University of Southern California Dissertations and Theses

Studies Of Relationships Between Insulin And Cyclic Nucleotide Phosphodiesterase In Normal And Diabetic Rats

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Studies Of Relationships Between Insulin And Cyclic Nucleotide Phosphodiesterase In Normal And Diabetic Rats

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Content STUDIES OP RELATIONSHIPS BETWEEN
INSULIN AND CYCLIC NUCLEOTIDE PHOSPHODIESTERASE
IN NORMAL AND DIABETIC RATS
by
John Glenn Hemington
A Dissertation Presented to the
FACULTY OP THE GRADUATE SCHOOL
UNIVERSITY OP SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OP PHILOSOPHY
(Biological Sciences)
August 1971
I
72-6062
HEMINGTON, John Glenn, 1938-
STUDIES OF RELATIONSHIPS BETWEEN INSULIN
AND CYCLIC NUCLEOTIDE PHOSPHODIESTERASE
IN NORMAL AND DIABETIC RATS.
University of Southern California, Ph.D.,
1971
Chemistry, biological
University Microfilms, A X E R O X Company, Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED
UNIVERSITY O F SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 9 0 0 0 7
This dissertation, written by
John G lenn Hemington
under the direction of h.is... Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by The Gradu
ate School, in partial fulfillment of require
ments of the degree of
D O C T O R O F P H I L O S O P H Y
Dean
Da/e...Sep.tember..l971____
DISSERTATION COMMITTEE
PLEASE NOTE:
Some Pages have i n d i s t i n c t
p r i n t . Filmed as re ceiv ed .
UNIVERSITY MICROFILMS
For Sue,
who
taught me to live and
to love.
ii
ACKNOWLEDGMENTS
It gives me great pleasure to finally acknowledge
those individuals so necessary to the realization of this
dissertation. I especially wish to thank Dr. Arnold S.
Dunn for his personal kindness and help during my graduate
years. He exemplifies all that is best of the academician
by creating an atmosphere of intellectual freedom and
creativity.
Since many of the biochemical aspects of this
research were unfamiliar to me, some individuals contrib
uted significantly to my understanding of the methods and
ideas central to this dissertation. Drs. M. Michael
Appleman and W. Joseph Thompson were essential. This work
could not have been done without their advice, counseling,
and technical expertise.
Drs. Dinesh Kumar and Edward R. Arquilla also con
tributed suggestions on methods and interpretations which
were invaluable.
I also wish to acknowledge Drs. Peter M. Shugarman
and Robert M. Chew for their guidance and help during
these recent years.
In my eleventh hour as a graduate student I wish to
thank my colleagues over the years: Drs. Dan Schaeffer,
iii
Dan Fertig, and Wayne Barcelona. Their friendship and
advice has sustained me. Finally, to those friends with
whom I worked so closely, Tom Russell, Maymie Chenoweth,
Karen Bever, Wes Terasaki, Pat Lissner, and Anita Larks,
they have been invaluable and tolerant during our many
hours of discussions and helpful in many areas of this
research.
iv
TABLE OP CONTENTS
Page
ACKNOWLEDGMENTS..........................................iii
LIST OP TABLES......................................... vii
LIST OP FIGURES........................................ viii
LIST OP ABBREVIATIONS................................ x
Chapter
I. INTRODUCTION................................... 1
General
Circulating and Tissue Source of Insulin
like Activity
Insulin Binding
Metabolic Pate of Insulin
Role of Insulin and Cyclic AMP in
Regulation of Metabolism
Mechanisms for Insulin Effects on
Cyclic AMP Levels
Cyclic Nucleotide Phosphodiesterase
II. MATERIALS AND METHODS........................ 25
Animal Sources and Maintenance
Whole Animal Studies
Tissue Preparations
Chromat ography
Assays
Statistical Analysis
III. RESULTS....................................... 34
Whole Animal Studies
Studies of Tissue Preparations
Plasma
Liver
Other Tissues
v
Chapter Page
IV. DISCUSSION..................................... 91
Liver Cyclic Nucleotide Phosphodiesterases
Insulin and Cyclic Nucleotide
Phosphodiesterases
Insulin, Insulin-Binding, and Insulinase
V. SUMMARY AND CONCLUSIONS........................ 102
REFERENCES.............................................. 104
vi
LIST OP TABLES
Table
1. Characteristics of Diabetic Rats and
Comparisons Between Normal and Diabetic
Rat Plasma Parameters ........................
2. Cyclic Nucleotide Phosphodiesterase Activ
ity and Immunoinsulin Content of Normal
and Diabetic Rat Liver Preparations .........
3. Separation of Immunoinsulin Activity From
Enzyme Activity and Recovery of Insulin
Prom Rat Liver Gel Filtration Concentrates. .
LIST OP FIGURES
Figure Page
1. Percent Change in Plasma Glucose, Insulin,
Cyclic AMP Hydrolysis, and Cyclic GMP
Hydrolysis During a Glucose Tolerance Test
in Normal Fasted Rats........................ 37
2. Chromatographic Profiles of Rat Plasma. . . . 4l
3. Cyclic GMP Hydrolysis by Rat Plasma......... 43
4. Cyclic AMP Hydrolysis by Rat Plasma......... 45
5. Results of Agarose Gel Filtration of Rat
Kidney......................................... 51
6. Chromatographic Profiles on Agarose Gel of
a Liver Preparation Stored for 16 days
at 4°C......................................... 53
7. Cyclic AMP Hydrolysis by Fraction II
Enzyme in Liver.............................. 56
8. Cyclic AMP Hydrolysis by Partial Separated
Fraction III Enzyme in Liver................. 58
9. Stages in the Appearance of Fraction III
Enzyme in Liver.............................. 61
10. Appearance of Fraction III Enzyme and
Relationship Between Phosphodiesterase
Forms and Immunoinsulin Content............. 64
11. Separation of Immunoinsulin and
Phosphodiesterase Activity by Gel
Filtration..................................... 68
12. Comparison of Normal and Diabetic Liver
Chromatographic Profiles...................... 70
viii
Figure Page
13. Relationships Among Various Activities
From a Single Liver Preparation ............. 75
14. Separation of Insulin and Other Proteins
By Gel Filtration of Concentrated Liver
Phosphodiesterase Preparation ............... 79
15. Activity Profiles After Gel Filtration
of a Kidney Preparation...................... 84
16. Activity Profiles After Gel Filtration
of Two Kidney Preparations. . . ........... 86
17. Activity Profiles After Gel Filtration
of a Red Blood Cell Preparation............. 89
ix
LIST OP ABBREVIATIONS
5'-AMP 5'-adenosine monophosphate
BSA Bovine serum albumin
CPM Counts per minute
Cyclic AMP Cyclic 3 5 ’-adenosine monophosphate
Cyclic GMP Cyclic 3’,51-guanosine monophosphate
°C
Degrees centigrade
E
V
Elution volume
E
vo
Void volume
B
Gram
ILA Insulin-like activity
Kg
kilogram
Km Michaelis-Menton constant
X
Lambda (10-^ liter)
yU Microunit
mg Milligram
mg# Milligrams per 100 milliliters
ml Milliliter
mM Millimolar
M Molar
NSILA Non-suppressible insulin-like activity
PDE Cyclic nucleotide phosphodiesterase
pH
-log hydrogen ion concentration
RBC Red blood cells
S.E.M. Standard error of the mean
Tris tris (hydroxymethyl) aminomethane
v Volume
xg • Times gravity
w Weight
xi
CHAPTER I
INTRODUCTION
Although insulin has been isolated and used suc
cessfully in the treatment of diabetes for 50 years, its
mechanism of action is unknown despite exhaustive
research. Insulin has been shown to reduce cyclic
31,5'-adenosine monophosphate (cyclic AMP) levels in
perfused livers (Jefferson, et al., 1968) and adipose
tissue (Butcher, et al., 1966). One mechanism suggested
for this effect is insulin activation of cyclic nucleotide
phosphodiesterase (PDE), the enzyme(s) which specifically
degrades cyclic AMP to 5*-adenosine monophosphate.
Insulin activation of this enzyme(s) has been reported
for adipose tissue, skeletal muscle, intact liver, and
partially purified phosphodiesterase from heart (Schultz,
Senft and Munske, 1966; Senft, et al., 1968, and Loten
and Sneyd, 1970).
This dissertation describes research on this rela
tionship between insulin and PDE in normal and diabetic
rats. These investigations show that multiple forms of
PDE, a high molecular weight insulin binding fraction,
presumably protein, and insulin-degrading activity
(insulinase) occur in the same agarose gel column frac
tions. Endogenous insulin can be separated from these
1
2
: activities by various methods. The introduction which
follows reviews pertinent literature concerned with
insulin and insulin effects on physiological processes
influenced by cyclic AMP levels.
General
; Diabetes mellitus is among the most prevalent of
metabolic diseases. With increasingly sensitive methods
■ of diagnosis it has been shown that diabetes exists in
: approximately 10% of the population (Butterfield, 1969).
Diabetes mellitus is characterized by an impaired glucose
tolerance test probably brought about by either lack of
insulin or failure of insulin action. Since insulin was
discovered in 1921 by Banting and Best (see Best, 1964),
there has been a continuously increasing interest in this
hormone. In the past decade, numerous reviews of current
understanding and research have been presented (e.g.,
Krahl, 1961; Williams and Ensinck, 1966; Prohman, 1969;
Ostman and Milner, 1969). A variety of other reviews
dealing with more limited aspects of insulin metabolism
: or effects have also appeared (e.g., Segal, 1964; Luken,
1964; and Steiner, 1966). ;
Recently cyclic AMP has been implicated in the
I mechanism of action of insulin. Prom the time that cyclic ,
3
AMP was described by Rail, Sutherland, and Berthet (1957),
scientific literature concerning this nucleotide has
increased in a logarithmic fashion (Yost and Rickenberg,
1971). Cyclic AMP has also been the subject of numerous
reviews (e.g., Robison, Butcher, and Sutherland, 1968;
Greegard and Costa, 1970; and Hardman, Robison and
Sutherland, 1971); some reviews have been limited to car
bohydrate metabolism and insulin action (e.g., Sneyd,
Corbin, and Park, 1968; Park, et al., 1969; and Sutherland
and Robison, 1969).
Sutherland and associates have developed the
"second messenger" hypothesis which unifies many hormonal
effects on cellular metabolism via control of intracellu
lar levels of cyclic AMP. In this hypothesis, a hormone,
the first messenger, activates membrane bound adenyl
cyclase by an unknown mechanism and cyclic AMP, the second
messenger, is then formed from ATP. This in turn activates
the appropriate metabolic system. In order to return the
system to the metabolic state existing prior to hormonal
activation, cyclic AMP is hydrolyzed to 5'-AMP by specific
cyclic nucleotide phosphodiesterases (Sutherland and
Robison, 1966).
n
Circulating; and Tissue Sources of
Insulin-like Activity
Since the introduction of the radioimmunoassay for
insulin (Yaldw and Berson, i960) there has been consider
able uncertainty as to the state of insulin-like activity
(ILA) in the blood. ILA is composed of at least two
forms: free or typical insulin and non-suppressible
insulin-like activity ("bound" or "atypical" ILA). The
majority of research and the different viewpoints regard
ing these different forms is reviewed in Ostman and Milner
(1969). The distinction between the two is based primar
ily on their ability to react with the insulin antibody.
Insulin will react with the insulin antibody and can be
measured by both immunoassays and bioassays. Biological
effects (e.g., glucose uptake, glycogen deposition) of
this insulin can be suppressed by excess anti-insulin
serum. Non-suppressible insulin-like activity (NSILA)
does not react with insulin antibody and the biological
activity cannot be suppressed by insulin antibodies.
NSILA can only be measured by bioassay methods.
The liver has assumed central importance as the
site of the formation of NSILA. Antoniades (1965) has
developed the hypotheses that NSILA is formed in liver
from insulin secreted by the pancrease. Gershoff, Welch,
and Antoniades (1970) report the rapid loss of insulin
5
concomitant with the appearance of NSILA in the perfused
rat liver, which they interpret as conversion of insulin
into NSILA. However, no direct conversion of ILA to NSILA
has been demonstrated (Froesch, Jakob, and Labhard, 1969).
