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Inhibition by phlorizin of insulin- and protease-stimulated glucose utilization in isolated adipose cells
BIOCHIMICA
252
ET BIOPHYSICA
ACTA
BBA 55352
INHIBITION
BY PHLORIZIN
GLUCOSE UTILIZATION
J. F.
KUO,
I.
K.
DILL
AND
OF INSULIN- AND PROTEASE-STIMULATED
IN ISOLATED ADIPOSE CELLS
C.
E.
HOLMLUND
Chemotherapy Research N.Y. (U.S.A.)
Section, Lederle Laboratories,
(Received
1967)
March
rxth,
American
Cyanamid
Company,
Pearl River,
SUMMARY I. Utilization of extracellular glucose, determined by its conversion to CO, and total lipid, has been employed to study the effect of phlorizin on isolated adipose cells incubated with and without insulin, a-chymotrypsin, trypsin and pronase (Streptomyces griseus protease, Type VI). 2. In dilute glucose medium, the effect of insulin on glucose utilization is to lower the apparent Km, and also to increase the v,,,. Phlorizin competitively inhibited glucose utilization without affecting intracellular metabolism, including the reactions involved in lipolysis and oxidation of X-labeled cellular materials. 3. Phlorizin at 0.92 mM inhibited glucose utilization by isolated adipose cells to the same extent in the presence and absence of insulin, cr-chymotrypsin or trypsin. Moreover, the degree of inhibition was essentially identical for both parameters measured (CO, production and lipid synthesis), and remained constant over a wide range of concentrations of these agents. It appears that phlorizin blocks only the process of glucose entry, and does not affect the mechanisms whereby glucose utilization is increased by insulin or proteolytic enzymes.
INTRODUCTION
Phlorizin, a polyphenolic b-n-glucoside, is known to inhibit sugar transport in several tissue preparations 1- 7. Insulin-stimulated sugar uptake is also inhibited by phlorizin6~B~8~s. Increasing attention has been drawn recently to the insulin-like activity of several proteolytic enzymes+‘6 and phospholipasesla-18. In the present report, the inhibitory effect of phlorizin on insulin- and protease-stimulated glucose utilization by isolated adipose cells have been compared in order to determine the extent to which these agents resemble each other. In particular, these experiments were conducted to explore the concept that the process for glucose uptake stimulated by various agents, possibly via different mechanisms, may be identical. Biochim. Biophys.
Acta, I++ (1967) 252-258
PHLORIZINANDGLUCOSEUTILIZATION
2.53
MATERIALSANDMETHODS The epididymal fat pads of male Sprague-Dawley rats, weighing IIO - 140 g, have been used in this study. The procedure for preparing and incubating isolated adipose cells were essentially the same as described by RODBELL~~with some modification’*. Sliced fat pads were digested for I h with collagenase in Krebs-Ringer bicarbonate medium made to 5 mM with glucose. The dispersed tissue was filtered through nylon cloth and the isolated adipose cells were washed three times with the same buffer with or without glucose (I mM). The weight of free adipocytes in the final suspension was estimated by subtracting the weight of residual tissue on nylon cloth from the pad weight before digestion. Unless otherwise stated, the incubation mixture consisted of I ml of the bicarbonate medium containing 4% bovine serum albumin (Fraction V), free adipocytes ranging from 33 to 52 mg, 0.2 & [l*C,]glucose and sufficient unlabeled glucose to make a concentration of I mM. Each treatment was incubated in duplicate to quadruplicate in each experiment, and each experiment was carried out at least two times to insure its reproducibility. Only the data from one experiment are reported. Procedures for extracting the incubated cells and determining CO, and total lipid were the same as reported elsewhere14~*9 except that CO, was absorbed with water-saturated phenylethylamine. Collagenase was obtained from Worthington, while epinephrine (DL-arterenolHCl, lot 52666), a-chymotrypsin (crystalline, lot 53251) and trypsin (crystalline, lot 44370) were purchased from Calbiochem. Insulin (24 l.U./mg, lot 74B-IOZO), ACTH (121 units/mg, lot 14B-0600), pronase (Streptomyces griseus protease, Type VI, repurified, lot 85B-2360) and bovine serum albumin (Fraction V, lot 105B-1520) were botained from Sigma. D-[l*C,]Glucose, 14.3 mC/mmole, was a product of New England Nuclear Corp. RESULTS Data pertaining to the effect of phlorizin on adipose cell metabolism, as measured by the conversion of extracellular glucose to CO, and total lipid as well as free fatty acid release are presented in Table I. Phlorizin clearly inhibited the utilization of glucose by adipocytes both in the presence and absence of insulin, norepinephrine, ACTH and caffeine.The fat mobilization process stimulated by the lipokinetic agents, however, was not significantly affected by phlorizin at 2 mM. Since glucose transport is known to be the major rate-limiting step in glucose uptake and the principal site of insulin action in epididymal adipose tissueao, it is likely that phlorizin specifically inhibited glucose entry without significantly interfering with intracellular activity. In order to further verify this point, an experiment was conducted with l*C-prelabeled adipocytes incubated in the same medium, but lacking extracellular glucose. The results were consistent with the above interpretation, since phlorizin failed to inhibit the conversion of %-labeled cellular materials into ‘*CO, in the presence and absence of insulin, ACTH and norepinephrine. It appears also that phlorizin does not significantly affect the metabolism of total lipid. In a separate experiment the radioactivity of the lipid fraction was found to be relatively unchanged after incubation for 2 h in the absence of extracellular glucose. Hormone-mediated lipolysis in the absence of glucose was also independent of phlorizin action. Biochim.
