THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 11, Issue of April 15, pp.

4 S 4 9 7 2 , 1987
0 1987 by The American Society of Biological Chemists, Inc Printed in U.S.A.

The Hypoglycemic Sulfonylureas Glyburide and Tolbutamide Inhibit

Fatty Acid Oxidation by Inhibiting Carnitine Palmitoyltransferase*
(Received for publication, August 11, 1986)

George A. Cook
From the Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163

The hypoglycemic sulfonylureas glyburide and tol- tissue uptakeof glucose by stimulating glycolysis in the skel-
butamide were found to be excellent inhibitors of the etal muscle (7), liver (8, 9), and heart (10, 11); and ( b ) they
rat liver, heart, and skeletal muscle carnitine palmi- decrease hepatic output of glucose by inhibiting gluconeo-
toyltransferases, but glyburide was by far the most genesis (8). Inprimarycultures of hepatocytes, glyburide
potent inhibitor. Carboxytolbutamide, a sulfonylurea increased insulin-stimulated glycogen synthesis without af-
that has no hypoglycemic effect, produced little or no fecting eitherinsulinbinding or degradation (9). Even in
inhibition of the enzyme from the three tissues exam- severely diabetic rats, direct, extra-pancreatic effects of sul-
ined. Fasting decreased the degree of inhibition of fonylureas have been found, e.g. in perfused rat hearts, the
carnitine palmitoyltransferase by the sulfonylureas, stimulation of glycolysis by tolbutamide was even greater in
and in genetically diabetic BB Wistar rats, a decrease diabetic rats compared with non-diabetic controls(10).
in sensitivity was also clearly demonstrated. Initial
rate kinetics of the inhibition of carnitine palmitoyl- Recently it was discovered that tolbutamide, in addition to
transferase indicated that glyburide inhibits noncom- having directinhibitory effects on gluconeogenesis in the
petitively with respect to palmitoyl-CoA while inhibi- isolated perfused liver, also inhibited ketogenesis (12), sug-
tion by malonyl-CoA was cooperatively competitive. gesting that sulfonylureas block the oxidation of fatty acids,
Inhibition by malonyl-CoA was noncompetitive with either directly or by inhibiting hepatic lipolysis. In light of
respect to carnitine, but inhibition by glyburide was the fact that Tanet al. (10) have concluded that tolbutamide
uncompetitive. These studies indicate that the hypo- can switch the energy metabolism of hearts of both normal
glycemic sulfonylureas inhibit carnitine palmitoyl- and diabetic rats from a preferential dependence on fattyacid
transferase by a mechanism that is much different from oxidation to an increased utilization of glucose as an energy
inhibition by malonyl-CoA, but are, nevertheless, po- source for contraction, it appears that the heart also might be
tent inhibitors of the enzyme. These results have im- susceptible to inhibition of fatty acid oxidation by sulfonylu-
portant implications for energy metabolism in the liver reas.
and heart in relation to the use of sulfonylureas and Thestudiesreported here were initiatedtoascertain
for understanding the mechanism by which the sulfo- whether the sulfonylureas glyburide and tolbutamide could
nylureas act to lower blood glucose, but there are also inhibit directly the mitochondrial carnitine palmitoyltrans-
important implications of these results on the study of ferases of rat liver or muscle. Carnitine palmitoyltransferase
the metabolic regulation of fatty acid oxidation. is the key regulatoryenzymein thefatty acidoxidation
pathway and is regulated in part by the tissue content of
malonyl-CoA, a physiological inhibitor of the enzyme (13),
and in part by a modulation of the enzyme that alters the
The hypoglycemic sulfonylureas are a group of drugs that inhibitory effects of malonyl-CoA (14). An interesting aspect
are currentlyused in the treatmentof non-insulin-dependent of the control of carnitine palmitoyltransferase in relation to
(type 11) diabetes. Because several studies (1-4) have shown regulation of blood glucose is the recently discovered change
that the sulfonylureas stimulate pancreatic insulin secretion, that is brought about in the enzyme following treatment of
it is thought that the primary mechanism by which these rats with insulin which results in a decreased capacity for
drugs act to lower blood glucose is by increasing circulating hepatic fattyacid oxidation (15). These studies indicated that
insulin levels. However, there is some question whether there insulin lowered fatty acid oxidationrates by rapidly increasing
is a sustained effect of the sulfonylureas to stimulate insulin the affinity of carnitinepalmitoyltransferase formalonyl-
secretion over a period of several weeks or months of treat- CoA. The results presented here indicate that glyburide and
ment even though they areeffective in lowering blood glucose tolbutamide are potent inhibitors of carnitine palmitoyltrans-
over long periods of time (5, 6). Also, more recent studies ferase and thatglyburide, the more potent hypoglycemic drug,
have shown that there are several extra-pancreatic effects of is also the more potent inhibitor of carnitine palmitoyltrans-
the sulfonylureas that may be involved in the production of ferase. The sulfonylureas, however, inhibit carnitine palmi-
their hypoglycemic effects. These glucose-lowering effects of toyltransferase by a mechanism quite different from inhibi-
the sulfonylureas are primarilyof two types: (a)they increase tion by malonyl-CoA.
* This work was supported by an Established Investigatorshipand EXPERIMENTALPROCEDURES
a Grant-in-Aid from the American Heart Associationwithfunds
contributed in part by the Tennessee Affiliate and the Memphis Male Wistar rats weighing 200-250 g were fed ad libitum (Purina
Chapter of the American Heart Association.Thesestudies were Lab Chow for Rats, Ralston Purina Co., Richmond, IN) or fasted for
further supported by a grant from The Upjohn Company, Inc. The 72 h. Diabetic rats were male BB Wistar spontaneously diabeticrats
costs of publication of this article were defrayed in part by the that were maintained on insulin after development of diabetes until
payment of page charges. This article must therefore be hereby 48 h before rats were used for experiments. All animals were allowed
marked “aduertisement” in accordance with 18 U.S.C. Section 1734 free access to water. Diabetic rats were checked for the presence of
solely to indicate this fact. ketone bodies inthe urine (determined with Bili-Labstix, Miles

