Factors influencing phosphoenolpyruvateformation in isolated rabbit liver mitochondria

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Authors Simpson, Donald Paul, 1943-

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FACTORS INFLUENCING PHOSPHOENOLPYRUVATE

FORMATION IN ISOLATED

RABBIT LIVER MITOCHONDRIA

by

Donald Paul Simpson

A Thesis Submitted to the Faculty o f the

COMMITTEE ON BIOCHEMISTRY (GRADUATE)

In P artia l F u lfillm e n t o f the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 9 7 4

STATEMENT BY AUTHOR

This thesis has been submitted in p a rtia l fu lf i l lm e n t o f requ ire­ments fo r an advanced degree at The U niversity o f Arizona and isdeposited in the University L ibrary to be made available to borrowers under rules o f the L ibrary.

B r ie f quotations from th is thesis are allowable w ithout special permission, provided that accurate acknowledgment o f source is made. Requests fo r permission fo r extended quotation from or reproduction o f th is manuscript in whole or in part may be granted by the head o f the major department or the Dean o f the Graduate College when in his judgmentthe proposed use o f the material is in the in terests o f scholarship. Ina ll other instances, however, permission must be obtained from the author.

SIGNED?

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

MERLE S. OLSON DateAssociate Professor o f Biochemistry

ACKNOWLEDGMENTS

The author wishes to express his appreciation to the Depart­

ment o f Biochemistry, College o f Medicine, fo r financ ia l support and

use o f laboratory fa c i l i t ie s which made his research and thesis

possible.

Thanks are given to Dr. Merle S. 01 son, Associate Professor o f

Biochemistry fo r guidance and support in th is e f fo r t .

Special appreciation is given to Ms. Candice Corley fo r her

e d ito r ia l assistance and to his w ife , Suzanne, whose patience and under­

standing were above and beyond the ca ll o f duty.

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS . . v

LIST OF TABLES . . . ......... ................ v i i

ABSTRACT ................ v i i i

1. INTRODUCTION ............................................. . . . . . . . . 1

Experimental Rationale ..................................................... . . . . 20

2. METHODS AND MATERIALS.............................. . . . 23

Mitochondrial Iso la tion Procedure ............................................. 23Miscellaneous Procedures- . .................................................. 24

3. RESULTS ....................................... 27

4. DISCUSSION ............................ 53

LIST OF REFERENCES..................................................................................... 64

tv

LIST OF ILLUSTRATIONS

Figure Page

1. The G lyco ly tic and Gluconeogenic P a thw ays................................ 2

2. The Two Proposed Substrate-Shuttle Mechanisms fo r MammalianL iver Mitochondria I l lu s tra t in g the D iffe ren t Compartmen- ta tio n fo r Phosphoenolpyruvate Synthesis .................... . . 7

3. The E ffect o f Uncoupler (FCCP) T itra tio n on the Rate o fOxygen Consumption (Panel A) and on the Oxidation- Reduction State o f the Intram itochondrial Pyridine Nucleotides (Panel B) .................................................................. 28

4. The E ffect o f Uncoupler (FCCP) T itra tio n on ATP Synthesis . 29

5. The E ffect o f Uncoupler (FCCP) T itra tio n on Phosphoenol-pyruvate Synthesis ...................................................................... 30

6. The E ffect o f Oligomycin in the Presence, and Absence, o fUncoupler (FCCP) on ATP S yn th e s is . 35

7. The E ffect o f Oligomycin in the Presence, and Absence o fUncoupler on Phosphoenol pyruvate S y n th e s is , 36

8. The E ffect o f Oligomycin on Phosphoenolpyruvate Synthesis . 38

9. Oxygen Consumption o f ADP Stimulated Respiration, in theAbsence and Presence o f Oligomycin ....................................... 39

10. The E ffect o f Arsenite on Phosphoenolpyruvate Synthesis . . 41

11. The E ffect o f Uncoupler (FCCP), Oligomycin and Calcium onthe Rate o f Phosphoenolpyruvate Formation in 2-Methyl-1 ,4-Naphthoquinone Plus Rotenone-Treated Rabbit L iver Mitochondria .......................................................... 44

12. The E ffect o f 8-Hydroxybutyrate on Phosphoenol pyruvateSynthesis from a-Ketoglutarate in the Presence o f Uncoupler (FCCP) . 45

13. The In h ib itio n o f Phosphoenolpyruvate Formation by theOxidation o f Octanoate, Acetyl ca rn itine and Pa lm ity l-ca rn itine in the Presence o f a-Ketogl utarate andUncoupler (FCCP) . 48

v

viLIST OF ILLUSTRATIONS—Continued

Figure Page

14. The E ffect o f the Oxidation o f Octanoate, Acetyl ca rn itineand Palm itylCarnitine on C itra te Formation in Uncoupler (FCCP) ...................................... 50

15. The Rates o f Phosphoenolpyruvate Formation in Uncoupler(FCCP) Mitochondria in the Presence and Absence o f Glutamate and Cystiene S u lf in ic Acid (CSA) . ................ 52

LIST OF TABLES

Table Page

1. Enzymes o f the G lyco ly tic and Gluconeogenic Pathways . . . . 4

2. Metabolic Effectors o f the G lyco ly tic and GluconeogenicPathways.............................................. 12

v i i

ABSTRACT

In th is study the regulation o f phosphoenolpyruvate formation in

iso lated rabb it l iv e r mitochondria was investigated. I t was shown that

an oxidation o f mitochondrial pyrid ine nucleotides noted in iso la ted rab­

b i t l iv e r mitochondria was essential to fa c i l i ta te maximum rates o f

phosphoenolpyruvate synthesis. The production o f phosphoenolpyruvate was

stimulated by the uncoupler p-trifluoromethoxyphenyl hydrazone carbonyl

cyanide (FCCP) from a-ketoglutarate and malate even when the respira tory

chain-linked oxidative phosphorylation was blocked w ith oligomycin but

was in h ib ited in the presence o f arsenite . Transphosphorylation via

nucleoside diphosphokinase was capable o f supplying the energy demands o f

phosphoenolpyruvate synthesis when adequate levels o f adenosine triphos­

phate prevailed.

Phosphoenolpyruvate production was shown to be in h ib ite d by the

oxidation o f p a lm ity lca rn itin e , ace tlyca rn itine and octanoate using

a-ketoglutarate as a source o f energy and 4-carbon units fo r phosphoenol-

pyruvate synthesis. This in h ib it io n was a ttrib u te d to competition fo r

oxalacetate by c itra te synthase resu lting from elevated levels o f

acetylCoA produced in 3-oxidation.

The s ign ificance o f competition by c itra te synthase and glutamic-

oxalacetic transaminase fo r available intram itochondrial bxalacetate was

evaluated. Competition fo r oxalacetate by these enzymes was correlated

w ith reduced rates o f phosphoenolpyruvate synthesis.

v i i i

CHAPTER 1

INTRODUCTION

The study o f carbohydrate synthesis in l iv e r preparations cur­

ren tly covers a period o f th ir ty or more years. Many investigators have

studied the nature and role o f the enzymes responsible fo r gluconeo-

genesis. Less than a decade ago, the discovery o f the enzyme, pyruvate

carboxylase (U tte r and Keech, 1963), seemed to complete the series o f

reactions tha t could account fo r glucose formation from noncarbohydrate

sources in l iv e r at the enzyme le ve l. Since that time, detailed reviews

have been published on th is subject (Krebs, 1963; Newsholme and Gevers,

1967; Scrutton and U tte r, 1968; Marco and Sols, 1970; Exton, 1972).

In mammals, the purpose o f gluconeogenesis is to provide glucose

fo r the body during periods o f starvation or under conditions where a

carbohydrate deficiency ex is ts . Gluconeogenesis serves as a pathway fo r

the re u til iz a tio n o f la c ta te , glycerol and certa in amino acids. In the

kidney, th is pathway is responsible fo r counteracting acidosis resu lting

from prolonged sta rva tion .

For v isu a liz in g , and at the same time lim it in g , the scope o f th is

discussion, a s im p lifie d scheme o f g lycolysis and gluconeogenesis is

il lu s tra te d in Figure 1. The metabolic pathway which converts glucose to

pyruvate is termed glycolysis and is the primary pathway o f carbohydrate

catabolism in most c e lls . Figure 1 il lu s tra te s the sequence o f reactions

by which la c ta te , pyruvate, and glycerol are converted to glucose.

NADNADHATP

ATP. Glycogen Synthesis

Glucose Glucose- 6 - Phosphate

Pentose Phosphate Pathway

Fructose - 6 - Phosphate

ATP

Fructose-1,6- Diphosphate

Dihydroxyacetone Phosphate 3 - Phosphoglycer aldehyde

ATPNADNADH

oi-G lycerol Phosphate 3-Phosphoglycerote

ATP

Glycerol 2-Phosphoglycerate

Phosphoenolpyruvate

GTPy C 0 2

ADP

NAD + Loctote NADH -P- Pyruvate

LEGEND

oxidized pyridine nucleotide ADP reduced pyridine nucleotide P-jadenosine triphosphate GTP

adenosine diphosphate inorganic phosphate guanosine triphosphate

Figure 1. The G lyco ly tic and Gluconeogenic Pathways. - - Id e n tif ic a ti o f enzymes catalyzing the steps are lis te d in Table 1.

Table 1 l is ts a ll the established enzymes o f g lycolysis and gluconeo-

genesis including the reactants and products involved in each step. Each

reaction is designated to e ith e r be active in glucose formation, in i ts

degradation or both. A comparison o f Table 1 and Figure 1 may be helpful

to c la r ify the proposed metabolic steps in gluconeogenesis.

The l iv e r , kidney and ce lls o f the small in te s tin e are the major

s ites o f glyconeogenes is in mammals. These tissues are capable o f cata­

lyzing both g lycolysis and gluconeogenesis. The b ra in , heart and

skeleta l muscle contain some o f the enzymes o f gluconeogenesis but the

enzymatic potentia l o f glucose formation is lim ite d , hence these are

considered nongluconeogenic tissues (Scrutton and U tte r, 1968; Newshoi me

and Severs, 1967; Exton, 1972).

In the g ly c o ly tic scheme, there are three steps which are thermo­

dynamically unfavorable and as a re su lt are not reversib le under physio­

log ica l conditions. The operation o f sp e c ific gluconeogenic enzymes

which are thermodynamically favorable is necessitated fo r glucose syn­

thesis to occur. These steps involve the conversion o f glucose-6-

phosphate (G6P) to glucose, fructose-1,6-diphosphate (FDP) to fructose-6-

phosphate (F6P) and pyruvate to phosphoenolpyruvate (PEP). The enzymes

involved in these conversions are g lucose-6-phosphatase (G6Pase), which

reverses g lucose-6-phosphate formation by a hyd ro ly tic reaction and

fructose diphosphatase (FDPase), which reverses the fructose-1,6-

diphosphate (FDP) formation again by hydro lysis. F in a lly , the reversal

o f pyruvate production from phosphoenolpyruvate in the pyruvate kinase

reaction is accomplished through a cycle consisting o f the conversion o f

4

Table 1. Enzymes o f the G lyco ly tic and Gluconeogenic Pathways. — Reac­tants and products o f each enzyme reaction are lis te d to the r ig h t o f the enzyme catalyzing tha t reaction. These enzymes are unique to g lyco lys is , unique to gluconeogenesis, or common to both pathways and are designated as g ly c o ly t ic , glucoeno- genic or both.