The early literature dealing with the extraction of
ILA from numerous plant and animal tissues has been
reviewed by Best (see Best, 1964). Best discarded all
these early reports of extra-pancreatic insulin because
of what he judged to be inadequate assays and extraction
techniques. Recently, more than 100 cases of tumors
accompanied by hypoglycemia have been reported. Insulin
like activity (determined by bioassay) has been extracted
from many of these tumors, including hepatomas. ILA could
not be detected, however, in these tumors by immunoassay,
(August and Hiatt, 1958; Lipsett, et al., 1964). Pansky,
House, and Cone (1965) have extracted ILA from thymus
glands which is both immuno- and bio-assayable. They did
not find immunoinsulin in calf liver. Arquilla, et al.,
{1970) have reported immunoassayable insulin in both
nuclear and non-nuclear fractions of mouse liver. The
liver content of ILA was reduced in alloxan diabetic mice.
Williams and Ensick (1966) have extracted NSILA from many
animal tissues. Beck, et al. (1966a) extracted immuno
insulin from kidney and liver tissues after intravenous
infusion or addition of insulin to tissue preparations.
6
Injected insulin produced higher tissue concentrations of
immunoinsulin than did in vitro addition of insulin.
Most of this immunoinsulin activity was found in mito
chondrial and microsomal fractions.
Prom the results of these studies, at least two
forms of insulin-like activity occur in the circulation
and can be extracted from extra-pancreatic tissues. The
form that is detectable by both immuno- and bio-assays is
generally agreed to be free insulin. There has been no
unanimity of results on the source, nature, or physiologi
cal significance of non-suppressible insulin-like activity
Insulin Binding
Early evidence for insulin binding to tissues was
presented by Stadie, et al. (1949). Since then most
studies of insulin binding and metabolism have relied on
the use of iodinated insulin preparations. Conflicting
results have appeared apparently due to variations in the
preparation of iodinated insulin (see Izzo, et al., 1966)
Mortimore and Tietze (1959) suggested that two separable
processes are involved in insulin metabolism in liver:
binding and degradation. They perfused rat livers at 3°
and 37°C with 1^'LI-insulin. At 37°C, iodoinsulin was
1 0*1
rapidly degraded:40$ of the I appeared as TCA non-
precipitable CPM after a single pass through the liver.
At 3°C, however, iodioinsulin was retained by the liver
and not degraded until the temperature was increased. At
both temperatures the rate of insulin degradation as
131
measured by TCA non-precipitable I was decreased by
addition of unlabeled insulin to the perfusion fluid.
Freychet, Roth, and Neville (1971) described the
preparation of biologically active monoiodoinsulin and
demonstrated binding to isolated fat cells and purified
125
plasma membranes from liver. ^I-insulin binding was
inhibited 20-90$ by unlabeled insulin at physiological
-9 -6
concentrations ranging from 10 ^ to 10 M. House and
Weidemann (1970) compared insulin binding activity in
three plasma membrane fractions from rat liver. They
found maximal binding and maximal phosphodiesterase activ
ity [bis(p-nitrophenyl) phosphate substrate] in a single
fraction which they suggested is from liver parenchymal
cells. Edelman and others (1963, 1966) reported specific
131
binding of I-insulin to rat muscle membranes and dis
placement with thiol compounds.
The insulin receptor has been most studied in fat
cells. Fong, et al. (1962) demonstrated increased
131
release of I-insulin from rat hemidiaphragms and fat
pads after treatment with thiol compounds (including 2-
mercaptoethanol). Minemura and Crofford (1969) found that
rat epidymal fat cells treated with p-chloromercuibenzene-
sulfonic acid exhibited increased glucose transport and
inhibited lipolysis. They concluded that these insulin
like effects result from a combination of the reagent with
sulfhydryl containing elements at or near the cell surface
Kono (1969a, 1969b) treated fat cells with trypsin and
destroyed the insulin sensitivity of the cells (increased
glucose transport and inhibition of lipolysis). Beyond
30 minutes after the trypsin treatment insulin responsive
ness returned; recovery was inhibited by puromycin and
cycloheximide. He suggested that the insulin effector
system contains a rapidly renewable peptide element on the
surface of the cell. Cuatrecasas (1969) bound insulin to
agarose polymers; the complex still had biological activ
ity (increased glucose utilization and suppressed
lipolysis) when added to isolated fat cells. He states
that the primary action of insulin is at the cell membrane
since the complexed insulin could not enter the fat cell.
Recently, Cuatracasas (1971) has described some properties
of the insulin receptor in fat cell and hepatoma tissue
culture cells. He emphasizes the importance of correcting
for non-specific binding which is not temperature depen
dent or saturable; specific binding is both temperature
dependent and saturable. The receptor is highly specific
and tightly binds insulin (dissociation constant
5-6 x 10""'^). He was able to quantitatively recover total
binding activity and insulin after homogenization and con
cluded that insulin was not bound inside the cell or
chemically altered by binding. Park, Crofford and Kono
(1968) found that insulin is rapidly bound to fat cells
and was not released but slowly metabolized. Insulin
binding was prevented by trypsin treatment and maleimide,
which presumably blocks sulfhydryl groups. Glucose trans
port and adenyl cyclase activity were not affected by
trypsin, indicating that the insulin receptor is a sepa
rate entity.
Once released into the circulation, the first step
in the action of insulin is to bind to a specific receptor
in the plasma membranes of the target cells. This binding
may involve some form of specific interaction with
sulfhydryl groups of the receptor protein. Whether or not
insulin is degraded in this process is not known. The
insulin-receptor complex may then initiate by some unknown
mechanism, changes in processes at other cellular sites.
Metabolic Pate of Insulin
The metabolic fate of radioidoinsulin has been
extensively studied in numerous species. The liver has
| clearly been established as a dominant site of disposition
10
of Insulin and has been considered the prime organ In
maintaining insulin homeostasis (Izzo, et. al., 1966;
Elgee, Williams, and Lee, 1954). The amount of insulin
removed by the liver from the portal circulation appears
to be a function of the amount released by the pancreas
since the percent of insulin removal increases 2-3 fold
during periods of increased insulin secretion (Field,
Webster, and Drapanas, 1968; Kadin, et al., 1971).
The importance of the kidney in insulin metabolism
has been demonstrated by the high levels of injected
iodoinsulin found there (Izzo, et al., 1966; Elgee,
et al., 1954). Studies of renal arterial-venous insulin
concentration differences (Chamberlain and Stimmler, 1967)
suggest that insulin is either completely reabsorbed or
destroyed in the proximal tubule and that insulin is uti
lized in renal metabolism. Proximal tubular reabsorption
of intact insulin has also been reported by Beck and
Fedynskyj (1967) based on both immunological and auto
radiography studies.
Additional evidence for the movement of insulin
into and across both kidney and liver cells has been
reported by other investigators. Lopez-Ouijada and Goni
(1967) and Daniel and Henderson (1968) report high levels
of immunoinsulin and iodoinsulin in bile. The insulin
concentration in bile is greater than found in the portal
11
circulation. Jeffcoate (1968) has studied the inter
ference of bile acids with the immunoassay of insulin and
concludes that only a part of the immunoreactive insulin
found in bile is genuine insulin. Beck, et al. (1966b),
126
after injecting a mixture of ^I-insulin and native
126
insulin, find both immunoassayable and ■'I counts in
tissue preparations of kidney and liver. Insulin added
to homogenates was not concentrated by liver particles
(presumably mitochondria and microsomes) brought down
between 700 x g and 105,000 x g. Lee (1957) has reviewed
the disposition of iodinated insulin in tissues and
together with his results suggests that intact insulin
molecules can penetrate liver and kidney cells. Lee and
Wiseman (1959) studied the intracellular fate of ^'*'1-
insulin in rat liver after intravenous injections. They
found the majority of radioactivity was associated with
mitochondrial and microsomal fractions. This radioactivity
was completely precipitable with TCA and could not be
removed by washing the fractions. Furthermore, the bound
181
I-insulin was less susceptible to degradation by
endogenous or added insulinase activity.
That insulin bound to antibodies is protected from
insulin degrading activity has been reported by several
other investigators. Yalow and Berson (1957) found that
bound insulin is protected from insulinase activity in
12
liver homogenates. Varandani and Tomizawa (1965) reported
that insulin bound to antibodies is protected from puri
fied beef liver glutathione-insulin transhydrogenase.
Brush and Kitabchi (1970) studied the uptake and
131
release of I-insulin in rat diaphragm. Cell debris
from homogenates (700 x g) contained 5 and 25 times as
much radioactivity as mitochondria and microsomes,
respectively. Most radioactivity was found in the
100,000 x g supernatant; 70$ of this was degraded insulin.
Unlabeled insulin diluted the radioactivity in only the
supernatant fraction which was precipitable by 60$
ammonium sulfate. Based on Sephadex G-50 gel filtration,
they suggested an insulin-specific binding component in
this fraction. Two insulin-degrading enzymes were found,
one in the supernatant and one in the cell debris fraction.
Both were active without added glutathione suggesting that
the enzymes are not glutathione-insulin transhydrogenase.
Similar enzymes have been found in rat skeletal muscle,
liver, and brain (Brush and Cheng, 1968) and are probably
proteolytic in nature.
Insulinase activity is widely distributed in
tissues; the greatest activity per gram of tissues occurs
in homogenates of liver. Very little activity is found
in brain and red blood cells (Mirsky, 1957). Tomizawa and
Halsey (1959) partially purified an insulin-degrading
13
enzyme from beef liver. Katzen and Stelten (1962) have
characterized and named this enzyme glutathione-insulin
transhydrogenase. This enzyme cleaves disulfide bonds
and does not hydrolyze peptide bonds, as is the case with
insulin degradation in adipose tissues (Rudman, et al.,
1966; Williams and Ensinck, 1966).
Mahler and Szabo (1967) after either in vivo or in
vitro alloxan treatment greatly reduced insulinase activ
ity of kidney, normally an insulin insensitive organ
(Levine, 1955). Under these conditions, kidney slices
responded to insulin by increased glucose uptake. They
suggest that insulin acts intracellularly and effects are
not normally seen because of the rapid inactivation of
insulin.
Based on the results of these studies, insulin can
penetrate into liver, kidney, and muscle cells. Whether
or not some specific transport system is involved is
unknown. The intracellular insulin is probably bound to
some cellular component and protected from insulinase
activity present in these tissues.
14
Role of Insulin and. Cyclic AMP
In Regulation of Metabolism
According to Sneyd, Corbin, and Park (1968):
In perfused rat liver Insulin Inhibits gluconeogene-
sis, glycogenolysis and potassium output whereas
glucagon and epinephrine stimulate these processes
and at the same time raise tissue cyclic AMP levels.
Insulin has been found to lower cyclic AMP in
livers perfused with glucagon, and it appears to
lower the cyclic AMP in livers perfused with buffer
alone. Furthermore, the cyclic AMP level in the
livers of alloxan diabetic rats is considerably
higher than in controls. .. . .It seems likely that
this lowering of liver cyclic AMP levels produced
by insulin accounts for many of its actions.
The regulation of glycogen synthesis and breakdown
is the best model of the second messenger hypothesis and
the role of cyclic AMP at the molecular level (Krebs,
et__al., 1966; Villar-Palasi and Larner, 1970; Schaeffer,
1969). The classical antagonistic effects of insulin and
glucagon on glucose production from hepatic glycogen has
been demonstrated in perfused liver studies (e.g., Miller,
1961; Menahan and Wieland, 1969) and by whole animal
isotope studies (e.g., de Bodo, et al., 1963). These
antagonistic effects have been extended to opposite effect
on intracellular cyclic AMP levels (Exton and Park, 1968;
Exton, et al., 1970). The activation and inactivation
of the liver glycogen synthetase system by insulin and
glucagon, respectively, has been reported by Bishop and
15
Larner (1967). Jungas (1966) has observed both Increases
In glycogen synthetase (I form) and decreases In phos-
phorylase a In response to insulin in adipose tissue.
However, Glinsmann and Mortimore (1968) compared the
effects of insulin and glucagon on glucose output,
glycogen levels and phosphorylase activity in the perfused
rat liver after a period of equilibration and obtained
conflicting results. Insulin alone had only a small
effect on hepatic glucose production, however, after
glucagon infusion, insulin caused a marked reduction in
the glucagon stimulated glucose output. These effects
were not related to phosphorylase activity since insulin
alone produced only a small reduction in phosphorylase
activity and had no effect on glucagon activated
phosphorylase.
Exton, et al. (1970) have recently reviewed the
evidence for the control of gluconeogenesis by hormones
and the evidence for cyclic AMP as the mediator of the
observed hormonal effects. Infusion of glucagon, epine
phrine or cyclic AMP in perfused liver produces a
"crossover point" in the levels of gluconeogenic inter
mediates between pyruvate and phosphenolypyruvate indicat
ing that these hormones effect some rate limiting step at
this point in the pathway. This pathway involves intra-
and extra-mitochondrial reactions, and they suggest that
16
cyclic AMP might somehow act via a "cascade" as In the
case with phosphorylase activation or effect mitochon
drial transport systems.
