Chapter 17
9224 words
Genetically programmed defects in beta-cell function
Aparna Pal and Anna L Gloyn
Abstract
A significant level of insight into the machinery and workings of the pancreatic beta-cell originates
from the study of naturally occurring mutations in genes that encode the various components.
Identifying these mutations has been important not only for tailoring treatment towards the
specific subtype of diabetes associated, but also for highlighting the importance and pivotal role
of a number of elements along the pathway of glucose stimulated insulin secretion within the
pancreatic beta-cell.
Key words: beta cell, mutation, glucose transporter 2 (GLUT2), KATP channel, glucokinase,
maturity onset diabetes of the young (MODY), hyperinsulinaemic hypoglycaemia (HH), neonatal
diabetes, endoplasmic reticulum stress
Aparna Pal and Anna L Gloyn
Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology & Metabolism,
University of Oxford, UK. Email: anna.gloyn@drl.ox.ac.uk
17.1 Introduction
The pancreatic beta-cell and insulin secretion pathway are central to the pathophysiology of
diabetes. Although the vast majority of diabetes is categorised as type 1 or type 2 diabetes,
approximately 5% of cases have other specific causes including monogenic diabetes i.e. diabetes
resulting from the mutation of a single gene. Most of these naturally occurring mutations affect
components of the beta-cell with consequences severe enough to commonly cause development
of diabetes in childhood or adolescence. Identifying the genes affected in monogenic beta-cell
dysfunction has lent considerable insight into the regulation of insulin secretion as well as guiding
more accurate and relevant clinical management of patients. It is increasingly clear that defining
these forms of beta-cell dysfunction according to underlying genetically programmed defects is a
more accurate and informative way of studying these disorders, rather than according to clinical
phenotype which in these instances often overlap despite different genetic aetiologies.
Susceptibility genes for the more common forms of T2D are also providing novel insights into
beta-cell function, indeed emphasising its key role in diabetes pathogenesis (over and above
insulin resistance) and will be dealt with in detail elsewhere (see chapter 16). It is interesting to
note the overlap between genes involved in monogenic forms of beta-cell dysfunction and T2D.
For a number of genes (e.g. KCNJ11, GCK) there are clear examples of an allelic spectrum
where genetic variants influence glycaemic control with differing degrees of severity (Glaser et al.
1998; Gloyn et al. 2003a; Gloyn et al. 2004; Gloyn et al. 2005; Hattersley et al. 1992; Njolstad et
al. 2001; Thomas et al. 1996; Weedon et al. 2005).
This chapter will trace the pathway of glucose from its uptake through metabolism and stimulation
of insulin secretion and via each beta cell component and its affecting naturally occurring
mutations demonstrate the key role that each play in the biology and system of the pancreatic
beta-cell.
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17.2 The pancreatic beta-cell, insulin secretion and the main targets of genetically
programmed defects
Figure 1 is a schematic representation of the pancreatic beta-cell showing the main components
involved in glucose stimulated insulin secretion as well as the sites of the main mutations
affecting beta-cell function.
Insert Figure 1 here (COLOUR)
17.3 Glucose Transporter 2 (GLUT 2) and Fanconi Bickel Syndrome
GLUT2 is a transmembrane carrier protein enabling passive movement of glucose across cell
membranes. Although widely expressed (liver, hypothalamus, small intestine, renal tubular cells)
its main focus here is as the principle transporter of glucose into the pancreatic beta-cell. Along
with the other 6 members of the family of glucose transporter proteins(GLUT1 – GLUT7), GLUT2
employs a facilitative transport mechanism – glucose is transported passively down a
concentration gradient and in contrast to ‘active’ transport is energy independent (Santer et al.
1998). Other features in common with its facilitative glucose transporter family are the 12
transmembrane components of GLUT2 and the fact that extracellular glucose binding induces a
conformational change in the protein leading to intracellular release of glucose (Figure 1) (Oka et
al. 1990). Thus GLUT2 demonstrates substrate specificity and saturation kinetics and is therefore
particularly vulnerable to mutations affecting its transmembrane structure.
Although initially thought to be the glucose sensor of the cell, in reality the amount of extracellular
glucose taken up by the beta cell bears no relation to the amount actually metabolised by the cell
(Efrat 1997). Whereas phosphorylation by glucokinase imposes a rate-limiting step on glycolysis
in the beta-cell (see section 17.4), transport into the cell by GLUT2 is a highly efficient system
due to its high Vmax and KM for glucose (Gould et al. 1991) which therefore provides an unrestricted
supply of glucose for metabolism in the beta-cell.
