Leucine oxidation in diabetes and starvation: Effects of ketone bodies on branched-chain amino acid oxidation in vitro

Leucine Oxidation in Diabetes and Starvation: Effects of Ketone Bodies on Branched-Chain Amino Acid Oxidation In Vitro Harbhajan

S. Paul and Siamak

Both starvation and diabetes markedly increase the rate of a-decarboxylation of leucine by gartrocnemius muscle homogenate of rat. Since hyperketonemia is common to both these conditions, the effect of acetoacetate and P-hydroxybutyrate on the rate of a-decarboxylation of leucine by tissue homogenotes of fed rats was investigated. The rate of leucine decarboxylation by muscle homogenate increased markedly when l-20 mM acetoacetate was added to the incubation medium ( 109% increase at 20 mM). In contrast, &hydroxybutyrate was without significant effect until its concentration in the medium was increased to 30 mM (24% increase). Acetoacetate also increased a-decarboxylation of valine, but had no effect on other amino acids, namely, alanine and glutamate. There was o significant correlation between the rates of leucine decarboxylation and the endogenous concentrations of acetoacetate in the gastrocnemius muscle of fed, starved, and diabetic rats (r = 0.83,

A. Adibi

p < 0.01). The stimuiatory effect of acetoacetate on leucine decarboxylation was also observed when homogenates of other skeletal muscles, namely, soleus, vastus lateralis, and rectus abdominis were used. The rate of cr-decarboxylation of leucine by the other tissue homogenates was not altered (heart and diaphragm), was decreased (liver), or was slightly increased (kidney) by the addition of acetoacetate. The energy-yielding potential of acetoacetate did not appear responsible for enhancing the leucine decarboxylation rate by the muscle. Acetoacetate had no significant effect on the rate of ieucine transamination, nor did it stimulate the uptake of cycloleucine by muscle mitochondria. Acetoacetate, however, markedly increased (over 100%) the rate of a-decarboxylation of a-ketoisocaproate by the muscle mitochondria. These results suggest that acetoacetate may have a metabolic role in regulation of the skeletal muscle decarboxylation of branched-chain amino acids in rats.

B

RANCHED-CHAIN AMINO ACIDS (leucine, isoleucine, and valine), which comprise a substantial portion of the daily amino acid requirement of man, affect a variety of metabolic processes.’ The markedly elevated plasma concentrations of these amino acids in clinical conditions such as starvation,2-5 diabetes mellitus,6m8 and obesity 2*9 have suggested that the metabolism of branched-chain amino acids is altered by nutritional and hormonal factors. Oxidation is a major pathway for the utilization of branched-chain amino

From the Gastrointestinal and Nutrition Unit of Montejiore Hospital. University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication April 4, 1977. Supported by Grant AM-15855Jrom the National Institute of Arrhritis. Metabolism, and Digestive Diseases, and Grant S-76fiom the Health Research and Services Foundation. Presented in part at the National Meeting oJ the American Federation for Clinical Research, Atlantic City, N.J., May 2. 1976 (Clin Res 24t367a. 1976). and at the Annual Meeting of the Federation of the American Society for Experimental Biology, Anaheim, Calij. April 12. 1976 (Fed Proc 35.257, 1976). Reprint requests should be addressed io Siamak A. Adibi, M.D., Ph.D., Montejiore Hospital. 3459 Fifth Avenue, Pittsburgh, Pa. 15213, 0 1978 by Grune & Stratton. Inc. 0026-0495/78/2702-0007$02.00/0 Metabolism, Vol.27, No. 2 (February), 1978

185

PAUL AND ADIBI

186

acids by tissues. Indeed, impairment of oxidation of these amino acids by hereditary enzymatic defects leads to severe metabolic derangements.]” The initial steps in oxidation are transamination to the corresponding ketoacids and the subsequent decarboxylation of ketoacids to their acyl-coenzyme A (CoA) derivatives.” These two reactions are catalyzed by branched-chain amino acid transaminases and branched-chain ketoacid dehydrogenases, respectively.” The skeletal muscle appears as the major site for both transamination and decarboxylation of branched-chain amino acids. The data suggesting this have been recently reviewed.’ In vivo studies in rats have shown that oxidation of leucine, as measured by the production of 14C02 in the expired air after the intraperitoneal or intragastric injection of i4C-leucine, is increased in starvation,‘2,‘3 In vitro studies in rats have shown that starvation increases the transamination’” and a-deof leucine by the skeletal muscle (gastrocnemius) and leucine carboxylationis oxidation by the diaphragm.16 In the present experiments, we have investigated the effect of chemically induced diabetes on a-decarboxylation of leucine by the rat tissues in vitro. Having discovered that tissue decarboxylation of this amino acid is increased by chemically induced diabetes, it became pertinent to investigate the role of ketone bodies in the regulation of branched-chain amino acid oxidation in tissues. The latter investigation was prompted by the fact that hyperketonemia is common to conditions of both starvation and diabetes. Furthermore, no chemical agent that may be responsible for adaptive changes in branched-chain amino acid oxidation has yet been identified. It is relevant to note that Buse et a1.i7~iShave recently reported that diabetes also increases the decarboxylation of leucine by rat hemidiaphragm and sciatic nerve. MATERIALS AND METHODS Animals and Their Care Male

Sprague-Dawley

were used throughout

rats weighing

tioned quarters (temperature libitum.