4968
 Palrnitoyltransferase
Carnitine
Sulfonylureas
Inhibit 4969
Laboratories, Elkhart, IN), and only those animals that had urine 100
I I I I
ketone bodies 280 mg/dl were used.Stomach andintestinal contents
were checked at the time of removal of organs for experimentsto be
60
certain that diabetic and ad libitum fed rats were well fed and that
fasting rats had not inadvertentlyreceived food. c
Mitochondria fromthe liver, heart, or skeletal (quadriceps)muscle 0
wereisolatedby the method of Johnson and Lardy (16) with the .,-.
‘L2 60

modificationspreviouslypublished (14-15).Skeletal musclemito- 8

c
chondria were isolated exactly as previously reported for heart (14- I
40
15). The final mitochondrial pellet was resuspended to a concentra-
tion of 10 mg/ml in 0.25 M sucrose, 1 mM EDTA, 3 mM Tris, pH 7.2.
Protein was determined by a biuretprocedure (17).Respiratory
control ratios (la),determined with 10 mM glutamate and 0.5 mM
malate as substrates, were 5 or greater for all preparations used. The
outer carnitine palmitoyltransferase was measured as described pre-
viously (15).Each assay contained, ina total volume of 1 ml, 82 mM
sucrose, 70 mM KC1, 35 mM Hepes,’ 35 mM imidazole, 5 mM reduced I I I I I
-6 -5 -4 -3 -2
glutathione, 2 mg of bovine serum albumin, L-carnitine (0.4 pCi of L- 10 10 10 10 10
[methyl-3H]carnitine/pmol) at the concentrations indicated, 1 pg of
antimycin A, palmitoyl-CoA and inhibitors at concentrations indi- Drug Concentration
cated, 2 mM ATP; and 2 mM MgC12. ATP and MgC1,wereadded FIG. 1. Inhibition of carnitine palmitoyltransferaseby su1-
because of a previousreport that acyl-CoA concentrations were fonylureas. Mitochondria from rat liver were assayed for the outer
maintained better in their presence (19). Reactions were carried out carnitine palmitoyltransferase as described under “Experimental Pro-
at pH 7 and 30 “C for 5 min following a 5-min preincubation period cedures’’using 0.1 mM palmitoyl-CoA and 0.1 mM carnitine. The
in the presence of all components except carnitine, which was added effects of glyburide (e),tolbutamide (O),and carboxytolbutamide(H)
to initiate the reaction. are presented as means f S.E.forfour different preparations of
Solubilized carnitine palmitoyltransferase was prepared from mi- mitochondria. Specific activityof the outer carnitine palmitoyltrans-
tochondria isolated as described above. The mitochondrial pellet of ferase measured at optimum concentrations of0.5 mM carnitine and
Johnson and Lardy (16)was resuspended indistilled waterto rupture 0.1 mM palmitoyl-CoA was 8.3 f 0.5 nmol/min/mg protein.
the mitochondria and then centrifuged at 45,000 X g for 30 min to
release soluble proteins and substrates and sediment mitochondrial dria with the sulfonylureasfor 10 min resulted in loss of
membranes. This pellet was resuspended in 0.5% Triton X-100 to inhibition, indicating that these compounds are reversible
solubilize membrane proteins before centrifugingat 105,000 X g for
1 h. The supernatant solution was used for carnitine palmitoyltrans- inhibitors. These results also suggest that the sulfonylureas
ferase inhibition experiments. do not require conversion to another molecular form, such as
Potassium glyburide, sodium tolbutamide, and carboxytolbutamide a coenzyme-A derivative, for their inhibitory effects.
weregiftsfrom The Upjohn Co. Malonyl-CoA, palmitoyl-CoA, re- The datashown in Fig. 1indicate thatcarboxytolbutamide,
duced glutathione, imidazole,
Hepes,L-carnitine
hydrochloride, a metabolite of tolbutamide which has nohypoglycemic activ-
EDTA, ATP, and essentially fatty acid-free bovine serum albumin ity (8, Z l ) , also does not inhibit carnitine palmitoyltransfer-
werepurchasedfromSigma. ~-[methyl-~H]Carnitine hydrochloride