Many studies of the action of insulin on the regu
lation of glycogen metabolism are consistent with the
hypothesis that regulation is mediated via control of
intracellular levels of cyclic AMP. Insulin reduces
cyclic AMP concentrations which in turn inactivates the
enzymatic cascades terminating in reduced phosphorylase a
and increased glycogen synthetase (I form). However,
conflicting reports have appeared. Whether or not this
reduction of intracellular cyclic AMP levels caused by
insulin can be extended to include other metabolic effects
of insulin (e.g., transport, gluconeogenesis) has yet to
be established.
A variety of studies have demonstrated insulin
effects on transport and intracellular events (mediated by
intracellular cyclic AMP levels) and that these processes
can be separated. In adipose tissue, insulin stimulates
glucose uptake and inhibits lipolysis. In a glucose free
medium (i.e., no glucose transport), insulin will inhibit
lipolysis in fat cells (Jungas and Ball, 1963;
Chlouverakis, 1967). Furthermore, Halperin and Robinson
(1971) reported that insulin increases pyruvate conversion
to fatty acids. They suggested that insulin increases the
17
binding affinity (lowers the apparent Km) of pyruvate or
some precusor in a rate limiting step in the pathway.
Park, et al. (1969) reported that insulin alone lowered
- cyclic AMP levels but did not inhibit lipolysis, however,
after epinephrine stimulation insulin did reduce cyclic
AMP and glycerol release. Further, insulin was shown to
increase glucose transport with no change in cyclic AMP
levels while epinephrine increased cyclic AMP levels with
no change in transport of glucose.
In rat diaphragm, Huijing, et al. (1969) reported
in vitro inhibition of glycogen synthetase phosphatase by
glycogen; no hormonal effects (epinephrine or insulin) on
percent glycogen synthetase (I form) were noted. Both
in vitro and in vivo insulin stimulated glucose uptake,
however, only in vivo did insulin increase the percent
. glycogen synthetase I. Craig, Rail, and Larner (1969)
observed increases in synthetase I activity after insulin
treatment with no change in cyclic AMP levels. They also
reported an insulin effect on glycogen synthetase after
epinephrine treatment with no reduction in the elevated
cyclic AMP levels. Goldberg, et al. (1967) found that
; insulin increased both the percent of glycogen synthetase
: (I form) and intracellular levels of cyclic AMP.
These results are difficult to reconcile to a
; single mechanism of insulin action, mediated by reduced
i cyclic AMP levels. The well documented effects of
18
Insulin on glucose transport In fat cells and muscle may
be Independent of cyclic AMP concentrations. Glinsmann
and others (1969s 1970) suggest that the perfusate con
centration of glucose, which Is freely permeable In liver
(Cahill, et al., 1958), Is sufficient to regulate
hepatic glucose production. Higher concentrations of
glucose increased the percent of glycogen synthetase (I
form) and decreased phosphorylase a activity with no change
in cyclic AMP concentrations. These results suggest that
insulin effects on glycogen metabolism may be independent-
of cyclic AMP concentrations under certain conditions.
Mechanisms for Insulin Effects on
Cyclic AMP Levels
Cyclic AMP concentrations could be reduced by
insulin inhibition of adenyl cyclase or insulin activation
of cyclic nucleotide phosphodiesterase. Experiments
investigating these two mechanisms have produced incon
sistent results. Jungas (1966) reported that insulin
I reduced adenyl cyclase activity by 30$. Pain, Kovacev,
and Scow (1966) found that insulin partially inhibited
lipolysis induced by adrenocorticotrophic hormone and
i theophylline treatment of fat cells. They concluded that
I insulin exerted effects on both the formation and breakdown
I 19
of cyclic AMP. Numerous studies have failed to find
insulin effects on adenyl cyclase in fat cells (e.g.,
; Vaughan and Murad, 1969; Rodbell, 1967). However, recent
studies on adenyl cyclase activity in liver have reported
significant insulin inhibition. Ray, Tomasi and Marinetti
(1970) have reported inhibition of basal and glucagon
stimulated adenyl cyclase activity by insulin. Hepp (1971),
reported that insulin and NSILA both inhibit glucagon but
not fluoride stimulated adenyl cyclase in physiological
concentrations that are dose dependent.
A second mechanism which would reduce cyclic AMP
: levels is the activation of cyclic AMP phosphodiesterase.
Schultz, Senft, and Munske (1966) report stimulation of
PDE by insulin in liver. However, Menahan, Hepp, and
Wieland (1969) were unable to detect any changes in liver
PDE activity of insulin perfused liver or upon addition of
insulin in vitro to PDE assays systems. Studies of PDE
from adipose tissue have also produced inconsistent results.
Loten and Sneyd (1970) compared PDE activity in fat pad
and fat cell homogenates with and without insulin.
Insulin increased PDE activity only in intact tissue and
had no effect when added to homogenates. Belcher, Merlino,'
: and Ro’Ane (1968) reported no effect of hyper- or hypo-
; insulinism on PDE activity of adipose tissue. Hepp,
et al., (1969) also found no effect of insulin on adipose
tissue PDE.------------ ---.........- i
20
Schultz, et, al. (1966) reported that PDE activity
in livers from alloxan diabetic rats is reduced and that
insulin enhanced this reduced activity. Kupiecki (1969)
also reported decreased PDE activity in homogenates of
pancreas and adipose tissue in spontaneously diabetic
mice. Miiller-Oerlinghausen, et al. (1968) compared liver
and adipose tissue PDE activity in acute and chronic
insulin treated alloxan diabetic rats. Experiments were
also carried out on fasted rats and animals made acutely
insulin deficient by injection of insulin antibody. They
observed no decreases in PDE activity due to insulin lack.
Insulin may also act to increase the biosynthesis
of PDE. Wool, et al. (1967, 1968) have described the
effects of insulin on protein biosynthesis in muscle.
They hypothesize that insulin forms a "translation factor"
(not cyclic AMP) which associates with and activates
ribosomes such that they are more competent to synthesize
14
proteins. Insulin increased the incorporation of C
amino acid into protein by 62$ within 5 minutes when
added in vitro to ribosomes prepared from diabetic animals
Insulin.'s effect is on an intracellular process and is
independent of glucose transport. Senft, et al. (1968)
found that liver PDE activity from alloxan diabetic rats
increased to a maximum at 30 minutes and then gradually
declined to normal levels at 240 minutes after insulin
21
Injection. Treatment with actinomycin D significantly
prevented this insulin activation of liver phosphodiester
ase. They suggested that the in vivo effect of insulin
was primarily due to increased enzyme synthesis. Steiner
(1966) has reviewed the adaptations of hepatic enzymes
concerned with intermediate metabolism in response to
insulin and altered metabolic states. Wimhurst and
Manchester (1970) and Chang, Schneider and Kalamazoo (1971)
have compared the changes in some key hepatic enzymes in
normal and streptozotocin diabetic rats. Diabetic rats
showed elevated levels of gluconeogenic enzymes (e.g.,
pyruvate carboxylase) and depressed levels of glucose-
catabolizing enzymes (e.g., glucokinase). The variable
response of these enzymes toward insulin treatment in
diabetic rats suggests complex and independent mechanisms
of regulation.
Most plasma proteins are synthesized in the liver
(Miller and Bale, 1954; Peters, 1964). Enzymes that are
specific to plasma are also syntehsized in the liver.
However, it is most difficult to determine the site of
biosynthesis of enzymes which occur in plasma and are
also widely distributed in other tissues (Hess, 1963),
such as cyclic nucleotide phosphodiesterase (see Thompson,
1971). Miller and John (1970) find the insulin is
essential for plasma protein biosynthesis by isolated
22
perfused rat livers. Diabetes results in a significant
reduction in total plasma protein, and especially the
concentration of plasma albumin (Sunderman, 1964).
The results of these studies indicate that insulin
may act by several different mechanisms to decrease intra
cellular cyclic AMP levels. Inhibition of adenyl cyclase
and direct activation of phosphodiesterase are more con
sistent with the observation that insulin can reduce
cyclic AMP levels rapidly (Park, et al., 1969; Moskowitz,
Harwood, and Krishna, 1971). Reports of reduced PDE in
diabetic rats and insulin activation of PDE after 30
minutes may be due to a defect in, or stimulation of,
protein synthesis.
Cyclic Nucleotide Phosphodiesterase
Studies of the enzymes which degrade cyclic AMP
have been recently reviewed by Cheung (1970) and Thompson
(1971). Multiple forms of PDE, which may be specific for
pure base cyclic nucleotides, have been reported for a
variety of tissues (see Thompson, 1971). Liver has been
reported to contain only a single form of phosphodiester
ase. The Km for cyclic AMP hydrolysis by this form has
— ft
been reported as 6.2 x 10" M (Menahan, et al., 1969) and
23
: 9.25 x 10"^M (Thompson and Appleman, 1971b). However,
based on differences In the ratio of hydrolysis of cyclic
AMP and cyclic GMP and kinetic data, Beavo, Hardman, and
Sutherland (1970) suggested the presence of multiple forms
- of PDE in liver and other tissues. Campbell, Pottinger,
and Oliver (1970) reported two classes of PDE in liver and
kidney. One class hydrolyzes purine cyclic nucleotides
and the other pyrimidines. Liver tissue PDE exhibited
the highest rate of cyclic '3*,5* thymidine monophosphate
hydrolysis. These forms were separated on polyacrylamide
gel electrophoresis.
Attempts to determine the molecular weights of
PDE are particularly interesting because the results are
so divergent. The estimated molecular weights values have
varied from 40,000 (Jard and Bernard, 1970) to greater
than 5,000,000 (Thompson and Appleman, 1971a, 1971b).
Thompson and Appleman (1971b) have obtained consistent
molecular weights of 400,000 and 200,000 for two fractions
: in five rat tissues. Cheung (1970) states: "It is likely
that phosphodiesterase may be composed of subunits and
that different conditions may give use to different sub
unit structures and thus different molecular weights.
Multiple forms of PDE in frog erythrocytes have
■ been reported by Rosen, et al. (1970). Patterson,
Hardman, and Sutherland (1971) suggested two forms of PDE
24
in rat red blood cells based on anaraolous kinetics of
cyclic AMP hydrolysis. They also found PDE in rat plasma.
They suggested that the observed increase in PDE activity
on storage of blood is due to destruction of formed ele
ments, especially platelets.
Sung, et al. (1971) reported multiple forms of
PDE in gastric mucosa and the loss of activity upon gel
filtration presumably due to loss of an activator. The
activation of cyclic AMP hydrolysis by bovine brain enzyme
has been extensively studied by Cheung (1970). He has
reported two types of activation: a small protein (40,000
molecular weight) and various proteolytic treatments. The
—4
Km of the enzyme he has studied is approximately 10 M,
presumably this is the form analogous to fraction II
reported by Thompson and Appleman (1971a). Jard and
Bernard (1970) reported multiple forms of PDE in kidney
which differed by 40,000 molecular weight. They interpret
this difference as due to the presence of an activator.
Since cyclic 3*,5*-nucleotide phosphodiesterases
degrade cyclic AMP, the regulation of this enzymatic activ
ity may be an important physiological mechanism for the
control of cyclic AMP levels. ,
CHAPTER II
MATERIALS AND METHODS
Animal Sources and Maintenance
Male albino Sprague-Dawley rats (200-350g) were
obtained from three different sources: Simonsen Animal
Laboratories, Gilroy, California; BioScience Animal Labora
tories, Oakland, California; and Mission Laboratory Supply
Inc., Rosemead, California. Rats were housed at the
University of Southern California Vivarium and maintained
on Purina Lab Chow and water ad libitum.
Rats were made diabetic with intra-peritoneal
injections (50 or 100 mg/kg) of streptozotocin (mfg. by
Ben Venure Laboratories, obtained through NIH, Bethesda,
Maryland). Diabetic rats were given 0.45% saline to drink
and used 2-22 days after onset of diabetes. Animals used
for tissue preparations were fed ad libitum.
Whole Animal Studies
Polyethylene cannulae were implanted in the ascend
ing aorta and anterior vena cava while under pentobarbital
anesthesia (50 mg/kg) according to the method of Popovic
26
and Popovic (I960). Animals were used 4-5 days post
operative after they returned to at least 90$ of their
pre-surgical weight. Rats were fasted 8-10 hours prior to
the experiment, usually begun at 9:00 A.M. The cannulae
permitted removal of serial blood samples (0.25 ml) and
injection of solutions with a minimum of stress to the
animals. After an initial sample(s) was taken, the rats
were subjected to two treatments: injections, usually
intra-arterially of 1 ml saline or 1 ml 50 mM Tris-acetate
buffer, pH 6.0, 3.75 mM 2-mercaptoethanol, followed by 2 ml
of 0.45 M glucose.