During

Diabetes weight)

starvation,

was induced

injected

280-300

these experiments.

Miller

Laboratories,

Allison

The rats were housed

in individual

cages in air-condi-

approximately

g (Zivic

24°C).

The rats were fed Purina

by the administration

of a single dose of streptozotocin was freshly

(pH 4.5). The control rats were injected with the citrate of streptozotocin,

the diabetic

(3-4

for

weight and normal continued,

rats were maintained

10 days. During

growth

was resumed.

dissolved

Chow

(65 mg/kg

in 0.05

M

buffer alone. To minimize

on daily injections

this period,

the rats

citrate

ad

the

zinc insulin

initial

insulin

96 hr after the last insulin

loss in body

treatment

injection.

of the diabetic rats at the time of sacrifice was 524 + 21 mg/lOO

body buffer

the toxic efTects

of protamine

recovered

At the end of this period,

and all the rats were sacrificed

concentration

Laboratory

Pa.)

rats received only water ad libitum.

in the tail vein, Streptozotocin

lU/rat/day)

Park,

was dis-

Blood

ml (mean

glucose + SEM.

6 rats).

Tissue Preparation The rats were killed between 9:00 a.m. and ll:OO a.m. each day by stunning One or more of the following

tissues was quickly

medium (100 mM heart, diaphragm,

1 mM KCI, 5 mM MgSO,, liver, kidney, gastrocnemius

rectus abdominis

muscle. All subsequent

muscle tissue was finely minced ported previously.*’

Kidney

removed

ATP,

I

mM

and decapitation.

and placed in ice-cold EDTA.

50 mM

Chappell-Perry

Tris-HCI,

pH 7.419):

muscle, soleus muscle, vastus lateralis

steps were carried

and homogenized

out in the cold room

in Chappell-Perry

cortex and liver were also homogenized

medium

(I:9

muscle, and

(o”-4°C). w/v)

in Chappell-Perry

The as re-

medium

LEUCINE

OXIDATION

IN DIABETES

AND STARVATION

(I:9

w/v) employing a Potter-Elvehjem homogenizer. enates instead of intact tissue preparations for studies discussed.”

187

The advantages of using tissue homogof leucine oxidation have been previously

Subcellular Fractionation of Muscle as modified by Ernster and Nordenbrand,** was used for fracThe procedure of Hogeboom,*’ tionation of subcellular components of the muscle. The crude gastrocnemius muscle homogenate was centrifuged at 600 g for 10 min. The resulting pellet was resuspended in Chappell-Perry medium and centrifuged again at 600 g for IO min. The pellet obtained after the second centrifugation was designated as the 600-g pellet. The supernatants from the first and second centrifugations were combined and centrifuged at 14,000 g for IO min to obtain a mitochondrial pellet. The mitochondrial pellet was twice suspended in Chappell-Perry medium and recentrifuged. The initial supernatant obtained from 14,000g centrifugation was combined with the two supernatants from the mitochondrial wash and designated the postmitochondrial fraction.

Measurement of Amino Acid Oxidation The rate of leucine oxidation by tissues was investigated by measuring the rate of “COz production when L-l-‘4C-leucine was incubated with tissue homogenates. The selection of incubation conditions, including the use of L-l-‘4C-leucine rather than L-U-14C-leucine, was based on the results of our previously published and unpublished studies2’ which showed that CO2 released from leucine oxidation by rat tissue homogenates is limited to a-decarboxylation. Incubation studies were carried out in duplicate in 25ml Erlenmeyer flasks containing center wells fitted with tubes. The reaction mixture, in a final volume of 5.0 ml, contained the following: 619 rmoles NaCI, 10.6 rmoles MgS04. 2.7 pmoles KH2P04, 109.4rmoles Na2HP04, 8.7 rmoles NaH2P04, 1@mole L-leucine, 5 pmoles a-ketoglutarate, 10 pmoies NAD, l#Zi L-l-‘4C-leucine, and 50 mg homogenized tissue (6--S mg protein). As established in our previous studies2’ these incubation conditions were chosen to maintain a rate of leucine oxidation comparable to that of tissue slices. It is pertinent to note that enhancement of leucine oxidation by starvation, which has been previously demonstrated by in vivo studies in rats’2”3 and by in vitro studies in gastrocnemius can also be well reproduced under these incubation condimuscle slicesI and diaphragm,16 tions.” The concentration of leucine in the incubation mixture (0.2 mM), which is a physiologic concentration,23 was selected on the basis of the previous evidence that at this concentration the rate of leucine decarboxylation by the muscle is maximal.*’ The reactions were initiated by the addition of tissue homogenates. The flasks were sealed with serum caps and maintained in a metabolic shaker for 60 min. At the end of each incubation, the reaction was terminated by injecting 0.5 ml 5.0 N H2SO.t through the sealed serum cap into the incubation medium. At the same time, 0.5 ml hydroxide of Hyamine-IOX was injected into the center well tubes to trap 14C0,. After 2 hr, the flasks were opened, and the center well tubes were removed and placed in vials containing IO ml Liquifluor-toiuene scintillation mixture. The radioactivity in each vial was determined in a liquid scintillation spectrometer as previously reported.*’ To determine background oxidative activity, tissue homogenates were boiled for 1 min and then studied as above. This background activity was subtracted from each amount of 14C02 produced when leucine was incubated with nonboiled tissue homogenate. The amount (nanomoles) of leucine oxidized was calculated from the amount of radioactivity determined in each vial and corrected for the specific activity of leucine in the incubation medium. The oxidation of L-alanine, L-glutamic acid, and L-valine by the muscle, liver, or kidney homogenates was investigated using similar incubation conditions as described for leucine oxidation. except that leucine was replaced by the appropriate I-‘4C-labeled and unlabeled amino acid. The following initial concentrations of the amino acids were present in the incubation medium: L-alanine, 2.0 mM; L-glutamic acid, 1.0 mM; and L-valine, 0.2 mM. These concentrations were chosen to approximate the tissue concentrations of these amino acids2’