was obtained from Amersham Corp. Normal Wistar rats wereob- ase. Carboxytolbutamide is produced by the oxidation of a
tained from Harlan Industries (Indianapolis, IN). BB Wistar spon- methyl group t o a carboxyl group and therefore represents a
taneously diabetic rats were provided by Dr. Solomon S. Solomon of minimal modification of the overall structure of tolbutamide.
the University of Tennessee, Memphis, and the Veterans Adminis- However, this small change in structure is apparently able to
tration Research Service. eliminate both the hypoglycemic effects and the inhibitory
effects on carnitine palmitoyltransferase, possibly because the
RESULTS carboxyl group is near oris a part of the point of attachment
Tolbutamide and glyburide were excellent inhibitors of the of the sulfonylurea to the carnitine palmitoyltransferase.
activity of carnitine palmitoyltransferase that is expressed in A decreasein the sensitivity of hepatic carnitinepalmitoyl-
intact mitochondria (on the outside of the mitochondrial inner transferase to inhibition by malonyl-CoA occurs during fast-
membrane), butglyburide wasmuch more potentas an inhib- ing (14,22) and with the onset of diabetes (15,23).In current
itor than tolbutamide(Fig. 1).The concentration of tolbutam- experimentshepaticcarnitinepalmitoyltransferase in rats
ide that produced50% inhibition of the enzyme was 35 times fasted for 72 h was inhibited somewhat less by 200 PM
greater than the concentrationof glyburide producing equiv- glyburide and 4 mM tolbutamide than the enzyme from fed
alent inhibition (1.4 mM and 40 wM,respectively). Glyburide rats (66 f 8 and 59 -+ 5%, respectively, in fasting rats com-
is a much more potent hypoglycemic agent than tolbutamide, pared with 86 f 2 and 72 f 4% in fed rats, n = 4).Experiments
and these observations indicate that the potency of the two were conducted with the sulfonylureas to determine whether
compounds for inhibition of carnitine palmitoyltransferase diabetes also diminished their inhibitory effects. Mitochon-
occurs in a very similar ratio to the concentrations producing dria isolated from BB Wistar spontaneouslydiabetic rats were
their hypoglycemic actions; however, comparingtheexact compared with mitochondria from their nondiabetic litter-
potencies of thecompoundsfortheseactionsis difficult mates. Both glyburide and tolbutamide were less effective
becauseglyburide binds more tightly than tolbutamide to inhibitors of carnitine palmitoyltransferase in diabetic rats
albumin (20). The percentage inhibition of enzyme activity (Fig. Z), indicating that the phenomenon occurring during
was not modified by increasing the timeof preincubation (5- fasting and diabetes that causesloss of sensitivity to inhibi-
20 min) of mitochondria with tolbutamide or glyburide or by tion is not restricted to inhibition by malonyl-CoA.
addingtheinhibitorsafterpreincubation of mitochondria Because the ability of malonyl-CoA to inhibit hepatic car-
with palmitoyl-CoA at the timeof starting the reaction by the nitine palmitoyltransferase isgreatly decreased after freezing
addition of carnitine (no preincubation with inhibitor). Wash- and thawing mitochondria (24), the inhibitory effects of the
ing mitochondria by centrifuging and resuspending in mito- sulfonylureas were tested on carnitine palmitoyltransferase
chondrial isolation medium after preincubating the mitochon- of frozen-thawed mitochondria. Fig. 3 indicates that freezing
and thawing had little effect on the inhibition of carnitine
’ The abbreviation usedis:Hepes, 4-(2-hydroxyethyl)-l-pipera- palrnitoyltransferase by glyburide or tolbutamide when as-
zineethanesulfonicacid. sayed underconditions identical to those for Fig. 1. It is
4970
100
I I I I I loo -
Palmitoyltransferase
Carnitine
Sulfonylureas
Inhibit
palmitoyl-CoA and carnitine substrates. Malonyl-CoA at 20