Legend

ATP adenosine triphosphate OAA oxalacetateADP adenosine diphosphate GTP guanosine triphosphateG6P g lucose-6-phosphate ITP inosine triphosphateF6P fructose-6-phosphate GDP guanosine diphosphateFDP fructose diphosphate IDP inosine diphosphateDHAP dihydroxy-acetone phosphate NAD+ oxidized pyridineGASP glyceraldehyde-3-phosphate nucleotide1,3-DPGA 1,3-diphosphoglyceric acid NADH reduced pyridine2PGA 2-phosphoglycerate nucleotide3PGA 3-phosphoglycerate Pi inorganic phosphatePEP phosphoenolpyruvatePyr pyruvate

Enzyme Reactants Products Pathway

Hexokinase Glucose, ATP

G6P, ADP Both

Phosphoglucoisomerase G6P F6P Both

Phosphofructoki nase F6P, ATP FDP, ADP G lyco ly tic

Fructose diphosphatase FDP F6P, P. Gluconeogenic

A1 dolase FDP DHAP,GASP

Both

Triose phosphate isomerase DHAP GASP Both

Glyceraldehyde-3-phosphatedehydrogenase

GASP, NAD+, Pi

1 ,3-DPGA, NADH

Both

Phosphoglycerate kinase 1,3-DPGA, ADP

3PGA, ATP Both

Phosphoglyceromutase 3PGA 2 PGA Both

Table 1--Continued

Enzyme Reactants Products Pathway

Enolase 2PGA PEP Both

Pyruvate kinase Pyr, ATP PEP, ADP G lyco ly tic

Phosphoenolpyruvate carboxykinase

OAA,GTP(ITP)

PEP, C0? GDP(IDPJ

Gluconeogenic

Pyruvate carboxylase Pyr, C09 ATP ^

OAA, ADP, pi

G1uconeogenic

pyruvate to phosphoenolpyruvate which proceeds via oxalacetate (OAA) as

an intermediate.

The enzyme involved in the in i t ia l step in the synthesis o f car­

bohydrate in l iv e r from precursors at the level o f pyruvate is pyruvate

carboxylase. This regulatory enzyme is active only in the presence o f

its a c tiva to r, acetylCoA, and catalyzes the carboxylation o f pyruvate to

oxalacetate (U tte r, 1970). This enzyme, together with phosphoenol-

pyruvate-carboxykinase constitutes a two-reaction sequence which, can

synthesize phosphoenolpyruvate from pyruvate (U tte r, 1970). In a recent

review (Scrutton and U tte r, 1968), i t was discussed tha t the formation o f

phosphoenolpyruvate from pyruvate in chicken, sheep and ra bb it live rs

occurred in the mitochondrial compartment. The phosphoenolpyruvate

formed was then released to the cytosol. However, i t was also shown tha t

mitochondria from species such as the ra t or mouse were unable to syn­

thesize phosphoenolpyruvate. These differences were explained by the

d iffe re n t in tra c e llu la r locations fo r the enzymes involved in phos­

phoenol pyruvate formation from pyruvate. The enzyme phosphoenolpyruvate-

carboxykinase (PEPCK), which catalyzes the conversion o f oxalacetate to

phosphoenol pyruvate in the mitochondria o f chicken (Mendicino and

U tte r, 1962) and rabb it l iv e r (Nordlie and Lardy, 1963), is found only in

the cytoplasm o f ra t or mouse (Shrago and Lardy, 1966) and in both the

mitochondria and cytoplasm of guinea pig (Garber and B a lla rd , 1969;

Brech, Shrago and Wilken, 1970). I t should be apparent tha t the m ito­

chondrial pathway o f phosphoenolpyruvate formation in the rabb it is not

operative in such species as the ra t or mouse. Figure 2 il lu s tra te s the

7

C Y TO P LA S M MITOCHONDRION

PYR

PYR

OAA 4- C4-PrecursorsM A LM A U

OAAPEP

PEPGLUCONEOGENESIS +

B CYTO PLASM MITOCHONDRION

PEP 4 ----------

8GLUCONEOGENESIS

C4- Precursors

LEGEND

PYRMALOAA

pyruvate malate oxalacetate

PEPASP(^-Precursors

phosphoenolpyruvate aspartatefour carbon precursors o f

oxalacetate

Figure 2. The Two Proposed Substrate-Shuttle Mechanisms fo r MammalianLiver Mitochondria I l lu s tra t in g the D iffe ren t Compartmentation fo r Phosphoenolpyruvate Synthesis.

two proposed pathways fo r phosphoenolpyruvate synthesis. In the top

scheme, phosphoenolpyruvate is formed in the mitochondrion and is trans­

ported to the cytoplasm where i t continues to glucose-6-phosphate. In

th is scheme, oxalacetate (OAA) may also be reduced to malate (Mai) via

malate dehydrogenase which la te r d iffuses to the cytoplasm. In the

scheme d ire c tly below th is , the oxalacetate formed w ith in the m ito­

chondria by pyruvate carboxylase may e ith e r be converted to malate

through reduction or to aspartate by transamination w ith glutamate or

both. The malate formed may then fre e ly d iffuse from the mitochonrida to

the cytosol where i t is reoxidized to form the extramitochondrial

oxalacetate. Aspartate may also fre e ly d iffuse out o f the mitochondria

in to the cytosol where i t w i l l undergo transamination w ith a-ketoglutarate

generating oxalacetate fo r the cytoplasmic formation o f phosphoenol-

pyruvate. The presence o f malate dehydrogenase glutam ic-oxalacetatic

transaminase (GOT) in both the mitochondria and cytosol is consistent

w ith the demands proposed by th is scheme (Marco and Sols, 1970). The

evidence derived from measuring the rates o f conversion o f iso to p ica lly

labeled malate and aspartate to phosphoenolpyruvate suggests th is pathway

is operative at least in ra t l iv e r (Scrutton and U tte r, 1968; Marco and

Sols, 1970). In e ith e r scheme, the malate dehydrogenase reaction favors

the formation o f malate; therefore, malate would be expected to be the

main supply o f the four-carbon precursor fo r phosphoenolpyruvate, more so

than asparate. The advantage o f th is formulation has been pointed out by

Lardy, Veneziale and G ab rie lii (1970). Since malate is a precursor o f

phosphoenolpyruvate, i t may supply both the four-carbon acids and the

reducing equivalents required fo r the reduction o f 1 ,3-disphosphoglycerate

in the cytosol, whereas aspartate, through transamination, would supply

only the four-carbon acid. The phosphoenolpyruvate generated by e ith e r

o f these two schemes is then converted to fructose diphosphate by the

d ire c t reversal o f the cy toso lic enzymes involved in the g ly co ly tic path­

way. Glycerol enters the glyconeogenic pathway at the level o f the

triose phosphates as shown in Figure 1.

The irre ve rs ib le enzymatic hydrolysis o f fruc tose-1 ,6-diphosphate

to y ie ld fructose-6-phosphate is catalyzed by fructose diphosphatase.

This enzyme is in h ib ite d by adenosine monophosphate and is maximally

active when the concentration o f adenosine triphosphate is re la tiv e ly

high (Scrutton and U tte r, 1968). The subsequent reversib le step gener­

ates g lucose-6-phosphate and the enzyme which catalyzes th is conversion

is phosphoglucoisomerase. The g lucose-6-phosphate formed during

gluconeogenesis may now e ith e r be directed to glycogen synthesis or the

pentose phosphate pathway. However, in tissues such as the l iv e r and

kidney, glucose-6-phosphate may be dephosphorylated to form free glucose.

The glucose formed is released to the blood to maintain glucose levels in

the peripheral tissues. The enzyme which catalyzes th is irre ve rs ib le

hydrolysis o f the 6-phosphate group is g lucose-6-phosphatase.

Glucose can be formed from a va rie ty o f noncarbohydrate precur­

sors which enter the gluconeogenic pathway at d iffe re n t leve ls . A

deta iled l i s t o f gluconeogenic precursors and a consideration o f pathways

by which these precursors are converted to intermediates o f gluconeo­

genesis has been presented by Krebs.(1963), Scrutton and U tte r (1968% and

10

Exton (1972). The source o f carbon fo r gluconeogenesis is dependent upon

the d ie tary status and the physical a c t iv ity o f the animal. In the

fasted animal, amino acids w i l l constitu te a major source o f the carbon

fo r glucose formation (Lardy e t a l . , 1970). The amino acids which can

serve as precursors o f phosphoenolpyruvate and therefore o f glucose are

termed glycogenic amino acids. I t is in te res ting to .note tha t many o f

the amino acid degradation products are intermediates in the tr ic a rb o x y lic

acid cycle (T.C.A. cycle). This suggests another source o f carbon fo r

gluconeogenesis. The capacity to synthesize glucose from tr ica rb o xy lic

acid intermediates has been established (Krebs, 1963; Scrutton and U tte r,

1968; Exton, 1972). I f the animal is in a fed state and exercising,

large quantities o f pyruvate and lac ta te w i l l be produced in muscle and

are transported by the blood to the l iv e r and kidneys where they are used

fo r gluconeogenesis.

The gluconeogenic flu x in l iv e r has been shown to respond to both

d ie tary and hormonal s tim u li (Krebs, 1963; Newshoi me and Gevers, 1967;

Scrutton and U tte r, 1968; Exton, 1972). The regulation o f conditions

which stim ulate the gluconeogenic f lu x such as elevated blood glucose

during muscle exercise, carbohydrate starva tion and hormonal disorders

such as diabetes remain unclear. These conditions may be experimentally

reproduced by adm inistration or withdrawal o f the appropriate substrate

or hormone and have been extensively studied in order to id e n tify the

reaction(s) involved (Krebs, 1963; Newshoi me and Gevers, 1967; Scrutton

and U tte r, 1968; Exton, 1972). The more recent studies have u tiliz e d the

iso la ted gluconeogenic tissue such as tissue s lices or perfused

npreparations o f the .whole organ. Scrutton and U tter (1968) have compared

the rates of.gluconeogenesis observed in perfused ra t l iv e r and kidney

with the rates in whole animals and have shown them to be in the same

range. This suggests that the perfused preparations approximate the in

vivo s itu a tio n .

Glycolysis and gluconeogenesis are opposite processes which are

not allowed to occur to a major extent at the same time in the same c e ll.

Considerable emphasis has been placed on the p o s s ib ility tha t the control

o f gluconeogenesis may be exerted at one or more o f the enzyme reactions

which overcome the energy barrie rs preventing the d ire c t reversal o f

g lyco lys is . This lin e o f reasoning is ju s t i f ie d by the observations tha t

the l iv e r content o f these enzymes in vivo increases a fte r some hours or

days under conditions o f enhanced gluconeogenesis (Weber, 1967), and tha t

the a c t iv it ie s o f these enzymes, e .g ., pyruvate carboxylase and fructose

diphosphatase have been shown to be regulated by th e ir cofactor require­

ments (Scrutton and U tte r, 1968). Table 2 l is t s known activators or

in h ib ito rs which assume the regulatory ro le o f switching-on or sw itching-

o f f key enzymes o f e ith e r pathway, thereby preventing the fu t i le forma­

tion o f glucose and i ts subsequent degradation from occurring in the same

c e llu la r compartment. Table 2 does not include a ll the activators and

in h ib ito rs fo r the enzymes lis te d , but includes only those which are

thought to operate in the in ta c t l iv e r under physiological conditions. A

more detailed l i s t o f e ffectors and a comprehensive discussion o f th e ir

action has been published (Krebs, 1963; Newshoime and Severs, 1967;

Scrutton and U tte r, 1968; Exton, 1972).

Table 2. Metabolic Effectors o f the G lyco ly tic and Gluconeogenic Pathways.

12

Legend

ADP adenosine diphosphate G6P glucose-6-phosphateAMP adenosine monophosphate F6P fructose-6-phosphateATP adenosine triphosphate FDP . fructose diphosphatePj inorganic phosphate

Enzyme Activators Inh ib ito rs

I . G lyco ly tic

Phosphofructokinase ADP, AMP, G6P, F6P, P.

