1006 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
The MODY1 gene HNF-4α regulates selected
genes involved in insulin secretion
Rana K. Gupta,1,2 Marko Z. Vatamaniuk,1,2 Catherine S. Lee,1,2 Reed C. Flaschen,1,2
James T. Fulmer,1,2 Franz M. Matschinsky,2,3 Stephen A. Duncan,4 and Klaus H. Kaestner1,2
1Department of Genetics, 2Institute for Diabetes, Obesity, and Metabolism, and 3Department of Biochemistry and Biophysics,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. 4Department of Cell Biology, Neurobiology and Anatomy,
Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Mutations in the gene encoding hepatocyte nuclear factor-4α (HNF-4α) result in maturity-onset diabetes of
the young (MODY). To determine the contribution of HNF-4α to the maintenance of glucose homeostasis
by the β cell in vivo, we derived a conditional knockout of HNF-4α using the Cre-loxP system. Surprisingly,
deletion of HNF-4α in β cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in
impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the
mutant β cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60%
reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays
that the Kir6.2 gene is a transcriptional target of HNF-4α. Our data provide genetic evidence that HNF-4α
is required in the pancreatic β cell for regulation of the pathway of insulin secretion dependent on the ATP-
dependent potassium channel.
Maturity-onset diabetes of the young (MODY) is a mendelian form
of type 2 diabetes characterized by an autosomal dominant mode
of inheritance, early onset, and impaired glucose-stimulated insu-
lin secretion. MODY can result from mutations in at least 6 differ-
ent genes. One of these encodes the glycolytic enzyme glucokinase
(MODY2), which is an important glucose sensor, while all the
others encode transcription factors: hepatocyte nuclear factor-4α
(HNF-4α) (MODY1); HNF-1α (MODY3); insulin promoter factor 1
(IPF1/pancreatic duodenal homeobox 1 [Pdx-1]) (MODY4); HNF-1β
(MODY5); and neurogenic differentiation factor 1 (NeuroD1)
(MODY6) (1). Genetic and biochemical studies have revealed that
many of these transcription factors participate in a transcriptional
regulatory network in both the liver and pancreas.
A hierarchy among MODY genes has been derived from the
molecular analysis of MODY, as mutations in both the gene
encoding HNF-4α (MODY1) and the binding site for the protein
HNF-4α in the HNF-1α promoter cause diabetes (2, 3). In addi-
tion, more recent studies have demonstrated that mutations in
the β cell–specific promoter (P2) of HNF-4α in humans are associ-
ated with increased risk of type 2 diabetes (4–6). Therefore, it has
been proposed that HNF-1α and HNF-4α form a regulatory loop
in the adult β cell and that this regulatory loop is essential for β
cell function (7, 8). Furthermore, it was hypothesized that haplo-
insufficiency for either gene can cause a breakdown in the regula-
tory loop, ultimately resulting in diabetes (9). However, much of
the information about the role of HNF-4α in the pancreas so far
has been based on expression and biochemical data. For example,
when a dominant negative form of HNF-4α was overexpressed in
insulinoma cells, several genes involved in glucose metabolism
as well as HNF-1α were differentially expressed, suggesting that
HNF-4α regulates β cell glucose metabolism through the regula-
tion of HNF-1α and several glycolytic and mitochondrial genes
(9, 10). Most recently, Odom and colleagues combined chroma-
tin immunoprecipitation with promoter microarrays to identify
over 1000 human promoter elements bound by HNF-4α in pan-
creatic islets, suggesting that HNF-4α may function to regulate
multiple pathways in the β cell (11). Although it has been shown
that HNF-4α is required for maintenance of the expression of
HNF-1α and important metabolic genes in the liver, it remains
unknown whether this relationship holds true in the pancreatic β
cell or whether HNF-4α is essential for β cell glucose metabolism
in vivo. Understanding the relationship between MODY genes
and their specific functional targets in vivo may identify a com-
mon mechanism of pathogenesis and lead to a novel approach
for improving β cell function.
MODY1 patients fail to secrete insulin adequately in response to
glucose challenge (12). This observation, along with other recent
biochemical studies, suggests that HNF-4α plays a role in pan-
creatic development and/or in the regulation of β cell function.
