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This is to certify that the

dissertation entitled
Studies elucidating the mechanisms of delta—9-THC

-mediated protection from streptozotocin-induced
diabetes

presented by

Xinguang Li

has been accepted towards fulfillment
of the requirements for

Ph.D. Pharmacology & Toxicology

degree in

 

 

—Environmental Toxicology

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6/01 cycincmaeouopssms

 

STUDIES ELUCIDATING THE MECHANISMS OF Ag-THC-MEDIATED
PROTECTION FROM STREPTOZOTOClN-INDUCED DIABETES

By

Xinguang Li

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Pharmacology & Toxicology

Institute for Environmental Toxicology

2000

ABSTRACT

STUDIES ELUCIDATING THE MECHANISMS OF Ag-THC-MEDIATED
PROTECTION FROM STREPTOZOTOClN-INDUCED DIABETES

By

Xinguang Li

Ag-Tetrahydrocannabinol (Ag-THC) is a well-established immune
suppressive agent, but has not been evaluated in models of immune-mediated
diabetes. The overall objective of the present study was to investigate the
protection by Ag-THC from the development of autoimmune diabetes. Multiple
low dose streptozotocin (MLDSTZ)-induced diabetes in CD-1 mice has been
utilized as an experimental model to study beta-cell pathogenesis in autoimmune
diabetes. it was demonstrated in the current investigation that 11-day treatment
with Ag-THC was able to attenuate the MLDSTZ-induced elevation in serum
glucose and loss of pancreatic insulin in CD-1 mice. Additionally, MLDSTZ-
induced insulitis and increases in IFN-y, TNF-oc and lL-12 mRNA expression
were all reduced by co-administration of AQ-THC. In separate studies, Ag-THC
given after completion of STZ treatment was found equally effective in protecting
CD-1 mice from MLDSTZ-induced diabetes. Additionally, results from
comparative studies showed that the BBC3F1 mice treated with MLDSTZ
exhibited an absence of insulitis, but demonstrated moderate hyperglycemia and
insulin loss. The less severe diabetic state in BGC3F1 mice was not altered by
AQ-THC, suggesting that MLDSTZ treatment can initiate a diabetic state without

an inflammatory response in pancreatic islets. Accordingly, these studies

indicate that AQ-THC is capable of attenuating the severity of the autoimmune
response in the experimental model of immune-mediated diabetes.

The protection from MLDSTZ-induced hyperglycemia by Ag-THC
treatment could also be attributed to a possible insulinotropic effect of the
cannabinoid on CD-1 mice. Studies toward an understanding of the direct effect
of A9-THC on pancreatic beta-cell function indicated that AQ-THC was able to
stimulate insulin release from CD-1 mouse islets and rat-derived insulin-
producing RINm5F cells. Closely associated with Ag-THC-stimulated insulin
release was a calcium influx-induced elevation in intracellular calcium and an
activation of CaM kinase II. Subsequent analysis of pancreatic islets and
Rle5F cells identified mRNA expression for the cannabinoid receptor, CB1.
Furthermore, the CB1 receptor antagonist, SR141716A was shown to block A9-
THC-stimulated insulin release and elevation in intracellular calcium. These
results indicate that Ag-THC-stimulated insulin release is mediated through an
elevation of intracellular calcium through the opening of calcium channels via a
CB1-dependent mechanism. In conclusion, the present study indicates that a
suppression of cell-mediated immune response against pancreatic beta-cells and
possibly a direct stimulatory effect on insulin release from beta-cells may be

involved in AQ-THC-attenuated hyperglycemia produced by MLDSTZ treatment.

To my parents, Chengxiao Li and Xiuqi Zhang
To my husband, Wei Sun
To my daughter, Lily

for their love, their sacrifice and their support

ACKNOWLEDGEMENTS

I would like to give my deepest gratitude to my academic advisor, Dr.
Lawrence Fischer for his masterful guidance, financial support and patience. The
scientific discipline he provided allowed me to find the right direction for my
graduate study and will greatly benefit my future career as well. I would also like
to give my sincere appreciation to Dr. Norbert Kaminski. His excellent insight, his
support and his encouragement really means very much throughout my graduate
work. I felt particularly fortunate to have both him and Dr. Fischer as my research
mentors.

Many thanks go to my committee members Dr. Jack Harkema and Dr.
Patricia Ganey for their shared expertise, their interest, and their
encouragement. I want to especially thank our department chairperson, Dr.
Kenneth Moore for his support and his help.

The whole Kaminski lab and the Fischer lab, especially Robert Crawford,
Robin Condie, Courtney Sulentic, Amy Herring, Barbara Faubert and Tony Jan,
have been invaluable for their constructive suggestions. A special note of
gratitude should go to Robyn Johnson, Kathy Campbell, and Amy Porter for their
cheerful technical assistance. I would also like to thank my fellollow students Hyn
Youn Cho for her friendship throughout the years.

Finally, I would like to give my heartful appreciation to my husband, Wei
Sun. Without his help, support and burden sharing, the degree and dissertation

would have been beyond my reach.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................... IX
LIST OF FIGURES .............................................................................................. X
CHAPTER I
INTRODUCTION ................................................................................................ XII
I.1. Preface ................................................................................................ xiii
|.2. Insulin and Insulin-producing cells ....................................................... xiii
l.2.A. Morphology and physiology of the pancreatic islets ......................... xiii
l.2.B. Functions of insulin—producing cells: insulin synthesis and insulin
release ............................................................................................ xv
I. 2. B. i. Insulin synthesis ................................................................... xv
I. 2. B.‘ Ii. Insulin release and calcium-dependent signaling .............. xviii
I. 3. Streptozotocin- induced diabetes ....................................................... xxiv
I. 3. A. Diabetes: general background ....................................................... xxiv
I.3.B. Streptozotocin: structure and biological effects .............................. xxvi
I.3.C. Direct treptozotocin cytotoxicity ..................................................... xxix
I.3.D. Multiple low dose streptozotocin-induced diabetes model ............. xxxii
I.3.D.i. Genetic susceptibility ........................................................ xxxiii
I.3.D.ii. Cell-mediated immune response ..................................... xxxv
|.4. Cannabinoid compounds and Ag-THC ............................................. xxxix
l.4.A. Cannabinoids: structure and biological effects .............................. xxxix
I. 4. B. Cannabinoid receptors: 081 and C82 ............................................ inv
I. 4. C. Intracellular signal transduction ...................................................... xlvii
l ..4 D. Immune modulation by Ag-THC ........................................................... Ii
I.5. Summary .............................................................................................. Iiv
CHAPTER II
EXAMINATION OF THE IMMUNOSUPPRESSIVE EFFECT OF
A°-THC IN MLDSTZ-INDUCED DIABETES MODEL ........................................ LVI
”.1. Summary ............................................................................................ Ivii
“.2. Introduction ........................................................................................ Iviii
".3. Materials and methods ........................................................................ lix
II.3.A. Chemicals ........................................................................................ lix
”.33. Animals ............................................................................................. Ix
II.3.C. Treatment ........................................................................................ 60
II.3.D. Quantitative competitive RT-PCR analysis. ..................................... 63
II.3.E. Histopathological examination. ........................................................ 65
II.3.F. Statistical analysis ............................................................................ 66
“.4. Results ................................................................................................ 66

vi

II.4.A. Effects of 11 day AQ-THC treatment on MLDSTZ-induced

hyperglycemia and loss of pancreatic insulin in CD-1 mice ............ 55
“.48. Effects of 11 day Ag-THC treatment on MLDSTZ-induced
hyperglycemia and loss of pancreatic insulin in B603F1 mice ....... 60
II.4.C. Effects of Ag-THC treatment on MLDSTZ-induced insulitis
in 00-1 and B6C3F1 mice .............................................................. 61
II.4.D. Effects of 11 day Ag-THC treatment on MLDSTZ-induced changes
in mRNA expression of selected cytokines in CD-1 mice ............... 64
II.4.E. Effects of Ag-THC treatment post administration of MLDSTZ
in CD-1 mice ................................................................................... 68
”.5. Discussion .......................................................................................... 75
CHAPTER III
CANNABINOID RECEPTOR CB1-MEDIATED INSULIN RELEASE
FROM ISOLATED PANCREATIC ISLETS AND RINMSF CELLS ........... 84
”L1. Summary ........................................................................................... 85
III.2. Introduction ........................................................................................ 86
"L3. Materials and methods ...................................................................... 88
|II.3.A. Materials ......................................................................................... 88
|II.3.B. Preparation of mouse pancreatic islets ........................................... 88
|II.3.C. Cells ................................................................................................ 89
”L30. Measurement of insulin release ...................................................... 89
|II.3.E. Determination of cannabinoid receptor mRNA expression ............. 90
|II.3.F. Measurement of [Cam]i .................................................................... 92
|II.3.G. Statistical analysis .......................................................................... 93
III.4. Results ............................................................................................... 93
III.4.A. Stimulation of insulin release from mouse pancreatic islets and
RINm5F cells by Ag-THC treatment ................................................. 93
”L48. Ag-THC-induced increase in [Ca2*]i in RINm5F cells ....................... 93
III.4.C. Attenuation of A9-THC-induced increase in [Ca2*]i and insulin
release by the calcium channel blocker verapamil .......................... 96
III.4.D. Thapsigargin and ryanodine exert no effect on the elevation in
[Ca2+]i induced by Ag-THC ............................................................. 100

”LS.

III.4.E. Effects of CaM kinase II inhibitor KN93 and PKC inhibitor
bisindolylmaleimide on the stimulation of insulin release

by AQ-THC ..................................................................................... 100
III.4.F. Identification of cannabinoid receptor mRNA expression in

isolated pancreatic islets and RINm5F cells ................................. 103
I ”46. Effects of cannabinoid receptor antagonists on Ag-THC-

stimulated insulin release .............................................................. 106
III.4.H. Effects of cannabinoid receptor antagonists on Ag-THC-

induced [Ca2*]i increase in RINm5F cells ...................................... 106
Discussion ....................................................................................... 1 13

vii

CHAPTER IV
OVERALL SUMMARY AND CONCLUSION .......................................... 120

BIBLIOGRAPHY ............................................................................................... 129

viii

LIST OF TABLES

Table ”-1. Histopathological evaluation of the protective effect of
AQ-THC on MLDSTZ-induced insulitis in CD-1 mice ..................................... 67

LIST OF FIGURES

Figure I-1. Diagram showing some important factors in glucose-
stimulated insulin release ........................................................................ 9

Figure I-2. Chemical structure of streptozotocin and Vacor ......................... 17

Figure I-3. Chemical structure of Ag-THC and cannabinoid
receptor antagonists ............................................................................. 29

Figure I-4. Scheme for the putative mechanism responsible for
MLDSTZ-indcued autoimmune diabetes .................................................... 30

Figure "-1. Scheme of the Ag-THC and STZ treatment regimens ................... 50

Figure "-2. Effect of Ag-THC treatment on MLDSTZ-induced
elevation of serum glucose in CD-1 mice and B6C3F1 mice .......................... 57

Figure "-3. Effect of Ag-THC treatment on pancreatic insulin in
MLDSTZ-induced diabetes in CD-1 mice and B6C3F1 mice .......................... 59

Figure "-4. Representative photomicrograph of islets in
pancreatic tissue .................................................................................. 63

Figure "-5. Representative photomicrograph of immunohistochemical
staining of islets in pancreatic tissue obtained from CD-1 mice ...................... 66

Figure "-6. Effect of Ag-THC treatment on IFN-y RNA expression

in MLDSTZ-induced diabetes in CD-1 mice ............................................... 70
Figure "-7. Quantitative analysis of cytokine steady state mRNA

expression after A9-THC treatment in MLDSTZ-induced diabetes ................... 72
Figure "-8. Effect of Ag-THC post MLDSTZ-treatment on serum glucose

and pancreatic insulin in MLDSTZ-induced diabetes in CD-1 mice .................. 74
Figure "-9. Scheme for the putative mechanism responsible for the protective
effect of Ag-THC on the MLDSTZ-induced diabetes model ............................ 76
Figure III-1A. Effect of Ag-THC on insulin release from

mouse pancreatic islets ......................................................................... 94
Figure III-1 B. Effect of Ag-THC on insulin release from RINm5F cells .............. 95

X

Figure III-2. Effect of Ag-THC on [Ca2+], in RINm5F cells .............................. 97

Figure III-3A. Effect of calcium channel blocker verapamil
on A9-THC-induced increase in [Ca‘P']i in RINm5F cells ................................. 98

Figure III-3B. Effect of calcium channel blocker verapamil on the
stimulation of insulin release by Ag-THC in RINm5F cells .............................. 99

Figure III-4A. Effect of thapsigargin on Ag-THC-induced
increase in [Ca2+], ............................................................................... 101

Figure III-4B. Effect of ryanodine on Ag-THC-induced increase in [Ca2*]i ....... 102

Figure III-5A. Effect of CaM kinase II inhibitor KN93 on the
stimulation of insulin release by AQ-THC .................................................. 104

Figure III-SB. Effect of PKC inhibitor bisindolylmaleimide on the
stimulation of insulin release by Ag-THC .................................................. 105

Figure Ill-6. mRNA expression of cannabinoid receptor in
isolated pancreatic islets and RINm5F cells ............................................. 107

Figure III-7A. Effect of SR141716A on Ag-THC-stimulated insulin
release from mouse pancreatic islets ...................................................... 108

Figure Ill-7B. Effect of SR141716A on Ag-THC-stimulated
insulin release from RINm5F cells .......................................................... 109

Figure III-7C. Effect of SR144528 on AQ-THC-stimulated
insulin release from RINm5F cells .......................................................... 110

Figure III-7D. Effect of cannabidiol on insulin release from RINm5F cells ...... 111

Figure III-8. Effects of SR141716A on Ag-THC-induced
increase in [Ca2*]i in RINm5F cells .......................................................... 112

Figure III-9. CB1 receptor mediated Ag-THC-stimulated insulin release
through calcium-dependent signaling pathways ........................................ 119

xi

CHAPTER I
INTRODUCTION

l.1.Preface

Current diabetes research is focused primarily on understanding the
mechanisms of diabetes development and pursuing the potential for improved
therapeutic treatments. Among those established therapeutic treatments,
immune suppression has been previously demonstrated to be a potentially useful
approach for immune-mediated diabetes. One well-known immunosuppressive
agent currently under study is A9-tetrahydrocannabinol (Ag—THC). Ag-THC is the
major psychoactive component of marijuana and alters a wide variety of
biological effects including those associated with immune responses and release
of insulin. The present study is undertaken to investigate the potential protective
effect of Ag-THC on an immune-mediated diabetes model that employs multiple
low dose streptozotocin (MLDSTZ) administration. This experimental model
employed in the present investigation will further provide valuable insight into the
mechanism by which certain environmental chemicals are capable of initiating
immune-mediated diseases. To provide background and rationale for these
studies, the remainder of the introduction section will first review the morphology
and physiology of insulin-producing cells with particular emphasis on the
regulation of insulin release. Furthermore, an overview of the literature relevant
to STZ and the MLDSTZ-induced diabetes model will be presented. Finally, the

therapeutic effects of Ag-THC and the related mechanisms will be introduced.

l.2.lnsulin and Insulin-producing cells

l.2.A.MorphoIogy and physiology of the pancreatic islets

2

Pancreatic islets are small spherical collections of endocrine cells
scattered throughout the pancreas. Islets comprise approximately 1-2% of the
mass of the pancreas. The remaining 98-99% of the pancreas is composed of
the exocrine, ductal, connective and neural tissues (see review in (Bonner-Weir
and Smith, 1994; Fischer, 1997)).

The pancreatic Islets contain four major cell types, each of which is
responsible for the synthesis and release of specific islet peptide hormones.
Alpha-cells produce glucagon, beta-cells produce insulin, delta-cells produce
somatostatin, and PP-cells produce pancreatic polypeptide. Generally, insulin-
producing beta-cells are located in the center of the islets, and comprise
approximately 70% of the islet mass. Alpha-cells, delta-cells and PP-cells are
located around the islet periphery and make up approximately 30% of the islet
mass.

Individual islets have extensive networks of highly fenestrated capillaries
allowing for rapid transfer of the islet hormones across the capillary endothelium
into the circulation. Insulin, as one of the islet hormones, stimulates the uptake
and utilization of glucose at various sites throughout the body, thereby lowering
blood glucose concentration. Glucagon stimulates the mobilization and release
of glucose, thereby raising blood glucose levels. Glucagon also stimulates the
release of insulin, while insulin appears to inhibit the release of glucagon.
Additionally, somatostatin inhibits the release of both glucagon and insulin. The
net result of these tight regulations is that the concentration of blood glucose

fluctuates only in a narrow range within normal physiological limits, despite

fasting and food intake.

Approximately 20% of the pancreatic blood flow is in the endocrine islets
even though it represents only 1-2% of the total tissue mass. For larger islets,
which obtain the majority of the islet blood flow, the arterial supply enters the islet
at a discontinuity or break In the mantle cells. Capillaries radiate into the center
of islet cells ending in collecting venules located at the periphery. As a result, the
arterial blood containing a potential toxic substance may enter the islets and first
contact insulin-producing beta-cells in the center of the islets. This blood flow
pattern and the relatively high blood flow rate in the pancreatic islets may
contribute to the high sensitivity of insulin-producing beta-cells to certain
chemicals such as streptozotocin (STZ). As the blood moves from the islet core
to the mantle, glucagon and somatostatin cells would be the next targets
exposed to a potentially toxic chemical. This exposure and a possible change in
normal insulin levels on insulin-producing cells due to chemical-induced effects
could subsequently alter the normal release of glucagon and somatostatin.

Therefore, an anatomical explanation for beta-cell selective toxicity is possible.

I.2.B.Functions of insulin—producing cells: insulin synthesis and
insulin release
I.2.B.i.lnsulin synthesis
The process from insulin genes to the formation of mature Insulin
molecules in secretory granules is referred to as insulin synthesis (Steiner and

Tager, 1979; Permutt.M.A, 1981; Permutt et al., 1984; Steiner et al., 1985;

4

Selden et al., 1987; Steiner, 1967). In general, the insulin gene is transcribed
into preproinsulin mRNA (PPImRNA) by RNA polymerase II. The primary
transcript of PPI is processed within the nucleus to form mature PPI mRNA
through a post-transcriptional process, which involves an addition of a 7-
methylguanosine residue at the 5' end, polyadenylation at the 3' end, and the
splicing out of one or two introns. The mature PPImRNA leaves the nucleus, is
bound to ribosomes and translated to form PPI. PPI has a very short half-life of
approximately 1 min, indicating that it is rapidly processed to form proinsulin.
After the pre portion of the proinsulin is removed from the nascent polypeptide,
the resulting proinsulin molecule is sequestered into the membranous vesicles of
rough endoplasmic reticulum (ER). Subsequently, the proinsulin molecules are
transferred in small membrane-bound vesicles to the Golgi region. There,
packaging of proinsulin into secretory granules is initiated. As the granules
mature, C-peptide is cleaved from the proinsulin molecule to produce insulin by
two types of proteolytic enzymes: a trypsin-Iike protease and a carboxypeptidase
B-Iike-enzyme. The insulin molecule then crystallizes with zinc to form the dense
core of the mature insulin secretory granules (Emdin et al., 1980). Afterwards,
release of insulin from the secretory granules occurs by exocytosis and the
insulin crystal is discharged to the extracellular space.

