208 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26002

EFFECTS OF B A C I L L U S S U B T I L I S PROTEASE, T Y P E V I I I
( S U B T I L O P E P T I D A S E A) ON ISOLATED A D I P O S E CELLS

FRUCTOSE U T I L I Z A T I O N AND ITS I N H I B I T I O N BY AVENACIOLIDE,

P I E R I C I D I N A AND PUROMYCIN

J. F. KUO
Experimental Therapeutic Research Section, Lederle Laboratories, A merican Cyanamid Co.,
Pearl River, N . Y . (U.S.A.)
(Received April iSth, 1968)

SUMMARY

I. It was found that Bacillus subtilis protease, Type V I I I (subtilopeptidase A,

EC 3.4.4.I6), like insulin, stimulated fructose utilization (oxidation and lipogenesis)
by isolated adipose cells. The optimum concentration was comparable to that required
for maximal stimulation of glucose utilization, about 5 #g/ml, and higher enzyme
concentrations resulted in a reduced stimulation. Insulin and B. subtilis protease
induced a higher Vmax for the utilization of glucose and fructose. Insulin, but not
B. subtilis protease, lowered the apparent Km values of either glucose or fructose
utilization to about one half of the basal values.
2. The basal and the elevated oxidation of fructose was not affected by pahnitic
acid, although its conversion to fatty acid was greatly reduced, and its conversion to
glyceride-glycerol was stimulated. The protease, like insulin, reduced the oxidation
of palmitic acid and enhanced its esterification in the presence of fructose.
3- From the evidence obtained through the use of avenaciolide, piericidin A
and puromycin, it is suggested that the basal and the insulin- or the protease-stimulat-
ed glucose and fructose uptake pathways may be different in isolated adipose cells,
and that glucose and fructose uptake m a y again not be effected through identical
mechanisms. Moreover, the kinetic data also suggested that B. subtilis protease
stimulated glucose and fructose utilization by a mechanism quite distinct from that
of insulin.

INTRODUCTION

FROESCH AND GINSBERG 1 reported an active fructose metabolism by adipose

tissue. FAIN2, employing dexamethasone and 2-deoxy-D-glucose as inhibitors, and
FAIN 3 and GUTMAN, AUTOR AND LYNN 4, using puromycin and actinomycin D as
inhibitors, proposed the existence of multiple processes for the basal and the insulin-
stimulated glucose and fructose entry in adipose tissue and isolated adipose cells.
Several proteolytic enzymes that mimic certain insulin effects on gross glucose and
palmitic acid utilization 5-7 and lipolysis 8 in isolated adipose cells have been reported

Biochim. Biophys. Acta, 165 (1968) 2o8-2I 7

B. sub,ills PROTEASE AND FRUCTOSE METABOLISM ~09

previously. Avenaciolide ~, an antifungal lactone, and piericidin A (ref. io), a structural

analog of coenzyme Q, have been shown to affect, to some different degrees, the basal
and the insulin- or the protease-stimulated glucose utilization. In the present report,
the influence of Bacillus sub,ills protease, Type VIII (subtilopeptidase A, EC 3.4.4.16)
and other proteolytic enzymes on fructose utilization, and their relationship with
glucose metabolism in isolated adipose cells were studied and compared with insulin.
The effects of avenaciolide and piericidin A on fructose and glucose utilization, either
in the presence and absence of insulin or proteolytic enzymes, were also studied and
compared with puromycin.

