Annals of Medicine.

2005; 37: 213–221

Molecular mechanisms of mitochondrial diabetes (MIDD)

JOHANNES A. MAASSEN, GEORGE M. C. JANSSEN & LEEN M. ’T HART

Department of Molecular Cell Biology LUMC, Leiden University Medical Centre, Leiden, The Netherlands

Abstract
Mitochondria provide cells with most of the energy in the form of adenosine triphosphate (ATP). Mitochondria are
complex organelles encoded both by nuclear and mtDNA. Only a few mitochondrial components are encoded by mtDNA,
most of the mt-proteins are nuclear DNA encoded. Remarkably, the majority of the known mutations leading to a
Ann Med Downloaded from informahealthcare.com by Laurentian University on 03/21/13

mitochondrial disease have been identified in mtDNA rather than in nuclear DNA. In general, the idea is that these
pathogenic mutations in mtDNA affect energy supply leading to a disease state. Remarkably, different mtDNA mutations
can associate with distinct disease states, a situation that is difficult to reconcile with the idea that a reduced ATP production
is the sole pathogenic factor. This review deals with emerging insight into the mechanism by which the A3243G mutation in
the mitochondrial tRNA (Leu, UUR) gene associates with diabetes as major clinical expression. A decrease in glucose-
induced insulin secretion by pancreatic beta-cells and a premature aging of these cells seem to be the main process by which
this mutation causes diabetes. The underlying mechanisms and variability in clinical presentation are discussed.

Key words: A3243G, MIDD, mitochondrial diabetes, mutations, tRNA

For personal use only.

Introduction: Physiology of glucose is suppressed by inhibition of glycogenolysis and

homeostasis
In diabetes, the complex interplay between the rates
of glucose appearance and glucose disappearance in
the circulation is deregulated and a state of persistent
hyperglycaemia develops. This review will discuss
underlying mechanisms leading to mitochondrial
diabetes.
Glucose homeostasis in vivo is maintained by an
interaction between glucose disposal and glucose
appearance as outlined in Figure 1. The rate of
glucose disposal to most tissues is largely insulin
independent and mediated by glucose 1 (Glut1)
transporters. The flux of glucose to these tissues
through these transporters is determined by the
actual glucose concentration in the blood. Only
muscle and adipose tissue are regulated by insulin
with respect to glucose uptake because these tissues
express the insulin-sensitive Glut4 transporter. The
number of Glut4 transporters on the cell surface of
these tissues is increased by an increase in serum
insulin. As a result insulin enhances glucose disposal
in muscle and fat and thereby contributes to
lowering of circulating glucose levels (Figure 2).
The liver is also regulated by insulin. At high
insulin levels, the production of hepatic glucose
gluconeogenesis (Figure 1). The combination of
these insulin-dependent processes in muscle, fat
and liver results in a lowering of blood glucose (1).
In the normal situation, fasting glucose levels are
kept at approximately 5.5 mmol/l. Diabetes develops
if the balance in the body between glucose disposal
and glucose appearance is deregulated leading to
persistently elevated glucose concentrations. A
number of pathophysiological mechanisms can
lead to diabetes. These include secretion of an
inappropriate amount of insulin by the pancreas.
Alternatively, the pancreas may produce large
amounts of insulin but due to the presence of insulin
resistance, a state in which tissues respond poorly to
insulin, glucose levels remain high.
The concentration of circulating insulin is deter-
mined by a complex glucose sensing system in
pancreatic beta-cells which senses the concentration
of glucose in the circulation (2,3). At high glucose
levels, insulin secretion is enhanced (Figures 1 and
3). All these physiological mechanisms may be
affected by the presence of diabetogenic mutant
mitochondria and this review will discuss recent
developments on the identification of the disease
mechanism.

Correspondence: J. A. Maassen, Wassenaarseweg 722333 AL Leiden, The Netherlands. Fax: +31 71 527 6437. E-mail: j.a.maassen@lumc.nl
ISSN 0785-3890 print/ISSN 1365-2060 online # 2005 Taylor & Francis Ltd
DOI: 10.1080/07853890510007188
 214 J. A. Maassen et al.