Leucine Transaminase Assay Leucine transaminase activity was assayed by a slight modification of the previously reported procedure.‘4 Approximately 1 g of liver, gastrocnemius muscle, or kidney cortex was homogenized

188

PAUL AND ADIBI

in 3, 6, or 9 ml, respectively, activity

was determined

contained

homogenates

25 pmoles sodium pyrophosphate

cy-ketoglutarate, acetoacetate

I5 pmoles

for kidney,

muscle,

I

L-leucine.

as indicated.

gether with unlabeled addition

of 0.1 M sodium phosphate

in whole

and liver

To separate leucine from its products

as reported

Instrument

0.50

transaminase

volume

of

phosphate,

1.0 ml

I5 rmoles

ml tissue homogenate.

by the addition

of radioactive

and

leucine

to-

respectively.

The

reaction

was terminated

by the

after transamination, (analytic

the total volume of each terminated

grade, 50 W-8.

were eluted with deionized

200 400 mesh, Hi

distilled water until

Co.,

La Grange.

counting

form) 10.0 ml

vial containing

III.) and the radioactivity

was determined

previously.‘4

Boiled tissue homogenates,

Each enzyme

of leucine

pyridoxal

One ml of each eluate was placed in a scintillation

10.0 ml Instagel (Packard

Leucine

reaction was carried out at 37°C for 3, 6. and 30 min

was placed on a I4 x 0.5 cm Dowex

eluate was collected.

7.4).

acid.

column and the products of transamination

activity.

L-l-‘4C-leucine.

homogenates,

trichloroacetic

(pH

8.6). 0.1 ymole

was initiated

leucine. The enzymatic

of 0.1 ml 55”,;, (w/v)

reaction

(pH

pCi

The reaction

buffer

of tissues. The final reaction

transaminated

eluate and corrected

studied as above,

assay

was corrected

were used for the determination

for the background

was calculated

from

the

activity.

amount

of

radioactivity

for the specific activity of leucine in the incubation

of background

The amount

(nanomoles)

determined

in the

medium.‘”

Measurement of cY-Ketoisocaproate Oxidation The rate of a-ketoisocaproate duction

when

subcellular

oxidation

I-‘4C-Lu-ketoisocaproate

fractions

was determined

was incubated

of the muscle. Incubation

by measuring

with

the rate of “CO,

appropriate

studies were carried

out in duplicate

similar to those described for leucine. The only difference was the substitution (0.20 mM)

for leucine (0.20 mM)

pro-

tissue homogenates

or

by methods

of o-ketoisocaproate

in the reaction mixture.

Succinate Dehydrogenase Assam The specific activity of succinate dehydrogenase sured according

to the method

in the subcellular

by observing

of Banner”

fractions

ferricyanide

of muscle was mea-

reduction

at 412 nm in a

Zeiss spectrophotometer.

Protein Concentration Protein concentration

in homogenates

or subcellular

Lowry et al.25 using bovine serum albumin

fractions

was measured

by the method

of

as the standard.

Determination of Ketone Bodies Acetoacetate

and D-@-hydroxybutyrate

muscle of fed, fasted, and diabetic of liver, kidney,

and muscle tissue was quickly

had been previously mortars

containing

additions

cooled

acid. After

transferred

in liquid

liquid nitrogen.

of liquid nitrogen)

perchloric

nitrogen.

reweighing,

to pH 556 with 20:~; (w/v)

in the liver, kidney,

The

frozen

the powdered

KOH,

to preweighed

for the determination were determined butyrate

beakers containing

for homogenization of potassium

Pa.) was removed

of ketone

bodies.

spectrophotometrically

oxidation

by centrifugation,

Acetoacetate by the method

and

clamps

(l/4

w/v).

cold

acid and protein

fluid was adjusted was removed (60

of NADH.26

and the supernatant D-&hydroxybutyrate

frequent

6”;,, (w/v)

Denatured

perchlorate

of Williamson

I g that

to separate

(with

with the perchloric

fluid was shaken for 30 set with Florisil

to remove flavins and to decrease the nonenzymatic

Scientific Co., Pittsburgh,

to a fine powder

tissues were mixed

and the precipitate

metal

tissues were transferred

at 10,000 g for 20 min at 0°C. The supernatant

The yellow supernatant

and gastrocnemius

with ether, and about

and pressed between

The tissues were pulverized

to chilled Dual1 glass tissue grinders

centrifugation.

removed

and were transferred

was removed by centrifugation

g/ml)

were determined

rats. Rats were lightly anesthetized

by

100 mesh. 0. I Florisil

(Fisher

fluid was used concentrations

et a1.27 using fl-hydroxy-

dehydrogenase.