It is generally accepted that there are multiple levels of control in the
regulation of insulin synthesis. Glucose as the principle physiological regulator of
insulin synthesis and release appears to modulate many steps of this process. It

has been previously shown that glucose directly modulates PPImRNA levels in

cultured mouse, rat and human islets ( Rafiq et al, 2000; Giddings et al., 1985;
Hammonds et al., 1987; Jarrett et al., 1967). Low glucose concentrations (2.8-
3.3 mM) lead to decreases in the levels of PPImRNA, whereas high glucose
concentrations (17-28 mM) allowed the maintenance of PPImRNA levels or
increased PPImRNA levels after culture at low glucose (Permutt and Kipnis,
1972; Hammonds et al., 1987). The increased PPImRNA levels by high glucose
are due to either alteration of insulin gene transcription and/or stabilization of
PPImRNA. In addition, both initiation and elongation involved in the translational
aspects of PPI synthesis are also enhanced by high glucose (Welsh et al.,
1986). Furthermore, glucose—stimulation has been shown to alter proinsulin
synthesis and conversion of insulin without changing the levels of PPImRNA
(Itoh et al., 1978; Rafiq, 2000). The effects of other agents on insulin synthesis
have also been documented. Nutritional substances (Welsh et al., 1986; Prentki
et al., 1994), cyclic adenosine 3'5' monophosphate (CAMP) (Nielsen et al., 1985),
dexamethasone (Welsh et al., 1988), and cholera toxin (Welsh et al., 1988) can
stimulate insulin gene transcription in insulin-producing cells. On the other hand,
cyproheptadine (Miller et al., 1993), STZ (90mg/kg) (Permutt et al., 1984), FK506
(Tamura et al., 1995), cytokines (e.g. IFN-y and TNF-a) (Sternesjo et al., 1995),
and fasting (Giddings et al., 1991) have been shown to decrease insulin
synthesis.

The pancreatic beta-cell is the major site for insulin synthesis and insulin
release in amounts sufficient to regulate blood glucose (Itoh et al., 1978;

Giddings et al., 1985). Until recently, it was believed that insulin synthesis and

6

insulin release was limited to beta-cells of the pancreatic islets. However, it has
now been demonstrated that extrapancreatic tissues such as human placenta,
mouse and rat brain, and the yolk sac of the developing rat fetus also may
synthesize insulin (Devaskar et al., 1993; Liu et al., 1985; Muglia and Locker,
1984). The physiological role of insulin synthesis in these non-pancreatic tissues

is unclear at this time.

I.2.B.ii.lnsulin release and calcium-dependent signaling

Insulin stored in mature secretory granules is released by exocytosis,
which is a multistage process involving transport of granules to the plasma
membrane, their docking, priming and finally their fusion with the plasma
membrane (Howell and Tyhurst, 1982). Once formed, granules enter a large
cytoplasm granule storage pool from which only a few percent are secreted per
hour. More recently formed granules are selected for earlier release, while older
granules accumulate and, if not secreted, eventually fuse with Iysosomes which
recycle the granule constituents. The above description is of the stimulated
pathway of insulin release, which is required for attenuating high glucose levels
and other nutrient stimulus (Prentki et al., 1997). Basal level insulin release
occurs via a constitutive pathway that does not involve secretory granules. This
pathway releases relatively small amounts of insulin that is insufficient for
controlling rising glucose levels associated with food consumption or increased
metabolic demands (Malaisse, 1972).

Glucose is the major physiological stimulus for insulin release.

Hyperglycemia stimulates the release of insulin, whereas hypoglycemia
stimulates the release of glucagon (Shi et al., 1996). Insulin release by glucose
stimulation is regulated by several critical components that are currently
identified in pancreatic beta-cells (Fig.I-1) (Henquin J.C., 1994). Specifically,
metabolism of glucose in the pancreatic beta-cells is initiated primarily by the
rate-limiting enzyme glucokinase. This glucose metabolic signal results in an
increase of the ATP/ADP ratio, which closes ATP-sensitive K+ channels (Km
channels) in the plasma membrane. This closure decreases the K“ conductance,
which allows the depolarization of the plasma membranes in the pancreatic beta-
cells (Roenfeldt et al., 1992; Findlay and Dunne, 1985). For a long time,
depolarization of the cell membrane was a property believed to be confined to a
small group of highly sophisticated cells such as nerve and muscle cells in which
the role and need of electrical signaling was obvious (Kostowski and Tarchalska,
1972; Cohen, 1972). However, during the 19608 and 1970s it became obvious
that a number of endocrine cells share this capacity. They utilize changes in their
membrane potential in order to transduce changes in their environment to
acceleration of hormone secretion (Bernardis and Frohman, 1971; Phelps and
Sawyer, 1977). The pancreatic beta-cell is one of the examples. Dean and
Matthews provided the first evidence for glucose-stimulated membrane
depolarization in the insulin-producing beta-cells (Dean and Matthews, 1968;
Dean and Matthews, 1970). When this depolarization is large enough, voltage-
dependent calcium channels become activated. The initial depolarization up to

the threshold potential causes the opening of a few voltage-dependent calcium

 

 

 

 

 

 

 

 

 

Glucagon

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Protein [mp
kinase A “—
IX 91 GLP-l
GIP
Protein fl
kinase E
\ DAG Insulin Secretion

 

 

 

 

 

 

 

Figure I-1. Schematic representation of the major mechanisms by which
stimulants and inhibitors regulate insulin release: -: inhibition; +; stimulation; Ach:
acetylcholine; DAG: diacylglycerol; CaM kinase: calcium/calmodulin dependent
kinase; IP3: inositol 1,4,5-triphosphate; PIP2: phosphotidylinositol 4,5-
biphosphate; GLP-1: glucagonlike peptide; 6le gastric inhibitory polypeptide

(Modified from reference - Henquin, 1994).

channels, which in turn causes a bigger depolarization and the opening of
additional calcium channels. The calcium influx via opening of calcium channels
produces a transient elevation of the intracellular Ca2+ concentration ([Ca2*],)
followed by a series of reactions including protein phosphorylation. These events
culminate in the exocytosis of the insulin-containing granules (Ashcroft and
Hughes,1990)

In addition to glucose, a variety of stimulants such as hormones,
neuropeptides, and sulfonylurea drugs can also stimulate insulin release from
pancreatic beta-cells (Figl-1) (Prentki et al., 1997; Neye and Verspohl, 1998). In
certain stimulus-secretion coupling, a calcium-independent mechanism (e.g.
ATP-dependent) has been proposed and reported as an alternative signaling
pathway (Sakuma et al., 1995; Lang et al., 1998). However, the [Caz*]i plays a
fundamental role in most secretagogue-induced release of insulin (Zawalich et
al., 1998; Ashcroft & Rorsman, 1989; Wollheim and Pozzan, 1984). Metabolism
of glucose and sulfonylurea drugs stimulate release of insulin by producing an
Increase in [Ca2"]i through a calcium influx into pancreatic beta-cells, via voltage-
dependent calcium channels (Porksen et al., 1996; Seri et al., 2000). In addition,
stimulants like carbachol or 12-O-tetradecanoylphorboI-13-acetate (TPA), may
mobilize Ca2+ from intracellular calcium stores, resulting in an increase in the
[Ca2+]i and insulin exocytosis (Zawalich et al., 1998; Tang et al., 1995). Current
evidence suggests that release of Ca” from intracellular compartments in
pancreatic beta-cells can be triggered by activation of various intracellular Ca2+

release channels (Ilkova, 1994). Insulin-producing cells may contain either or

10

both types of intracellular Ca2+ release channels: 1) inositol 1,4,5-trisphosphate
(IP3) receptor/ Ca2+ release channel; and 2) ryanodine receptor/CaZ*-induced
Ca2+ release channel (Lange and Brandt, 1993; Takasawa et al., 1998; Holz et
al., 1999). To determine the involvement of calcium in a particular cell function,
various Ca2+ channel blockers or inhibitors have been commonly utilized. Garcia
et al. employed verapamil in reducing the movement of calcium into the cells
from the extracellular matrix and indicated a calcium influx involved in the
adrenoceptor-stimulated insulin release (Garcia et al., 1992; Ohta et al., 1993).
Inhibitors for ER Ca2*-ATPase such as thapsigargin (Aizawa et al., 1995), or
inhibitors acting at ryanodine receptor-operated channels for Ca2*-induced Ca2+
release (Holz et al., 1999) have also been commonly employed to study the
regulation of Caz+ transport in beta-cells.

The mechanisms by which most secretagogues stimulate insulin release
involve a calcium-dependent signaling pathway. However, the precise regulatory
mechanism by which a sufficient increase in [Ca2+], is translated into the release
of insulin is still largely unknown. Current reports implicated that calcium should
perhaps be regarded as an initiator rather than a determinant of exocytosis
(Easom, 1999). It has been suggested that the amplitude of the exocytotic
responses depended to a greater extent on the activity of protein kinases and
phosphatases than the actual [Ca2*]i (Braiman et al., 1999; Miura et al., 1998;
Cui et al., 1998). For example, agents which increase intracellular cAMP levels,
such as glucagon and glucagon-like peptide-1 (GLP-1), were able to potentiate

glucose-stimulated insulin secretion almost 10-fold while modestly calcium influx

11

and [Ca2+],, suggesting a protein kinase A (PKA)«dependent mechanism
(Gromada et al., 1997). However, no stimulation of exocytosis was observed in
the absence of glucose, indicating that glucose metabolism was required for the
PKA—dependent phosphorylation. In addition, calcium-dependent exocytosis was
also enhanced by agents which activated protein kinase C (PKC), such as
acetylcholine (Hughes et al., 1990) and the phorbol ester 4-8-phorbol-12-B-
myristate-13-a -acetate (PMA) (Howell et al., 1990). In general, conditions which
promote protein phosphorylation lead to enhancement of secretion. Conversely
one would expect that agents which produce the activation of protein
phosphatases inhibit exocytosis. Indeed, this seems to be the mechanism by
which the inhibitory hormones and neurotransmitters somatostatin, galanin and
adrenaline suppress glucose-stimulated insulin secretion. The action of these
compounds is mediated by activation of an inhibitory (pertussis toxin-sensitive)
G-protein and culminates in the activation of the protein phosphatase
calcineurin. Accordingly, numerous Ca2*—dependent protein kinases and
phosphatases have been demonstrated within secretory cells, and many of
these are implicated in the secretory process.

A particular important member in the calcium-dependent protein kinase
family is the group of Ca2*/CaM-dependent kinases (CaM kinases). The
prototype of this group is smooth muscle myosin light-chain kinase (MLCK),
which phosphorylates myosin regulatory light chain in a CaZVCaM-dependent
manner resulting in activation of actomyosin ATPase required for muscle fiber

contraction (Walsh et al., 1982; Kilhoffer et al., 1992). A CaM kinase distinct from

12

MLCK was identified in a rabbit brain extract (Endo and Hidaka, 1980) and many
CaM kinases have subsequently been discovered. They are classified into two
groups based on their ability to phosphorylate the endogenous protein target: 1)
multifunctional enzymes: CaM kinase IN, II, and IV; and 2) specific enzymes:
MLCK and CaM kinase III (Kishi et al., 1998; Sobieszek, 1994; Wenham et al.,
1994; Nairn and Picciotto, 1994). Of these kinases, several have been identified
in pancreatic beta-cells including MLCK, CaMII and CaMKIlI (Wenham et al.,
1994; Niki et al., 1993; Mohlig et al., 1997). It has been demonstrated that the
loss of secretory responsiveness of islets to Ca” is accompanied by reductions
in calcium-dependent phosphorylation of endogenous islet substrates for CaM
kinase II (Norling et al., 1994). This suggests an important role for CaM kinase II
in beta-cell stimulus-secretion coupling. The inhibitors of CaM kinase II, such as
KN93 have been useful in studying the activation mechanism of CaM kinase II.
Treatment with KN93 (up to 10 pM) suppressed glucose-induced insulin release
in isolated rat pancreatic islets in a dose-dependent manner without affecting its
basal secretion (Wenham et al., 1994). By contrast, insulin release stimulated by
TPA, a direct activator of PKC, was not affected by KN93. Taken together, Ca2+
plays a critical role in insulin release and CaM kinase u may be involved in the

Ca2*-mediated insulin release.

I.3.STZ-induced diabetes
I.3.A.Diabetes: general background

Diabetes mellitus represents a complex variety of disorders characterized

13

by the inability to regulate blood glucose levels within normal physiological limits
(Walsh et al., 1982). At the molecular level, diabetes can occur from impaired
insulin secretion and/or insulin action. On this basis, diabetes has been classified
into type I (insulin-dependent) and type II (non-insulin-dependent) diabetes
mellitus (see review in Joslin’Diabetes, Krall et al., 1994). Type I diabetes often
occurs at a young age, while type II diabetes occurs later in life. Since type II
diabetes results primarily from impaired insulin-induced signaling rather than
insulin secretion from the beta-cell, it is not the focus of the present study.

Type I diabetes results from an almost complete destruction of pancreatic
beta-cells with maintenance of the alpha-cells and delta-cells within islets of
Langerhans (Gepts, 1965). In parallel with dramatic developments in genetics
and immunology during recent years, it has become clear that type I diabetes
develops as a consequence of the selective destruction of pancreatic insulin-
producing cells by autoimmune mechanisms. Although the etiology responsible
for type I autoimmune-based diabetes has not yet been definitively elucidated; it
is currently believed that some environmental factors are one of the elements
that contribute to the disease and may trigger its onset. These factors could
include mycobacteria, viral infections, stress, sex hormones and exposure to
xenobiotics such as certain drugs, food constituents, and environmental toxic
chemicals (Cahill and McDevitt, 1981; Craighead, 1978; Yoon, 1990). The first
case was reported from a clinical investigation involving individuals who ingested
rodenticide Vacor and later became diabetic (Karam et al., 1980; Prosser and

Karam, 1978). Other known beta-cell toxicants include the marine antifoulant

14

triphenyltin fluoride (Manabe and Wada, 1981), antiserotonergic drug
cyproheptadine (Fischer et al., 1973), the experimental diabetogenic agent
alloxan (Veleminsky et al., 1970), and the nitrosourea compound STZ (Rakieten
et al., 1963). Among these compounds, the rodenticide Vacor and STZ represent
the group of N-nitrosamines (Fig.I-2). N-nitrosamines are present in a variety of
food constituents and also can be formed in the body after ingestion of
conventional food such as dried bacon, spinach, and nitrate/nitrite-rich drinking
water (Brown, 1999; Scanlan, 1983). Contamination of nitrosamines has also
been found in rubber nipples for babies’ bottle and many other rubber product
(Havery and Fazio, 1982; Fajen et al., 1979). Accordingly, these compounds
pose a potential threat to humans either through trace contaminants in the
environment or through exposure to formation of these agents in the body, and
therefore have generated considerable public concern because of their potential
to cause diabetes and their known ability to produce cancer. The better
understanding of their biological effects and the underlying mechanisms may
contribute to both environmental toxicology studies as well as clinical diabetes

research.

I.3.B.Streptozotocin: structure and biological effects

STZ was first discovered in 1959 by the UpJohn company (Wiggans et al.,
1959). It was used as a broad-spectrum antibiotic as well as an antitumor agent
for malignant beta-cell tumors. After its diabetogenic effect was found in rats and

dogs in 1963 (Rakieten et al., 1963), STZ has been widely used to produce an

15

animal model of experimental diabetes. The effective dose of STZ in inducing
diabetes ranges from 50 to 200 mg/kg depending upon the species, such as
rats, mice, dogs, Chinese hamsters, monkeys, miniature pigs, and rabbits
(Lazarus and Shapiro, 1972). Certain species such as guinea pigs and Syrian
hamsters are much more sensitive to STZ than to alloxan, another commonly
used diabetogenic chemical (Veleminsky et al., 1970).

The structure of STZ has been determined to be the nitrosamide
methylnitrosourea linked to the C-2 position of D-glucose and exists as either the
a- or 8- anomers in the pyranose form (Fig.l-2). STZ is very unstable in solution
even at acid pH, and should be administered promptly after being dissolved in
citrate buffer at pH 4.5. The in vivo life span of STZ is less than 15 min (Aganival,
1980)

It has been suggested that the plasma membrane glucose transporter is
involved in the entry of STZ into pancreatic beta-cells. The a-anomer of STZ,
similar to the a-anomer of glucose, showed greater effect on insulin secretion
(Malaisse, 1991; Rossini et al., 1977). In addition, cells that have lost their
responsiveness to glucose (e.g. RlNr cells) also have lost their sensitivity to STZ
toxicity (LeDoux and Wilson, 1984). It has also been demonstrated that
analogues of STZ whose glucose moieties were replaced by manonose,
galactose, or alpha-O-methylglucose were nondiabetogenic (Bannister, 1972;
Kawada et al., 1986). Moreover, studies with 1“C-STZ showed that considerably
more STZ was taken up by the beta-cell than nitrosamide methylnitrosourea

(MNU) which does not have the glucose moiety (Wilson et al., 1988). These

16

0 N
</ \)CH2— NH
OH OH OH -— '

020

NH I
O = (I:
H3C "‘ N " N0
Streptozotocin Vacor

Figure l-2. Chemical structures of N-nitrosamine compounds:

streptozotocin and rodenticide Vacor.

l7

results indicated that the glucose moiety of STZ allows pancreatic beta-cells to
uniquely recognize and transport STZ to some critical cellular compartment,

where the methylnitrosourea moiety is responsible for the cytotoxic activity.

I.3.C.Direct STZ cytotoxicity

The preferential uptake of STZ into pancreatic beta-cells causes
preferential damage in this cell type. Once inside the cell, STZ is able to
spontaneously decompose, without metabolism, to form an isocyanide
compound and a methyldiazohydroxide (Tjalve, 1983). The isocyanide
component is able to either carbamolyate various cellular components or
undergo intramolecular carbamoylation. The methyldiazohydroxide decomposes
further to form a highly reactive carbonium ion. The carbonium ion is able to
alkylate various cellular components such as DNA and protein or to react with
H20 to from methanol which can subsequently enter the carbon pool. It is
notable that when considering the cytotoxic mechanism of STZ, it becomes
apparent that the drug, besides accumulating in certain cells, has a damaging
effect on many surrounding macromolecules. Outside the pancreas, major
cytotoxic effects caused by STZ have been seen in the liver, on the immune
system and renal function etc (Sutherland, 1977; Feldman, 1977; Durand, 1980).

The understanding of the direct cytotoxic actions of STZ on pancreatic
beta-cells has been greatly aided by the putative mechanism suggested by
Okamoto et al.(Okamoto, 1985). Okamoto and his colleagues proposed that the

carbonium ions formed by the decomposition of STZ reacted with nucleophilic

18

centers in DNA by a unimolecular reaction. The predominant site for the
alkylation reaction was at N7 position of guanine (LeDoux et al., 1986). Lesions
in DNA were subsequently removed by excision repair, which required the
activation of poly ADP-ribose polymerase (PARP) to form poly ADP-ribose with
nicotinamide adenine dinucleotide (NAD) as a substrate (Wilson et al., 1988).
Afterwards, the extensive NAD depletion in affected cells caused decreased
energy metabolism, protein biosynthesis, a cessation of cellular functions and
cell death (Yamamoto et al., 1981; Okamoto, 1981; Okamoto, 1985). As a
support of this hypothesis, it has been demonstrated that inhibitors of PARP
such as nicotinamide, 3-aminobenzamide and thymidine suppressed the
consumption of NAD, consequently preventing STZ-induced beta-cell death and
diabetes (Schein and Bates, 1968; Yonemura et al., 1984; Bolaffl et al., 1987).
Although the Okamoto mechanism has been widely accepted, recent
studies have demonstrated that the toxic action of STZ is more complex than
over-activation of one enzyme-PARP. LeDoux and coworkers showed that
equimolar concentrations of STZ and its nitrosamide moiety MNU caused the
same extent of DNA alkylation, DNA strand breaks and PARP activation in
insulin-producing cells in vitro, but the depletion of NAD from the same
concentration (1mM) of MNU and STZ was significantly different (LeDoux et al.,
1986). For example, while 1mM of MNU treatment caused a 50% depletion in
NAD concentration, 1mM of STZ treatment resulted in a 90% decreases of NAD.
This result indicated that the additional NAD depletion from STZ exposure is due

to factors other than PARP overactivation which is unique to STZ. Consistent

19

with this finding, it was demonstrated by Wilson et al. that STZ was able to
induce an alkylation not only in DNA, but also in the mitochondrial enzymes that
were necessary for the generation of adenine triphosphate (ATP) (Wilson et al.,
1988). The fall in ATP generation impaired the resynthesis of NAD and therefore
caused a further decrease in the NAD content. These observations suggested
that the combination of NAD depletion and impaired NAD synthesis allow STZ to
rapidly destroy pancreatic beta-cells.