Biophys.
Acta,
144 (1967)
252-258
J. F. KUO, I. K. DILL, C. E. HOLMLUND
254 TABLE
I
EFFECTS
ACTH,
OF
PHLORIZIN
ON
NOREPINEPHRINE
THE
AND
METABOLISM
OF
ISOLATED
ADIPOSE
CELLS
INCUBATED
WITH
INSULIN,
CAFFEINE
Free adipocytes were incubated in r ml Krebs-Ringer bicarbonate medium (pH 7.4) containing 0.2 yC n-[i”C,]glucose (I pmole) at 37’ for 2 h. Each treatment was performed in quadruplicate. The values given in the tables are means (f S.E.). For further experimental details see the text. Additives
Phlorizin, _
2
CO,
mM Total lipid
(pmoles glucose/g cells per 2 h) None Insulin (1000 punits/ml) ACTH (0.5 pg/ml) Norepinephrine (0.5 @g/ml) Caffeine (2 mM)
Free fatty acid
(A pequivlg cells per 2 h) 0.03
0.0 *
_.___
+ CO,
0.29 + 0.06
0.77 j
I.7
0.96 & 0.02 0.42 f 0.01
2.35 $- 0.05 0.97 * 0.04
-3.0 f 0.1 81.0 -& 2.8
0.49 f 0.02 0.15 + 0.01
I.01 -+ 0.04 0.24 * 0.03
86.0 & 4.0 78.0 f 3.3
Totalipid
___ (pmoles glucose/g cells per 2 h)
.____ Free fatty acid (A pequiv Is cells per 2 h)
0.06 + 0.02
0.22 *
0.01
0.6 + 1.0
0.05 0.04
0.65 f
0.04
0.21
i
0.03
0.0 f 2.5 82.8 * 3.0
0.21
&
0.01
0.22
f
0.07 i
0.06 & 0.03 0.06 + 0.01
0.16 & 0.03
86.4 h 2.8 76.8 + 3.1
3.5 3.0 2.5 i
1
V
Fig. I. Lineweaver-Burk plots of effect of phlorizin on conversion of extracellular glucose to CO, (A) and total lipid (B) by isolated adipose cells incubated with or without insulin. All experimental conditions were essentially the same as Table I except that the amount of o-[W,]glucose employed was altered to maintain a constant specific activity of 0.093 ,uC/pmole. If added, the amounts of insulin and phlorizin were IOOO punits and 0.92 pmole/ml of incubation mixture. Each treatment was performed in quadruplicate and mean values are presented in the figures. Velocity (v) is expressed as pmoles glucose converted per g cells per 2 h. 0, control: l , insulin; x , phlorizin ; A, insulin + phlorizin.
Fig. I separately shows the effect of phlorizin (0.92 mM) on the conversion of extracellular glucose to CO, (A) and total lipid (B) in the presence and absence of insulin as a function of glucose concentration. The kinetic constants calculated from the data are summarized in Table II. The similarity in the kinetics and the values of the apparent Km'ssupport the contention that the measured metabolic parameters reflect the processes of glucose transport through the cell membrane. The kinetics of the conversion of glucose to CO, plus total lipid (figure not shown) were also found to be similar to that measured by the two individual parameters (Fig. I). Phlorizin competitively inhibited glucose utilization by adipocytes (Kc= 2.3. IO-*-2.6.1o-~M), presumably by binding to the same site of the transport system as glucose. It is Biochim. Biophys.
Acta. 144 (1967) 252-258
PHLORIZIN TABLE
AND GLUCOSE UTILIZATION
255
II
SUMMARY
OF KINETIC
CONSTANTS
OF GLUCOSE
UTILIZATION
BY
ISOLATED
ADIPOSE
CELLS
Constants K,i, Kmz. Km,, K,,, and vriraxr.v,,,~~~. vn,ax8. and vaiax~, represent Michaelis constants and maximum velocities of glucose conversion by adipocytes of (I) no additive (control), (2) insulin (1000 ~units/ml), (3) phlorizin (0.92 mM) and and phlorizin at the aforementioned concentrations, respectively. Kt represents constant of phlorizin competitively inhibiting glucose utilization. Conversion of D-[14C,]glucose to
Constants
2’
rgi
ma z&q
Km,
,the apparent in the presence (4) both insulin the inhibition
(Ml
(pmoles/g cells per %l%il. vmaxa (pmoles/g cells per 2 h) vmaXl (pmoles/g cells per 2 h) vmsXS
2
h)
CO, (Fig. rA)
Total lipid (Fig. IB)
1.0. 4.5 . 5.0.
10-a
1.1
10-d
4.5 . 10-a 5.0.