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GLUT2 was first isolated from human liver and kidney cDNA libraries (Fukumoto et al. 1988) and
subsequently also localized to the rat pancreatic beta cell (Orci et al. 1989). The human
pancreatic islet identical counterpart was demonstrated in the same year (Permutt et al. 1989)
and the architecture of the 11 exon gene (SLC2A2 also referred to as GLUT2) that encodes this
glucose transporter on chromosome 3 was defined by Takeda et al in 1993 (Takeda et al. 1993).
The importance of GLUT2 in carbohydrate metabolism is illustrated by the rare glycogen storage
disease, Fanconi Bickel syndrome (FBS) caused by inactivating homozygous mutations within
SLC2A2. FBS is a rare autosomal recessive disorder, first described in 1949 (Fanconi 1949)
whose clinical features include hepatomegaly secondary to glycogen accumulation, glucose and
galactose intolerance, fasting hypoglycaemia, a characteristic proximal tubular nephropathy and
severe short stature (Santer et al. 1998). This phenotype, characterised by glycogen excess in
liver and kidney cells, emphasises the role of GLUT2 in glucose output as well as uptake: in its
absence, glucose produced from gluconeogenesis in the liver, and from tubular reabsorption in
renal cells, is trapped and therefore stored as excess glycogen. Interestingly loss of GLUT2
function in humans does not affect insulin secretion significantly (Leturque et al. 2009). In
contrast Glut2-null mice display a lethal diabetic phenotype (Guillam et al. 1997). This is probably
explained by the difference in the extent of GLUT2 expression, most markedly in the pancreas,
between rodents and man (De Vos et al. 1995). In addition there may be another compensatory
transporter activated on loss of GLUT2 in human pancreatic beta cells, but this requires further
investigation (Leturque et al. 2009).
Treatment of FBS is symptomatic and includes a low-sugar, galactose restricted diet, frequent
small meals and replacement of fluids and electrolytes (Santer et al. 1998). Thus FBS and this
naturally occurring loss of the principle pancreatic beta-cell transporter system is interesting in its
manifestation as a glycogen storage disease (as all other diseases in that group are caused by
enzymatic defects of glycogenolysis) rather than as a more typical diabetic phenotype.
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17.4 Glucokinase and defects in glucose homeostasis
Glucokinase (GCK) catalyses the first rate limiting step in glucose metabolism through its
phosphorylation of glucose on carbon 6 to form G6P. It is a member of the hexokinase family of
4 enzymes, and recognised as the pancreatic beta-cell ‘glucose sensor’ as its kinetics allow rate
of glucose phosphorylation to vary over a range of physiological glucose concentrations (4—
15mmol/l) (Matschinsky 2002). [GCK has a lower affinity for glucose than the other three
hexokinases and its activity is localised to fewer cell types (liver, pancreas, small intestine and
brain): therefore the other hexokinases bear the brunt of glucose phosphorylation for glycolysis
and glycogen sythesis in most tissues. The key distinguishing features of GCK which permit its
specific function as glucose sensor in the beta-cell are firstly its lower affinity for glucose than the
other hexokinases and secondly the lack of inhibition by its product G6P which allows its
continued stimulation of insulin release amid accumulating product (Matschinsky 1996).
GCK is encoded by the 12 exon gene GCK on chromosome 7 and consists of a monomeric
protein of 465 amino acids. The crystal structure, only relatively recently defined in detail
(Kamata et al. 2004), vastly aids comprehension of the biochemical consequence of the various
GCK mutations: there is a large and a small domain between which lies a deep cleft where
glucose binds. As GCK binds glucose and ATP it undergoes a conformational change which
approximates the large and small domains, resulting in a closed, active conformation. The GCK
structure occurs in closed, open and ‘super-open’ conformations which define two catalytic cycles
(slow and fast). The characteristic sigmoidal response of GCK to glucose is due to the ratio
between these two catalytic cycles (Kamata et al. 2004).
Genetically programmed defects in GCK include heterozygous inactivating mutations that cause
a subtype of Maturity-onset diabetes of the young (MODY), homozygous or compound
heterozygous inactivating mutations causing permanent neonatal diabetes mellitus (PNDM) and
finally heterozygous activating mutations which cause Hyperinsulinaemic Hypoglycaemia (HH)
(Froguel et al. 1992; Glaser et al. 1998; Hattersley et al. 1992; Njolstad et al. 2001). These
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defects in GCK all alter the efficiency of glucose binding and phosphorylation in the beta-cell
leading to increased or decreased glucose stimulated insulin secretion and result in clinically
appreciable hyperglycaemia or hypoglycaemia.