::H68:H
PM showed a very strong cooperative effect on the hepatic
enzyme, and at the higher concentrations of palmitoyl-CoA
the competitive nature of the inhibition was observed (Fig.
4A). These effects havebeenreported previously (14). In
contrast to the inhibitory pattern of malonyl-CoA, 100 WM
glyburide exhibited nocooperativity and appeared to decrease
the V,, of the enzyme without affecting the K , for palmitoyl-
CoA, suggesting that glyburide is a noncompetitive inhibitor
20 0
40 50 100 IS0 2W 20
4 0 0 1 2 3 4 with respect to palmitoyl-CoA.While malonyl-CoA was a
much better inhibitor at lower concentrations of palmitoyl-
[Glyburide], uM [Tolbutamide], mM
CoA, glyburide was a better inhibitorat higher palmitoyl-CoA
FIG. 2. Comparison of the inhibitory effects of glyburide concentrations. When palmitoyl-CoA was kept constant and
and tolbutamide in genetically diabetic BB Wistar rats and carnitine was the variable substrate, both malonyl-CoA and
their nondiabetic littermates. Inhibition of carnitine palmitoyl-
transferase by glyburide, panel A, and tolbutamide, panel B, was glyburide decreased the VmaXof carnitine palmitoyltransfer-
compared in liver mitochondria from diabetic (0)and nondiabetic ase, but while malonyl-CoA did not affect the K , for carnitine,
(0)BB Wistar rats under the same conditions as in Fig. 1. Results glyburide lowered the carnitineK , (Fig. 4B), suggesting that
are means f S.E.for four animals in each group. Specific activity of malonyl-CoA is a noncompetitive inhibitor with respect to
the outer carnitine palmitoyltransferaseof diabetic and nondiabetic carnitine, but glyburide is an uncompetitive inhibitor with
rats, measured underthe optimum conditions indicatedin Fig. 1, was respect to carnitine. Because of the high concentration of
13.3 f 0.6 and 7.6 k 0.5 nmol/min/mg protein, respectively.
palmitoyl-CoA used in this experiment and the competitive
nature of malonyl-CoA with respect to palmitoyl-CoA, 20 PM
malonyl-CoA was a relatively poor inhibitor of the enzyme,
100 with muchgreaterinhibition being exhibited by 100 FM
glyburide. The suspectedmode of inhibition by glyburide was
confirmed by additional kinetic experiments in which double-
80
h
reciprocal plots were generated. The plot of l / u versus 1/
4
.e [palmitoyl-CoA] had lines intersecting on the x axis, indicat-
>
.r(
60 ing noncompetitive inhibition by glyburide with respect to
4 palmitoyl-CoA (Fig. 5 A ) . The plotof l / u versus l/[carnitine]
had parallel lines, indicating uncompetitive inhibitionby gly-
40 buride with respect to carnitine (Fig. 5 B ) .
Because the kinetic data on inhibition of carnitine palmi-
&? toyltransferase indicated quite different mechanismsof inhi-
20 bition by malonyl-CoA and thesulfonylureas, the conclusion
that they must bind to different sites on the enzyme seems
unavoidable. It is extremely unlikely, if not impossible, that
two such different molecules could bind to the same site and
0 2 4 6 8 produce such completely different typesof inhibition. Inorder
[Inhibitor], mM to gain more insight into this question, inhibition of carnitine
palmitoyltransferase that had been solubilized by detergent
FIG. 3. Inhibition of carnitine palmitoyltransferase (CPT) extraction from mitochondrialmembranes was tested for
in frozen and thawed rat liver mitochondria. Inhibition by
glyburide (O), tolbutamide (O), andcarboxytolbutamide (D) was inhibition by tolbutamide, carboxytolbutamide, andmalonyl-
compared in rat liver mitochondria that had been frozen at -80 “C, CoA. Carnitine palmitoyltransferase that had been solubilized
thawed,then refrozen and thawed. Assays were conductedunder with detergent has been reported to be insensitive to inhibi-
conditions identicalto those in Fig. 1.Results are presented as means
* S.E. for three preparations of mitochondria. The specific activity I I I I I I I
of these preparations measured at 2.5 mM L-carnitine and 0.15 mM 8