Cycloleucine Uptake by Muscle Mitochondria Mitochondria without

20 mM

prepared

from

acetoacetate

the gastrocnemius

in 5.0 ml of medium

muscle were incubated

in duplicate

with the same composition

with

as described

or

above

LEUCINE

OXIDATION

IN

DIABETES

AND

STARVATION

189

for leucine oxidation. Instead of leucine, 0.20 mM cycloleucine (aminocyclo-pentane-1-carboxylic acid) and I.0 nCi I-‘4C-cycloleucine were added to the incubation mixture. Cycloleucine is a nonmetabolizable analogue of leucine and has been previously used as a model for the studies of leucine uptake by tissues.‘s After 60 min of incubation, the incubation mixture was centifuged at 14,000 g for 1 min to obtain the mitochondrial pellet. The mitochondrial pellet was homogenized in 2 ml of 37; (w/v) HCIO,. One ml of the clear supernatant fluid obtained by centrifugation was added to 10 ml Aquasol (New England Nuclear Corp., Boston, Mass.) for the determination of radioactivity. validated.29

This method

of amino

acid uptake

by the mitochondria

has been previously

Materials L-l-‘4C-leucine (50.6 mCi/mmole), mCi/mmole), and L-l-‘4C-cycloleucine

L-l-‘4C-valine (45.6 (29.97 mCi/mmole)

mCi/mmole). L-l-‘4C-alanine (58.2 were purchased from New England

Nuclear. L-l-‘4C-glutamic acid (23.0 mCi/mmole) was purchased from Amersham/Searle Corp., Arlington Heights, Ill. I-‘4C-a-ketoisocaproate (0.80 mCi/mmole) was synthesized by New England Nuclear. Radioactive purity of the synthesized material was checked by the manufacturers by scanning paper chromatography as outlined by Wohlhueter and Harper.30 Radiochemical purity of the synthesized compound was established to be 99%. Unlabeled a-ketoisocaproate sodium salt, acetoacetic acid lithium salt, DL-P-hydroxybutyric acid sodium salt, P-hydroxybutyrate dehydrogenase, NAD. NADH. and ATP were purchased from Sigma Chemical Co., St. Louis, MO. Streptozotocin was kindly supplied by Dr. W. E. Dulin of the Upjohn Co.. Kalamazoo, Mich.

Statistics Student’s or paired t tests, correlation coefficient (r), tion were used for the statistical analyses of the data.3’

and

least-squares

regression

computa-

RESULTS

Eflect of Caloric and insulin Deprivation on Leucine Oxidation and Concentration of Ketone Bodies in Tissues Starvation for 4 days significantly increased the rate of decarboxylation of leucine by the muscle homogenate, but was without effect on the rate of decarboxylation of this amino acid by the kidney and liver homogenates (Fig. 1). In contrast, in diabetic rats insulin deprivation for 4 days markedly increased the rate of decarboxylation of leucine in all three tissues. Starvation increased the concentrations of acetoacetate and D-fl-hydroxybutyrate in liver, muscle, and kidney (Table 1). Maximal levels were attained by the second or third day of starvation. The concentrations of acetoacetate and D-@-hydroxybutyrate in muscle, liver, and kidney were significantly greater (p < 0.01) after 4 days of insulin deprivation in diabetic rats than after 4 days of caloric deprivation in nondiabetic rats. Effect of Ketone Bodies on Amino Acid Oxidation As shown in Fig. 2, addition of acetoacetate (e.g., 20 mM) had a much greater effect on the rate of cY-decarboxylation of leucine by gastrocnemius muscle homogenate (1097, increase) than by homogenates of the liver (3203 decrease) and kidney (24% increase). There were almost linear increases in the rate of leucine decarboxylation by the gastrocnemius muscle when acetoacetate in the incubation medium was varied from I-20 mM. In contrast to acetoacetate, ,&hydroxybutyrate was without a significant effect on leucine decarboxylation by muscle homogenates until its concentration was raised to

190

PAUL AND ADIBI

2.6

P< 0.01

2.4 2.2 2.0 1.8 1.6 I .4 1.2 1.0 0 P(O.01

2.4 2.2

LIVER

2.0 1.8 1.6 1.4 1.2 I.0 0 26 24

r -

22

-

20

-

KIDNEY

P
Rater of a-decorboxylation of leucine by Fig. I. tissue homogenates of fed, &day starved, ond 4-day insulin-deprived (diabetic) rats. Each value represents the mean f SEM of 1O-l 2 rots. Statistical significance for the difference between fed and treated rots is shownas*,p
30 mM. The stimulation by e-hydroxybutyrate was considerably less than that by acetoacetate (109?,, versus 24”,,). Addition of acetate, as high as 10 mM, had no significant effect on the rate of leucine decarboxylation by the gastrocnemius muscle homogenate (1.68 + 0.08 versus I .74 f 0.09 nmoles/mg protein/60 min, mean + SEM in four rats). Additional studies were performed to determine whether the effect of ketone bodies on muscle oxidation is unique to the gastrocnemius muscle. Under identical conditions to those described for the gastrocnemius muscle, the addition of 20 mM acetoacetate also significantly increased the rate of leucine

LEUCINE

OXIDATION

IN

DIABETES

AND

191

STARVATION

Table 1. Concentrations

(Mean

f SEM) of Ketone Bodies in the

Tissues of Fed, Forted, ond Diabetic Rats Acetoacetate Nutritional

State

(~~moles/g

fresh

weight

D-/jl-Hydroxybutyrate of tissues)

(~moles/g

fresh

weight

of tissues)