EXPERIMENTAL PROCEDURE

The epididymal fat pads used in the preparation of isolated adipose cells for
this stlldy were obtained from male Sprague-Dawley rats (11o-15o g) that had been
fed Purina laboratory chow. The procedures for preparing and incubating isolated
adipose cells for determining the incorporation of 1~C into CO~, total lipid, fatty acid,
and glyceride-glycerol were essentially the same as described by RODBELL11, with
some modificationsS, 7. l~C-Labeled glycogen was determined by the method of GUT-
~IAX, AUTOR AND LYNN~. The esterification of palmitic acid into total cell lipid was
determined by the method previously described 7. Unless otherwise stated, the in-
cubation mixture consisted of I ml Krebs-Ringer bicarbonate buffer (pH 7.4) con-
taining 4 o/~ dialyzed bovine serum albumin, free adipocytes ranging from 35 to 45 rag,
and a specified amount of radioactive substrate as indicated in the figures and Table I.
The weight of adipose cells was estimated by subtracting the weight of residual
tissue on nylon cloth after filtering the collagenase-treated pads from the initial
tissue weightS, ~2. The gas phase was O2-CO., (95:5, v/v). Each treatment was in-
cubated in triplicate in each experiment, and each experiment was carried out 2-4
times to insure its reproducibility. Only the data from one experiment are reported.
Collagenase was purchased from Worthington; ~-chymotrypsin (crystalline)
and Ii-~Clpalmitic acid (29.6 mC/n~mole)were from Calbiochem; insulin (~4 intern.
units/mg), S~reptomyces griseus protease (Type VI, repurified), B. sub,ills protease
(Type VIII, subtilopeptidase A, crystalline) were from Sigma; uniformly ~C-labeled
glucose (14. 3 mC/mmole) and fructose (0.5 mC/o.5o5 rag) were from New England
Nuclear, and bovine serum albumin (Fraction V) was from Armour.

RESULTS

The dose-response curves of insulin and B. subtilis protease on the conversion

of radioactive fructose to CO 2 (A) and fatty acid (B) by isolated adipose cells are
presented in Fig. i. Like the effects of B. sublilis protease on the utilization of glucose 7,
it showed a maximal stimulation (1. 7- and 2.i-fold for CO 2 production and fatty acid
synthesis, respectively) at a concentration of about 5/~g/ml. At higher concentration
(20/~g/ml), the stimulatory effects were reduced. Insulin at high concentration (2000
/~units/ml) showed no reduction in stimulation. The maximal stimulation of fructose
utilization by insulin and B. subtilis protease was found to be lower than that of
glucose. Lipogenesis from uniformly labeled I14C~fructose (A) and from I14C!glucose
(B), as a function of their concentrations, in the presence and absence of insulin and

B.iochim. Biophys. Acta, 165 (1968) 2o8-217

21o j.F. KUO

B. subtilis protease was compared in Fig. 2. Total lipid synthesis from fructose
proceeded at a linear rate up to the highest substrate concentration tested (5 mM),
with a small but significant stimulation by insulin and B. subtilis protease. In contrast,
as the glucose concentration increased, the rate of lipogenesis approached a plateau
value. Isolated adipose cells, up to a sugar concentration of 5 raM, metabolized
glucose faster than fructose in the absence of insulin or B. subdlis protease, but at
5 mM sugar, the basal rate of lipogenesis from fructose became comparable to that

Insulin ( p u n i t s / r n l )
~ 400 800 2000 0 400 800 I t ~ O0
~I I I I l

' ~ ' B' ' ' ' •

'
~ --~-~]
~_~..~_~ 400 ~
250

2OO Boo

~ ~5o
--
~
U o
~ 100 o Insulin
c~ • B. s u b t i l / s p r o t e a s e
.Q ~ ~00
~6~ 50
N ~
~ O

o& , I
4 6
I
B
I I
10
j}.~20 OIQ I
2
I
4
I
6 8
I I
10
ij 2~ )
B. s u b t i l i s p r o t e a se ( }~g/rnl )

Fig. I. C o m p a r i s o n s of t h e effects of insulin, B. subtilis protease on t h e utilization of u n i f o r m l y

14C-labeled fructose b y isolated adipose cells. Free a d i p o c y t e s were i n c u b a t e d for 2 h in I ml
K r e b s - R i n g e r b i c a r b o n a t e buffer (pH 7.4) c o n t a i n i n g 4 ~/o dialyzed bovine s e r u m a l b u m i n , 0. 5 ffC
(20 ffmoles) [14Cn~fructose, a n d specified a m o u n t s of insulin a n d B. s~btilis p r o t e a s e as indicated.
E a c h t r e a t m e n t was p e r f o r m e d in triplicate a n d t h e m e a n s ( + S.E., expressed as vertical bars)
are presented. T h e b a s a l r a t e s (without additives) of CO~ p r o d u c t i o n (A) a n d f a t t y acid s y n t h e s i s
(B) from 2o m M fructose were, respectively, 0.54 a n d 0.25 # m o l e / g cells per 2 h.