Tissue Preparations
Following sodium pentobarbital anesthesia (50 mg/
kg), blood was withdrawn in heparinized syringes from the
renal vein. Livers (some perfused in situ) were removed,
weighed and cooled on ice. Plasma was separated from
whole blood by centrifugation at 4300 x g for 10 minutes.
Phosphodiesterase activity was measured in the plasma
directly or after gel filtration. Prior to chromatography,
plasma was adjusted to pH 6.0 with 1 M acetic acid and
centrifuged for 20 minutes at 20,000 x g to remove
precipitates.
27
The preparation and assay of cyclic nucleotide
phosphodiesterase from liver were performed according to
the method of Thompson and Appleman (1971a), with slight
modification. All procedures were carried out near 4°C.
Liver was homogenized in 8 volumes (w/v) of homogenizing
medium in a Sorval Omni-Mixer (Ivan Sorvall Inc., Newton,
Conn.) at maximum speed for approximately 2 minutes. The
homogenizing medium contained 10.9# sucrose, 36.5 mM
Tris-acetate buffer, pH 6.0 and 2.82 mM 2-mercaptoethanol.
The pH of the homogenate was adjusted to 6.0 with 1 M
acetic acid, sonicated (Biosonik III, Bronwill Scientific,
Rochester, New York) for 15-20 minutes at a setting of 39
(standard probe) and centrifuged for 20 minutes at 20,000
x g. The supernatant was treated with ammonium sulfate
(50# saturation) and centrifuged for 20 minutes at 20,000
x g. The precipitate was resuspended in a minimum volume
of 50 mM Tris-acetate buffer, pH 6.0, 3.75 mM 2-mercapto
ethanol. Following overnight dialysis against the same
buffer, the precipitate formed was removed by centrifuga
tion and the supernatant was fractionated by agarose gel
filtration. The 50 mM Tris-acetate buffer described above
was used in all agarose gel chromatographic procedures.
Rat kidney PDE preparations were also assayed for
insulin and insulinase activity and compared with results
from liver. The kidney phosphodiesterase was prepared
28
from frozen kidney tissue. Red blood cell and brain
tissue were prepared as described for liver.
Chromat ography
Tissue preparations were fractioned with Biogel
A-5m, A-1.5m and A-0.5m (BioRad Laboratories, Richmond,
California). The molecular weight exclusion limits for
6 6 6
these gels are 5 x 10 , 1.5 x 10 and 0.5 x 10 , respec
tively. Biogel A-5m and A-1.5m columns were either
2.2 x 170 cm (bed volume of 475 ml) or 2.2 x 150 cm (bed
volume of 425 ml). Thompson and Appleman (1971b) have
estimated the molecular weights of both forms of PDE by
identical chromatographic procedures. Their values are
assumed in all molecular weight determinations. Molecular
weight determinations based on gel filtration methods are
determined from E /E ratios, where E equals the elution.
v v0 * v M
volume of the molecular species in question and E equals
vo
the elution volume (or void volume) of material completely
excluded from the gel. The molecular weight of the
unknown is determined from a plot of E /E vs the
v vQ
logarithm of the molecular weights of known species.
Since the values are a ratio determined for each column,
they are essentially independent of column height and flow
29
rate. The flow rates for Blogel columns varied from
12-15 ml/hr.
Values for the comparison of agarose gel fractlona- :
tlon results from different liver preparations were deter
mined by measuring the area of activity profiles by plani
metry (Keuffel and Esser Co., Hoboken, New Jersey). Biogel
column fractions were concentrated with an Amicon Ultra
filtration cell using a UM20E membrane (20,000 molecular
weight "cut-off," Amicon Corp., Lexington, Mass.).
Separation of insulin from concentrated enzyme
preparations was on Sephadex G-75 (Pharmacia Fine Chemicals,
Inc., Piscataway, New Jersey), using a 0.167 M acetic acid
medium.
Assays
The assay for cyclic nucleotide phosphodiesterase
activity (Thompson and Appleman, 1971a) is a two-step
radiochemical enzyme assay. Tritium labeled cyclic AMP
or cyclic GMP (normally, 1.25 and 2.5 x 10“^M, respec-
; tively) is incubated for 10 minutes at 30°C with enzyme
; aliquots in an initial volume of 0.4 ml. The PDE reaction
i is stopped by boiling for 2.5 minutes. The 5'-AMP formed ;
; by hydrolysis of cyclic AMP by PDE is converted into
i i
j__adenqsi_ne by excess nucleotidase from snake venom j
30
(0.1 ml of 1 mg/ml of Ophlophagus hannah) in a second
incubation at 30°C for 10 minutes. This reaction is
stopped by a 1 ml slurry of BioRad AGI-X2 resin, pH 5.0.
After centrifugation a 0.5 ml aliquot, containing tri-
tiated adenosine is counted by liquid scintillation
(Nuclear Chicago, Unilux II). The scintillant solution
contained 375 g napthalene, 22.5 g 2,5 diphenyloxazole
(PPO), 1.125 g 1-4 bis [2-(4-methyl-5-phenyloxazole)]
benzene (P0P0P)/3 liters dioxane. One unit of phosphodi
esterase equals one picomole of cyclic nucleotide hydro
lyzed per minute by 0.2 ml enzyme aliquots in an initial
assay volume of 0.4 ml. Samples were diluted to a range
where the assay is linear, approximately 20-30$ of maximal
substrate hydrolysis. Results are expressed as units/ml
or units/fraction. Enzymatic activities of a given pre
paration are compared using the ratio of cyclic GMP
hydrolysis to cyclic AMP hydrolysis (G/A). Substrate
concentrations for kinetic analysis ranged from 0.8-100 x
10"6M for cyclic AMP PDE and 5-200 x 10“7M for cyclic GMP
PDE.
Insulin was assayed using a radioimmunoassay "kit"
(Schwarz/Mann BioResearch, Inc., Orangeburg, New York).
In this procedure guinea pig insulin antibody and rabbit
anti-guinea pig serum are added together and incubated
with aliquots of tissue preparations (100-200X) for 6 hours
31
| at 4°C. ' 1 '^I-insulin is added and after an 18 hour Incu
bation at 4°C, the insulin-antibody complex is collected
on membrane filters on a pyrex microanalysis filter holder
(Millipore Corporation, Bedford, Massachusetts). The
membranes (oxoid, 0.45 grade, mfg. Oxoid Ltd., Southwark
Bridge Road, London), were dissolved in Wu’s scintillant
(Wu, 1964). Results (expressed as microunits/ml) are
determined by comparison with standard curves.
Insulinase was measured by two different procedures.
In the first method 100 microunits of standard human
. insulin (Schwarz/Mann) was added to sample aliquots (200X)
and incubated overnight at 4°C. The insulin remaining at
the end of incubation was assayed immunologically as
described above. Results are expressed as the difference
between microunits added and microunits remaining after
1 p r
incubation. In the second method, 3I-insulin (Schwarz/
Mann) was added to sample aliquots (100-200X) and incubated
for 10 minutes at 30°C. The reaction was stopped with
1 ml of 5 % TCA. After centrifugation, 0.5 ml aliquots of
the supernatant were counted as in the PDE assay. There
was no quenching by the TCA. Results are expressed as
■ percent of the initial ^ ^ 1 CPM (corrected for blank) not
; precipitated by the TCA.
Insulin was extracted from concentrated Biogel
; fractions by an acid-ethanol extraction procedure
suggested by Arquilla (1971). Concentrated column frac
tions (1.5 ml) were reduced to pH 3.0 with 1 M acetic
acid. The protein solution was precipitated and washed
twice with acidic ethanol. The combined acid-ethanol super
natants were adjusted to pH 5.2 with 4 N ammonium hydroxide
and 4 volumes of a 3^5 (v/v) mixture of 95$ ethanol and
ether is added and mixed. Insulin was precipitated from
this mixture at -65°C overnight. After centrifugation,
the precipitate was washed in acetone and dissolved in
40 mM Na^POjj buffer, pH 7.4, 0.154 M NaCl, containing
0.1$ BSA. Insulin was assayed in this buffer by the radio
immunoassay procedure described above.
Protein was measured by the Lowry method (Lowry,
et al., 1951). Results are expressed as mg/ml. Total
heme pigments in plasma were measured by the benzidine
method in Cartwright (1968). Results are expressed as
mg/100 ml (mg$). Plasma was diluted and deproteinized by
the zinc-barium technique of Somogyi (1952) and plasma
glucose determined by the glucose-oxidase procedure modi
fied by Meites and Bohman (1963). Urine-glucose was
measured with Clinistrix test strips (Ames Company, Inc.,
Elkhart, Indiana). Results are determined by degree and
rate of color change of the test strip.
33
Statistical Analysis
All statistical calculations were done on the
Olivetti Underwood Programma 101 electronic desk computer
(Olivetti Underwood Corporation, New York, New York). The
type and source of the programs used were as follows:
correlations for linear regression (r) obtained through
Dr. Robert M. Chew (1971), the t-test for the significance
of the difference between two sample means from Statisti
cal Analysis Manual (Olivetti Underwood Corporation). The
level of significance (P value) was taken from the
"Student’s" t-distribution table, appendix 2 (Bailey,
1959).
CHAPTER III
RESULTS
Whole Animal Studies
Recent studies have suggested that cyclic AMP may
act as part of a feedback mechanism to regulate insulin
release (Bayliss, 1966). After epinephrine or glucagon
stimulation, cyclic AMP is released into the incubation
medium from fat and liver cells (Montague and Cook, 1970;
Moskowitz and Pain, 1970; and Schaeffer, 1971) and into
the perfusate of perfused liver (Lewis, Exton, and Park,
1971). Levine and others (1969, 1970) have shown that
high concentrations of exogenous cyclic AMP stimulate
insulin release from the pancreas. Experiments were car
ried out to determine if increments in endogenous plasma
insulin concentration can increase plasma phosphodiesterase
activity which then would reduce circulating levels of
cyclic AMP as part of this postulated feedback mechanism.
Normal fasted rats were infused with 2 ml of 0.45 M
glucose (162 mg) via an indwelling arterial cannula. All
animals (weight range 201-305 g) were given the same glu
cose load regardless of weight. Serial plasma samples :
were collected via a vena caval cannula prior to and at
35
intervals up to 45 minutes following administration of the
glucose load. Samples were assayed for glucose, insulin,
and phosphodiesterase activity.
Figure 1 compares the percent changes in plasma
glucose, insulin, cyclic AMP, and cyclic GMP hydrolysis
during glucose tolerance tests. Control blood samples
were taken prior to glucose infusion. The average control
values for glucose, insulin, cyclic AMP hydrolysis, and
cyclic GMP hydrolysis are 98.4 ± 2.7 mg$, 36.1 ± 4.2 yU/ml,
7.5 ± 1.1 units/ml, and 60.4 ± 15.2 units/ml, respectively.
These control values are expressed as 0%> change (Figure 1).
Experimental points represent the mean ± the standard
error of the mean (indicated by bar length). The numbers
listed over the 5 minute point represents both the number
of animals used in each experiment and the number of plasma
samples at that time. Differences in numbers at subse
quent times are due to variation in sample regimen. At
5 minutes after the glucose load, the plasma glucose con
centration increased to a maximal value of 1 5 0 % over con
trol levels and then gradually declined to below the
control level at 30 minutes. Plasma insulin levels also
reached a maximum value ( 8 7 % increase) at 5 minutes after
; glucose administration. There is no significant change in
either cyclic AMP or cyclic GMP hydrolysis. Cyclic AMP PDE
J activity shows greater variability than the other
FIGURE 1
PERCENT CHANGE IN PLASMA GLUCOSE (1A), INSULIN (IB),
CYCLIC AMP HYDROLYSIS (1C) AND CYCLIC GMP HYDROLYSIS
DURING GLUCOSE TOLERANCE TESTS IN NORMAL FASTED RATS
Experimental points were taken at the times indi
cated after intra-arterial infusion of 2 ml of 0.45M
glucose (162 mg). The numbers above the 5 minute points
are the number of animals used for each study and the
number of samples at that time. Control values, taken
before the glucose load, were set equal to 0% change.
Initial values were 98.4 ± 2.7 mg$, 36.1 ± 4.2 pU/ml,
7.5 ± 1.1 units/ml and 60.4 ± 15.2 units/ml for glucose,
insulin, cyclic AMP hydrolysis, and cyclic GMP hydrolysis,
respectively. Standard error of the mean is shown by the
bar length at each point.