However, the exact role of HNF-4α in the maintenance of β cell
function has not to our knowledge been determined in vivo until
now. Because targeted disruption of HNF-4α in mice results in
early death due to defective gastrulation (13, 14), genetic analysis
of the function of HNF-4α in the adult pancreas has thus far been
precluded. To determine the role of HNF-4α in the β cell and in
the maintenance of glucose homeostasis in vivo, as well as its con-
tribution to the molecular etiology of MODY, we have derived a
conditional knockout of HNF-4α using the Cre-loxP system. Dele-
tion of HNF-4α in β cells resulted in impaired glucose tolerance
but, surprisingly, also in fasting and fed hyperinsulinemia. The
data presented here reveal an unpredicted role for HNF-4α in the
regulation of the pathway of insulin secretion dependent on the
Nonstandard abbreviations used: BHK, baby hamster kidney; EMSA, electropho-
retic mobility-shift assay; Foxa2, forkhead box a2; fura-2AM, fura-2 acetoxymethyl-
ester; HNF-4α, hepatocyte nuclear factor–4α; KATP channel, ATP-dependent potas-
sium channel; MODY, maturity-onset diabetes of the young; NeuroD1, neurogenic
differentiation factor 1; Pdx-1, pancreatic duodenal homeobox 1; PP, pancreatic
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 115:1006–1015 (2005).
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
ATP-dependent potassium channel (KATP channel) and demon-
strate that HNF-4α is not required for the maintenance of HNF-1α
expression in the adult β cell.
β cell–specific deletion of HNF-4α. In order to obtain mice lacking
HNF-4α in pancreatic β cells, we mated HNF-4αloxP/loxP mice to
mice containing a transgene with Cre recombinase under con-
trol of the rat insulin 2 promoter (Ins.Cre) (15). The resulting
HNF-4αloxP/+; Ins.Cre offspring were then bred to HNF-4αloxP/loxP
homozygotes to obtain HNF-4αloxP/loxP; Ins.Cre mutants and the
littermate groups HNF-4αloxP/+, HNF-4αloxP/loxP, and HNF-4αloxP/+;
Ins.Cre (Figure 1A). HNF-4αloxP/loxP; Ins.Cre mice were born with
the expected mendelian distribution and no significant differ-
ences in size or appearance were observed at birth or in adult mice
(control = 28.6 ± 1.3 grams; mutants = 30.7 ± 1.8 grams; n = 6–8
mice; 5 months of age; P = NS) compared with littermate controls
(HNF-4αloxP/loxP and HNF-4αloxP/+).
To evaluate the specificity and efficiency of Cre-mediated dele-
tion of HNF-4α, we first used PCR analysis of genomic DNA to
determine if the floxed exon 2 of the HNF-4α gene was excised in
freshly isolated islets of HNF-4αloxP/loxP; Ins.Cre and HNF-4αloxP/loxP
mice (Figure 1B). Primers were designed to amplify a 450-bp prod-
uct only detectable when the floxed HNF-4α gene was deleted. The
PCR analysis indicated that gene ablation occurred in approxi-
mately 70% of cells in the islets of HNF-4αloxP/loxP; Ins.Cre mice.
Concordantly, mRNA levels of HNF-4α in isolated islets of HNF-
4αloxP/loxP; Ins.Cre mice were reduced by approximately 63% com-
pared with those of controls (Figure 1C). Given that 30–40% of islet
cells are non–β cells that express HNF-4α, this degree of reduction
in whole islets suggests deletion in greater than 90% of β cells. We
confirmed the β cell specific inactivation of HNF-4α by examin-
ing the expression of HNF-4α protein by immunohistochemistry.
HNF-4α is normally found in all cell types of the islet and through-
out most of the acinar tissue (Figure 1D). Consistent with pre-
vious reports of the use of the Ins.Cre-transgenic line, we found
that approximately 90% of pancreatic β cells in the HNF-4αloxP/loxP;
Ins.Cre mice had lost HNF-4α expression by 2–3 weeks after birth,
while the expression of HNF-4α was maintained throughout the
remainder of the islet as well as in surrounding exocrine tissue
(Figure 1E) (15–18). Staining of adjacent sections for insulin and
glucagon confirmed the continued presence of HNF-4α protein in
α cells, demonstrating the specificity of the Ins.Cre-mediated gene
ablation within the pancreas (data not shown).
