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This is to certify that the
thesis entitled

HORMONAL CONTROL OF TUBULIN CONTENT
AND POLYMERIZA‘I‘ION IN RAT LIVER

presented by
Rosalyn M. Zator

has been accepted towards fulfillment
l of the requirements for

.__M;S_.___degree in MS try

W4

Major professor

MM

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Place in book return to remove
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HORMONAL CONTROL OF TUBULIN CONTENT
AND POLYMERIZATION IN RAT LIVER

By
Roselyn Mary Zator

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry

1979

ABSTRACT

HORMONAL CONTROL OF TUBULIN CONTENT
AND POLYMERIZATION IN RAT LIVER

BY
Roselyn Mary Zator

The starved-refed rat and the streptozotocin induced
diabetic rat were used as models to study microtubule reg-
ulation. The method of Pipeleers and co-workers (J‘,le1
giglg‘zfi, 3&1-350. 1977). which includes a colchicine
binding assay, was used to quantitate soluble free and
polymerized tubulin. During starvation and diabetes. the
percent of liver tubulin polymerized was decreased com-
pared to control levels. Refeeding starved rats and ins
jecting insulin into diabetic rats resulted in an increase
in the degree of tubulin polymerization. Tubulin content
and polymerization changed in the starved-rated rat liver
due to polymeric tubulin while in the diabetic rat liver
the change was due to free tubulin. Fluctuations in the
degree of polymerization in the starved-refed rat liver
may be correlated to VLDL secretion from the liver.

Plasma glucose and insulin levels. and liver cAMP and
cGMP levels did not appear to directly affect tubulin
content or polymerization. The method of quantitating
ig;zizg free and polymerized tubulin was investigated to
verify that actual isolation of the in give situation

had been accomplished.

DEDICATION

To my parents. Henry and Henrietta Zator and

my loving husband. Dennis M. Wojcik for their

constant love, patience. and understanding.

ii

ACKNOWLEDGEMENTS

I wish to express my appreciation to Dr. William W.
Wells for his understanding. guidance, patience, and
financial support throughout these studies. Sincere
thanks goes to my committee members. Dr. Philip Filner
and Dr. Steve Heidemann for their suggestions.

I would also like to express my eternal gratitude
and appreciation to Jeffrey A. Nickerson without whose
guidance. support, encouragement. and friendship none
of this would have been possible.

Special thanks to James Kurtz. Christine Collins,
and Greg Doll for their intellectually stimulating
conversations.

Last but not least I would like to thank my ever so
patient husband and family for their everlasting moral
support.

iii

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . .
LIST OF FIGURES . . . . . . 0
LIST OF ABBREVIATIONS . o . .

INTRODUCTION. 0 o o . . . . .

Objectives . . . . . . .

Literature Search. . . . .

Bibliography e e o e e

CHAPTER I: TUBULIN CONTENT AND

THE STARVED-REFED

Abstract 0 o o e e e 0
Introduction . . . . .
Materials and Methods.
ROSUltS. e e o e o o 0
Discussion . . . . . .
Bibliography 0 e o e 0

CHAPTER II: EXAMINATION OF THE
‘ QUANTITATING FREE AND POLYMERIZED

TUBULIN. . o . .

Abstract 0 e o o o e 0
Introduction . . . . .
Methods and Materials.
ROSUItBo o e e o e o 0
Discussion . . .
Bibliography . .

CHAPTER III: LIVER TUBULIN CONTENT AND

POLYMERIZATION IN THE

INDUCED DIABETIC RAT. . 0

Abstract 0 e e o e e 0
Introduction . . . . .
Materials and Methods.
RGSU1tSe o o o o o o 0
Discussion . . .
Bibligraphy. . .

I I I I I I I I I v
I I I I I I I I I vi
0 e o o o o o e 0V111
I I I I I I I I I 1
I I I I I 1
. . o 2
I I I I 18
POLYMERIZATION IN
RAT LIVER . . . . 26
I I I I I I I I I 26
I I I I I I I I I 2?
I I I I I I I I I 28
I I I I I I I I I 33
I I I I I I I I I “8
I I I I I I I I I 57
METHOD OF
I I I I I I I I I 60
I I I I I I I I I 60
I I I I I I I I I 61
I I I I I I I I I 62
I I I I I I I I I 6+
I I I I I I I I I 73
I I I I I I I I I 76
STREPTOZOTOCIN
. . . . 77
I I I I I I I I I 77
I I I I I I I I I 78
I I I I I I I I I 79
I I I I I I I I I 81
I I I I I I I I I 91
I I I I I I I I I 94

LIST OF TABLES

CHAPTER I
I. Food Consumption and Body and Liver‘Weights
of Starved Rats Refed a High Glucose Diet . . 38
II. Liver Protein. DNA, and Protein/DNA in
Starved-Refed Rats. o o e o o o e o e o o e o 40
III. The Degree of Tubulin Polymerization and
Amount of Tubulin per Protein in the Starved-
Refed Rat Liver e e o e o e o o o o e e o e e “3
CHAPTER II
I. The Effect of Tissue Dilution on the Degree
of Tubulin Polymerization . . . . . . . . . . 65
II. The Effect of Tissue Dilution on the Percent
of Tubulin.Polymerized and Total Tubulin
content I I I I I I I I I I I I I I I I I I I 66
CHAPTER III
I. Protocol of Experiment and Body Weight of
RatSe e e e e o o o o e e o e e o o e e o o e 82
II. Plasma Glucose and Insulin Levels. Liver

Protein Content. and Total Liver Tubulin
Based on Protein Content. . . . . . . . . . . 83

CHAPTER I

I.

LIST OF FIGURES

Protocol Used for Determining the Amount of
Free and Polymerized Tubulin in the Starved-
Refed Rat Liver. e o o o o e e o o e o o e e 31

II. Kinetics of Colchicine Binding Activity in
MTS and TS o o e o e o o o e o o o e e e o o 35
III. Liver Free and Polymerized Tubulin and the
Degree of Tubulin Polymerization in the
Starved-Refed Rat. o e o o o o e e o o e o 0
IV. Free and Polymerized Tubulin Based on DNA in
the Starved-Refed Rat Liver. e o o o e o o o 7
V. Plasma Glucose and Insulin Levels in the
Starved‘Refed Rate 0 o e o e e o o o o o e e 50
VI. Liver cAMP and cGMP Levels in the Starved- '
Refed Rate a o o e e e o e e o o e o e o e e 52
CHAPTER II
I. The Effect of Tissue Dilution on the
Linearity of Colchicine Binding Activity in
MTS and TS I I I I I I I I I I I I I I I I I 67
II. The Effect of Tissue Dilution on the
Linearity of Colchicine Binding.Activity in
MTS and T5 0 e o e e e o o o o e e o o o e 0
III. Colchicine Binding of Liver Tubulin in MTS
or TSI I I I I I I I I I I I I I I I I I I I 70
IV. Colchicine Binding of Brain Tubulin in MTS
or TSe e o e e e o o o e o e o e o o o e o e 71
v. In Vitro Polymerization Studies Using MTS

BEerr and.Assembly Buffer with a Tubulin
Concentration of 600 ug/ml . . . . . . . . . 72

vi

 

 

vii

CHAPTER'III

I.

II.

III.

The Degree of Tubulin Polymerization in the
Streptozotocin Induced Diabetic Rat Liver. . . 86

The Amount of Free and Polymerized Tubulin
per Gram Liver in the Streptozotocin
Induced Diabetic Rat . . . . . . . . . . . . . 88

The.Amount of Free and Polymerized Liver
Tubulin per Mg DNA in the Streptozotocin
Induced Diabetic Rat . . . . . . . . . . . . . 90

DEAE
GTP
ATP
EGTA

GDP
GMPPCP
MAPS
GMPPNP
cAMP
cGMP
VLDL
MES
DMSO
TCA

BW

LIST OF ABBREVIATIONS

Diethylaminoethyl
Guanosine-S'-triphosphate
Adenosine-S'-triphosphate

Ethylene glycol bis ( - aminoethyl ether)
N. N. N', N' tetraacetic acid

Guanosine-5'-diphosphate
5'-guanylylmethylene diphosphonate
Microtubule associated proteins
Guanyl-S'-yl-imidodiphosphate
Cyclic adenosine monophosphate
Cyclic guanosine monophosphate
Very low density lipoprotein
2-(N-morpholino)ethanesulfonic acid
Dimethylsulfoxide

Trichloroacetic acid

Body‘weight

viii

 

 

INTRODUCTION

Objectives and Organization

The regulation of tubulin polymerization is not well
understood. Tubulin polymerization. in vitro. has been

 

extensively investigated. Though several mechanisms have
been elucidated; a controversy still exists concerning
these mechanisms. There is. therefore. a need to identify
model systems in 1119 in which tubulin content and poly-
merization can be experimentally manipulated and studied.
The starved-refed rat and the streptozotocin induced dia-
betic rat before and after insulin treatment are two such
systems.

The purpose of these studies is to:

1. Identify model systems in which tubulin con-
tent and polymerization can be experimental-
ly manipulated.

2. Elucidate the role of microtubules in these
systems.

3. Determine whether physiological factors con-
trol microtubule dependent processes through
alterations in the equilibrium between free

and polymerized tubulin.

2
Chapter 1 deals with the regulation of tubulin con-
tent and polymerization in the starved-refed rat liver.
Chapter 2 examines the method used to measure free and
polymerized tubulin. Chapter 3 deals with the regulation
of tubulin content and polymerization in the streptozoto-
cin induced diabetic rat with and without insulin treat-

ment.

Wm

Micrctubules have been implicated in a wide variety of
cellular processes such as secretion. intracellular trans-
port. mitosis, cell motility. maintenance of cell shape.
and integrity of cell membrane receptors. The mechanism
by which microtubules participate in these processes may
involve the dynamic equilibrium between free and polymer- -
ized forms of tubulin, the major microtubule protein.

More specifically. microtubular involvement in movement
may be regulated by the control of tubulin polymerizaticn~
(1-5). However. the factors or conditions influencing
assembly and disassembly are not well understood. A
major obstacle to studying the regulation of microtubules
is the lack of a reliable and valid assay for microtubules
or tubulin.

With the aid of tritiated colchicine (6), Borisy and
Taylor (3,?) found that colchicine binding activity was

highest in sources known to be rich in microtubules. such

 

3
as mitotic spindles, cilia, sperm tails, and brain. and
concluded that the colchicine binding protein was a subunit
of microtubules. Weisenberg. Borisy, and Taylor (8) were
the first to isolate and purify the colchicine receptor
from supernatant extracts from porcine brain by ammonium
sulfate fractionation and ion-exchange chromatography on
DEAE Sephadex. The protein was subsequently identified as
the microtubule protein (9,10).