~L A i , l l , B ' ' ' ' '

0n,,s,m
/ .....

//

~ O ~ i t i , , 1~
0 1 2 3 4 5 0 2i 3~ 4~ .5~
Hexose~ raM)
Fig. 2. C o m p a r i s o n s of lipogenesis from u n i f o r m l y 14C-labeled fructose (A) a n d glucose (B) b y
isolated adipose cells. E x p e r i m e n t a l conditions were essentially t h e s a m e as for Fig. i e x c e p t t h a t
cells were i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of [14C6]fructose (o.2 #C/ffmole) or [14C6]glucose
(0.2 ffC/#mole), a n d a specified a m o u n t of insulin or B. subtilis protease. E a c h t r e a t m e n t was
p e r f o r m e d in triplicate a n d t h e m e a n s (~- S.E., expressed as vertical bars) are presented.

l~iochim. Biophys. Acta, 165 (1968) 2o8-217

B. subtilis PROTEASE AND FRUCTOSE METABOLISM 211

from glucose. The stimulated lipogenesis from glucose in response to insulin and B.
subtilis protease was greater than that of fructose at all sugar concentrations tested.
A kinetic analysis of the oxidation of uniformly ~C-labeled fructose (A) and
glucose (B) by isolated adipose cells, as shown in Fig. 3, indicated that the apparent
Km for fructose oxidation is 4 ° times higher than that for glucose; i.e., 2.9" IO-~ M

3.0 A 3.0 B

2.5 2.5

2.0 2.C
Y~
1.5 1.5
IX
J J
1.C 1.0 J
_ x ~ . x...~.-- x ~ ' ~ x ~
_~..__~..~...~_.--e..-
O.~ 0.5

I I I I0 I I I I
0 0.25 0.50 0.75 1.0 O~ O.5O 1.00 1.50 2.00
y[S] ( M - l x 10~) Y[53 (M-'xlO3)

F i g . 3. L i n e w e a v e r - B u r k plots of oxidation of uniformly 14C-labeled fructose (A) and glucose (B)

b y isolated adipose cells in the presence and absence of insulin and B. subtilis protease. Experi-
mental conditions were essentially the same as for F i g . I. The specific activities of [14Crlfructose
and [14C~]glucose used in this experiment were o.o25 and o.2 f f C / f f m o l e , respectively. E a c h treat-
m e n t w a s performed in triplicate and the means (S.E. less than ~ 6 % ) are presented. The oxida-
tion rate (v) and the m a x i m u m velocity (Vmax) are expressed as ffmoles sugar converted per g
cells per 2 h.

Kra (M) vraaz Additive Km (M) vmax

2.9" IO - a 1.17 O, None 7 . 1 . lO -4 0.83

1.4" lO -2 1.67 O, Insulin 3.8- IO -4 2.27
2. 7. lO -2 1.43 × , P r o t e a s e B. subtilis 6 . o - lO -4 1.67

versus 7.1" IO-~ M. Insulin lowered the apparent Km values of either sugar to about
one half of their basal values. B. subtilis protease, on the other hand, failed to induce
an increased affinity for either sugar. In a previous report, B. subtilis protease and
~-chymotrypsin, in contrast to insulin, also showed no significant effect on lowering
the apparent Km for glucose utilization in adipose cells 7. The Vmaxof the basal fructose
oxidation was calculated to be higher than that for glucose (1.17 versus 0.83 ffmoles/g
cells per 2 h), but the Vmax values of the insulin- and the protease-stimulated glucose
oxidation were shown to be greater than the values for fructose. This is due to the
fact that insulin and the protease stimulated glucose oxidation to a greater degree
than they did oxidation of fructose. Lipogenesis from either sugar was also measured
in the same experiment. Even though data are not shown, it was found that the
apparent Km values comparable to those from oxidation experiments were obtained
for the basal and the insulin- or the protease-stimulated processes, respectively. The
relative magnitude of the Vmax of lipogenesis of the basal and the stimulated processes
was also found to be similar to those obtained from the measurement of sugar oxida-