The Ins.Cre-transgenic mouse used in this study has been report-
ed to excise loxP targets ectopically in the central nervous system.
This issue is of potential significance, as the hypothalamus plays
an important role in glucose homeostasis. However, this ectopic
activity of the Ins.Cre transgene is only relevant if the loxP-flanked
gene to be targeted is expressed in the brain. Multiple expression
studies of HNF-4α have failed to detect any expression of this gene
in the neuroectoderm during development, or in the hypothala-
mus or other brain regions in the adult mouse, making it extreme-
ly unlikely that HNF-4α plays a role in the central nervous system
(19, 20). Fasting corticosterone levels, which are controlled by
the hypothalamic-pituitary-adrenal axis, in mutant mice are not
changed (control, 245 ± 62 ng/ml; mutant, 277 ± 49 ng/ml; n = 5
mice; P = NS), supporting the notion that hypothalamic function
is maintained in HNF-4αloxP/loxP; Ins.Cre mice. Thus, it is unlikely
that any excision of the floxed HNF-4α allele in the brain would
contribute to the phenotype of HNF-4αloxP/loxP; Ins.Cre mice. In
addition, our studies on isolated islets described below demon-
strate a specific requirement for HNF-4α in the β cell.
β cell deletion of HNF-4α results in hyperinsulinemia and impaired
glucose tolerance in vivo. Next we determined the effect of HNF-4α
deficiency in the β cell on glucose homeostasis in vivo by mea-
suring fed and fasting blood glucose levels in the HNF-4αloxP/loxP;
Ins.Cre mice. Compared with controls, HNF-4α mutants exhib-
ited a mild decrease in blood glucose levels in both the fed and
fasting states (Figure 2A). To determine if this difference in blood
Derivation of β cell–specific HNF-4α knockout mice. (A) HNF-4αloxP/loxP
mice in which exon 2 was flanked by loxP sites were bred to Ins.Cre-
transgenic mice expressing Cre recombinase under control of the rat
insulin promoter. The resulting HNF-4αloxP/+; Ins.Cre offspring were
then mated with HNF 4αloxP/loxP homozygotes to obtain HNF-4αloxP/loxP;
Ins.Cre mutants and their littermate controls: HNF-4αloxP/+, HNF-
4αloxP/loxP, and HNF-4αloxP/+; Ins.Cre. (B) Primers 1, 2, and 3 (red, blue,
and green in A) were used for PCR genotyping of isolated islets from
HNF-4αloxP/loxP; Ins.Cre and HNF-4αloxP/loxP mice. In the absence of
Cre, amplification by primers 1 and 2 results in a 620-bp product. Cre-
mediated excision of exon 2 results in a 450-bp product amplified by
primers 1 and 3. Quantification of the bands shows that deletion occurs
in approximately 70% of all islet cells (note that non–β cells make up
20–30% of the islet cell numbers). (C) Concordant with the results in
B, mRNA levels of HNF-4α were reduced by 63% in mutant islets,
as determined by quantitative PCR using primers specific to exon 2.
*P < 0.05; n = 3 per group. (D and E) Immunostaining of pancreatic
sections from adult control (D) and mutant (E) mice using an antibody
against HNF-4α indicates that the number of β cells expressing HNF-4α
protein is reduced by approximately 85–90% (arrow) in the mutant
mouse. Non–β cells in the islet mantle still express HNF-4α protein
(arrowhead) in the mutant mouse. Thus, HNF-4α is deleted efficiently
and specifically in pancreatic β cells. Magnification, ×200.
1008 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
glucose was a result of changes in circulating levels of insulin
and glucagon, we measured plasma levels of these hormones.
We found that in both the fed and fasting states, plasma insu-
lin was significantly elevated in the mutants compared with the
littermate controls (Figure 2B). In contrast, fasting plasma levels
of glucagon were unchanged (Figure 2C). Therefore, the ratio of
plasma insulin to plasma glucagon was approximately 70% high-
er in HNF-4αloxP/loxP; Ins.Cre mice, which accounted for the lower
glucose levels observed in these mice (Figure 2D). Together, these
results demonstrate that HNF-4α in the β cell contributes to the
maintenance of glucose homeostasis.
