

KCI, and had K, for glucose of 6.6 x lo-;M and 1.3 x lO_‘M, respectively (data not shown). There was no evidence for any hexokinase isozyme which eluted after Peak 2, had a K, for glucose less than 5 x lo-“M, or was inhibited by O.lM glucose, these being characteristics of hexokinase III.‘-I.‘” Peaks I and 2 were both heat-labile in the absence of glucose, peak 2 being somewhat more so, as shown in Fig. 3. As with rat hexokinases,‘.“.Y the time course of heat inactivation was not first order, but consisted of rapid and slow components. Both peaks were protected by 0.1 M glucose, especially with respect to the early rapid phase of inactivation (Fig. 3). Peak 2 was more susceptible to inactivation by trypsin than was peak 1, and was protected by 0.1 A4 glucose (Fig. 4). The absolute activity of human peak 1 was 6.0 i I .5 mU/g of fat (mean & SE: n = 7), and that of peak 2 was 14 * 4 mU/g (mean * SE: n = 8). By comparison, pooled epididymal fat from two 270-g rats yielded hexokinase I and 11 activities of 25 and 73 mU/g fat, respectively. With human fat the data obtained so far show no evident relationship between the relative activities of peaks 1 and 2, and the ages or degrees of obesity of the donors. The aggregate data show peak 2 comprising 66 i 5 percent (mean l SE; n = 10) of the total hexokinase activity. DISCUSSION

In the work reported here, care was taken to obviate the problem of the lability of hexokinase II by the use of surgical rather than postmortem specimens of adipose tissue, and by the inclusion of glycerol as a stabilizing agent in buffers.‘“.,‘Z

1104

VICTOR R. LAVIS

PEAK 2

PEAK 1 * l~~~_O k \ L b

O.lM GLUCOSE

80-\

a

-\ I \

\ \

;

a

80-

z k 0

40-

!i w

20.

- \ \ \ _ \

‘\, “t,

O.lM GLUCOSE

‘.

\ +-a

\

NO GLUCOSE

A\

._

e--A

0

NO GLUCOSE

E a

1

0-f

0

20

40

1

0

80

MINUTES

INCUBATION

20

40

,

80

AT 45 o

Fig. 3. Heat stability of hexokinases in adipose tissue. Peaks 1 and 2 were pooled after DEAE-cellulose chromatography as described under “Methods”, and heated to 45”, in the presence of 5% (v/v) glycerol. At the times indicated on the abscissas, aliquots were withdrawn for hexokinase assay. Ordinates: Hexokinase activity, expressed as percent of activity prior to heating. Each point represents the mean of two experiments done with different specimens (donors: women aged 24 and 52 yr). Symbols: a-0, 0.1 M glucose present during heating;A-A, no glucose present during heating.

Separation of hexokinases by DEAE-cellulose chromatography rather than by electrophoresis permitted recovery of isozymes for quantification and partial characterization. In the present work, there were two peaks of hexokinase activity separable on DEAE-cellulose. Peaks 1 and 2, herein described, closely resemble hexokinases I and II from other species and from other human tissues, with respect to the concentration of KC1 required for elution from DEAE-cellulose, and Km for glucose 1.3.31 PEAK 1

PEAK 2

,oo NO GLUCOSE 60

60

40

20

0 0

10

MINUTES

20

30

0

10

20

30

INCUBATION WITH TRYPSIN, 0.1 mg/ml at 23’

Fig. 4. Proteolytic stability of hexokinases in adipose tissue. Peaks 1 and 2 were pooled after DEAE-cellulose chromatography as described under “Methods”, and incubated with 0.1 mg/ml trypsin (Nutritional Biochemicals Co.) at 23”. in the presence of 2.5% (v/v) glycerol. At the times indicated on the abscissas, 0.1 ml aliquots were withdrawn and added to hexokinase assay tubes containing 25 pg lima bean trypsin inhibitor (Worthington). Ordinates: hexokinase activity, expressed as percent of activity prior to addition of trypsin. Each point represents the mean of triplicate assays in one experiment (donor: 46-yr old man). Symbols: o - D , 0.1 M glucose present during incubation with trypsin: n - 0, no glucose present during incubation with trypsin.