palmitoyl-Coh (determinedto be optimal conditions for these prep-

*
arations) was 31 3 nmol/min/mg protein.
6

interesting also that the maximum inhibition far exceeded

50%, which was the limit previously reported for inhibition 4

by malonyl-CoA in disrupted mitochondria (24). The inhibi-

tion curves for the sulfonylureas indicate thatmore than 80%
2
of the enzyme activity could be inhibited under the assay
conditions used here. Carboxytolbutamide had no inhibitory
effect on the frozen-thawed enzyme. The specific activity of - 0
0 .2 .4 6 .8 IO
the frozen-thawed preparation (Fig. 3) was four times that of
[Palmitoyl-CoA], uM [Carnitine], mM
the outer carnitine palmitoyltransferase activity(Fig. I), in-
dicating that both the inner and outer carnitine palmitoyl- FIG. 4. Comparisonof the inhibitory effects ofmalonyl-CoA
transferaseactivities were measured inthefrozen-thawed and glyburide in rat liver mitochondria. Inhibition by glyburide
preparation and that the sulfonylureas inhibit both inner and (e)and malonyl-CoA (D) was compared with activity in the absence
of inhibitors (0).Carnitine palmitoyltransferase ( C P T )was assayed
outer activities. at 0.5 mM carnitine in panel A and at 0.1 mM palmitoyl-CoA in panel
In order to compare the inhibition of carnitine palmitoyl- B. Other conditions are described under “Experimental Procedures.”
transferase by malonyl-CoA and the sulfonylureas, we tested Results arepresented as means * S.E. for three preparations of
glyburide and malonyl-CoA at several concentrations of the mitochondria.
 Palmitoyltransferase
Carnitine
Sulfonylureas
Inhibit 4971
an explanation of this method). The solubilized enzyme was
not inhibitedby carboxytolbutamide or tetradecylglycidic acid
in the absence of added coenzyme A, Mg2+, or ATP; the
addition of these substrates resulted in inhibition by tetra-
decylglycidic acid, but had no effect on the results obtained
with tolbutamide or carboxytolbutamide (results not shown).
The rat heart and skeletal muscle carnitine palmitoyltrans-
ferases are very much more sensitive than the liver enzyme
to inhibition by malonyl-CoA (14,19),but when the enzymes
from the heart and skeletal muscle were assayed under con-
ditions identical to those in Fig. 1, 100 p M glyburide inhibited
the heart enzyme by 57 f 6% ( n = 4) and the skeletalmuscle
enzyme by 60 f 7% ( n = 3), indicating that the heart, skeletal
muscle, and liver enzymes were very similar in their response
ll[Palmitoyl-CoA], uM“ l/[Carnitinel, m ~ ”
to inhibitionby glyburide. Carboxytolbutamide had very little
FIG. 5 . Inhibition of rat liver carnitine palmitoyltransfer- inhibitory effect oncarnitinepalmitoyltransferases from
ase (CPT)by glyburide. Carnitine palmitoyltransferase was as-
sayed without inhibitor (O),with 50 p M glyburide ,).( or with 100 heart and skeletalmuscle (less than 10% inhibition).
p~ glyburide (m) under the conditions described under “Experimental
Procedures,” except that in panel A carnitine was 0.2 mM and in DISCUSSION
panel B palmitoyl-CoA was 40 p ~ The . experiments illustrated here The data presented here indicate that sulfonylureas
the are
are representative of three different preparations of rat liver mito- potent, reversible inhibitors of liver, heart, and skeletal mus-
chondria.
cle carnitinepalmitoyltransferases. Glyburide, which is a
much more potent hypoglycemic drug than tolbutamide, is

loo1
80
also a muchbetter inhibitorof carnitine palmitoyltransferase,
but carboxytolbutamide, which is an inactive metabolite of
tolbutamide,hasno effect on enzymeactivity.Two other
inhibitors of carnitine palmitoyltransferase, (+)-decanoylcar-
nitine (25)and pentaenoic acid (26),are known to inhibit
hepatic gluconeogenesis by inhibiting fatty acidoxidation.
The studies of Menahan and Wieland (27)have established
that maximal rates of hepatic gluconeogenesis require the
40 oxidation of fatty acids to meet the energy demand. A recent
investigation has demonstrated that the carnitinepalmitoyl-
transferase inhibitor 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2