Muscle

Fed

0.06

f 0.02

(9)*

0.05 l 0.01

(7)

0.08

f 0.03

(5)

0.34

f 0.047

(6)

Fasted 2 days

0.13

f 0.09

(4)

0.13

f 0.021

(6)

0.84 0.69

+ 0.1 lt f 0.16t

(6)

Fasted 3 days Fasted 4 days Diabetic

0.14 0.35

i i

0.06 0.08t

(7)

0.69

zt 0.17t

(7)

(6)

0.96

* 0.107

(12)

Fasted

1 day

(8)

Liver

Fed

0.10

i

0.02

(7)

0.14

+ 0.02

(10)

0.48

f 0.08t

(7)

0.90

f 0.20t

(11)

Fasted 2 days

0.61

zk 0.117

(6)

1.46 zt 0.197

(6)

Fasted 3 days

0.55

f 0.137

(8)

1.24 f 0.24t

(8)

Fasted 4 days

0.42

+ 0.107

(6)

Diabetic

1.34 * 0.257

(11)

2.10

* 0.327

(13)

(10)

Fasted

1 day

1.10 + 0.217

(8)

Kidney Fed

0.13

f 0.03

(6)

0.09

f 0.02

0.25

zt 0.08

(6)

0.42

k 0.057

(6)

Fasted 2 days

0.44

f 0.067

(6)

1.05 * 0.09t

(6)

Fasted 3 days

0.40

f 0.06t

(6)

0.90

f 0.15t

(8)

Fasted 4 days

0.42

zt 0.05t

(5)

0.92

+ 0.187

(6)

Diabetic

0.83

zt 0.13t

(12)

2.21

k 0.327

(12)

Fasted

*Number tSignikontly Ip

<

1 day

of rats used for each determination. different

from the controls,

p < 0.01.

0.05.

decarboxylation by the homogenates of vastus lateralis, soleus, and rectus abdominis, but was without effect on rates for diaphragm and heart (Fig. 3). The addition of 20 mM P-hydroxybutyrate was without significant effect on the rate of leucine decarboxylation by the homogenates of the gastrocnemius, vastus lateralis, soleus, rectus abdominis, and diaphragm, but markedly decreased that of the heart. Therefore, among the skeletal muscles, the gastrocnemius muscle did not appear to be unique in its response to acetoacetate. It should be noted that without the addition of ketone bodies, the heart exhibited a much higher rate of leucine decarboxylation than other muscle homogenates examined. The rate of leucine decarboxylation by diaphragm was also higher than that of the four skeletal muscle homogenates examined. Rates of leucine decarboxylation by the gastrocnemius and soleus muscles were comparable but were higher than that of either vastus lateralis or rectus abdominis (p < 0.01). ,&Hydroxybutyrate and, less effectively, acetoacetate both decreased the rate of leucine decarboxylation by the liver homogenate (Fig. 2). Addition of acetoacetate stimulated leucine oxidation by the kidney, while P-hydroxybutyrate had an inhibitory effect (Fig. 2). Mixtures of acetoacetate (0.62-5.0 mM) and &hydroxybutyrate (1.87- 15.0 mM), simulating physiologic proportions (1:3), increased the rate of leucine decarboxylation by the muscle homogenate and decreased that of liver and kidney homogenates (data not shown).

192

PAUL

F

2’ B ‘0 :

0.6

AND ADIBI

._

f

3.02.8

-

;

2.6

-

b E c

22

-

2.0

-

2.4 - MUSCLE

oL

’

0

’

2

’

4

’

6

a

8

’

IO

I

’

e a

12 14 16 18=4-26

concentration

of ketones

28

30

(m M)

Fig. 2. Rates of rr-decarboxylation of leucine by tissue homogenates of fed rats when various concentrations of acetoacetate or +%hydroxybutyrote were added to the reaction mixture. Each value represents the mean + SEM of 6-8 rats. The increases in the rate of ieucine decarboxylation by the muscle were statistically sign&ant when acetoacetate was increased to 2 mM or higher ( p < 0.01) and when P-hydroxybutymte was increased to 30 mM ( p < 0.05). In liver, the decreases in the rate were statistically signifKant when acetoacetate or @-hydroxybutyrate was increased to 5 mM or higher (p < 0.01). The alterations in the rate with kidney were statistically significant when acetoacetate was increased to 5 mM or higher (p < 0.01) and when @-hydmxybutymte was increased to 1 mM ( p < 0.05) or higher ( p < 0.01).

Examination of the data presented in Figs. 1 and 2 and Table 1 indicate that the increases in tissue oxidation of leucine in starved and diabetic rats could be attributed to increases in ketone body concentration only in the muscle. To determine whether there was indeed a relationship between the ketone levels in the muscle and the capacity of this tissue to oxidize leucine, the rates of leucine oxidation by the muscle of fed, starved, and diabetic rats were plotted against the total concentration of acetoacetate in this tissue (Fig. 4). A regression line

LEUCINE

OXIDATION

IN DIABETES

Gastrocnemius

193

AND STARVATION

Soleus

m

Without

m

With

acetoocetate

m

With

p- hydroxybutyrate

Vastus lateralis

ketone

Rectus abdominis

Fig. 3. Rates of a-decarboxylation of leucine by the addition of ketone bodies, with addition of acetoocetate Each value represents the mean f SEM of 6-R rats. The in rates of leucine decarboxylation in the presence or .,p < 0.01.