ATP, c it ra te , FDP

Pyruvate kinase FDP ATP, alanine

I I . Gluconeogenic

Pyruvate carboxylase acetlyCoA ATP, alanine

Phosphoenolpyruvate carboxy kinase

tryptophan q u in il ic acid

Fructose diphosphatase AMP, FDP

Glucose-6-phosphatase c it ra te , ATP, ADP

13

Many investigators have observed a lte ra tions in the gluconeogenic

rate fo llow ing the adm inistration o f various hormones. As a consequence,

the hormonal influence on glucose synthesis has received considerable

a tten tion . I t has been shown tha t glucagon stimulates glucose formation

from lac ta te (S truck, Ashmore and Wieland, 1966; Williamson, Kreisberg

and Fe lts , 1966; Krebs, Gascoyne and Nottom, 1967; Ross, Hems and Krebs,

1967; Exton and Park, 1968; Exton, Corbin and Park, 1969; Garrison and

Haynes, 1973), pyruvate (Williamson et a l . , 1966; Krebs e t a ! . , 1967;.

Ross e t a l . , 1967; Exton and Park, 1968; Exton et a l . , 1969; Garrison and

Haynes, 1973).and alanine (Williamson e t a l . , 1966; Exton e t a l . , 1969;

Garrison and Haynes, 1973). Evidence from these studies have led Exton

(1972) to speculate tha t the s ite at which glucagon in te racts with

gluconeogenic pathway is pyruvate carboxylase. However, Veneziale (1971)

using ra t l iv e r preparations has demonstrated that glucagon is also

capable o f s tim u la ting gluconeogenesis from fructose and more recently

from D-glyceraldehyde and dihydroxyacetone phosphate (Veneziale, 1972).

The gluconeogenic response to glucagon may be duplicated a lte r ­

na tive ly through the e ffects o f fa t ty acid oxidation. The mechanism o f

action o f fa tty acid oxidation on gluconeogenesis is not ye t understood

in d e ta il, but i t seems well established tha t intermediates aris ing from

fa tty acid oxidation are also capable o f stim ula ting glucose synthesis.

I t has been shown experimentally using in h ib ito rs o f fa t ty acid oxidation

such as a-bromopalmitate (Sauer, Mahadevan and Erf 1 e , 1971; Mahadevan and

Sauer, 1971), 4-pentenoic acid (Corredor, Brendel and Bressler, 1967;

Brendel and Bressler, 1970) and (+ )-acy lca rn itine derivatives (D e lis le

14

and F r itz , 1967) were shown to decrease gluconeogenesis. Williamson

(1967) proposed a mechanism which explains the re la tionsh ip between fa t ty

acid oxidation and gluconeogenesis. This mechanism involves the ac tiva ­

tion o f pyruvate carboxylase by acetlyCoA, a d ire c t product o f fa tty acid

oxidation. Williamson concluded tha t s tim ula tion o f pyruvate carboxylase

by acetylCoA is the most important step responsible fo r increasing

gluconeogenic rates and tha t the hepatic l ip o ly t ic action o f glucagon is

a secondary factory (Williamson, 1967). More recently , evidence has been

presented (Williamson, Jakob and Scholz, 1971) which takes in to account

tha t possibly the high rate o f recycling between pyruvate and phosphoenol-

pyruvate coupled with the changes in the cytoso lic redox state o f the

pyrid ine nucleotides which d ire c tly e ffects the g lyce ra ldehyde-3-phosphate

dehydrogenase step may also be responsible fo r co n tro lling glucose '

formation.

I t has been shown tha t the level o f cyc lic AMP in perfused liv e rs

may be increased by adding glucagon to the perfusate and decreased by the

addition o f in su lin (Exton and Park, 1968; Williamson, 1967;

Menahan and Wieland, 1969). I t has been postulated (Exton and Park,

1968) tha t cyc lic AMP appears to accelerate the rate lim it in g steps

assumed to be located in the conversion o f pyruvate to phosphoenol-

pyruvate. Therefore, cyc lic AMP may function as an in tra c e llu la r messen­

ger in the expression o f the effects o f glucagon and in su lin on gluconeo­

genesis. The controversy between the action o f glucagon, e ithe r d ire c tly

or through cyc lic AMP and fa tty oxidation on the regulation o f glucose

synthesis has not been solved. Exton and Park (1967), based on

15

calculated y ie lds o f ATP from pyruvate oxidation, have concluded tha t

glucose formation from pyruvate could proceed in the absence o f l iv e r

l ip id breakdown. However, Menahan and Wieland (1969) have presented

evidence in support o f the conclusion tha t maximal rates o f gluconeo-

genesis from pyruvate cannot proceed w ithout the support o f fa t ty acid

oxidation.

The ro le o f corticostero ids in the regulation o f gluconeogenesis

has been extensively studied and reviewed (Krebs, 1963; Newsholme and

Gevers, 1967; Scrutton and U tte r, 1968; Exton, 1972). Using perfused

l iv e rs . Exton e t a l . (1969) compared the gluconeogenic rate between fasted

and adrenalectomized ra ts . I t was observed that adrenalectomy in rats

caused a reduction in the gluconeogenic rate from lac ta te or pyruvate

w ith no a lte ra tio n in the rate from fructose. Eisenstein (1965, 1967)

found tha t the addition o f co rticosteriods to the perfusate in adrenal-

ectomi zed ra t l iv e r perfusion enhanced glucose formation from alanine,

lacta te and pyruvate. No e ffe c t was observed in perfused liv e rs of

normal ra ts . In kidney cortex s lice s , a comparison o f fasted and

adrenalectomized rats showed decreased rates o f gluconeogenesis from

alanine, pyruvate and lac ta te . Normal gluconeogenic rates were restored

when corticos te ro id was administered p r io r to sa c rifice (Henning,

Huth and Seubert, 1964). Elevated rates o f gluconeogenesis were observed

when the kidney s lices from adrenalectomized rats were incubated w ith

co rtiso l in v it ro (Seubert, Henning and Schoner, 1968). M a lle tte , Exton

and Park (1969) noted tha t corticostero ids given to adrenalectomized rats

in vivo th ir ty minutes p r io r to s a c r if ic e , f u l ly restored the promotion

16

o f gluconeogenesis from lacta te i f glucagon was added to the perfusate.'

Addition o f co rticoste ro id to the perfusate was only p a r t ia l ly e ffe c tive

in stim ulating glucose synthesis. Since then, 0j i , Shreeve and Tashjian

(1971) have shown that the addition o f hydrocortisone in whole l iv e r o f

normal, in ta c t rats has an accelerating e ffe c t on gluconeogenesis. The

glucocorticoids stim ulate protein catabolism in peripheral tissues,

releasing amino acids fo r uptake by the l iv e r , and thus, add to the

supply o f precursors fo r gluconeogenesis (Oji e t a l . , 1971). Oji e t a l .

(1971) suggests tha t the gluconeogenic action o f g lucortico ids in the

early time period a fte r hormone adm inistration is exerted p r in c ip a lly

through regulation o f flow o f substrate to the l iv e r and w ith in the

l iv e r c e ll. In addition to supplying substrates such as pyruvate,

lac ta te , oxalacetate, and malate, there is an increased a v a ila b il i ty o f

fa t ty acids which in conjunction w ith an adequate supply o f gluconeogenic

precursors provides a strong and rapid stim ulation o f enhanced hepatic

gluconeogenesis, th is is in agreement w ith Williamson (1967). The

in trahepatic mechanism fo r early ac tiva tion o f gluconeogenesis appears

to agree w ith others, in tha t enhanced flow Of substrate to the l iv e r , .

accompanied w ith fa t ty acid oxidation, allows glucocorticoids to

increase the mitochondrial NADH/NAD+ ra tio . The flow o f hydrogen from

fa tty acid oxidation would proceed through extramitochondrial malate

and la te r to the reduction o f diphosphoglycerate in the cytosol. Oji

e t a l . (1971) proposed tha t an action o f glucocorticoids d ire c tly on the

l iv e r in vivo, i f only fo r a short time, is required fo r the fu l l

expression o f the gluconeogenic e ffe c t.

17

I t has been .shown by Shrago and Lardy (1966) tha t phosphoenol-

pyruvate carboxykinase a c t iv ity was increased by g luco rtico lds , fas tin g ,

glucagon, lacta te or the induction o f diabetes by a lloxan. This a c t iv ity

was due to increased phosphoenolpyruvate carboxykinase synthesis. More

recently Exton (1972) has shown that in su lin suppresses phosphoenol-

pyruvate carboxykinase a c t iv ity but, only when a source o f carbohydrate

is availab le. Apparently, carbohydrate is the major d ie ta ry component

e ffec ting the phosphoenolpyruvate carboxykinase synthesis in l iv e r and

in su lin is required fo r i t s action (Exton, 1972). The stim ulation of

phosphoenolpyruvate carboxykinase synthesis probably may be an important

component o f the regulation o f gluconeogenesis in l iv e r . Growth hormone

(ACTH) and epinephrine have been shown by Exton (1972) to increase the

hepatic uptake o f spec ific amino acids in v ivo . I t is not known

whether or not these e ffec ts are exerted d ire c tly on the l iv e r , or

directed to changes o f plasma levels o f the amino acids or possibly

through changes in levels o f other hormones (Exton, 1972).

Mendicino and U tter (1962), Mendicino e t a l . (1968), and

Mendicino and Kratowich (1972) have presented evidence in mitochondria

iso la ted from kidney, fo r fructose diphosphatase, fo r the presence o f a

bound enzyme system which is capable o f ina c tiva ting fructose diphos­

phatase. Their resu lts show tha t the a c t iv ity o f the regulatory enzyme

present in kidney mitochondria which inactiva tes fructose diphosphatase

is very sensitive to the steady state ra tio o f ATP/ADP in the mito­

chondria. The regulatory enzymes appear to be protein kinases and phos-

phoprotein phosphatases which function by in terconverting active and

inactive forms o f some o f the g ly co ly tic enzymes. The degree o f

s p e c if ity o f these enzymes fo r the regulatory enzymes involved has not

been c la r if ie d . Studies of the levels o f phosphoenolpyruvate carboxy-

kinase in subcellu lar components in l iv e r o f d iffe re n t species by

Nordlie and Lardy (1963) revealed d iffe re n t patterns o f d is tr ib u tio n

among the species studied. In rabb it l iv e r , phosphoenolpyruvate

carboxykinase could only be detected in mitochondria. This was fu rth e r

investigated by Gamble and Mazur (1967) and Davis and Gibson (1969) who

also concluded tha t phosphoenolpyruvate was formed exclusive ly in rabb it

l iv e r mitochondria and was then translocated to the cytosol in order to

continue the gluconeogenic sequence. More recently, Johnson, Ebert

and Ray (1970) demonstrated the cytoplasmic presence o f phosphoenol-

pyruvate carboxykinase in ra bb it l iv e r and has presented evidence tha t

the synthesis o f th is enzyme was inducible under conditions o f fasting

and diabetes. The cytoso lic presence o f th is enzyme in rabb it l iv e r

was also noted by Garber and Hanson (1971) who in agreement w ith

Johnson e t a l . (1970), concluded tha t the cytoso lic a c t iv ity o f phos­

phoenol pyruvate carboxykinase was m arginally s ig n if ic a n t in the liv e rs

from fed rabbits but a fte r fasting 48 hours, the cy toso lic a c t iv ity o f

th is enzyme was induced s ix - fo ld while the a c t iv ity o f the mitochondrial

enzyme remained unchanged. In no instance however, did the cytoso lic

a c t iv ity o f phosphoenolpyruvate carboxykinase equal or exceed the in tra -

mitochondria! a c t iv ity o f the enzyme. This observation suggests tha t

the intram itochondrial formation o f phosphoenolpyruvate may be s ig n if ic a n t

fo r gluconeogenesis. The mechanism o f phosphoenolpyruvate formation in

19

mitochondria and i t s sign ificance to gluconeogenesis remains unclear in

species in which phosphoeno1 pyruvate carboxykinase is in both the

cytosol and the mitochondria. I t seems l ik e ly , tha t in the ra bb it,

s im ila r to the guinea pig and ra t (Nordlie and Lardy, 1963), only the

cytoso lic a c t iv ity o f phosphoenolpyruvate carboxykinase responds

adaptively to gluconeogenic demands (Garber and Hanson, 1971). The

induction o f the cytoso lic form o f phosphoenolpyruvate carboxykinase in

response to fasting rabb it l iv e r (Johnson e t a l . , 1970; Garber and

Hanson, 1971) suggests tha t some proportion o f the overa ll gluconeogenic

f lu x proceeds by way o f th is enzyme. Previous studies w ith rabb it l iv e r

considered the formation o f phosphoenolpyruvate to occur only in the

mitochondria. I t has been postulated (Gamble and Mazur, 1967; Davis and

Gibson, 1969) tha t the amount o f phosphoenolpyruvate formed in and

libera ted by rabb it l iv e r mitochondria is only a small fra c tio n o f the

to ta l carbon leaving the mitochondrion as phosphoenolpyruvate, malate,

aspartate, and c itra te . This observation is d i f f ic u l t to ju s t i f y , in

l ig h t o f the complexity involved in measuring the mitochondrial output

in the in ta c t l iv e r . However, i t should be apparent from an e a r lie r

discussion, tha t some malate and aspartate formation must occur not only

to supply cytoso lic NADH fo r gluconeogenesis, but also, to provide the

four-carbon acids fo r the cy toso lic formation of phosphoenolpyruvate.