Furthermore, it has been shown that sublethal concentrations of STZ
treatment caused a decrease of NAD levels to 50% of control levels in pancreatic
beta-cells. The change in NAD concentrations was restored to normal values
overtime, however, the treated cells exhibited an insulin secretory defect in
response to glucose stimulation (Bolaffi et al., 1986). Thus, it indicated that
sublethal concentration of STZ produced a permanent defect in stimulated
insulin secretion, which is independent of NAD content.

In conclusion, STZ exerts direct cytotoxic effects on pancreatic beta-cells.
It is capable of inducing the alkylation of cellular components leading to beta-cell
death. Furthermore, it can produce some nonlethal alterations in pancreatic beta-
cell functions. Therefore it is speculated that these alterations include some
conformational changes of membrane proteins associated with glucose transport
and metabolism, which may further be recognized as “non-sell” by the immune
system and play a potential pathogenic role through activation of an

inflammatory response.

20

I.3.D.Multiple low dose streptozotocin-induced diabetes model

An autoimmune etiology for type I diabetes was initially suggested by the
finding of inflammation in the islets of patients with a recent onset of diabetes.
This finding stimulated the efforts to develop experimental animal models of type
I diabetes, in which inflammation was a prominent feature of the prediabetic and
early diabetic pancreas.

Historically, the MLDSTZ-induced autoimmune diabetes model provided,
researchers with one of the few experimental tools for studying the role of the
immune system in beta-cell pathogenesis. When administered in a single high
dose, STZ caused the complete destruction of pancreatic beta-cells within 24
hours, resulting in a rapid onset of hyperglycemia. However, five daily
subdiabetogenic doses of STZ leads to a gradual onset of hyperglycemia one or
two weeks later, as first observed in male CD-1 mice by Like and Rossini, (Like
and Rossini, 1976). The lymphocytic cell infiltration of the islets of Langerhans
(insulitis) has been observed in this MLDSTZ model, suggesting an involvement
of immune components.

Subsequently, two spontaneously occurring diabetogenic rodent models,
the Bio-Breed (BB) rat (Nakhooda et al., 1977) and the non-obese diabetic
(NOD) mouse (Makino and Tochino, 1978), have also become available for type
I diabetes research. The understanding of the pathogenesis of type I diabetes
has been greatly aided by studies of these experimental animal models. The
MLDSTZ-induced autoimmune diabetes model particularly employed in the

current investigation offers several additional advantages. First, in insulitis-prone

21

strains such as CD-1 mice, the MLDSTZ model allows "synchronization" of beta-
cell destruction such that the inflammatory events occur on a predictable
timescale. Second, the MLDSTZ-induced diabetes model offers the possibility of
a better understanding of the mechanism by which certain environmental
chemicals, such as STZ, are capable of initiating immune-mediated diseases.
Additionally, in contrast to the high mortality induced by single high doses of
STZ, protracted administration of smaller STZ dosages in the MLDSTZ model

yields a more stable diabetic condition.

I.3.D.i.Genetic susceptibility

Type I diabetes has long been known as a hereditary disease on the
basis of the relatively high rate of familial transmission. The MLDSTZ model also
requires genetic predispositions (Le et al., 1985). At least three gene groups
have been found to influence MLDSTZ-induced diabetes. First, there is a strong
influence of gender difference on diabetes development in the MLDSTZ model
(Rossini et al., 1978). It was demonstrated that females appeared to be largely
resistant to the effects of MLDSTZ. The resistance to MLDSTZ treatment were
overcome by increasing the dose of STZ (Rossini et al., 1978). Castration and
administration of estrogen, androgen or testosterone further confirmed sex
dependency in the MLDSTZ model (Rossini et al., 1978; Maclaren et al., 1980;
Paik et al., 1982; Kromann et al., 1982; Leiter, 1982). In addition, age
differences, which are often related to the changes in the sex hormone levels,

were closely associated with susceptibility to the MLDSTZ treatment (Kuttler and

22

Schneider, 1982; Reddy and Sandler, 1995). The gender influence seems to
affect the pancreatic beta-cells directly, and not through the immune system
(Hahn et al., 1981). For example, it was observed that islet cells of male mice
were more prone to MLDSTZ-induced cytotoxicity than islet cells of female mice
(Ganda et al., 1976). However, since sex hormones are known to strongly
influence autoimmunity, it is possible that there is an additional gender influence
on the MLDSTZ-initiated islet inflammation.

The second important susceptibility locus has been linkedito the major
histocompatibility complex (MHC). By studying congenic-resistant mouse strains
(differing only at H-2) in the MLDSTZ model, Kiesel and Kolb discovered the
diabetes susceptibility genes within the H-2 complex on chromosome 17 (Kiesel
and Kolb, 1982; Kiesel et al., 1983). These genes were later mapped to the left
of the H-2 region, near H-2 K and la (Wolf et al., 1984; Weber et al., 1983;
Weber et al., 1984). In the mouse, the H-2 complex contains important immune
regulatory loci (Kromann et al., 1982). It was shown that administration of la (H-
2) antibodies was able to attenuate the development of MLDSTZ-induced
diabetes (Kiesel et al., 1983; Kolb, 1987; Kiesel et al., 1989), indicating that H-2
region genes may play an important role in the MLDSTZ-induced diabetes
model.

However, when the backgrounds of different inbred strains were analyzed,
the MHC associations were not consistent. This indicated that the MLDSTZ
sensitivity must be controlled by at least one non-MHC gene (Kiesel et al., 1983;

Le et al., 1985; Wolf et al., 1984). Indeed, the effects of H-2 genes may be

23

overridden or at least strongly modified by genes outside the H-2 complex. Leiter
et al. (Le and Leiter, 1986) showed that one non-MHC gene is associated with
MLDSTZ sensitivity and functions opposite to the MHC-linked gene. It was
further demonstrated that non-MHC genes associated with sensitivity to
endogenous androgen were involved in the susceptibility to MLDSTZ-induced
diabetes (Le et al., 1985). Whether the non-MHC genes affect the pancreatic

beta-cells or the immune system remains unclear.

I.3.D.ii.CeIl-mediated immune response

Although there has been considerable effort directed toward
characterizing the role of the immune component in the MLDSTZ model, many
observations yield a highly complex and often controversial picture. The precise
nature of the immune reaction remains unclear.

It is known that the initiation and progression of MLDSTZ-induced
autoimmune diabetes depends on both genetic susceptibility and neoantigen
exposure. However, identification of the target neoantigen has been a major
challenge in studies of immune-mediated diabetes and the MLDSTZ model.
Several candidate neoantigens have been described, but none has convincingly
’ been shown to be the “real antigen”. Studies by Klinkhammer and Krzystyniak
suggested that the “non-self” antigen in the MLDSTZ model might be presented
at the beta-cell surface (Klinkhammer et al., 1988; Krzystyniak et al., 1995). The
popliteal lymph node T lymphocytes were isolated 12 days after priming

BALB/cByJ females with a single injection of one subdiabetogenic dose of STZ

24

in complete Freund’s adjuvant. The primed T cells harvested were then cultured
with BALB/cByJ islet cell monolayers pretreated with non-cytotoxic levels of STZ
or control medium alone. A specific T-cell Iymphoproliferation was observed with
only the STZ pretreated islet cells, suggesting that the beta-cell surface proteins
altered by STZ may indeed be immunogenic (Klinkhammer et al., 1989). This
observation also addressed the critical involvement of T-cells in this
subdiabetogenic dose of STZ treatment.

The observations to date do not support a primary role for humoral
immunity in the MLDSTZ model. Although some preliminary reports suggest the
presence of islet cell surface antibodies (Huang and Taylor, 1981 ), there is a lack
of experimental evidence to prove organ specificity of these antibodies. In
addition, B—lymphocyte deficient mice retain their susceptibility toward MLDSTZ
treatment (Blue and Shin, 1984).

Most studies have directly or indirectly implicated a cell-mediated immune
response in the MLDSTZ model. Initial studies (Rossini et al., 1978)
demonstrated that injections of anti-T-lymphocyte serum (ALS) for 5 weeks (0.25
ml, 3 times daily) retarded development of hyperglycemia during the course of
the treatment, although diabetes developed shortly after termination of
treatment. Later, Paik and coworkers showed that “Balb/cBom-nu/+” male mice
were susceptible to MLDSTZ treatment, whereas “Balb/cBom-nu/nu” (athymic)
mice were resistant unless reconstituted with T-cell enriched splenocytes from
euthymic donors (Paik et al., 1982). Further studies showed that lethal irradiation

eliminated sensitivity of “Balb/cBom-nu/+” male mice to MLDSTZ treatment,

25

while reconstitution of T-cell enriched splenocytes was also able to restore the
sensitivity (Paik et al., 1982). Overall, these studies strongly supported the
diabetogenic potential of T-cells and cell-mediated immune response in the
MLDSTZ-induced diabetes model. Nonetheless, the striking difference in
susceptibility between euthymic mice and athymic mice in MLDSTZ-induced
diabetes model has not been uniformly observed in other mouse colonies
(Beattie et al., 1980; Nakamura et al., 1984). The genetic heterogeneity among
the experimental animals might attribute to this difference.

Cell-mediated immune response and humoral immune response are
mediated by different TH-cells, which represent a subpopulation of antigen-
activated T-cells, capable of releasing cytokines. TH-cells can be divided into two
distinct subsets based on their cytokine profiles. TH 1 cells, which secrete IFN-y
and |L-2, are primarily associated with cell-mediated immunity. Conversely TH-2
cells, which are characterized by the secretion of |L-4, lL-6 and lL-10, are
predominantly involved in humoral immunity. Most studies on the two
spontaneous diabetic animal models (NOD mice and BB rats) suggest that a
dominant activation of TH-1 cells over TH-2 cells is a key determinant in
establishing autoimmune diabetes (Katz et al., 1995; Trembleau et al., 1995;
Liblau et al., 1995; Maclaren and Alkinson, 1997; Rabinovitch, 1994; Rabinovitch
et al., 1996). It was demonstrated that NOD mice carrying a segment of
chromosome flanking the disrupted IFN-y receptor gene from original 129 ES
cells are resistant to development of diabetes (Kanagawa et al., 2000). In

addition IFN-y transgenic animals, in which the expression of the IFN-y is

26

directed by the insulin promoter, developed inflammatory lesions, islet antigen-
specific T-cell responses, and beta-cell destruction (Sarvetnick et al., 1990). IFN-
y also showed synergism with tumor necrosis factor a (TNF-a) to destroy beta-
cells in vitro (Pukel et al., 1988). Furthermore, lL-1, which was not cytotoxic by
itself to islet beta-cells (Oschilewski et al., 1986), exerted strong beta-cytolytic
action in combination with IFN-y (Pukel et al., 1988).

Although there is a paucity of observations, activation of TH-I cells has
also been implicated in the MLDSTZ model (Herold, 1996). Previous studies
have shown that mice given STZ and lFN-y were significantly more
hyperglycemic than those given STZ alone (Campbell et al., 1988). Additionally,
anti-lFN-y mAb was capable of attenuating the development of MLDSTZ-induced
diabetes (Campbell et al., 1988). It has been demonstrated that lFN-y alone in
vitro failed to induce class II MHC (Ia) antigens on CBA mouse islets, however,
high concentrations of lFN-y plus TNF-a were able to induce the H-2 la
expression (Campbell et al., 1985). Following MLDSTZ treatment, H-2 Ia-positive
cells around and within islets were observed by immunohistochemistry (Farr et
al., 1988). It is likely that that MLDSTZ treatment induced the expression of H-2
K or la in pancreatic beta-cells, as a consequence of local increases in TH 1 cell-
related cytokines. These results strongly suggest that the local accumulation of
TH 1 cell-associated cytokines, especially lFN-y, plays a pathogenic role in the
MLDSTZ-induced diabetes model.

In conclusion, all these findings indicate that the functional imbalance

27

between TH 1 cells and TH 2 cells and the paradigm of TH 1-cell-mediated

immune response apply to the MLDSTZ-induced diabetes model (Fig I-3).

l.4.Cannabinoid compounds and A°-THC

For many centuries, marijuana has been used both recreationally and
medicinally. The considerable therapeutic potential of marijuana has been
documented as early as the fourth century BC. During the rule of Emperor Chen
Nung, the Chinese used marijuana for the treatment of malaria, constipation,
rheumatic pains, absentmindedness, and female disorders. Marijuana is by far
the most widely used illicit drug with an estimated 20 million regular users in
North America and Europe. Many thousands of patients with AIDS, multiple
sclerosis, and other illnesses are also illegally self-medicating with cannabis in
the belief that it provides them with a therapeutic benefit [see review (Taylor,

1998”.

l.4.A.Cannabinoids: structure and biological effects
The mechanism for the various actions of marijuana remained unclear until
chemical analysis of plant extracts revealed a principal active ingredient. A9-
Tetrahydrocannabinol (A9-THC), the major psychoactive component in marijuana,
was first isolated as "toxic red oil" in the nineteenth century. The chemical
structure of Ag-THC was not fully established until 1964 (Fig.l-4) (Mechoulam,
1964). To date, more than 60 related cannabinoid compounds have also been
found in marijuana preparations. Structure activity relationship studies have

28

 

 

 

 

 

 

 

Figure l-3. Scheme for the putative mechanism responsible for the multiple low
dose streptozotocin-inducued autoimmune diabetes. M¢zmacrophage; Th1: T
helper 1 cells; Th2; T helper 2 cells; To: cytotoxic T cell; Ag: antigen; MHCII;

MHC class II molecule.

29

 

SR141716A SR144528

Figure l-4. Chemical structures of AQ-THC, CB1 antagonist SR141716A, and

C82 antagonist SR144528.

30

determined at least four structural requirements for cannabimimetic activity
(Razdan, 1986). A benzopyran structure provides the backbone for
cannabinoids. In addition, the length of the alphatic side chain determines the
potency of cannabinoids. A three carbon side chain seems to be the minimal
requirement while the nine carbons or more leads to a reduction in activity.
Furthermore, branching of the aromatic side chain can enhance potency. Lastly,
the attachment of an alicyclic ring to the benzopyran backbone at the 3,4-
position is critical for cannbimimetic activity. Several synthetic cannabinoid
compounds, such as CP-55,940, HU-210/211, WIN-55,211, possess the
essential structural requirements for activity and demonstrate high potency.
Regardless, among all the natural and synthetic cannabinoid compounds, A9-
THC is one of the analogues that have been most extensively studied.

Anecdotal information about the health benefits of AQ-THC was put to
scientific scrutiny early in the nineteenth century. Especially in the past three
decades, extensive studies have been conducted investigating the clinical
effectiveness of both smoked and oral AQ-THC treatment (Voth and Schwartz,
1997; Chait and Zacny, 1992). Many patients prefer smoking Ag-THC to an oral
preparation due to a more rapid onset of the inhaled preparation. However,
treatment by smoking AQ-THC has many difficulties in clinical trials, particularly in
controlling dosing and the inherent hazards of smoking (Mendelson and Mello,
1984). These problems can be largely overcome by the use of an oral

preparation. Availability of Ag-THC in its pure form and the development of

31

structural analogues have led to controlled studies characterizing the biological
profile of Ag-THC.

Ag-THC has been shown to produce a wide variety of biological effects in
animals and humans. The primary effect of Ag-THC is an alteration of central
nervous system (CNS) function. In general, Ag-THC produces changes in mood,
decreases psychomotor skills, modifies cognition and memory (Carchman et al.,
1979; Pertwee, 1988). Another well-characterized effect of Ag-THC is the
suppression of the immune system, and a detailed description of this effect will
be provided in the subsequent section. While the majority of cannabinoid
research is presently focused on the CNS and immune system, Ag-THC is also
likely to have significant and highly relevant modulation on the reproductive
system and the endocrine system. The AQ-THC-induced effects on reproductive
system have been shown to involve secretion of prolactin, follicle-stimulating
hormone and Ieutinizing hormone and ACTH (Wenger et al., 1999; Smith et al.,
1978). Several studies have also demonstrated a reduction of spermatogenesis,
a regression of Leydig cells, and an alteration of testosterone production by A9-
THC treatment (Hatoum et al., 1981; Dalterio et al., 1981). In addition, the study
conducted by Laychock et al. has shown that Ag-THC is capable of stimulating
the basal release of insulin as well as potentiating the insulin secretory response
produceded by glucose in rat pancreatic islets (Laychock et al., 1986). This
observation in vitro suggests an alteration of endocrine pancreatic cells functions

by Ag-THC, may occur in vivo.

32

A9-THC has been utilized as a treatment for a diverse variety of diseases.
These medical ailments include pain, migraine, epilepsy, glaucoma,
hypertension, nausea associated with chemotherapy, and the discomforts of
child birth (Voth and Schwartz, 1997; Holdcroft et al., 1997; Pertwee, 1988;
Ungerleider and Andrysiak, 1985; Gong et al., 1984). Recently, the use of A9-
THC as an appetite stimulant has been indicated in patients with cachexia or
wasting disease observed in AIDS victims (Gorter, 1999; Mechoulam, 1999;
Nelson et al., 1994). These studies indicate that low doses of oral A9-THC are
sufficient to produce appetite stimulation and anti-emesis in the absence of
significant psychotropic effects. Altogether, the current findings support the
therapeutic use of Ag-THC. Furthermore, California, Arizona, Alaska, Nevada,
Oregon, and Washington have recently legalized the physician-prescribed
medicinal use of marijuana, which has renewed interest in the potential
therapeutic uses of this drug (Dow and Meyers, 1981). However, the therapeutic
effects of Ag-THC have been accompanied by significant side effects associated
with CNS activity. Therefore, the development of synthetic compounds that are

therapeutic and lack undesirable side effect is important.

l.4.B.Cannabinoid receptors: CB1 and 632

Due to the lipophilic nature of cannabinoids, nonspecific intercalation and
disruption of the plasma membrane has historically represented a putative
mechanism to explain the actions of cannabinoid compounds. The
pharmacological evidence for the possible existence of cannabinoid receptors

33

emerged slowly over last two decades. Binding studies performed In neuronal
tissue demonstrated specific and saturable binding by cannabinoid compounds
(Harris et al., 1978). In addition, stereoselective differences in biologic activity
were observed among cannabinoid enantiomer pairs although the degree of
lipophilicity was equivalent between (+) and (-) isomers (Thomas et al., 1990).
Furthermore, it was demonstrated that cannabinoids were able to inhibit
adenylate cyclase activity and cAMP formation (Felder et al., 1992; Kaminski et
al., 1992; Melck et al., 1999). This was a significant finding considering the
specific association of adenylate cyclase and G-protein coupled receptors. All
these observations were ultimately supported by the isolation and cloning of
cannabinoid receptors, CB1 and CB2. CB1 was cloned in 1990 from a rat brain
cDNA library (Matsuda et al., 1990). The sequence of the protein indicates that it
differs from other neurotransmitter receptors and a high affinity ligand was shown
to be Ag-THC. More than 90% of the amino acid sequence of the receptor is
identical among human, rat, and mouse CB1. However, there appearto be some
species differences in the chromosomal localization of the CB1 receptor gene
(Pertwee, 1998). The gene for the bovine CB1 receptor has been localized to
chromosome 9, whereas the murine gene is found on chromosome 4. The
second cannabinoid receptor, CB2, was cloned from a human cDNA library, and
it shared 44% identity to CB1 (Munro et al., 1993). Despite significant differences
in the amino acid sequence between the two forms of cannabinoid receptors,
both receptors have amino acids characteristic of a G-protein-coupled receptor

(Mallet and Beninger, 1998; Lay et al., 2000; Glass and Northup, 1999) (Felder

34

and Glass, 1998; Klein et al., 1998). Besides, most natural and synthetic
cannabinoid receptor ligands, including Ag-THC, exhibit similar binding affinity for
CB2 and CB1, with some exceptions such as cannabinol, a plant-derived
cannabinoid, which exhibits greater binding affinity for CBZ (Munro et al., 1993).
No additional receptor subtypes have been identified to date.