Tubulin is a dimer having a mass of 100,000-130,000
Daltons and a 320,w of 68. When denatured in guanidine
hydrochloride or sodium dodecyl sulfate two subunits of
approximately the same size are found. The dimer binds one
mole of colchicine and two moles of GTP (8.11); of the two
nucleotide binding sites only one is exchangeable. Col-
chicine binds tubulin, inhibits assembly, and reverses
assembly causing disassembly in 3119 and in 31333 (11,13,
14). Colchicine binding activity of tubulin is unstable
and decays rapidly in vitgg. Agents which stabilize the
activity include colchicine itself, GTP, Mg*2, lyophil-
lization, moderate ionic strength. pH 6.7-6.8, and vinblas-
tine (8.12.15). Recently Margolis and Wilson (14) proposed
that colchicine blocks microtubule assembly by binding to
tubulin which then binds to the growing end of the micro-
tubule during assembly and blocks further assembly. Studies
(16,17,129), showing protein molecules in mammalian brain
interacting with the colchicine site in tubulin, indicate

that this site may have a significant role in the in vivo

 

regulation of tubulin polymerization.

L.

The highly specific and essentially irreversible bind-
ing of colchicine to tubulin (15) has allowed it to be
used as a tool to assay for tubulin. Tritiated-colchicine
binding assays must include a step to separate the colchicine-
tubulin complex from free colchicine. Gel filtration. ionic
adsorption. and adsorption of the free colchicine on acti-
vated charcoal have been used. The gel filtration method
involves the separation of tubulin-colchicine complex and
free colchicine via the column (3.18). The second method
depends on the adsorption of the highly acidic tubulin-
colchicine complex to the DEAE groups on the paper and
filtering or washing away the free colchicine (8.19).

With the activated charcoal method. free colchicine is
adsorbed by the charcoal. thereby accomplishing separation
of the free colchicine and colchicine-tubulin complex (20).
Other methods have been designed to assay for tubulin with-
out the use of the drug colchicine. for instance antibodies
have been used in radioimmuncassays as well as immuno-
fluorescent probe assays (21.24). Electron microscopy has
also been used to quantitate microtubules (25).

In an attempt to understand ;p_zixg microtubule as-
sembly. ig gitgg polymerization systems have been developed.
‘Weisenberg (26) was the first to achieve polymerization
in zijgg in a temperature dependent system. Borisy and
Olmsted (27) confirmed his results. Weisenberg showed that
in vitro polymerization of rat brain tubulin requires Mg+2.
ATP or GTP. EGTA to chelate CaIZ, and 37 C. Shelanski.

 

5
Gaskin. and Cantor (28) subsequently) designed a method
to purify tubulin based on its ability to polymerize
and depolymerize. They also showed that microtubule
assembly could occur in the absence of added nucleotides
in the presence of 1M sucrose or #M glycerol. Micretubule
polymerization in extracts of mammalian tissue has been
measured using viscometry. electron microscopy. turbidity.
and optical diffraction (13.26.28-31). Microtubule
assembly is dependent on temperature. pH. Ca+2 concen-
tration. Mg+2 concentration. ionic strength. and tubulin
concentration. In polymerization studies. preparation
from different species behave differently. The age of
the animal may influence polymerization ability (32.
33). Besides tubulin from brain. tubulins from platelets
(3h-37). bovine renal medullary tissue (38). drosophila
embryo (39). Ehrlich ascites tumor cells (#0). and HeLa
cells (hl.h2) have been polymerized in ziggg.

Guanine nucleotides have been found bound to
tubulin (8.43). Tubulin contains two binding sites for
GTP. an exchangeable site which readily exchanges
phosphate with the medium. and the nonexchangeable site
which binds tightly and does not exchange with the
medium. GTP is normally required for tubulin poly-
merization (26.29.u4.u5). In the presence of high con~
centrations of sucrose or glycerol. GTP was not required

for polymerization (28). Though no depolymerization

 

 

6
occurred in glycerol and occurred in sucrose only under
abnormal conditions. Occupancy of the exchangeable site
by GTP is necessary for polymerization but polymerization
may occur in the presence of GDP and ATP by a transphos-
phorylase reaction (46). A controversy exists about whether
hydrolysis of GTP is required for polymerization. Inves-
tigators have seen polymerization in the presence of the
nonhydrolyzable analogs. GMPPCP and GMPPNP (47.48). The
hydrolysis of GTP to GDP may not be required for microtubule
polymerization but rather for the subsequent depolymerization
of the microtubule formed (45.47.49).

Tubulin purified through several cycles of polymeriza-
tion and depolymerization has been found to contain a small
fraction of other proteins called Microtubule Associated
Proteins (MAPS) (50-53.68). The MAPs (53) which can be
separated from tubulin by chromatography techniques have
been found to influence the rate and extent of polymeriza-
tion ip,gitgg (51.55-58). Brain tubulin can be polymerized
in the absence of MAP8 but a high concentration of tubulin
is required (59.60). A class of MAP3 has been identified
as High-MolecularAWeight proteins (BMW) of subunit molecular
weight 275.000 to 350.000. These HMW proteins have been
visualized by electron microscopy as a filamentous coating
on the surface of microtubules. in gitgg and ig,z§zg (55.
58.61.62). These HMW proteins may bridge microtubules to
each other or to other structures such as cellular organelles.

They may have a motile function and be directly involved in

7
the translocation of particles and organelles (55). Sherline.
Lee. and Jacobs (63) have found that the EMT proteins asso-
ciated with microtubules are preferentially adsorbed by
secretory granules and that they may provide the link be-
'tween tubulin and the secretory granule. This is evidence
for microtubule involvement in intracellular movement of
secretory granules. Recently Nunez and cc-workers (17)

have found that HMW proteins and tau proteins competitively
inhibit colchicine binding to tubulin. These proteins may
bind at or near the same site as colchicine or modify the
affinity of the colchicine binding site and therefore may
play a role in the regulation of microtubule function and
assembly. Some investigators believe that polymerization

is promoted by a mixture of 4 or 5 polypeptides of molecular
weights between 50.000 and 70.000 Daltons which they col-
lectively call tau (52.64-66). Recent observations show
that fluorescein-conjugated antiserum to pig brain tau pro-
tein stains a colcemid-sensitive network in interphase mouse
embryo fibroblasts and mitotic spindles in dividing cells.
suggesting some ig_zizg function for tau protein in the
microtubule (67).

Several mechanisms of microtubule assembly have been
proposed. Kirschner and co-workers (25) proposed that
microtubules consist of proteins which were initially both
in the ring form and the tubulin dimer. Rings and tubulin
dimers sequentially form ribbons. sheets. and then a cylin-

drical structure containing the 13 protofilaments. the

 

. ag~ ”y.“ . “h-.."-

8

microtubule. Vallee and Borisy (68) have investigated the
forms found in tubulin preparations through analytical sedi-
mentation techniques. Their findings are that 308 oligomers,
are tubulin double rings with HM" proteins. 398 oligomers
are 308 rings stripped of their BMW proteins. 188 oligomers
are tubulin with HMW proteins but not in a ring form. and
208 oligomers are single rings with tau. These oligomers
do not have to be intermediates for microtubule assembly.
moreover it appears that multiple pathways to the formation
of microtubules may exist depending on the protein composi-
tion of the preparation and the nature of the oligomers
present (68).

It has been reported that tubulin preparations contain
a cAMP dependent protein kinase as well as a cAMP indepen-
dent protein kinase. both of which phosphorylate tubulin
and MAP8. Goodman and co-wcrkers (73) preposed that puri-
fied brain tubulin is both a cAMP dependent protein kinase
and a substrate for this enzymatic activity,tand that phos-
phorylation was related to polymerization. Scifer (70)
because of his unsuccessful attempt to separate protein
kinase activity from tubulin agreed that cAMP dependent
protein kinase was intrinsic to the tubulin itself. Recent-
ly Ikeda and Steiner (36) have found cAMP independent pro-
tein kinase activity in platelet tubulin that seems to be
an intinsic property for its lack of separation from the
colchicine binding activity. However Eipper (72) and
Shigekawa and Olsen (74) separated tubulin and the protein

9
kinase by gel filtration chromatography. Sloboda and co-
workers (53) did likewise using sucrose gradient ultracen-
trifugation. The method used to purify tubulin determined
whether tubulin was itself phosphorylated by the associated
protein kinase. Goodman and co-workers (73) using the
Weisenberg procedure (8) obtained ig,zitgg.phosphorylation
while Sloboda and co-workers (53) using the Shelanski method
(28) did not see phosphorylation. Sloboda and co-workers
(53) have also found cAMP dependent phosphorylation of HMW
proteins which may be related to their role in microtubule
structure and function. Rappaport and co-workers (75)
achieved phosphorylation of MAP8 but found that an inhibitor
of cAMP dependent protein kinase did not affect ig,zi§gg
polymerization. They concluded that phosphorylation of
MAPS did not affect microtubule assembly.

Tubulin prepared by the polymerization-depolymerization
method of Shelanski and co-workers (28) has been shown to
contain other enzymatic activities. besides cAMP stimulated
protein kinase. which may affect microtubule structure and
function. Among these are a nucleosidediphosphate kinase
activity (122). a diglyceride kinase activity (80). a phos-
pholipase C activity (81). and a tubulin d-subunit tyros-r
ylating activity (82).

The inhibitory affect of colchicine on microtubule
assembly has made it useful as a tool for studying the
involvement of the microtubules in various cell processes.

Microtubules have been implicated in the secretion or release

10

of substances by various cell types including insulin from..
the pancreas (77-79). thyroid hormone from the thyroid (83.
84). growth hormone from the pituitary (85). VLDL from the
liver (86.92-94). protein from lacrimal glands (95). amylase
from the pancreas (96). amylase from.the parotid gland (97.
98). glycoprotein from the salivary gland (99). catechola-
mines from.the adrenal medulla (100.101). and lyscsomal
enzymes from phagocytes (102-104). Colchicine did not seem
to be poisoning other cellular functions such as glycolysis.
gluconeogenesis. maintenance of energy charge. lipid bio-
synthesis. protein synthesis. or urecgenesis so presumably
its effects on secretion were solely due to microtubule
disruption (86). Supporting this was the fact that inves-
tigators correlated inhibition of secretion with microtubule
disappearance (85.86.88.9l.96.98). Other evidence that
colchicine effects were solely due to microtubule disruption
was actual isolation of colchicine-tubulin complex and sim-
ilarities in the time course of colchicine binding. micro-
tubule disruption. and inhibition of secretion (78.84.85.95).