t~iochim, t~iophys. Acta, 165 (1968) 2 o 8 - 2 1 7

212 J.F. KUO

tion. It appears, therefore, that regardless of the metabolic parameter measured, the
kinetic data reflect the initial step in the sugar utilization (i.e. transport process) by
adipose cells. Moreover, transport process is usually considered as the rate-limiting
step in the sugar utilization by adipose tissue 2 and isolated adipose cells n, 1~.
The effects of several proteolytic enzymes including B. subtilis protease on
some parameters of glucose and fructose utilization by isolated adipose cells were
studied and compared with the effect of insulin (Fig. 4). A sugar concentration of
2 mM, a value approximating the apparent Km for glucose, and a higher sugar
concentration of 2o mM, a value approximating the apparent Km for fructose, were
chosen for both sugars. Stimulatory effects of insulin and B. subtilis protease were
more pronounced for glucose at the lower concentration; however, the effects were
more pronounced for fructose at the higher concentration, especially in its conversion
to C02 (A) and fatty acid (C). In Fig. 4 (D), the proteases, like insulin, were shown to
stimulate glycogen synthesis from glucose in adipose cells. Glycogenesis from fructose,
however, was not significantly enhanced by either insulin or B. sub,ills protease. The
synthesis of glyceride-glycerol (B) from fructose was not stimulated to a significant
degree.

2.0

~.5

~ I.Q
~ 0.5
,.~
~'~ ~

-~ Z4 .C ,~Control
U~
~] Insulin, 2ooo punits/mt i

~ 2.c [~B. subtilis proteese, 5pg/ml

O_
[2IS. griseus proteose, 5pg/ml
~ ~.~ ~e-Chymotrypsin , lOpg/ml

~ ~.2~
S o.s
~ 0.4
~ 0
2 mM 20mM 2raM 20raM 2ram !OmM 2ram OmM
['4C~ FPuctose [14C6]Fructose [!4C~3 G l u c o s e [~4C6] Fructose
Fig. 4. C o m p a r i s o n s of c o n v e r s i o n of u n i f o r m l y 14C-labeled fructose a n d glucose to CO,~ (A),
glyceride-glycerol (B), f a t t y acid (C) a n d glycogen (D) b y isolated adipose cells i n c u b a t e d in t h e
presence a n d absence of i n s u l i n a n d several proteolytic e n z y m e s . E x p e r i m e n t a l conditions were
essentially t h e s a m e as for Fig. I. T h e specific a c t i v i t y of I14C~lglucose a n d [14C~]fructose ~vas b o t h
0.I ffC/ffmole. E a c h t r e a t n l e n t was p e r f o r m e d in triplicate a n d t h e m e a n values ( ~ S.E., ex-
pressed as vertical bars) are presented,

Although it was observed that the basal utilization of 2 mM uniformly laC-

labeled glucose (oxidation and lipogenesis) and that the process stffnulated byinsulin
or by B. subtilis protease were not affected by an equimolar concentration of fructose,
its utilization was greatly reduced by 2o mM fructose, particularly in the presence of
insulin or B. subtilis protease (Table I). In a parallel experiment (Fig. 5), utilization
of 2o mM uniformly l~C-labeled fructose was influenced by added glucose as low as