bodies

Diaphragm

Heart

homogenates of various muscles without (20 mM) or &hydroxybutyrate (20 mm). statistical sign&once for the difference absence of ketone bodies is shown as

expressing the best relationship between the two variables was drawn by the method of least squares. A highly significant correlation between the concentration of acetoacetate in the muscle and the capacity to oxidize leucine was obtained with a correlation coefficient of 0.83 (p < 0.01). To determine whether the effect of ketone bodies on amino acid oxidation was unique to leucine or included other amino acids, the rates of decarboxylation of alanine, glutamate, and valine were determined in the presence and absence of 2 and 10 mM acetoacetate and &hydroxybutyrate (Table 2). The selection of these amino acids was based on earlier reports which showed that rat diaphragm can oxidize them.16 Acetoacetate (10 mA4) significantly increased the rate of valine decarboxylation by the gastrocnemius muscle, but both acetoacetate and @-hydroxybutyrate were without effect on the rate of decarboxylation of alanine and glutamate by this tissue. With liver homogenates, acetoacetate significantly increased the rate of decarboxylation of alanine and glutamate, but there was a significant decrease for valine. In contrast, fi-hydroxybutyrate reduced the rate of decarboxylation of all these amino acids by the liver, alanine oxidation being most severely affected (a IO-fold decrease). Among the amino acids tested with kidney, the oxidation of alanine was most severely decreased (4-7-fold) by ketone bodies. The inhibition of alanine oxidation was apparent at both low (2 mM) and high (10 mM) concentrations of /3_hydroxybutyrate. Site of Action of Acetoacetate

on Leucine Oxidation

In Vitro

Acetoacetate-induced changes in the rate of a-decarboxylation could be the result of the effect of acetoacetate on either leucine

of leucine transaminase

PAUL

AND

ADIBI

.

Fig.

4.

Rates

boxylation

of

gastrocnemius (o),

fasted

days;

3

-40 ,50 .60 of acetoacetate

.70

,80

Table

2.

Effect

Muscle,

of Ketone Liver,

Kidney

on the

Oxidation

Homogenates

Rate

(Mean

Substrate

NOW

L-Alonine (2.0 mM)

acid

L-Voline (0.2 mM

*Significantly

tp < 0.01.

Acetoocetote,

2 mM

insulin-deprived function

a

sue.

of

The

in

the

(m) endogof

same

regression and of

least

line

drawn

tiswas

by

the

(I =

squares

p < 0.01).

of Amino

Acids

4 Fed

by the

Rats)

Acetoacetate,

10 mM

Liver

f

1.8

81.7

+ 3.3

56.3

f

1.4

51.5

f

3.9

102.2

i: 6.1*

26.0

i

4.1 t

zt 3.77

13.7 i

i

27.7

51.8

f

2.3

120.9

56.0

ztz 2.7

34.6

&Hydroxybutyrote,

10 mM

48.9

;t 2.9

NOW Acetoacetate,

2 mM

Acetoacetate,

10 mM

Kidney

56.3

2 mM

2.4t

8.0 f 0.97

0.87

_t 1.6t

8.2 f 0.51

1 19.4 * 7.9

136.2

* 8.9

293.6

f

11.3

115.9

f

7.8

180.0

zt 10.1”

265.6

i

12.0

126.5

rt 5.9

174.0

*

250.1

i

9.1*

&Hydroxybutyrate,

2 mM

117.7i

6.6

128.7

zt 8.5

240.1

*

11.61

d-Hydroxybutyrate.

10 mM

124.1

3.2

69.4

i

10.07

193.2

* 7.8t

2.4 f 0.1

0.71

l

0.04

12.5 i

1.4

2.3 f 0.2

0.61

f 0.04

12.0 *

1.5

3.1 i

Acetoocetate,

2 mM

Acetoocetate,

10 mM

i

10.1*

0.2*

0.51

* 0.03t

10.2 *

1.3

e-Hydroxybutyrate,

2 mM

2.2 * 0.1

0.56

f 0.04*

9.7 *

1.1

p-Hydroxybutyrate,

10 mM

2.4 t 0.1

0.40

* 0.027

7.4 zt 0.8*

different

from the control,

p < 0.05.

2

days),

concentrations

computed

SEM,

a,

4

as

0-Hydroxybutyrate,

NOW

)

day; C,

4-day

Muscle

Addition

(1.0 mM)

1

Rate of a-Decorboxylotion

1 J4 C-labeled

L-Glutomic

+

fed

and

0.83,

Bodies

and

the

of

rats

method

(pmol/g)

by

days;

