An Update on the pathogenesis of Diabetes

Mellitus
M.F. Al Homsi and ML Lukic*
Department of Pathology and Medical Microbiology (Immunology Unit*)Faculty of Medicine and Health
Sciences, UAE University, Al Ain, United Arab Emirates

Introduction

The term "Diabetes mellitus" encompasses a heterogeneous group of disorders

characterized by insulin hyposecretion and/or insensitivity. Type 1 (insulin-dependent)
diabetes mellitus is thought to have an autosomal recessive inheritance and an autoimmune
pathogenesis, exhibited by lymphocytic insulitis. The latter may be triggered by viruses or
chemical agents. Type 2 (non-insulin-dependent) diabetes mellitus has a stronger genetic
association. It is associated with obesity. Hyalinization and fibrosis are seen in the
pancreatic islets of Langerhans, and the peripheral target cells are deficient in insulin
receptors.This paper summarizes current knowledge regarding pathology and
pathophysiology of both types of primary diabetes mellitus.

Pathogenesis of type I diabetes mellitus (type 1 DM)

Type 1 DM is a chronic autoimmune disease associated with selective destruction of

insulin-producing pancreatic β-cells. The onset of clinical disease represents the end stage of β-
cell destruction leading to type 1 DM. Several features characterize type 1 DM as an autoimmune
disease: [a] presence of immuno-competent and accessory cells in infiltrated pancreatic islets; [bl
association of susceptibility to disease with the class II (immune response) genes of the major
histocompatibility complex (MHC; human leucocyte antigens HLA); [c] presence of islet cell
specific autoantibodies; [d] alterations of T cell mediated immunoregulation, in particular in CD4+
T cell compartment; [el the involvement of monokines and TH1 cells producing interleukins in the
disease process. [f] response to immunotherapy; and [g] frequent occurrence of other organ specific
autoimmune diseases in affected individuals or in their family members. The mechanisms that
cause the immune system to mount a response to this small population of highly specialized cells
have been intensively studied. We shall summarize the recent evidence obtained in the clinical and
experimental studies on imununogenetics and immunopathogenesis of type 1 DM. These new
findings will justify the statement made by Shields Warren in 1930 "The pancreas in diabetes is not
simply the scarred field of an old battle-ground, but is the actual field of conflict. It does not
submit without a struggle to injury. This conception explains the gradual development of the
disease through slow but progressive destruction of the islands and the failure of regeneration to
make good the losses."

Immunogenetic determinants:

A variety of gene loci have been studied to determine their association with type 1 DM. The early
studies suggested that the B8 and B15 of HLA class I antigens were increased in frequency in the
diabetics compared to the control group. However, more recently the focus has shifted to the class
II HLA-DR locus.1 It was found that DR3 and DR4 were more prevalent than HLA-B in type 1
DM than HLA-B.2 Finally alleles of HLA DQ locus have been implicated in disease susceptibility.
Restriction fragment length polymorphism (RFLP) analysis and direct sequencing, using the
polymerase chain reaction (PCR) to amplify specific DNA sequences, have improved our
understanding of the HLA complex and of the involvement of HLA alleles in disease susceptibility.
Evidence was put forward indicating that the ability to confer susceptibility or resistance to type 1
DM resides within a single amino acid residue of the HLA-DQ β -chain. The use of locus-specific
oligonucleotide probes derived from HLA DQ β -chain sequences has helped also to clarify the
relationship between DR4 subtypes and type IDM associated DQ alleles. It was found that only
those DR4-positive haplotypes which carry the DQW8 allele at the HLA DQ locus are associated
with type 1 DM. Comparison of DQ- β -chain sequences from type 1 DM probands and controls
showed that haplotypes which were positively associated with the disease differ from those which
are negatively associated by the amino acid of position 57 in the first domain of HLA-DQ β -chain.
The positively associated haplotypes, have alanine, valine or serine at position 57.3 In
nonsusceptibility haplotypes aspartic acid was found at position 57.3,4 However, several
observations do not support the “position 57" hypothesis. Most important is definitely the findings
that specificities DQW4 and DQW9 have an aspartic acid in position 57; in Japanese patients type
1 DM is highly associated with DQW4 and DQW9.3 This indicates that other mechanisms must be
involved to account for the susceptibility to type 1 DM in some groups. The observed association
between type 1 DM and HLA has been interpreted as a consequence of a functional involvement of
HLA class II molecules in type 1 DM. The involvement of the DQ β -chain itself or the DQ α/β
heterodimer could indicate that the antigen presentation function of class II molecules is
relevant for type 1 DM susceptibility.