Biochim. Biophys. Acta, 165 (1968) 2o8-2I 7

B. subtilis PROTEASE AND FRUCTOSE METABOLISM 213

TABLE I
EFFECTS OF FRUCTOSE ON" THE UTILIZATION OF UNIFORMLY IIC-LABELED GLUCOSE BY ISOLATED
ADIPOSE CELLS INCUBATED IN THE PRESENCE AND ABSENCE OF INSULIN AND B. subtilis PROTEASE
E x p e r i m e n t a l c o n d i t i o n s were t h e same as for Fig. I e x c e p t t h a t a d i p o s e cells were i n c u b a t e d w i t h
II~Cnlglucose (0.2 #C, or i # m o l e / m l ) in t h e presence of v a r y i n g c o n c e n t r a t i o n s of u n l a b e l e d
fructose as i n d i c a t e d . E a c h t r e a t m e n t was p e r f o r m e d in t r i p l i c a t e a n d t h e m e a n s 4- S.E., e xpre s s e d
as # m o l e s glucose c o n v e r t e d per g cells per 2 h, are present e d. The v a l u e s in p a r e n t h e s e s refer to °/o
r a t e of glucose u t i l i z a t i o n in t h e absence of a d d e d fructose.

A dditives Parameters Utilization of EleCt]glucose

--Fructose + Fructose, I m M + Fructose, 2o m M

None CO 2 0.43 4- o.oi 0.43 4- o.oI (IOO) o.o 7 4- o.oi (16)

T o t a l lipid 0.69 ± 0.02 0.68 4- 0.02 (99) o. I I ~_ o.oi (16)

Insulin CO2 1-97 ± o.oi 1.98 -i- 0.02 (IOI) o.14 4- o.oi (7)
2000 # u n i t s / m l T o t a l lipid 2.82 4- 0.07 2.81 4- 0.02 (IOO) o.2I 4- O.Ol (7)

B. subtilis CO 2 1.14 ± o.oi 1.13 4- o.oi (99) 0.07 4- o.oi (6)

protease, 5 # g / m l Totallipid 1.61 4- 0.07 1.62 4- o.oi (IOO) o.12 4- 0.02 (7)

3001 A 500[~S 150 - C

240~" o Contcol 400 I~0~

~ L~ eZnsu,in,2000punits/rm L \
@~ x BIsubt~h~ p r o t e o s e
~0 0~ 180~ 5 pg/ml 30 ~
o 3

& o
W5
~ o 120 ~00 6C
~

.9
~c~B 6 I00 ~2"'-'- -- ~ 3C
.~ Z
-- ~
5~ ol ~ ~ I I ' ' ~ ~ I I I ~

o ~ ~ ~ ,4 (3 ~ 9 .3 4 o 1 ~ ~ ,4
Glucose (mM)
Fig. 5. Effects of unlabeled glucose on the conversion of u n i f o r m l y 14C-labeled fructose t o CO s (A),
f a t t y acid (13), a n d g l y c e r i d e - g l y c e r o l (C) b y i s o l a t e d a d i p o s e cells in t h e presence a n d a bs e nc e of
i n s u l i n a n d B. subtilis protease. E x p e r i m e n t a l c o n d i t i o n s were s a m e as for Fig. I. The a m o u n t of
[14C~]fructose used w a s 20/~moles (0. 5/~C) per ml of i n c u b a t i o n m e d i u m . The c o n t r o l r a t e s (with-
o u t a d d e d glucose) of c o n v e r s i o n of r a d i o a c t i v e fructose to CO s, f a t t y acid, a n d g l y c e r i d e - g l y c e r o l
were, r e s p e c t i v e l y , o.78, 0.27 a n d 1.15 # m o l e s / g cells pe r 2 h. E a c h t r e a t m e n t w a s p e r f o r m e d in
t r i p l i c a t e a n d t h e m e a n s (4- S.E., e x p r e s s e d as v e r t i c a l bars) are p r e s e n t e d .