The tissue and cell-type distribution of CB1 and CB2 has not yet been
comprehensively characterized. CB1 receptors are primarily detected in the brain
which correlates well with the known effects of cannabinoids on memory,
perception, and the control of movement (Howlett, 1998). Initially it was simply
thought that the CB1 receptor was localized in the CNS, whereas the C82
receptor was localized peripherally; however, it is gradually emerging that the
distinction is not so simplistic. In addition to the brain, the CB1 receptor has also
been suggested to be present peripherally in guinea pig small intestine, testis,
the mouse urinary bladder and vas deferens, vascular smooth muscle cells, and
pre-synaptically on sympathetic nerve terminals (see review (Felder and Glass,
1998)). RT-PCR analysis has detected mRNA for CB1 receptor in spleen cells,
adrenal gland, heart, lung, prostate, bone marrow, thymus, tonsils and uterus
(Felder et al., 1995; Das et al., 1995). CB2 receptors are found in the spleen and
thymus, in immune cells (B-cells, monocytes, T-cells, etc) and possibly in primary
cultures of rat microglia (Schatz et al., 1997; Bouaboula et al., 1993; Munro et
al., 1993; Kaminski etal., 1992; Gerard et al., 1991;Gerard et al., 1990).

Recently developed cannabinoid receptor antagonists have allowed

investigators to examine the physiological role of the cannabinoid system by

35

blocking receptor function with the application of a specific antagonist. The CB1
receptor antagonists, epitomized by the compound SR141716A (developed by
the French company Sanofi), first became available in 1994 (Rinaldi et al., 1994).
A CB2 receptor selective antagonist, SR144528 has also been reported recently
(Rinaldi et al., 1998; Barth and Rinaldi, 1999). A number of studies have used
these specific antagonists in the past few years, especially the CB1 antagonist
(Mallet and Beninger, 1998; Lay et al., 2000; Coutts et al., 2000; Beardsley and
Martin, 2000). It was demonstrated that appetite stimulation, one of the C81-
mediated effects, could be blocked by treatment with SR141716A (Colombo et
al., 1998; Chaperon and Thiebot, 1999). Studies with SR141716A in
cannabinoid-induced hypotension and bradycardia also showed the antagonism.
Additionally, SR141716A completely blocked the pain-relieving effects of AQ-THC
in various animal models of pain, while treating solely with SR141716A showed
no effect on the baseline sensitivity to pain stimuli in these animal studies. This
finding is consistent with the studies using recently-developed CB1‘" mice

(Reibaud et al., 1999; Mascia et al., 1999).

I.4.C.lntracellular signal transduction

Many of the intracellular effects mediated by C81 and 082 receptors can
be explained by their ability to inhibit adenylate cyclase via pertussis toxin—
sensitive Gi-proteins. Cannabinoid ligands bind to G,—protein-coupled receptors
CB1 or C82, and the binding signal might be further transmitted from the cell

surface to the nucleus through the cAMP signaling pathway (Condie et al., 1996;

36

Schartz et al., 1992; Kaminski et al., 1994; Felder et al., 1995). This molecular
mechanism has been suggested in neuroblastoma cells, astrocytoma cells,
cultured neurons, brain tissue cells, splenocytes, peripheral blood mononuclear
cells, uterine membranes, and heterologous hosts expressing cloned CB1 or
C82 (Felder et al., 1995; Welch et al., 1998; Felder and Glass, 1998; Lu et al.,
2000; Jarai et al., 1999; Ho et al., 1999). However, as might be expected with
their low sequence homology, 081 and C82 receptors also vary considerably in
their coupling to signal transduction pathways. It has been recently shown that
CB1 but not CBZ receptors are able to couple positively to adenylate cyclase in
astrocytes (Felder et al., 1998).

Cannabinoid agonists are also able to modulate the release of arachidonic
acid (AA) and the inhibition of AA re-acylation. The mechanism of the
cannabinoid agonist—mediated production of AA and eicosanoids has been
somewhat controversial. Recently it has been shown that Ag-THC and
endogenous cannabinoid anandamide stimulated AA in cortical astrocytes and
that this could be blocked by SR141716A (Shivachar et al., 1996). However, the
CB1 receptor expression in astrocytes has not been clearly demonstrated.
Antisense oligonucleotide probes forthe CB1 and C82 receptors have also been
shown to block AA release (Hunter and Burstein, 1997), thereby providing
additional evidence that in some cells, CB1 may mediate eicosanoid production.
It is unclear whether these effects are of physiological significance in the
response to cannabinoids in vivo. Moreover, cannabinoid receptor ligands have

been shown to activate the mitogen-activated protein (MAP) kinases (Bouaboula

37

et al., 1996). This activation is believed to occur via G-proteins, but perhaps not
via a CAMP—dependent mechanism. MAP-kinase activation may be an
intermediate step in the cannabinoid receptor-mediated induction of certain
transcription factors (Glass and Dragunow, 1995).

The calcium-dependent signaling also plays a role In cannbinoid-mediated
cellular effects. Results from numerous pharmacological studies indicated that
Ag-THC produced many of their CNS effects by depressing calcium channel
activity (Twitchell et al., 1997; Pan et al., 1996). For example, cannabinoids
reduced synaptically-evoked [Cap-“ji signals (Shen and Thayer, 1998) and
inhibited presynaptic glutamate release through actions on voltage-dependent N-
type calcium channels (Levenes et al., 1998). The modulation of [Ca2+]i by
cannabinoids possibly involves cannabinoid receptors. Studies in neuroblastoma
cells showed that cannabinoid compounds inhibited a w-conotoxin-sensitive
high-voltage-activated calcium channel, an effect that was blocked by the
administration of pertussis toxin and independent of the formation of cAMP
(Caulfield and Brown, 1992). The inhibition of N-type calcium channels may
account for some of the decreases of neurotransmitter release from specific
neuronal cells. In addition, the CB1 receptor also seems to modulate the activity
of Q-type calcium channels. When expressed in mouse ArT20 cells (Vogel et al.,
1993), the CB1 receptor, but not the cloned C82, modulated Q-type calcium
channel activity when exposed to either WIN 55,212-2 (100nM) or anandamide
(300nM) (Felder et al., 1995). It was initially believed that L—type calcium

channels were insensitive to cannabinoid compounds. However, a more recent

38

study by Gebremedhin et al. indicated that the synthetic cannabinoid,
WIN55,212-2 (10-100nM), induced a concentration-dependent inhibition of peak
L-type calcium current in cat cerebral cells (Gebremedhin et al., 1999). This
effect was further abolished by pretreatment with C81 antagonist, SR141716A.
The enhancement in [C321, by treatment with cannabinoid compounds
has been reported infrequently compared to the inhibitory effects of these
compounds. In neurooblastoma x glioma hybrid NG108-15 cells and N18TGZ
cells, CB1 receptor mediated an induction of a rapid increase in [Ca2*]i by
treatment of Ag-THC or the endogenous cannabinoid ligand 2-
arachidonylglycerol (2-AG). The increase in [Ca”], was abolished by
pretreatment of the cells with SR141716A (Sugiura et al., 1997). Furthermore,
several studies suggested that A9-THC altered [Ca2+], by the formation of IP3. IP3
formation has been shown to enhance the release of calcium from ER, a major
organelle for buffering of {Caz}. It has been demonstrated that WIN55,212-2 and
HU-210 enhanced NMDA-evoked calcium signals, which resulted in calcium
release from intracellular IFS-sensitive calcium stores. This effect was blocked by
pretreatment of SR—141716A. Similarly, in smooth muscle cells, Ag-THC
treatment caused calcium mobilization from IP3-sensitive intracellular Ca2+ stores
through a thapsigargin-sensitive channel (Filipeanu et al., 1997). This calcium
release from internal stores following Ag-THC treatment was blocked by
SR141716A. Calcium influx from extracellular matrix was also observed in their
studies; however, it appeared to be CB1-independent. Because Ca2+ is an
important intracellular second messenger, cannabinoid effects on [Ca2*]i could

39

have important implications for the functions of a variety of cell types or systems.

l.4.D.lmmune modulation by A°-THC

The immunosuppressive effect is a well-established characteristic of
cannabinoid compounds, as shown by their ability to inhibit lymphocyte
proliferation, antibody production, macrophage action, natural killer cell activity
and cytokine secretion. Although cannabinoids seem to alter the functional
competence of nearly every immune cell type, T-cells may represent one of the
most sensitive immunological targets for this group of compounds. In light of this
aspect, the study of immunosuppression by A9-THC has predominantly focused
on T-cell functions and T-cell dependent immune response.

T-cells play a critical role in cell-mediated immunity and are responsible
for antitumoricidal and antiviral activities. A number of studies have reported
cannabinoid-mediated inhibition of T-cell function as assessed by a variety of
functional endpoints. T-cell blastogenesis is a mechanism important for clonal
expansion of reactive T-cells during an immune response. This response was
significantly decreased by cannabinoids under in vitro conditions. Pross and
coworkers have demonstrated that both AQ-THC and its metabolite, 11-OH-A9-
THC, markedly inhibited mouse splenic and thymic T-cell blastogenic responses
(Pross et al., 1987). Similarly, the sRBC lgM antibody forming cell (AFC)
response, which requires both accessory T-cells and antigen-presenting cells,
was inhibited by Ag-THC (Schatz et al., 1993). Conversely, AFC responses to

DNP-ficoll or the polyclonal B-cell activator-LPS, which do not involve T-cells,

40

were unaffected. In addition, it has been shown that the greatest magnitude of
suppression by Ag-THC was achieved when it was administered at times around
antigen sensitization (Schartz et al., 1993). This observation suggested that
cannabinoid-induced immuno-suppression was mediated either through the
inhibition of early an T-cell activation event or by interference with antigen
presentation. However, T-cell proliferation to immobilized anti-CDS+ does not
require antigen-presenting cells, yet is inhibited by Ag-THC (Schartz et al., 1993).
These results suggested a direct inhibitory effect on T-cells by treatment with A9-
THC.

Several recent studies indicated that A9-THC suppressed cellular immunity
by inhibiting TH1-cell activity, including cytokine production. It has been reported
that lL-2 transcription Is altered by cannabinoid compounds as detected by
significant decreases in IL-2 mRNA and protein (Condie et al., 1996).
Splenocytes stimulated with PHA, LPS, or Con A produced significantly less IF N-
y in the presence of AQ-THC than untreated controls (Blanchard et al., 1986). In
separate studies, mice were treated with either AQ-THC or the vehicle
dimethylsulfoxide (DMSO) and infected one day later with an intracellular
pathogen, Iegionella pneumophila (Newton et al., 1994). The splenocytes from
these mice were then stimulated in culture with mitogen for 24 hr, and the
resulting supernatant was assayed for IFN-y protein. The results showed that the

splenocytes treated with Ag-THC were deficient in IFN-y production when

compared to the control. Suppression of serum lFN-y levels in rodents has also

41

been observed following Ag-THC injection (Cabral et al., 1986b; Cabral et al.,
1986a). Additionally, A9-THC has been shown to decrease the responsiveness of
T-cells to lL-2 (Kawakami et al., 1988). Furthermore, Ag-THC treatment of
splenocyte cultures and stimulation with various antigen preparations
suppressed cytokines that are associated with cell-mediated immune response
such as |L—12, lL-15 and lFN-y (Klein et al., 1998; Zheng et al., 1992). Based on
the current findings, it appears that cannabinoid treatments are able to direct the
cytokine network away from TH 1-cell mediated immunity.

The CAMP signaling cascade is involved in the alterations of the
expression of immmunoregulatory genes, cell cycle arrest, and
immunosuppression by cannabinoid treatments. Once cAMP is produced, it
binds to the regulatory subunits of CAMP dependent kinase (PKA) and facilitates
the release of activated catalytic subunits which phosphorylate numerous target
immune-regulatory proteins leading to suppression of the immune system.
Previous reports have established that cannabinoid treatment suppresses the
forskolin-induced increases in cAMP (Jeon et al., 1996; Little and Martin, 1991).
The suppression of cAMP has been linked to the cannabinoid-induced decrease
in lL-2 production (Condie et al., 1996) and nitric oxide production (Jeon et al.,
1996). In an in vitro system, membrane permeable CAMP analogues have been
shown to reverse certain inhibitory effects produced by cannabinoids on
leukocyte functions (Kaminski et al., 1994). Likewise, pertussis toxin blocked the
inhibitory responses induced by cannabinoids on certain immune responses

(Kaminski et al., 1994).

42

Taken together, considerable evidence exists for the immune inhibitory
effects of Ag-THC. Those findings indicate that T-cells appear to be particularly
sensitive to inhibition by A9-THC. Therefore there is a great potential that AQ-THC
may suppress the cell-mediated immune response in specific immune-based
diseases and produce a protective effect. The studies conducted on the CNS
autoimmune disorder-experimental allergic encephalomyelitis (EAE) have
demonstrated that AQ-THC and several other cannabinoid compounds markedly
decreased the incidence and severity of this disease in an autoimmune
experimental model of EAE (Wirguin et al., 1994). However, there is relatively
limited information in the cannabinoid literature concerning the potential

protective effect of these compounds on immune-mediated disorders.

I.5.Summary

In summary, STZ produces a direct cytotoxic effect on pancreatic beta-
cells. When STZ is administered in multiple low doses, it augments a TH-1 cell-
associated immune response directed towards beta-cells in genetically
susceptible animals. Therefore, the model of MLDSTZ-induced diabetes has
been used to study the mechanism for immune-mediated diabetes and search
for potential improved treatment.

Early attempts on the treatment of immune-mediated diabetes
indiscriminately inhibited cell proliferation [imuran(Carter et al., 1993);
cyclosporine A (Iwakiri et al., 1986)], often leading to serious side effects such as

pancreatic beta-cell cytotoxicity (Kolb et al., 1985; Sestier et al., 1985; Iwakiri et

43

al., 1986). Consequently, extensive efforts have focused on the compounds that
are capable of inhibiting the immune system, but do not produce cytotoxic effects
on insulin-producing cells. One of those compounds currently under study is A9-
THC. AQ-THC inhibits nearly every aspect of immune function, especially T-cell-
associated immune response. The immunosuppressive effect of Ag-THC
presents a potential to inhibit the immune process against pancreatic beta-cells
in the MLDSTZ-induced diabetes, and attenuates the diabetes development.
Furthermore, Ag-THC produces a direct stimulatory effect of insulin release from
endocrine insulin-producing cells, which possibly decrease MLDSTZ-induced
hyperglycemia and contribute to the protective effect of A9-THC in that model.
The overall hypothesis of the present study is that A9-THC treatment attenuates
the development of MLDSTZ-induced diabetes. The studies to test this

hypothesis are described in detail in the following chapters.

44

CHAPTER II
EXAMINATION OF THE IMMUNOSUPPRESSIVE EFFECT OF A°-THC IN

MLDSTZ-INDUCED DIABETES MODEL

45

ll.1.Summary

Ag-Tetrahydrocannabinol (Ag-THC) is capable of modulating a variety of immune
responses, but has not been evaluated in models of immune-based diabetes.
The objectives of the present study were: (a) to investigate the effect of A9-THC
in an established model of multiple low dose streptozotocin (MLDSTZ)-induced
autoimmune diabetes; and (b) to determine the contribution of the immune
response in the MLDSTZ model. CD—1 mice were treated with 40 mg/kg STZ for
5 days in the presence or absence of A9-THC treatment. A9-THC administered
orally in corn oil at 150 mg/kg for 11 days attenuated, in a transient manner, the
MLDSTZ-induced elevation in serum glucose and loss of pancreatic insulin.
MLDSTZ-induced insulitis and increases in IFN-y, TNF-cx and |L-12 mRNA
expression were all reduced on Day 11 by co-administration of A9-THC. In
separate studies, six doses of A9-THC given after completion of STZ treatment
was found equally effective in attenuating MLDSTZ-induced diabetes. Studies
performed using B6CSF1 mice showed moderate hyperglycemia and a
significant reduction in pancreatic insulin by MLDSTZ in the absence of insulitis.
In addition, MLDSTZ produced a less pronounced hyperglycemia compared to
CD-1 mice that was not attenuated by A9-THC. These results suggest that
MLDSTZ can initiate direct beta-cell damage thereby augmenting the destruction
of beta-cells by the immune system. Moreover, these results indicate that A9-
THC is capable of attenuating the severity of the autoimmune response in this

experimental model of autoimmune diabetes.

46

ll.2.lntroduction

Over three decades of investigations have established that plant-derived
cannabinoids, including A9-THC, are capable of modulating a wide variety of
immune responses including innate, humoral and cell-mediated (Kaminski, 1996;
Klein et al., 1998). One critical, although not exclusive, property that may
significantly contribute to the immunomodulatory activity of cannabinoids is their
ability to inhibit T-lymphocyte activation and subsequently the expression of T-
lymphocyte derived cytokines including IL-2 and IFN-y (Schatz et al., 1992;
Condie et al., 1996; Klein et al., 1998). In addition to T-Iymphocyte derived
cytokines, cannabinoids also inhibit a number of pro-inflammatory cytokines
including TNF-a, lL-1, and lL-6 as well as lL-12, a macrophage-derived TH1
promoting factor, all of which likely contribute to a general decrease in immune
competence associated with A9-THC administration (Srivastava et al., 1998;
Zheng et al., 1992; Fischer-Stenger et al., 1993; Newton et al., 1998).

In light of the immunosuppressive effects of cannabinoid compounds, the
present study investigated the ability of A9-THC to repress the onset of
autoimmune diabetes induced by administration of multiple low doses of
streptozotocin (MLDSTZ). MLDSTZ-induced diabetes has been used as a
common model of Type I diabetes for studying autoimmune processes
associated with pancreatic beta-cell pathogenesis and potential therapeutic
interventions (Herold et al., 1996; Like and Rossini, 1976). STZ when
administered at high doses (200 mg/kg) produces rapid destruction of insulin-

47

producing beta-cells by direct cytotoxic action resulting in permanent
hyperglycemia (Yamamoto et al., 1981). In contrast, Like and Rossini using male
CD-1 mice showed that when injected as five 40 mg/kg daily doses, STZ induced
a gradual onset of hyperglycemia with lymphocytic infiltration into the pancreatic
islets (insulitis). Using this modified dosing regimen, insulitis was observed 5~6
days after the last STZ injection (Like and Rossini, 1976). There are two potential
components leading to beta-cell damages in this model of chemical-initiated
autoimmune disease. One is the direct cytotoxic action of STZ and the other is
the immune-based response resulting in the loss of insulin-producing beta-cell
function. The direct cytotoxic effect of STZ can be attributed to its ability to
alkylate critical cell components (Yamamoto et al., 1981), and the immune-based
response has been shown to involve activation of T-lymphocytes (Vallera et al.,
1992 and Herold et al., 1996). However, the relative contributions of the direct
toxic effect of STZ and the immune-based component in the MLDSTZ model
have not been clarified. The objectives of the present studies were two-fold: (a)
to determine whether the immunomodulatory activity of A9-THC would be
protective in an established model of chemical-initiated autoimmune disease,
MLDSTZ-induced diabetes; and (b) to further characterize the contribution of the
immune response versus STZ direct cytotoxicity in the onset of diabetes after

MLDSTZ treatment.

II.3.Materials and methods

ll.3.A.Chemicals

48

All reagents were purchased from Sigma Chemical Co. (St. Louis, MO)
unless stated otherwise. The reagents used for RT-PCR were of molecular
biology grade and were purchased from Promega Co. (Madison, WI) unless
othenNise indicated. Ag-THC was provided by the National Institute on Drug

Abuse National Institute of Health, Rockville, MD).

ll.3.B.Animals

The studies have been performed according to the Guide for the Care and
Use of laboratory Animals as adopted by the National Institute of Health. Virus
antibody free, 8 to 9 week old, male CD-1 mice or 86C3F1 mice were obtained
from Charles River Laboratory (Dortage, Ml), one week before use. On arrival,
mice were transferred to standard filter topped cages containing sawdust
bedding. Five mice per cage were housed in a pathogen-free facility at 21-24°C
constant temperature, 40~60% relative humidity and in a 12 hour light/dark
cycle. Animals were allowed access to standard laboratory animal chow (Purina

Certified Laboratory Chow) and water ad Iibitum.

ll.3.C.Treatment

Figure ”-1 illustrates, schematically, the Ag-THC and STZ treatment
regimens. The dosing regimen for STZ consisted of 5 consecutive daily 40 mg/kg
doses administered i.p., as reported previously in CD-1 mice (Like and Rossini,
1976). This dosing regimen was confirmed in preliminary studies to induce

diabetes with peak insulitis occurring on Day 11. For comparative purposes,

49

mm x'
11dTHC TTTTT

T Tlli
“THC TTTTTT

Figure "-1. Scheme of the STZ, 11 day Ag-THC (11dTHC) and 6 day A9-THC
(6dTHC) treatment regimens employed in the experiments. Arrows indicate a

single daily dose.