This ta c i t ly implies tha t some intram itochondrial mechanism(s) must

e x is t which control the formation o f one precursor re la tiv e to the other.

20

Experimental Rationale

The production o f phosphoenolpyruvate from a number o f sub­

strates appears to be influenced by three facto rs. The f i r s t fac to r

involves an energy requirement. In rabb it l iv e r , the high energy

compound GTP is essential fo r the conversion o f oxalacetate to phos­

phoenol pyruvate via GTP dependent phosphoenolpyruvate carboxykinase. The

GTP required fo r th is process may be formed w ith in the mitochondrion,

e ith e r from the ATP pool by the nucleoside diphosphokinase reaction

or by the substrate level phosphorylation.of GDP to GTP during succinyl-

CoA conversion to succinate. Garber and Ballard (1970) were able to

demonstrate in guinea pig l iv e r mitochondria, the rate o f phosphoenol-

pyruvate synthesis was dependent upon the i ntrami tochondrial ATP/ADP

ra tio . Presumably, the ATP/ADP ra tio d ire c tly determines the in tra ­

mi tochondrial concentration o f GTP via the equilibrium constant o f near

un ity fo r the nucleoside diphosphokinase reaction. This suggests tha t

the enzymatic behavior o f nucleoside diphosphokinase determines the

a v a ila b il i ty o f GTP from ATP and, therefore , regulates the rate o f

phosphoenolpyruvate synthesis. In order to evaluate the importance

o f th is energy requirement in rabb it l iv e r mitochondria, the levels o f

phosphoenolpyruvate produced from various substrates were measured and

compared w ith measured levels o f ATP. A .corre la tion between the mito­

chondrial ATP levels and phosphoenolpyruvate synthesis was then made.

The second fac to r pertains to the a lte ra tions o f the oxida tion-

reduction state o f the mitochondrial pyrid ine nucleotides. I t has been

demonstrated tha t under gluconeogenic conditions, there is a d e fin ite

21

4-a lte ra tio n in the NAD /NADH ra tio in the mitochondria o f ra t (Williamson,

1967), in guinea pig (Garber and Ba llard , 1970), and in ra bb it (Garber

and Hanson, 1971) which d ire c tly e ffects the rates o f malate oxidation

due to the displacement o f the malate dehydrogenase equ ilib rium . This

s h if t in the redox state o f the pyrid ine nucleotides has been shown to

d ire c tly e ffe c t phosphoenolpyruvate production. The influence o f the

redox state o f the pyrid ine nucleotides on mitochondrial phosphoenol-

pyruvate synthesis was evaluated through the use o f an uncoupler-

t i t r a t io n in which the energy component o f phosphoenolpyruvate formation

was not l im it in g . The ra tiona le being, the uncoupler would increase the. y

intram itochondria l concentration o f NAD+ through accelerated electron

trans fe r. The elevated NAD /NADH ra tio would promote an increase in the

level o f oxalacetate due to the displacement o f the malate dehydro­

genase equ ilib rium . Subsequently, increasing the a v a ila b i l i ty o f th is

substrate would favor phosphoenolpyruvate formation. D iffe ren t concen­

tra tio n s o f uncoupler (FCCP) were used to produce various changes in

the NAD+/NADH ra tios . The e ffe c t o f th is redox s h if t was then assessed

by measuring phosphoenolpyruvate formation from various substrates in

the presence o f uncoupler (FCCP). Additional experiments were performed

to fu rthe r evaluate the e ffec ts o f the mitochondrial redox state on

phosphoenolpyruvate synthesis.

The th ird fa c to r which influences the production o f phosphoenol-

pyruvate in rabb it l iv e r mitochondria involves the competition fo r

mitochondrial oxalacetate by c itra te synthase and glutam ic-oxalacetic

transaminase. The competitive influence exerted by c it ra te synthase was

22

evaluated using pal m ity lca rn itin e , ace ty lca rn itine and octanoate in the

presence o f a-ketoglutarate and uncoupler (FCCP). Under these conditions

the oxidation o f pal m ity lca rn itin e and octanoate via 3 -ox idation and the

conversion o f acetyl ca rn itine to the CoA derivative would increase the

mitochondrial content o f acetylCoA. In the presence o f each cosubstrate

corresponding increases in c itra te formation were correlated w ith con­

comitant decreases in phosphoenolpyruvate production. These results

ind icate tha t c itra te synthase e ffe c tiv e ly competes w ith phosphoenol-

pyruvate carboxykinase fo r available oxalacetate. In these experiments

a-ketoglutarate served both as a source o f GTP and as a carbon source

fo r oxalacetate. In order to estimate the competition exerted by

glutam ic-oxalacetic transaminase fo r oxalacetate, rates o f phosphoenol-

pyruvate were measured in the presence and absence o f glutamate in one

case, and in the presence o f cystiene s u lf in ic acid in another. As in

the previous experiments a-ketoglutarate was included to meet the energy

requirement o f phosphoenolpyruvate, carboxykinase and as a source o f

carbon fo r oxalacetate. In the presence o f glutamate or cystiene

s u lf in ic acid, s ig n if ic a n tly lower rates o f phosphoenolpyruvate synthesis

were observed in uncoupled (FCCP) mitochondria. This reduction in the

rate o f phosphoenolpyruvate formation was correlated w ith the competitive

influence exerted by glutamic-oxalacetate transaminase fo r the available

oxalacetate.

CHAPTER 2

METHODS AND MATERIALS

Mitochondrial Iso la tion Procedure

Rabbits were stunned by a sharp blow to the back o f the head, and

the abdomen was rap id ly opened, the l iv e r perfused w ith a solution o f

0.25 M sucrose and the l iv e r was removed. Liver s lices weighing approxi­

mately 1 0 gm. were placed in a previously ch ille d 1 0 0 ml beaker con­

ta in ing 20 ml o f 225 mM mannitol, 75 mM sucrose and 50 piM ethylene glycol

b is - (g-aminoethyl ether) - N, N1-te traaceta te (EGTA), th is was referred

to as homogenizing media. The l iv e r s lic e was then swirled in a cold

solution and the so lu tion was decanted o f f . The l iv e r s lic e was then

chopped in to small fragments w ith p rech illed scissors and these fragments

were then transferred d ire c tly to 100 ml o f homogenizing media. Using

a power driven Teflon pestal a l iv e r homogenate was accomplished, the

homogenate was completed w ith approximately 3 or 4 strokes o f the

Teflon pestal. The Potter-E lveljem tissue grinding tube was then f i l le d

to the brim (to ta l volume, 90 ml) w ith homogenizing so lu tion . The

d ilu ted homogenate was equally d is tribu ted by volume in 4 centrifuge

tubes. The homogenate was centrifuged a t 1800 rpm fo r 10 minutes in a

Sorvall Model RC2B preparative centrifuge. The supernatant was collected

and centrifuged at 6500 rpm fo r 20 minutes. Subsequently, the fa t ty

layer which collected on the top o f the supernatant was removed with

absorbent tissue paper. The supernatant was ca re fu lly discarded and the

23

24

p e lle t was then resuspended in a f in a l volume o f 1 0 ml o f a prechi lie d

solution containing 225 mM mannitol and 75 mM sucrose which is termed

washing so lu tion . The best technique was to add 5 ml o f the washing

solution to the p e lle t and then gently break the p e lle t loose w ith a

Teflon s t ir r in g rod. The f in a l 5 ml o f washing so lution was then added.

In order to ensure the p e lle t was completely resuspended, a 10 ml blow­

out p ipette was used to gently draw the volume o f resuspended p e lle t up

and subsequently th is volume was permitted to drain w ithout force in to

the centrifuge tube. The resuspended p e lle t was then centrifuged at

8000 rpm fo r 10 minutes. This process o f resuspending the p e lle t

fo llow ing a 8000 rpm centrifuge was done tw ice , on the th ird and fin a l

cen trifuga tion , the p e lle t was resuspended in 1 ml volume o f wash

so lu tion and the contents o f the other centrifuge tubes were combined.

The combined suspension which contains iso la ted mitochondria in the wash

so lu tion was then transferred to a prechi lie d 10 ml Potter-E lveljem

tissue grinder. Using a Teflon pestal the mitochondria were gently

homogenized with no more than two strokes o f the pesta l. The Potter-

E1veljem tissue grinding vessel containing the mitochondria was placed

in ice u n til used.

Miscellaneous Procedures

Oxygen consumption was measured in a 8 ml glass reaction chamber

using a Clark-type oxygen electrode. Oxygen rates are reported as nmoles

oxygen/min/mg mitochondrial pro te in . The.incubation medium used in the

oxygen electrode chamber contained 50 mM T ris -ch lo rid e . The pH o f the

incubation media was adjusted to pH 7.1 - 7.2 w ith 1 M hydrochloric acid.

25

A portion o f th is media was then transferred to a large te s t tube which

was placed in a 27°C water bath and aireated. Absorption measurements

o f the intram itochondrial reduced pyrid ine nucleotides were accomplished .

using a Perkin-Elmer Model 356 dual beam/split beam spectrophotometer

using the wavelength p a ir 340-374 nm. Samples o f the mitochondrial

reaction mixture were withdrawn from the oxygen electrode chamber w ith ,

a 1 ml p ipe tte . The pepette which, was used to co lle c t the samples was

la te r ca librated so that the exact volume o f sample discharged was

known. The protein was precip ita ted w ith perchloric acid ( fin a l con­

centration 6 % w/v). Following cen trifuga tion (10,000 rpm fo r 10 minutes)

the samples were neutralized w ith 3 M potassium carbonate plus 0.5 M

trie thano l amine p r io r to measurement o f various intermediates. Metabolic

intermediates and nucleotides were measured using the enzymatic-

fluorom etric procedures described by Williamson and Corkey (1968). The

assay o f phosphoenolpyruvate was accomplished by coupling the two

enzymatic reactions, pyruvate kinase and lacta te dehydrogenase.

Quantitative amounts o f phosphoenolpyruvate may be determined by

fo llow ing the increase in fluoresence o f NADH formed in the la te r

reaction. The levels o f c itra te were measured by coupling c itra te

lyase and-malate dehydrogenase, in th is case, quantita tive amounts o f

c itra te are determined by fo llow ing the decrease in fluoresence due to

the oxidation o f NADH. Quantitative amounts of ATP were determined by

coupling the enzyme reactions hexokinase and glucose-6 -phosphate

dehydrogenase. A decrease in fluoresence is observed through the reduction ' +

o f NADP . Enzymes used in these assays were obtained from Boehringer

Mannheim Corporation. Mitochondrial protein concentrations were estimated

using a b iu re t procedure o f Layne (1957).