10-S
110-3
CO, + total lipid (figure not shown) I.1
10-S
2.3 . 10-a
2.3 . 10-d
2.6.
I.5
2.8
4.5
2.3
4.5
4.5
. 10-a
4.5 . 10-1 4.8 . 10-3 10-I
6.4
9.0
‘3.9
interesting to note that inhibition by phlorizin can be relieved by the stimulatory effect of insulin in a manner of non-competitive inhibition kinetics. The apparent K,,, I * IO+ to 4.5 - IO-~M in the presfor glucose utilization was lowered fro& I.O.IO-s-~ 2.4 A f
o Lipid, +Phlorizin 1 CO,, Control 0 COP, +Phlorizin
$yzg l
Lipid (av. inhibition: 66.0%)
1
o CO,
(av. inhibition : 67.7 %I
601‘ 501 0
I 100
I 200
I 300
I 400
I 500
.O
f % 0
Insulin (j.U/ml)
2
U-Chymotrypsin
(pg/ml)
Fig. 2. Inhibition by phlorizin of the insulin-stimulated utilization of glucose. Experimental conditions were essentially the same as Table I except that free adipocytes were incubated with or without phlorizin (0.92 mM) in the presence of varied insulin concentrations as indicated. Each treatment was performed in quadruplicate and mean values are presented in the figures. Fig. 3. Inhibition of phlorizin of a-chymotrypsin-stimulated utilization of glucose. Experimental conditions were essentially the same as Fig. 2. Each treatment was performed in duplicate and mean values are presented in the figure. Biochim. Biofihys. Acta, 144 (1967) .252-258
256
J. F. KUO, I. K. DILL, C. E. HOLMLUND
ence of insulin, while the constant increased to 5.0. IO-~M in the presence of phlorizin (with or without insulin). The vmaxwas not affected by phlorizin, but increased to 1.4 - 1.6 fold in the presence of insulin and insulin plus phlorizin, respectively. Certain proteolytic enzymes mimic insulin in their action on isolated adipose cellP+. It is therefore interesting to know whether phlorizin also inhibits the enhanced glucose utilization elicited by these enzymes. The results presented in Figs. 3A, 4A and gA clearly demonstrate that the stimulatory effects of cr-chymotrypsin,
0 Lipid,+Phlorizin l
CO.2 , Control o Lipid,+Phlorizin l
CO2
,
Control
0 CO,, + Phlorizin
*Lipid (av. inhibition: 71.3 %) oCOs Iav. inhibition: 69.0%)
c 0 P
Trypsin (pglml)
Pronase (IJglrnl)
Fig. 4. Inhibition of phlorizin of trypsin-stimulated utilization of glucose. Experimental conditions were essentially the same as Fig. a. Each treatment was performed in duplicate and mean values are presented in the figure. Fig. 5. Inhibition by phlorizin of pronase-stimulated utilization of glucose. Experimental conditions were essentially the same as Fig. a. Each treatment was performed in triplicate and mean values are presented in the figure.
trypsin and pronase on glucose utilization, like insulin, were subject to phlorizin inhibition. These observations also suggest that the insulin-like activity of these proteases may be due to the creation of favorable states for glucose uptake, or glucose transport, via a process similar to that mediated by insulin. Stimulation of glucose utilization by phospholipase C in adipocytes was also reported to be inhibited by phloretinla, the aglycone of phlorizin. It was found that the degree of inhibition by phlorizin over the entire range of insulin concentrations remained constant, and was identical for both parameters measured: 66.3% (CO,) vs. 66.0% (total lipid), see Fig. 2B. These data further substantiate the contention that phlorizin affects only the processes of glucose entry without affecting the fate of glucose metabolism or insulin action. This conclusion is B&him.
Biophys.