Mutations in Glucokinase (GCK) cause Maturity Onset Diabetes of the Young subtype GCK
(GCK-MODY) (formerly known as MODY 2)
MODY is thought to account for approximately 1-2% of diabetes (Frayling et al. 2001; Ledermann
1995) and is an autosomal dominantly inherited form of diabetes characterised by young age of
onset and pancreatic beta-cell dysfunction. The first MODY gene to be identified was GCK
(Froguel et al. 1992; Hattersley et al. 1992) and now over 600 mutations in GCK have been
described worldwide (Osbak et al. 2009). All inactivating GCK mutations are associated with a
mild fasting hyperglycaemia, the majority of patients having fasting blood glucose levels within a
narrow range of 6-8mmol/l, which is in contrast to all other forms of diabetes. This phenotype is
perhaps to be expected given the fact that the heterozygous mutations simply cause reduced
activity of the enzyme and a higher threshold for insulin release (on account of the compensation
provided by the wild type GCK allele) which is stimulated consistently nonetheless and accounts
for the relatively benign natural history of GCK-MODY. Patients do not have accelerated
deterioration of beta-cell function, rarely need treatment– the majority being treated with diet
alone, and perhaps most importantly in terms of morbidity and mortality in diabetes, patients with
GCK-MODY rarely develop microvascular or macrovascular complications (Ellard et al. 2008).
Permanent Neonatal Diabetes Mellitus due to GCK mutations (GCK-PNDM)
In contrast to the mild phenotype described above, homozygous or compound inactivating GCK
mutations lead to the severe condition of PNDM. This rare form of diabetes diagnosed within the
first six months of life and homozygous mutations in GCK are now recognised as a rare cause of
this condition. GCK-PNDM was first described in 2001 by Njolstad and colleagues and
functional studies of the mutant GCK by the same group showed the enzyme’s activity to be
below 0.2% of that of the wild-type (Njolstad et al. 2001). To date only eight isolated cases of
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PNDM due to GCK mutations have been reported, either homozygous or compound
heterozygous for a missense, frameshift or nonsense mutation leading to complete absence of
glucokinase activity (Njolstad et al. 2001; Njolstad et al. 2003; Porter et al. 2005; Rubio-Cabezas
et al. 2008; Turkkahraman et al. 2008). The severity of GCK-PNDM may vary depending on the
amount of activity retained by the mutant enzyme (Porter et al. 2005): the missense R397L
mutation gives rise to a milder phenotype with the mutated GCK still able to stimulate insulin
release, although not enough to avoid the need for supplemental insulin. All GCK-PNDM patients
have required treatment with insulin for their diabetes although there is promising evidence in at
least one case for the use of sulphonylureas (in addition to insulin) to augment improved
glycaemic control (Turkkahraman et al. 2008).
Hyperinsulinaemic Hypoglycaemia due to GCK mutations (GCK-HH)
Heterozygous gain of function mutations in GCK increase the affinity for binding glucose
effectively reducing the threshold glucose that triggers insulin release, thereby leading to
inappropriate over secretion of insulin despite hypoglycaemia. The ensuing condition is
Hyperinsulinaemic Hypoglycaemia (HH) and is also known as Persistent Hyperinsulinaemic
Hypoglycaemia of Infancy (PHHI), Hyperinsulinaemia of Infancy (HI) and congenital
hyperinsulinaemia of infancy (CHI). Although the most common genetic cause for HH are
mutations in the potassium channel genes (see section 17.6), twelve causal activating mutations
in GCK have been reported to date (Barbetti et al. 2009; Christesen et al. 2002; Christesen et al.
2008; Cuesta-Munoz et al. 2004; Glaser et al. 1998; Gloyn et al. 2003b; Meissner et al. 2009;
Sayed et al. 2009; Wabitsch et al. 2007). Interestingly the vast majority of these mutations occur
in an allosteric activator site where small synthetic molecular activators which are currently under
development for the treatment of T2D bind (Christesen et al. 2002; Gloyn et al. 2003b; Grimsby et
al. 2003; Osbak et al. 2009).
The severity of hypoglycaemia in GCK-HH depends on the specific mutation: some cause
possibly fatal episodes but the majority result in mild asymptomatic hypoglycaemia (Osbak et al.
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2009). Treatment is either by taking regular small meals but most require pharmacological
intervention with the potassium channel activator diazoxide (Gloyn et al. 2003b; Sayed et al.
2009; Wabitsch et al. 2007).
The spectrum of clinical phenotypes ranging from hyperglycaemia to severe hypoglycaemia
caused by genetically programmed defects in GCK in the pancreatic beta cell has given
considerable insight into the workings of the system for glucose stimulated insulin secretion here.
In addition the functional characterizations of these inactivating and activating mutations has
increased our knowledge of the mechanics of biochemical activation, structure and regulation of
GCK which thus shows promise as a drug target for the treatment of common multifactorial T2D
as well as these more rare monogenic beta-cell dysfunction disorders.