A. G lucose
150
100
50
I
C. Cyclic AMP Hydrolysis
! F ioo
|
j
i
D. Cyclic GMP Hydrolysis
F ioo
15 25 35
Minutes After Glucose Load
45
parameters. The variation is not due to the assay method
or due to RBC hemolysis since there was no correlation
between PDE activity and plasma heme pigment concentrations
(r = -0.234, P > 0.10, N = 31). Whether or not the varia
tion is due to disruption of other formed elements, e.g.,
platlets, in the plasma was not determined (Patterson,
et al., 1971).
These results show that there is no relationship
between the amount of circulating insulin and PDE activity
in plasma. This suggests that plasma PDE is not activated
by insulin and is not involved in a feedback mechanism
regulating the release of insulin via control of cyclic
AMP levels in the circulation.
Studies of Tissue Preparations
Plasma
The results presented in this dissertation as well
as a preliminary report by Patterson, et al. (1971) show
that plasma does contain phosphodiesterase activity.
Hardman, et al. (1971) discussed the possible importance
of understanding the sources and factors which regulate
extra-cellular fluid levels of cyclic nucleotides. They
cite studies of a variety of disease states and hormonal
39
treatments which influence blood and urine cyclic AMP
levels. Body fluids, especially blood, are convenient
tissue sources and since extra-cellular cyclic nucleotide
levels may reflect changes in intracellular concentrations,
studies were undertaken to characterize plasma cyclic
nucleotide phosphodiesterase(s) which effect extra-cellu
lar cyclic AMP levels.
The average amount of cyclic AMP PDE activity found
in pooled plasma obtained from 4 rats was 3.38 units/ml
and 16.58 units/ml for cyclic GMP PDE activity (G/A ratio =
4.92). The results of agarose gel fractionation (Biogel
A-5m) of this plasma sample is presented in Figure 2.
Since the activity profiles of cyclic AMP and cyclic GMP
hydrolysis are different, it is probable that these two
activities are due to two separate enzymes. The estimated
molecular weights of the cyclic AMP and cyclic GMP enzymes
obtained from the E /E ratios (1.56 and 1.46) are 500,000
0
and 700,000, respectively. These molecular weight values
are larger than those previously reported for other
tissues (Thompson and Appleman, 1971b). No significant
immunoinsulin activity was detected in the fractions
tested.
Kinetic studies of cyclic AMP and cyclic GMP
hydrolysis by plasma are presented in Figures 3 and 4.
The estimated Michaelis constants for cyclic GMP and cyclia
FIGURE 2
CHROMATOGRAPHIC PROFILES OF RAT PLASMA
Activity profiles of cyclic AMP and cyclic GMP
hydrolysis and immunoinsulin activity on agarose gel
(Biogel A-5m) obtained from 5 ml pooled rat plasma. A280
for each fraction is given. Fractions were assayed for
immunoinsulin activity where indicated. Enzyme prepara
tion and assays are described in Chapter II.
O Cyclic G M P s u b stra te
x Cyclic AM P su b s tra te
A Im m u n o in su lin
1 .0-
O
CO
L i _
0.6—i
0.2-
50 30 40 60
Fraction N u m b er
70 90 80
42
FIGURE 3
CYCLIC GMP HYDROLYSIS BY RAT PLASMA
Kinetic analysis of cyclic AMP hydrolysis by whole
rat plasma. Enzyme preparation and assays are described
in Chapter II.
60
50
40
80
O 60
30
x 40
20
20
- 0.02 0.01 0.03 0.05
/ S X I 0 7
0.07 0.0S
40 80
[Cyclic G M P] x I 0 “ 7
120 160 200
U )
FIGURE 4
CYCLIC AMP HYDROLYSIS BY RAT PLASMA
Kinetic analysis of plasma phosphodiesterase after
partial purification by gel filtration. Enzyme prepara
tion and assays are described in Chapter II.
40 -
150
O 100
> 50
Km= 7 x 10
- 0.2 0.3. 0.5
/ S x 10 6
0.7
20
[Cyclic AMP] x 10 6
V J l
HS...
— f i / r
AMP hydrolysis are 5 x 10“ M and 7 x 10” M, respectively.
The double reciprocal plot is linear for cyclic GMP
hydrolysis and non-linear (negatively cooperative) for
cyclic AMP hydrolysis, in agreement with the kinetics
reported for this enzyme in other tissues. The negative
cooperatively may, however, be due to the summation of
velocities from two or more forms of PDE as suggested by
Thompson and Appleman (1971a).
The results presented in Table 1 show that strepto-
zotocin diabetes produces significant changes in plasma
PDE activity. Results are presented for fed and fasted
normal rats and fed diabetic rats. Diabetic rats were
sacrificed at varying times after streptozotocin treatment.
All diabetic animals exhibited increased plasma glucose
concentration and intense glucosuria. All diabetic rats,
with the exception of the animal sacrificed two days after
streptozotocin treatment, exhibited significant weight
loss. Plasma insulin levels were significantly reduced to
H 6 % of the value for normal fed rats. Reduced plasma
insulin due to streptozotocin agrees with the results of
Junod, et al. (1969) and Chang, et al. (1971). Plasma
cyclic AMP and cyclic GMP phosphodiesterase activity
decreased significantly in diabetic rats examined 8-22
days after streptozotocin treatment. Although the 2 day
post-streptozotocin diabetic rat exhibited hyperglycemia
TABLE 1
CHARACTERISTICS OP DIABETIC RATS AND
COMPARISONS BETWEEN NORMAL AND DIABETIC RAT PLASMA PARAMETERS
Normals
fed ad libitum
t-statistic
(fed normal vs diabetic)
P value
Glucose Insulin
yU/ml
cyclic GMP
hydrolysis
units/ml
cyclic AMP
hydrolysis
units/ml
4.544
<0.002
2.143
<0.05
a) All exhibited heavy glucosuria.
b) Days used after streptozoticin treatment.
3.414
<0.01
G/A
Tno.)
mean
S.E.M.
fasted
(no.)
mean
S.E.M.
(3)
139.5
±4.3
' (34)
98.4
±2.7
(6)
41.5
±3.9
(10)
36.1
±4.2
(32)
3.15
±0.30
(5)
7.5
±1.1 .
(32)
22,65
±1.86
(5)
60.5
±15.2
(32)
8.02
±0.70
(5)
8.05
±0.87
£ 1 b
Diabetics days
1 2
2 8
3 21
5 22
6 22
weight
change
1
-24
-36
-45
-49
374
444
451
442
473
25
19
20
25
3-73
1.24
1.84
1.75
1.41
23.22
3.93
6.03
4.58
5.40
6.22
3.16
3.28
2.61
3.83
mean
S.E.M.
436.8
±16.6
22.2
±1.6
2.0
±0.45
8.63
±3.67
3.82
±0.63
4.568
<0.001
1J8
and glucosuria, there was no difference in plasma cyclic
AMP or cyclic GMP hydrolysis. The ratios of cyclic GMP
hydrolysis to cyclic AMP hydrolysis in plasma (G/A) also
were significantly decreased in diabetic rats, indicating
that cyclic GMP hydrolysis decreases to a greater extent
than cyclic AMP hydrolysis.
From the results of these studies, phosphodiesterase
activity does exist in plasma. Furthermore, the gel fil
tration studies, the negatively cooperative kinetics of
cyclic AMP hydrolysis and the decline in the G/A ratio in
diabetics suggest that multiple forms of PDE exist in
plasma. There is a significant decrease in cyclic nucleo
tide phosphodiesterase activity from diabetic rat plasma.
Liver
Any physiologically important effect of insulin on
the activity of cyclic nucleotide phosphodiesterase to con
trol cyclic AMP levels should be evident in liver. The
liver is an important organ in the regulation of blood
glucose levels via control of glycogen metabolism (mediated
by cyclic AMP levels) and gluconeogenesis, and the
metabolic disposition of insulin (see Chapter I). Studies
were undertaken therefore to study PDE in liver. Liver
49
has been reported to contain only the high Michaelis con
stant, high molecular weight enzyme (Thompson and Appleman,
1971b).
The agarose gel (Biogel A-1.5m) profiles of phos
phodiesterase activity from a sonicated supernatant prepa
ration of kidney are presented in Figure 5 so that they
may be compared with results obtained from a liver prepa
ration which was stored for 16 days at 4°C (Figure 6).
Thompson and Appleman (1971b) designated the three peaks
of kidney PDE activity (Figure 5) as fraction I (column
fraction 37: E„/E„ = 1.0), fraction II (column fraction
v vQ
53: Ev/Ev = 1-43) and fraction III (column fraction 68:
V Evc =
According to Thompson and Appleman (1971b), kidney
fraction II hydrolyzes both cyclic AMP (Km 3.50 x 10“^M)
— f i
and cyclic GMP (Km 9.25 x 10“ M) and has an estimated
molecular weight of 400,000. Kidney fraction III is more
specific for cyclic AMP hydrolysis (Km 4.65 x 10"^M) and
is negatively cooperative. The estimated molecular weight
is 200,000.
As shown in Figure 6 there are two peaks of phos
phodiesterase activity in this liver profile, analogous to
kidney fractions II and III. There is no liver fraction
analogous to kidney fraction I. Since the E /E ratios
v v0
of the peaks of phosphodiesterase activity in this liver
FIGURE 5
RESULTS OF AGAROSE GEL FILTRATION (BIOGEL A-1.5m)
OF RAT KIDNEY
Activity profiles for cyclic GMP hydrolysis and
cyclic AMP hydrolysis are shown. E../E ratios are given
v vQ
in parenthesis above the peaks of phosphodiesterase activ
ity. Flow rate was 11.1 ml per hour; 75 mg protein
fractionated. A280 is shown for each fraction. Enzyme
preparation and assays are described in Chapter II.
(1 . 43)
o Cyclic GMP substrate
x Cyclic AMP substrate
-20
-15
0.5-
(1 . 83)
-10
0.3-
-5
1 0 .1 -
35 T 45 55 65
Fraction number
75 85 95 105
U1
FIGURE 6
CHROMATOGRAPHIC PROFILES ON AGAROSE GEL (BIOGEL A-1.5m)
OF A LIVER PREPARATION STORED FOR 16 DAYS AT 4°C.
Activity of fraction II (cyclic GMP hydrolysis)
and fraction III (cyclic AMP hydrolysis) forms of phos
phodiesterase are shown. Ev/Ev ratios are shown in
v o
parenthesis. Flow rate was 11.7 ml/per hour; 4l mg protein
fractionated, ^ qq is shown for each fraction. Enzyme
preparation and assays are described in Chapter II.
o C yclic GMP s u b s t r a t e
(1.86) x Cyclic AM P s u b s t r a t e
v
- 12
(1.48)
-8
0.2-
L -4
0.1-
85 95
V V
65 75
F raction n u m b e r
45 55 35 105
54
preparation (1.48 and 1.86) are similar to the E /E
v v0
ratios in kidney, the estimated molecular weights are the
same as those reported for kidney fractions II and III
(i.e., 400,000 and 200,000, respectively). It is also
evident from Figure 6 that fraction III is more specific
for cyclic AMP hydrolysis; fraction II appears to hydrolyze
only cyclic GMP, in this case.
The results of kinetic studies on cyclic AMP hydrol
ysis by liver fractions II and III are shown in Figures 7
and 8. Unlike previous reports of cyclic AMP hydrolysis
by liver preparations and other tissue fraction II forms
(Menahan, et al., 1969; Thompson and Appleman, 1971b), the
double reciprocal plot of cyclic AMP hydrolyses suggests
positively cooperative catalytic control (Figure 7). This
is interpreted as evidence for interaction between multiple
sites or sub-units (Atkinson, 1969). This kinetic behav
ior indicates an additional mechanism for regulation of
intra-cellular cyclic AMP levels. The kinetics of cyclic
AMP hydrolysis by fraction III (Figure 8), is similar to
results obtained with fraction III from kidney. The
apparent Michaelis constant for liver cyclic AMP hydrolysis
is 3 x 10-^M which is well with the range of Michaelis
constants reported for fraction III from five rat tissues
(Thompson and Appleman, 1971b), and agrees with the Km
reported by Menahan, et al. (1969). The non-linear
FIGURE 7
CYCLIC AMP HYDROLYSIS BY FRACTION II ENZYME IN LIVER.
Kinetic analysis of cyclic AMP hydrolysis by frac^
tion II enzyme after partial purification by Biogel A-5m
fractionation of sonicated supernatant. Preparation and
assays are described in Chapter II.