50

similar studies were conducted in 86C3F1 mice, a mouse strain to our
knowledge not previously used for studies of STZ-induced diabetes. The
86C3F1 mouse strain has been commonly used in toxicology studies and was
therefore employed in the present study to provide additional txicology-related
information concerning genetic bases for susceptibility and resistance to STZ.
Each treatment group in an experiment contained five non-fasted mice. A naive
(NA) control group that received no treatment was utilized as a control for the
vehicle treatment group. The vehicle (VH) group received 0.2 ml of 0.1M, pH 4.5
citrate buffer (vehicle for STZ), i.p., for 5 consecutive days (Days 1-5) and 0.2 ml
of corn oil (vehicle for Ag-THC) by oral gavage for the same duration as Ag-THC
treatment. Two different AQ-THC treatment regimens were employed in these
studies. The Ag-THC treatment regimen utilized predominantly in these
experiments consisted of 11 consecutive daily doses of 150 mg/kg spanning
Days 1-11 (11dTHC). For several studies, a modified AQ-THC treatment regimen
was utilized which consisted of 6 consecutive daily doses of 150 mg/kg spanning
Days 6-11 (6dTHC). Lastly, a STZ + AQ-THC co-administration treatment group
was employed in which mice received STZ on Days 1-5 and Ag-THC on Days 1-
11 (STZ + 11dTHC) or STZ on Days 1-5 and Ag-THC on Days 6-11
(STZ+6dTHC). Preliminary dose-response experiments demonstrated that oral
administration of AQ-THC at 150 mg/kg/day produced a marked attenuation of the
STZ-induced hyperglycemia in the absence of any overt signs of CNS

depression. It is important to emphasize that although oral administration of A9-

51

THC solubilized in corn oil results in poor absorption of the drug, it represented
the least stressful route of administration since repeated dosing was required.

All mice were sacrificed by cervical dislocation at the end of the
experiment. Blood and tissue samples were collected without fasting the mice.
Blood samples were obtained directly from the heart. A peroxidase colorimetric
method was used to determine the serum glucose concentration of blood
samples (Sigma Diagnostic, St. Louis, MO). The pancreas was isolated and
divided into three portions. The duodenal portion of the pancreatic tissue was
used for determination of pancreatic immunoreactive insulin content using
radioimmunoassay (RIA) as previously described (Miller et al., 1990). The
splenic portion and the middle portion of the pancreas were used for total RNA
isolation and histopathological examination, respectively. Mice with serum blood
glucose concentration above 250 mg/dL were considered diabetic and above
400 mg/dL were considered as severely diabetic. Each experiment was repeated

on at least three different occasions.

ll.3.D.Quantitative competitive RT-PCR analysis.

The splenic portion of the pancreas was snap frozen in liquid nitrogen,
and then homogenized in 5 ml Trizol reagent (Life Technologies, Grand Island,
NY) per 100 mg tissue for isolation of total RNA according to the manufacturers’
directions. Quantitative RT-PCR was performed as outlined in Gilliland et al.
(Gilliland et al., 1990), except that a recombinant RNA (rcRNA) was used as an

internal standard (IS) instead of genomic DNA. Internal standards were

52

generated as previously described (Vanden Heuvel et al., 1993) and contain
specific PCR primer sequences for lFN-y, lL-12, or TNF-a and a spacer gene of
rat B-globin. All isolated RNA samples were treated with RNAse-free DNAse to
eliminate any potential genomic DNA contamination prior to RT-PCR. Lack of
DNA contamination was confirmed by the absence of target gene product
following PCR amplification in the absence of reverse transcriptase (GIBCO
BRL, Grand Island, NY). An internal standard (IS) was generated as a competitor
RNA with a rat B-globulin sequence as its spacer gene. This method avoids
sample to sample variation of reference gene expression, as well as gene to
gene differences in amplification efficiency. Primer sequences for lFN-y are:
forward primer GGATATCTGGAGGAACTGGC, reverse primer, GAG
CTCATTGAATGC'ITGGC; for IFN-y IS: froward primer, TAATACGAC

TCACTATAGGGGATATCTGGAGGAACTGGCAAGCCTGATGCTGTAGAGCC.

 

reverse primer, I II III I III III I I I I IGAGCTCATTGAATGCTTGGCAACCT
GGATACCAACCTGCC; for lL-12: forward primer, CAGTACACCTGCCACAA
AGGA, reverse primer, GTGTGACCTTCTCTGCAG ACA; for IL-12 IS: fonNard
primer, TAATACGACTCACTATAGGCAGTACACCTGCCAAAGGAAAGCCT G A
TGCTGTAGAGCC, reverse primer, TT'I‘I'TI'TTFITITITITTGTGTGACCTTCT
CTGCAGACAAACCTGGATACC AACCTGCC; for TNF-oc are: fonNard primer,
TCTCATCAGTTCTATGGCCC, reverse primer, GGGAGTAGACACAAGGTAC
AAC; for TNF-a IS: forward primer, TAATACGACTCACTAAGGTCTCAT

CAGTTCTATGGCCCAAGCCTGATGCTGTAGAGCC, reverse primer, l l l l I l l

53

 

II I I I I I I I I IGGGAGTAGACACAAGGTACAACAACCTGGATACCAACCTGC

C; for IL-4 are: forward primer, AACGAGGTCACAGGAGAAG, reverse primer,
GCTTATCGATGAATC CAGGC; primer sequences for lL-2 have been reported
previously by Condie et al. (Condie et al., 1996). Briefly, 100 ng (for TNF-a) or
400 ng (for lL-2, IL-4, |L-12 and lFN-y) of pancreatic total RNA were used for RT-
PCR analysis. Samples were cycled 35 times; each cycle consisted of 94°C for
15 sec, 59°C for 30 sec and 72°C for 45 sec. PCR products were visualized by
ethidium bromide staining and quantified by assessing the absorbency using a
Gel-doc 1000 video imaging system (BioRad, Hercules, CA). The amount of
target cytokine mRNA present in the samples was determined according to the

method described by Gilliland et al. (Gilliland et al., 1990).

ll.3.E.Histopathological examination.

The middle portion of the pancreatic tissue from each mouse was
immediately immersed in 10% neutral-buffered formalin and reserved for
histopathological examination. The fixed tissue samples were embedded in
paraffin and cut into 3~5 pm sections. Sections of pancreatic tissue stained with
haematoxylin and eosin (H&E) were graded without knowledge of the identity of
the samples. Each pancreas was graded and placed into one of the following
categories: none (no inflammation, normal islet architecture); moderate-insulitis
(lymphocytic infiltration surrounding but little infiltration into the islets); severe
insulitis (massive lymphocytic infiltration into the islets with islet destruction).

Sections of pancreatic tissue were also examined by immunohistocytochemistry

54

and light microscopy for the presence of CD3 cells using an anti-C03 antibody
(DATO, Glotrup, Denmark). "Images in this thesis/dissertation are
presented in color."
ll.3.F.Statistical analysis
All data are reported as mean values i SE. Statistical analysis was
performed by a parametric analysis of variance. Tukey's test was used to

compare the treatment groups and p<0.05 was defined as statistical significance.

II.4.Results

ll.4.A.Effects of 11 day A°-THC treatment on MLDSTZ-induced
hyperglycemia and loss of pancreatic insulin in CD-1 mice

STZ-treated CD-1 mice demonstrated typical diabetic symptoms such as
polydipsia, polyuria, and positive urine glucose [>250 mg/dL when tested by
Uristix strips (Bayer Co., Elkhart, IN)] starting on Day 9 (data not shown). The
serum glucose (Fig.ll-2) and pancreatic insulin (Fig.ll-3) for the CD-1 mice in
each of the different treatment groups was determined on Day 11 unless
indicated otherwise. Serum glucose in the STZ (40 mg/kg/day) treatment group
was observed to be in excess of 400 mg/dL, indicating severe diabetes. Mice
receiving STZ in combination with Ag-THC (150 mg/kg/day) given on Day 1
through Day 11 exhibited significantly lower blood glucose concentrations than
the STZ treatment group, i.e. 312.1:156 mg/dL (STZ+11dTHC) versus
447.0i9.7 mg/dL (STZ). No severe hyperglycemia or positive urine glucose was
observed in the STZ+11dTHC treatment group. However, compared to the VH

55

FIGURE "-2. Effect of Ag-THC treatment on MLDSTZ-induced elevation of
serum glucose in CD-1 mice and BSC3F1 mice. Untreated naive (NA)
mice were utilized as a control for the vehicle (VH) treatment group. The
streptozotocin (STZ) treatment group received 5 consecutive 40mg/kg i.p.
injections on Days 1-5. The 11dTHC treatment group received 11
consecutive doses of Ag-THC (150 mg/kg) by oral gavage on Days 1-11.
The STZ+11dTHC treatment group received 5 consecutive STZ
(40mg/kg) i.p. injections on Days 1-5 and 11 consecutive doses of Ag-THC
(150 mg/kg) by oral gavage on Days 1-11. Serum glucose was measured
on Day 11 and Day 17 (see insert). The results are expressed as the
mean i SE, n=15. A significant difference of p<0.05 is indicated where “a”
denotes mean values that are different compared to the VH group for CD-
1 mice and “b” denotes mean values that are different compared to the
STZ group for CD-1 mice; for B6C3F1 mice, “a' ” denotes mean values

that are different compared to the VH group.

56

 

 

 

 

 

 

(1p/6w)asoonle POOIB

STZ STZ

11dTHC

VH

11dTHC

Treatment

FIGURE 3. Effect of Ag-THC treatment on pancreatic insulin in MLDSTZ-
induced diabetes in CD-1 mice and BGC3F1 mice. The treatments were
performed as described in Figure 2 legend. Pancreatic insulin was
measured on the samples taken on Day 11. The results are expressed as
the mean t SE, N=15. A significant difference of p<0.05 is indicated
where “a” denotes mean values that are different compared to the VH
group for CD-1 mice and “b” denotes mean values that are different
compared to the STZ group for CD-1 mice; for 86C3F1 mice, “a' ”denotes

mean values that are different compared to the VH group.

58

 
   
  

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Treatment

11dTHC

group (192.0i27.2 mg/dL), STZ+11dTHC mice attained a mild hyperglycemic
state with serum glucose concentrations above 250 mg/dl. No significant
difference in serum glucose was observed among the NA, VH and 11dTHC
treatment groups. Additional measurements were obtained on Day 17, 6 days
after Ag-THC administration was terminated on Day 11, to determine whether the
protective effect of Ag-THC was persistent (data shown in Fig.ll-2 insert). The
results indicated that serum glucose, in contrast to results obtained on Day 11
was severely elevated on Day 17 in the STZ+11dTHC treatment group and
equal to that in the STZ given alone treatment group.

Pancreatic insulin in MLDSTZ-treated and control animals measured on
Day 11 indicated that the treatment alone lowered pancreatic insulin content to
less than 25% of control. STZ+11dTHC treated mice exhibited higher pancreatic
insulin content than mice in the STZ only treatment group, i.e. 95.3:154 ng/mg
pancreatic tissue (STZ+11dTHC) versus 35.1:85 ng/mg pancreas tissue (STZ)
(Fig.ll-3). No significant difference in pancreatic insulin was observed among the

NA, VH and 11dTHC treatment groups.

|I.4.B.Effects of 11 day A°-THC treatment on MLDSTZ-induced
hyperglycemia and loss of pancreatic insulin in B6C3F1 mice

Identical studies to those described above for CD-1 mice were performed
using B6C3F1 mice. Mice in the STZ treatment group did not show signs of

polydipsia, polyuria, and positive urine glucose on Day 11 (data not shown), but

60

were mildly hyperglycemic when compared to the VH group (Fig.ll-2). Mice in the
STZ+11dTHC treatment group showed no statistically significant differences in
serum glucose from mice in STZ group. Similar results were observed when
pancreatic insulin content was used as an index of STZ-induced destruction of
pancreatic beta-cells (Fig.lI-3). Pancreatic insulin in the STZ group was
decreased from vehicle control, i.e. 49.5i12.1 ng/mg pancreatic tissue (STZ)
versus 189.4:10] ng/mg pancreatic tissue (VH). Mice in the STZ+11dTHC
treatment group showed no statistically significant differences in insulin content

from mice in the STZ treatment group.

ll.4.C.Effects of A’-THC treatment on MLDSTZ-induced insulitis in CD-
1 and BGC3F1 mice

Histopathological evaluation of pancreatic tissue was performed to
examine the development of insulitis. The evaluation of tissues from CD-1 mice
(Fig. “-4) demonstrated that the pancreatic islets of mice in the NA, VH, and
11dTHC-treatment groups were normal, i.e. few mononuclear cells present in the
islets. In addition, Ag-THC treatment significantly attenuated the insulitis induced
by MLDSTZ as evidenced by significantly fewer numbers of infiltrating
lymphocytic cells. Lymphocytic cells, when present were primarily localized
around the periphery of the islets as a result of the MLDSTZ + 11THC treatment.
Compared to the mice in the STZ treatment group, disruption of islet
cytoarchitecture and the presence of occasional necrotic cells within the islets

were less pronounced in the STZ+11dTHC treatment group. Morphologically, the

61

FIGURE "-4. Representative photomicrographs of islets in pancreatic
tissue obtained on Day 11: (A) VH/CD-1 mice; (B)11dTHC/CD-1mice; (C)
STZ/CD-1 mice; (D) STZ+11dTHC/CD-1 mice; (E) VH/BGC3F1 mice; (F)
STZ/BBC3F1 mice. For histopathology examination, pancreatic tissue was
placed in 10% buffered formalin at sacrifice, and tissue sections were

stained with H&E. (CD-1 mice: 100X ; 86C3F1 mice: 240x ).

62

 

63

STZ+6dTHC treatment group was similar to the STZ+11dTHC treatment group,
i.e. a less pronounced degree of insulitis as compared to the STZ treatment
group (data not shown). A semi-quantitative evaluation of the histopathology
results is provided in Table 1. It indicates that the STZ+11dTHC group showed a
moderate degree of insulitis as compared to the STZ group. Histopathology of
tissues from BBC3F1 mice in contrast to results from CD-1 mice indicated that
MLDSTZ treatment showed moderate to no insulitis when compared to vehicle
controls (Fig.ll-4). Due to this observation, further studies in which cytokine
levels in MLDSTZ-treated animals were measured employed CD-1 mice that
showed severe insulitis on Day 11. The results in Figure ”-5 illustrate that the
majority of inflammatory cells infiltrating the pancreatic islets are T-lymphocytes
as measured by immunohistochemical staining. Pancreatic tissue sections were
stained with anti-CD3+ antibody. The islets from the VH control group showed no
CD3+ positive cells. Conversely, the pancreatic islets from STZ-treated mice
showed marked infiltration of inflammatory cells, the majority of which were CD3+
positive cells. Compared to the STZ group, the STZ+11dTHC treatment group
exhibited markedly fewer CD3" positive cells within the islets, although some

were observed on the periphery of islets.

ll.4.D.Effects of 11 day A°-THC treatment on MLDSTZ-induced
changes in mRNA expression of selected cytokines in CD-1 mice
To determine the profile of cytokines produced in pancreatic tissue during

MLDSTZ-induced insulitis, RT-PCR was employed to evaluate total pancreatic

64

FIGURE "-5. Representative photomicrographs of immuno-histochemical
staining of islets in pancreatic tissue obtained from CD-1 mice on Day 11,
100x. Pancreatic tissue was fixed with 10% buffered formalin. Tissue
sections shown in (A)VH, (C)STZ, and (D)STZ+11dTHC were incubated
with anti-CD3” and tissue section shown in (B)STZ was incubated with

control serum.

65

 

 

 

66

 

 

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fol

TABLEI
Histopathological evaluation of the protective effect of Ag-THC on MLDSTZ-

induced insulitis in CD-1 mice. (Values indicate numbers of mice)

 

 

TREATMENT GROUP
ZESUIZEIEIEE NA VH 1 1dTHC STZ STZ+1 1dTHC
NONE 14 15 15 0 3
MODERATE 1 O O 3 1O
SEVERE O O 0 12 2

 

The results are compiled from three separate studies (N=15). Each pancreas
was prepared for light microscopy then examined and placed into one of the

following categories:

a . . . .
' None: no Inflammation, normal Islet architecture;

Moderate-insulitis: some lymphocytes infiltration surrounding but little
infiltration into the islets
Severe insulitis: massive lymphocytes infiltration into the islets with islet

destruction.

67

 

 

 

RN.

ST}

ex;

W9

an

IIII

Ire

RNA in treated and control CD-1 mice. Qualitaive RT-PCR analysis showed that
STZ induced significant increases in lL-12, lFN-y and TNF-a in mRNA
expression on Day 11, as compared to the VH group; whereas, lL-2 and lL-4
were not detectable by RT-PCR analysis (data not shown). Quantitative RT-PCR
analysis was performedto analyze selected cytokines. The time course of lFN-y
mRNA expression was determined to examine the onset of insulitis. A significant
increase in lFN-y steady state mRNA expression was observed in the STZ
treatment group as compared to vehicle controls on and after Day 9 (Fig.ll-6). On
Day 11, A9-THC treatment reduced the STZ-induced IFN-y (Fig.ll-7a), lL-12 (Fig.
ll-7b) and TNF-a (Fig. Il-7c) steady state mRNA expression when 'compared to
the STZ group. No significant difference in the magnitude of mRNA expression
was observed for any of the measured cytokines among the NA, VH and

11dTHC groups.

ll.4.E.Effects of A°-THC treatment post administration of MLDSTZ in
CD-1 mice

An abbreviated and delayed A9-THC treatment schedule was employed in
which mice received 6 consecutive daily treatments with A9-THC (150 mg/kg/day)
beginning on Day 6, one day after termination of the STZ treatment. A protective

effect was observed similar to that produced by 11-day A9-THC treatment. Serum
glucose concentrations in the STZ+6dTHC group were 286.5:74 mg/dL versus

447.6:10.1 mg/dL in the STZ treatment group (Fig.ll-8). Similarly, pancreatic

68

FIGURE "-6. Effect of AQ-THC treatment on lFN-y RNA expression in
MLDSTZ-induced diabetes in CD-1 mice. The treatments were performed
as described in Figure 2 legend. RT-PCR analysis for lFN-y was
performed on the total pancreatic RNA isolated from the tissue samples
taken from mice on the days indicated above. The results are expressed
as the mean i SE, N=3; A significant difference of p<0.05 is indicated
where “a" denotes mean values that are significantly different compared to

the VH group.

69

  
 

  

  
        
    

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FIGURE "-7. Quantitative analysis of cytokine steady state mRNA
expression after A9-THC treatment in MLDSTZ-induced diabetes. The
treatments were performed as described in Figure 2 legend. RT—PCR
analysis for lFN-y (a), lL-12 (b) and TNF-a (c) was performed on the total
pancreatic RNA isolated from the tissue samples taken on Day 11. “ND”
indicated |L-12 mRNA of NA, VH and 11dTHC groups were below the
level of quantitative measurements. The results are expressed as the
mean 1 SE, N=15; A significant difference of p<0.05 is indicated where “a”
denotes mean values that are significantly different compared to the VH
group and “b” denotes values that are significantly different compared to

the STZ only group.