17.5 Mitochondrial mutations impairing beta-cell function and Mitochondrial Diabetes and
Deafness (MIDD)
Mitochondria are membrane bound organelles found in most eukaryotic cells and their key role is
in producing the majority of a cell’s chemical energy in the form of ATP. Mitochondrial
dysfunction results in reduced oxidative phosphorylation and ATP synthesis and has most
pronounced effect in high energy consuming tissues such as muscle, nerves and the pancreatic
beta-cell. They are unique in their genetics and inheritance: they carry their own circular DNA
that contains 37 genes and inheritance is exclusively maternal as mitochondrial DNA is present in
oocytes but not spermatozoa.
Mutations in mitochondrial DNA are a rare cause of beta-cell dysfunction accounting for 0.5-3%
diabetes (Katulanda et al. 2008; Maassen et al. 2004; Murphy et al. 2008; Waterfield 2008). By
far the most common mutation to cause beta-cell dysfunction and diabetes occurs in the gene
that encodes tRNA leucine, leading to substitution of guanine for adenine (A→G) at position 3243
(Goto et al. 1990). This mt.3243 A>G mutation leads to self-dimerization of the tRNALeu
molecule causing impaired amino acid delivery to the ribosome and reduced protein synthesis:
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this leads to a reduction in oxidative phosphorylation and ensuing beta-cell dysfunction
(Wittenhagen and Kelley 2002). The tRNA Leu 3243 mutation was originally identified in patients
with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) (Goto et al. 1990) although diabetes is not actually part of this syndrome: this
association was made later (Kadowaki et al. 1994) hinting at the range of phenotypes associated
with this mutation. This variation in phenotype is due to different heteroplasmy loads across
tissues and between individuals: heteroplasmy is the variable expression of wild and mutant
mitochondrial DNA, with severest phenotypes having highest levels of heteroplasmy (Koga et al.
2000). The beta-cell dysfunction and diabetes associated with the mt.3243 A>G mutation is
known as Maternally inherited diabetes and deafness (MIDD) and was first described in 1992
(van den Ouweland et al. 1992). As the name suggests key clinical features are presence of
diabetes and deafness, and a family history amongst maternal relatives. The organs involved are
manifest as those with highest metabolic rate such as muscle, kidney, brain, retina, cochlea and
endocrine pancreas. Treatment varies widely according to the organ affected but most MIDD
patients, although initially treated with diet or oral hypoglycaemics, will require insulin within a
couple of years of diagnosis of diabetes (Murphy et al. 2008).
17.6 The KATP channel and defects in glucose homeostasis
ATP-sensitive potassium (KATP) channels control potassium flux across cell membranes thus
determining membrane potential and connects metabolism within the cell to electrical activity.
Increased metabolism and therefore intracellular ATP:Mg-ADP ratio closes the KATP channel
leading to membrane depolarization, increasing electrical activity which can trigger events
including muscle contraction and hormone release. The role of the KATP channel in pancreatic
beta-cell function and insulin secretion was elucidated in 1984 (Ashcroft et al. 1984) and the
importance of its role here is illustrated by the fact that mutations in the genes encoding the
various channel components result in a spectrum of hypo- and hyperglycaemia disorders
including transient neonatal diabetes mellitus (TNDM), PNDM and HH (Aguilar-Bryan et al. 1995;
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Babenko et al. 2006; Flanagan et al. 2009; Gloyn et al. 2004; Gloyn et al. 2005; Proks et al. 2006;
Thomas et al. 1996).
The beta-cell KATP channel is an octameric complex of four inner pore-forming Kir6.2 subunits and
four regulating outer sulphonylurea receptor 1 (SUR1) subunits (Shyng and Nichols 1997). Kir6.2
is encoded by KCNJ11 on chromosome 11 and consists of a single exon encoding this 390
amino acid protein (Inagaki et al. 1995). The SUR1 subunit is encoded by the gene ABCC8 which
is interestingly only~ 4.5kb from KCNJ11: ABCC8 is significantly larger consisting of 39 exons
and spanning greater than 100kb (Aguilar-Bryan et al. 1995).
Genetically programmed defects in the Kir6.2 or SUR1 KATP channel subunits cause a range of
clinical phenotypes most obviously demonstrating a relationship between severity of phenotype
and degree of membrane hyperpolarization (Babenko 2008; Gloyn et al. 2005; Tarasov et al.
2006). KCNJ11 mutations cause a spectrum of phenotypes ranging from TNDM and PNDM
through to the severe syndrome of developmental delay, epilepsy and neonatal diabetes (DEND)
(Gloyn et al. 2004; Gloyn et al. 2005). This variability of phenotype is thought to be due to a
combination of variation in Kir6.2 expression across tissues, mutation severity and compensatory
mechanisms (Hattersley and Ashcroft 2005; Inagaki et al. 1995; Karschin et al. 1997; Shyng et al.