CHAPTER 3

RESULTS

In order to establish the re la tionsh ip between the oxidation-

reduction s ta te , the energetic state o f the rabb it l iv e r mitochondrial

suspension and the rate o f phosphoenolpyruvate production from c i t r ic

acid cycle intermediates, the experiments described in Figures 3-5 were

performed. An uncoupler t i t r a t io n (FCCP) was used to vary the res­

p ira tion ra te , the oxidation-reduction state of the intram itochondrial

pyridine nucleotides and the intram itochondrial ATP le ve l. The pre­

cursor o f 4-carbon units fo r phosphoneolpyruvate synthesis was malate

as oxalacetate is not an optimal precursor due to i t s re la tiv e ly poor

penetration o f the mitochondrial membrane. As a source o f energy, e .g .,

GTP, fo r the synthesis o f phosphoenolpyruvate, the oxidation o f

a-ketoglutarate was used. The generation o f GTP fo r phosphoenolpyruvate

synthesis is probably the sole source o f GTP or ATP fo r th is reaction

since the mitochondria are uncoupled w ith FCCP during the incubation.

Malonate was included in the incubation so tha t a-ketoglutarate oxidation

did not serve as a means o f providing 4-carbon un its , e .g ., oxalacetate

fo r the phosphoenolpyruvate synthetic reaction, phosphoenolpyruvate

carboxykinase.

The e ffe c t o f FCCP on the rate o f oxidation o f a-ketoglutarate

and malate in the presence o f malonate is shown in Figure 3, panel A.

In th is experiment oxygen consumption was measured in a 9.0 ml glass

27

FCCPFC C P

No Additions

Absorbance Decrease

3 4 0 - 3 7 8 nm

No Additions

Substrates :o -K etoglutaro te

Malate and Malonote

-W M-I Minute

- - 0 2 - 0 — FCCP ( ,3 3 /jM)

Figure 3. The E ffect o f Uncoupler (FCCP) T itra tio n on the Rate o f Oxygen Consumption (Panel A) and on the Oxidation-Reduction State o f the Intramitochondrial Pyridine Nucleotides (Panel B). - - The to ta l incubation volume was 6.0 ml and contained 50 mM sucrose, 100 mM KC1, 5 mM KP04 , 20 mM T ris -ch lo ride (pH 7.2), 1 mM malonate, 3 mM malate and 1.5 mM a-ketogl utarate. Respiration was in it ia te d with uncoupler at concentrations noted in the corresponding panels o f Figure 3.

rx>00

FCCP Concentrations

OlE

CL

5.083 xiM

.33 /uM

Minutes

Figure 4. The E ffect o f Uncoupler (FCCP) T itra tio n on ATP Synthesis. — The reaction conditions were iden tica l with those described in Figure 3.

5

Figure

30

jioXc0)2

CLcnE

XCLLUCLtoaoEc

2.4-

2 .0 -

1.6 -

1.2 -

.8 -

.4 -

FCCP Concentrations

0 T0

---------1--------- p .

6

Minutes

1—1-----T“—i—"nr8 10 12

5. The E ffect o f Uncoupler (FCCP) T itra tio n on Phosphoenol-pyruvate Synthesis. - - The reaction conditions were iden tica l with those described in Figure 3.

31

reaction chamber using a Clark-type oxygen electrode. Oxygen rates are

reported as nmoles oxygen/min/mg o f prote in. The rate o f resp ira tion

was increased as the concentration was increased. Concentrations o f

uncoupler exceeding 0.33 yM had no fu rth e r a lte ra tio n in the resp ira to ry

rate and, in fa c t, caused an in h ib it io n o f resp ira tion . The absolute

rates o f oxygen consumption ranged from 3.7 to 42.8 nmoles oxygen/min/mg

protein in the experiment to which 0.33 yM FCCP was added.

The e ffe c t o f FCCP on the oxidation-reduction state o f the in tra -

mi tochondrial nicotimamide pyrid ine nucleotide is shown in Figure 3,

panel B. Absorption measurements o f the in tram itochondria l, reduced

pyrid ine nucleotides were accomplished using a Hatachi-Perkin-Elmer

Model 356 dual beam/split beam spectrophotometer using the wavelength

pa ir 340-347 nm. Experiments performed to te s t the e ffec ts o f uncoupler

on the oxidation-reduction state o f the intram itochondrial pyridine

nucleotides were conducted on the same mitochondrial preparation and

under the iden tica l conditions as were used in the oxygen electrode

experiments shown in panel A o f Figure 3. Upon addition o f uncoupler a

rapid oxidation o f the pyrid ine nucleotides was observed. Increasing

concentrations o f FCCP caused a s h if t in the steady state in tram ito ­

chondrial NAD+/NADH ra tio to a more oxidized leve l. In a previous

analysis o f the metabolic a lte ra tions in liv e rs o f fasted rabbits com­

pared with fed rabb its , a s h if t toward oxidation o f the in tram ito ­

chondrial nicotimamide coenzymes was demonstrated (Garber and Hanson,

1971). In the present l iv e r mitochondrial experiments the pyrid ine

32

nucleotide oxidation upon uncoupler addition was consistent with the

a lte ra tions seen in the resp ira tion rate in panel A.

During the oxygen electrode experiment outlined in Figure 3,

samples (1 ml) were rap id ly withdrawn, the protein prec ip ita ted with

perch loric acid (6 % w/v) and analysis fo r ATP were performed using the

procedure outlined in Materials and Methods (See Figure 2). As shown

in Figure 4, as the concentration o f FCCP was sequentia lly increased,

the intram itochondria l ATP levels decreased in the separate incubations.

Also, i t should be noted tha t at the highest uncoupler concentrations,

the ATP level in the ind iv idua l experiments decreased during the 8-10

minute incubation period. Even though the ATP level which is normally

maintained by resp ira to ry chain-linked phosphorylation was very low in

the experiment to which 0.33 uM FCCP was added as w il l be seen la te r ,

th is experiment had the highest rate o f phosphoenolpyruvate formation.

As long as a source o f GTP was ava ilab le , in th is case a-ketoglutarate

oxidation, the energetic state as indicated by the ATP level may not be

an accurate monitor o f the energetic potentia l fo r phsophoenolpyruvate

formation in th is synthetic system. The GTP levels were not measured

in these experiments as the techniques fo r measuring th is nucleotide

are not presently sensitive enough to be used rou tine ly in mitochondrial

experiments.

Analyses fo r phosphoenolpyruvate were performed using the samples

collected in the above experiment and the resu lts are shown in Figure 5.

A very slow rate o f phosphoenolpyruvate formation was observed in the

experiment to which no uncoupler was added. This incubation had a slow

rate o f resp ira tion , v ir tu a l ly no oxidation o f intram itochondria l

pyridine nucleotides but had the highest ATP leve l. On the other hand,

the experiment to which the highest concentration o f uncoupler was added

had a very rapid rate o f phosphoenolpyruvate synthesis, a rapid rate' t'- ■ r ,

o f resp ira tion , maximal oxidation o f the intram itochondrial pyridine

nucleotides but the lowest ATP level o f a ll other incubations. The

elevated rates o f phosphoenolpyruvate synthesis may be explained by

a lte ra tions in the re la tive oxidation-reduction state o f the pyridine

nucleotides and, hence, the steady state level o f oxalacetate. Increases

in the NAD+/NADH ra tios have been shown to elevate the level o f oxal-

acetate due to the displacement o f the malate dehydrogenase equilibrium

(Garber and Ballard, 1970; Garber and Sa lgan ico ff, 1973; Arinze, Garber

and Hanson, 1973). The higher level o f oxalacetate read ily fa c il ita te s

the conversion o f oxalacetate to phosphoenolpyruvate via phosphoenol-

pyruvate carboxy kinase as long as an adequate source o f GTP is present,

e .g ., a-ketoglutarate oxidation.

Additional experiments were performed using the oxidative phos­

phorylation in h ib ito r , oligomycin, in the presence and absence o f un­

coupler to evaluate the e ffe c t o f both the redox state and the energetic

state o f the intram itochondria l formation o f phosphoenolpyruvate from

malate and a-ketoglutarate. Oligomycin acts to e ffe c tiv e ly in h ib it ATP

synthesis in the oxidative phosphorylation sequence in addition to

preventing the access o f high energy bonds formed in the oxidation o f

a-ketoglutarate to the uncoupler stimulated mitochondrial ATPase. The

e ffe c t o f oligomycin in the presence o f uncoupler on the ATP levels o f

th is phosphoenolpyruvate synthesizing system can be seen in Figure 6 .

Oligomycin added to the rabb it l iv e r mitochondria in the absence o f

uncoupler leads to a s ig n if ic a n t decrease in the ATP level over the 10

minute incubation. Under these c o n d it io n s ,e .g ., in the absence of

uncoupler, the pyrid ine nucleotides would be fu l ly reduced both in the

presence and absence o f oligomycin. Under these conditions, as can be

seen in Figure 7, re la tiv e ly slow rates o f phosphoenolpyruvate formation

were observed. When uncoupler, FCCP, was added to the system, the ATP

levels (Figure 6 ) were even more depressed both in the presence and

absence o f oligomycin. Rapid and nearly iden tica l rates o f phosphoenol-

pyruvate formation were observed under these conditions. Hence, under

incubation conditions where a re la t iv e ly low intram itochondrial ATP

level was observed but where the redox state was oxidized and where

the resp ira tion rate was rapid, maximal rates o f phosphoenolpyruvate

formation were observed. This experiment confirms the conclusion drawn

in the experiments shown in Figure 3-5 tha t the primary influence on the

rate o f phosphoenolpyruvate formation from malate is the set o f the redox

state and the concentration o f oxalacetate. These resu lts also i l l u s ­

tra te tha t the ATP level per se is not a primary concern as long as a

source o f GTP is ava ilab le . Also, il lu s tra te d in the experiment shown

in Figures 6 and 7 is the fa c t tha t oligomycin addition which should

prevent access o f GTP through the nucleoside diphosphokinase reaction

to the mitochondrial ATPase did not s ig n if ic a n tly e ffe c t the rate o f

phosphoenolpyruvate formation. This observation indicates a rather

c'eyov_

CLC7>E\Q.

CZ)<y"oEc

10 —

8 -

6 -

4

2 H

0

No Additions

OligomycinPC CP

Oligomycin plus POOP

0

i4

I8 10 1 2

Minutes

Figure 6 . The E ffect o f Oligomycin in the Presence, and Absence, o f Uncoupler ( FCCP) on ATPSynthesis. — The to ta l incubation volume was 6.0 ml and contained 50 mM sucrose, 100 mM KOI, 5 mM KPO4 , 20 mM T ris -ch lo ride (pH 7 .2), 1 mM malonate, 3 mM malate and 1.5 mM a-ketoglutarate. The concentration o f oligomycin was 10 ygm/ml and uncoupler (FCCP) was 0.33 yM.

CO<_n

nmol

es

PE

P/m

g Pr

otei

n X

10

36

1 2 -

O ligom ycin plus FCCP

FCCP1 0 -

8 4

No Additions

6 4

Oligomycin

2 4

Minutes

Figure 7. The E ffect o f Oligomycin in the Presence, and Absence o fUncoupler on Phosphoenolpyruvate Synthesis. - - The reaction conditions were iden tica l with those described in Figure 6 .

in te res ting and d iffe re n t compartmentation o f the nucleotide diphos-

phokinase and phosphoenolpyruvate carboxykinase.

Additional experiments were performed to fu rth e r compare the

re la tive contribution o f the mitochondrial oxidation-reduction state o f

the pyrid ine nucleotides and the energy state on the production o f

phosphoenolpyruvate. The results o f these experiments are shown in

Figure 8 . In these experiments the substrates used were the same as

those used in experiments previously discussed except ADP was used to

stim ulate resp ira tion rather than FCCP. Under these conditions the

resp ira to ry chain i t s e l f is the rate lim it in g fac to r. A consideration

o f the energy contribution under these conditions suggest tha t the

transphosphorylation o f GDP from ATP derived via resp ira to ry linked

oxidative phosphorylation did not s ig n if ic a n tly enhance the rate o f

phosphoenolpyruvate synthesis above the level provided by substrate

level phosphorylation alone. As shown in Figure 9, the rate of ADP

stimulated resp ira tion was rapid and rates o f phosphoenolpyruvate

formation were maximal. The primary influence on phosphoenolpyruvate

formation in th is system may be accounted fo r through the oxidative

s h if t o f the intram itochondrial pyrid ine nucleotides. In order to show

tha t ATP levels were not responsible fo r a lte ra tions in phosphoenol­

pyruvate production, oligomycin was included in the incubation mixture.