71

mRNA (mplecular/100 ng total RNA)

100000~
80000-
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1 1dTHC
Treatment
72

STZ STZ+11dTHC

FIGURE "-8. Effect of Ag-THC post MLDSTZ-treatment on serum glucose
and pancreatic insulin in MLDSTZ-induced diabetes in CD-1 mice.
Untreated naive (NA) mice were utilized as a control for the vehicle (VH)
treatment group which received 0.2 ml citrate buffer, i.p., for 5 consecutive
days and 0.2 ml corn oil, by oral gavage, for 6 consecutive days. The STZ
treatment group received 5 consecutive 40mg/kg i.p. injections on Days 1-
5. The 6dTHC treatment group received 6 consecutive dose of Ag-THC
(150 mg/kg) by oral gavage on Days 6-11. The STZ+6dTHC treatment
group received 5 consecutive STZ (40mg/kg) i.p. injections on Days 1-5
and 6 consecutive dose of AQ-THC (150 mg/kg) by oral gavage on Days 6-
11. Serum glucose and pancreatic insulin were measured on Day 11. The
results are expressed as the mean t SE, n=15. A significant difference of
p<0.05 is indicated where “a” denotes mean values that are different
compared to the VH group and “b” denotes mean values that are different

compared to the STZ group.

73

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Treatment

insulin content in the STZ+6dTHC group was 106.2:212 ng/mg pancreatic
tissue, which was significantly different from 35.1i3.5 ng/mg pancreatic tissue in

STZ group (Fig.ll-8).

ll.5.Discussion

The results presented in the current study indicate Ag-THC can exert a
transient attenuation from MLDSTZ-induced autoimmune diabetes. Both
MLDSTZ-induced elevation of serum glucose and loss of pancreatic insulin are
significantly diminished by Ag-THC co-treatment in CD-1 mice (Fig.lI-2, Fig.ll-3).
MLDSTZ-induced insulitis is also significantly attenuated as evidenced by
decreases in CD3 inflammatory cells in the pancreatic islets and in mRNA
expression for IL-12, IFN-y, and TNF-a (summarized in Fig.ll-9). The protective
effect of Ag-THC in this autoimmune model of diabetes is similar to previous
results from studies in which treatments with Ag-THC, AB-THC and the synthetic
cannabinoid compound, HU-211, were reported to significantly reduce the
incidence and severity of experimental autoimmune encephalomyelitis in rats
(Wirguin et al., 1994; Achiron et al., 2000; Lyman et al., 1989). These results
indicate that cannabinoid compounds are able to diminish the severity of
autoimmune responses in experimental models of autoimmune diseases.

The inhibition of T-lymphocyte activation by cannabinoid compounds has
been extensively demonstrated (Kaminski, 1996; Schatz et al., 1992; Condie et

al., 1996; Klein et al., 1998). Based on previous findings, the protective effect of

75

 

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76

Ag-THC on MLDSTZ-induced insulitis may involve two possible mechanisms:
either through suppressing T-lymphocyte infiltration into the pancreatic islets
and/or by directly inhibiting T lymphocyte and macrophage function. Consistent
with either mechanism, a marked decrease in inflammatory cells, including CD3
cells was apparent in mice treated with both Ag-THC and STZ as compared to
those treated with STZ only. In addition, the reduction of lFN-y mRNA expression
is consistent with the attenuation of MLDSTZ-induced diabetes by Ag-THC
through inhibition of a TH1-mediated immune response. Based on the profile of
lymphocyte cytokine production, T helper cells are divided into at least two
distinct subsets, TH1 cells and TH2 cells. IL-12 produced by macrophages can
stimulate IL-2 and lFN-y secretion that helps to initiate cell-mediated immunity.
Most results from studies using animal models of spontaneous autoimmune
diabetes suggest a dominant activation of TH1 over TH2 cells as being a key
determinant in the pathogenesis of autoimmune diabetes (Maclaren and
Alkinson, 1997; Rabinovitch, 1994). In the present study, total pancreatic mRNA
was analyzed for the expression of TH1-associated cytokines including lL-2, IFN-
y, lL-12 and the TH 2 cytokine, lL-4 on Day 11, six days after the last STZ
treatment. The TH1 cytokines, specifically lFN-y, IL-12 and the proinflammatory
cytokine, TNF-a, showed lower steady state mRNA expression associated with
AQ-THC co-treatment as compared to the STZ only treatment group indicating a
reduction in inflammatory response (Fig.ll-7). Expression of |L-2 could not be

detected at the peak of MLDSTZ-induced insulitis in any of the experimental

77

treatment groups. The presence of TH1 cytokines such as IFN-y, IL-12 on Day 11
in the STZ-treated animals suggested that lL-2 maybe expressed prior to Day 11
as suggested by the studies of Herold et al. (Herold et al., 1996). The lack of
detection in the limited sampling protocol used in this study does not permit
speculation on the possible role of |L-2 and IL-4 in the MLDSTZ-induced
diabetes model.

The protective effect of Ag-THC on MLDSTZ induced diabetes could also
be accomplished by acting at target sites other than the immune system.
Cannabinoid compounds produce a number of biological effects on different cell
types and organs systems. Therefore, alternative explanations for Ag-THC-
mediated protection from MLDSTZ-induced diabetes were considered. The
protective effect of Ag-THC could be mediated by several different putative
mechanisms, such as: 1. inhibition of STZ uptake by beta-cells therefore
blocking the direct cytotoxic action of STZ; 2. direct stimulation of glucose
metabolism (Sanchez et al., 1998) resulting in reduced hyperglycemia; 3. direct
effects on pancreatic beta-cell functions including increased insulin production
and secretion as previously suggested (Laychock et al., 1986). One or more of
these AQ-THC-mediated effects may account for the protective mechanism in the
MLDSTZ model; however, several of these possibilities appear unlikely based on
observations made in the present investigation and previously by others. For
example, the AQ-THC treatment was also protective even when Ag-THC treatment

was initiated 24 hr after administration of the last dose of STZ (Fig.ll-8). Due to

78

the very short half-life of STZ (approximately 15 min), STZ would have been
cleared from the bloodstream and any direct STZ beta-cell toxicity would have
been initiated (Like and Rossini, 1976). Thus interference by Ag-THC with STZ
uptake or its initial action on the beta-cell appears unlikely. Furthermore, the
notion that AQ-THC increases glucose metabolism to reduce hyperglycemia also
seems to be contrary to our findings because Ag-THC treatment alone did not
alter serum glucose and pancreatic insulin. The possibility that Ag-THC directly
affects pancreatic beta-cell function(s) appears to be a plausible explanation for
protection from hyperglycemia because there is evidence that insulin secretion
can be stimulated by this cannabinoid. A study conducted by Laychock and
colleagues showed that A9-THC stimulated the basal release of insulin and also
potentiated glucose-stimulated insulin release in isolated rat pancreatic islets
(Laychock et al., 1986). These actions would reduce hyperglycemia and may
attenuate the loss of pancreatic insulin in the MLDSTZ + THC treated mice
utilized in the experiments reported here. Based on this limited information, it is
possible that immunosuppression alone may not account for the ability of Ag-THC
to attenuate MLDSTZ-induced diabetes in CD-1 mice.

Although Ag-THC attenuated MLDSTZ-induced diabetes in CD-1 mice,
complete and permanent protection was not achieved. The lack of complete
protection may be attributed to several factors. One possible explanation may be
STZ produces irreversible cytotoxic damage to beta-cells that is insensitive to

modulation by Ag-THC. To date, there has been considerable speculation

79

regarding the role of the immune response in the MLDSTZ model and whether or
not damage to the beta-cells by the leukocytes is sufficient in itself to produce
cell death (Beattie et al., 1980). It has been shown that STZ alone can directly
Induce the lethal alkylation of cellular components and also can cause some
nonlethal alterations in pancreatic beta-cell function (Sarvetnick et al., 1990;
Maclaren and Alkinson, 1997). These alterations may include conformational
changes in . membrane proteins associated with glucose transport and
metabolism. These or other alterations may further be recognized as “non-self”
bythe immune system and thus contribute to the immune-based beta-cell
damage. Therefore, a suppression of either the direct cytotoxic effect by STZ on
beta-cells or the subsequent inhibition of the immune response by A9-THC may
only result in partial protection. The partial protective effect by Ag-THC treatment
on MLDSTZ-induced diabetes in the present study is similar to the effects
previously observed using other Immunosuppressive treatments (Sai P. et al.,
1991 and Herold et al., 1996). For example, the results reported by Herold et al.
(Herold et al., 1996) showed that anti-IFN-y mAb treatment only partially
attenuated the MLDSTZ-induced elevation in blood glucose. A second factor that
may explain the partial protective effect by A9-THC is that it is a relatively weak
immunosuppressant. Moreover, the results obtained on Day 17 (data shown in
Fig.lI-2 insert) further demonstrated that the attenuation of MLDSTZ-induced
diabetes by 11-day treatment of Ag-THC is transient, which is similar to the

effects produced by other immunosuppressive treatments. The termination of A9-

80

THC treatment on Day 11 may allow for the restoration of immune competence
and subsequent infiltration of T-lymphocytes into the damaged islet. Further
studies will be required to more fully elucidate the reason why a permanent
protection was not achieved by Ag-THC treatment in this model.

It is widely established that different mouse strains exhibit markedly
different sensitivity to STZ treatment. For comparative purposes studies were
also performed in BGC3F1 mice, a mouse strain not previously utilized in the
MLDSTZ model of diabetes. In contrast to CD-1 mice, B6C3F1 mice exhibited
minimum islet inflammation after MLDSTZ treatment (Fig.lI-4). In addition, the
hyperglycemia was less pronounced after MLDSTZ treatment and the protection
by Ag-THC was also less extensive than that observed in CD-1 mice. The
decreased sensitivity of the B6C3F1 mice to MLDSTZ-induced hyperglycemia
and loss of pancreatic insulin (Fig.lI-2, Fig.ll-3), as compared to CD-1 mice, can
be explained in part by a less robust immune response in the pancreas (Fig.ll-4).
However, the mild diabetic state produced in MLDSTZ-treated B6CBF1 mice
which occurred in the absence of insulitis may reflect the direct cytotoxic action
of MLDSTZ on pancreatic beta-cells. The premise that the direct toxic effects of
STZ make a substantial contribution to the MLDSTZ model is further supported
by the results of Gerling et al. from studies demonstrating that MLDSTZ was able
to induce diabetes in NOD immune incompetent scid/scid mice (Gerling et al.,
1994). Based on their results, lymphocytic infiltration was not observed and
therefore was not required for diabetes development in those animals. The

hyperglycemia induced in NOD scid/scid mice by MLDSTZ is likely due to direct

81

toxicity of STZ on beta-cells. Accordingly, these observations from different types
of experiments suggest that sensitivity of beta-cells to the direct (i.e. non-
immune) toxicity of STZ can elicit diabetogenic responses from laboratory
animals given MLDSTZ treatment.

In conclusion these studies strongly suggest that the mechanism
responsible for MLDSTZ-Induced autoimmune diabetes involves at least two
major elements. One element is associated with a robust immune response that
is mounted against pancreatic cells. Most likely, it is the autoimmune component
that is most effectively modulated by A9-THC treatment as suggested by our
results in CD-1 mice. Of the two mouse strains examined, CD-1 mice exhibited
the greatest magnitude of insulitis, the highest level of hyperglycemia and also
the greatest magnitude of protection from Ag-THC co-administration. The second
element appears to be more closely associated with the direct toxic action of
STZ. This component of STZ-mediated diabetes is less sensitive to modulation
by Ag-THC as suggested by our studies in BBC3F1 mice. These studies showed
that 86C3F1 mice exhibited significant hyperglycemia and pancreatic insulin loss
but very mild inflammation in pancreatic islets after MLDSTZ treatment and also
were relatively insensitive to the protective actions of Ag-THC. The data
presented in this report and by others, as discussed above, permit speculation
regarding the degree of participation by the direct and the immune-based
elements of MLDSTZ-induced diabetes in CD-1 mice. It appears likely that as
much as one-half of the diabetogenic effects of MLDSTZ may be attributed to the

direct, non-immune related toxic actions that are known to be characteristic of

82

STZ treatment in laboratory animals. For this reason use of the MLDSTZ model
is appropriate for investigation of chemical-induced autoimmune response
directed against the endocrine pancreas but it may be less appropriate for

explanation of genetically based autoimmune diabetes.

83

CHAPTER III
CANNABINOID RECEPTOR CB1-MEDIATED INSULIN RELEASE FROM

ISOLATED PANCREATIC ISLETS AND RINM5F CELLS

84

lll.1.Summary

Ag-Tetrahydrocannabinol (Ag-THC) modulates a wide variety of biological
functions. Among these, it has been reported that Ag-THC treatment of rat
pancreatic islets stimulated the release of insulin. The objective of the present
studies was to further characterize the molecular mechanism responsible for A9-
THC-stimulated insulin release and to determine whether this biological activity is
mediated through cannabinoid receptors. The present studies demonstrate that
Ag-THC treatment stimulated insulin release from isolated mouse pancreatic
islets and from the rat-derived insulin producing RINm5F cells. Closely
associated with Ag-THC-stimulated insulin release was a rapid but transient
elevation in intracellular calcium. Interestingly, the calcium channel blocker,
verapamil abrogated both the Ag-THC-induced elevation in intracellular calcium
and release of insulin. In contrast, pretreatment with either thapsigargin or
ryanodine, as inhibitors of calcium release from intracellular calcium stores, did
not alter the elevation of intracellular calcium by Ag-THC. Moreover, the inhibitor
of CaM kinase II, K93, but not the PKC inhibitor, bisindolylmalemide, diminished
Ag-THC-stimulated insulin release. Analysis of both primary pancreatic islets as
well as RINm5F cells by RT-PCR identified mRNA expression for the central
cannabinoid receptor, CB1. Interestingly, the CB1 receptor antagonist,
SR141716A, blocked Ag-THC-stimulated insulin release from both isolated
mouse islets and from RINm5F cells. In addition, pretreatment of RINm5F cells

with SR141716A blocked the rise in Ag-THC-induced elevation in intracellular

85

calcium. Collectively, these results demonstrate that Ag-THC-stimulated insulin
release is mediated through an elevation of intracellular calcium through the

opening of calcium channels via a CB1-dependent mechanism.

lll.2.lntroduction

Stimulation of insulin secretion by pancreatic beta-cells in response to
most secretagogues involves changes in intracellular calcium concentration
[Ca2+]i ( Seri et al., 2000; Fischer et al.,1999; Maechler et al., 1999; Lange and
Brandt, 1993). Glucose and other nutrients that stimulate insulin release produce
an elevation in [Ca"”’]i via mechanisms that induce membrane depolarization and
a calcium influx via the opening of voltage sensitive calcium channels and
through the release of calcium from intracellular stores (Takasawa et al., 1998;
Zawalich et al., 1998; Safayhi et al.,1997; Porksen et al., 1996; Tang et al.,
1995). A variety of calcium-sensitive intracellular regulatory elements involved in
the insulin secretory process have been identified. One of the most extensively
characterized is the calcium/calmodulin dependent kinase, CaM kinase II.
Numerous studies have demonstrated an increase in CaM kinase II activity
under conditions where insulin release was induced (Mohlig et al., 1997; Norling
et al., 1994). Likewise, the inhibition of CaM kinase II activity has been closely
correlated to an inhibition of insulin release (Wenham et al., 1994). Collectively,
CaM kinase II is widely established as an important transducer of calcium
signaling and intimately involved in the phosphorylation of proteins that regulate

insulin granual excytosis.

86

Ag-Tetrahydrocannabinol (Ag-THC), the primary psychoactive constituent
of marihuana, modulates a wide variety of biological functions (Wenger et al.,
1999; Voth and Schwartz, 1997; Smith et al., 1978) . Among these, it has been
reported that Ag-THC treatment of isolated rat pancreatic islets stimulated the
release of insulin (Laychock et al., 1986). Although the mechanism responsible
for Ag-THC-stimulated insulin release in the aforementioned study was not
established, it is notable that these studies were conducted prior to the
identification and cloning of the two major forms of cannabinoid receptors, CB1
and C82 (Matsuda et al., 1990; Munro et al., 1993) . CB1 is predominantly
expressed within the central nervous system (Howlett, 1998), but has also been
identified in peripheral tissues including the lung, testis, hearts, spleen, and small
intestine (Felder and Glass, 1998; Rice, 1997; Pertwee et al., 1996). The second
and more recently characterized cannabinoid receptor subtype, C82, is not
expressed In the brain but has been identified in a number of peripheral tissues
with the immune system being best characterized (Schartz et al., 1997; Munro et
al., 1993; Kaminski et al., 1992). It has now been established that certain
cannabinoids mediate at least part of their biological activity through one or both
of these cannabinoid receptors. Both CB1 and C82 are G-protein coupled
receptors and have been demonstrated to modulate a number of different signal
transducing elements including adenylate cyclase, ERK MAP kinases and most
important to the present investigation intracellular Caz+ (Bouaboula et al., 1996;
Condie et al., 1996; Schartz et al., 1992; Kaminski et al., 1994). The objectives

of the present study were two-fold: 1) to investigate whether calcium-dependent

87

signaling pathways are responsible for Ag-THC-stimulated insulin release; and 2)

to determine whether this biological activity is mediated through cannabinoid

receptors.

|II.3.Materials and methods

lll.3.A.Materials

All reagents were purchased from Sigma Chemical Co. (St. Louis, MO)
unless otherwise identified. RPMI-1640 and other cell culture reagents were
purchased from BRL Life Technologies (Gaitherburg, MD). Ag-THC and
cannabidiol (CBD) were provided by the National Institute on Drug Abuse
(National Institute of Health, Rockville, MD). Stock solutions of A9-THC and CBD
were prepared in ethanol and added directly to the culture medium to achieve
the desired experimental concentrations. Cannabinoid receptor CB1 antagonist
SR1141716A and C82 antagonist SR144528 were also obtained from the
National Institute on Dmg Abuse (National Institute of Health, Rockville, MD) and
prepared in DMSO. RT-PCR reagents were purchased from Promega Co.
(Madison, WI). Research grade collagenase from Clostn'dium histolyticum was
obtained from Boehringer Mannheim (GmbH, Gemany). Fura-2 acetoxymethyl-
ester (Fura-2/AM) was obtained from Molecular Probes (Eugene, OR). The CaM
kinase II inhibitor (KN93) was purchased from Calbiochem-Novabiochem Corp.

(La Jolla, CA).

lll.3.B.Preparation of mouse pancreatic islets

88

Mouse pancreatic islets were prepared from 8 to 9 week old male CD-1
mice (Charles River Laboratory, Dortage, Ml). Upon arrival, mice were randomly
transferred to standard filter topped cages containing sawdust bedding. Five
mice per cage were housed in a pathogen-free facility, which was maintained at
21-24°C constant temperature, 40~60% relative humidity and a 12 hour
light/dark cycle. Mice were provided standard laboratory animal chow (Purina
Certified Laboratory Chow) and water ad Iibitum. Isolated pancreatic islets from
CD-1 mice were prepared by collagenase digestion (Gotoh et al., 1985) and
were maintained in culture overnight in complete RPMl-1640 medium
supplemented with 10% heat-inactivated fetal calf serum, 200 ug/ml penicillin

and 200 mU/ml streptomycin to recover from the isolation procedure.

lll.3.C.Cells

Rat insulinoma-derived RINm5F cells were generously provided by Dr.
Paolo Meda (Geneva, Switherland). The cells were cultured in a humidified
atmosphere of 95% O2 and 5% CO2 at 37°C in complete RPMl-1640 medium
which is supplemented with 10% heat-inactivated fetal calf serum, 200 pg/ml
penicillin and 200 mU/ml streptomycin (GIBCO, Grand Island, NY). The medium
was changed every other day, and the cells (passage 40-60) were utilized for

experiments on Day 5 after plating.

|II.3.D. Measurement of insulin release

Cultured pancreatic islets were distributed into polypropylene centrifuge

89

 

 

 

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tubes in batches of 10 islets per treatment group. The islets were preincubated
for 15 min with 1ml of oxygenated Krebs-Ringer bicarbonate (KREB) buffer
supplemented with 11.4 mM glucose and 0.01% bovine serum albumin (BSA)
(fraction V; fatty acid free) at 37°C in a COzincubator (95% O2 and 5%). Similarly,
RINm5F cells were transferred into 6-well culture plates containing fresh RPMI-
1640. The release of insulin from islets was determined by measuring insulin in
aliquots of culture supernatant sampled after a 30 min incubation with A9-THC or
vehicle (0.1% ethanol) unless indicated otherwise. In experiments where
cannabinoid receptor antagonists, calcium channel blockers or kinase inhibitors
were employed, the cultures were preincubated with these respective agents, or
the vehicle control (0.1 % DMSO), for 30 min prior to treatment with Ag-THC.