2000).
Neonatal Diabetes Mellitus caused by KCNJ11 mutations
KCNJ11 mutations most commonly manifest as PNDM accounting for up to 64% cases (Gloyn et
al. 2004). This is the more severe form of neonatal diabetes (compared to TNDM) where
reduced insulin secretion results in lowered birth weight and hyperglycaemia and diabetes that
persists beyond 12 months of age. The most common KCNJ11 mutation, R201H, causes PNDM
through a 40-fold lowering of ATP sensitivity of the KATP channel and prolonged opening leading
to reduced insulin secretion (Gloyn et al. 2004). The most severe phenotype, DEND, associated
with KCNJ11 mutations typically cause the greatest KATP channel ATP insensitivity (Proks et al.
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2004). Involvement of extrapancreatic tissues in DEND, in contrast to other phenotypes, may be
explained by the highly activating nature of the causal mutations (Proks et al. 2005a).
Interestingly a single mutation may not always be associated with a single phenotype: the V59M
mutation is associated with both isolated diabetes and intermediate DEND (Gloyn et al. 2004;
Tammaro et al. 2008) lending weight to the existence of yet to be discovered compensatory
mechanisms. The mildest phenotype associated with KCNJ11 mutations is TNDM where
diabetes remission usually occurs within 3-6 months. Functional studies of three KCNJ11
mutations (G53R, G53S and 1182 V) showed approximately a four-fold reduction in KATP channel
ATP sensitivity demonstrating they are functionally less severe than the R201H mutation which
causes PNDM (Gloyn et al. 2004; Gloyn et al. 2005).
Hyperinsulinaemic Hypoglycaemia caused by KCNJ11 mutations
The heterogeneity of HH is illustrated by the range of causal mutations. In addition to GCK-HH
(section 17.4), a total of 24 KCNJ11 mutations have been reported (Flanagan et al. 2009). These
cause HH by severely reducing KATP channel activity in the beta-cell membrane (Nestorowicz et
al. 1997).
Neonatal diabetes Mellitus caused by ABCC8 mutations
TNDM and PNDM are also caused by ABCC8 mutations, affecting the SUR1 subunit of the KATP
channel, which are found in approximately 27% of PNDM patients in whom no KCNJ11 mutation
is identified (Ellard et al. 2007). The underlying mechanism here is accentuation of the effect of
Mg-ADP on the KATP channel resulting in beta-cell hyperpolarization and inhibition of insulin
secretion (Babenko 2008). ABCC8 mutations have also been identified as a rarer cause of the
more severe phenotype of DEND and iDEND: the F132L mutation has been shown to alter the
gating of the KATP channel as well as the sensitivity to Mg-ADP causing prolonged opening and
severe membrane hyperpolarization (Proks et al. 2004; Proks et al. 2005b; Proks et al. 2007).
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Hyperinsulinaemic Hypoglycaemia caused by ABCC8 mutations
ABCC8 mutations are the most common cause of HH and over 150 mutations have been
described (Flanagan et al. 2009). They have been functionally divided into two classes (Ashcroft
2005); class I refers to an absent resultant protein at the membrane surface and class II refers to
a channel that is present but persistently closed. Class I mutations lead to reduced protein levels
or faulty trafficking of the channel (Taschenberger et al. 2002): class II mutations prevent KATP
channel activation by reducing channel stimulation by Mg-ADP (Huopio et al. 2002). Generally
the class I mutations have a more severe phenotype than the class II mutations which may be
milder due to a partial response to MgADP (Flanagan et al. 2009).
The underlying molecular diagnosis in NDM and HH has implications for treatment
The clinical implications of identifying an underlying KATP channel mutation in neonatal diabetes
are significant given that many patients have now been successfully transferred from insulin to
sulphonylurea therapy after the first confirmatory study in 2006 (Pearson et al. 2006). Even at the
more severe end of the spectrum in DEND and iDEND some response to high-dose
suplhonylurea therapy has been demonstrated (Koster et al. 2008; Mlynarski et al. 2007).
In contrast to GCK-HH, diazoxide is not effective in HH due to KCNJ11 and ABCC8 mutations,
which is perhaps to be expected given that the target for this drug is the KATP channel itself.
Octreotide (a somatostatin analogue) has been used with some success in children (Glaser et al.
1993). Partial pancreatectomy is reserved for those who do not respond to medical treatment.
17.7 Defects in glucose homeostasis due to mutations in genes encoding beta-cell
transcription factors
Five of the eight causal MODY gene mutations occur in transcription factors (Bell et al. 1991;
Frayling et al. 1997; Horikawa et al. 1997; Malecki et al. 1999; Stoffers et al. 1997; Yamagata et
al. 1996a). The study of these naturally occurring mutations has increased our understanding of
the genes and interlinking pathways required for normal function of the pancreatic beta-cell.