S25r
I6OO-1
1200-
140 r
O 105
800J
35
- 0 . 1 0.2 0.4 0.6
/ S X I0 " 6
20 40 60
[ c A M P ] x 10'
80 1.00
FIGURE 8
CYCLIC AMP HYDROLYSIS BY PARTIAL SEPARATED
FRACTION III ENZYME IN LIVER.
Kinetic analysis of cyclic AMP hydrolysis by par
tially purified lower molecular weight liver phosphodies
terase. Preparation and assays are described in
Chapter II.
200r
120 ISO
W)
120-
80 80
= 3.0 x I0“ s
40
40
0.4
I/S X I0‘6
-0.4 -0.2 0.2 O.S 0.8
80 40 60
[CAMP] X I0~6
100 20
59
portion of the double reciprocal plot indicates negatively
cooperative catalytic control (Conway and Koshland, 1968).
However, since the liver fraction III enzyme was not
purified further, it is possible that the non-linear
double reciprocal plot may be due to contamination by
fraction II enzyme, as discussed by Thompson and Appleman
(1971a).
This fraction III form of cyclic nucleotide phos
phodiesterase is not readily apparent. Three characteris
tic "stages" in the appearance of this form (Figure 9)
have been designated under the conditions of this study.
In the first stage (Figure 9, Stage I), the chromatographic
profiles of cyclic AMP and cyclic GMP hydrolysis by
sonicated supernatant are nearly coincident, and cyclic
AMP hydrolysis only is not evident. This stage is similar
to the results reported by Thompson and Appleman (1971b).
After ammonium sulfate precipitation of sonicated super
natant, dialysis and fractionation; however, a shoulder of
only cyclic AMP hydrolysis appears between fractions 65-80
(Figure 9, Stage II). This shoulder of cyclic AMP
hydrolysis represents the second stage in the appearance
of fraction III PDE. Storing ammonium sulfate precipitated:
and dialysed liver preparations at 4°C for varying periods
of time results in the appearance of a discrete peak of
fraction III activity upon fractionation; this is
60
FIGURE,9 . .
STAGES IN THE APPEARANCE OF FRACTION III ENZYME IN LIVER.
All profiles are from the same preparation on the
same column (Biogel A-5m) after various treatments. Stage
I shows cyclic AMP hydrolysis and cyclic GMP hydrolysis of
sonicated supernatant (50.4 mg protein); Stage II after
ammonium sulfate precipitation and dialysis (120 mg pro
tein); Stage III after storage for 6 days at 4°C (72 mg
protein). Flow rate averaged 13.9 ml/hr. Preparations
and assays are described in Chapter II.
Units / fraction Units / tract ion Units / fraction
PO PO
o o
~ pj-
ro
ro ro
co
co
co O
o l
O"
62
designated the third stage in the appearance of the lower
molecular weight form. The third stage after storage for
6 days at 4°C is shown in Figures 9 and 10 (lower panel).
After ammonium sulfate precipitation of rat liver
preparations, every chromatographic profile that was
assayed for both cyclic AMP and cyclic GMP hydrolysis
(11 examples) revealed either a shoulder or a discrete
peak of cyclic AMP hydrolysis only. In all cases where
fraction III appeared as a discrete peak after storage at
4°C (4 examples) the amount of cyclic AMP hydrolysis rela
tive to cyclic GMP hydrolysis in fraction II is reduced,
compared to the same ratio prior to the appearance of
fraction III (see Figures 9 and 10). In every case where
distinct peaks of cyclic AMP and cyclic GMP hydrolysis by
fraction II phosphodiesterase occur (9 examples), the peak
of cyclic AMP hydrolysis chromatographs slightly ahead
(8-10 mis) of the peak of cyclic GMP hydrolysis. The
average difference of the E /E ratios between these two
o
peaks, based on assays of the same fractions, is 0.081.
If the E /E ratio of the peak of cyclic GMP hydrolysis
v vQ
is assumed to be 400,000, then the estimated molecular
weight of the peak of cyclic AMP hydrolysis is 440,000. The!
difference (40,000) corresponds to the molecular weight
of the protein activator reported by Cheung (1970). These
results suggest that fraction II is composed of two
FIGURE 10
APPEARANCE OF FRACTION III ENZYME AND RELATIONSHIP
BETWEEN PHOSPHODIESTERASE FORMS AND
IMMUNOINSULIN ACTIVITY
Cyclic AMP hydrolysis appears as a shoulder of the
total PDE activity after ammonium sulfate treatment (upper
panel); fraction III enzyme appears as a distinct peak
after storage for 6 days at 4°C (lower panel). Immuno-
insulin activity occurs between the fraction II and frac
tion III forms. Preparation and assays are described in
Chapter II.
g Cyclic GMP substrate 70-
x Cyclic AMP substrate
a I rn mu noinsulin
■50-
-40
30-
-20
i
10"
o
l
i
-20
20- -10
Fraction number
different forms of PDE, one which may preferentially
hydrolyze cyclic AMP and one which is more specific for
cyclic GMP, or that some small molecular weight component
facilitates, perhaps in a positively cooperative fashion,
the hydrolysis of cyclic AMP by fraction II. The results
also suggest that the lower molecular fraction is formed
from the higher molecular weight fraction which is more
specific for cyclic AMP hydrolysis.
Preliminary studies of the role of endogenous cyclic
AMP in the possible regulation of insulin release (see
above) led to the observation that partially purified liver
phosphodiesterase also assayed positively for immunoinsulin.
Since insulin was reported to activate PDE (see Chapter I),
it was conceivable that insulin was bound to PDE in the
process of activation. In order to clarify this observa
tion, further experiments were carried out. These studies
involve analysis of agarose gel activity profiles for PDE,
insulin and insulinase activity; comparisons between
normal and diabetic rats, and studies of methods to
isolated endogenous insulin from high molecular weight
fractions, presumably insulin binding protein.
The activity profiles after agarose gel fractiona
tion (Biogel A-5m) of PDE and immunoinsulin activity from
a normal rat liver preparation are shown in Figure 10.
Immunoinsulin activity peaks between the two forms of PDE,
and is not coincident with either form. The estimated j
; 66
; molecular weight of this Immunoinsulin peak Is 300,000.
This immunoinsulin activity can be further separated from
PDE activity by additional gel filtration (Biogel A-0.5m)
of concentrated fractions from the larger Biogel A-5m
columns (Figure 11). These results indicate immunoinsulin
is not bound to the PDE since the two activities are not
coincident.
Figure 12 compares agarose gel (Biogel A-5m)
activity profiles from equal amounts of protein (150 mg)
obtained from normal and diabetic rat livers which were
prepared and assayed in parallel. The peak fractions of
activity profiles are not directly comparable since the
two preparations were fractionated on different columns.
The only striking difference between the normal and diabet
ic profiles is the marked reduction in immunoinsulin con
tent of the diabetic liver. Table 2 is a compilation of
the cyclic nucleotide phosphodiesterase activity and
immunoinsulin content prepared from 6 normal and 3 diabetic
rat liver preparations. All preparations in this table
are results for the second stage in the appearance of
fraction III PDE, i.e., after ammonium sulfate precipita
tion. The immunoinsulin content of diabetic rat livers is ■
significantly reduced to 53$ of the content of normal
rats. There is no difference in total cyclic AMP hydrol-
! ysis between normal and diabetic rat livers. These
FIGURE 11
: SEPARATION OF IMMUNOINSULIN AND PHOSPHODIESTERASE
ACTIVITY BY GEL FILTRATION.
Chromatographic profiles of protein (A2g0)s cyclic
i
: AMP hydrolysis and immunoinsulin activity on Biogel A-0.5m
: from agarose gel (Biogel A-5m) concentrates from liver.
: Preparations and assays are described in Chapter II.
x Cyclic AMP s u b s t ra te
A I m m u n o in s u l in
o 60 - 33 30
0.9-
c o 40- cn 20
c:
0.6-
20- £ 10
I — 5
0.3-
V
' /
w ,
20 22
Fraction n u m b e r
FIGURE 12
COMPARISON OF NORMAL AND DIABETIC LIVER
CHROMATOGRAPHIC PROFILES.
Chromatographic profiles of cyclic AMP hydrolysis,
immunoinsulin and insulinase activity measured immunologi-
cally on agarose gel (Biogel A-5m) from normal (A) and
diabetic (B) rat livers. Equal amounts of protein (150 mg)
were applied to different columns. Preparations and assays
(done in parallel) are described in Chapter II.
Normal
D iabetic
90
80
to
60 60
X Cyclic AMP substrate
A Immunoinsulin.
© Insulinase
1 50
•a
50
CO
40
® 20
CO
“ O
o
-C
n
y
30 30
20
D_
»v
tA
v0 FRACTION NUMBER
o FRACTION NUMBER
TABLE 2
CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITY AND
IMMUNOINSULIN CONTENT OP NORMAL AND
DIABETIC RAT LIVER PREPARATIONS
Inmunoinsulin cyclic AMP cyclic GMP
yU/mg Substrate Substrate
units/mg units/mg
NORMAL -----------------------------------------------------
1.
216.3 2.59
--
2.
228.7 2.38
—
3- 266.9 4.47 4.89
4. 196.8 4.41 4.64
5. 281.1
3.73
4.96
6. 270.0 6.24 6.43
mean
243.3 3.97 5.23
±S.E.M.
±13.9 ±0.58 ±0.41
DIABETIC
1. 92.0 3.16
_
2.
131.7 4.00 4.96
3. 165.6 3.12 6.90
mean 129.8
3.43 5.93
±S.E.M.
±21.3 ±0.29 —
"t" statistic 4.468 0.840
P value <0.01 >0.10
72
results also suggest that there Is no difference in total
cyclic GMP hydrolysis between normal and diabetic rat
livers.
These results indicate that the peak of immuno
insulin activity is specific since no other fractions in
the liver profiles react positively with the radioimmuno
assay for insulin. Both the circulating levels of insulin
(Table 1) and the immunoinsulin content of livers from
diabetic rats are reduced to approximately the same extent;
: 46% for plasma insulin and 53$ for liver immunoinsulin.
These results support the hypothesis that the immunoinsulin
detected in agarose gel fractions is some form of native
■ insulin.
Preliminary studies also showed that when insulin
was incubated with partially purified liver PDE, the
insulin was no longer detectable by the immunoassay. Sub-
125
sequent studies with I-insulin have shown that insulin
appears to be degraded at a "first-order" reaction rate;
1?R
that the amount of I-insulin degraded is related to the
; concentration of partial purified liver PDE and that there
125
is no degradation of ^I-insulin after the liver prepara-
! tions are boiled for 2.5 minutes. These criteria suggest
' that the insulin-degrading activity is enzymatic (i.e.,
insulinase), and that phosphodiesterase preparations con-
j !
! tain this insulinase activity. Insulinase activity will
73
interfere with the immunoassay for insulin by degrading
125
added ^I-insulin such that it can no longer compete for
antibody binding sites. Consequently, the amount of radio
activity detected in a given assay is reduced, indicating
the presence of insulin. Because of this interference, it
is necessary to distinguish between positive immunoassays
for insulin due to the presence of native insulin and that
due to insulinase aativity which occur in the same liver
phosphodiesterase preparations.
In Figure 12, insulinase activity, measured by the
disappearance of added standard insulin, is apparently not
affected by streptozotocin induced diabetes. In both
normal and diabetic profiles, insulinase activity does not
appear as a single peak as do PDE and immunoinsulin activ
ities. In the preparation from normal rat liver, insulin
ase activity peaks in fraction 50, decreases until frac
tion 54 and then increases again to a second peak in
fraction 64. This "notch" pattern is characteristic of
liver insulinase activity when measured by this method and
occurs in all liver preparations tested. As can be seen
in Figures 12 and 13 (B and C), the "notch" in insulinase
activity is coincident with the rise in immunoinsulin. In
most cases, the immunoinsulin peak occurs in the same
fraction as the lowest point of the "notch." When insulin
ase activity profiles are determined by measuring the ,
FIGURE 13
RELATIONSHIPS AMONG VARIOUS ACTIVITIES FROM
A SINGLE LIVER PREPARATION.
Profiles of protein (A2qq) and phosphodiesterase
activity (13A) and immunoinsulin (12B). Insulinase was
measured by two methods: disappearance of exogenous human
125
insulin, detected immunologically (yU) and by J I CPM
remaining in the TCA supernatant ($)(13C). After homogeni'
125
zation, 3000 yU of I-insulin were added. Resulting
profiles of ‘ ^'’ i CPM and ^^1 CPM/mg protein are shown
(13D). Preparations and assays are described in Chapter
II.