Abbreviations Key message

ADP adenosine diphosphate N Some mtDNA mutations lead to pancreatic
ATP adenosine triphosphate beta-cell dysfunction and diabetes
Glut glucose transporter
Melas mitochondrial myopathy,
encephalopathy, lactic acidosis defect in mitochondrial diabetes resides in the
and stroke-like episodes pancreatic beta-cells where somehow the function-
MIDD maternally inherited diabetes and ality of the glucose sensor or the capacity of the cells
deafness to secrete insulin is impaired. Actually, this gradual
MODY maturity onset diabetes of the decline in functionality resembles an enhanced
young ageing of the pancreatic beta-cell. Upon ageing, in
PCR polymerase chain reaction
TMRA thiamine responsive
megaloblastic anaemia
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Involvement of mitochondrial DNA mutations

in the development of diabetes
When we made our initial discovery on a family with
mitochondrial diabetes for which we proposed the
name ‘maternally inherited diabetes and deafness’
(MIDD) (4) we reasoned that diabetes in these
For personal use only.

individuals may develop by an inappropriate oxida-

tive disposal of glucose in peripheral tissues leading
to enhanced lactate fluxes to the liver thereby lead-
ing to hyperglycaemia via gluconeogenesis (5).
Subsequent studies showed, however, that this may
be only one contributing factor but that the main
Figure 2. Glucose disposal into cells. Glucose is taken up by cells
by different glucose transporters, Glut1 and Glut4 being most
prominent. Glut1 is more or less constitutively present on the
surface of cells and contributes to basal glucose uptake. In fat and
muscle an additional glucose transporter Glut 4 is present. At low
insulin levels, this transporter is inside the cell and not active in
transport. After a rise in insulin, the transporter translocates to the
cell surface and allows cells to take up more glucose.

Figure 1. Overview of steps contributing to maintenance of

correct glucose levels in the body. Glucose can emerge in the
circulation either from food or from hepatic glucose production
whereas tissues consume glucose. In muscle and fat, glucose
uptake is strongly stimulated by insulin. Other tissues take up
glucose independently of insulin. Furthermore, elevation of
insulin levels inhibits hepatic glucose output. The concentration
of glucose in the circulation is sensed by the pancreatic beta-cells
which start to secrete more insulin in response to a rise in blood
glucose.
Figure 3. Mechanism whereby pancreatic beta-cells sense glucose
and regulate insulin secretion. Glucose is taken up in pancreatic
beta-cells and converted into glucose 6 phosphate (G6K) by a
specific hexokinase isoform glucokinase (GK). After metabolism
in glycolysis and mitochondria, the resulting glucose-induced
increase in ATP/ADP ratio closes a specific K-channel in the
pancreas. The subsequent depolarisation of the plasma membrane
opens a Ca-channel which triggers the release of vesicles contain-
ing the insulin. LDH5lactate dehydrogenase.
 Mitochondria and diabetes 215

Table I. Overview of mitochondrial DNA point mutations associated with diabetes mellitus

hetero-(het) or homo- Diabetes as prominent associating

Mutation plasmic (hom) mutation phenotype Reference

T3200C, 16S rRNA gene s, hom 2 (50)

A3243G, tRNA(Leu,UUR) m, het + (37)
C3254G, tRNA(Leu,UUR) s, het + (51)
C3256T, tRNA(Leu,UUR) s, het + (52,53)
T3264G, tRNA(Leu,UUR) s, het + (54)
T3271C, tRNA(Leu,UUR) m, het 2 (55,56)
G3316A, ND1 m, hom + (57,58)
T3394C, NADH dehydr m, hom + (59)
T3398C, NADH dehydr m, het + (60)
G4284A tRNA(Ile) s, het + (61)
7472insertC, tRNA(Ser,UCN) m, het 2 (62)
8281 heterogeneous length variant s, het + (in preparation)
A8296G tRNA(Lys) m, het + (63)
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A8344G tRNA(Lys) m, het 2 (64)

T8356C tRNA(Lys) m, het 2 (65)
A10438G tRNA(Arg) m, het 2 (66)
C12258A tRNA (Ser,AGY) s, het 2 (67)
T14577C s, het + (68)
A14693G tRNA(Glu) s, hom + (69)
T14709C, tRNA(Glu) m, het 2 (70,71)
T16189C, hypervariable region m, het 2 (72)

s5found in single pedigree; m5seen in multiple pedigrees.