The sequence of protein synthesis. intracellular trans-
port. and secretion is well established. Proteins are syn-
thesized on polysomes. attached to the membrane of the endo-
plasmic reticulum. transported to the interior of the rough
endoplasmic reticulum. then to the smooth endoplasmic reti-
culum and finally to the Golgi apparatus. The Golgi appara-
tus then migrates towards the cell membrane fuses with it

and releases its contents. VLDL are also secreted via the

ll
Golgi-derived secretory vescicles. With the administration
of colchicine. protein (92-94) and lipoprotein granules
(87.90) accumulated in Golgi-derived secretory vescicles
implying that colchicine was inhibiting the release of
contents from.the Golgi. Chambert-Guerin and cosworkers
(95) working with rat lacrimal glands suggested that
colchicine inhibited the secretory processeby dis-
turbing intracellular migration of secretory granules
rather than by interfering with the discharge step.
A weak and/or delayed inhibitory effect of high colchi-
cine concentrations has been demonstrated on the secre-
tory processes that follow hormonal stimulation (83.97)
while the release of VLDL or other proteins was inhibited
by low colchicine concentrations (86-88.92.94). The dif-
ference between systems may be an indication of inherent
differences in cell type. secretion processes. or micro-
tubules. Recently Reaven and Reaven (105) have seen that
an increase in VLDL secretion can occur without any demon-
strable change in hepatocyte assembled microtubule or
tubulin content and questioned whether microtubules
are involved in hepatic VLDL secretion. Redman and co-
workers (93) observed that colchicine inhibited protein
synthesis in cells exposed for more than one hour and
this inhibition affected both secretory and nonsecretory
proteins. Investigators have also shown that colchicine
concentrations which cause the virtual disappearance of

microtubules gave only a 35-45% inhibition of release or

 

 

12
secretion (85,104,106). These observations imply that
a change in tubulin content or polymerization may not be
the controlling factor in these secretory processes.
Speculations have been made that colchicine is affecting
secretory processes by a mechanism independent of micro-
tubules (91.105).

To better understand the function and regulation of
microtubules in the cells investigators have been looking
at the subcellular distribution of the colchicine binding
protein. Investigators have shown that a substantial a-
mount of brain tubulin was in the particulate fraction
of brain homogenates (15.107). Bhattacharyya and
Wolff (108) found that in brain 45-50% of the total
homogenate colchicine binding activity was in the
cytoplasm while in the thyroid the percentage was 70-75.
The remaining colchicine binding activity was associated
with membrane bound tubulin. which on the basis of
colchicine binding properties and immunological cross-
reactivity resembled soluble tubulin. This solu-
bilized membrane tubulin can polymerize to form micro-
tubules (109). While colchicine binding activity in
membrane containing brain fractions resembled that of
soluble tubulin. colchicine binding activity in liver
membrane containing fractions differed considerably from
tubulin (110). Ninety-eight percent of the colchicine
binding activity in platelets was in the soluble

fraction rather than in membranes and granules (37).

13

In the islets of langerhans. colchicine binding activity
was predominantly confined to the post microsomal super-
natant fraction (105.000 g for 60 minutes) (78). The
reason for the different distribution of colchicine
binding activity in the various cell types is not known.

In order to study microtubule regulation. in gig-9,.
attempts were made to isolate the in,ziyg situation of
polymerized and free tubulin. Koehn and Olsen (33)
measured the forms of tubulin in brain by disassembling
all microtubules to obtain the total tubulin content
while determining free levels by homogenizing tissue in
a glycerol-MES stabilizing buffer. centrifuging. and
assaying tubulin in the supernatant. They found that
the distribution of tubulin between the polymeric and
free state varied with protein concentration. possibly
due to ip_gitgg polymerization. This conclusion may
be warranted because the tubulin concentrations studied
were in the range of the critical concentration for
polymerization of purified tubulin. iglyipgg. Rubin
and Weiss (113) measured the extent of microtubule 389-
sembly in ovary cells. by first disassembling micro-
tubules with a cold buffer followed by colchicine binding
to determine total tubulin content. Polymerized tubulin
was measured by homogenizing cells in a glycerol buffer.
pelleting the microtubules. resuspending the pellet to
disassemble the microtubules. centrifuging. and assaying

the supernatant for colchicine binding activity. The

l4
assay was not sensitive to cold conditions but an in-
crease in the percent of tubulin polymerized was seen
with an increase in dibutryl cAMP. Patzelt and co-
workers (114) were the first to characterize livers?
tubulin. They measured the total microtubule content
by depolymerizing microtubules in the cold in.the absence
of glycerol. Free tubulin was measured by homogenizing
tissue in a cold glycerol buffer. centrifuging. and
[assaying the supernatant. The amount of tubulin polyp
merized was 40% of the total. In comparison to 15$
which was attained by Reaven and co-workers (115) using
a combined morphometric and biochemical approach to
identify and quantitate microtubules. Pipeleers and co-
workers (116) have also devised a method to assay poly-
merized and free forms of tubulin. Liver tissue was
homogenized in a glycerol-BMSO containing buffer and
the homogenate centrifuged to pellet the intact micro-
tubule. The supernatant was assayed for free tubulin
using a colchicine binding assay. The pellet was re-
suspended in a cold sucrose buffer to disassemble the
intact microtubule. The suspension was centrifuged and
the supernatant was assayed using a colchicine binding
assay. The amount of total tubulin was the calculated
sum.of the free and polymerized forms. The percent of
total tubulin that was.in the polymerized form.wa8rv35.
Eichhorn and Peterkofsky (106) measured total and poly;

merized tubulin in 3T3 cells. To measure polymerized

 

 

15
tubulin. cells were sonicated in a glycerol-DMSO con-
taining buffer. centrifuged to sediment the intact micro-
tubules. pellet resuspended in cold phosphate-magnesium
buffer with 0.5% triton x-1oo, sonicated. centrifuged.
and the supernatant was assayed for colchicine binding
activity. Total tubulin was measured by disrupting all
microtubules into soluble tubulin by sonicaticn at 0‘0.
Forty-seven percent of the total tubulin was polymerized.
These proceeding methods basically use a glycerol buffer
to stabilize the microtubule allowing for the separation
of polymerized tubulin from free tubulin. Cold tem-
peratures are used for their depolymerization affects.
Finally a colchicine binding assay is used to quantitate
tubulin.

Investigators have looked at the influence of phys-
iclogical states on tubulin content and polymerization.
In pancreatic islets. during fasting total tubulin con-
tent and the percent of tubulin polymerized was decreased
compared to controls (11?). A 30% dextrose solution diet
resulted in an increase in the percent of tubulin poly-
mlrized over that of controls. This study supports the
theory that tubulin polymerization is involved in the
insulin secretory response to a glucose stimulus. In
another study (118). starved rats showed a decrease in
total liver tubulin and the percent of tubulin polymerized.
Feeding rats a 30% dextrose solution diet increased the

degree of liver tubulin polymerization over that of con-

 

16
trols without affecting the total tubulin content.
Pipeleers and co-workers (118) found that the total
tubulin content per DNA in the liver of genetically
obese mice was increased over their lean litter mates
in the fed state but in the fasted state tubulin content
was the same. Genetically obese mice had a lower per-
centage of their tubulin in the polymeric form in both
fed and fasted conditions. Pipeleers and co-workers
also looked at tubulin content and polymerization in
lymphocytes and platelets (118). Phytohemagglutin-
stimulated lymphocytes had an increase in total tubulin
content over controls. The increase was due to an in-
crease in polymerized tubulin. Storage of platelets re-
sulted in a decrease in total tubulin and even greater
reduction in the polymerized form. Normal values of the
percentage of tubulin polymerized in these tissues were
liver 30-35%. islets 35-40%. lymphocytes'V60%. and
plateletsaV90%. From these studies it has been shown
that total tubulin content and its degree of poly-
merization can be modulated independently by a wide
variety of physiological factors.

The function of microtubules in the cell may be
involved in their attachment to cell membrane compo-
nents. Various studies have shown that local anesthetics
interfere in microtubule function. possibly by depoly-
merization of the microtubule (106). Recently in-

vestigators have shown that these anesthetics do not

 

l7
bind to tubulin or cause depolymerization though they
may be involved by detaching microtubules from the cell
membrane (106). Electron microscopy has shown that
microtubules were absent immediately beneath the cell
‘membrane. Studies of “capping“ of various cell types in
the presence of concanavaliaaA.and colchicine indicate
that membrane proteins may be "anchored" in place by

microtubules (120 . 121) .

9.

10.

ll.

12.

13.

l4.

l5.
l6.

 

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CHAPTER I

TUBULIN CONTENT AND POLYMERIZATION
IN THE STARVED-REFED RAT LIVER

ABSTRACT

The regulation of liver tubulin content and polymeri-
zation. ig_zizg. is not well characterized. By looking at
a system where these features can be experimentally manipu-
lated, a better understanding of tubulin regulation may be
attained. Accordingly. young male rats of the Holtzman
strain were starved for 3 days and rated a high-glucose con-
taining diet. At intervals during the 48 hour rereading
period. rats were killed and the liver was assayed for free
and polymerized tubulin (1). After‘j days starvation. plasma
glucose and insulin levels were decreased and liver any?
and cGMP levels were elevated compared with fed controls.
Following refeeding. plasma glucose and insulin levels rap-
idly increased reaching a peak at 6 hours for glucose and
1 hour for insulin. though the latter did not exceed those
of control rats. Liver CAMP levels decreased in the refed
rats to those of normal rats by 3 hours,_while liver cGMP
levels decreased below control levels and did not approach

26*

27

control levels until 36 hours after refeeding. Starved rats
showed a significant decrease in both free and polymerized
liver tubulin. Eighteen hours after refeeding. the level
of liver polymerized tubulin approached control levels.
while the free tubulin levels failed to increase throughout
the 48 hours. The degree of tubulin polymerization was de-
pressed during starvation and increased significantly after

refeeding to values above those of controls.

INTRODUCTION

In this study. the starved-refed rat liver was used to
study regulation of tubulin content and in 1112 polymeriza-
tion. It has long been recognized that liver function is
hormonally regulated. During starvation of the rat. plasma
glucose and insulin levels decrease and plasma glucagon
levels increase. In the liver. the rates of glycolysis.
protein synthesis. lipogenesis and lipoprotein secretion
decrease while the rates of gluconeogenesis and fatty acid

‘ig-oxidation are greatly increased (3.4). Liver cAMP concen-
trations are increased during starvation but a controversy
regarding cGMP levels remains (5.6.7).

Refeeding starved rats a diet rich in glucose causes
an increase in plasma glucose and insulin levels and a de-
crease in glucagon concentrations. After refeeding. the rates
of protein synthesis. lipogenesis. and lipoprotein release
increase in the liver (8.9.10). Microtubules are thought

to be involved in and required for the secretion of VLDL

28

and plasma proteins from the liver (ll-18.44). Therefore.
this system. in which the secretory processes can be manip-
ulated. is one in which the regulation of microtubule func-
tion might profitably be studied.

Preparations of microtubule protein contain a cyclic
AMP dependent protein kinase which can phosphorylate tubulin
and MAPs (19). Cyclic AMP dependent phosphorylation of
microtubule components may play a role in microtubule assem-
bly or function 12.1122 (19.20). Reciprocal effects of cAMP
and cGMP on the microtubule-dependent release of lysosomal
enzymes from polymorphonuclear leukocytes have been found
(21.23). It is possible that cyclic nucleotides affect
secretory processes via an effect on microtubules.