acetoacetate

.20 .30 concentration

ru-decar-

muscle (o,

A,

enous

-10

of

leucine

LEUCINE

OXIDATION

IN DIABETES

195

AND STARVATION

or decarboxylation of a-ketoisocaproate (transamination product of leucine), or on both of these reactions. To investigate this problem, the activity of leucine transaminase (nmoles transaminated/mg protein/60 min, mean f SEM in 6 rats) in tissues was determined in the presence or absence of acetoacetate. As shown previously by several groups of investigators,’ the specific activity of leucine transaminase of the gastrocnemius muscle (301 f 19) and kidney (423 f 30) was markedly greater than that of the liver (21 + 1). Acetoacetate (40 mM) did not significantly alter the activity of leucine transaminase in either the muscle or kidney homogenates, but significantly reduced that of liver homogenate (13 + 1; p < 0.01). These studies indicate that the activating effect of acetoacetate on leucine oxidation by the muscle and kidney could not be attributed to an effect on leucine transaminase in these tissues, but the inhibitory effect of acetoacetate in the liver could be due to its effect on leucine transaminase in this tissue. To investigate this problem further, the effects of a wide range of concentrations of acetoacetate on the rate of decarboxylation of cY-ketoisocaproate by muscle, liver, and kidney homogenates were determined (Fig. 5). Among the three tissues, the kidney showed the highest and muscle the lowest rates of decarboxylation of a-ketoisocaproate. Acetoacetate increased the rates for all three tissues. There were linear increases in the rates of decarboxylation of a-ketoisocaproate by the liver and muscle over the 5-40 mM range of acetoacetate concentrations. the maximal increases being 140”,/, and lOOS/,, respec-

Fig. 5. Rates of a-decarboxylation of cu-ketoisocaproate by tissue homogenates of fed rats with various concentrations of added acetoacetate. Each value represents the mean + SEM of 6-8 rats. The rater with muscle and liver homogenates were significantly increased over the ranger of concentrations of acetoacetate shown in the figure (p < 0.01). With kidthe rate ney homogenate, became significantly increased when acetoacetate was increased to 15 mM or higher ( p < 0.05).

0

5

IO

15 20

25

30

35

concentroi~on of acetoocetote (mM)

40

45

50

196

PAUL

Table 3.

Subcellular

Distribution

of Rat Muscle a-Ketoisocaproate

a-Ketoirocaproate Subcellular

Total

Fraction

600-g

(Per Cent

pellet

Mitochondrio Postmitochondria

Dehydrogenare

Activity

Whole

Specific

Activity?

4.62

63.0 22.0

2.07

0.7

0.12

2.0

0.04

88.8

density/mg

Activity

Whole Homogenate)

39.85

decarboxylated/mg

in optical

(Per Cent

Activity

25.1

*Nanomoles tDecreose

Activity*

protein/M)

ADIBI

Dehydrogenore

63.0

Per cent recovery a-ketoisocaproate

Total

Specific

Homogenate)

Dehydrogenase Succinate

AND

0.45

87.0 protein/60

min.

min.

tively. In contrast, the effect of acetoacetate on kidney decarboxylation of cu-ketoisocaproate was modest-maximally, only a 20”; increase. These studies indicate that the in vitro effect of acetoacetate on leucine oxidation by the muscle and kidney (Fig. 2) could be attributed to its effect on a-ketoisocaproate dehydrogenase in these tissues. Although there is information on the subcellular distribution of Lu-ketoisocaproate dehydrogenase activity in the liver, 30,32its subcellular distribution in the muscle has not yet been studied. The data presented in Table 3 provide evidence that the principal cellular site for oxidation of a-ketoisocaproate is in the mitochondria. The distribution of activity in the muscle subcellular fractions parallels that of succinate dehydrogenase, which is a known mitochondrial marker enzyme. The large total activity in the subcellular fraction designated as the “600-g pellet” was presumably due to entrapped mitochondria that could not be removed by washing. A large fraction of succinate dehydrogenase activity was also recovered in the “600-g pellet” fraction. The specific activity of cu-ketoisocaproate dehydrogenase in the postmitochondrial fraction was very low and further distribution studies in the microsomal and soluble fractions were not carried out. Acetoacetate also increased the rate of decarboxylation of a-ketoisocaproate (0.20 mM) by isolated muscle mitochondria. For example, in the presence of acetoacetate (40 mM), over 1000,, increase in the rate was observed (41 =+=3 versus 85 + 5 nmoles/mg protein/60 min, mean + SEM in 5 rats, p < 0.01). Finally, to investigate whether the enhancement of leocine decarboxylation by the muscle homogenate was the result of increased leucine uptake by the mitochondria, the rates of uptake of cycloleucine were determined in the presence and absence of 20 mM acetoacetate with mitochondria isolated from the gastrocnemius muscle. The rate of cycloleucine uptake was not altered by acetoacetate (7.89 f 0.59 versus 7.71 + 0.97 nmoles cycloleucine/mg mitochondrial protein/60 min, mean f SEM in 7 rats). DISCUSSION

The results of the present studies on the oxidation of leucine by homogenates of the gastrocnemius muscle, liver, and kidney, together with results of Buse et al.“~” on the oxidation of leucine by the isolated rat diaphragm and sciatic nerve, show that diabetes enhances oxidation of this amino acid in all the tissues studied thus far. In contrast, under similar experimental conditions, starva-