Acta,
144 (1967) 252-258
PHLORIZINANDGLUCOSEUTILIZATION
257
consistent with proposals made by FRERICHS ANDBALLS. Similar observations were also made with cc-chymotrypsin (Fig. 3B) and trypsin (Fig. 4B) ; the degree of inhibition of 67.7% (CO,) Y.S.69.8% (total lipid) for the former, and 69.0% (CO,) vs. 71.3% (total lipid) for the latter. However, pronase, a bacterial protease with an unusually broad substrate specificity, behaved somewhat differently (Fig. 5B). Although phlorizin inhibition of pronase-stimulated conversion of glucose to lipid remained quite constant over all the enzyme concentrations tested, this was not the case with CO, production. With increasing concentration of pronase the inhibition by phlorizin of CO, production from glucose increased. A possible explanation of these observations is given in DISCUSSION. DISCUSSION The results from the experiments with free adipocytes incubated in the presence of glucose (Table I) and with 14C-prelabeled cells incubated in glucose-free medium clearly demonstrate that the primary action of phlorizin is the inhibition of glucose entry into the cells. This conclusion is consistent with that of other workerse~‘, even though different parameters of measurement were employed for determining glucose uptake. The similarity in the kinetics of glucose conversion to CO, and total lipid (Fig. I), the comparable value of the apparent Km calculated therefrom (Table I), and the identical degree and constancy of phlorizin inhibition over a wide range of concentrations of insulin (Fig. 2B), a-chymotrypsin (Fig. 3B) and trypsin (Fig. 4B) support this concept. Moreover, based on these observations, the following conclusions may also be advanced: (I) the glucose uptake pathways stimulated by insulin as well as by cr-chymotrypsin and trypsin may be indistinguishable from the preexisting ones, and (2) E-chymotrypsin and trypsin, therefore, mimic insulin in their gross effect on glucose uptake by free adipocytes. From studies with 3-O-methylglucose and L-glucose, it has been concluded that the glucose uptake stimulated by phospholipase C (ref. 3) as well as by proteolytic enzymes (i.e. trypsin, a-chymotrypsin, pronase and Bacillus subtilis protease, Type VIII)* is a carrier-mediated, stereospecific process. It is interesting to note that the presence of both insulin and phlorizin resulted in a combined kinetics, in which the lower substrate affinity (i.e. higher Km) caused by phlorizin and the higher uUmax (bu t not the lower Km) caused by insulin were retained (Fig. I). The kinetic data seem to indicate further that phlorizin inhibits only the process of glucose uptake, and does not affect the mechanism whereby insulin enhances this process. FRERICHS AND BALLS reported a similar conclusion from their study with adipose tissue. Pronase resembles trypsin, a-chymotrypsin and insulin in all parameters tested, except with regard to the extent of inhibition by phlorizin of CO, production, which increased with increasing pronase concentration (Fig. 5B). Since pronase is capable of hydrolyzing a greater number of peptide bonds than a-chymotrypsin or trypsin, it is possible that at higher concentrations modifications of plasma membrane structure were more extensive than required solely for facilitating glucose entry. Under these specific circumstances phlorizin may be able to affect to a greater extent the * J. F. Kuo, I. K. DILL ANDC. E.
HOLMLUND.
unpublished
B&him.
observations.
Bioph.ys.
Ada,
144 (1967) 252-258
M3
J. F. KUO, I. K. DILL, c. E. HOLMLUND
intracellular activity. Since pronase, like insulin 21, has been found to stimulate YO, production from [x-l*G]glucose (and to specifically effect increased incorporation of the radioactivity into the fatty acid moiety)*, it is possible that phlorizin exerts its inhibition on %O, production by interference with the oxidation of C-I of glucose via the pentose cycle. FRERICHS AND BAI.L~ have shown that phlorizin is without effect in adipose tissue homogenates on hexokinase, glucosed-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, or on the overall conversion of glucose to triose phosphate. It seems, therefore, that lactonase may be the site of phlorizin inhibition, since it is the only enzyme participating in the steps prior to decarboxylation after glucose entry into the shunt pathway which has not been yet excluded from phlorizin inhibition by the above authors. REFERENCES I D. W. HAND, P. A. SANDFORDAND D. H. SMYTH, Nature, zag (1966) 618. 2 A. K. SEN AND W. F. WIDDAS, J. Physio?., 160 (1962) 404. 3 L. LACKO AND M. BURGER, Biochim. Biophys. Acta, 79 (1964) 563. 4 I. BIHLER, H. M. CAVERT AND R. B. FISHER, J. Physiol., 180 (1965) 168. 5 F. C. BATTAGLIA, K. L. MANCHESTERAND P. J. RANDLE, Biochim. Biophys. Acta, 43 (1960) 50. 6 H. FRERICHSAND E. G. BALL, Biochemistry, 3 (1964) 981. 7 0. B. CROFFORDAND A. E. RENOLD, J. Biol. Chem., 240 (1965) 3237. 8 D. M. KELLER AND W. D. LOTSPEICH,J. Bid. Chem., 234 (1959) 995. g W. D. LOTSPEICHAND A. H. WHEELER, Am. J. Physiot., 202 (1~62) 1065. IO R. E. DAILEY, P. E. WOOD, R. A. HINES AND J. H. ROE, PYOC.Sot. Exptb. Biol. Med., IIQ (1965)
306.
II P. RIESER, Lanced, 7394 (1965) 1070. 12 P. RIESER AND C. H. RIESER. Proc. Sot. Exfitl. Bid. Med., 116 (1964) 66~. 13 P. RIESER, Federation Proc., 25 (1966) 441. _ 14 J. F. Kuo, C. E. HOLMLUND, I. K. DILL AND N. BOHONOS, Arch. Biochem. Biophys., (1966) 269. 15 J, F. KUO,C. E. WOLMLUND AND I. K.DILL, Life Sci., 5 (1966)2257. 16 M. BLECHER.Biochem. Biophys. Res. Commun., 21 (1~65) 202. 17 M. RODBELL, .J. Biol. Chem., 241 (1966) 130. 18 M. BLECHER, B&hem. Biophys. lies. Commun., 23 (1~66) 68. 19 M. RODBELL, J. Biol. Chem., z3g (1964) 375. 20 0. B. CROPFORDAND A. E. RENOLD, J. Biol. Chem., 240 (1~65) 14. 21 J. P. FLATT AND E. G. BALL, J. Biol. Chem., 239 (1964) 675. * J. F. Kuo,
I. K. DILL AND C. E. HOLMLUND, unpublished observations.
B~och~m. Bioehys.