In this study we were able to manipulate many physio-
logical variables in the rat by starving and refeeding.
By measuring liver tubulin levels and the degree of tubulin
polymerization we could try to correlate physiological
variables with microtubule assembly in;zizg. .Among the
physiological variables measured were plasma glucose and

insulin levels. and liver cyclic nucleotide levels.

MATERIALS AND METHODS

Animal Treatment. Male Roltzman rats weighing approximately
180 grams were housed in groups of 3 or 4 in plastic cages
containing wood chips. All animals were housed in an air-
conditioned room at 23°C under controlled lighting (lights

6 A.M.-6 P.M.). A control group was fed a commercial chow

 

 

 

29
and water. ad libitum. throughout the experiment. Animals
were starved for 3 days with free access to water and refed
a high-glucose containing diet. 20% casein. 68.9% glucose
monohydrate. 5% corn oil. 5%‘Wesson salt mixture. 0.1%
choline chloride. and 1% vitamin mixture (24).
Samplg Prepgggtion.£gg Tubulinwggggy. Rats were killed by
decapitation. Livers were excised and quickly immersed in
MTS buffer containing 50% glycerol. 5% DMSO. 1.0 mM GTP.*+-
0.5 mM MgClz. 0.5 mM EGTA. in 10 mM phosphate buffer pH 6.95
and immediately homogenized with a loosely fitting Teflon
pestle (l). The homogenate was centrifuged (to pellet the
microtubules) at 40.000 rpm for 45 minutes at 30°C. The
amount of free tubulin in the supernatant fraction (SN 1)
was measured by a colchicine binding assay (25). The pellet
was resuspended in ice-cold TS buffer containing 0.25 M
sucrose. 0.5 mM MgClz. 1.0 mM GTP in lOmM phosphate buffer
pH 6.95 and centrifuged at 40.000 rpm for 30 minutes at
4°C. The supernatant (SN II) was assayed for tubulin
(original polymerized tubulin) by a colchicine binding
assay (Figure I).
Colchicine Binding.5§§gy. Colchicine binding activity was
determined using a modification of Pipeleers and co-workers
(1) method. Aliquots (100 ad) of the supernatant fractions
were incubated with 25.nl of a 40.uM 3H-colchicine solution
(0.25 Ci/mmole) at 37°C for 3 hours when MTS was used and
1 hour when TS was used. A 5 mg/ml charcoal in water sus-
pension (500 ml) was then added. to adsorb the free colchi-

cine. The suspension was mixed thoroughly. allowed to settle

30

Figure I: Rats were sacrificed and livers were removed.
A piece of liver was homogenized in MTS buffer
and centrifuged. The supernatant was assayed
for free tubulin using a colchicine binding
assay. The pellet was resuspended in TS buffer
(cold) and centrifuged. The supernatant was
assayed for tubulin (original polymerized
tubulin) using a colchicine binding assay.

 

 

31

 

TUBULIN ASSAYl

PE. ham. --- Homogenate

{

Pellet
resuspend In TS but.

at 0° for IS min.

 

‘ lO0,000xg
. 0°, 30min.
Pellet Supernate
discard

 

V

[attJ-colchicine
incubate lhr.,37°

V

*

 

activated charcoal

count aliquot of supernate
jPOLYME RlZEDl

 

 

' in MTS but.
Liver ' %

centrifuge, l,OOOxg,

count aliquot ofsupernate

Ioo ,OOOxg
30", 45min.

 

Supernate
[3H]"'colchicine“r
incubate 3hrs.,37°

V

activated charcoal

V

 

’. 5min.

 

IFREEI

 

centrifuge,l,000xg,5min.

 

I 0.0.PIPELEERS.OQOI.(I977)
J. Cell Biol. 74.34I'350

*EH} colchicine=8.0uMl _
2.5on” uCi/mmol.

 

 

‘ Figure I:

H

Protocol Used for Determining the Amount of Free and '-

Palymerized Tubulin in the Starved-Refed Rat Liver.

 

32
for 5 minutes. then centrifuged for 5 minutes. A 500‘pl
aliquot of the supernatant was added to 9 ml of Brays scin-
tillation cocktail (26) and radioactivity detected in a
Beckman Liquid Scintillation System. Colchicine binding
activity was converted to tubulin equivalents by assuming
a 1:1 molar ratio of colchicine binding to tubulin and a
molecular weight for tubulin of 110.000 (27). The colchi-
cine binding activity was measured in SN I and SN II to
quantitate respectively the amount of tubulin originally
present in the free and polymerized forms (Figure 1).
Protein Determination. Liver tissue was homogenized in
phosphate buffered saline and protein concentration was
determined by the method of Lowry and co-workers (28)
which was modified for automated analysis as described by
Mak and Wells (24).
DNA Determination. DNA content was measured in liver homo-
genates (phosphate buffered saline) by the Ethidium Bromide
method as described by Kurtz and Wells (29).
Sample Prepggation Egg Glucose. Insulin. and Cyclic
Nucleotides. Rats were ether-anesthetized and a hepari-
nized syringe was used in cardiac puncture to collect blood
for glucose and insulin analysis. Livers were quick frozen
between stainless steel tongs cooled in liquid nitrogen.
The tissue was later used to measure cyclic nucleotides.
Glucose Determinations. Plasma glucose was determined by
using the Computer-Directed Gilford 3500 Analyzer and the
Gilford/Worthington reagent glucose kits.

33
Insulin Determination. An Insulin Radioimmunoassay kit

(Amersham..Arlington Hts. Illinois) was used to measure
plasma insulin.

ngl 2 Nucleotide Determinations. Freeze clamped liver
samples were deproteinized by TCA extractions. TCA was
then.removed by extractions with water-saturated other.
cAMP and cGMP Radioimmunoassay kits (Amersham..Arlington
Hts. Illinois) were used to measure cyclic nucleotides
levels.

Colchicine Binding Qggzgg. Male Holtzman rats weighing
”250 grams were decapitated and livers were removed. See
§amplg Preparation {23 Tubulin.£§§gy and Colchicine Binding
Agggy_for methods of measuring free and polymerized tubulin
by the colchicine binding assay. Modifications of the col-
chicine binding assay were: SN 1 incubated with 3H-colchi-
cine for 5 hours and SN II incubated with 3H-colchicine

for 3 hours. every 15 minutes samples were assayed for
colchicine binding activity.

Statistics. Statistical Analyses were performed using

the two-tailed t-test (35).

RESULTS

In order to use the colchicine binding assay quanti-
tatively. experiments were done to determine. in both
buffers. the time at which maximum.binding of colchicine
to tubulin occurred. Figure II indicates maximum binding

of colchicine in MTS (glycerol-DMSO buffer) occurs after

Figure II: MTS and TS samples were incubated with
H-colchicine. At time intervals aliquots
were assayed for colchicine binding
activity. Colchicine binding activity is
expressed as a percent of maximal binding.

35

wk DZ< m...<< Z. >._._>_._.U<
02.02_m uZ_U_IU._OU ".0 mU_._.mZ_v_ u: memu:

mthZ_<<

oom CNN ova O—N 02 05 ON— 00 00 on
_ _ _ a _ _ _ _ _ _

 

 

 

 

oNlaNIs
aNIDIHo'Ioo %

36
3 hours and in TS (sucrose buffer) after 1 hour.

To analyze liver cyclic nucleotide levels accurately.
the liver was quick frozen by freeze clamping. Rats used
for cyclic nucleotide analysis were other anesthesized
and heart puncture was performed.on them to collect blood
for glucose and insulin analysis. Ether anesthesia may
interfere with tubulin polymerization andfreeze clamping
the liver may interfere with the tubulin analysis. so a-
nother set of rats (which were treated similarly to the
ether anesthesized rats) were decapitated and used for the
tubulin assay. Even though the data in Table I is from
the decapitated rats. it is a fair representation for both
sets of rats.

Protein and DNA levels were measured in order to ex-
press tubulin content in terms of these parameters. Also.
by measuring liver protein and DNA content the effects of
starving and refeeding on these parameters could be ob-

. served. The amount of protein per liver is decreased
during starvation and increased to normal levels by 6
hours after refeeding (Table II). As seen in Table II.
the DNA content of liver appears to increase with re-
feeding contrary to earlier reports which state that

DNA content does not change with dietary state in an adult
rat (30.31). This reproducible increase may be a growth
phenomenon or an artifact of the Ethidium Bromide Assay.
In the liver of young adult mammals. most liver cells

are binucleated (34). Many liver cells. therefore are

37

Table I: Male Holtzman rats were starved for 3 days and
then refed a high glucose diet. Each value is
the mean 1 standard deviation of 3 animals.

38

 

 

m.ma as. w 50.: ea w mam .mus we
o.~n ma. w mo.: m n mad .ma: on
m.a~ wm.a fl am.: m n mod .mas em
m.ea «o. H om.s as H com .mas ma
a.sa mm. u am.n o n mom .mas ma
«.0 am. u ma.n mm H one .mas o
m.n no. u oo.m me u and .ma: n
m.H 3N. h om.N w H “ma .9: H
0 ma. u mn.m ma w and mas» o
I. as. u mn.n me n mum pone souusoso
Amy pom ham sopam Asmmooa\msaawv va pawn wsauoomom

pawn omaao><

passe; oo>fig

passe: seam

aspu< mane

 

wean mucosao swam m couom spam uo>uupm
Ho unawaoz no>wg was keen use soavassmsoo coon

H mqm<a

39

Table II: Male Holtzman rats were starved for 3 days
and then refed a high glucose diet. Rats were
sacrificed and livers were removed. Livers
were assayed for protein and DNA content.
Each value is the mean‘: standard deviation of

3 animals.

40

 

 

~n.mfi n Hm.sn ms.e fl Ha.a~ mm. « ms.H .mus we
mm.na u ma.om me.s w sH.aH so. u so.H .mas on
on.oa w ma.as em.~ N ma.o~ mm. N am. .mus em
mn.n M an.nn ao.e M om.~m so. u ao.a .mp2 we
sm.m M m~.mn am.s u em.m~ ma. fl nH.H .mas NH
an.a M Hm.an ma.o u sa.s~ mm. a He. .mae o
ma.a u no.3m mo.~H M oa.a~ om. n mm. .mus m
Ha.aa M aa.on ao.s u am.ma mo. n co. .h; H
mm.ma u am.em mm.e H ms.mH No. N me. asap 0
mo.o M on.mo ms.o H nn.mH em. a mn.a peas emm-zogo
3%.. man. gamma... gamma“.

 

apex comomlvo>uapm 2“
<zn\saovoum was .<zn .saopoum mo>ag

HH mqm<s

41
polyploid. Infrequency of cell division in the adult
leads to a slowly increasing degree of polyploidy with
389 (32.33.34). Therefore. in young and adult liver
there is evidence for increases in DNA content. Protein/
DNA ratios are decreased for the first day after re-
feeding due to the increase in DNA (Table II).