Following "epitope selection" approach to explain autoimmune phenomena Nepons5 have

suggested a model wherein HLA class II alleles affect IDDM susceptibility as follows: a)
the array of class II dimers encoded by any individual's HLA complex, vary in their
affinities for a specific peptide that can induce autoimmunity to β-cells; b) only certain
class II dimers, the products of "susceptibility genes," actually promote autoimmunity to β-
cells after binding that peptide, c) an individual is susceptible if the product of a
susceptibility gene binds the peptide more strongly than the products of the
nonsusceptibility genes present in that individual. Thus, in this model the products of
certain HLA alleles are associated with type 1 DM because they bind and present specific
peptides so as to induce an immune response to pancreatic β-cells. Sheeny6 had recently
argued an alternative model in which immunological tolerance is the primary determinant
of type 1 DM susceptibility. He suggested that the HLA alleles negatively associated with
type 1 DM (e.g. DR2, DQWI) code products with high affinity for certain β-cell peptide or
peptides needed to establish and maintain tolerance to β-cells, whereas the alleles that are
common in IDDM (e.g. DR3, DR4 and DQW8) produce products that have low affinity
for the tolerogenic peptide(s) or that bind the peptide(s) in the wrong orientation or
configuration for establishing tolerance. Thus, the susceptible HLA genotypes would he
those that "tolerize" ineffectively because of weak or otherwise ineffective binding of
tolerogen. To this it may be added that HLA related differences in the production of
inflammatory and down-regulatory cytokines may play a role in susceptibility to type 1
DM as recently argued in the regulation of autoaggression in the central nervous system.7
β -Cell specific autoantigens:

The nature of autoantigen(s) responsible for the induction of type 1 DM is unknown.