I raM. Compared to the relatively little inhibition by glucose (up to 2o o/o) /

of the
basal oxidation of fructose (A), the stimulated process was completely abolished by
the added glucose at 4 raM. This was so also for the synthesis of glyceride-glycerol
from fructose (C), which was least stimulated b y insulin or the protease in the absence
of glucose. It is most striking to note that the basal rate of f a t t y acid synthesis from
fructose (B) was augmented about 5o % b y 2 mM glucose, whereas the stimulated
f a t t y acid synthesis induced by insulin and the protease was greatly reduced in the
presence of 4 mM glucose.

l?iochim. Biophys. Acta. 165 (1968) 2o8-217

214 j . F . KUO

The effects of palmitic acid on some parameters of fructose utilization are

presented in Fig. 6. Palmitic acid at 2 mM exerted little or no effect on the oxidation
400
B

300

200

~ IOO

o
~) 0
~ D
m ~ 400
2~ [~ ~TIControl 1~
Olnsulin.~OOOuunits/ml I I
~ o 300 [ ] B. subtilis proteese, 5 pg I rnl [ ~. -X-
uE I'~S.gti . . . . peot..... 6 pglml I ~
,~,~ _ - . ,
~ ~ ~oo
g8
~~ ioo
~
3 o
S~ o
O 2 0 ~
P a l m l t l c acid (miVl)
Fig. 6. Effects of p a l m i t i c acid on t h e conversion of u n i f o r m l y l~C-labeled fructose to CO~ (A),
total lipid (B), f a t t y acid (C), a n d glyceride-glycerol (D) b y isolated adipose cells i n c u b a t e d in t h e
presence a n d absence of insulin a n d several p r o t e o l y t i c e n z y m e s . T h e a m o u n t of ~l¢C~]fructose
u s e d was 20 ffmoles (I ffC) per i m l i n c u b a t i o n m e d i u m . T h e b a s a l r a t e s (control, w i t h o u t a d d e d
p a l m i t i c acid) of conversion of fructose to CO s, t o t a l lipid, f a t t y acid, a n d glyceride-glycerol were,
respectively, 0.97, 1.66, 0.40 a n d 1.26 ffmoles/g cells per 2 h. E a c h t r e a t m e n t was p e r f o r m e d in
triplicate a n d t h e m e a n s ( ± S.E., expressed as vertical bars) are p r e s e n t e d .

Insulin ( p u n i t s / m l )
0 250 500 750 1000 0 250 500 750 lO00
I00 ' ' ' I B ! ~ I I

30C
o Insulin
8O • B. subtil/s p r o t e a s e
25C

6O 200

? 4o ~5C

I0C

g~ 2o
~ 5C

-
2~_
5~ I
4
I
6
1
8
I
I0
1~--I0
~ 2~ % ~ I
~ ~
I I
8
|
10
i' ' ~ I0

[3. subtllls p r o t e a s e {~9/rnl)

Fig. 7. C o m p a r i s o n s of t h e effects of i n s u l i n a n d B. subtilis protease on t h e utilization of [I-14C]-

p a l m i t i c acid b y isolated adipose cells in t h e presence a n d absence of u n l a b e l e d fructose. E x p e r i -
m e n t a l conditions were t h e s a m e as for Fig. I e x c e p t t h a t free a d i p o c y t e s were i n c u b a t e d w i t h
20 /lmoles u n l a b e l e d fructose, i /*mole (o.2 #C} [I-14C]palmitic acid, a n d specified amoun%s of
insulin a n d B. subtilis p r o t e a s e as indicated. E a c h t r e a t m e n t w a s p e r f o r m e d in triplicate a n d t h e
m e a n s ( ± S.E., e x p r e s s e d as vertical bars) are p r e s e n t e d . T h e b a s a l r a t e s of o x i d a t i o n (A) a n d
esterification (B) of [I-l*C]palmitic acid were, respectively, o.o 4 a n d 4.25 # m o l e s / g cells p e r 2 h.