Starving and refeeding rats causes a change in liver
tubulin content and polymerization. After 3 days
starvation the percent of tubulin polymerization is de-
creased compared to controls. Refeeding elicited a
significant increase in the degree of polymerization
which is increased significantly beyond that of controls
after 12 hours (Table III. Figure III). During star-
vation a significant decrease in total liver tubulin is
observed which is expressed in a decrease in both the
free and polymerized pools. Total liver tubulin refers
solely to soluble free and polymerized tubulin. In these
experiments we did not measure membrane-bound tubulin.
and. therefore we do not know if the amount of tubulin
in this fraction (membrane-bound) changed during starving
and refeeding. In later experiments we will attempt to
measure membrane-bound tubulin. Liver polymerized
tubulin increases after refeeding and approaches control
levels after 18 hours. while free tubulin stays depressed
throughout the refeeding period (Figure III). The ng of
tubulin per mg DNA profile closely resembles that of
tubulin content per liver (Figure IV). The forms of

Table III:

42

Livers of experimental animals were assayed
for free and polymerized tubulin and total
protein. The degree of polymerization is
calculated by dividing the amount of poly-
merized tubulin by the sum of the free and
polymerized tubulin x 100. The forms of
tubulin are expressed on a protein basis.
Each value is the mean 2 standard
deviation of 3 rats.

* Significantly different from starved
values at P<.O5.

43

 

 

ea. H ma.a mo. H ea. so. H as. .om.~ H sm.me .uus as
ea. H m:.a ea. H mm. so. H em. .m~.a H Hm.so .mus on
ma. H m~.H No. H as. as. H ms. .m~.o H em.ae .mus em
mo. H sa.a oo. H #5. ca. H as. .Nn.n H mm.ss .mhs ms
nu. H oo.a oa. H as. no. H we. .sn.H H ao.an .us: «H
«H. H mm. as. H me. as. H mm. oa.a H no.2: .mag w
on. H HA.H ea. H HA. on. H mm. oo.H H sa.as .mos n
on. H «m.a no. H om. on. H ~o.a oo.a H Ho.ms .a: H
an. H mn.a as. H ow. ma. H ma. ma.s H om.~s mas» o
as. H mo.a ea. H as. on. H mm. mm. H s~.s: sou-:o:o
saopoum ma :Hopoum ms saopoum MB

max”. gunman“? a. flaw... .“MmmmHM. “mean“

 

no>HA Pam somomuuo>uuvm on» ma swopoam mom :«asnsa Ho

Hssos< ess nowadaduoshaom seasnss Ho nonwoa one

HHH mqm<9

 

 

44

Figure III: Liver Free and Polymerized Tubulin and the
Degree of Tubulin Polymerization in the
Starved-Refed Rat. Each value is the mean

1 standard deviation of 3 rats.

III- Significantly different from control
values at P<.05.

45

pengeuMIod %

wt

«.92: ozEmwmwm mm...m< MEI.
m_ N. m

N¢ mm Om .VN

saoaooEmk
o zo:.<>m<5

N _

 

 

 

ueNtoEboa

mm>_._ .._<._.O._. zo Qmm<m
2339:. mm>_u_ ._.<m

 

.

o

 

,1
I o._

O

 

 

(JeAII/bw) UIInan.

 

46

Figure IV: Free and Polymerized Tubulin Based on DNA
in the Starved-Refed Rat. Each value is
the mean 3: standard deviation of 3 rats.

47

fimaéezammmmm mm._.u_< m2;
ms NV mm Om ¢N m_ N_

m

Amaeaooamn.

O zo_._.<>m<._.m
m N _ o O

 

_ _ _ _ _ _ _

m ..../.H\

/\ueNtoE>_oa

<20 20 Qmm<m
2339:. mm>3 ._.<m

m/UVA
\wq

 

VA/H/

_ _ _

ON

1/

7
ix
»

 

 

(VNG film/5W) Ullnqnl

48

tubulin expressed per mg of protein do not change sig-
nificantly over the course of the experiment. though it
appears that with respect to protein content. the free
tubulin pool is decreasing after 6 hours of refeeding
while the polymerized pool is increasing (Table III).

Plasma glucose and insulin levels and liver cyclic
nucleotide levels were measured to investigate their re-
lationship to tubulin content and polymerization. Plasma
glucose and insulin levels increase rapidly after re-
feeding reaching a peak at 6 hours for glucose and 1 hour I
for insulin. though the latter did not exceed that of
control rats (Figure V). After 3 days of starvation
liver cAMP and cGMP levels are elevated above those of fed
controls. Liver cAMP levels decrease to normal by 3 hours
after refeeding. Liver cGMP levels do not approach con-
trol levels until 36 hours after refeeding (Figure VI).
Changes in plasma glucose and insulin levels and liver
cyclic nucleotide levels did not appear to correlate to

changes in tubulin content and polymerization.

DISCUSSION

In our experiments using the whole rat system we did
not detect a direct correlative relationship between in-
sulin or glucose and tubulin content and polymerization:
though a relationship may be detectable in a hepatocyte

system.

 

“9

Figure V: Plasma Glucose and Insulin Levels in the
Starved-Refed Rat. Each value is the mean
t standard deviation of 3 animals.

50

1mm '7 Nl'lnSNI

ON
9»
00

cm

we.

mevmzammmmm Kuhn; NE;
NV on on VN m. N_

m

2839mm...
0 20.2225

mm

o

 

 

omoo:_el\

.<2m<u_n_

I.

In

 

_

 

 

on

00.

02

CON

1P/5W 38000-19

51

Figure VI: Liver cAMP and cGMP Levels in the Starved-
Refed Rat. Each value is the mean i
standard deviation of 3 animals.

52

(enssu qseu b/low d) dwgo

0.

ON

Om

7.3.: ozEmwmwm awhmd. ME;

(“38525;

 

 

0

0mm

com

0
IO
[x

m¢ 0.? mm Om ¢N m_ N_ o O ZO_._.<>m<._.m
m N _ O
_ _ _ _ _ _ _ _ _ _ _ q
I!
: \ “”2200 EL
a a w r x
/ \W\ \\
u \ x \H
as?» \x m
x x
k\
mmo_._.0m:032 030>0 mm>_n_ ._.<m

 

 

 

(enssu use” b/low d) dWVO

53

It has been reported that high intracellular cAMP
concentrations inhibit lysosomal enzyme secretion from
leukocytes similar to colchicine inhibition of the same
secretory process (21—23). However. high cGMP levels en-
hance the secretory processes in these cells. High cAMP
concentrations may affect secretion by depolymerizing
microtubules while high cGMP levels may enhance in xix;
polymerization and secretion. In the starved-refed rat
liver, however, correlations between cyclic nucleotide
concentrations and the extent.of tubulin polymerization
in zizg were not observed. From our experiments we were i
not able to determine whether cAMP may be exerting a
regulatory effect on microtubules through a cAMP-depen-
dent protein kinase. However. the lag between cAMP
concentration decreases and increases in the degree of
tubulin polymerization after refeeding imply that there
is no direct, immediate effect.

Our results concerning tubulin content and poly-
merization were similar to those of Pipeleers and co-
workers (2). They found a decrease in total tubulin and
in the degree of polymerization in starved rat livers.
Rats which were maintained exclusively on a 30% dextrose
drinking solution had a larger percent of liver tubulin
polymerized than controls. The amount of total liver
tubulin in dextrose fed rats was greater than that of
starved rats but both groups had less than controls.

Pipeleers and co-workers (2) suggested that changes in

54
tubulin polymerization may influence or reflect intracel-
lular transport or secretion. i.e. release of albumin and
lipoprotein from the liver.

Pipeleers and co-workers (43) also measured free and
polymerized tubulin in rat pancreatic islets to study
microtubule involvement in the glucose stimulated insulin
secretion from the cells. In starved rats. total pan-
creatic islet tubulin and the degree of tubulin polymer-
ization decreased. Control rats and rats maintained ex-
clusively on a 30% dextrose solution had comparable total
tubulin values. though the percent of total tubulin that
was polymerized was larger in dextrose fed rats than in
control rats. Insulin secretion from the pancreatic
islets was also measured in the experiment. The insulin
secretory response to glucose in pancreatic islets paral-
leled the change in tubulin polymerization. Therefore.
the degree of tubulin polymerization may be an impor-
tant regulatory factor in insulin secretion.

If polymerized tubulin is required for cellular
events involving microtubules, then during refeeding
microtubules are involved in some cellular event that
does not occur during starvation. As Table I indicates
the liver increased in size throughout the experiment.
The appearance of a fatty liver has been seen 24 hours
after refeeding a high-glucose containing diet. Inves-
tigators have found that the enzymes of liver lipogenesis

are induced after refeeding starved rats a high carbohydrate

. 55

diet (8.24.36-39). Studies have also shown that a delay
of 2h-48 hours is required for the full effect of refeeding
to be seen. including high rates of lipogenesis (36.38-42).
Lipoprotein release is stimulated by glucose feeding and
decreased during starvation (3.9.10). These observations
imply that 2#-48 hours after refeeding the liver contains
a large amount of lipid. a significant amount in the form
of VLDL. Microtubules have been implicated in the release
of VLDL from the liver (ll-15). The functional role of
microtubules in this system may then be the secretion of
VLDL from the liver. The increase in the percent of u
total tubulin that is polymerized may reflect the need for
microtubules in the secretion process. Assaying VLDL and
plasma proteins in the liver and plasma during starving-
refeeding would help to confirm this relationship. The
starved-refed rat liver provided us with a model system

in which the in gigg regulation of tubulin content and
polymerization could be studied. The factors responsible
for the modulation of tubulin content and polymerization
in this system still need to be determined. although we
can rule out cyclic nucleotides. insulin. and glucose
level effects.

It should be noted that in the colchicine binding assay
ug of tubulin was calculated from the umoles of colchicine
bound. This was done by assuming that 1 mole of colchicine
bound 1 mole of tubulin dimer. But. in fact we have seen
in a preliminary study that this may not be the actual

stoichiometry. Therefore, the colchicine binding assay

56
may not be exactly quantitative. but can be used to in-
dicate relative amounts of tubulin within this experiment
(we can compare values of tubulin content from one group

of rats to another group in this experiment).

 

9.

10.

11.

12.

13.

14.

BIBLIOGRAPHY

‘ Pipeleers. D. G.. Pipeleers-Marichal. M. A.. and

Kipnis. D. M. (1977). g; Cell Biol. 23, 341.

Pipeleers. D. G.. Pipeleers-Marichal. M. A.. and
Kipnis. D. M. (1977). £4,0ell Biol. 23, 351.

Heimberg. M.. Weinstein. 1.. Klausner. H.. and
Watkins. M. L. (1962). Amer, g; Physiol. 202. 353.

Seitz. H. J.. Muller. M. J.. Krone. w.. and
Tarnowski. W. (1977). Archives 2; Biochemistry and

Biophysics lg}. 6n7.

Steiner. A. L.. Pagliara. A. 8.. Chase. L. R.. and
Kipnis. D. M. (1972). g; Biol. Chem. 242. 1114.

Ichihara. K. and Murad. F. (1979). Arch. Biochem.
Biophys. l 4. 292.