LEUCINE

OXIDATION

IN DIABETES

AND STARVATION

197

tion increased leucine oxidation only in the muscle (Fig. 1). This observation is not surprising in view of the fact that insulin deficiency has a more profound catabolic effect than caloric deprivation. For the past several years, since we found that starvation increases leucine oxidation by the skeletal muscle, we have been investigating various metabolites and hormones that might affect oxidation of branched-chain amino acids in vitro. In a previous study, using a cell-free preparation of the gastrocnemius muscle, we found that glucose, palmitic acid, insulin, CAMP, glucagon, and epinephrine had no effect on a-decarboxylation of leucine.” However, studies in the same muscle preparation showed that hexanoate and octanoate stimulate and decanoate inhibits oxidation of leucine.20 Similar results with hexanoate and octanoate were also obtained by Buse et a1.33 using rat diaphragm and heart preparations. Although these observations are of biochemical interest, they may not be physiologically relevant since there is very little accumulation of these short-chain fatty acids in mammalian tissues. The results from the present experiments show that branched-chain amino acid oxidation in vitro is markedly affected by ketone bodies, especially acetoacetate. Although a physiologic role for acetoacetate as the regulator of branched-chain amino acid oxidation cannot be proposed until this effect has been established by studies in vivo, several lines of evidence suggest that the present observations may have physiologic relevance: (1) Both starvation and diabetes, which increased muscle concentration of acetoacetate (Table l), also increased muscle oxidation of leucine (Fig. I). (2) There was a significant correlation between the rates of leucine oxidation and the endogenous concentrations of acetoacetate in the gastrocnemius muscle (Fig. 4). (3) Acetoacetate concentrations as low as l-2 mM added to the skeletal muscle homogenate of fed rats increased oxidation of leucine (Fig. 2). The principal problem in implicating acetoacetate in the regulation of branched-chain amino acid oxidation in vivo is the observation that the concentration of acetoacetate that substantially stimulates leucine oxidation by the muscle (Fig. 2) is considerably greater than the concentration of acetoacetate found in the muscle of starved and diabetic rats (Table 1). However, the present method of measurement does not take into account the possibility that there could be compartmentalization of acetoacetate within the muscle cells and the concentration of acetoacetate near or within the mitochondria may be much higher than the concentration of this ketone body in other parts of the muscle cell. Currently there is no suitable method for testing this hypothesis and, therefore, the above question remains unresolved. The biochemical specificity of acetoacetate modulation of skeletal muscle oxidation of branched-chain amino acids in vitro was established by the following observations: (1) The skeletal muscle oxidation of other amino acids was not affected by ketone bodies (Table 2). (2) Acetoacetate had a much greater effect than /3-hydroxybutyrate on the oxidation of branched-chain amino acids (Fig. 2). (3) Rates of decarboxylation of leucine by heart and diaphragm were not affected by acetoacetate (Fig. 3). The present studies also define the subcellular site and the step in the oxidative catabolism of leucine, which is affected by acetoacetate in the skeletal muscle. Acetoacetate enhanced

198

PAUL AND ADIBI

the mitochondrial oxidation of a-ketoisocaproate, which is the second reaction in the oxidative catabolism of leucine.” Further elucidation of the biochemical mechanism of acetoacetate effect will have to await the isolation and purification of a-ketoisocaproate dehydrogenase from the muscle mitochondria. In fact, ketone bodies might be useful as a chemical tool in biochemical characterization of this enzyme. The effect of acetoacetate on leucine oxidation does not appear to be related to its energy yielding potential, based on the following evidence: (1) Acetoacetate stimulated the branched-chain amino acid oxidation in the skeletal muscle, while it inhibited the oxidation of these amino acids in the liver (Fig. 2). (2) /3-Hydroxybutyrate, which has the same energy-yielding potential as acetoacetate. was much weaker in the stimulation of branched-chain amino acid oxidation in the skeletal muscle, and acetate was without an effect in this regard. (3) Neither &hydroxybutyrate nor acetoacetate is metabolized by the liver and. therefore, could not serve as an energy source in this tissue. (4) Our previous studies2’ showed that the addition of glucose or palmitate, which have more energy-yielding potential than ketone bodies, does not affect the oxidation of leucine by the gastrocnemius muscle homogenate. In comparison to the skeletal muscle, the in vitro effect of ketone bodies on branched-chain amino acid oxidation by the liver appears to be more complex. Although the addition of ketone bodies to the liver homogenates of fed rats reduced the oxidation of branched-chain amino acids by this tissue, the oxidation of leucine by the liver of starved rats was not significantly altered, and such oxidation by the liver of diabetic rats was actually increased (Fig. 1). These apparently contradictory observations may be accounted for by the two opposing effects of ketone bodies on the oxidation steps of branched-chain amino acids in the liver. There may be a balance between an inhibitory effect on the transamination of leucine and a stimulatory effect on the oxidation of a-ketoisocaproate. The effect of P-hydroxybutyrate on leucine oxidation by an isolated rat diaphragm has been previously investigated in two independent laboratories. Buse et a1.33 found that P-hydroxybutyrate significantly inhibited the release of 14C02 from labeled leucine, while Odessey and Goldberg34 did not observe any significant effect. Due to fundamental differences in experimental conditions, such as cellular transport and intracellular concentrations, the comparison of results of experiments using intact muscle preparation (isolated diaphragm) with those using a cell-free preparation of skeletal muscle (homogenates) may not be appropriate. Nevertheless, the results of the present experiment with P-hydroxybutyrate are consistent with the observation of Odessey and Goldberg.34 We found that oxidation of leucine by the homogenate of diaphragm was not significantly affected by the addition of /3-hydroxybutyrate to the incubation medium (Fig. 3). In the present experiment, the most pronounced effect of P-hydroxybutyrate was the inhibition of oxidation of amino acids, especially alanine, in the liver and kidney (Table 2). This inhibition of alanine oxidation by the addition of @-hydroxybutyrate is consistent with reports that during starvation and other ketotic states pyruvate oxidation is reduced.35 37

199

LEUCINE OXIDATION IN DIABETES AND STARVATION

ACKNOWLEDGMENT We would like to thank

Jacqueline

A. Peterson

for her skillful technical

assistance.

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200

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