Fraction num bei
Insulinase
(jusU adderi-
assayed) 7 ml*
Specific activity
(cpm / m g) x 10“2
Immunoinsulin
( ,ull / ml)
Phosphodiesterase
(units/m!)
CM ro
C M
O o
C J 1
cn
125
I-insulin remaining in the TCA supernatant the "notch"
is absent. With the exception of the "notch," insulinase
activity profiles measured by both methods can be super
imposed (Figure 13C). The difference between these two
activity profiles of insulinase activity suggests that
endogenous insulin is bound in some form, and protected
from insulinase activity present in the same fractions.
Several studies have shown that when bound, insulin is pro
tected from insulinase activity (see Chapter I). Insulin
ase measured by the degradation of exogenous iodinated
insulin has a single peak of activity. Insulinase measured
by the disappearance of exogenous human insulin, detected
immunologically, should have the same result. The "notch"
is interpreted as bound insulin which the insulinase could
not degrade, but which can still be bound to insulin
antibodies.
In order to determine if a specific insulin binding
125
component was present in this fraction, ^I-insulin was
added to liver preparations, fractionated, and examined
125
for I radioactivity. The normal rat liver preparation
shown in Figure 13 was perfused with saline in situ prior
to the preparation. After homogenization, 0.5 ml of
1
I-insulin (3000 yU) was added and the normal preparation
continued. In Figure 12D the resulting column profile of
' L2^I CPM/ml is shown. Specific activity (Figure 12D) is
77
expressed as CPM/mg protein determined from the A^ q q curve
(Figure 12A). Unlike the immunoinsulin activity, specific
activity of ‘ '"^I-insulin binding peaks in fraction 61.
Similar studies with partially purified liver PDE prepara-
126
tion, i.e., binding of ^I-insulin followed by chroma
tography, did not reveal a peak of specific activity
associated with the phosphodiesterase activity. The
results of these studies suggest that insulin may bind to
a specific high molecular weight component in liver.
126
However, the I-insulin binding is not coincident with
immunoinsulin activity.
If insulin is bound to some high molecular weight
component of liver, it should be possible to separate
endogenous insulin from enzymatic activity and higher
molecular weight proteins. Various methods, all utilizing
acidic conditions, were successful in this separation:
chromatography on Sephadex G-75, acid-ethanol extraction
and ultrafiltration. Figure 14 is a comparison of the
chromatographic properties of a standard insulin solution
(Eli Lilly, Glucagon low) with an acidified (pH 3.0) con
centrated agarose gel fraction from liver. Both insulin
peaks were detected immunologically and have similar
chromatographic properties. The high molecular weight
proteins (200,000-400,000), indicated by absorbance at
280 nm, are excluded from the gel. Enzymatic activity
FIGURE 14
SEPARATION OF INSULIN FROM OTHER PROTEINS BY
GEL FILTRATION OF CONCENTRATED LIVER
PHOSPHODIESTERASE PREPARATIONS
Comparison of chromatographic properties on
Sephadex G-75 of immunoinsulin activity from concentrated
agarose gel fractions (A) and a solution of standard
insulin (Lilly, Glucagon low)(B). Medium is 0.167 M
acetic acid.
1.5 ml conc. a g a r o s e gel fra c tio n , pH 3 . 0
0.8
-30 -j0.6 o
oo
CM
-0.4<
Im m unoinsulin
A b s o rb an ce , 2 8 0
-20
-10 0.2
Lilly, insulin, g l u c a g o n low
•No d e t e c t a b le a b s o r b a n c e at 2 8 0 nm
-30
-20
-10
FRACTION NUMBER
i 80
■ (PDE and insulinase) is not detectable in the immunoinsulin
peak in Figure l^A. Immunoinsulin from acidic liver PDE
concentrates also has similar chromatographic properties
125
on Sephadex G-75 as ^I-insulin.
Table 3 is a compilation of the results of insulin
and insulinase assays of concentrated agarose gel fractions
from nine different rat liver phosphodiesterase prepara-
! tions. The concentrates were assayed prior to and after
acid treatment or after acid-ethanol extraction. After
these treatments there was no detectable insulinase or PDE
activity. However, the solutions still assayed positively
for immunoinsulin.
The results of these experiments indicate that endo
genous insulin is bound in some form to a high molecular
weight protein. In spite of the presence of insulinase
activity in the same fractions, immunoinsulin can be
detected as a discrete peak of activity. Endogenous
insulin can also be extracted from this protein by various
methods.
I Other Tissues
I
! I
The previous section described relationships among
insulin, insulinase and PDE on agarose gel column profiles :
I from liver. Kidney preparations were analyzed in a similar.
TABLE 3
SEPARATION OP IMMUNOINSULIN ACTIVITY PROM ENZYME
ACTIVITY AND RECOVERY OF INSULIN PROM
RAT LIVER GEL FILTRATION CONCENTRATES
LIVER
CONCENTRATE
Protein
mg/ml
Imraunoinsulln
yU/ml
Insulinase
% non-precipitable
Acidified
Concentrate*
Acid-
ethanol extract*
CPM Insulin
yU/ml
Insulin
yU/ml
1. 0.8
9
22.4 2
2. 4.8
47 34.7
82 66
3- 4.7 46
36.3 36
__
4. 4.4 54 39.0
43
——
5-
4.2 42 37.4
25 25
6. 5.2
33 37.2 26 48
7-
4.6 14 22.8
55
8. 4.6 14
28.3
26 38
9.
2.6 40 34.8 80
47
*A11 preparations tested for phosphodiesterase and insulinase activity
(% TCA non-precipitable CPM) were negative.
82
j fashion. Comparisons among the results from liver and
other tissues serve to aid in the interpretations of the
interrelationships between these various activities. The
results from three kidney preparations, assayed for immuno
insulin and insulinase are shown in Figures 15 and 16.
In Figure 15, the immunoinsulin peak is reduced
(58 yU/mg) compared to the results from liver preparations,
including diabetics. Insulinase, measured immunologically,
: has only a single peak of activity. There is no "notch"
in this profile as found in all liver preparations. Simi
larly, in Figure 16a , the total of two immunoinsulin peaks
is reduced (70 yU/mg) in comparison to the results from
the liver. There is no immunoinsulin or insulinase activ
ity in column fractions associated with the fraction I
form of kidney phosphodiesterase. The peak of insulinase
activity measured by the disappearance of added standard
insulin (Figure 15) and by the degradation of exogenous,
125
I-insulin (Figure 16B) is nearly coincident with the
peak of the higher molecular weight form of phosphodiester
ase.
Since most liver preparations had whole blood as a
; contaminating tissue in the preparation of cyclic nucleo-
i tide phosphodiesterase, it is important to determine the
; contribution, if any, of red blood cells to the results.
; The results of plasma preparations of PDE have been
i j
I __________________________________ , ___________ I
FIGURE 15
ACTIVITY PROFILES AFTER GEL FILTRATION
(BIOGEL A-5m) OF A KIDNEY PREPARTION
Profiles of protein (A2Qq), cyclic AMP hydrolysis,
immunoinsulin and insulinase measured immunologically are
shown. Preparation and assays are described in
Chapter II.
x Cyclic AMP substrate
A Immunoinsulin
© Insulinase juU
80
70
CD
C \ J
5 50
CO
0. 8
5 30
20 0.4
/\
I — I
^
85 35 45 55
Fraction N um ber
65 75
CO
FIGURE 16
ACTIVITY PROFILES AFTER GEL FILTRATION
(BIOGEL A-1.5m) OF TWO KIDNEY PREPARATIONS
Cyclic AMP hydrolysis and cyclic GMP hydrolysis
are shown in both 16A and 16B. Immunoinsulin activity is
shown in 16A and insulinase measured as % non-precipitable
CPM is shown in 16B. Preparations and assays are described
in Chapter II.
Vf
Fraction N um ber
□ Insulinase %
60
40-
40
L l _
30-
C CL
20-
30
Fraction Number
87
described (see above). Phosphodiesterase was prepared
from washed packed red blood cells (2.3 g) by the same
method used to study liver tissue. The results of agarose
gel fractionation (Biogel A-0.5m) of this preparation are
presented in Figure 17. The activity profiles of cyclic
AMP and cyclic GMP are consistent with studies of other
tissues. Multiple forms of PDE exist in rat erythrocytes.
The immunoinsulin content is greatly reduced (6 U/mg)
compared to other tissues. The peak of immunoinsulin
activity is nearly coincident with the higher molecular
weight form of PDE.
The tissues other than liver which were assayed for
immunoinsulin and insulinase are blood (plasma and RBC),
kidney and brain (not shown). These results help to clar
ify the interrelationships between immunoinsulin, insulin
ase and phosphodiesterase detected in agarose gel column
profiles of liver. It Is possible to conclude that
immunoinsulin and insulinase activities are not associated
with either form of PDE. Fraction I phosphodiesterase,
eluted in the void volume of kidney and brain preparations
does not have either activity. In fact, brain tissue did
not have these activities anywhere in the column profile.
In liver, these activities also did not coincide on
column profiles. These studies also suggest differences
between immunoinsulin and insulinase activities. In the
FIGURE 17
ACTIVITY PROFILES AFTER GEL FILTRATION
(BIOGEL A-0.5m) OF A RED BLOOD CELL PREPARATION
Profiles of cyclic AMP hydrolysis, immunoinsulin
and protein (A^qq) are shown. 13.5 mg protein fractionated.
Preparation and assays are described in Chapter II.
O Cyclic GMP substrate
x Cyclic AMP substrate
A Immunoinsulin
40
0.3
co
0.2 ™
A
v\ r-fft
23 27
CO
M O
Fraction N u m b er
kidney preparations (Figures 15 and 16) there was no
"notch" and the peaks of activity were more nearly coinci
dent with the 400,000 molecular weight form of PDE. These
results suggest that kidney tissue does not contain bound
insulin and that insulinase activity elutes more rapidly
than immunoinsulin activity on agarose gel columns. The
reduced immunoinsulin content of kidney tissue may indicate
the extent of interference of insulinase activity in the
radioimmunoassay for insulin.
No immunoinsulin activity was detected in plasma
and the amount in red blood cells is not sufficient to
account for the content of immunoinsulin in liver. This
suggests that the results from liver are not due to con
tamination by circulating insulin.
CHAPTER IV
DISCUSSION
Recent investigations have centered on whether the
ability of insulin to alter intracellular levels of cyclic
AMP is the single primary action that will account for
insulins effect on such diverse metabolic processes as
glucose transport, glycogen metabolism, and protein
synthesis. Glucagon, epinephrine and other hormones
increase the formation of cyclic AMP, an important intra
cellular effectof of metabolic processes. Insulin is one
of a few hormones which acts to decrease cyclic AMP levels.
The intracellular concentration of cyclic AMP at any moment
represents a balance between the activities of adenyl
cyclase (rate of formation) and cyclic nucleotide phos
phodiesterase (rate of degradation). In addition, intra
cellular levels of cyclic AMP may also be influenced by
transport rates to extra-cellular compartments. Studies
reviewed in Chapter I indicate that insulin may activate
phosphodiesterase both in vitro and in vivo. Since
enzymatic degradation is an intracellular mechanism
influencing cyclic AMP levels, the regulation of PDE
activity may be related to the mechanism of action of
insulin. Because of this relationship, it is important
to understand the properties of this enzyme.
92
Liver Cyclic Nucleotide Phosphodiesterases
The results of this study indicate that multiple
forms of cyclic nucleotide phosphodiesterase exist in
plasma, red blood cells, and liver. This result agrees
with other studies of these same tissues and studies of
PDE in many other tissues. The presence of multiple forms
in liver is consistent with reports by Beavo, et al.
(1970, 1971) and Campbell, et al. (1970) and Terasaki
(1971). However, these results are quite different from
earlier studies using essentially identical methods
(Thompson and Appleman, 1971b). There is only one minor
difference between the two methods; the homogenizing
medium in this study contained tris-acetate buffer and
2-mercaptoethanol (instead of water) and 10.9 % sucrose.
Consequently, the liver preparations in this study always
had excess sulfhydryl groups present. Reports by Cheung
(1970) have suggested the importance of 2-mercaptoethanol
for phosphodiesterase catalytic activity and structural
integrity. Cheung found that incubation of phosphodiester
ase with 2-mercaptoethanol converted a higher molecular
weight form, in the first peak of a Sepharose *JB column,
into a lower molecular weight second peak. He also
reported that enzymatic activity is inhibited by
p-hydroxymercuribenzoate. This inhibition was reversed
93
by 2-mercaptoethanol and suggests that sulfhydryl groups
are necessary for enzymatic activity.