Pointer. R. M.. Butcher. F. R.. and Fain. J. N. (1976).
g&_Biol. Chem, 251. 2987.

Gibson. D. M.. Lyons. R. T.. Scott. D. P.. and Mute.
Y. (1972). Adva Enz. Regul.plg. 187.

Eaton. R. P. and Kipnis. D. M. (1969). Amer. g; Physiol:
312. 1153-

Quarfordt. S. R.. Frank. A.. Shames. D. M.. Barman. M.
and Steinberg. D. (1970). g; Clin, Invest, Q2. 2281.

Le Marchand. Y.. Singh. A.. Assimacopoulos-Jeannet. F..
Orci. L.. Rouiller. C.. and Jeanrenaud. B. (1973).
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Stein. 0. and Stein. Y. (1973). Biochim. Biophys, Acta
39g, l#2.

Orci. L.. Le Marchand. Y.. Singh. A.. Assimacopoulos-
Jeannet. P.. Rouiller. C.. and Jeanrenaud. B. (1973).
Nature 2h4. 30.

Stein. 0.. Sanger. L.. and Stein. Y. (l97h). g;.Cell
Biol, Q. 90.

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Singh. A.. Le Marchand. Y., Orci. 4L.. and Jeanrenaud. B.
(1975). Eur. J. Clin. Invest. 5.4 5.

 

 

Re dman. C. M.. Banerjee. D.. Howell. R.. and Palade.
G. E. (1975). J. Cell Biol. 66. 42

Redman. C. M.. Banerjee. D.. Manning. C.. oHuang. C. Y..
and Green. K. (1978), J. Cell Biol. 11.4 0.

 

 

Le Marchand. Y.. Patzelt. C.. Assimaco oulos-Jeannet. F..
Rouiller. C.. and Jeanrenaud. B. (197“), J. Clin,

Invest, 52. 1512.

Sloboda, R. D.. Rudolph. S. A.. Rosenbaum. J. L.. and
Greengard. P. (1975). Proc. Natl, Acad. Sci. 22. 17?.

Piras. R. 1and Piras. M. M. (1975). Proc. Natl, Acad,
Sci, Z_.1ll.

Weissmann. G.. Goldstein. 1.. Hoffstein. S.. and Tsung. ,
P. K. (1973). Ann, N; 1, Acad, Sci. 253. 750. g

Zurier. R. B.. Hoffstein. S.. and Weissmann. G. (1973).
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Zurier. R. B.. Weissmann. G.. Hoffstein. S.. Kammerman.
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Sherline. P.. Bodwin. C. K.. and Kipnis. D. M. (1974).
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Weisenberg. R.. Borisy. G.. and Taylor, E. (1968),
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Thomson. R. Y.. Heagy. F. C.. Hutchison. W. C.. and
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Harrison. M. F. (1953). Biochem. g, 55. 204.
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Togiani. R. and Pucinelli. E. (1970), Histochemie
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CHAPTER II

EXAMINATION OF THE METHOD OF QUANTITATING
FREE AND POLYMERIZED TUBULIN

ABSTRACT

Experiments were done to validate the method. de-
signed by Pipeleers and co-workers (1), of quantitating
free and polymerized tubulin by a colchicine binding as-
say. Neither MTS (glycerol-DMSO buffer) nor TS (sucrose
buffer) interferred with the colchicine binding activity
of tubulin. At high tissue concentrations and, therefore.

tubulin concentrations the possibility of in vitro poly-

 

merization was investigated. Purified tubulin did not

polymerize in MTS in vitro. High tissue concentrations

 

resulted in lower apparent total tubulin values than more
dilute concentrations but the same polymerized tubulin
values. We suggest that naturally occurring molecules
are competing with colchicine to bind tubulin at the high

tissue concentrations in the MTS fractions.

60

61
INTRODUCTION

The method of Pipeleers and co-workers (1) was used
to measure free and polymerized tubulin by a colchicine
binding assay. Pipeleers and co-workers (1). in designing
the method. conducted various experiments to validate '
their procedure: among them linearity. kinetics. and
decay rate of the colchicine binding activity in both
buffers. To measure the in gigg extent of polymerization.
they chose an initial tissue/buffer of 1:13 to 1:80 to
prevent the polymerization of tubulin in vitro. Tubulin

 

is known to polymerize in vitro at high concentrations

 

(above the critical concentration) and glycerol is

known to stimulate this in vitro polymerization. There-

 

fore. by keeping the tissue/buffer low, in vitro poly-

 

merization may be prevented (2,3.#).

The incubation period of the colchicine binding assay
we used was different than that of Pipeleers and co-
workers (1). In some experiments the degree of tubulin
polymerization was higher than reported by Pipeleers and
co-workers (1). The amount of total tubulin assayed by
the colchicine binding assay did not always equal the
calculated sum of the free and polymerized tubulin. In
view of these discrepancies concerning the degree of poly-
merization and the amounts of tubulin. we chose to further
investigate the method.

To evaluate the importance of the tissue to buffer

dilution. free and polymerized tubulin was measured at

62
different tissue dilutions. The degree of tubulin poly-
merization was determined at the various tissue dilutions.
The question of whether colchicine binding activity in-
creased linearly with tissue concentration in both buffers
was investigated. The discrepancy between experimental
and calculated total tubulin levels was investigated.
Glycerol has been shown to bind tubulin (5,6). Therefore.
MTS may interfere with the colchicine binding assay. Ex-
periments were done to resolve the question of inter-
ference of the colchicine binding activity of tubulin by
MTS or TS. In an effort to determine whether MTS was
causing ip zippg polymerization during the experiment.
ip pippg polymerization experiments were done in the

presence of MTS.

METHODS AND MATERIALS

Tissue Dilution Experiments. Male Holtzman rats weighing
tvzso grams were decapitated and livers were removed.
Tissue pieces ofIVl00.N200.~300.~#00. and~500 mg were
homogenized in 2.5 mls of MTS at room temperature. Free
and polymerized tubulin were measured in these samples by
the procedure described in Chapter I (see Sample Eggp-
aration gpp_Tubulin ggggy and Colchicine Binding Agpgx).
To assay for total tubulin. a sample of liver was home-
genized in cold TS and incubated at #°C for 1 hour. then
centrifuged at #°C for #5 minutes at #0.000 rpm. The

63
supernatant was assayed for total tubulin by the colchicine
binding assay described in Chapter I.
Colchicine Binding 2; Bpggp Egg nggp Tubulin gp MTS 9; TS.
Male Holtzman rats weighing/VZSO grams were decapitated
and livers and brains were removed. Brain or Liver was
homogenized in cold PBS. incubated for a i hour at #°C.
and centrifuged at #°C for #5 minutes at #0.000 rpm. The
supernatant was diluted with TS or MTS. 2:1 for liver
samples and 5:1 for brain. Aliquot sizes of 5. 15. 30.
60, 80. and 100 m1 of both MTS and TS samples were assayed
for tubulin by the colchicine binding assay (see Chapter I.
Colchicine Binding gpggy).
IQ Zippg Polyperization Studies. Twice-cycled brain
tubulin (7) was used in these studies. Lg vitro poly-

 

merization of tubulin was done in Assembly Buffer (20 mM
MES pH 6.5. 70 mM NaCl. 0.5 MM MgClg. 1 mM EGTA) or MTS
(glycerol-DMSO buffer) with cycle 2 tubulin at a final
concentration of 600 ng/ml or 1200 ng/ml. Before use.
cycle 2 tubulin was centrifuged at 6.500 g for 5 minutes
at #°C to remove particles in order to reduce background
light scattering. Samples were made in the cold and
placed in the 37°C water-jacketed chamber of a Gilford
2#00 multi-sample spectrophotometer. Nucleotide additions
(GTP) were made just before assembly was begun. Assembly
was followed by measuring light scattering at 350 nm (7).

6#
RESULTS

In order.to determine if and if so. why an increase
in tissue concentration caused an increase in tubulin poly-
merization. various tissue concentrations were assayed
for free and polymerized tubulin. Table I indicates that
the degree of tubulin polymerization increases at tissue
concentrations greater than'Vl30 mg tissue per ml MTS.
Table II shows again the increase in the degree of poly-
merization with decreasing tissue dilution and shows that
the calculated sum of free and polymerized tubulin equals
the experimental value of total tubulin at the higher
tissue dilution. At the lower tissue dilution the amount
of measured total tubulin (calculated as the sum of free
and polymerized tubulin) is less than the value obtained
at the higher tissue dilution. The amount of polymerized
tubulin is the same for both tissue dilutions but the a-
mount of free is less at the lower tissue dilution.

In order to better investigate the problem of in-
creasing tubulin polymerization with increasing tissue
concentration. the colchicine binding activities measured
in both the MTS and TS fractions were plotted vs.tissue
concentrations. Colchicine binding activity increases
linearly with tissue concentrations in TS over the whole
range of dilutions tested (Figures I & II). In contrast.
the colchicine binding activity in MTS does not increase
linearly after a tissue concentration ofINl3O mg per ml

MTS (Figures I & II). Figures I & 11 suggest that at a

the Degree of Tubulin Polymerization

TABLE I

The Effect of Tissue Dilution on

 

Experiment I

Experiment II

 

 

mg of Liver/ % Tubulin mg of Liver/ % Tubulin

ml of MTS Polymerized m1 of MTS Polymerized
37.6 39.73 36.0 32.35
60.8 41.67 52.8 28.15
88.0 37.60 88.0 35.08
122.# 38.26 128.0 33.96
183.2 #5.0# 185.2 #1.13
204.0 52.78 208.0 #7.81

 

 

In Experiments I and II. free and polymerized tubulin were

measured as described in Chapter I.

66

TABLE II

The Effect of Tissue Dilution on the Percent of
Tubulin Polymerized and Total Tubulin Content

 

 

 

Rat # Dilution of % Tubulin MTS+TS mg Total
Liver Polymerized ug Tubulin/ Tubulin/
m ml mg Tissue mg Tissue
(calculated (measured
sum) directly)
203.2/MTS 57.86 299
#6.#/MTS 36.83 #67
I
188.0/Ts #86
52.0/Ts 471
zoo.o/MTs #8.?9 zuo
36.0/Mrs 26.69 397
II
39.2/TS #16

 

Free and polymerized tubulin were measured as described in
Chapter I. Total tubulin was measured by disassembling
microtubules in cold TS buffer. and measuring tubulin by a
colchicine binding assay. Two tissue dilutions were used

to measure free and polymerized tubulin and the approximately
same tissue dilutions were assayed directly for total
tubulin. Total tubulin values calculated from the sum of
free and polymerized tubulin were compared to experimentally
determined total tubulin values.

6?