In th is case as shown in Figures 8 and 9, the rates o f resp ira tion were

slow and levels o f phosphoenolpyruvate’formation were s ig n if ic a n tly

lowered. However, even in the presence o f oligomycin, GTP was supplied

via the oxidation o f a-ketoglutarate which implies the energy demands

o f phosphoenolpyruvate carboxykinase were met. The most p lausib le

nmol

es

PE

P/m

g Pr

otei

n X

10

38

1 0 - ADP

No Additions

Oligomycin plus ADP

Minutes

Figure 8 . The E ffect o f Oligomycin on Phosphoenolpyruvate Synthesis. — In these experiments ADP was used to stim ulate respira tion rather than uncoupler ( FCCP). The concentration of oligomycin used was 10 pgm/ml. The to ta l incubation volume was 6.0 ml and contained 50 mM sucrose, 100 mM KCL, 5 mM KPO4 , 20 mM T ris -ch lo ride (pH 7 .2 ), 1 mM malonate, 3 mM malate and 1.5 mM a-ketoglutarate.

nmol

es

Oxy

gen

cons

umed

39

ADR

2 2 -

20-1

8 -i

16 -

14 -

No Additions12 -

10 - Oligomycin plus ADR

122 8 100 6

Minutes

Figure 9. Oxygen Consumption o f ADR Stimulated Respiration, in theAbsence and Presence o f Oligomycin. - - The reaction conditions were iden tica l with those described in Figure 8 .

40

explanation o f low levels o f phosphoenolpyruvate synthesis under these

conditions is tha t the redox state o f the intram itochondria l pyrid ine

nucleotides were h ighly reduced. A major consequence o f th is reductive

s h if t is the reversal o f the malate dehydrogenase equ ilib rium toward

the formation of malate. Under these conditions the a v a ila b il i ty o f

oxalacetate fo r phosphoenolpyruvate synthesis would be s ig n if ic a n tly

reduced.

In an attempt to evaluate an energy e ffe c t on phosphoenolpyruvate

production the experiments shown in Figure 10 were performed. This

experiment i l lu s tra te s the importance o f energy derived from substrate

level phosphorylation o f phosphoenolpyruvate synthesis. In the presence

o f FCCP (0.33 yM) and arsenite (1 mM) the oxidative s h i f t in the pyrid ine

nucleotides was observed. Arsenite was included to in h ib it a-keto-

g lu tarate oxidation, thereby e lim inating the major source o f GTP derived

through the conversion of succinolCoA to succinate. In the uncoupled

system there would be no resp ira to ry linked oxidative phosphorylation,

hence, no net ATP production. Endogenous levels of ATP would be

dissipated through uncoupler stimulated ATPase a c t iv ity . The lack o f

ATP a v a ila b il i ty rules out the generation o f s u ff ic ie n t amounts o f GTP

derived through transphosphorylation via nucleotide diphosphokinase.

When oligomycin was included w ith FCCP and arsenite , ATPase a c t iv ity is

presumably in h ib ite d , therefore, endogenous levels o f ATP might be

u t il iz e d through transphosphorylation o f ATP to GTP via nucleotide

diphosphokinase. This was not observed to have any e ffe c t, since the

rate of phosphoenolpyruvate synthesis was not s ig n if ic a n tly enhanced.

In the experiment where FCCP was not included, the rate o f resp ira tion

41

12 -

p Oligomycin plus FCCP

oxc

"o>

oCLO'E\

CLUJCLv>

oE

10

8 -

6 —

4 —

2 -

FCCP

No Additions

FCCP plus ArseniteFCCP plus Oligomycin

plus Arsenite

Oligomycin plus Arsenite

0 -8

Minutes

n r10

I12

Figure 10. The E ffect o f Arsenite on Phosphoenolpyruvate Synthesis. - - The dotted lines i l lu s t ra te the rate of phosphoenolpyruvate synthesis in a system under iden tica l cond itions. The reac­tion conditions fo r the experiments il lu s tra te d in Figure 10 were iden tica l to those described in Figure 6 , except arsenite , 1 mM, was included where indicated.

was inh ib ited and the intram itochondria l NAD+/NADH ra tio was low, these

conditions exemplify an an energy depleted state with a h igh ly reduced

intram itochondrial redox s ta te . When no additions are made, the

endogenous supply o f substrates were presumably exhausted and low rates

o f phosphoenolpyruvate formation were observed. In Figure 10, the dotted

lines i l lu s t ra te the rate o f phosphoenolpyruvate synthesis produced in

a system under the same conditions in the presence o f uncoupler (FCCP)

and uncoupler (FCCP) plus oligomycin but w ithout the addition o f arsenite .

The resu lts were taken from the experiment shown in Figure 7 which was

performed under iden tica l conditions as the experiments in Figure 10.

This experiment il lu s tra te s the importance o f energy in terms o f GTP

derived via substrate level phosphorylation on phosphoenolpyruvate

synthesis.

Additional experiments were performed to evaluate the e ffe c t o f

mitochondrial energy levels on phosphoenolpyruvate formation. In order

to do th is a system was required which would regulate the mitochondrial

energy level somewhat independently o f the rate o f resp ira tion . The

system selected u t il iz e d the compounds 2-methyl-l,4-napthoquinone

(Vitamin K^) and rotenone. In these experiments, malate was included to

supply a source o f oxalacetate. In th is system reducing equivalents

from malate are shunted around the f i r s t and most t ig h t ly coupled phos­

phorylation s ite o f the resp ira tory chain using 2-methyl-l ,4-naptho­

quinone (0.83 yM). The f i r s t phosphorylation s ite was blocked by

rotenone (0.83 pM). However, the remaining s ites o f the resp ira to ry-

1 inked phosphorylation are functional and coupled with resp ira tion .

43

Intram itochondrial pyridine nucleotides in th is experiment were nearly

completely oxidized, thus e lim inating a possible redox e ffe c t on the

malate:oxalacetate ra tio . As shown in Figure 11, phosphoenolpyruvate

formation was highest w ith malate (3 mM) alone. Adequate levels o f

intram itochondrial ATP were presumably generated to supply energy fo r

phosphoenolpyruvate synthesis in th is incubation. The remaining three

experiments shown in Figure 11 u t i l iz e uncoupler (FCCP), calcium and

oligomycin, an in h ib ito r o f oxidative phosphorylation. In the presence

o f oligomycin, the formation o f ATP via oxidative phosphorylation was

blocked, and in the case of calcium add ition , high energy intermediates

normally used in ATP synthesis were used fo r the energy-linked calcium

uptake. Under the conditions of th is experiment the resp ira to ry rates

o f the four experiments were nearly iden tica l as were the oxidation-

reduction states o f intram itochondrial pyrid ine nucleotides as measured

by the absorbance o f the mitochondria a t 340 nm.

The in h ib ito ry influence o f g-hydroxybutyrate on phosphoenol-

pyruvate formation in uncoupled (FCCP) mitochondria can be seen in

Figure 12. The rate o f phosphoenolpyruvate synthesis was s ig n if ic a n tly

reduced when g-hydroxybutyrate (5 mM) was included in the incubation

mixture. The metabolism o f g-hydroxybutyrate by the l iv e r involves only

i t s oxidation to acetoacetate thereby generating mitochondrial reduced

pyrid ine nucleotides. Presumably, the results are a d ire c t re fle c tio n

o f a redox mediated e ffe c t on the oxalacetate level o f the mitochondria.

As mentioned previously, a lower oxidized pyrid ine nucleotide to reduced

44

Fi gure

<u 6 —

oXc<u

eCLcn

Q_UJCLv>Q)OEc

8 -

4 -

2 -

No A dd itions

FCCPO ligom ycin

C o**

i— r T10

1-

12

Minutes

11. The E ffect o f Uncoupler (FCCP), Oligomycin and Calcium on the Rate o f Phosphoenolpyruvate Formation in 2-Methyl-1,4- Naphthoquinone Plus Rotenone-Treated Rabbit L iver M ito­chondria. - - The to ta l incubation volume was 6.0 ml and contained 50 mM sucrose, 100 mM KC1, 5 mM KPO4 , 20 mM T ris - chloride (pH 7.2), 3 mM malate, 0.83 yM rotenone, 1.6 pM2-methyl-1 ,4-naphthoquinone, 0.33 pM FCCP, 10 pgm/ml oligomycin and 80 pM calcium chloride.

45

CT>E\

CLLUCL</)

_0)oE

2 .0 -

1.8 -

1.6 -

5 1.4 Hx c 53 o

CL

a - Ketoglutarafc

^ 1.2 -

1.0

8 -

6 -

a -K e tog lu ta ra te plus p - Hydroxybutyratc

4 -

2 -

0

0

Minutes

Figure 12. The E ffect o f 6 -Hydroxybutyrate on Phosphoenolpyruvate Syn­thesis from a-Ketoglutarate in the Presence o f Uncoupler (FCCP). - - The reaction mixture contained 50 mM sucrose,100 mM KC1, 5 mM KPO,, 20 mM T ris -ch lo ride (pH 7 .2), 1.5 mM a-ketog lu tarate , 5 mM 3-hydroxybutyrate and 0.33 pM FCCP.

46pyridine nucleotide ra tio would exert an e ffe c t on the malate dehydro­

genase equilib rium which under these conditions would not favor the

formation of oxalacetate and the oxalacetate level would become the rate

lim it in g consideration.

I t is generally accepted that under certa in gluconeogenic con­

d itions such as fas ting , the l iv e r is exposed to high levels o f free

fa t ty acids. The l iv e r responds to high fa t ty acid uptake in several

ways but o f primary importance is the u t i l iz a t io n of these fa t ty acids

in g-oxidation. The oxidation o f the long hydrocarbon chains of fa t ty

acids occurs in the mitochondria and is the main source o f energy fo r

the gluconeogenic response. As shown in previous experiments, the

oxidation-reduction state o f the mitochondrial oxidized to reduced

pyridine nucleotide ra tio in iso la ted rabb it l iv e r mitochondria exerts

a major influence in the production o f phosphoenolpyruvate from

a-ketoglutarate and malate in the presence o f uncoupler (FCCP). In

these studies a comparison was made on the in h ib ito ry e ffe c t o f the

increasing the level o f reducing equivalents generated in mitochondria

on the rates o f phosphoenolpyruvate production from 4 -carbon substrates.

The substrates fo r 3 -oxidation selected were two ca rn itine deriva tives,

pa lm ity lca rn itine and acetyl ca rn itin e , the th ird substrate was the

e ight carbon fa t ty acid, octanoate. Substrate selection was based on

the widely accepted fac t tha t ca rn itine and i ts acyl derivatives pene­

tra te and cross the inner mitochondrial membrane to reach the locus o f

3 -oxidation which is normally inaccessible to long chain fa t ty acids and

th e ir coenzyme-A derivatives. In the case o f octanoate, the oxidation

47

o f th is short chain fa t ty acid is carnitine-independent. Again, the

depletion o f endogenous high energy phosphate was accomplished by the

presence o f uncoupler (FCCP), while a-ketoglutarate was included to

meet the GIF requirement o f phosphoenolpyruvate carboxykinase. In

these experiments, malate was not included in the incubation mixture.