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Hepatocyte nuclear factor 1 homeobox A (HNF-1 alpha), Hepatocyte nuclear factor 1 homeobox
B (HNF-1 beta), Hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Neurogenic differentiation 1
(NeuroD1), Insulin promoter factor1 (IPF1) are all transcription factors regulating several genes in
a tissue specific manner in the beta-cell (Mitchell and Frayling 2002). The precise underlying
mechanism by which mutations in these transcription factors cause diabetes is unknown but a
range of in vitro and in vivo studies, largely in rodents, has highlighted their importance in
pancreatic beta-cell development and differentiation as well as in the regulation of many genes
involved in glucose stimulated insulin secretion (Byrne et al. 1996; Chen et al. 1994; Nammo et
al. 2002; Pontoglio et al. 1998; H. Wang et al. 2000; Wild et al. 2000; Yamagata et al. 2002).
Insert Figure 2 here (COLOUR OR BLACK AND WHITE)
Mutations in Hepatocyte Nuclear Factor 1 alpha (HNF1-alpha) cause Maturity Onset
Diabetes of the Young subtype HNF1A (HNF1A-MODY) (formerly known as MODY 3)
HNF-1alpha was previously well known as a liver-specific transcription factor, but its role in
diabetes pathophysiology and the pancreatic beta-cell was uncovered after a genome wide
linkage scan (Yamagata et al. 1996b). HNF1A is encoded by a 10 exon gene on chromosome
12, and is a homeoprotein containing a DNA binding domain through which the protein binds to
its target DNA sequence as a dimer (Chouard et al. 1990). It has 90% amino acid homology in its
DNA binding domain with HNF1B and the two transcription factors bind to the same target DNA
sequence (Rey-Campos et al. 1991). HNF1A interacts with many other transcription factors and
has several beta-cell specific targets underlying its crucial role in normal beta-cell function (Figure
2): it shares a transcriptional feedback loop with HNF4A and although HNF1A expression is
restricted by HNF4A in hepatocytes, it is an upstream regulator in pancreatic beta-cells
(Yamagata 2003). In the beta-cell HNF1A activates both the GLUT2 gene and the L-type
pyruvate kinase (PKL) gene ( a rate-limiting enzyme of glycolysis) by binding to their promoter
regions (H. Wang et al. 1998; Yamagata et al. 2002). HNF1A is also involved in the regulation of
mitochondrial enzymes as well as organization of pancreatic islets through its regulation of E-
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cadherin, an adhesion molecule (Yamagata et al. 2002; Yamagata 2003). Thus HNF1A has
multiple roles in pancreatic beta-cells (Figure 2) perhaps making it a relatively common site of
defects in diabetes subtypes.
Over 200 HNF1A mutations have been described with a common mutation at codon Pro291
(Pro291fsinsC) (Ellard and Colclough 2006): most mutations are localised along the DNA-binding,
dimerisation and transactivation domains of the protein (Mitchell and Frayling 2002). HNF1A is
the most commonly mutated gene in MODY accounting for approximately half of all cases (Ellard
and Colclough 2006). Patients present in childhood or as young adults, have deteriorating betacell function over time and develop both microvascular and macrovascular complications.
Patients respond well to sulphonylureas, which act downstream of many of the targets of HNF1A,
and are the first-line choice of medication for HNF1A MODY (Pearson et al. 2003; Shepherd et al.
2003).
Mutations in Hepatocyte Nuclear Factor 1 beta (HNF1-beta) cause Maturity-onset diabetes
of the Young subtype HNF1B (HNF1B-MODY) (formerly known as MODY 5)
HNF-1beta is another homeodomain containing transcription factor which functions as a
homodimer or heterodimer with HNF-1 alpha. Spontaneous mutations are not uncommon and
heterozygous deletions on chromosome 17 encompassing the HNF1B gene account for around a
third of known mutations (Bellanne-Chantelot et al. 2005). It is thought that beta-cell dysfunction
due to HNF1B mutations is due to defects in pancreatic development (Haumaitre et al. 2005).
The phenotype associated with HNF1B mutations also includes progressive nondiabetic renal
dysfunction (Nishigori et al. 1998; Yamagata 2003) reflecting the high level of expression in the
kidneys. Unlike the other MODY subtypes, patients with HNF1B-MODY are not sensitive to
sulphonylureas and insulin treatment is most often required (Ellard et al. 2008).