B$ochim. Biophys. Acta, I65 (1968) 2o8--217

B. subtilis PROTEASE AND FRUCTOSE METABOLISM 215

of fructose (A) by adipose cells incubated in the presence and absence of insulin and
several proteolytic enzymes including B. subtilis protease. F a t t y acid synthesis (C)
was greatly reduced in the presence of added palmitic acid, and conversely, the
glyceride-glycerol synthesis (D) was stimulated. Palmitic acid also caused a small
but significant increase in overall synthesis of total cell lipid (B).
Fig. 7 presents the effects of fructose on the utilization of [I-14C1palmitic acid
in the presence of varying concentrations of insulin or B. subtilis protease. Oxidation
(A) of extracellular palmitic acid decreased, whereas its esterification into total lipid
(B) increased, as insulin concentration increased. The effects of B. subtilis protease
were similar until the enzyme concentration exceeded about 5/~g/ml (at which con-
centration a maximal stimulation of fructose utilization was noted; see Fig. i). In
the presence of higher enzyme concentration (where a reduction in stimulation of
fructose utilization became evident; see Fig. I), the oxidation of palmitic acid in-
creased sharply, whereas its esterification decreased accordingly. In a previous
report, a similar observation was made for the protease on the relationship of glucose
and palmitic acid metabolism 7, and it has been concluded that utilization of exogene-
ous fatty acid is secondary to that of sugar in the presence of insulin and B. subtilis
protease.
The effects of avenaciolide and piericidin A are compared with those of puro-
mycin on the basal and the insulin- or the protease-stimulated oxidation of glucose
and fructose (Fig. 8). As previously observed for glucose9, avenaciolide selectively
inhibited stimulated fructose oxidation without affecting the basal process. Piericidin
A inhibited the basal oxidation of fructose and glucose to a smaller degree than the
stimulated ones. Insulin still caused an augmentation of glucose oxidation over the
[14C6] IMexos e --I~r~4C_~ C 0 2

8
F TNone •
~ ] I n s u l i n , ~ O 0 0 ~units t ml
? ,oc ~;'~.B.subtilis protease, 5Ng ml
r/A ~
.~_ : ....

.~ 8o
-~
=o
~ 60

~: 40

_~
~o 2 o

~
8 o
Glucose Fructose Glucose Fructose Glucose Fructose
~ ~ m ,

Avenaciolide (10 #glint) Piericidin A (20pg/rnl) Puromycin(275~Jg/rnl)

Fig. 8. Effects of avenaciolide, piericidin A, a n d p u r o m y c i n on t h e o x i d a t i o n of u n i f o r m l y I~C-
labeled glucose a n d fructose to CO 2 b y isolated adipose cells i n c u b a t e d in t h e presence a n d absence
of insulin a n d B. subtilis protease. E x p e r i m e n t a l conditions were essentially t h e s a m e as for Fig. I.
T h e a m o u n t s of linCh]glucose a n d ElaC6]fructose u s e d were, respectively, 0.2 # C (I #mole) a n d
0. 5 /~C (20 /,moles) p e r ml i n c u b a t i o n m e d i u m . T h e control r a t e s (without a d d e d inhibitors) of
glucose oxidation, e x p r e s s e d as /*moles/g cells p e r 2 h, in t h e presence of no a d d i t i v e (basal),
insulin, a n d B. subtilis p r o t e a s e were o.41 , 2.15, a n d 1.57, respectively. T h e c o r r e s p o n d i n g r a t e s
for fructose were 1.o3, 2.3 ° a n d 1.77 , respectively. E a c h t r e a t m e n t w a s p e r f o r m e d in triplicate
a n d t h e m e a n s (4- S.E., e x p r e s s e d as vertical bars) are presented.

Biochira. Biophys. Acta, 165 (1968) 2o8-217

216 J.F. KUO

control in the presence of piericidin A; e.g. the rates of glucose oxidation b y adipose
cells incubated with piericidin A, expressed as/2.moles/g cells per 2 h, in the presence
of no addition (control), insulin, and the protease, were respectively, o.18, o.3o, and
o.16. Both insulin and the protease, however, failed to stimulate fructose oxidation
in the presence of piericidin A; e.g. the rates were o.45 and o.39 compared with the
control rate of o.42. Puromycin has been reported to selectively inhibit the basal
glucose and the insulin-stimulated fructose utilization ~, ~. This has been confirmed in
the present study, and it has been found that puromycin inhibited to a much greater
extent (about 8o %) the protease-stimulated glucose oxidation than the insulin-
stimulated process (about 15 %). On the other hand, puromycin inhibited either the
insulin- or the protease-stimulated fructose oxidation to a similar extent (about
45 %)- Although data are not shown, the effects of avenaciolide, piericidin A, and
puromycin on the conversion of glucose and fructose to total lipid and f a t t y acid were
qualitatively similar to that of their conversion to CO~., as shown in Fig. ~.