The experiment shown in Figure 13 demonstrates tha t phosphoenol-

pyruvate formation was in h ib ite d by the oxidation o f octanoate, acety l-

ca rn itine and pa1 m ity lca rn itin e using a-ketoglutarate as the source o f

energy and 4 -carbon units fo r phosphoenolpyruvate synthesis. In an

attempt to explain these re su lts , consideration must be directed toward

the redox state o f the pyrid ine nucleotides as well as substrate

a v a ila b il i ty in terms o f oxalacetate. The energy component is not a

fac to r in th is system, since oxidation o f a-ketoglutarate is more than

s u ff ic ie n t to meet the energy requirements o f phosphoenolpyruvate

carboxykinase. Decreases in the mitochondrial oxidized to reduced

pyrid ine nucleotide ra tio by the addition o f substrate fo r 3 -oxidation may

decrease the a v a ila b il i ty o f mitochondrial oxalacetate the immediate

precursor o f phosphoenolpyruvate. The reduction o f oxalacetate a v a il­

a b i l i t y may be mediated through the displacement o f the malate dehydro­

genase equ ilib rium . A lower NAD+/NADH ra tio when e ithe r acetyl ca rn itine

or octanoate were added (data not shown). Hence, i t is u n like ly that the

strong in h ib it io n o f phosphoenolpyruvate synthesis shown in Figure 13

could be due to a displacement o f the malate dehydrogenase equ ilib rium and

a subsequent deprivation o f the mitochondrial phosphoenolpyruvate car­

boxykinase o f i t s substrate, oxalacetate. The rates o f

48

10-

_ 8 -

oXc0)oqZcnE\

CLLUCLto<DOEc

6 -

4 -

2 -

No Add it ions

Octonoote

-a Accty lcarn itine

Palmitylcarnitine

0i

4

Minutes

Figure 13. The In h ib itio n o f Phosphoenolpyruvate Formation by theOxidation o f Octanoate, Acetyl ca rn itine and P a lm ity lca rn itine in the Presence o f a-Ketoglutarate and Uncoupler (FCCP). - - The incubation mixture contained 25 mM sucrose, 100 mM KOI,5 mM KPO4 , 20 mM T r is -c h i0 ride (pH 7.2), 1.6 mM a-ketoglutarate, 6 mg/ml, defatted bovine serum albumin,20 yM L (-)-p a lm ity lc a rn itin e , 0.66 yM acetyl ca rn itine ,0.66 yM octanoate and 0.33 yM p-trifluoromethoxyphenyl hydrazone o f carbonyl cyanide (FCCP).

49

phosphoenolpyruvate formation fo r both acetyl ca rn itine and octanoate are

s ig n if ic a n tly higher than fo r p a lm tty lca rn itine . The re la tive e ffec ts

o f acetyl ca rn itine and octanoate on phosphoenolpyruvate synthesis are

probably due to the fa c t tha t a ll o f the three co-substrates added

above re su lt in the production o f acetylCoA which could serve as a trap

o f oxalacetate via the c itra te synthase reaction, thus depriving the

system o f oxalacetate fo r phosphoenolpyruvate synthesis. That th is

may be the case is indicated by the sequential increase observed in

c itra te formation as the rate o f phosphoenolpyruvate formation was

diminished as shown in Figure 14.

In previous experiments a b r ie f consideration alluded to the

p o s s ib ility tha t a mechanism(s) e x is t which may p re fe re n tia lly d ire c t the

metabolic u t i l iz a t io n o f oxalacetate through one pathway over another.

In rabb it l iv e r mitochondria under gluconeogenic conditions there are two

enzyme reactions which p o te n tia lly compete w ith phosphoenolpyruvate

carboxykinase fo r the intram itochondria l oxalacetate. These two enzymes

are c itra te synthase and glutam ic-oxalacetic transaminase. The la te r is

the p rin c ip le route fo r the metabolism o f glutamate in mitochondria under

gluconeogenic conditions, e .g . , s ta rva tion . The reaction glutamaic-

oxalacetic transaminase is the pyridoxal phosphate-dependent trans­

amination invo lv ing glutamate, a-ketog lu tarate , oxalacetate and aspartate.

I n i t ia l ly glutamate and oxalacetate serve as co-substrates and are in te r ­

converted to a-ketoglutarate and aspartate in th is reaction. The

interconversion o f a-ketoglutarate and aspartate is an important lin k

which d ire c tly couples the metabolism o f glutamate and carbohydrate

50

Palmifylcarnitino

Acetylcarniline

Ocianoafe o No Additions

2 -

0 J i r 1-----1----- 1----- 1-----1 r0 2 4 6 8

Minutes

Figure 14. The E ffect o f the Oxidation o f Octanoate, Acetyl ca rn itine and Pal m ity lca rn itin e on C itra te Formation in Uncoupler (FCCP) Mitochondria. - - The reaction mixture was iden tica l with tha t described in Figure 13.

formation. Experimentally the primary in te re s t was directed towards

evaluating the competition o f glutamic-oxalacetate transaminase fo r the

available oxalacetate w ith the phoephoenolpyruvate carboxykinase

reaction, under conditions which had previously been shown to favor

phosphoenolpyruvate synthesis, i . e . , uncoupler (FCCP) and a-ketoglutarate.

As shown in Figure 15, glutamate in the presence o f uncoupler (FCCP)

and a-ketoglutarate e ffe c tiv e ly competes fo r the oxalacetate produced

under these conditions, since levels o f phosphoenolpyruvate are s ig n i­

f ic a n t ly lower in the presence o f glutamate than in i t s absence.

Cystiene s u lf in ic acid (CSA), which also competes fo r available

oxalacetate producing a th io l de riva tive and aspartate was shown also to

fa c i l i ta te a greater competition fo r oxalacetate than did glutamate.

This conclusion was based on the lower leve ls o f phosphoenolpyruvate

measured in the presence o f cystiene s u lf in ic acid (CSA) re la tive to

glutamate.

nmol

es

PEP/

mg

Pr

otei

n X

!0"'

52

2 6 -No Additions

2 4 -

2 2 -

2 0 -

18 -

1 6 -

Glulomote (1.66 mM)

1 4 -

12-

1 0 - CSA(l.66mM)

8 -

6 —

4 -

2 -

0 4 862Minutes

Figure 15. The Rates o f Phosphoenolpyruvate Formation in Uncoupler(FCCP) Mitochondria in the Presence and Absence o f Glutamate and Cystiene S u lf in ic Acid (CSA). - - The reaction mixture contained 50 mM sucrose, 100 mM KC1, 5 mM KPO4 , 20 mM T ris - chloride (pH 7.2), 1 mM a-ketoglutarate, 1.66 mM cystiene s u lf in ic acid and 1.66 mM glutamate. The uncoupler (FCCP), 0.33 yM was used to in i t ia te resp ira tion .

CHAPTER 4

DISCUSSION

The enzymatic mechanisms and control aspects o f hepatic gluco-

neogenesis appear to be w idely d ive rs ifie d in animal species. A notable

va ria tion in mammalian hepatocytes is the in tra c e llu la r d is tr ib u tio n o f

phosphoenolpyruvate carboxykinase, a key enzyme in the gluconeogenic

sequence. The mitochondrial form o f the enzyme appears to be immuno-

chemically (Ballard and Hanson, 1969) and physiochemically (D iesterhaft,

Shrago and Sallach, 1971) d is t in c t from the cytoso lic form. In rabb it

l iv e r i t has been shown tha t the cy toso lic form of the enzyme responds

adaptively to gluconeogenic demands such as starvation (Garber and

Hanson, 1971) diabetes (Johnson e t a ! . , 1973) and hormone adm inistration

(Exton, 1972) while the mitochondrial a c t iv ity o f phosphoenolpyruvate

carboxykinase remains la rge ly unchanged (Arinze, Garber and Hanson,

1973). The exact mechanism o f gluconeogenesis is unclear in species such

as the ra bb it in which phosphoenol pyruvate carboxykinase is localized in

both the cytosol and the mitochondria. Any hypothesis attempting to

explain the regulation o f phosphoenolpyruvate production or glucoeno-

genesis must consider the compartmentation o f these two enzymes. In the

present study the energetic requirements and co n tro llin g features o f

mitochondrial phosphoenolpyruvate formation in terms o f a lte ra tions in

the redox state and substrate a v a ila b il i ty were investigated. The

53

54

metabolic sign ificance o f intram itochondria l phosphoenolpyruvate

synthesis in terms o f these regulatory features is discussed.

I t is generally accepted that gluconeogenesis occurs in d ie ta ry

and disease states which promote rapid m obilization and oxidation o f

fa t ty acids. As a consequence the level o f free fa t ty acids in the

l iv e r is elevated under the va rie ty o f conditions in which gluconoe-

genesis is observed. In recent years long chain fa t ty acids have been

shown to stim ulate gluconeogenesis in l iv e r preparations o f various

animal species (Scrutton and U tte r, 1968; Marco and Sols, 1970); i t

has also been suggested tha t the e ffects o f various hormones in stimu­

la tin g gluconeogenesis may be mediated through the release o f fa tty

acids. The gluconeogenic e ffe c t o f glucagon and epinephrine in perfused

l iv e r may be secondary to th e ir stim u la ting the release o f cAMP which

in turn may activa te a lipase in e ithe r l iv e r or adipose tissue resu lting

in the release o f free fa t ty acids in s itu (Exton, 1972). The s ig n i­

ficance o f these observations has led to the hypothesis tha t under con­

d itions which promote gluconeogenesis the concentration o f hepatic free

fa t ty acids is s u ff ic ie n t to stim ulate gluconeogenesis by.the activa tion

o f pyruvate carboxylase due to elevated levels of acetylCoA.

I t has been demonstrated many times in preparations o f rabb it

l iv e r mitochondria tha t the production o f phosphoenolpyruvate from

various substrates could be stimulated by uncouplers such as d in itro -

phenol (DNP) and oleate. (Stanbury and Mudge, 1954; Mudge, Newberg and

Stanbury, 1954; Nordlie and Lardy, 1963; Davis and Gibson, 1969; Garber

and Hanson, 1971). The marked s im ila r it ie s observed in the gluconeogenic

55

response e lic ite d by free fa t ty acids and uncouplers promoted the

extensive use o f uncouplers in the investiga tion o f the regulation o f

mitochondrial phosphoenolpyruvate formation.

In th is present study maximum rates o f phosphoenolpyruvate

formation were observed from trial ate and a-ketoglutarate in the presence

o f uncoupler (FCCP). Under these conditions, the formation o f phos­

phoenol pyruvate is promoted in the presence o f malate and a-ketoglutarate.

Mai ate served as a source of OAA, the immediate precursor o f phos­

phoenol pyruvate, while the oxidation o f a-ketoglutarate supplied the

energy requirements o f phosphoenolpyruvate carboxykinase, namely GTP.

Data from in i t ia l studies previously c ite d , reported lower rates o f

phosphoenolpyruvate formation were observed from malate in the presence

o f succinate even though ATP levels were high and unchanged. This

suggested tha t the oxidation-reduction state o f the pyrid ine nucleotides

assumed a ro le in the regulation o f phosphoenolpyruvate production.

Under physiological conditions gluconeogenesis presumably is accompanied

by d e fin ite a lte ra tions in the intram itochondrial NAD /NADH ra tio . This

s h if t in the redox state o f the pyrid ine nucleotides has been shown to

d ire c tly e ffe c t phosphoenolpyruvate production in ra b b it l iv e r pre­

parations (Davis and Gibson, 1969; Garber and Hanson, 1971; Johnson e t al,,

1973). Also in the present study, sequential a lte ra tio n in the in tra - +

mitochondrial NAD /NADH ra tio was shown to regulate the ra te o f phos­

phoenol pyruvate synthesis. A d e fin ite co rre la tion was observed between

the oxidation-reduction state o f the mitochondrial pyrid ine nucleotides

or the rate o f resp ira tion and the rate o f phosphoenolpyruvate synthesis.