Acta, 144 (1967) 252-258
1I 7
252
ET BIOPHYSICA
ACTA
BBA 55352
INHIBITION
BY PHLORIZIN
GLUCOSE UTILIZATION
J. F.
KUO,
I.
K.
DILL
AND
OF INSULIN- AND PROTEASE-STIMULATED
IN ISOLATED ADIPOSE CELLS
C.
E.
HOLMLUND
Chemotherapy Research N.Y. (U.S.A.)
Section, Lederle Laboratories,
(Received
1967)
March
rxth,
American
Cyanamid
Company,
Pearl River,
SUMMARY I. Utilization of extracellular glucose, determined by its conversion to CO, and total lipid, has been employed to study the effect of phlorizin on isolated adipose cells incubated with and without insulin, a-chymotrypsin, trypsin and pronase (Streptomyces griseus protease, Type VI). 2. In dilute glucose medium, the effect of insulin on glucose utilization is to lower the apparent Km, and also to increase the v,,,. Phlorizin competitively inhibited glucose utilization without affecting intracellular metabolism, including the reactions involved in lipolysis and oxidation of X-labeled cellular materials. 3. Phlorizin at 0.92 mM inhibited glucose utilization by isolated adipose cells to the same extent in the presence and absence of insulin, cr-chymotrypsin or trypsin. Moreover, the degree of inhibition was essentially identical for both parameters measured (CO, production and lipid synthesis), and remained constant over a wide range of concentrations of these agents. It appears that phlorizin blocks only the process of glucose entry, and does not affect the mechanisms whereby glucose utilization is increased by insulin or proteolytic enzymes.
INTRODUCTION
Phlorizin, a polyphenolic b-n-glucoside, is known to inhibit sugar transport in several tissue preparations 1- 7. Insulin-stimulated sugar uptake is also inhibited by phlorizin6~B~8~s. Increasing attention has been drawn recently to the insulin-like activity of several proteolytic enzymes+‘6 and phospholipasesla-18. In the present report, the inhibitory effect of phlorizin on insulin- and protease-stimulated glucose utilization by isolated adipose cells have been compared in order to determine the extent to which these agents resemble each other. In particular, these experiments were conducted to explore the concept that the process for glucose uptake stimulated by various agents, possibly via different mechanisms, may be identical. Biochim. Biophys.
Acta, I++ (1967) 252-258
PHLORIZINANDGLUCOSEUTILIZATION
2.53
MATERIALSANDMETHODS The epididymal fat pads of male Sprague-Dawley rats, weighing IIO - 140 g, have been used in this study. The procedure for preparing and incubating isolated adipose cells were essentially the same as described by RODBELL~~with some modification’*. Sliced fat pads were digested for I h with collagenase in Krebs-Ringer bicarbonate medium made to 5 mM with glucose. The dispersed tissue was filtered through nylon cloth and the isolated adipose cells were washed three times with the same buffer with or without glucose (I mM). The weight of free adipocytes in the final suspension was estimated by subtracting the weight of residual tissue on nylon cloth from the pad weight before digestion. Unless otherwise stated, the incubation mixture consisted of I ml of the bicarbonate medium containing 4% bovine serum albumin (Fraction V), free adipocytes ranging from 33 to 52 mg, 0.2 & [l*C,]glucose and sufficient unlabeled glucose to make a concentration of I mM. Each treatment was incubated in duplicate to quadruplicate in each experiment, and each experiment was carried out at least two times to insure its reproducibility. Only the data from one experiment are reported. Procedures for extracting the incubated cells and determining CO, and total lipid were the same as reported elsewhere14~*9 except that CO, was absorbed with water-saturated phenylethylamine. Collagenase was obtained from Worthington, while epinephrine (DL-arterenolHCl, lot 52666), a-chymotrypsin (crystalline, lot 53251) and trypsin (crystalline, lot 44370) were purchased from Calbiochem. Insulin (24 l.U./mg, lot 74B-IOZO), ACTH (121 units/mg, lot 14B-0600), pronase (Streptomyces griseus protease, Type VI, repurified, lot 85B-2360) and bovine serum albumin (Fraction V, lot 105B-1520) were botained from Sigma. D-[l*C,]Glucose, 14.3 mC/mmole, was a product of New England Nuclear Corp. RESULTS Data pertaining to the effect of phlorizin on adipose cell metabolism, as measured by the conversion of extracellular glucose to CO, and total lipid as well as free fatty acid release are presented in Table I. Phlorizin clearly inhibited the utilization of glucose by adipocytes both in the presence and absence of insulin, norepinephrine, ACTH and caffeine.The fat mobilization process stimulated by the lipokinetic agents, however, was not significantly affected by phlorizin at 2 mM. Since glucose transport is known to be the major rate-limiting step in glucose uptake and the principal site of insulin action in epididymal adipose tissueao, it is likely that phlorizin specifically inhibited glucose entry without significantly interfering with intracellular activity. In order to further verify this point, an experiment was conducted with l*C-prelabeled adipocytes incubated in the same medium, but lacking extracellular glucose. The results were consistent with the above interpretation, since phlorizin failed to inhibit the conversion of %-labeled cellular materials into ‘*CO, in the presence and absence of insulin, ACTH and norepinephrine. It appears also that phlorizin does not significantly affect the metabolism of total lipid. In a separate experiment the radioactivity of the lipid fraction was found to be relatively unchanged after incubation for 2 h in the absence of extracellular glucose. Hormone-mediated lipolysis in the absence of glucose was also independent of phlorizin action. Biochim.
