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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