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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
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
INTRODUCTION
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
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
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 )
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.
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)
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-
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~
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,
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.
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)
& 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 .
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
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 ,
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
ACKNOWLEDGEMENTS
REFERENCES