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most individuals a gradual decline of insulin secre-

tion takes place (6), especially when risk factors are
present such as obesity. In carriers of the A3243G
mutation the rate of this decline seems enhanced (7).
Approximately 85% of carriers of the A3243G
mutation will develop diabetes before the age of 70
indicating that we are dealing with a high penetrance
diabetogenic mutation. The mutation is strictly
maternally transmitted and mostly all children from
an affected mother will become carriers of the
mutation. Next to the A3243G mutation, many
other point mutations, mostly in tRNA genes, have
been identified to represent a risk factor for diabetes,
next to various other co-morbidities. They are listed
in Table I. Their frequency is much lower compared Figure 4. Overview of the pedigree in which apparently a de novo
to the A3243G mutation. Also, some nearly homo- A3243G mutation has occurred.
plasmic mutations have been suggested to modify
the risk for diabetes or diabetes-related parameters. testing did confirm the relationship within the
These variants are also summarised in Table I. family. It is remarkable that high heteroplasmy levels
It is unclear why the A3243G mutation is such a were present in the proband, 55% in mouth mucosa
hotspot for the development of a mutation. For cells and 18% in leucocytes, indicating that in one
example, studies in Finland based on haplotype step during embryogenesis heteroplasmy levels can
analysis indicate that in the Finnish population the greatly increase (Figure 4) (10).
mutation has emerged at least nine times (8). Deletions in mitochondrial DNA also associate
Furthermore, it has been shown that the A3243G with diabetes, mostly with a juvenile-onset, type 1-
mutation accumulates in individuals in response to like diabetes. (Pearson syndrome, Kearns Sayre
environmental stress upon ageing such as hypergly- syndrome) (11,12). These diseases are extremely
caemia (9). We recently identified a Turkish family rare. Affected children die generally before adult-
in which apparently in the affected proband the hood. In most cases these deletions are sporadic with
A3243G mutation is present as de novo mutation as little risk for the other siblings (13,14). A large
all other family members were negative. Paternity deletion in mtDNA of 10.4 kb originally described
 216 J. A. Maassen et al.

by Ballinger, associates with type 2-like diabetes
as in A3243G carriers (15). However, only one
pedigree has been identified with this deletion. This
deletion remarkably shows maternal transmission. It
is unclear why deletions in mtDNA involved in
Pearson/Kearns Sayre syndrome lead to such a rapid
loss of insulin secretion whereas point mutations in
tRNA and other deletions lead to a gradual loss of
insulin secretion at a more elderly age.
Pathobiochemistry of the A3243G mutation
The consequences of the A3243G mutation for the
function of the mt tRNA have been examined in
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heteroplasmic mutations have been described and
most heteroplasmic mutations are associated with a
disease state (reviewed in (11)). Furthermore,
biochemical studies on cells repopulated with
mutant mitochondria via the cybrid cell technology,
as originally developed by Attardi and co-workers
(18) often show clear biochemical defects in those
cells such as defects in oxygen consumption. These
findings support the notion that these mutations are
indeed pathogenic.
A number of homoplasmic variants have been
identified in mtDNA and there has been much
speculation whether they contribute to a particular
clinical phenotype such as diabetes. In case these
mutants affect only mildly cellular metabolism they

multiple studies. As such the mutation reduces

are not detected as being pathogenic by the cybrid
charging of the tRNA (Leu, UUR) but not to such
technology. If these genetic variants have a high
an extent that mitochondrial protein synthesis is
carrier frequency in the general population, one may
fully inhibited (16). Subsequent elegant studies by
use genetic association as an alternative approach to
the group of Florentz et al. have identified the
show an involvement of these mtDNA variants in
domains in tRNA that are needed for optimal
vulnerability for diabetes. In this approach, a large
charging of the tRNA by Leu-tRNA synthase (17).
population is analysed for the presence of the
We are currently analysing the consequences of the
mtDNA variant as is the distribution of the diabetes
presence of mitochondria with the A3243G muta-
state in this cohort. If the population used is large
For personal use only.