DISCUSSION

In contrast to insulin, which lowered the apparent Km values of glucose and

fructose oxidation by isolated adipose cells, respectively, from 7.1. IO 4 M to 3.8'
io -t M, and from 2.9" IO -~M to 1.4" IO-'° M, B. subtilis protease failed to induce
adipose cells to display a higher affinity towards these hexoses. Nevertheless, B.
subtilis protease, even though not quite as active as insulin, caused a considerably
higher Vmax for either sugar (Fig. 3). Similar observation was made on glucose and
fructose utilization in isolated adipose cells as stimulated by an antifungal antibiotic.
deoxyfrenolicin (J. F. K~'o, unpublished data). It seems that there exists a fundamen-
tally different mechanism through which a facilitated hexose uptake is induced by
insulin or by B. subtilis protease, presumably at the plasma membrane level. Limited
proteolytic modification of the lipoprotein structure of the membrane has been
considered a cause for such effects by several proteases simulating insulin effects in
adipose cells ~ :. The stimulated glucose entry, and hence its utilization, in response to
B. subtilis protease and ~-chymotrypsin, could not be differentiated from that of
insulin by studies with phlorizin t~, or with 3-O-methylglucose and L-glucose~.
The effects of piericidin A on adipose cells, as shown in Fig. 8, are of interest in
that (~) it abolished the enhancement of glucose utilization caused by B. s~,btilis
protease, but not by insulin, and (2) it inhibited the protease-stimulated glucose
utilization to a greater degree than the protease-stimulated utilization of fructose.
The first point, coupled with the above-mentioned observation (Fig. 3), suggests that
the stimulated glucose uptake induced by insulin and B. subtilis protease are distin-
guishable. The second point m a y indicate that the processes of facilitated uptake of
glucose and fructose ~nay also be functionally distinct. The latter contention is in line
with recent proposals of I?AINe,a and GUTMAN,AUTOR AND LYNN4 that there exist
multiple processes for the basal and the insulin-stimulated glucose and fructose entry
in adipose cells. The effects of puromycin (Fig. 8) in the present study not only
confirm the finding of the above authors, but also support the above-mentioned
contention based on the data of inhibition by piericidin A. Puromycin inhibited the
protease-stimulated glucose utilization without exerting a significant effect on that
process stimulated by insulin. Moreover, the protease-stimulated fructose oxidation

Biochim. Biophys. dcta, 165 (1968) 2o8-217

B. subtilis PROTEASE AND FRUCTOSE MGTABOLISM 217

was affected to a much smaller extent by puromycin than the protease-stimulated

glucose oxidation. The selective effects of avenaciolide in inhibiting the insulin- and
the protease-stimulated oxidation of glucose and fructose, but not the basal processes
(Fig. 8), further reveal the possible differences between the basal pathways for sugar
uptake and the pathways stimulated by insulin or by the protease.
It has been reported that piericidin A inhibited NADH and succinate oxidase 14.
An ultimate effect of its action on sugar metabolism, therefore, would be a reduced
ATP synthesis via oxidative phosphorylation. Whether the degree of inhibition by
piericidin A on the basal and the stimulated uptake of glucose and fructose reveals a
requirement of ATP for those specific processes is still unclear.

ACKNOWLEDGEMENTS

I wish to acknowledge Mrs. I. K. DILL for her expert technical assistance,

Piericidin A was a kind gift from Dr. K. FOLKERS of the Stanford Research Institute.
and avenaciolide was obtained originally from Dr. F. H. STODOLA of Northern
Utilization Research and Development Division, U.S. Department of Agriculture.

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Biochim. Biophys. ~lcta, 165 (1968) 2o8-217