1
2oL
carboxylate) not only inhibits fatty acid oxidation but also
stimulates glycolysis (28).The mechanism for stimulation of
glycolysis may be by the activation of pyruvate dehydrogen-
0 ase, since recent investigations have shown that oxidation of
1 fatty acids inactivates pyruvate dehydrogenase in liver (29)
0 100 200 300 and heart (30) by modulating pyruvate dehydrogenase kinase
[Inhibitor] , uM activity. The proposed mechanism is that inhibition of car-
nitine palmitoyltransferase inhibits oxidation of fatty acids
FIG. 6. Comparison of the effects of malonyl-CoA and tol- causing the mitochondrial [NAD+]/[NADH] ratio to become
butamide on detergent-solubilized carnitine palmitoyltrans-
ferase from rat liver mitochondria. Carnitine palmitoyltransfer- more oxidized and the mitochondrial [acetyl-CoA]/[CoA] ra-
ase was assayed at 40 p~ palmitoyl-CoA and 0.5 mM carnitine to tio to be decreased. Such changesin these mitochondrial ratios
determine the effect of malonyl-CoA (m), and at 0.1 mM palmitoyl- inactivatepyruvate dehydrogenase kinaseand lead toin-
CoA and 0.1 rnM carnitine to determine the effects of tolbutamide creased conversion of pyruvate dehydrogenase to the active
(0).Data are reported as means k S.E. for three different prepara- form (29).These considerationssuggest that at leasta part of
tions of enzyme. The specific activity of these preparations, measured
at optimal concentrations of 0.15 mM palmitoyl-CoA and 2.5 mM the hypoglycemic action of the sulfonylureas could be ex-
carnitine, was 89 k 8 nmol/min/mg protein.This enzyme preparation plained by the inhibition of carnitine palmitoyltransferase
was not inhibited by tetradecylglycidic acid in the absence of CoA, which would result in ( a ) inhibition of hepatic gluconeogenesis
Mg2+, and ATP. by decreasing energy supply and ( b ) stimulation of glycolysis
by increasing the relative amount of the active form of pyru-
tion by malonyl-CoA (24).We also found no inhibitionof the vate dehydrogenase. This idea is supported by the recentwork
solubilized enzyme by malonyl-CoA (Fig. 6),but this prepa- of Tutwiler et al. (31)which indicated that tetradecylglycidic
ration of enzyme could be completely inhibited by tolbutam- acid, when administered to animalsin uiuo, not only inhibited
ide. Intheseexperiments,inhibition by malonyl-CoA was ketogenesis but also lowered blood glucose.
tested at a low palmitoyl-CoA concentration (40 PM) and The sulfonylureas areapparentlydifferent from other
inhibition by tolbutamide and carboxytolbutamidewas tested known carnitine palmitoyltransferase inhibitorslike malonyl-
at a low carnitine concentration (0.1 mM) so that conditions CoA and tetradecylglycidic acid, which inhibit in the form of
in each case (non-saturating concentrationsof the competing the coenzymeA ester (32-33),and they are also different
substrate) would be mostconducive toinhibition by that from oxfenicine, which must be converted to thefree carbox-
particularinhibitor.Maximuminhibition, which was esti- ylic acid form before inhibition will occur (34).The mecha-
mated by extrapolatingthe line obtained by plotting (% nisms by which these compoundsproduce their inhibitory