However, two recent lines of evidence are relevant to this issue. In an animal model of
type 1 DM, multiple low dose streptozotocin (MLD-STZ)-induced diabetes, Herold and
colleagues have shown that transplantation of isolated islets into the thymuses of adult
mice will induce immunologic tolerance to the subsequent induction of autoimmune
diabetes.8 These findings suggest that the non-responsiveness to induced autoimmune
diabetes was due to T cell maturation in the presence of islet antigens. This assumption is
supported by the findings that autoimmune diabetes may he prevented, in an animal model
of spontaneous type 1 DM, diabetes prone Bio Breeding (BB) rats, by intrathymic
transplantation of islet 9,10 Thus, islet antigen-reactive T cells have been either depleted or
inactivated in the thymuses with grafted islets. Failure to delete these T cells during
normal development may reflect the inaccessibility of islet antigens for antigen presentation
by MHC class II positive accessory cells because antigens are not shed or are present in
low concentration. It was already proposed that tolerance to islet cell autoantigens is
maintained simply by lack of efficient autoantigens presentation.11 It was also
demonstrated that the inactivation of different antigen specific T cell subsets depends on
self antigen concentration in the circulation.12,13 Antigens involved in type 1 DM include
64kD antigen, glutamic acid decarboxylase (GAD) and cytoplasmic islet cell antigens.
Cytoplasmic islet cell antibodies (ICA), bind cytoplasmic islet cell components on sections
of human pancreas and the 64kDa antibodies precipitate a 64kDa protein from islet cell
extract. Whereas 64kDa antibodies were shown to be β-cell specific within the islet, some
ICA-positive sera have been described to react with all islet cells. The target antigen of the
64kDa antibodies was identified as the enzyme GAD. In order to analyze the fine
specificities of ICA peripheral blood lymphocyte from prediabetic individuals and patients
with newly diagnosed type 1 DM have been immortalized by EBV transformation. 14 Islet
cell specific b-cell line producing IgG antibodies that bound to cytoplasmic islet cell
antigen(s) were established. Surprisingly all (six) monoclonal antibodies produced by these
lines, recognized GAD as target autoantigen. Thus, GAD may be a major target antigen in
type 1 DM. It was recently documented that antibodies to GAD are sensitive markers for
diabetes development but may also be present in genetically susceptible individuals who
are unlikely to develop disease.15 Limited trypsin diagestion of the islet 64,000-M antigen
yields three major poly-peptide products. It appeared that the presence of antibodies to
37,000M fragments were the best markers for diabetes development in genetically
susceptible twins.15 Antibodies reacting with insulin can be also detected in the clinically
latent prediabetic period. However, it appears that insulin autoantibodies have a lower
sensitivity as a marker for diabetes development than GAD antibodies or ICA. The
relative contributions of the above mentioned autoantigens to the induction and/or
perpetuation of disease remains to be clarified. It is clear, however, that the identification
of autoantigens in type 1 DM is essential both for diagnostic purposes and for potential
immunotherapeutic intervention in the disease process.

Environmental factors:
Despite strong evidence for an association with genetic factors, the concordance rate for
type 1 DM is surprisingly low in identical twins. This observation suggests that
susceptibility to the disease, rather than the expressed disease, is inherited. The lack of
100% concordance in identical twins for type I DM has contributed to a search for
environmental factors associated with the disease. The only clearly defined environmental
factor increasing the risk for developing type 1 diabetes is congenital rubella infection.,
where up to 20% of such children later in life develop diabetes. This observation, in
addition to the findings that an aminoacid sequence of DQ-bchain is also present in the El
envelope protein of rubella virus would support viral antigen mimicry as an etiological
factor in type I DM. The role of environmental factors is also suggested by an analysis of
immune responses to cow's milk proteins.16 Nearly all type 1 DM patients have antibodies
to a peptide of bovine serum albumin and demonstrate a T-cell response to it, compared to
only about 2% of control.

Immunopathogenesis:

The, pathogenesis of selective b-cell destruction within the islet in type 1 DM is difficult to
follow due to marked heterogeneity of the pancreatic lesions. At the onset of overt
hyperglycemia a mixture of pseudoatrophic islets with cells producing glycogen (a cells),
somatostatin (d cells) and pancreatic poly-peptide (PP cells), normal islets, and islets
containing both b-cells and infiltrating lymphocytes and monocytes may be seen. It should
be noted that lymphocytic infiltration is found only in the islet containing residual b-cells.
It is likely that the chronicity with which type 1 DM develops reflects this heterogeneity of
islet lesions. In contrast to this chronicity in the natural history of the disease, b-cells are
rapidly destroyed when pancreas is transplanted from identical twin donors into their long-
term diabetic twin mates in the absence of immunosuppression. In these cases massive
insulitis develops rapidly with infiltrating T lympocytes indicating an anamnestic
autoimmune reaction. In addition this observation also indicates that the chronic time
course in type 1 DM (but not in a transplanted pancreases) is a consequence of down
regulatory phenomena taking part in immunopathogenesis of the disease. These down
regulatory mechanisms have been analysed in the animal models of type 1 DM. The
existence of three animal models for type 1 DM, the BB rat, NOD mouse and MLD-STZ-
induced diabetic mouse, has improved ability to understand the processes leading to b cell
destruction. However, since all conclusions drawn from animal models are based on the
assumption of an analogy with human disease, the analogy needs detailed validation.
Activation of islet antigen - specific CD4+ T cells appear to be absolute prerequisite for
the development of diabetes in all animal models of type 1 DM.16 CD4+ islet specific T-cell
clones derived from diabetic NOD mice, when injected into prediabetic or
nondiabetesprone Fl mice, induce insulitis and diabetes. It was also reported that CD4+ T
cells are sufficient to induce insulitis while CD8+ T cells contribute to the severity of the
damage.17 These findings together with the evidence that insulitis in chronic graft versus
host disease may occur in the absence of CD8+ T cells suggest that CD4+ T cells may be
the only immunocompetent cells required in the disease process.18 However, it seems that
only one subset of CD4+ T cells is responsible for disease induction. CD4+ T cell bearing
alloantigen RT6 are absent in diabetes prone BB rats and appear to protect AO rats from
MLD-STZ induced diabetes.19,20 Down-regulation of diabetogenic autoimmune response
by the spleen cells derived from animals treated with adjuvants could also be explained by
CD4+ T cell subsets interplay.21 Preliminary results by Lafferty's group (to be published)
indicate that pretreatment with adjuvant does not block the autoimmune response per se,
but rather may deviate the response from TH-1 type to TH-2 type cytokine profile.
Indeed, high level of THI type cytokines IL-2 and interferon g are found to correlate
or/and to enhance induction of autoimmune diabetes in experimental models.22-24 The TH-1
type cells, and in particular their product IFN-g, activate macrophages. In animal models
of type 1 DM electron microscopic studies of pancreata showed that macrophages are the
first cell type invading the islets.25 In vitro studies and studies on perfused pancreas
suggest that Interleukin 1 (IL-1) and tumor necrosis factor (TNFa), two cytokines mainly
produced by macrophages, induce structural changes of b-cells and suppression of their
insulin releasing capacity.26,27 However, it seems that IL-1 and TNF do not contribute
appreciably to the cytotoxic activity of macrophages.28 Interferon g is also a powerful
activator of macrophages for nitric oxide synthesis. Recently, evidence has been provided
indicating that NO synthase activity is involved in diabetes development.29 These data
indicated, for the first time, that nitric oxide may be a pathogenic factor in autoimmunity
and suggested a possibility that a new class of immunopharmacological agents, capable of
modulating nitric oxide secretion may be tested in the prevention of type 1 DM
development.30

Pathogenesis of type 2(non-insulin-dependenO diabetes mellitus (type 2 DM)

Type 2 DM has a greater genetic association than type 1 DM. The 100% concordance
rate in identical twins is thought to be overestimated, due to a selection or reporting bias.
A population based twin study in Finland has shown a concordace rate of 40%.
Environmental effect may be a possible reason for the higher concordance rate for type 2
DM than for type 1 DM.31 Type 2 DM affects 1-2% of caucasians,32 but it is much higher
in some ethnic groups such as Pima Indians33 and Arabs,34 an approaches 50% in South
India. This indicates that genetic factors are more important than environmental factors.
Except for maturity onset diabetes of the young (MODY), the mode of inheritance for
type 2 DM is unclear.

MODY, inherited as an autosomal dominant trait, may result from mutations in

glucokinase gene on chromosome 7p. Glucokinase is a key enzyme of glucose
metabolism in beta cells and the liver.35,36 MODY is defined as hyperglycemia diagnosed
before the age of twenty-five years and treatable for over five years without insulin in
cases where islet cell antibodies (ICA) are negative and HLA-DR3 and DR4 are
heterozygous. MODY is rare in Caucasians, less than 1 %, and more common in blacks
and Indians, more than 10% of diabetics. Chronic complications in MODY were thought
to be uncommon but later were found to be more common, indicating its heterogeneity.
Considering MODY as a separate entity may masquerade its association with specific
genetic diseases; and without a definite genetic marker, it should be treated as type 1
DM.37 Identification of a nonsense mutation in the glucokinase gene and its linkage with
MODY was reported for the first time in a French family, implicating a mutation in a
gene involved in glucose metabolism in the pathogenesis of type 2 DM .38 Later, sixteen
mutations were identified in 18 MODY families. They included 10 mutations that
resulted in an amino acid substitution, 3 that resulted in the synthesis of truncated
protein, and 3 that affected RNA processing. Hyperglycemia in these families was
usually mild and began in childhood, whereas the hyperglycemia of MODY families
without glucokinase mutations usually appeared after puberty.35