Suspecting abnormal regulation of insulin secretion in HNF-4α–
deficient β cells, we performed glucose tolerance tests on 3- to
5-month-old HNF-4αloxP/loxP; Ins.Cre mice and littermate controls.
After glucose injection, the elevation in blood glucose levels was sig-
nificantly higher in HNF-4α mutants, indicating impaired glucose
tolerance in these animals (Figure 2E). As glucose intolerance can
result from decreased peripheral insulin sensitivity or impaired glu-
cose stimulated insulin secretion, we measured plasma insulin levels
at various time points after glucose challenge. We found that despite
a significantly higher basal level of plasma insulin prior to glucose
injection (mutant, 0.21 ± 0.02 ng/ml, n = 11; control, 0.14 ± 0.02
ng/ml, n = 11; P < 0.05), plasma insulin levels failed to increase in
the mutant mice at the same rate as in controls after injection (Fig-
ure 2F). In particular, HNF-4α mutants lacked a robust first-phase
insulin secretory response. In order to investigate the possibility
that peripheral insulin resistance contributes to the glucose intol-
erance of HNF-4α mutants, we performed insulin tolerance tests.
Both groups of mice showed similar insulin responses (Figure 2G),
concordant with the fact that HNF-4α is not deleted in the major
insulin-responsive tissues in our model. These results indicate that
while HNF-4α mutants have higher basal plasma insulin levels, they
fail to secrete sufficient insulin in response to exogenous glucose
administration and thus suffer from dysregulated insulin secretion
and impaired glucose tolerance.
Loss of HNF-4α in the β cell does not affect islet architecture or β cell
mass at 4 months of age. Several mechanisms can account for the dys-
regulation of insulin secretion in vivo, including changes in islet
architecture or β cell mass, defective glucose sensing and metabo-
lism, or a combination of these factors. For example, disruption of
HNF-1α in mice results in defective glucose sensing and the failure
of isolated islets from these animals to properly respond to glucose
(21, 22). More recent studies have shown that heterozygous dele-
tion of Pdx-1 in mice results in a decrease in β cell mass due to an
increase in islet apoptosis (23).
To determine the effect of HNF-4α deficiency in the β cell on
islet architecture, we performed indirect immunofluorescence
using antibodies raised against the pancreatic hormones insulin,
glucagon, somatostatin, and pancreatic polypeptide (PP) to label
4 major islet cells types: β cells, α cells, δ cells, and PP cells, respec-
tively. We found that both the control mice (Figure 3, A and C)
and HNF-4αloxP/loxP; Ins.Cre mice (Figure 3, B and D) contained
all 4 pancreatic islet cell types, with the insulin-producing β cells
centrally located within the islet and the less-frequent α cells,
δ cells, and PP cells (not shown) located on the periphery. In addi-
tion, using point-counting morphometry, we determined the β cell
mass in both HNF-4αloxP/loxP; Ins.Cre mice and littermate controls
at 4 months of age and found no significant difference between
the 2 groups (control, 0.93 ± 0.17 mg, n = 6; mutant, 1.04 ± 0.22
mg, n = 5; P = NS) (Figure 3E). Together, these results indicate that
HNF-4α is not required in the β cell for the maintenance of islet
architecture or β cell mass at this age, suggesting that the defect in
insulin secretion is a consequence of loss of β cell function rather
than β cell differentiation.
Diminished first-phase insulin secretion from isolated islets of HNF-4α
mutant mice. We performed insulin secretion studies of perifused
Deletion of HNF-4α in β cells results in hyperinsulinemia and impaired
glucose tolerance in vivo. (A) In the fed state (Fed) and after an over-
night (16-hour) fast (Fast), blood glucose concentrations are decreased
in HNF-4αloxP/loxP; Ins.Cre mice compared with littermate controls. (B)
Plasma insulin levels are elevated in HNF-4α mutants in both the fed
and overnight-fasted states. (C) Fasting plasma glucagon levels in the
mutants were indistinguishable from controls. (D) The ratio of plasma
insulin to plasma glucagon is elevated 70% in HNF-4αloxP/loxP; Ins.Cre
mice. (E) Glucose tolerance test. After an overnight fast, 3- to 5-month-
old HNF-4αloxP/loxP; Ins.Cre mice and littermate controls were chal-
lenged with 2 grams of glucose per kilogram of body weight. The blood
glucose elevation is significantly higher in HNF-4αloxP/loxP; Ins.Cre mice
than in controls, indicating impaired glucose tolerance in the HNF-4α
mutants. (F) Following glucose injection (3 g/kg body weight), HNF-4α
mutants exhibit a diminished first-phase insulin secretory response in
comparison to controls. (G) Insulin tolerance test. Mutant and control
mice that had fasted for 4 hours were injected with 0.75 units of insulin
per kilogram of body weight. The insulin sensitivity of HNF-4α mutants
is indistinguishable from that of controls. *P < 0.05 by Student’s t test
or ANOVA; n = 8–13 animals per group for each experiment.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
islets to define the mechanistic defect in HNF-4αloxP/loxP; Ins.Cre
mice. We treated islets with 0–30 mM glucose, which elicited a
robust insulin secretory response in control islets (Figure 4A).