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Mutations in Hepatocyte Nuclear Factor 4 alpha (HNF-4 alpha) cause Maturity-onset
diabetes of the Young subtype HNF4A (HNF4A-MODY) (formerly known as MODY 1)
The transcription factor HNF-4alpha is a member of the steroid hormone receptor family and
binds to DNA as a homodimer. HNF-4alpha is thought to regulate similar pathways to HNF1alpha which may be due to the fact that HNF-4alpha is a downstream regulator of HNF-1alpha
in pancreatic beta-cells and a positive feedback loop involving both exists (Figure 2) (Yamagata
2003).
Mutations causing beta-cell dysfunction occur in all exons and the pancreatic promoter (Ellard
and Colclough 2006) but are much less common than HNF1A mutations (Ryffel 2001). Clinical
features associated are similar to HNF1A-MODY in adults but HNF4A mutations have also been
found to account for a form of neonatal HH that resolve and later develops into MODY (Kapoor et
al. 2008; Pearson et al. 2007), suggesting a differing role for these transcription factors in fetal
and neonatal life. In addition to neonatal HH, HNF4A mutation carriers are often macrosomic
aiding distinction from HNF1A-MODY where neonates are generally of normal birth weight
(Pearson et al. 2007).
Mutations in Insulin Promoter Factor 1 (IPF1) cause Maturity Onset Diabetes of the Young
subtype IPF1 (IPF1-MODY) (formerly known as MODY 4)
IPF1 is another central transcription factor in the pancreatic beta-cell where it regulates
transcription of GLUT2 and GCK, mediates glucose stimulated insulin gene transcription as well
as having a pivotal role in pancreatic development (Jonsson et al. 1994; Macfarlane et al. 1997;
Ohlsson et al. 1993; Waeber et al. 1996). Heterozygous IPF1 mutations are a rare cause of
MODY (Chevre et al. 1998) and homozygous and compound heterzygous mutations are a very
rare cause of PNDM due to pancreatic agenesis (Stoffers et al. 1997).
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Mutations in Neurogenic differentiation 1 (NeuroD1) cause Maturity Onset Diabetes of the
Young subtype NEURO1 (NEURO1-MODY) formerly known as MODY 6
Neurod1 is a transcription factor involved in regulating GLUT2, GCK and insulin gene
transcription (Kim et al. 2008). Mutations in NEUROD1 have only been described in three
families and are a very rare cause of MODY (Malecki et al. 1999).
17.8 Mutations in Carboxy Ester Lipase (CEL) cause Maturity-onset diabetes of the Young
subtype CEL (CEL-MODY)
Mutations in CEL have been described in individuals from families that conform to the MODY
phenotype (Raeder et al. 2006), however strictly speaking this is MODY due to a defect of the
exocrine pancreas and not a defect in the pancreatic beta cell.
17.9 Endoplasmic reticulum (ER) Stress as a cause of beta-cell death and defects in
glucose homeostasis
With the recent discovery of mutations in the insulin (INS) gene causing neonatal diabetes and
subsequent functional studies to determine the molecular mechanism behind the mutations it is
clear that ER stress plays an important role in pancreatic beta-cell dysfunction.
Mutations in the insulin (INS) gene as a cause of Neonatal Diabetes and Maturity Onset
Diabetes of the Young
Insulin gene (INS) mutations have been identified as causing PNDM and rarely MODY (Bonfanti
et al. 2009; Edghill et al. 2008; Molven et al. 2008; Stoy et al. 2007). The mutations stop
disulphide bonds forming thus preventing normal folding of proinsulin within the endoplasmic
reticulum (ER) in the pancreatic beta-cell. The ER is sensitive to accumulation of unfolded
proteins and has a specific unfolded protein response (UPR) to alleviate this stress. Failure of
the UPR to clear unfolded proteins results in beta-cell apoptosis (Yoshida 2007). Treatment of
patients with INS mutations is with insulin therapy in order to reduce endogenous insulin
production and protect the ER from accumulation of unfolded insulin (Stoy et al. 2007).
16
This model of accumulating misfolded proinsulin causing ER stress is supported by the Akita
mouse model, which is a mouse model of MODY that develops diabetes as a consequence of
beta-cell dysfunction (J. Wang et al. 1999). In this model a tyrosine for cysteine substitution at
position 96 (C96Y) causes production of abnormal proinsulin: this collects within the ER causing
ER stress which leads to beta-cell death (Allen et al. 2004; Yoshinaga et al. 2005).