tion in cybrid cell lines on nuclear gene expression

(i.e. nw2000) this approach is sensitive to detect
patterns in order to understand the pleiotropic
relatively small in vivo effects of the gene variant but
consequences of this mutation for the phenotype.
it is also vulnerable to artefacts in case of genetic
A microarray-based approach covering all known
admixture. This method is only applicable in case of
genes was applied. The presence of the A3243G
common mutations with allele frequency w0.10
mutation affects gene expression of gene clusters and
such as the common variant at 16189 (19,20).
some individual genes. A distinct gene cluster such
During a genetic analysis of a pedigree with
as for complex 1 is coordinately affected in expres-
diabetes and deafness we observed a novel mutation
sion by the mutation (Roshan Tafrechi, manuscript
which comprises a heteroplasmic heterogeneity in
in preparation). Remarkably, the maximal rate of
the sequence of mtDNA at 8281 and 568 (G.M.C.
cytosolic protein synthesis also gets reduced by this
Janssen, manuscript submitted). We were unable to
deregulated expression in the cybrid cells. If this also
detect this mutation in 200 additional pedigrees
happens in beta-cells it may explain the attenuated
excluding it as being a common polymorphism.
response of insulin synthesis and secretion by
Cybrids were developed to detect functional con-
pancreatic beta-cells periods of high demand for
sequences of this length variant but no abnormalities
insulin. Also the somewhat dystrophic phenotype of
in oxygen consumption and mt-protein synthesis
many carriers of the A3243G mutation may be
were detected. Thus, it was unclear whether we are
related to the suboptimal rate of protein synthesis by
dealing with a neutral polymorphism. We developed
the patient’s cells. One may speculate that not only
a novel method to further analyse functional con-
the A3243G-related change in ATP/ADP ratios, as
sequences of this mtDNA mutation. This approach
discussed below, is responsible for decreased insulin
is based on measuring the serum-induced prolifera-
secretion but that a reduced rate of insulin synthesis
tion rate of cybrids with different types of mutant
may also be a contributing factor.
mtDNA. When cybrids from the same clonal donor
cell with the different 8281-heterogeneities were
cultured, there was a clear proliferative disadvantage
How to analyse other mtDNA mutations in
for cells with the 8281-heterogeneity indicating that
diabetes patients for pathogenicity?
the mutation is certainly not neutral. Table I
MtDNA is highly polymorphic. Most deviations summarizes the mutations in mtDNA that have
from the published sequence are homoplasmic and been implicated in the literature to disease states
are probably true polymorphisms. Pathogenic muta- with diabetes as co-morbidity. One should realise
tions tend to be heteroplasmic. A number of these that with most mutations, diabetes is a co-morbidity