Biophys.
Acta,
144 (1967)
252-258
J. F. KUO, I. K. DILL, C. E. HOLMLUND
254 TABLE
I
EFFECTS
ACTH,
OF
PHLORIZIN
ON
NOREPINEPHRINE
THE
AND
METABOLISM
OF
ISOLATED
ADIPOSE
CELLS
INCUBATED
WITH
INSULIN,
CAFFEINE
Free adipocytes were incubated in r ml Krebs-Ringer bicarbonate medium (pH 7.4) containing 0.2 yC n-[i”C,]glucose (I pmole) at 37’ for 2 h. Each treatment was performed in quadruplicate. The values given in the tables are means (f S.E.). For further experimental details see the text. Additives
Phlorizin, _
2
CO,
mM Total lipid
(pmoles glucose/g cells per 2 h) None Insulin (1000 punits/ml) ACTH (0.5 pg/ml) Norepinephrine (0.5 @g/ml) Caffeine (2 mM)
Free fatty acid
(A pequivlg cells per 2 h) 0.03
0.0 *
_.___
+ CO,
0.29 + 0.06
0.77 j
I.7
0.96 & 0.02 0.42 f 0.01
2.35 $- 0.05 0.97 * 0.04
-3.0 f 0.1 81.0 -& 2.8
0.49 f 0.02 0.15 + 0.01
I.01 -+ 0.04 0.24 * 0.03
86.0 & 4.0 78.0 f 3.3
Totalipid
___ (pmoles glucose/g cells per 2 h)
.____ Free fatty acid (A pequiv Is cells per 2 h)
0.06 + 0.02
0.22 *
0.01
0.6 + 1.0
0.05 0.04
0.65 f
0.04
0.21
i
0.03
0.0 f 2.5 82.8 * 3.0
0.21
&
0.01
0.22
f
0.07 i
0.06 & 0.03 0.06 + 0.01
0.16 & 0.03
86.4 h 2.8 76.8 + 3.1
3.5 3.0 2.5 i
1
V
Fig. I. Lineweaver-Burk plots of effect of phlorizin on conversion of extracellular glucose to CO, (A) and total lipid (B) by isolated adipose cells incubated with or without insulin. All experimental conditions were essentially the same as Table I except that the amount of o-[W,]glucose employed was altered to maintain a constant specific activity of 0.093 ,uC/pmole. If added, the amounts of insulin and phlorizin were IOOO punits and 0.92 pmole/ml of incubation mixture. Each treatment was performed in quadruplicate and mean values are presented in the figures. Velocity (v) is expressed as pmoles glucose converted per g cells per 2 h. 0, control: l , insulin; x , phlorizin ; A, insulin + phlorizin.
Fig. I separately shows the effect of phlorizin (0.92 mM) on the conversion of extracellular glucose to CO, (A) and total lipid (B) in the presence and absence of insulin as a function of glucose concentration. The kinetic constants calculated from the data are summarized in Table II. The similarity in the kinetics and the values of the apparent Km'ssupport the contention that the measured metabolic parameters reflect the processes of glucose transport through the cell membrane. The kinetics of the conversion of glucose to CO, plus total lipid (figure not shown) were also found to be similar to that measured by the two individual parameters (Fig. I). Phlorizin competitively inhibited glucose utilization by adipocytes (Kc= 2.3. IO-*-2.6.1o-~M), presumably by binding to the same site of the transport system as glucose. It is Biochim. Biophys.
Acta. 144 (1967) 252-258
PHLORIZIN TABLE
AND GLUCOSE UTILIZATION
255
II
SUMMARY
OF KINETIC
CONSTANTS
OF GLUCOSE
UTILIZATION
BY
ISOLATED
ADIPOSE
CELLS
Constants K,i, Kmz. Km,, K,,, and vriraxr.v,,,~~~. vn,ax8. and vaiax~, represent Michaelis constants and maximum velocities of glucose conversion by adipocytes of (I) no additive (control), (2) insulin (1000 ~units/ml), (3) phlorizin (0.92 mM) and and phlorizin at the aforementioned concentrations, respectively. Kt represents constant of phlorizin competitively inhibiting glucose utilization. Conversion of D-[14C,]glucose to
Constants
2’
rgi
ma z&q
Km,
,the apparent in the presence (4) both insulin the inhibition
(Ml
(pmoles/g cells per %l%il. vmaxa (pmoles/g cells per 2 h) vmaXl (pmoles/g cells per 2 h) vmsXS
2
h)
CO, (Fig. rA)
Total lipid (Fig. IB)
1.0. 4.5 . 5.0.
10-a
1.1
10-d
4.5 . 10-a 5.0.
10-S
110-3
CO, + total lipid (figure not shown) I.1
10-S
2.3 . 10-a
2.3 . 10-d
2.6.