Media insulin was assayed using a radioimmunoassay as previously
described (Fischer et al., 1996). An aliquot of KREB buffer or culture medium
was removed and stored at —20°C until assayed. Release of insulin was
normalized to milligrams of total cellular DNA content (measurement described in
Fischer et al., 1996).

Viability of cells was assessed by trypan blue exclusion. In addition, the
release of RINm5F cells of lactate dehydrogenase (LDH) into the medium was
also used as the further indicator of cytotoxicity (measurement according to the

method of Bergmeyer and Bemt, 1974).

|II.3.E. Determination of cannabinoid receptor mRNA expression

Trizol reagent (3ml) (Life Technologies, Grand Island, NY) was added to

90

500 to1000 cultured pancreatic islets or 1x107 RINm5F cells for total RNA
isolation. All isolated RNA samples were DNAse treated to eliminate any
potential genomic DNA contamination prior to the RT-PCR. Briefly, RNA were
reverse-transcribed simultaneously into cDNA in 20 pl reaction buffer containing
oligo (dT)15 and Molony Murine Leukemia Virus reverse transcriptase. Then a
PCR mix consisting of 10x PCR buffer (20 mM ammonium sulfate, 67 mMTris,
6.7 pMEDTA, 8 mg/ml BSA, and 5 mM 2-mercaptoethanol), 4 mM MgCIz, 6 pmol
each of forward and reverse primers, and 1.25 units of Taq DNA polymerase
was added to the cDNA samples. Samples were then heated to 94°C for 4 min
and cycled at a 94°C denaturing step for 15 sec, a 59°C annealing step for 30
sec, and a 72°C elongation step for 30 sec, after which an additional extension
step at 72°C for 5 min was included. Specifically, for the detection of CB1 and
C82 transcripts, 400 ng RNA and 35 amplification cycle. Primer sequences for
mouse CB1 are: fonlvard primer GGATATCTGGAGGAACTGGC, reverse primer,
GAGCTCATTGAATGCTTGGC; for mouse CB2 primer: froward primer,
CAGTACACCTGCCACAAAGGA, reverse primer, GTGTGACCTTCTCTGCAGA
CA. Primer sequences for rat CB1 are: fonNard primer CTGTGGAGGAACGGAT
ATGC, reverse primer, TTGAAGTCATGCTTGAGCGC; for mouse C82 primer:
froward primer, GAACCTGCCAGTACCAACAAG, reverse primer, CTTCGACTG
TGTGCAACTCGA. The RT-PCR product was determined by densitometric
analysis of ethdium bromide-stained agarose gel (3%, Nusieve; agarose=3:1)
using the Gel Doc 1000 analysis system (BioRad Laboratories Inc., Herculese,

CA).

91

lll.3.F.Measurement of [Caz‘L

[Ca2+], was measured in Fura-2/AM loaded cells using a SPEX FIuorolog-2
Spectrofluorometer (SPEX Industries, Inc., Edison, NJ.) RINm5F cells were
aliquoted at density of 2x104 cells/ml in 25 cm2 flasks. After 5 days of culture, the
cells were detached from the flask by treatment with trypsin, washed twice with a
modified KREB buffer, adjusted to a density of 1 X 106 cells/ml and incubated
with 1uM Fura-2/AM at 37°C. After 30min incubation, the loading buffer was
removed and the suspended cells were rinsed twice with fresh KREB buffer.
Fura-2/AM-loaded cells (1 X 10" cells/ml) were added to a cuvette and the
temporal changes in [Ca2+],-induced fluorescence by various chemical treatment
were measured. Fluorescence emission at 505 nm was monitored at room
temperature with constant stirring, using a dual wavelength spectrofluorometer
system with excitation at 340 and 380 nm. At the end of scanning, 0.1% Triton
X-100 was added to determine the maximum fluorescence (Rmax) and then
0.015M EGTA was added to obtain minimum fluorescence (Rmin). These
measurements were used in computer-based calculations of [Ca2+], to obtain the
representative graphs. The [Ca”], was calculated from fluorescence intensity
readings using the following equation: [Ca2*]i = Kd*Q(R-Rm,,,)/(Rma,,-R). R is the
ratio of emission intensities at 340 and 380 nm excitation. Kd is the dissociation
constant of the Ca2*/Fura-2 complex and equal to 1.45 X107; and Q is the ratio of
the 380 nm fluorescence under conditions of minimum and maximum [Ca2*] ,

conditions (Shao et al. 1998).

92

lll.3.G.Statistical analysis

Data are presented as the mean :I: SEM from at lease three experiments
conducted in triplicate (n=9). Treatment groups were compared with the vehicle
control groups using an analysis of variance and Dunnett's two-tailed t-test, with

p< 0.05 considered statistically significant.

III.4.Results

Ill.4.A.Stimulation of insulin release from mouse pancreatic islets
and RINm5F cells by A°-THC treatment

Mouse pancreatic islets and insulin-producing RINm5F cells were
employed to examine the direct effects of Ag-THC treatment on insulin release.
Figure Ill-1 shows that a 30 min treatment with Ag-THC stimulated the release of
insulin from both mouse islets (Fig.lll-1A) and RINm5F cells (Fig.llI-1 B) in a

concentration-dependent manner during a 30-min treatment period. At the
highest concentration tested, Ag-THC (15 pM) produced an approximately 3-fold
increase in insulin release by RINm5F cells, as compared to control. The
treatment of RINm5F cells or pancreatic islets with Ag-THC at concentrations
equal or lower than 15pM was not cytotoxic as assessed by release of LDH into

culture mediums and/or trypan blue exclusion.

Ill.4.B.A°-THC-induced increase in [Ca”]. in RINm5F cells

One of the proximal events governing insulin release is a rapid increase in

93

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Ag-THC (uM)
Treatment Group

Figure Ill-1A. Effect of Ag-THC on insulin release from mouse pancreatic islets.
Islets were incubated in KREB buffer containing: no treatment (NA); vehicle
(VH); and Ag-THC as indicated. The insulin release into the medium was assayed
over a 30 min period and normalized per 50 islets. The results are expressed as
the mean t SE, n=9. A significant difference of p<0.05 is indicated where “*"

denotes mean values that are different compared to the VH control group.

94

15-

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Insulin Release (nglpg DNA)

 

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Treatment

Figure Ill-1 B. Effect of A9-THC on insulin release from RINm5F cells. Cultures
were incubated with: no treatment (NA); vehicle (VH); and A9-THC as Indicated.
The insulin release into the medium was assayed over a 30 min period and
normalized to DNA content. The results are expressed as the mean i SE, n=9. A
significant difference of p<0.05 is indicated where “*" denotes mean values that

are different compared to the VH group.

95

[Ca2+],. To determine whether AQ-THC-stimulated insulin release was being
mediated through a rise in [Ca2+],, [Ca2+]i was measured in Fura-2/AM-Ioaded
RINm5F cells. In control cells (Fig.lII-2), the free calcium level did not rise upon
addition of vehicle (0.1% ethanol) and remained constant throughout the
experimental period. In Ag-THC-treated cells (Fig.llI-2), calculated [Ca""‘ji was
elevated in approximately 50 sec and exhibited a maximal 3-fold increase within
300 sec after drug addition. The elevation in [Ca2*]i was rapid and sustained,
persisting for more than 300 sec. No further increase in [Ca2*]i was observed

within a 30 min time period (data not shown).

lll.4.C.Attenuation of A’-THC-induced Increase in [Caz‘]I and insulin
release by the calcium channel blocker verapamil

To further determine whether insulin release stimulated by Ag-THC was
associated with a rise in [Ca2*]i via influx through plasma membrane associated
calcium channels, the calcium channel blocker verapamil was utilized. The
results in Figure Ill-3A show that verapamil (40 pM) pretreatment abrogated the
increase of [Cam]i induced by A9-THC in RINm5F cells. In a separate experiment,
verapamil completely blocked the insulin release stimulated by A9-THC in
RINm5F cells (Fig.lll-3B). Verapamil treatment alone neither altered basal insulin
release (Fig.lll-3B) nor changed the basal level of [Ca2+], (Fig.lII-3A). These
results suggest that the source for the increase of [Ca2+]i induced by Ag-THC is

extracellular.

96

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Calculated [Ca2+]i (10'8M)

 

 

o 160 260 330 450 560 600

Time (sec)

Figure Ill-2. Effect of Ag-THC on [Ca2*]i in RINm5F cells. AQ-THC or vehicle (VH)
were administered at the arrow to the Fura-2/AM loaded cell suspension. [Ca"’*]i

was measured and determined as described in Materials and Methods. Data

shown are the representative of five experiments.

97

 

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5° I I

Verapamil Ag-THC

250‘
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0 100 200 300 400 500 600
Time (sec)

Figure III-3A. Effect of calcium channel blocker verapamil on AQ-THC-induced
increase in [Caz*]i in RINm5F cells. Fura-2/AM loaded cells were treated with 15
pM of A9-THC in the presence of vehicle (VH) or 40 pM verapamil. Arrows
indicate additions of treatments. [Ca2*]i was measured and determined as
described in Materials and Methods. Data shown are the representative of three

experiments.

98

 

 

 

 

 

 

 

 

 

 

 

 

 

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Treatment Group

Figure III-38. Effect of calcium Channel blocker verapamil on the stimulation of
insulin release by A9-THC in RINm5F cells. Cultures were incubated with
verapamil for 30 min prior to exposure to vehicle (VH) or A9-THC treatment for
additional 30 min. The insulin release into the medium was assayed and
normalized to DNA content. Data are expressed as the mean t SE, n=9. A
significant difference of p<0.05 is indicated where “a” denotes mean values that
are different compared to the corresponding vehicle control receiving no
verapamil treatment; and “b” denotes mean values that are different compared to

the corresponding control receiving verapamil treatment.

99

lll.4.D.Thapsigargin and ryanodine exert no effect on the elevation in
[Cap]l induced by A°-THC

To confirm that the rise in [Ca2+], induced by Ag-THC was not due to the
mobilization of calcium from intracellular calcium stores, additional studies were
performed utilizing thapsigargin. Control experiments confirmed the ability of
thapsigargin to deplete intracellular calcium stores and inhibit the ATP-
dependent Ca2+ reuptake as evidenced by the inability of carbachol, a known
releaser of Ca2*-stored in the ER, to increase [Ca2+]. after thapsigargin treatment.
Similarly, thapsigargin treatment alone rapidly increased [Ca2*].. Interestingly,
thapsigargin did not alter the increase of [Ca2+]i induced by Ag-THC in RINm5F
cells (Fig.lIl-4A). In a separate experiment, the increase in [Ca2”]i induced by A9-
THC was not altered in the presence of ryanodine (10 pM) pretreatment (Fig.lll-
4B). The latter is able to block Ca2+ release from intracellular Ca2+ stores through
ryanodine-sensitive Channels. These studies confirm that source for the Increase

of [Ca2+], induced by Ag-THC is extracellular.

lll.4.E.Effects of CaM kinase II inhibitor KN93 and PKC inhibitor .
bisindolylmalemide on the stimulation of insulin release by A9-THC

Two calcium regulated protein kinases, CaM kinase II and PKC, are well
established as being critically involved in the regulation of insulin release
(Gromada et al., 1999; Miura et al., 1998; Gregersen et al., 2000b; Tabuchi et

al., 2000). To further examine the putative involvement of CaM kinase II and/or

100

A. c_

VH Carbachol

I

1x1o‘M

Carbachol

Calculated [Cam]I F———)I
m

 

 

 

0 100 200 300 400 500 600 0 100 200 300 400 500 600

Time (sec) Time (sec)

Figure Ill-4A. Effect of thapsigargin on Ag-THC-induced increase in [Ca2“]i in
RINm5F cells. The ability of 2 pM thapsigargin (TG) to deplete intracellular
calcium stores and inhibit the ATP-dependent Ca2+ reuptake was confirmed by
treatment with 200 nM carbachol (A: vehicle, B: thapsigargin). In separate
experiments, fura-2/AM loaded cells were treated with 15 pM AQ-THC in the
presence of C: vehicle (VH) or D: 2 pM thapsigargin (TG). Arrows indicate
additions of treatments. [Ca2+], was measured and determined as described in

Materials and Methods. Data shown are the representative of three experiments.

101

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0 1 00 200 300 400 500 600

Time (sec)

Figure III-4B. Effect of ryanodine on Ag-THC-induced increase in [Ca2+], in
RINm5F cells. Fura-2/AM loaded cells were treated with 15 pM Ag-THC in the
presence of vehicle (VH) or 10 pM ryanodine. Arrows indicate additions of
treatments. [Ca2*]i was measured and determined as described in Materials and

Methods. Data shown are the representative of three experiments.

102

PKC in A9-THC-stimulated insulin release, experiments were performed utilizing
selective inhibitors of these two enzymes. As illustrated in Fig-III.5A, KN93, an
inhibitor for CaM kinase II, completely inhibited the stimulation of insulin release
by A9-THC whereas KN93 by itself did not Change the basal level (no Ag-THC
treatment) of insulin release. In contrast, the PKC inhibitor bisindolylmaleimide
produced no effect on Ag-THC-stimulated insulin release (Fig-lll.5B). As a control
to assess the efficiency and extent of PKC down-regulation by
bisindolylmaleimide, TPA-induced stimulation of insulin release (Tang et al.,
1998) was measured. TPA-induced stimulation of insulin release in the presence

of bisindolylmaleimide was reduced by more than 50%.

III.4.F.Identification of cannabinoid receptor mRNA expression in
isolated pancreatic islets and RINm5F cells

Although cannabinoid receptor expression has been identified in a variety
of cell types and tissues, insulin-producing cells have not been previously
examined for either CB1 or CB2. To investigate the potential involvement of
cannabinoid receptors in the insulinotropic action of Ag-THC, the expression of
both CB1 and CB2 mRNA was examined in CD-1 mouse pancreatic islets and
RINm5F cells using RT—PCR analysis (Fig-Ill.6). Figure III-6 involvement of
cannabinoid receptors in the insulinotropic action of A9-THC, the expression of
both CB1 and CB2 mRNA was examined in 00-1 mouse pancreatic islets and
RINm5F cells using RT-PCR analysis (Fig-Ill.6). Figure Ill-6 shows that CD-1
mouse pancreatic islets express mRNA for the CB1 receptor but not the CB2

103

 

 

 

 

   
        
      

 

 

 

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a
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KN93 (pM)
Treatment

Figure Ill-5A. Inhibition of AQ-THC-stimulated insulin release from RINm5F cells
by the CaM kinase II inhibitor, KN93. RINm5F cellswere incubated with KN93 or
vehicle (VH) for 30 min prior to exposure to Ag-THC treatment for additional 30
min. The insulin release into the medium was assayed and normalized to DNA
content. The results are expressed as the mean i SE, n=9. A significant
difference of p<0.05 is indicated where “a" denotes mean values that are
different compared to the vehicle control receiving no KN93 treatment; and “b”
denotes mean values that are different compared to the corresponding control

receiving Ag-THC treatment.

104

 

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15pM A°-TI-IC

 

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Insulin Release (nglug DNA)

Figure Ill-5B. Effect of bisindolylmaleimide (BIS), a PKC inhibitor, on Ag-THC-
stimulated insulin release from RINm5F cells. RINm5F cells were incubated with
bisindolylmaleimide or 0.1% DMSO for 30 min prior to exposure to vehicle (VH),
TPA, or Ag-THC for additional 30 min. The insulin release into the medium was
assayed and normalized to DNA content. Data are expressed as the mean i SE,
n=9. A significant difference of p<0.05 is indicated where “a” denotes mean
values that are different compared to the corresponding vehicle receiving no
bisindolylmaleimide treatment; “b” denotes mean values that are different
compared to the corresponding vehicle receiving bisindolylmaleimide treatment;
and “c” denotes mean values that are different compared to the corresponding

control (0.1% DMSO) receiving TPA treatment.

105

receptor. In contrast, mRNA for both CB1 and CBZ was readily detected in

RINm5F cells.

Ill.4.G.Effects of cannabinoid receptor antagonists on A°-THC-
stimulated insulin release

In light of the Identification of CB1 and C82 mRNA expression by insulin
producing cells, additional studies were performed to investigate the involvement
of cannabinoid receptors in the insulinotropic actions of Ag-THC. For these
experiments, the CB1 selective antagonist, SR141716A, and the C82 selective
antagonist, SR144528, were employed. Pretreatment of the mouse pancreatic
islets (Fig.llI-7A) and RINm5F cells (Fig.lll-7B) with the CB1 antagonist,
SR141716A (10 pM) inhibited AQ-THC-stimulated insulin release. In a separate
experiment, SR144528 (10 pM), a CB2 receptor-specific antagonist, did not
affect Ag-THC (15 pM)-stimulated insulin release from RINm5F cells (Fig.lll-7C).
As shown in figures III-7, neither 10 uM of SR 141716A nor 10 pM of SR144528
alone altered basal insulin release (no Ag-THC treatment). Furthermore,
cannabidiol, a plant-derived cannabinoid with low affinity for both CB1 and CB2,
was unable to induce insulin release from RINm5F cells at the same

concentration that Ag-THC exhibited a maximum release of insulin (Fig.llI-7D).

lll.4.H.Effects of cannabinoid receptor antagonists on A°-THC-

induced [Ca2*]. increase in RINm5F cells

106

Islet Islet Spleen RINm5Fcells

CBZ—_*
CB1——*

GAPDH

 

Lane 1 2 3 4 5

Figure Ill-6. RT-PCR analysis of CB1 receptor mRNA and C82 receptor mRNA
expression in the mouse pancreatic islets and RINm5F cells. RT-PCR analysis

was performed as described under “Materials and Methods". Lane1: mouse CB1; ‘
lane 2: mouse CB2; lane 3: mouse CB1; lane 4: rat CB1; lane 5: rat CB2.

Results are representative of three separate experiments.

107

 

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Figure Ill-7A. Effect of SR141716A on Ag-THC-stimulated insulin release from
mouse pancreatic islets. Islets were pretreated with SR141716A (SR) for 30 min
prior to exposure to 15 pM Ag-THC as indicated. Additional groups were
employed as control (VH: 0.1% DMSO and 0.1% ethanol; THC: 0.1%DMSO and
15 pM Ag-THC; SR10: 10pM SR141716A and 0.1% ethanol). The insulin release
into the medium was assayed and normalized per 50 Islets. Data are expressed
as the mean t SE, n=9. A significant difference of p<0.05 is indicated where “a”
denotes mean values that are different compared to the VH group; and “b"

denotes mean values that are different compared to the THC group.

108

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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A9-THC(uM)
Treatment group

Figure Ill-7B. Effect of SR141716A on Ag-THC-stimulated insulin release from
RINm5F cells. Cells were preincubated with CB1 antagonist SR141716A (SR)
for 30 min prior to exposure to Ag-THC or vehicle (VH) treatment as indicated.
The insulin release into the medium was assayed and normalized to DNA
content. Data are expressed as the mean i SE, n=9. A significant difference of
p<0.05 is indicated where “a” denotes mean values that are different compared
to the corresponding VH group receiving no Ag-THC; and “b” denotes mean
values that are different compared to the corresponding 0 pM SR group

receiving same concentrations of AQ-THC.

109

 

 

 

 

 

 

 

 

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Figure Ill-7C. Effect of SR144528 on AQ-THC-stimulated insulin release from
RINm5F cells. Cells were preincubated with C82 antagonist SR144528 for 30
min prior to exposure to Ag-THC or vehicle (VH) treatment. The insulin release
into the medium was assayed and normalized to DNA content. Data are
expressed as the mean 1- SE, n=9. A significant difference of p<0.05 is indicated

“*I!

where denotes mean values that are different compared to the corresponding

VH group.

110

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Treatment Group

Figure III-7D. Effect of cannabidiol on insulin release from RINm5F cells.
Cultures were incubated with: no treatment (NA); vehicle (VH); 15 pM
cannabidiol (CBD) and 15 pM Ag-THC as indicated. The insulin release into the
medium was assayed over a 30 min period and normalized to DNA content. Data
are expressed as the mean : SE, n=9. A significant difference of p<0.05 is

11*”

indicated where denotes mean values that are different compared to the

corresponding VH group.