Molecular genetic studies in type 2 DM, with the exception of MODY, have not been as
successful as in type 1 DM. Mutations in the insulin gene lead to the synthesis and
secretion of abnormal gene products, leading to what are called insulinopathies.39 Most
of the patients with insulinopathies have hyperinsulinemia, inherited in autosomal fashion,
heterozygous for normal and mutant alleles, and normally respond to exogenous insulin
administration. Most insulin gene mutations lead to (a) abnormal insulins. such as
insulins Chicago and Wakayama where the mutation leads to an amino acid replacement
at an important site for receptor interaction; or (b) the mutation may interfere in the
proinsulin processing to insulin.41,41 The association of the polymorphic (hypervariable) 5'
flanking region of the human insulin gene and type 2 DM is lacking in some population
groups, indicating that it may be one of many factors in a multifactorial disease. Even
MODY patients have shown no association with this region. It was mentioned earlier
that there is a strong association between HLA-DR3/4 and type 1 DM. It was also
reported that such an association is present with type 2 DM, rendering HLA-DR3/4
markers for beta cell destruction in these patients .34,42

Pancreatic abnormalities in islet secretory cells in type 2 DM are noted in beta, alpha and
delta cells of the islets. Defects involving insulin secretion include relative decrease in
basal secretion, decreased first and second phases of insulin response, glucose
insensitivity and amino acid hypersensitivity of insulin release. Number and volume of
beta cells are usually decreased to half of the normal, and, the alpha cell mass is increased
leading to hyperglucagonemia. The islets exhibit hyalinization and amyloid deposition,
containing islet amyloid polypeptide (IAPP) or amylin. This is a minor secretory peptide
of the beta cells, released along with insulin and C-peptide, but its role in the
pathogenesis of type 2 DM is not well understood .43 This amylin is thought to produce
insulin resistance.44 IAAP is reduced with progression of type 2 DM .45 Intimate contact
between beta cells and amyloid deposit in type 2 DM is noted by electron microscopy.46
Away from the islets in the exocrine pancreas, fatty infiltration and diffuse fibrosis are
evident. Defective islet cell function is the primary event which may be due to an
autoimmune reaction producing hyperglycemia in type 2 DM . 47

The insulin receptor gene is located on chromosome 19 and it encodes a protein having
alpha and beta subunits including the transmembrane domain and the tyrosine kinase
domain.48 Mutations affecting the insulin receptor gene have been identified and their
association with type 2 DM and type A insulin resistance is recognized. Type A insulin
resistance is hereditary and type B is an autoimmune disorder .49 Restriction fragment
length polymorphism (RFLP) analysis of the insulin receptor gene,50 erythrocyte glucose
transporter gene and HLA genes were not found useful as genetic markers for type 2
DM.

Insulin resistance is insufficient to cause overt glucose intolerance, but may play a
significant role in cases of obesity where there is known impairment of insulin action.
Insulin resistance per se may he a secondary event in type 2 DM, since it is also found in
non-diabetic obese individuals. Insulin secretion defect may be the primary event,
presenting as impaired pulsatile secretion of insulin. Hence, hyperglycemia is an inducer as
well as a consequence of impaired islet cell function and insulin resistance. Many factors
contribute to the insulin insensitivity. including obesity and its duration,51 age, lack of
exercise, increased dietary fat and decreased fibres, and genetic factors. Fish oil is found
to prevent insulin resistance in animals, but not in humans. It has a protective effect
against thrombosis and vasospasm in type 2 DM.52