and that other clinical abnormalities are more
prominent.
Biochemical mechanism involved in MIDD
Glucose is taken up by pancreatic beta-cells via
an insulin-independent pathway, thus circulating
plasma glucose concentrations determine the con-
centration of glucose available for metabolism within
the pancreatic beta-cell. Via glycolysis and mito-
chondrial metabolism, high glucose leads to high
ATP/ADP ratios within the cell. This ratio is sensed
by subunits of the ATP-sensitive potassium channel.
At high ATP/ADP ratios, the channel is closed
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leading to a change in the membrane potential over
the plasma membrane and opening of a Ca-channel.
The resulting influx of Ca-ions triggers the release of
insulin (Figure 3). Furthermore, Ca is a cofactor for
enzymes of the citric acid cycle and may enhance
ATP production by activating the citric acid cycle.
An additional possibility is that the rate of protein
synthesis in the beta-cells gets limited by the
presence of mutant mitochondria leading to reduced
For personal use only.
insulin synthesis at high insulin demand.
Any change in the biochemical pathway linking
glucose uptake and ATP generation has the potency
to alter the efficiency by which glucose stimulates
insulin secretion. A well known illustration for this
concept is the observation that heterozygous muta-
tions in the gene for glucokinase (the rate-limiting
gene of the glycolytic flux) results in diabetes called
‘maturity onset diabetes of the young’ (MODY-2)
(2,21) due to inappropriate insulin release. Pyruvate
dehydrogenase with thiamine as cofactor is also a
critical factor in the final step of the glycolytic flux
as judged from the existence of the syndrome of
thiamine-responsive megaloblastic anaemia syn-
drome TRMA, which associates with diabetes
(22). Furthermore, impairment of other components
in the glucose-sensing pathway (e.g. Ca), and
mitochondrial toxins such as azide or cyanide are
often associated with hyperglycaemia (23). As
pancreatic beta-cells lack lactate dehydrogenase all
the glucose that is taken up will be metabolized
through the mitochondria making the ATP/ADP
ratio a correct reflection of circulating glucose (24).
A number of in vitro studies have indicated that
removal of mitochondrial DNA from pancreatic
beta-cells leads to a loss of glucose-induced insulin
secretion (25). Though underscoring the importance
of proper mitochondrial function and in line with a
correct ATP/ADP ratio as being a determinant for
insulin secretion, these studies do not explain the
gradual loss of insulin secretion in humans with the
A3243G mutation. In addition mice were generated
Mitochondria and diabetes 217
Figure 5. Summary of the steps by which the A3243G mutation
may contribute to a reduced glucose-induced secretion of insulin.
with a deletion in mtDNA in pancreatic beta-cells.
These mice showed a number of abnormalities
which differed between type of mutation and back-
ground strain (26,27,28) Interestingly, not all the
strains developed diabetes.
Also other nuclear genes involved in mitochondrial
function have been implicated in changes in beta-cell
function (Figure 5). An example is the length variant
in the nuclear-encoded frataxin gene. Frataxin is a
protein involved in iron homeostasis within mitochon-
dria. Certain severe mutations are involved in
Friedreichs ataxia, a disease characterized by neuro-
logical abnormalities with diabetes as co-morbidity
(29). Non-pathogenic variants in the gene have been
shown to affect the insulin secretory response during
glucose stimulation of the pancreas in vivo in humans
indicating a critical role for mitochondrial iron
metabolism in insulin secretion (30). Furthermore,
additional signalling molecules, such as glutamate,
have been proposed in linking mitochondrial function
to the stimulation of insulin secretion (23).
The A3243G mutation interferes with the rate of
charging of the tRNA(Leu, UUR) (16,31) and may
also reduce the capability of cells to respond to an
enhanced need for protein synthesis. An unexplained
situation remains, however, that mutations in gluco-
kinase and also deletions in mtDNA are already
clinically manifest early in life, whereas diabetes
associated with the A3243G mutation becomes
manifest at the average age of 38 years. This has led
to the postulate that the A3243G mutation results in
an accelerated ageing of mitochondria rather than in a
direct mitochondrial defect. Apparently, partially
different biochemical mechanisms are responsible for
the early-in-life manifestation of glucokinase and
mtDNA deletion-associated diabetes on one hand
and the A3243G mutation on the other.
Clinical aspects of MIDD
Classically, mutations in mitochondrial DNA have
been seen in neuromuscular disorders. This is
 218 J. A. Maassen et al.
plausible as in non-dividing tissues there is no
selection against cells with high heteroplasmy during
cell division. Muscle and brain are examples of such
tissues and muscle shows in general high hetero-
plasmy levels. In addition, these tissues have a high
energy demand. Thus they are inferred to be most
sensitive to ATP depletion.
The clinical appearance of MIDD has been
discussed previously and recently an excellent over-
view has been published on French MIDD patients