111

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0 100 200 300 400 500 600

Time (sec)

Figure Ill-8. Effect of SR141716A on AQ-THC-induced increase in [Ca”], in
RINm5F cells. Fura-2/AM loaded cells were treated with 15 pM of A9-THC in the
presence of vehicle (VH) or 1 pM SR141716A (SR). Arrows indicate additions of

treatments. [Ca2+], was measured and determined as described in Materials and

Methods. Data shown are the representative of three experiments.

112

The role of the CB1 receptor in the elevation of [Ca2+], induced by Ag-THC
was examined using CB1 receptor antagonist SR141716A. At concentrations of
1 uM SR141716A blocked the rise in [Ca2+], induced by Ag-THC (15 pM) (Fig.lll-
8). SR141716A (1 and 10 pM) alone did not alter basal levels of [Ca2*]i (data not

shown).

lll.5.Discussion

Results from the present study demonstrate that Ag-THC treatment
produced a cannabinoid receptor-mediated increase in [Ca”], and a marked
increase in insulin release from isolated mouse pancreatic islets and rat
insulinoma-derived RINm5F cells (summarized in Fig.lII-9). These results are
consistent with and extend those in a previous report which indicated that insulin
was released from rat pancreatic islets treated with the cannabinoid (Laychock et
al., 1986). Based on their report, Ag-THC was able to increase both high glucose
(8.5 mM) stimulated and basal (glucose, 2.8 mM) insulin release from isolated
islets. RINm5F cells are known to be unresponsive to high concentrations of
glucose as a stimulant for insulin release (Halban et al., 1983), but do release
insulin as a result of treatment with other insulin secretagogues, eg. amino acids,
sulfonylurea drugs (Laychock, 1998; Leiers et al., 2000). The stimulation of
insulin release from in vitro preparations by Ag-THC treatment appears to be a
direct action on insulin-producing cells, which is independent of high glucose

concentrations. A comparison of the concentration-response curves for AQ-THC-

113

induced insulin release obtained in mouse islets and RINm5F cells in the present
study with that obtained using rat pancreatic islets (Laychock et al.,1986)
indicates that they are similar. The lowest concentration of Ag-THC eliciting
insulin release is in the 200-500 nM range with increasing hormone released as
the cannabinoid concentration was increased to 15-20 pM. This similarity of
response suggests a common mechanism of action for A9-THC in each cellular
preparation.

A previous report (Laychock et al., 1986) showed an involvement of
arachidonic acid (AA) release and possibly Iipoxygenase products may be
involved in Ag-THC-stimulated insulin release. In the present study, the
involvement of several other, important intracellular signaling pathways for
insulin release were investigated to provide additional insight into the mechanism
of action by Ag-THC. The results demonstrated a marked elevation of [Ca2*]i is
Closely associated with Ag-THC-stimulated insulin release in RINm5F cells. This
finding is consistent with the accepted view that [Ca2+], is an important
intracellular messenger regulating insulin release from pancreatic beta-cells (
Seri et al., 2000; Fischer et al.,1999; Maechler et al., 1999; Lange and Brandt,
1993). The primary source of the increase in [Ca2+], appears to be the opening of
verapamil-sensitive calcium channels presumably on the plasma membrane of
the cell. Inhibiting the rise in [Ca2*]i with verapamil pretreatment (Fig.lll-3A) is
consistent with the demonstrated inhibitory action of the calcium channel blocker

on Ag-THC-induced insulin release from RINm5F cells (Fig.lll-3B) and from

114

isolated mouse pancreatic islets (data not shown). This result is also concordant
with data indicating an influx of extracellular calcium through voltage-dependent
L-type channels or voltage-independent Channels is required for insulin release
stimulated by many nutrient and non-nutrient secretagogues (Safaihi et al., 1997;
Boyd,1992)

Ag-THC-induced increases in [Ca2*]i could also occur by mobilization of the
ion from major intracellular stores such as those in the endoplasmic reticulum
(ER) of insulin producing cells. Pretreatment of RINm5F cells with two different
inhibitors of Ca” mobilization from the ER, thapsigargin and high ryanodine
concentrations, did not block the Ag-THC-induced rise in [Ca2+],. This result is
consistent with little or no role for the ER, the major intracellular storage site for
Ca”, as a source of the AQ-THC-induced rise in [Ca”],. Other possible sources of
the cannabinoid-induced rise in [Ca2+],, including mitochondrial and nuclear
stores, will be investigated in future experiments directed toward further
elucidation of the calcium signaling pathway.

Previously published reports indicate that AQ-THC treatment can alter
[Ca2+], in a variety of different cell types. Ovenlvhelmingly, the majority of these
studies have demonstrated an inhibitory effect of Ag-THC on the opening of
several types of calcium channels (Twitchell et al., 1997; Pan et al., 1996; Shen
and Thayer, 1998). In addition, Ag-THC caused a concentration dependent
inhibition of calcium mobilization elicited by caerulein in the exocrine pancreas

(Laychock et al., 1988). Several reports have also shown that a rise in [Ca”], is

115

observed upon A9-THC treatment In several cell types or cell line. For example, a
treatment-related increase of [Ca2+]i in DDT1MF-2 smooth muscle cells has been
observed (Filipeanu et al., 1997) and neuroblastoma x glioma hybrid cell NG108-
15 cells and N18TG2 cells showed a rapid increase in [Ca”], (Sugiura et al.,
1997). Considering these and other reports, it is likely that cell and/or species
specific effects may be prominent In the calcium signaling pathways responsive
to Ag-THC.

Increases In [Ca2*]i after Ag-THC exposure can be expected to activate a
variety of calcium-dependent enzymes. Among those, CaM kinase II (Easom,
1999) and PKC (Howell et al., 1994) have been extensively studied and reported
to be activated in RINm5F cells In response to known insulin secretagogues
(Gromada et al., 1999; Miura et al., 1998; Gregersen et al., 2000b; Tabuchi et
al., 2000). By using selective inhibitors of these two kinases, KN93 for CaM
kinase II and bisindolylmaleimide for PKC, the results of the present study
suggest a possible role of CaM kinase II in the calcium-dependent signal
transduction mechanism involved in Ag-THC-stimulated insulin release and rule
out the possible involvement of calcium-dependent PKC (Fig.lll-SB). While there
is evidence showing that the degree of specificity of KN-93 can be questioned,
results from preliminary experiments indicate that KN-93 when used at 10 pM did
not block a Ag-THC-induced increase in [Ca"’*]i (data not shown) but as shown in
Fig.llI-5A did inhibit the cannabinoid-induced insulin release (Fig.lII-5A). Thus,

attributing the inhibitory effect of KN-93 on insulin release to a calcium channel

116

blocking action, similar to that possessed by another CaM kinase inhibitor KN-
62, is not possible (Li et al.,1992).

To date, the tissue and cell type distribution of CB1 and CB2 has not yet
been comprehensively characterized. The results of the present study have
clearly demonstrated mRNA expression of CB1 cannabinoid receptor in isolated
mouse pancreatic islets and RINm5F cells by RT—PCR analysis (Fig.lll-6). These
findings are the first to identify pancreatic islet-cells as another periphery location
for CB1 receptor/ligand interactions. Notably, mRNA expression for the CB2
receptor was detected in RINm5F cells but not in mouse pancreatic islets by RT-
PCR analysis (Fig.lll-6). The reason why RINm5F cells but not islet cells possess
CB2 receptor mRNA may be a reflection of either the transformed phenotype of
the Cloned cells or differences among rodent species. In addition, the possibility
of CB1 or CB2 expression In endocrine cells within islets other than the beta-
cells cannot be determined from present studies and will require further
examination.

Based on the identification of CB1 transcripts in mouse pancreatic islets
and CB1 and CBZ transcripts in RINm5F cells, further studies were performed to
examine the potential involvement of cannabinoid receptors In Ag-THC-
stimulated Insulin release. One striking observation was that the CB1 antagonist,
SR141716A, antagonized insulin release Induced by Ag-THC in both mouse
islets and in RINm5F cells. Moreover the antagonism by SR141716A of the A9-
THC-induced insulin response in both cell preparations was concentration

dependent, suggesting an involvement of CB1 receptor. Additional experiments

117

also demonstrated that SR141716A was capable of completely abolishing the A9-
THC—induced increase In [Ca"’*]i in RINm5F cells, further supporting our premise
that the induction of insulin release by pancreatic beta-cells in the presence of
cannabinoid treatment is critically linked to the elevation of [Ca2+],. Although it is
not possible to discern from the present studies whether CB1 directly or indirectly
couples to calcium channels in the pancreas-derived cell preparations,
nevertheless the results suggest that the influx of calcium that is induced by A9-
THC is regulated through a CB1-dependent mechanism. To our knowledge, this
is the first report demonstrating that a cannabinoid ligand Induces calcium influx
through a CB1-dependent mechanism. Experiments employing the C32
antagonist, SR144528, showed no effect on Ag-THC-stimulated insulin release
from RINm5F cells (Fig.lll-7C) ruling out the involvement of CB2 receptors.

The findings from this present Investigation allow for some interesting but
speculative hypothesis to explain previously reported observations. From a
recent Investigation conducted in this laboratory it was reported that CD-1 mice
treated daily with AQ-THC exhibited protection from streptozotocin-Induced
diabetes (Li et al.,1999). This protective effect was partially due to a suppression
of the Immune-mediated destruction of beta-cells initiated by multiple low dose
administration of streptozotocin. It Is tempting to speculate that the
secretagogue-like effect of A9-THC reported here on insulin-producing cells in
vitro could also partly account for the reduction in streptozotocin-induced

hyperglycemia observed in this model in association with Ag-THC administration.

118

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CHAPTER IV

OVERALL SUMMARY AND CONCLUSION

120

Diabetes mellitus is recognized as the world's most common metabolic
disorder, affecting people worldwide and of all age groups. The severity of
diabetes is reflected both in its prevalence and economic impact. It is therefore of
great importance to understand the mechanisms of diabetes and to work toward
the discovery of improved clinical treatments. These two objectives are also the
ultimate goal of the study described in this dissertation. Toward this end, a series
of experiments were designed to evaluate the ability of Ag-THC, a cannabinoid
compound, to attenuate the MLDSTZ-induced autoimmune diabetes. Results
obtained from these studies not only provided insight into the potential role of
environmental Chemicals in the triggering of type I diabetes, but also contribute
to a better understanding of the pharmacological and toxicological profiles of
cannabinoid compounds.

In the current investigation the effect of AQ-THC on MLDSTZ-induced
diabetes was first examined in CD-1 mice, which is a mouse model of
autoimmune diabetes previously established by Like and Rossini. The results
from the present studies demonstrated a partial and transient protective effect by
Ag-THC on the development of diabetes in this model. It was apparent from the
cytokine profile that Ag-THC Inhibited a TH1-mediated immune response thereby
attenuating pancreatic beta-cell damage initiated by MLDSTZ treatment.
However, In spite of these data, It remains unclear whether Ag-THC would be
capable of attenuating the development of diabetes in the spontaneous diabetes
animal model and human Type I diabetes patients. As mentioned earlier, the
MLDSTZ-Induced diabetes model represents a diabetic condition in which the

121

primary etiopathologic effect is produced by an environmental chemical. In this
regard, etiopathogenesis in the MLDSTZ model is different from that underlying
the disease in NOD mice, and BB rats which may involve a genetically
programmed loss of tolerance to beta-cell specific antigens. Evidently, there is
no single cause of type I diabetes in man, with disease induction representing a
genetic susceptibility interacting with environmental triggers, such as toxins in
the diet as well as pathogenic viruses. In order to clarify whether the protection
produced by AQ-THC Is unique to this chemical-initiated diabetes model, future
studies will be necessary to determine whether Ag-THC or other cannabinoid
compounds can attenuate the disease process in animal models that develop
diabetes spontaneously. However, to manipulate the timescale and the
incidence of the diabetes development in those spontaneous models Is much
more challenging, therefore the MLDSTZ model was employed here in these
initial studies aimed at evaluating the protective effect of AQ-THC in a
reproducible model of chemical-induced autoimmune diabetes.

The present study further examined the effect of Ag-THC in MLDSTZ-
induced diabetes in 86C3F1 mice for comparative purposes. The B603F1
mouse strain is a commonly used animal model in toxicology studies and was
therefore employed in the present study to provide additional txicology-related
information concerning genetic bases for susceptibility and resistance to
diabetogenic environmental toxins. In contrast to CD-1 mice, BBC3F1 mice
exhibited a less pronounced hyperglycemia and the absence of insulitis after
MLDSTZ treatment and the protection by A9-THC was also less extensive. This

122

observation is consistent with the probability that MLDSTZ treatment Initiated
direct Irreversible beta-cell damage in certain animal strains, even in the absence
of a robust Immune response. These data supported previously reported results
that different animal species and strains exhibit a markedly different sensitivity to
MLDSTZ treatment In eliciting diabetogenic response. Accordingly it indicated
that MLDSTZ-induced diabetes is a heterogeneous disorder which Is dependent
on genetic factors as well as environmental factors.

It Is intriguing that MLDSTZ-treated B6C3F1 mice exhibited an absence of
insulitis as well as minimal protection by AQ-THC co-treatment in developing the
diabetic phenotype. This is in contrast to CD-1 mice which exhibited strong
insulitis and significantly greater protection by A9-THC. These results lead to two
speculations. The first being that MLDSTZ-induced diabetes likely consists of
two simultaneously mechanisms: direct cytotoxic action of STZ to Insulin-
producing cells which is coupled with the induction of an immune response
against insulin-producing cells. It was further suggested that the protective action
of Ag-THC in STZ-induced diabetes was through the inhibition of the autoimmune
response directed against the Insulin-producing cells. As a mattter of fact, In the
case of chemical-induced immune response such as In the model of MLDSTZ-
induced diabetes, It is difficult to separate the functional component of cell
damage or cell loss attributed to the direct cytotoxicity of the toxin from the extent
of functional impairment produced by cell-mediated immunity. In order to
distinguish which of the two putative mechanisms (eg. direct alteration of beta-

Cells and immune response) may be modulated by Ag-THC treatment, studies

123

were designed to examine the effect of Ag-THC treatment post STZ
administration in CD-1 mice. Surprisingly, these studies demonstrated that A9-
THC treatment was protective even when cannabinoid treatment was not
initiated until the STZ treatment regimen was concluded. It is important to
emphasized that the actions of STZ are extremely rapid in light of the fact that
the half life of this compound in an aqueous environment is approximately 15
min. Therefore the protective effects produced by A9-THC treatment beginning
on Day 6 are not attributable to an Inhibition of STZ cell uptake, altered
metabolism or disruption of Its alkylating properties. Combined with the results
observed in B6C3F1 mice, these results further support the contention that both
the direct cytotoxic action of STZ and the Immune-based elements contributed to
the diabetogenic effects of MLDSTZ treatment. Overall, these findings add to our
current knowledge in assigning relative importance to the components
participating in the ultimate beta-cell destruction in the MLDSTZ model.
Furthermore, these studies contribute to our understanding of the mechanism by
which environmental toxins elicits immune-mediated responses.

In addition to the immunosuppressive effect produced by A9-THC, another
contributing factor considered was a direct stimulatory effect of A9-THC on
pancreatic insulin-producing cells. In agreement with a previous report by
Laychock et al. using Isolated rat pancreatic islets (Laychock et al., 1986), the
present study showed that Ag-THC stimulated Insulin release from both CD-1

mouse islets and rat derived insulin-producing cell line. Although It is well known

124

that A9-THC produces a variety of biological effects on different cell types and
organs systems, there was relatively little information ayailable regarding the
effects of cannabinoids on the pancreas, particularly on insulin-producing cells.
For example, before our study, it was unclear whether there Is an expression of
cannbinoid receptors in the pancreas; whether A9-THC could alter another
important insulin-producing cell function, insulin synthesis; and if so, what were
the intracellular signaling pathways involved in Ag-THC-induced insulin release.
As an initial attempt to facilitate a better understanding of this undeveloped area,
important signaling pathways were characterized In the current investigation to
provide additional information on the mechanism of the A9-THC-stimulated insulin
release observed in this study. It was suggested from the data obtained here that
Ag-THC-stimulated insulin release is mediated through an elevation of
intracellular calcium through the opening of calcium channels via a CB1-
dependent mechanism. These findings expand present knowledge and may
stimulate future studies concerning the pharmacological and toxicological profile
of cannabinoid compounds.

However, it is important to note that results from our in vivo studies
presented in Chapter II showed that A9-THC treatment alone did not alter serum
glucose and pancreatic insulin in CD-1 mice not treated with MLDSTZ. An
insulin-releasing effect of A9-THC on mice treated the cannabinoid alone would
be expected to produce hypoglycemia and possibly a reduction in pancreatic

insulin. Several considerations should be made when analyzing these results

125

obtained in vivo. First, when possible A°-THC-Induced hypoglycemia was
examined in vivo in the present investigation, non-fasting serum glucose
concentrations were measured In the experimental animals. This measurement
is valid to detect a possible diabetic state, but within the normal physiological
range, blood glucose can vary markedly in non-fasted animals. Therefore, to
help assess a more precise picture of a possible insulin stimulatory effect of A9-
THC occurring in vivo, a thorough analysis of glucose and insulin levels, such as
a glucose tolerance test (GTT) after Ag-THC treatment, should be conducted In
future studies. Second, it is possible that only hyperglycemic animals would
respond to Ag-THC treatment-induced insulin release under a very high glucose
condition. This could explain why control mice treated with A9-THC alone did not
exhibit elevated pancreatic Insulin or reduced blood glucose. However, this
interpretation is countered by a previous observation (Laychock et al., 1986) and
the results obtained from the current study, showing an insulinotropic effect of A9-
THC at lower (e.g. fasting) glucose levels. It should be recognized that a
biological effect in vivo Involves a more complex system of insulin release and
blood glucose regulation. For example, in non-diabetic normal animals, where
the net result of hormonal regulation is capable of maintaining a normal hormone
balance, a Ag-THC-induced insulin release may be counteracted by actions of
glucagon and somatostatin. Hence, the blood glucose concentration measured
in normal CD-1 mice may remain within normal physiological limits upon Ag-THC

treatment. Third, it is possible that the effect of A9-THC on pancreatic beta-cell

126

functions may only involve a stimulation of insulin release with no modulation of
insulin synthesis. If insulin synthesis was not also modulated by AQ-THC, the total
insulin content in the pancreas may remain unchanged after treatment with A9-
THC. Therefore, It Is not surprising that the pancreatic insulin content measured
in CD-1 mice in our in vivo study was not altered by Ag-THC treatment, neither
was the blood glucose concentration.

In conclusion, our results demonstrate that Ag-THC treatment attenuated
the hyperglycemia produced by MLDSTZ treatment in CD-1 mice. Moreover, the
attenuation of MLDSTZ-induced hyperglycemia was partly due to the inhibition of
the immune response by THC directed against the pancreatic Islets. In addition
to the Immune suppression, our studies also suggest that Ag-THC exerts direct
effects on insulin-producing cells by directly stimulating the release of Insulin.
Stimulation of insulin release by Ag-THC is mediated through a CB1 dependent
mechanisms which involves the rapid rise of [Ca”], through verapamil-sensitive
channels and the activation of CaM kinase II. In spite of these data, it is difficult
to determine whether the protective effect of Ag-THC on MLDSTZ-induced
diabetes is due to modulation of the immune system and/or due to direct
alteration of beta-cell function since these two mechanisms are not easily
separated in vivo. In order to further ascertain the relative contribution of each of
these two mechanisms other approaches will be necessary. For example, in vitro
studies to further examine the effect of Ag-THC on insulin release and insulin

synthesis In MLDSTZ-treated mouse islets may present a direction for the future

127

investigations. Furthermore, the CD-1 scid\scid mouse currently being developed
at Charles River Laboratory would also be useful for future studies to test the
intervention protocol with A9-THC in the MLDSTZ model, because it lacks the

immune component.

128

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