I.5
2.8
4.5
2.3
4.5
4.5
. 10-a
4.5 . 10-1 4.8 . 10-3 10-I
6.4
9.0
‘3.9
interesting to note that inhibition by phlorizin can be relieved by the stimulatory effect of insulin in a manner of non-competitive inhibition kinetics. The apparent K,,, I * IO+ to 4.5 - IO-~M in the presfor glucose utilization was lowered fro& I.O.IO-s-~ 2.4 A f
o Lipid, +Phlorizin 1 CO,, Control 0 COP, +Phlorizin
$yzg l
Lipid (av. inhibition: 66.0%)
1
o CO,
(av. inhibition : 67.7 %I
601‘ 501 0
I 100
I 200
I 300
I 400
I 500
.O
f % 0
Insulin (j.U/ml)
2
U-Chymotrypsin
(pg/ml)
Fig. 2. Inhibition by phlorizin of the insulin-stimulated utilization of glucose. Experimental conditions were essentially the same as Table I except that free adipocytes were incubated with or without phlorizin (0.92 mM) in the presence of varied insulin concentrations as indicated. Each treatment was performed in quadruplicate and mean values are presented in the figures. Fig. 3. Inhibition of phlorizin of a-chymotrypsin-stimulated utilization of glucose. Experimental conditions were essentially the same as Fig. 2. Each treatment was performed in duplicate and mean values are presented in the figure. Biochim. Biofihys. Acta, 144 (1967) .252-258
256
J. F. KUO, I. K. DILL, C. E. HOLMLUND
ence of insulin, while the constant increased to 5.0. IO-~M in the presence of phlorizin (with or without insulin). The vmaxwas not affected by phlorizin, but increased to 1.4 - 1.6 fold in the presence of insulin and insulin plus phlorizin, respectively. Certain proteolytic enzymes mimic insulin in their action on isolated adipose cellP+. It is therefore interesting to know whether phlorizin also inhibits the enhanced glucose utilization elicited by these enzymes. The results presented in Figs. 3A, 4A and gA clearly demonstrate that the stimulatory effects of cr-chymotrypsin,
0 Lipid,+Phlorizin l
CO.2 , Control o Lipid,+Phlorizin l
CO2
,
Control
0 CO,, + Phlorizin
*Lipid (av. inhibition: 71.3 %) oCOs Iav. inhibition: 69.0%)
c 0 P
Trypsin (pglml)
Pronase (IJglrnl)
Fig. 4. Inhibition of phlorizin of trypsin-stimulated utilization of glucose. Experimental conditions were essentially the same as Fig. a. Each treatment was performed in duplicate and mean values are presented in the figure. Fig. 5. Inhibition by phlorizin of pronase-stimulated utilization of glucose. Experimental conditions were essentially the same as Fig. a. Each treatment was performed in triplicate and mean values are presented in the figure.
trypsin and pronase on glucose utilization, like insulin, were subject to phlorizin inhibition. These observations also suggest that the insulin-like activity of these proteases may be due to the creation of favorable states for glucose uptake, or glucose transport, via a process similar to that mediated by insulin. Stimulation of glucose utilization by phospholipase C in adipocytes was also reported to be inhibited by phloretinla, the aglycone of phlorizin. It was found that the degree of inhibition by phlorizin over the entire range of insulin concentrations remained constant, and was identical for both parameters measured: 66.3% (CO,) vs. 66.0% (total lipid), see Fig. 2B. These data further substantiate the contention that phlorizin affects only the processes of glucose entry without affecting the fate of glucose metabolism or insulin action. This conclusion is B&him.
Biophys.