Wolfram and Wolcott-Rallison syndromes
ER stress within the pancreatic beta-cell is also thought to be the mechanism underlying the rare
genetic conditions of Wolfram syndrome (WFS) and Wolcott-Rallison syndrome (WRS). WFS,
also known as DIDMOAD, causes a syndrome that includes diabetes, optic atophy and deafness:
in WRS the main clinical features are diabetes, multiple epiphyseal dysplasia, osteopenia, mental
retardation or developmental delay, and hepatic and renal dysfunction. WRS is due to mutations
in EIF2AK3 which encodes a protein kinase-like ER kinase (PERK) (Delepine et al. 2000). PERK
is highly expressed in pancreatic beta cells and its role in detecting misfolded proteins is
supported by the fact that PERK knockout mice develop pancreatic beta-cell death due to
increased ER-stress (Harding et al. 1999; Harding et al. 2001). Wolfram syndrome is due to
mutations in WFS1 which encodes another transmembrane protein (Inoue et al. 1998; Strom et
al. 1998) and the WFS1 knockout mice also develop beta-cell death and diabetes due to ERstress (Ishihara et al. 2004; Riggs et al. 2005; Yamada et al. 2006)
17.11 Common genetic variants associated with T2D in genes implicated in monogenic
forms of beta-cell dysfunction
Genome wide association scans for T2D susceptibility have now revealed up to 20 robustly
implicated novel genetic variants (see chapter 16) (Prokopenko et al. 2008). Due to their
established role in monogenic diabetes the main beta-cell genes discussed above have been
cross-examined for harbouring common variants that influence T2D susceptibility (as well as the
rare penetrant mutations that cause the monogenic conditions described above). One of the first
to be identified was the E23K variant of KCNJ11 (Florez et al. 2004; Gloyn et al. 2003b). The
17
underlying causal molecular mechanisms linking this variant to diabetes pathophysiology has yet
to be defined precisely but recent functional studies have demonstrated the complexity of
translating association signals to clear mutational mechanisms (Hamming et al. 2009). This
illustrates the difference between rare mutations which have a large effect on protein function and
common genetic variants which have more subtle effects. A variant within the GCK islet promoter
(Weedon et al. 2005) and another in LD with this (rs4607517) have been identified associating
with fasting plasma glucose levels in the general population (Prokopenko et al. 2009). Most
recently the variant rs11920090 in SLC2A2 (encoding GLUT2) has been associated with fasting
hyperglycemia (Dupuis el al. Nature Genetics in press). These observations demonstrate that
critical components of the pancreatic beta-cell can exert their effects over an entire allelic
spectrum with the functional severity of the defect dictating the clinical phenotype.
Summary
Figure 1 is a simple representation of the main components of beta-cell glucose stimulated insulin
secretion. However the variety of mutational mechanisms affecting specific components and the
associated distinct phenotypes spanning severe hypoglycaemia, through to mild and then severe
hyperglycaemia illustrates a more complex interplay of biochemical pathways and structures.
The study of these naturally occurring beta-cell mutations has given much to our understanding of
the mechanics of this system and in particular is providing novel drug targets. An example of this
is the insight into GCK structure from GCK-HH mutations which have highlighted the allosteric
activator sight as a novel target for therapeutics aimed at attenuating GCK activity. The variation
in phenotype encompassing both mild and severe manifestations of disease despite identical
beta-cell components affected also highlights the existence of compensatory mechanisms at work
which have yet to be defined. The pancreatic beta-cell is unique in its efficient translation of
extracellular glucose to an individuals required level of insulin secretion: by the identification and
study of a growing number of mutations with beta-cell specific effects, we are appreciating the
complexity behind this system.
18
Acknowledgments
AP is a Medical Research Council (MRC) Clinical Training Fellow. ALG is a MRC New
Investigator (Grant Code 81696).
19
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Figure titles and legends:
Figure 1 The pancreatic beta-cell:- Location of mutations identified in humans which cause
monogenic forms of beta-cell dysfunction
Glucose enters the pancreatic beta-cell via the glucose transporter 2 (GLUT2). Inside the cell
glucose is phosphorylated by glucokinase (GCK) to glucose-6-posphate (G6P) in the first rate
limiting step of glucose metabolism. G6P is metabolized in the mitochondria which raises the
intracellular ATP: Mg-ADP ratio. This leads to closure of the ATP sensitive KATP channel which
causes depolarization of the beta-cell membrane. This activates voltage gated calcium channels
leading to an influx of Ca2+ which triggers insulin exocytosis.
Figure 2 The beta-cell transcription factor network
The HNF network in pancreatic beta-cells. HNF4A expression is mainly regulated by HNF1A.
HNF1B functions with HNF1A as a homodimer or heterodimer. Transcription factors in orange
boxes are known to be mutated in MODY subtypes.
ALTERNATE IF BLACK AND WHITE FIGURE 2 IS USED
The HNF network in pancreatic beta-cells. HNF4A expression is mainly regulated by HNF1A.
HNF1B functions with HNF1A as a homodimer or heterodimer. Transcription factors in grey
boxes are known to be mutated in MODY subtypes.
26