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use was on» a“ snares» no pssoas one .H novasso a“ cooaaomou
ma msofipsaau osmmdp msoams> cheap you censuses one; swamps»
mouaaosma°q was seam and .mss we was m.~ s“ couasomcso: one:
me>aa Ho mvssoss vsouemhan .ma was was ma hvd>fipo< msavsam

 

 

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was so was n.m\ao>3 .8 ems
con 8: com com
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Imp I on

 

 

 

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68

msmu
me: e
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.ma use was as apa>apo< mcaesam oneoagoaoo mo
synagogue on» so campuses .smaea mo scones one .HH assess

was he was m.m\ao>ag me am:

con 8: com com 03
_ _ _ _ _

 

 

 

 

 

xeyyng JO In/punog eutotqotoo JO setomn

69
large tissue to MTS ratio. colchicine binds to a constant
amount of tubulin independent of the amount of tubulin.

Liver or brain was homogenized in cold PBS to dis-
assemble microtubules. The homogenate was centrifuged and
the supernatant was diluted with MTS or TS 2:1 for liver
samples and 5:1 for brain. To determine if either buffer
was inhibiting the binding reaction of colchicine to
tubulin. colchicine binding activities in both buffers
were plotted vs.tubulin concentration. Figure III in-
dicates that the colchicine binding activity is propor-
tional to tubulin concentration in MTS and TS. The a-
mount of tubulin in both MTS and TS is theoretically the
same and Figure III supports this experimentally. showing
that neither buffer is inhibiting the colchicine binding
activity of tubulin. MTS and TS should contain the same
amount of brain tubulin but as seen in Figure IV less
colchicine binding activity is found in the MTS samples.
Therefore. in brain preparations MTS has an inhibitory
effect on the colchicine binding activity.

Ip pippg polymerization studies indicate that in the
presence of tubulin concentrations of 600 ug/ml (Figure V)
or 1200 ug/ml (not shown). MTS will not promote assembly
of microtubules. This is supportive evidence that MTS
(glycerol-DMSO buffer) is not causing Lg zippg polymeriza-

tion in our ip vivo studies.

Colchicine Bound (0PM)

12000

10000

8000

6000

#000

2000

70

 

.—

 

 

 

l J l 1 l
20 no 60 80 100

ml of Diluted Supernatant

Figure III: Colchicine Binding of Liver
Tubulin in MTS or TS. Liver was home—
genized in cold PBS to disassemble micro-
tubules. The homogenate was centrifuged
and the supernatant was diluted with TS
or MTS. Aliquots of the diluted super-
natants were assayed for colchicine
binding activity.

© MTS s TS

Colchicine Bound (CPM)

20000

16000

12000

8000 __

#000

71

 

_-

 

 

l I l J |

 

20 #0 60 80 100

ml of Diluted Supernatant

Figure IV: Colchicine Binding of Brain
Tubulin in MTS or TS. Brain was home-
genized in cold PBS to disassemble micro-
tubules. The homogenate was centrifuged
and the supernatant was diluted with TS
or MTS. Aliquots of the diluted super-
natants were assayed for colchicine
binding activity.

C] MTS 0T5

 

 

OD350

0.10

0.08

0.06

0.04

0.02

 

72

 

 

 

 

Figure V:

Time in Minutes

In V tro Polymerization Studies
Using M S Buffer and Assembly Buffer
with a Tubulin Concentration of

600 ug/ml.

A Assembly Buffer
B 600 lug/ml Tubulin in MTS

C 600 .ug/ml Tubulin in Assembly
Buffer with lmM GTP

73
DISCUSSION

Colchicine binding activity was not proportional to
tissue concentration in the HTS fractions. MTS may in-
terfere with the separation of free tubulin from micro-
tubules at high tissue concentrations and. therefore.
lower values of colchicine binding activity would be seen
in the MTS fractions. MTS because of its high glycerol
content may be causing gp yippg polymerization at these
high tissue concentrations. Figure V indicates that MTS
did not allow ;2 zippg polymerization at tubulin concen-
trations of 600 ug/ml. a concentration far above that seen

in our ip vivo studies. This suggested that ip vitro poly-

 

merization was not occurring in MTS during our gp pipe
studies. If either of the two possible mechanisms men-
tioned above were occurring. at high tissue concentrations
the amount of polymerized tubulin should be increased.

The sum of free and polymerized tubulin would be the same
at all tissue dilutions but the distribution of tubulin
between forms would be different. This was not the case.
There was a decrease in measured total tubulin and an in-
crease in the percent of total tubulin that was poly~
merized, which could be completely accounted for by a de-
crease in the measured concentration of free tubulin.

It appeared as if colchicine was not binding to all the
tubulin in the MTS fractions at high tissue concentrations.

The ability of colchicine to bind large amounts of tubulin

7#
in the presence of MTS is seen in Figure III. therefore
indicating MTS (glycerol-DMSO buffer) was not interfering
with the colchicine-tubulin binding reaction. We are
speculating that at high tissue concentrations. other
factors in the tissue besides tubulin are also more
lconcentrated and may be interfering with the colchicine
binding assay. Possibilities are proteases or esterases
which may destroy the tubulin molecule or the tubulin-
colchicine complex. Investigators have recently re-
ported endogenous protein molecules bind tubulin at the
colchicine binding site (8.9.11). These endogenous mole-
cules may be binding tubulin and inhibiting colchicine
binding. If the colchicine binding incubation in MTS
was prolonged. when using high tissue concentrations.
more colchicine binding activity was detected. If the
MTS fraction (free tubulin) was diluted with 320 or TS.
the binding of colchicine to tubulin proceeded at a
faster rate. The greater dilution or longer time period
may enhance tubulin binding to colchicine relative to
binding to endogenous proteins. ,

Recently. Ostlund and co-workers (10) investigated
Pipeleers and co-workers' method (1) and demonstrated
that glycerol and DMSO were responsible for apparent
reduced affinity of colchicine binding in MTS. when
colchicine concentrations of less than 20 uM were used.
At larger colchicine concentrations the effects of glycerol

and DMSO seem to be circumvented. 0stlund and co-workers

75
(10) suggested that glycerol-DMSO was competing with
colchicine to bind tubulin. therefore a higher concen-
tration of colchicine was needed to counteract the gly-
cerol-tubulin reaction. Figure III would seem to indi-
cate that MTS (glycerol-DMSO buffer) was not interfering
with colchicine binding to tubulin. but one has to recall
the sample was diluted 2:1 (MTS:PBS). therefore the PBS
may be diluting the glycerol-DMSO. Ostlund and co-workers
(10) have also shown that the reduced colchicine binding
in MTS can be remedied by diluting the MTS. If we con-
sider Ostlund and co-workers (10) results when interpreting
our results then the nonlinearity of colchicine binding
activity in MTS with respect to tissue concentration
may indicate a need for a higher colchicine concentration
at the higher tissue concentration (tubulin concentration)
to compete with the glycerol-tubulin reaction. In con-
clusion. even though we have presented two interpretations
to explain the reduced affinity of colchicine binding in
MTS. more work is needed to fully explain the phenomenon
i. e.. isolating the endogenous inhibitors or finding
glycerol or DMSO bound to tubulin in a way in which in-
hibits the colchicine binding activity. It also should
be noted that the reduced colchicine binding activity in
the higher tissue concentrations may be due to increased

protease or esterase activity.

10.

11.

BIBLIOGRAPHY

Pipeleers. D. G.. Pipeleers-Marchial. M. A.. .
Sherline. P.. and Kipnis. D. M. (1977). J Ce llB Biol,

LET-Fl.

Murphy. D. B.. Vallee. R. B.. and Borisy. G. G. (1977).
Biochemistry 16. 2598.

Koehn. J. A. and Olsen. R. W. (1978). Arch. Biochem.
Bio Rhys . 186’ ll“.

 

 

Gaskin. F.. Cantor. C. R.. and Shelanski. M. L. (l97#).
._I_. Mol, Biol. 82. 737.

Detrich III. H. W.. Berkowitz. S. A.. Kim. H.. and
gillizm. R. C. (1976). Biochem. Biophys. Res. Commun.
_§. 9 l. _

Eichhorn. J. H. and Peterkofsky. B. (1979). g, Cell
Biol. 8;, 26.

Nickerson. J. A. and Wells. W. W. (1978). Biochem.
Biophys. Res. Commun. 85. 820.

Nunez. J.. Fellous. A.. Franccn. J.. and Lennon. M.
(1979). Proc. Natl. Acad. Sci. USA 16. 86.

 

118#.

Ostlund.R . E.. Leung J. T.. and 5Vaerewyck Hajek. S.
(1979). Analytical Biochem, 2_.15.

Sherline. P.. Schiavone. H.. and Brocato. S. (1979).
Science 295. 593.

76

CHAPTER III

LIVER TUBULIN CONTENT AND POLYMERIZATION IN THE
STREPTOZOTOCIN INDUCED DIABETIC RAT

ABSTRACT

The streptozotocin induced diabetic rat was used as
a model to study microtubule regulation. The method of
Pipeleers and co-workers (1) was used to measure free and
polymerized tubulin in streptozotocin induced diabetic
rats before and after insulin injections. A significant
decrease in the degree of polymerization was observed in
diabetic rats compared to control rats. Twenty four
hours after a single insulin injection. the percent of
total tubulin that was polymerized tubulin increased
significantly over that of controls and diabetics. The
free form of tubulin was elevated in diabetic rats but
after an insulin injection decreased to control values.
The change in total tubulin concentration closely paral-
leled that of the free form, while the concentration of
polymerized tubulin remained unchanged throughout the
experiment. Plasma glucose and insulin levels confirmed
earlier reports concerning diabetic and insulin treated

rats.

77

 

78
INTRODUCTION

The starved-refed rat model system demonstrated that
the distribution of tubulin between the polymerizad and
depolymerized forms may be modulated by physiological
factors. The diabetic rat. before and after insulin in-
jections, is another system in which physiological fac-
tors may be manipulated and corresponding changes in
tubulin content and polymerization studied. During
diabetes hepatic gluconeogenesis and cAMP levels increase
and protein content, albumin synthesis and secretion, and
cGMP levels decrease (2,3,24,33). Plasma glucose levels
increase and insulin levels decrease (5.6). Injecting
insulin into diabetic rats results in a reversal of the
effects of diabetes. i.e. decrease in plasma glucose
levels and an increase in plasma insulin levels. In-
sulin mediates an increase in the rates of glycolysis.
the citric acid cycle. and fatty acid biosynthesis while
decreasing hepatic cAMP levels, hepatic long-chain acyl
CoA levels and serum free fatty acids (7). Insulin has
been shown to stimulate hepatic lipogensis (8-14) and may
increase triglyceride secretion from the liver (l#-16).
Treatment of diabetic rats with insulin has been shown to
restore rates of synthesis and secretion of albumin in
the liver (24.33). An extensive amount of research
supports the theory that microtubules are involved in the
secretion of VLDL and plasma proteins from the liver (34-

38,40-42). In view of the link between hepatic VLDL

79
and plasma protein synthesis and secretion and the diabe-
tic. insulin treated rat and the link between VLDL and
plasma protein secretion and microtubules. we studied
the effect of streptozotocin induced diabetes (insulin
treated) on microtubule polymerization.