In contrast, mutant islets lacked a first-phase secretory response
to glucose. In addition, mutant islets failed to terminate insulin
secretion when reexposed to buffer containing 0 mM glucose.
Depolarization with KCl at the end of each experiment confirmed
that the islets had remained viable throughout the experiment.
HNF-4α–deficient β cells exhibit a diminished response to glyburide.
Glucose-stimulated insulin secretion from the pancreatic β cell
occurs after the generation of ATP from the metabolism of glucose
through glycolysis and the Krebs cycle. The intracellular rise in the
ATP/ADP ratio leads to the closure of the KATP channels, calcium
influx, and subsequent activation of insulin secretion through cal-
cium-dependent pathways (24). This KATP-dependent pathway is
the best characterized mechanism leading to insulin secretion and
is essential for proper first-phase insulin release.
We investigated whether components of this pathway were
disrupted in HNF-4αloxP/loxP; Ins.Cre mice by first measuring ATP
levels in isolated islets stimulated with various concentrations of
glucose. We found that at physiological glucose concentrations
(2, 5, and 10 mM), ATP levels in HNF-4α deficient β cells were
indistinguishable from those of controls (Figure 4B), suggest-
ing that glucose metabolism is maintained in mutant β cells. To
determine if calcium influx occurs properly in response to glu-
cose, we performed calcium-imaging experiments using cultured
isolated islets. In response to 16.7 mM glucose, intracellular calci-
um increased rapidly in control β cells and then diminished when
the addition of exogenous glucose was stopped (Figure 4C). How-
ever, intracellular calcium increased at a slower rate in mutant
islets and failed to decrease as rapidly when glucose was stopped
(Figure 4D). To determine if other insulin secretagogues can trig-
ger proper calcium influx, we treated islets with the KATP chan-
nel blocker glyburide as well as with KCl to fully depolarize the
plasma membrane. In control islets, glyburide and KCl treatment
resulted in a strong and rapid rise in intracellular calcium (Figure
4C). In contrast, intracellular calcium increased at a slower rate
in mutant islets exposed to glyburide, but increased normally in
response to KCl (Figure 4D).
HNF-4α is required for potassium channel subunit Kir6.2 expression in
the pancreatic β cells. The observation that both KCl-induced cal-
cium influx and insulin release as well as glucose metabolism in
mutant mice are indistinguishable from those of controls sug-
gested a defect downstream of glucose metabolism but upstream
of the voltage-gated calcium channels. The diminished response
to glyburide pointed to a defect in KATP-channel function. There-
fore, we examined mRNA levels of the 2 essential subunits of the
channel, SUR1 and Kir6.2. While no statistically significant differ-
ences were observed in SUR1 subunit expression, mRNA and pro-
tein levels of Kir6.2 were downregulated by 40% and 60%, respec-
tively, in the islets of HNF-4αloxP/loxP; Ins.Cre mice (Figure 5, D and
F). Given that Kir6.2 and SUR1 are expressed in α cells as well
as β cells and that the Ins.Cre transgene mediates HNF-4α dele-
tion only in approximately 85% of β cells, the actual reduction in
Kir6.2 expression in HNF-4α–deficient β cells is likely to be greater
than 60%. This reduced expression of Kir6.2 provides a possible
molecular link between the loss of HNF-4α in the β cell and the
dysregulation of insulin secretion observed in vivo and in vitro in
HNF-4αloxP/loxP; Ins.Cre mice (see Discussion).