As noted previously, a lte ra tions in the intram itochondria l NAD+/NADH

ra tio may regulate the steady state concentration o f oxalacetate w ith in

the mitochondrial m atrix. This e ffe c t is presumably mediated through

the malate dehydrogenase equ ilib rium . A decrease in the proportion o f +NAD re la tive to NADH decreases the intram itochondria l level of oxal-

acetate, the immediate precursor o f phosphoenolpyruvate. This in turn

s ig n fica n tly decreases the rate o f phosphoenolpyruvate production by

s h ift in g the malate dehydrogenase equ ilib rium toward the formation o f

malate. In contrast, under conditions where the concentration o f NAD+

is increased by uncouplers through accelerated electron transfe r the

dynamic equilib rium o f the malate-oxalacetate couple would be shifted,

toward oxalacetate. Indeed the evidence from experiments in th is study

suggest tha t th is is the ,case. Under conditions where the concentration

o f NAD* was increased by uncoupler (FCCP) through accelerated electron

trans fe r, elevated rates of phosphoenolpyruvate synthesis were observed

as long as a source o f GTP was present.

Energy derived through the oxidation o f a-ketoglutarate was

adequate to sustain a high rate o f phosphoenolpyruvate synthesis.

Correspondingly oligomycin addition in the absence o f uncoupler but in

the presence o f a-ketoglutarate resulted in slow rates o f phosphoenol-

pyruvate formation. Under these conditions the decrease in the con­

centration o f NAD* re la tive to NADH would be associated w ith a

proportional decrease in the steady state level o f oxalacetate. This

could account fo r the slow rate o f phosphoenolpyruvate synthesis demon­

strated in these oligomycin experiments. In these experiments increases

57

in the extent o f oxidation o f mitochondrial pyrid ine nucleotides were

accompanied by corresponding decreases in the mitochondrial ATP leve ls .

Ind icating perhaps tha t the mitochondrial ATP concentration may not be

correlated w ith rates o f phosphoenolpyruvate formation observed in these

experiments. The influence o f the intram itochondria l redox state was

fu rth e r demonstrated by the addition o f 3 -hydroxybutyrate to l iv e r

mitochondria in the presence o f a-ketoglutarate and uncoupler. Under

these conditions 3 -hydroxybutyrate addition in it ia te d a reductive s h if t

in the intram itochondria l pyrid ine nucleotides resu lting in slow rates

o f phosphoenolpyruvate synthesis. I t is concluded from th is evidence,

tha t phosphoenolpyruvate synthesis in iso la ted rabb it l iv e r mitochondria

appears to be favored by a combination o f a rapid rate o f resp ira tion

and an increase in the extent o f oxidation o f the mitochondrial pyrid ine

nucleotides. In add ition , the intram itochondria l ATP level may not

s ig n if ic a n tly influence th is rate o f phosphoenolpyruvate formation as

long as source o f GTP is supplied. This oxidative s h if t in the redox

state o f the pyrid ine nucleotides may be an important event in switching

from carbohydrate u t i l iz a t io n to carbohydrate formation.

In th is study an evaluation o f the energy con tribu tion fo r

maximal rates o f phosphoenolpyruvate synthesis was attempted. The

contribu tion o f substrate level phosphorylation to phosphoenolpyruvate

synthesis was demonstrated with arsenite in combination w ith concen­

tra tions o f uncoupler (FCCP), malate, malonate and a-ketog lu tarate ,

previously shown to stim ulate phosphoenolpyruvate synthesis. Arsenite

was included in these experiments to block the oxidation o f

a-ketoglutarate. The resu lting reduction in the forward rate o f

a-ketoglutarate oxidation in the presence o f arsenite accompanied

s ig n if ic a n tly lower rates o f phosphoenolpyruvate synthesis in th is

system. Arsenite in the presence o f uncoupler showed no a lte ra tions in +

the NAD /NADH ra tio above tha t observed fo r uncoupler alone. Therefore,

suggesting that the reduced rate o f phosphoenolpyruvate synthesis may

be correlated w ith lower leve ls o f GTP produced via substrate level

phosphorylation linked w ith a-ketoglutarate oxidation. The observation

tha t phosphoenolpyruvate is e f f ic ie n t ly produced under these experi­

mental conditions even when the resp ira to ry chain-linked ATP synthesis

is completely blocked shows that substrate level phosphorylation linked

to the oxidation o f a-ketoglutarate is an e f f ic ie n t energy source fo r

phosphoenolpyruvate production. Any augmentation o f phosphoenolpyruvate

synthesis through transphorphorylation is d i f f ic u l t to d is tingu ish in

these experiments. A comparison o f the rates o f phosphoenolpyruvate

production w ith uncoupler plus oligomycin plus arsenite reveals no

s ig n if ic a n t d ifference. In both cases in the absence o f arsenite

maximal rates o f phosphoenolpyruvate synthesis were observed. These

two experiments were designed to d is tingu ish the con tribu tion o f trans­

phosphorylation to phosphoenolpyruvate synthesis. Presumably in the > .

presence o f oligomycin endogenous levels o f ATP may be available fo r

transphosphorylation since the uncoupler stimulated ATPase a c t iv ity

would be blocked. Previous measurements o f ATP levels in an iden tica l

system re flected diminished levels o f ATP in both uncoupler (FCCP) and

uncoupler (FCCP) plus oligomycin and the observed low rates o f ATP

formation may be in terpreted as energy lim it in g in each case. A con­

sideration o f the energy contribution under these conditions suggest

tha t the transphosphorylation o f GDP from endogenous levels o f ATP did

not s ig n if ic a n tly enhance the rate o f phosphoenolpyruvate synthesis

above the level provided by substrate level phosphorylation. Evidence

from e a r lie r studies (Garber and Hanson, 1971) suggests tha t rabb it

l iv e r mitochondria are more dependent on substrate level phosphorylation

fo r the generation o f GXP due to low level o f nucleoside diphospho-

kinase a c t iv ity . In these experiments phosphoenolpyruvate was shown to

be e f f ic ie n t ly produced from malate and a-ketoglutarate in the uncoupled

system even when resp ira tory chain-linked ATP synthesis was completely

blocked, showing tha t the substrate level phosphorylation linked to the

oxidation o f a-ketoglutarate is an e f f ic ie n t energy source fo r phos­

phoenol pyruvate formation.

Additional experiments in which the mitochondrial energy level

was regulated somewhat independently o f the rate o f resp ira tion by the

presence of 2 -m e th y l- 1 ,4 -napthoquinone reveal tha t adequate mito­

chondrial ATP levels were generated in the presence o f a high NAD+/NADH

ra tio . The energy supply was s u ff ic ie n t to promote phosphoenolpyruvate

synthesis in the presence o f malate alone. The GTP fo r phosphoenol-

pyruvate formation would be p rim a rily derived through transphosporylation.

This assumption is substantiated through a comparison o f rates of

phosphoenolpyruvate synthesis in the presence o f uncoupler, calcium and

an in h ib ito r o f oxidative phosphorylation. In each o f the three

experiments comparable resp ira to ry rates and intram itochondrial redox

60

states were observed. When resp ira to ry linked oxidattve-phosphorylation

was blocked as in the presence of oligomycin, the rates o f phosphoenol-

pyruvate synthesis were v ir tu a l ly abolished. In the case o f calcium

addition high energy intermediates normally used in ATP synthesis were

used fo r the energy-linked calcium uptake. In the presence o f calcium

a reduction of available ATP levels is re flected by the lack o f phos-

phoenolpyruvate formation as was the case also fo r uncoupler (FCCP).

Under each o f the conditions the common fac to r shared is tha t ATP was

not available fo r transphosphorylation, as a.consequence the GTP

requirement fo r phosphoenolpyruvate carboxykinase was not met and no

phosphoenolpyruvate formation was observed.

As noted previously the steady state level o f mitochondrial

oxalacetate may be determined by the malate dehydrogenase equilibrium

and i ts in te rac tion w ith the mitochondrial pyrid ine nucleotides and the

intram itochondrial level o f malate. The results o f th is study c le a rly

show in in ta c t rabb it l iv e r mitochondria tha t other metabolic pathways

also assume an important ro le in in fluencing the steady state con­

centration o f oxalacetate.

The experiments u t i l iz in g p a lm ity lca rn itin e , acetyl ca rn itine

and octanoate as sources o f acetylCoA indicated tha t the steady state

oxalacetate concentration may be affected by factors other than merely

the mitochondrial redox state o f the pyrid inq nucleotides. In these

experiments the energy component was not a fac to r since the energy

requirement in terms o f GTP was s a tis fie d via the oxidation o f

a-ketoglutarate. A previous explanation o f the low rate o f

phosphoenolpyruvate formation alluded to the p o s s ib ility tha t each o f

the B-oxidation cosubstrates supplied acetylCoA at a concentration which

may e ffe c tiv e ly compete fo r the available oxalacetate through the

c itra te synthase reaction. This la te r explanation is favored over the+influence o f redox change in view o f the minimal change in the NAD /NADH

ra tio observed in the presence o f each o f the cosubstrates and the fa c t

tha t c itra te synthesis was s ig n if ic a n tly stimulated under these

experimental conditions. In view o f these resu lts the in te rac tion o f

c itra te synthase competition fo r the available oxalacetate assumes a

prominent ro le . The resu lts o f these experiments c le a rly show tha t

pal m ity lca rn itin e which would be expected to provide the highest level

o f acetylCoA has the highest rate of c itra te formation and the slowest

rate o f phosphoenolpyruvate production. Correspondingly the reduced

rates o f phosphoenolpyruvate formation observed fo r acetyl ca rn itine and

octanoate are re flected in elevated rates o f c itra te synthesis. The

experiments indicate tha t c itra te synthase may e ffe c tiv e ly compete fo r

available oxalacetate when acetylCoA is read ily ava ilab le . The formation

o f c itra te may be regulated by the a v a ila b il i ty o f oxalacetate through

the oxidation-reduction state o f the mitochondrial pyrid ine nucleotides

which a ffects the malate to oxalacetate ra tio . In these experiments»

the absorbance measurements a t 340 nm revealed a decrease in the NAD*/

NADH ra tio through the addition o f uncoupler (FCCP) but only a minimal

change in the presence o f pal m ity lca rn itin e and no change in the presence

o f acetyl ca rn itine and octanoate above tha t observed w ith uncoupler

alone. I t is concluded from these resu lts tha t under metabolic

62

s itua tions such as high levels o f acetylCoA, additional competition fo r

oxalacetate by c itra te synthase may fa c i l i ta te fu rth e r reduction in

the rate o f phosphoenolpyruvate formation.

Another example o f the competition, fo r available oxalacetate or

a primary regulator o f phosphoenolpyruvate synthesis is at the level

o f glutamate-oxalacetic transaminase (GOT). The pyridoxal phosphate-

dependent transamination o f glutamate via th is enzyme represents an

important pathway o f flow of th is amino acid to glucose and emphasizes

the central ro le tha t continued glutamate u t il iz a t io n must play in

gluconeogenesis. The a c t iv ity o f th is enzyme appears to be correlated

w ith the glutamate to a-ketoglutarate ra tio . Increasing the ra tio o f

glutamate to a-ketoglutarate favors the net formation o f aspartate from

glutamate and oxalacetate. I t would be expected then under metabolic

conditions where the level o f oxalacetate might be c lose ly regulated

tha t additional competition fo r oxalacetate by GOT and phosphoenol-

pyruvate carboxykinase may fa c i l i ta te fu rth e r reduction in the rate o f

phosphoenolpyruvate formation. Prelim inary experiments in th is study

c lea rly demonstrate the competitive influence exerted by glutamate fo r

oxalacetate. A comparison of the rates o f phosphoenolpyruvate formation

in the presence and absence o f glutamate strong ly supports the o b li­

gatory e ffe c t o f oxalacetate a v a ila b il i ty in order fo r maximal phos­

phoenol pyruvate synthesis to occur.

In summary, the regulation o f phosphoenolpyruvate formation in

iso la ted rabb it l iv e r mitochondria appears to be influenced by at least

three metabolic fac to rs : ( 1 ) the oxidation-reduction state o f the '

in tram itochondrial pyrid ine nucleotides; (2 ) the energy fo r phos-

phoenolpyruvate carboxykinase; and (3) the extent o f competition fo r

the steady state level o f oxalacetate by other enzymatic reactions.

The in te raction o f these three co n tro llin g features is essential in

the mechanisms governing gluconeogenesis in rabb it l iv e r .

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