HEXOKINASE

ISOZYMES

OF SUBCUTANEOUS

ADIPOSE

TISSUE

1105

These results may be compared with the previously published studies of human adipose tissue. Neumann et al. I3 found one band of hexokinase activity on starch gels with the mobility of hexokinase I. They employed postmortem fat specimens. so it seems possible that the more labile hexokinase II was lost. Isozymes with the electrophoretic mobilities of hexokinases I and II have been obtained from fresh biopsy specimens by Strickland and Ellis,Y” Pilkis,!’ and by Bernstein.* Brown et aLZ1 found bands on starch gels that corresponded to hexokinases I, II, and III. Galton and Jones’” found multiple bands by acrylamide gel electrophoresis in a single surgical specimen. In none of these earlier studies of human fat were the hexokinases further characterized. The present data show no evidence for hexokinase 111in human adipose tissue, with the limit of detection of the hexokinase assay method being about 0.5 milliunits per gram fat. It is of course possible that a labile hexokinase III existed in the specimens, but was lost during tissue preparation. Other investigators have reported human adipose hexokinases with the electrophoretic mobility of rat hexokinase III,‘d,“’ but it is possible that their results reflected contamination of the specimens by blood cells.“..‘” There is some controversy regarding whether or not other mammalian adipose tissues contain hexokinase III. Several authors have published data showing only hexokinases I and II in rat adipose tissue;‘.” ” however, their assays were performed at glucose concentrations of 25 mM or greater, which might have concealed the substrate-inhibited hexokinase III. w.” More relevant are the published studies in which starch gels of rat fat homogenates were stained in the presence of 0.5 mM glucose. In four such studies,i.“_‘“.:~_’ only hexokinases I and II were found, while in two others’-‘!’ there was faint staining in the hexokinase III zone. It therefore seems reasonable to conclude that hexokinase III is at most a minor isozyme of both human and rat adipose tissue. I!’ Inactivation of human hexokinase peaks 1 and 2 by trypsin was similar to what has been reported for rat hexokinases I and II:‘. Although hexokinase II is generally agreed to be heat-labile in the absence of glucose,‘,:‘,; there is some controversy in the literature about the heat lability of hexokinase I from rats, Grossbard and Schimke” having reported it to be stable at 45” in the absence of glucose, while Bernstein and Kipnis’ and Katzen and Schimke’ published inactivation curves similar to those for human peak 1 reported here. It is noteworthy that Gustke:‘” has described a soluble heat-labile human placental isozyme which immunologically resembles hexokinase I. Possibly heat lability is a general characteristic of human hexokinase I. Because of the several similarities of the isozymes of human fat to those from rats, it is reasonable to identify peaks I and 2 as homologous with hexokinases I and II from other species. The hexokinase activities of human fat were only about 35% of those from rat fat, expressed per gram of fat. This difference probably reflects the interspecific difference in mass of inert lipid, which is about 0.6 pg/cell in subcutaneous fat from normal humans’“’ and 0.18 pg/cell in epididymal fat from 270 g rats.‘” Enzymatic activities are here reported on the basis of wet weight rather than tissue protein, because of large interspecimen variability in contamination of specimens by blood. As in fed young rats,‘.“,“.“’ the major hexokinase isozyme of human fat *Bernstein,

RS. personal

communication.