(31,32,33). Thus, only some general data are given
in the current review. Mitochondrial diabetes is
mostly a clinically unremarkable form of diabetes
with some patients becoming rapidly dependent on
insulin treatment and, before genetic testing, often
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diagnosed as type 1 patients. Other patients can be
managed by diet or oral hypoglycaemic agents for a
while and they are often diagnosed as having type 2
diabetes. Obesity is not seen as a major additional
factor. After many years of diabetes, all carriers tend
to become dependent on insulin.
The frequency of mitochondrial diabetes in the
diabetes population depends on the ethnic origin.
In the Netherlands we observed a frequency of
For personal use only.
approximately 0.7% among type 2 diabetics. Other
European countries, as emerged from a survey in the
UKPDS cohort, showed frequencies of 0.1%–0.2%
(34). In Japan frequencies of 1%–2% have been
reported (35) and in China, a frequency of 0.3% was
observed (36). A complicating factor in the inter-
pretation of the frequencies in these cohorts is the
possible presence of a selection bias. If one selects
for individuals with onset of diabetes at an elderly
age, as is typical for the type 2 diabetes, one may
select against patients with MIDD who have been
often classified as type 1 patients because of their
young age of onset. In contrast to what one would
expect, the rate at which diabetes develops as
reflected by age of onset is hardly dependent on
the degree of heteroplasmy of the mutation in
leucocytes. This observation fits with a situation
that multiple factors in the body contribute to the
disease process rather than only an impaired ATP
production. Impaired hearing is a characteristic co-
morbidity in carriers of the A3243G mutation which
helps in the identification of MIDD patients. It
seems again that this is a reflection of the accelerated
ageing process in these carriers. Furthermore, the
strong familial clustering with maternal inheritance
helps in diagnosing the disease.
Next to diabetes and impaired hearing a number
of other co-morbidities are sometimes seen to
associate with the A3243G mutation. Originally
the mutation was discovered in patients with the
MELAS syndrome (37). This is a neuromuscular
disease. Also cardiomyopathy (38), Alport-like
kidney failure and other co-morbidities have been
reported (39,40). Why this mutation exhibits such a
wide variability in clinical expression is unknown.
The distribution of the mutation load over individual
tissues and also the degree of mutation load may be
factors that contribute to the phenotype. Only a few
post mortem studies have examined this aspect and
the results are not conclusive (41,42,43). Also the
mtDNA haplotype does not seem to contribute to
the clinical phenotype (44). In our patient group
which consists of ,120 patients, neuromuscular
abnormalities are rare. However, our patients were
selected through endocrinology departments which
may introduce a selection bias. Furthermore, the
lack of a suitable animal model system for the
A3243G mutation is a major drawback in under-
standing the pathophysiology of this mutation.
The mechanism by which the impaired hearing is
associated with the A3243G mutation is at this
moment unclear. There are a large number of
different genes that associate with deafness (45–
47). In the inner hair cell of the ear a similar
coupling occurs between K-channels of the KCNQ4
type and Ca channels as in pancreatic beta-cells and
it is suggestive that in both organs this coupling may
be affected in a similar way by the A3243G mutation
(46).
For detection of the A3243G mutation in patients
which is generally based on Apa1 cleavage of a PCR-
fragment of mtDNA, leucocyte DNA is generally
appropriate. Heteroplasmy levels can be very low in
some patients, e.g. 1%–3% which requires the use of
sensitive detection techniques. It is advisable to
include also mouth mucosa DNA as this tissue has,
in general, higher heteroplasmy levels than leuco-
cytes (48,49).
MIDD patients show heteroplasmy levels in
leucocytes between ,1% and ,40%. We still do
not know whether heteroplasmy levels below ,1%
also are risk factors to develop MIDD as these low
levels were previously not detectable. In this respect
it is of interest that a new method was developed to
detect low levels of the A3243G mutation.
Remarkably, the authors observed that low hetero-
plasmy levels of the A3243G mutation emerged as
somatic mutation during ageing (9).
In summary
Several mitochondrial DNA mutations have the
tendency to make the human organism more
vulnerable to developing diabetes. Most of these
mutations are rare, only the A3243G mutation is
consistently detected in 0.1%–1.5 % of the diabetic

cases throughout the world. The exact mechanism
by which this mutation causes diabetes and other co-
morbidities is still unknown though the main defect
leading to diabetes seems to reside in a more rapid
deterioration of pancreatic beta-cells to secrete
sufficient amounts of insulin. Other factors such as
a change in lipid metabolism that may contribute to
the pathogenesis of diabetes cannot be excluded and
requires additional studies.
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