We used streptozotocin to induce diabetes in our
rats. Streptozotocin, N-methylnitroso earbamyglucos-
amine. is an antibiotic and antitumor agent derived from
Streptegyces achromoggnes (43,#4).. Rakietan and co-
workers (as) first demonstrated that the drug could selec-
tively destroy the pancreatic beta cell. with production
of permanent diabetes. Increasing the dose of strepto-
zotocin results in increased severity of the diabetes

(#6).

MATERIALS AND METHODS

Animal Treatment. All animals were housed in an air-
conditioned room at 23°C under controlled lighting (lights
6AM-6PM). Male Holtzman rats weighingIVZ50 grams were
housed in groups of 4 in plastic cages containing wood
chips. All groups were fed a commercial chow and H20, 2Q
libitum. throughout the experiments. Diabetes was induced
in rats by injection of streptozotocin (The Upjohn Co.)
(65mg/ml/kg BW in 0.1 M citrate buffer, pH “.5) into the
femoral vein within 10 minutes of solution preparation.
Seven days after injection, blood was collected from the

tail vein and analyzed for glucose levels. Only animals

80
with glucose levels of or above #00 mg% were used in the
experiment. Eight days after streptozotocin injection.
animals were injected with Regular Insulin i.p. (BU/100
grams BW) or Lente Insulin subcutaneously (lSU/lOO grams
BW). Some groups received daily injections of Lente
Insulin (l5U/100 grams BW) subcutaneously while the
single injection group received Regular Insulin i.p.
At time intervals after injection of insulin, rats were
sacrificed by decapitation and trunk blood was collected
in heparinized tubues. Livers were removed and free and
polymerized tubulin were measured using a colchicine
binding assay (see Chapter I for methods).
Glucose Determination. Plasma glucose was determined by
using the Computer-Directed 3500 Gilford Analyzer and the
Gilford/Worthington reagent kit.
Insulin Determination. An Insulin Radioimmunoassay kit
(Amersham) was used to measure plasma insulin.
Protein Determination. Liver tissue was homogenized in
PBS and protein concentration was determined by the method
of Lowry and co-workers (17) which was modified for auto-
mated analysis as described by Mak and Wells (39).
235 Determination. DNA content was measured in liver
homogenates (PBS) by the Ethidium Bromide method as des-
cribed by Kurtz and Wells (18).
Statistics. Statistical Analyses were performed using

the two-tailed t-test (9.7).

 

81
RESULTS

The protocol of the experiment is explained in Table
I. To study the effects of diabetes on tubulin content
and polymerizatin. rats were sacrificed 3. 8. and 14 days
after streptozotocin injection at which time free and
polymerized tubulin were measured. The eight day diabetic
rats were given insulin injections. Therefore, the 14 day
diabetic group would correlate with the 8 day diabetic
group, 6 days after insulin injection.

In order to verify the physiological state of the rat,
plasma insulin and glucose levels were measured. Plasma
glucose levels are elevated in the diabetic groups and
decrease to approximately control levels after insulin
injections (Table II). Twenty four hours after a single
insulin injection plasma glucose levels are elevated again
to those of diabetics. Repeated injections of insulin
for 6 days results in abnormally low plasma glucose levels.
Plasma insulin levels do not appear to be depressed in
diabetic rats as compared to citrate injected rats (Table
II). These results have been confirmed by other experi-
ments. Streptozotocin injected rats which are injected
with a single dose of insulin and allowed to become hyper-
glycemic again have lower insulin levels than diabetic
rats which have not been given insulin. The antibody
used in the radioimmuncassay to quantitate plasma insulin
may be cross-reacting with a plasma component which does

not have insulin-like activity. Diabetics' (streptozotocin

82
TABLE I

Protocol of Experiment and Body Weight of Rats

 

 

Explanation of Treatment Code Weight of Rats
Citrate injection C 281 i 33
Citrate injection, 14 hrs. 6 I (ip) C-I 290 i 8
3 days after streptozotocin 3D 255 i L?
8 days after streptozotocin SD _247 i 30
1“ days after streptozotocin lhD 285 i 22
8D, 1 hr. with Insulin (ip) lH-I 260 1 16
8D, 4 hrs. with Insulin (ip) #H-I 255 i 15
8D, 3 hrs. with Insulin (ip), QHZ-I 263 i 12
then another insulin injection (ip),

1 hr. later sacrificed

8D, 2# hrs. with Insulin (ip) 24-1 251 i 9
8D, 6 days with Insulin (ip) 6DI 275 i 9
8D, 6 days with Insulin (subcu) 6DR 313 1 3

given one insulin injection per day

 

Male Holtzman rats were in ected with streptozotocin (controls
were injected with citrate). Some rats were sacrificed at
time intervals after streptozotocin injection. Eight days
after stre tozotocin injection some rats were injected with
insulin (I . At time intervals after insulin injections
rats were sacrificed. A group of rats injected with citrate
were)injected with insulin and 1# hours later sacrificed
(3‘1 0

ip - interperitoneal

subcu - subcutaneous

 

83

 

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ms.H H ms.ma mm. H on.H m.oa H o.mm H.o~ H o.an am
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sm.a H se.ma mm. H mm.a 2.0n H n.oe m.w H o.m~a o

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HH mqm<a

8h
injected) plasma insulin levels return to control levels
despite the fact that the plasma glucose levels remain
elevated (19).

Liver tubulin content and polymerization can be
manipulated by injecting rats with streptozotocin and in-
sulin. The percent of total tubulin that is polymerized
is decreased significantly in the livers of streptozotocin
injected rats (Figure I)‘. Twenty four hours after a single
insulin injection the degree of polymerization is increased
significantly beyond 8 day diabetics and citrate controls.
The degree of liver tubulin polymerization 6 days after
a single insulin injection approaches diabetic values.
Total tubulin expressed per gram of liver or per gram
of DNA increases in diabetic rats and decreases after
insulin injection to that of control rats (Figures II &
III). No appreciable weight gain is seen in the liver 1
hour after insulin injection. Therefore, the decrease in
total tubulin per gram tissue is not due to an increase
in tissue weight. The change in total tubulin is due to
the change in amount of free tubulin since there is no
change in that of the polymerized form. The concentration
of free tubulin is increased in diabetics,.decreases-imme-
diately after insulin injections, then begins to increase
after 24 hours and approaches diabetic levels 5 days later.
Multiple injections of insulin did not significantly
change the results with respect to tubulin content or

85

Figure I: Each value is the mean-i standard deviation
of 4 animals.

¥ Significantly different from citrate in-
jegted rats (C) at a P<.05 by a two-tailed
t- est.

CJSignificantly different from 8 day diabetics
(80) at a P<.05 by a two-tailed t-test.

.86“

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masoao HdHSosHaeaxm
mac Haw

23m 783 . ES 75 9: am am To U

 

 

_ _ fl _ _ _ _ _ _ _ _

 

 

 

 

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1
87

Figure II: Each value is the mean 1: standard deviation
of 14» animals. ‘

*Significantly different from citrate in-
jected rats (C) at a P<.05 by a two-tailed
t'testo _

Asignifioantly different from 8 day diabetics
(8D) at a P<.05 by a two-tailed t-test.

 

 

88

.vam,0HHonan voosusH sHocHouopaosHm

on» Ga ao>HA some Mom :Hasnsa uosHuoszaom use seam mo vssos< one .HH mhswfim

madame asvsoaHuoaxm

m8 So 75% Hams: H43 75 9: am on

Huo

0

 

 

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omN_~_w<<>._O

 

 

 

 

a».

on

2:

omH

oom

 

 

mextq J0 S/uttnqnm :0 Sn

 

89

Figure III: The Amount of Free and Polymerized Liver
Tubulin per Mg DNA in the Streptozotocin
Induced Diabetic Rat. Each value is the
mean 1 standard deviation of 4 animals.

9O

mam

Haw

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91
polymerization.

Liver protein was measured in order to observe the
effects of streptozotocin and insulin injections on this
parameter, as well as being able to express tubulin content
in terms of protein content, The amount of protein in the
liver evidently does not decrease as much in diabetesg
as do other tissue constituents (Table II). After insulin
injections the amount of protein per mg tissue stays con-
stant. The amount of total tubulin per mg protein does
not change after streptozotocin injection. After insulin
injection the pattern of tubulin amount per mg protein
(Table II) resembles that of tubulin amount per mg DNA or
per gram liver (Figures II & III). A decrease in the
free form of tubulin per mg protein occurs 2b hours after
insulin injection and then, a subsequent increase to dia-

betic levels 5 days later.

DISCUSSION

As stated earlier microtubules have been implicated
in VLDL and plasma protein secretion from the liver. The
starved-refed rat system, because of its increased liver
lipogenesis, VLDL synthesis, and VLDL secretion, seemed
an ideal system in which to study microtubule regulation.
From our starved-refed rat studies, an increase in tubulin
polymerization appeared to correlate with the time at
which an increase in VLDL secretion would occur.

Studies with perfused liver or isolated hepatocytes

92
show that there is an impaired secretion of proteins in
diabetic rats (24,30,31). This defect (impaired secretion
of proteins) may be related to the marked disruption of
the rough endoplasmic reticulum (RER), which has been seen
in livers of streptozotocin injected rats (export proteins
are synthesized almost exclusively on the RER) (23,32).
Investigators have observed not only the extensive dis-
ruption of BBB, but also appearance of large lysosomes.
With chronic insulin treatment the ultrastructure of the
cell is restored (23). The rate of albumin synthesis and
secretion has been reported to be markedly reduced in dia-
betes (2h.33). Treatment of diabetic rats with insulin
restores rates of synthesis and secretion of albumin.
Insulin has been shown to stimulate hepatic lipogenesis
(8-14) and may increase triglyceride secretion from the
liver (14-16). In view of the link between hepatic albu-
min and triglyceride secretion and the diabetic rat, we
thought this would also be an ideal system to study micro-
tubule regulation.

In the diabetic rat, an insulin injection produced
an increase in the degree of tubulin polymerization. This
increase in the degree of tubulin polymerization, at a
time when an increase in hepatic VLDL and plasma protein
may occur, would suggest that microtubules may be involved
in secretion in the diabetic, insulin treated rat. The

problem was the increase in the degree of tubulin

 

93
polymerization was due to an decrease in the free form
rather than an increase in the polymerized form. For
this reason we are not able to discuss whether micro-
tubules are involved in the secretion process in the dia-
betic rat system._ Tubulin content and polymerization can
be independently modulated by physiological factors in
the diabetic rat, but further work must be done to eluci-
date these factors as well as to explain the role of
microtubules in this system.

We did not see a correlation between insulin or
glucose levels and tubulin content and polymerization in
the starved-refed rat. In view of the extreme changes in
glucose and insulin levels in the diabetic rat, a cor-
relation may be able to be detected in this system. We
failed to detect any correlation between insulin or

glucose levels and tubulin content and polymerization.

9.

10.

11.

12.
l3.

14.

15.

BIBLIOGRAPHY

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