1106

VICTOR R. LAWS

was hexokinase I1 (peak 2). These results may be compared with those of Bernstein et aLsx whose data show total hexokinase activity of about 50 mU/g wet weight in homogenates of fat cells from normal human subcutaneous tissue; this activity is about 2.5-fold greater than that reported here for hexokinases I and II. There are at least two plausible explanations for this difference. First, Bernstein et al.“X used adipocytes rather than tissue fragments, which contain variable amounts of fibrous material and blood. Second, DEAE-cellulose chromatography usually entails some loss of enzymatic activity.:’ Using differential heat stability to distinguish isozymes,7 Bernstein et al.“’ found that hexokinase II comprised about 30% of the total activity in normal human fat cells, compared to the value of 66% reported here. However, the heat Iabilities of hexokinases I and II are strongly dependent on conditions such as protein and glucose concentrations,‘.:j7 and may also differ among species, as discussed above. Therefore, the assignment of relative activities of human isozymes based on heat stability should not be regarded as definitive. The potential significance of these results derives from the relationship of hexokinase II to insulin action. Fasting and diabetes have been found to reduce total hexokinase activity in skeletal muscle,“,‘” cardiac muscle,“,‘fi.27 and adipose tissue”.7.“.‘7.‘s.22.*6 of rats.* When isozymes were examined, this adaptive change was shown to involve principally hexokinase II.1.~~.7.‘1-‘x.26,27Insulin, given in vivo or in vitro, can restore hexokinase II activity in cardiac and skeletal muscle, and adipose tissue, from fasted or diabetic rats.“.‘5.‘6.‘8,20.?a.3g Possibly part of this action of insulin is mediated by increased entry of glucose into cells. It has been suggested that glucose or one of its metabolites may stabilize intracellular hexokinase II by reducing its susceptibility to denaturation and/or proteo1ysis.‘“.27 Indeed, incubation of rat adipose tissue ‘5.26.3gor small intestine, 4’)or culture of human-liverderived cells’ with high concentrations of glucose alone increases hexokinase II activity. However, there are several kinds of evidence which suggest additional mechanisms for this action of insulin on hexokinase II, other than stabilization by intracellular glucose. First, this effect of insulin can be blocked by puromycin, cycloheximide, or Actinomycin D,‘5.‘X.3g implying a requirement for protein synthesis. However, protein synthesis is not required for the actions of insulin on glucose metabolism of adipose cells.4’ Second, when incubated with rat adipose tissue in vitro, insulin specifically enhances incorporation of labeled amino acids into protein(s) with the electrophoretic mobility of hexokinase II. 2o.3oThird, insulin in vitro further augments the hexokinase activity of rat fat pads over that produced by high glucose alone. 15.2”.“6.3gFourth, with rat fat pads, insulin in vitro increases hexokinase activity even in the absence of extracellular glucose.‘0,Y6.3g The augmentation by insulin of the activity of hexokinase II in rodent muscle and adipose tissue therefore appears to reflect actions over and above those related to glucose metabolism, and may be a useful model for study of the protein-synthetic effects of the hormone. It remains to be determined whether the hexokinases of human adipose tissue, like those of rats, are subject to hormonal regulation. In particular, it will be of interest to determine whether the effects of insulin on hexokinase II observed in rodents5. 1s.16.IR--Z0.22.26.R9 apply also to humans. *It should be noted that in other tissues. hexokinases may respond differently to insulin; example, hexokinase II activity is actually increased in the small intestine of diabetic rats (5).

for

HEXOKINASE

ISOZYMES

OF SUBCUTANEOUS

ADIPOSE

1107

TISSUE

ACKNOWLEDGMENT Thomas Johnson. Harry

Knutowski.

Jr.. and Gloria Tan provided valuable technical assistance.

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HM,

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characterization

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Ellis DA:

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28. Bray GA: The Obese Patient, Major Problems, in Smith LH, Jr, (ed): Internal Medicine. Philadelphia, W.B. Saunders, 1976, p 9 29. Lineweaver H, Burk D: The determination of enzyme dissociation constants. J Am Chem sot 56:658666, 1934 30. Gonzalez C, Ureta T, Sanchez R, et al: Multiple molecular forms of ATP: Hexose 6phosphotransferase from rat liver. Biochem Biophys Res Commun 16:347-352, 1964 31. Cayanis E, Balinsky D: Comparative kinetic properties of human hexokinases. Int J Biochem 6:741--749. 1975 32. Holroyde MJ, Trayer IP: Purification and properties of rat skeletal muscle hexokinase. FEBS Lett 62:215-219, 1976 33. Povey S, Corney G. Harris H: Genetically determined polymorphism of a form of hexokinase, HKIII, found in human leucocytes. Ann Hum Genet 38:407--415, 1975 34. Grossbard L, Weksler M. Schimke RT: Electrophoretic properties and tissue distribution of multiple forms of hexokinase in various mammalian species. Biochem Biophys Res Commun 24:32-38, 1966

VICTOR R. LAWS

35. Gustke HH: Properties of human placental hexokinase. Enzyme 20:292-304. 1975 36. Brook CGD, Lloyd JK. Wolf OH: Relation between age of onset of obesity and size and number of adipose cells. Br Med J 2:25%27, 1972 37. Greenwood MRC, Hirsch J: Postnatal development of adipocyte cellularity in the normal rat. J Lipid Res 15:474483, 1974 38. Bernstein RS, Grant N, Kipnis DM: Hyperinsulinemia and enlarged adipocytes in patients with endogenous hyperlipoproteinemia without obesity or diabetes mellitus. Diabetes 24~2077213, 1975 39. Hansen RJ, Pilkis SJ, Krahl ME: Effect of insulin on the synthesis in virro of hexokinase in rat epididymal adipose tissue. Endocrinology 86:57-65, 1970 40. Shakespeare P, Srivastava LM, Hubscher G: Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity. Biochem J 11 l:63-67, 1969 41. Beigelman PM, Schlosser G: Effects of puromycin upon isolated adipose tissue cell metabolism. Biochem Med 2:l IO-1 17. 1968