The results of experiments on liver PDE support the
observation by Thompson and Appleman (1971b), "that the
hydrolysis of cyclic nucleotides by phosphodiesterase may
be a complicated enzymatic process." Under the conditions
of this study, liver can contain both fraction II and
fraction III enzymes. The evidence that the fraction III
enzyme found in liver is similar to fraction III enzymes
reported for other rat tissues is based upon three
criteria: similar molecular weight (E /E ratio), similar
v v0
kinetics (negatively cooperative and an apparent Km of
3 x 10”^M) and specificity for cyclic AMP substrate.
However, the results of experiments on liver frac
tion II enzyme suggests that it is more complex than
previously reported. Cyclic AMP hydrolysis and cyclic GMP
hydrolysis by this fraction are not coincident. When the
same column fractions are assayed for hydrolysis of both
substrates, the peak activities differ by an estimated
molecular weight of 40,000. This difference in molecular
weight is especially suggestive. Three different investi
gators have noted 40,000 molecular weight units in studies
of PDE. Cheung (1970) estimated the molecular weight of a •
protein activator at 40,000. Because of the high Km
(10”^M), presumably he activated fraction II enzyme with
................. 94
this factor. Jard and Bernard (1970) and Terasaki (1971)
estimated the molecular weights of their smallest form of
PDE as 40,000. It is interesting to speculate that the
difference between the two forms in fraction II is due to
the presence of one of these 40,000 molecular weight units.
Thompson (1971) has described a component in the elution
volume of agarose gel columns which activates fraction II
enzyme. He suggests this component may be the activator
described by Cheung. The difference between the results
from liver reported here and those of Thompson (1971) may
be that in this study, liver fraction II retained the low
molecular weight component.
Further evidence that fraction II is composed of
two different forms is based on the lack of any relation
ship between the ratio of cyclic AMP hydrolysis to cyclic
GMP hydrolysis (G/A ratio). Presumably, if the enzyme
concentration in fraction II is the same, the G/A ratio
should be similar. By inspection of these results, this
is clearly not the case for liver or kidney tissue
preparations.
A kinetic analysis, which has not been duplicated,
suggests that cyclic AMP hydrolysis by fraction II is posi
tively cooperative (slightly sigmoidal substrate-velocity
plot). Very recently, Beavo, Hardman, and Sutherland
(1971) have reported that liver contains two PDE activities.
95
Rat livers homogenized with 40 mM tris-Cl (pH 7.5)» were
separated into two PDE fractions with ammonium sulfate:
that which precipitated between 25 and 40% saturation and
that which precipitated between 40 and 67# saturation.
The 25-^0# ammonium sulfate fraction was positively coopera
tive (slightly sigmoidal substrate-velocity plot) for both
cyclic GMP and cyclic AMP hydrolysis. In this study the
kinetics of cyclic GMP hydrolysis by fraction II was not
determined. These results suggest that fraction II con
tains two distinct forms of PDE with very similar molecular
weights, and that liver tissue can have a total of three
different forms of phosphodiesterase after fraction III
has appeared.
The relationship between liver fraction II and
fraction III supports the hypothesis of a subunit aggrega
tion relationship, as suggested by Thompson (1971). In
liver, the low molecular weight form may be a product of
the higher molecular weight fraction. The evidence for
this precursor-product relationship is based primarily on
the fact that cyclic AMP hydrolysis only (characteristic
of fraction III) appears first as a shoulder of the total
PDE activity. This stage suggests the existence of inter
mediate molecular weight forms or shapes between fractions
II and III. In addition, cyclic AMP hydrolysis, relative
to cyclic GMP hydrolysis in fraction II is reduced in
96
association with the appearance of fraction III. As men
tioned, Cheung (1970) has reported a conversion from a
higher molecular weight from into a lower molecular weight
form. Furthermore, it is possible that Thompson (1971)
has converted fraction III into fraction II. Treatment
of fraction III with 2 M sodium acetate or fractionation
at pH 8.4 changed the molecular weight or shape of frac
tion III such that the E / E ratio was more similar to the
o
higher molecular weight fraction II. It would be of
interest to determine if specificity for cyclic AMP is
also changed by these treatments.
This study has demonstrated that the low Km form of
PDE can, under appropriate conditions, appear in liver
preparations. The possibility exists that intracellular
levels of cyclic AMP can be regulated by the amount of
fraction III present and catalytically active at any
moment. Since all liver preparations of phosphodiesterase
contained insulin, it is interesting to speculate that
this somehow facilitated the appearance of fraction III,
presumably from fraction II. If so, this effect would be
consistent with the observation that insulin reduces !
cyclic AMP concentrations in liver. i
I
97
Insulin and Cyclic Nucleotide Phosphodiesterases
The results presented here do not support the hypo
thesis that insulin activates phosphodiesterase in plasma
or liver. Senft, et al. (1968) observed a correlation
between the blood glucose concentration (and presumably
circulating insulin levels) altered by feeding, starving,
and refeeding or glucose infusion, and the activity of
cyclic AMP phosphodiesterase in liver homogenates. In
this study, circulating insulin increased by infusion of
a glucose load did not increase the hydrolysis of cyclic
AMP or cyclic GMP by plasma phosphodiesterases. In rats
made diabetic by intraperitoneal injections of streptozo-
tocin, plasma and liver insulin levels were significantly
reduced yet there was no difference in total cyclic AMP or
cyclic GMP hydrolysis from livers prepared from these rats.
Also, insulin was added in vitro to liver homogenates and
partially purified liver PDE and no increase in phosphodi
esterase activity was observed. However, plasma PDE
activity, especially cyclic GMP hydrolysis, was reduced in
the plasma of diabetic animals.
The fact that plasma and not liver PDE activity is
reduced in diabetic rats may be related to an effect of
insulin on protein synthesis. The animal that was sacri
ficed 2 days after streptozotocin treatment exhibited
hyperglycemia but no decrease in plasma PDE activity.
98....
Since there was little difference in plasma insulin levels
among the diabetic rats, this suggests that the reduced
PDE levels in the rats used 8-22 days after streptozotocin
injection was not due to insulin lack. This difference
between plasma and liver PDE activity is even more curious
because the liver is the site of biosynthesis of most
plasma proteins. Whether or not plasma PDE is synthesized
in liver, however, has not been determined. According to
the concept of Hess (1963), if it is possible to establish
that plasma PDE is unique to plasma, then it is probably
synthesized in liver. The kinetic properties of plasma
phosphodiesterase are similar to other tissues studied;
however, the observed molecular weights are unique to
plasma. This suggests that plasma PDE is a unique form of,
this enzyme. If so, separate hepatic biosynthetic mecha
nisms for liver and plasma PDE may be involved. The rela
tively reduced insulin level in streptozotocin diabetic
rats may be sufficient to maintain normal synthesis levels
of liver PDE but not plasma PDE.
Since insulin has been reported to directly activate
PDE in vitro, then insulin must somehow be associated with
PDE in vivo, i.e., insulin must have access to PDE. The
question of the subcellular distribution of insulin has
been discussed in Chapter I. The evidence indicates that
insulin can act both at the cell membrane and at
99
intracellular sites. Studies of the subcellular distribu
tion of phosphodiesterases has been reviewed by Thompson
(1971). Adipose tissue, kidney and brain have fraction I
activity which is interpreted as a membrane association.
Fraction II appears to be cytoplasmic. Thus, insulin
could activate negatively cooperative PDE associated with
membranes or positively cooperative cytoplasmic PDE.
Insulin, Insulin-Binding, and Insulinase
The evidence that endogenous insulin is bound to some
high molecular weight fraction, probably a protein, is
based primarily on the specificity of the insulin immuno
assay-, the various methods of separating immunoinsulin
activity from other proteins, the interpretation of the
"notch" and the reduced insulin content of column profiles
from streptozotocin diabetic rat livers.
The immunoassay for insulin appears to be specific.
Tissue preparations of plasma, kidney, red blood cells,
brain, and liver were fractionated and assayed for insulin.
Only one peak of immunoinsulin activity was observed. This
activity was always located between phosphodiesterase
fractions II and III.
100
There are at least three different enzymatic insulin-
degrading activities which have been described. Two dif
ferent types exist in liver: glutathione-insulin trans-
hydrogenase and a form which does not require glutathione
or sulfhydryl groups for activity and is probably proteoly
tic. Since the insulinase in these profiles has not been
characterized and because 2-mercaptoethanol was always
present, it is not possible to determine if it corresponds
to either of the previously described forms in liver.
Insulinase measured by both methods should produce
the same result. When insulinase is measured by the dis
appearance of added standard insulin, a characteristic
"notch" in the activity profile is evident. Strictly
interpreted, the lowest point of the "notch" indicates
less insulin is gone than at the peaks on either side of
the "notch;" or conversely, more insulin is there. This
is interpreted as bound insulin that the antibody can
recognize.
The immunoinsulin peak between PDE fractions II and
125
III could be due to insulinase degrading added I-
insulin in the assay. Both unlabeled insulin and this
degradation would reduce the CPM in an assay, indicating
the presence of insulin. However, in the comparisons of
normal and diabetic rat livers, the insulinase activity
was the same, only the insulin content was reduced. The
101
results from analysis of rat kidney preparations may Indi
cate the extent of the insulinase interference. Kidney
preparations contain insulinase activity comparable to
liver; however, there is no "notch" when insulinase is mea
sured immunologically and the immunoinsulin content is very
reduced. Presumably, there is no insulin in these kidney
preparations since there is no "notch" and the immunoinsul
in is due to insulinase_interference. In all liver prepa
rations studied, the "notch" was present and the content of
insulin/mg of protein exceeded that found in kidney.
These studies show that bound insulin and insulin-
degrading activity occur in the same agarose gel column
fractions. The data suggest that these activities are
independent of one another and chromatograph together only
by coincidence. The results from kidney preparations sug
gest that there is no bound insulin, yet insulinase still
chromatographs between phosphodiesterase fractions II and
III. The insulinase also appears to be slightly larger
than the insulin-binding activity. The lowest point of the
"notch" is always slightly offset from the center of the
insulinase peak. Whether or not the insulin-binding and
insulinase represent a single unit must await further tests
using different separation methods and studies of the sub- ;
cellular distribution of both activities. However, both
may be important to the physiological action of insulin in
liver.
CHAPTER V
SUMMARY AND CONCLUSIONS
Recent investigations have shown that insulin
reduces intracellular levels of cyclic 3*,5 '-adenosine
monophosphate (cyclic AMP) and that insulin activates
cyclic nucleotide phosphodiesterase, the enzyme(s) that
hydrolyzes cyclic AMP. Because this activation may repre
sent a site of insulin action, relationships between
insulin and cyclic nucleotide phosphodiesterase were
studied in normal and streptozotocin diabetic rat plasma
and liver.
There was no difference between liver phosphodi
esterase activity prepared and assayed by the method of
Thompson and Appleman (1971a) from normal and diabetic
rats. Plasma phosphodiesterase was not activated by
increased insulin released during a glucose tolerance test,
however, both cyclic AMP and cyclic 3',5'-guanosine mono
phosphate hydrolysis were reduced in diabetic plasma.
Multiple forms of cyclic nucleotide phosphodiester
ase exist in plasma and liver. In liver, the previously
unreported lower molecular weight form with a high affinity
for cyclic AMP (apparent Michaelis constant 3 x 10”^M)
appears after ammonium sulfate precipitation and storage
at 4°C. This form exhibits physical and kinetic properties
similar to the lower molecular weight forms reported for
other tissues. Results suggest that the higher molecular
weight fraction in liver may be two different forms and
that the lower molecular weight is a product of the higher
molecular weight fraction.
Discrete peaks of immunoinsulin activity (molecular
weight 300,000) and enzymatic insulin-degrading activity,
occur on agarose gel column profiles between the high and
low molecular weight forms of phosphodiesterase in liver.
The immunoinsulin content of diabetic rat livers is reduced
when compared to normal rat livers. This immunoinsulin
activity presumably bound insulin, is not present in brain,
kidney, erythrocytes, and plasma preparations. Endogenous
insulin has been separated from the binding and enzymatic
activities by chromatography, acid-ethanol extraction, and
ultrafiltration.
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Creator Hemington, John Glenn (author)

Core Title Studies Of Relationships Between Insulin And Cyclic Nucleotide Phosphodiesterase In Normal And Diabetic Rats

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Degree Doctor of Philosophy

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Publisher University of Southern California (original), University of Southern California. Libraries (digital)

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Advisor Dunn, Arnold S. (committee chair), Appleman, M. Michael (committee member), Shugaram, Peter M (committee member)

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