Acta,
144 (1967) 252-258
PHLORIZINANDGLUCOSEUTILIZATION
257
consistent with proposals made by FRERICHS ANDBALLS. Similar observations were also made with cc-chymotrypsin (Fig. 3B) and trypsin (Fig. 4B) ; the degree of inhibition of 67.7% (CO,) Y.S.69.8% (total lipid) for the former, and 69.0% (CO,) vs. 71.3% (total lipid) for the latter. However, pronase, a bacterial protease with an unusually broad substrate specificity, behaved somewhat differently (Fig. 5B). Although phlorizin inhibition of pronase-stimulated conversion of glucose to lipid remained quite constant over all the enzyme concentrations tested, this was not the case with CO, production. With increasing concentration of pronase the inhibition by phlorizin of CO, production from glucose increased. A possible explanation of these observations is given in DISCUSSION. DISCUSSION The results from the experiments with free adipocytes incubated in the presence of glucose (Table I) and with 14C-prelabeled cells incubated in glucose-free medium clearly demonstrate that the primary action of phlorizin is the inhibition of glucose entry into the cells. This conclusion is consistent with that of other workerse~‘, even though different parameters of measurement were employed for determining glucose uptake. The similarity in the kinetics of glucose conversion to CO, and total lipid (Fig. I), the comparable value of the apparent Km calculated therefrom (Table I), and the identical degree and constancy of phlorizin inhibition over a wide range of concentrations of insulin (Fig. 2B), a-chymotrypsin (Fig. 3B) and trypsin (Fig. 4B) support this concept. Moreover, based on these observations, the following conclusions may also be advanced: (I) the glucose uptake pathways stimulated by insulin as well as by cr-chymotrypsin and trypsin may be indistinguishable from the preexisting ones, and (2) E-chymotrypsin and trypsin, therefore, mimic insulin in their gross effect on glucose uptake by free adipocytes. From studies with 3-O-methylglucose and L-glucose, it has been concluded that the glucose uptake stimulated by phospholipase C (ref. 3) as well as by proteolytic enzymes (i.e. trypsin, a-chymotrypsin, pronase and Bacillus subtilis protease, Type VIII)* is a carrier-mediated, stereospecific process. It is interesting to note that the presence of both insulin and phlorizin resulted in a combined kinetics, in which the lower substrate affinity (i.e. higher Km) caused by phlorizin and the higher uUmax (bu t not the lower Km) caused by insulin were retained (Fig. I). The kinetic data seem to indicate further that phlorizin inhibits only the process of glucose uptake, and does not affect the mechanism whereby insulin enhances this process. FRERICHS AND BALLS reported a similar conclusion from their study with adipose tissue. Pronase resembles trypsin, a-chymotrypsin and insulin in all parameters tested, except with regard to the extent of inhibition by phlorizin of CO, production, which increased with increasing pronase concentration (Fig. 5B). Since pronase is capable of hydrolyzing a greater number of peptide bonds than a-chymotrypsin or trypsin, it is possible that at higher concentrations modifications of plasma membrane structure were more extensive than required solely for facilitating glucose entry. Under these specific circumstances phlorizin may be able to affect to a greater extent the * J. F. Kuo, I. K. DILL ANDC. E.
HOLMLUND.
unpublished
B&him.
observations.
Bioph.ys.
Ada,
144 (1967) 252-258
M3
J. F. KUO, I. K. DILL, c. E. HOLMLUND
intracellular activity. Since pronase, like insulin 21, has been found to stimulate YO, production from [x-l*G]glucose (and to specifically effect increased incorporation of the radioactivity into the fatty acid moiety)*, it is possible that phlorizin exerts its inhibition on %O, production by interference with the oxidation of C-I of glucose via the pentose cycle. FRERICHS AND BAI.L~ have shown that phlorizin is without effect in adipose tissue homogenates on hexokinase, glucosed-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, or on the overall conversion of glucose to triose phosphate. It seems, therefore, that lactonase may be the site of phlorizin inhibition, since it is the only enzyme participating in the steps prior to decarboxylation after glucose entry into the shunt pathway which has not been yet excluded from phlorizin inhibition by the above authors. REFERENCES I D. W. HAND, P. A. SANDFORDAND D. H. SMYTH, Nature, zag (1966) 618. 2 A. K. SEN AND W. F. WIDDAS, J. Physio?., 160 (1962) 404. 3 L. LACKO AND M. BURGER, Biochim. Biophys. Acta, 79 (1964) 563. 4 I. BIHLER, H. M. CAVERT AND R. B. FISHER, J. Physiol., 180 (1965) 168. 5 F. C. BATTAGLIA, K. L. MANCHESTERAND P. J. RANDLE, Biochim. Biophys. Acta, 43 (1960) 50. 6 H. FRERICHSAND E. G. BALL, Biochemistry, 3 (1964) 981. 7 0. B. CROFFORDAND A. E. RENOLD, J. Biol. Chem., 240 (1965) 3237. 8 D. M. KELLER AND W. D. LOTSPEICH,J. Bid. Chem., 234 (1959) 995. g W. D. LOTSPEICHAND A. H. WHEELER, Am. J. Physiot., 202 (1~62) 1065. IO R. E. DAILEY, P. E. WOOD, R. A. HINES AND J. H. ROE, PYOC.Sot. Exptb. Biol. Med., IIQ (1965)
306.
II P. RIESER, Lanced, 7394 (1965) 1070. 12 P. RIESER AND C. H. RIESER. Proc. Sot. Exfitl. Bid. Med., 116 (1964) 66~. 13 P. RIESER, Federation Proc., 25 (1966) 441. _ 14 J. F. Kuo, C. E. HOLMLUND, I. K. DILL AND N. BOHONOS, Arch. Biochem. Biophys., (1966) 269. 15 J, F. KUO,C. E. WOLMLUND AND I. K.DILL, Life Sci., 5 (1966)2257. 16 M. BLECHER.Biochem. Biophys. Res. Commun., 21 (1~65) 202. 17 M. RODBELL, .J. Biol. Chem., 241 (1966) 130. 18 M. BLECHER, B&hem. Biophys. lies. Commun., 23 (1~66) 68. 19 M. RODBELL, J. Biol. Chem., z3g (1964) 375. 20 0. B. CROPFORDAND A. E. RENOLD, J. Biol. Chem., 240 (1~65) 14. 21 J. P. FLATT AND E. G. BALL, J. Biol. Chem., 239 (1964) 675. * J. F. Kuo,
I. K. DILL AND C. E. HOLMLUND, unpublished observations.
B~och~m. Bioehys.
Acta, 144 (1967) 252-258
1I 7