Insulin resistance in type 2 DM is not totally clear, it may involve reduced insulin receptor
number, it may be secondary to hyperinsulinemia and hyperglycemia,53 or it may result
from reduced tyrosine kinase activity 54,55,56 or even abnormalities distal to the receptor
involving glucose transporter proteins through a family of glucose transporter genes.57 The
GLUT2 gene, expressed in liver and pancreatic beta cells, and GLUT4, expressed in
skeletal muscle and adipocytes, are strong candidate genes for the genetic susceptibility to
type 2 DM (reviewed in 48a). Analysis of these two glucose transporter genes, in addition
to GLUTI, encoding for the brain/erythrocyte glucose transporter, has yielded, in
Caucasians, no association of any RFLP marker on haplotype with either type 2 DM or
obesity.58

Obesity has genetic as well as environmental causes. It has a strong effect on the
development of type 2 DM,59,60 as it is found in western countries 61,62 and some ethnic
groups such as Pima Indians. 63,64,65,66 Obesity is more than just a risk factor, it has a causal
effect in the development of type 2 DM against a genetic background. The evolution from
obesity to type DM results from a succession of pathophysiological events: (a)
augmentation of the adipose tissue mass, leading to increased lipid oxidation; (b) insulin
resistance noted early in obesity, revealed by euglycemic clamp, as a resistance to insulin-
mediated glucose storage and oxidation. blocking the function of the glycogen cycle;
(c) despite maintained insulin secretion, unused glycogen prevents further glucose
storage leading to type 2 DM; (d) complete b-cell exhaustion appears 1ater. 67

Type 2 DM patients have a characteristic shoulder, girdle-truncal obesity. When

compared with the gynoid., peripheral type obesity, patients who have android.,
central~type of obesity, show a higher rate of insulin resistance., type 2 DM,68
hyperlipidaemia., hypertension and premature mortality. Furthermore, the fat cells in
shoulder-girdle-truncal obesity expand and shrink in response to calorie intake; but in
pelvic-girdle obesity, the fat cells do not show such a response.

Nutrient composition has also been found to be a risk factor for developing type 2 DM,
where increased fat and decreased carbohydrate consumption have contributed to
 hyperinsulinemia of obesity. Dietary fibres, both soluble and insoluble, improve type 2
DM. It is also found that simple sugars do not directly cause diabetes. Deficiency of
micronutrients, such as chromium and copper, is found to be an important cause of type 2
DM in a minority of cases.

Stress has also been thought to induce type 2 DM. Electroconvulsive therapy has been
tried, inconclusively, to improve the disease in depressed patients.69 Actually,. obesity and
over-availability of food rather than stress, are the contributing factors to type 2 DM.
Therefore, when permanent change in dietary habits is established, some people should be
allowed to escape the "life-long" diagnosis of type 2DM.70

In conclusion. it is said that both types 1 and 2 DM have a genetic background; however,
we are unable to define the genetic lesion and the mode of inheritance of this disease.
Type 1 DM has a high discordance in identical twins, indicating that the nuclear gentic
component per se is not sufficient for full penetrance; therefore it is most likely due to
environmental changes. Type 2 DM exhibits several features of a degenerative disorder
and can thus possibly be attributed to a variety of degenerative processes associated with
defects in oxidative phosphorylation. Hence, the possibility that environmental factors
could preferentially affect the second human genome, the mitochondrial DNA, thus leading
to metabolic, immunologic, genetic and phylogenetic alterations.71

Acknowledgements

This paper was presented in part by one of us (MFH) at the 1992 meeting on 'Update in
Diabetes', Abu Dhabi, UAE. We thank Dr. M. Agarwal for the critical review of the
manuscript, and M. Channon and P.K. Ratnayaka for the secretarial assistance.

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