inhibition by tolbutamide)” versus [tolbutamide]” to infinite effects are all quite different. While malonyl-CoA is cooper-
inhibitor concentration was 106 f 7% ( n = 3) (see Ref. 27 for atively competitive with respect to palmitoyl-CoA and non-
4972 Palmitoyltransferase
Carnitine
Sulfonylureas
Inhibit
competitive with respect to carnitine, the sulfonylureas are BB/WorUtm by the Instituteof Laboratory Animals of the National
noncompetitive with respect to palmitoyl-CoA and uncom- Science Foundation. The designation indicates the source of the
petitive with respect to carnitine. The metabolite of oxfeni- substrain (Utm for the University of Tennessee, Memphis, and Wor
for the University of Massachusetts at Worcester) of the original
cine, hydroxyphenylglyoxylate, is competitive with carnitine colony of spontaneously diabetic rats (BB for the BioBreeding Lab-
and noncompetitive with palmitoyl-CoA (34). These obser- oratories of Ottawa, Canada). This colony was developed and main-
vations suggest that carnitine palmitoyltransferase isa com- tained by funds provided in part by the Tennessee Affiliate and the
plex enzyme that is capable of binding a number of different Memphis Chapter of the American Diabetes Association.
inhibitors at apparently different sites. Recent data (33, 35- REFERENCES
36) have suggested that malonyl-CoA binds at a site different 1. Buchanan, K.D.,Vance, J. E., Dinstl, K., and Williams, R.H. (1969)
from theactive site andpossibly on a regulatory subunit (33). Diabetes 18,ll-18
2. Blackward, W. G., and Nelson N. C. (1971)Diabetes 20,168-170
Also, the cooperativity of inhibition by malonyl-CoA seen by 3. Erwald, R., Hed, R., Nygren, A. Rojdmark, S., Sundbald, L., and Wiechel,
us (14-15) points tosome interaction between subunits of the K. L. (1971)Diabetes 20,686y690
Colwell, A. R., Jr., and Zuckerman, L. (1972)Diabetes 21,209-215
enzyme. It is thought that freezing and thawing or detergent 4. 5. h a v e n , G., and Dray, J. (1967)Diabetes 16, 487-492
treatment separates theregulatory and catalytic subunits and 6. Feldman, J. M., and Lebovitz, H. E. (1971) Diabetes 20, 745-755
7. Daniels, E. L., and Lewis, S. B. (1982)Endocrinology 110, 1640-1842
prevents inhibition by malonyl-CoA (33). The sulfonylureas 8. Matsutani, A., Kaku, K., and Kaneko, T. (1984)Diabetes 33,495-498
apparently behave in an entirely different manner from that 9. Fleig, W. E., Noether-Fleig, G., Fussgaenger, R., and Ditschuneit, H. (1984)
Diabetes 33,285-290
of malonyl-CoA since freezing and thawing had little effect 10. Tan, B. H., Wilson, G. L., and Schaffer, S. W. (1984)Diabetes 33, 1138-
11A2
on inhibition by tolbutamide or glyburide. Furthermore, a
11. Krimer, J. H., Lampson, W. G., and Schaffer, S. W. (1983)Am. J. Physiol.
preparation of carnitine palmitoyltransferase that was solu- 246, H313-H319
12. Patel, T. B. (1986)Am. J. Physiol. 260, E82-E86
bilized by detergent treatment could be completely inhibited 13. McGarry, J. D., and Foster, D. W. (1980)Annu. Reu. Biochem. 49, 395-
by tolbutamide, but malonyl-CoA did not inhibit thisenzyme 430

preparation even though assay conditionswere used for each 15. 14. Co&: G. A. (1984)J.Biol. Chem. 269,12030-12033
Gamble, M. S., and Cook, G. A. (1985)J. Biol. Chem. 260,9516-9519
inhibitorthat were mostconducive toinhibition by that 16. Johnson, D., and Lardy, H. (1967)Methods Enzymol. 10,94-97
17. Layne, E. (1957)Methods Enzymol. 3,450-451
particular inhibitor. 18. Estabrook, R. W. (1967)Methods Enzymol. 10,41-47
Results presented here indicate that there aresome differ- 19. McGarry, J. D., Mills, S. E., Long, C. S., and Foster, D. W. (1983)Biochem.
ences and some similarities between inhibition of carnitine 20. BryyE,J.214,21-28
K. F., and Crooks, M.J. (1976)Biochem. P h n r m o l . 26, 1175-
palmitoyltransferase by the hypoglycemic sulfonylureas and I1 I B
21. Thomas, R. C., and Ikeda, G. J. (1966)J. Med. Chem. 9,507-510
the other known inhibitors of this enzyme. Inhibition by the 22. Cook, G. A., Otto, D. A,, and Cornell, N. W. (1980)Biochem. J. 192,955-
sulfonylureas occurs by a much different mechanism than QKR
23. C&i: G. A,, Stephens, T. W., and Harris, R. A. (1984)Biochem. J. 219,
inhibition by malonyl-CoA, hydroxyphenylglyoxylate, or te- 337-339
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Acknowledgment-Grateful acknowledgment is due to Jeanine Tutwiler, G. F. (1984)J. Bwl. Chem. 269,9750-9755
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at the University of Tennessee, Memphis, which was the source of 37. Harano, Y., Kashiwagi, A,, Kojima, H., Suzuki, M., Hashimoto, T., and
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