Prior studies using forced overexpression of either wild-type or
a dominant negative version of HNF-4α in insulinoma cells had
suggested a range of HNF-4α targets, many of them involved in
glycolysis or mitochondrial function (10). Of these genes, only the
gene encoding L-pyruvate kinase was differentially expressed in
isolated islets of HNF-4α mutant mice (Figure 5C). Other impor-
tant metabolic genes such as those encoding GLUT2, glucokinase,
aldolase B, oxoglutarate dehydrogenase, and insulin, previously
identified as putative HNF-4α targets, were not differentially
expressed. The maintenance of normal expression of these genes is
consistent with the unchanged ATP production in response to glu-
cose in HNF-4αloxP/loxP; Ins.Cre mice described above (Figure 4B).
Previous studies have suggested that HNF-4α may exert its
function in the pancreatic β cell through the regulation of
HNF-1α expression (9, 10) and that the regulation of HNF-1α
by HNF-4α is a component of a transcriptional regulatory loop
that exists in the adult β cell (11). However, using quantitative
real-time PCR and Western blot analysis, we found that HNF-4α
is not required for the maintenance of HNF-1α mRNA or pro-
tein expression in the adult β cell (Figure 5, A and E). In addi-
HNF-4α is not required for the maintenance of islet architecture or β
cell mass. (A–D) Immunofluorescence detection of the pancreatic hor-
mones insulin, glucagon, and somatostatin, which label β cells, α cells,
and δ cells, respectively. Similar to controls (A and C), 3- to 5-month-
old HNF-4αloxP/loxP; Ins.Cre mice contain glucagon-positive α cells (B)
and somatostatin-positive δ cells (D). In addition, insulin-positive β cells
are centrally located in the islets of both controls and mutants, while
less-frequent α cells and δ cells are found along the periphery (A–D),
indicating normal islet architecture in the HNF-4αloxP/loxP; Ins.Cre mice.
Magnification, ×200. (E) Point-counting morphometry of 4-month-old
mice reveals no significant difference in β cell mass between controls
and mutants (control, 0.93 ± 0.17 mg, n = 6; mutant = 1.04 ± 0.22 mg,
n = 5; P = NS).
1010 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
tion, we did not find significant differences in mRNA levels of
other MODY genes, including HNF-1β, Pdx-1, and NeuroD1, or
those encoding other pancreatic transcription factors that regu-
late insulin secretion (Figure 5, A and B). Levels of mRNA in
the gene encoding Foxa2, an essential regulator of Kir6.2 expres-
sion, were not altered significantly (18, 25). However, expres-
sion of the nuclear receptor HNF-4γ was significantly reduced
by approximately 34%, indicating that HNF-4α is required for
normal expression of its related family member. Furthermore,
PPARα, a target of HNF-4α in the liver (26) postulated to play
a role in the β-oxidation of lipids in β cells (27), was downregu-
lated by approximately 70% in mutant islets (Figure 5B).
HNF-4α binds the Kir6.2 promoter and activates the Kir6.2 gene in
cotransfection assays. To evaluate whether HNF-4α can directly regu-
late Kir6.2 expression at the transcriptional level, we searched for
potential HNF-4α binding sites in the Kir6.2 promoter. A puta-
tive HNF-4α binding site was identified 2,300 bp upstream of the
transcriptional start site (Figure 6, A and B). In order to determine
if HNF-4α can bind this sequence of the Kir6.2 promoter, we per-
formed electrophoretic mobility shift assays (EMSAs). Incuba-
tion of wild-type liver nuclear extract with a radiolabeled oligo-
nucleotide containing the putative HNF-4α binding site sequence
resulted in a strong shift of the radioactive band (Figure 6C). The
addition of an antibody against HNF-4α generated a supershifted
band comparable to that achieved with the consensus site probe,
but was not observed with preimmune serum, indicating that the
bound protein was indeed HNF-4α. These results were also con-
firmed using an additional antibody against HNF-4α. To determine
if the cis-regulatory element in the Kir6.2 gene can function as an
HNF-4α–dependent enhancer, we performed cotransfection assays
with a 237-bp region of the Kir6.2 gene containing the HNF-4α
binding site cloned into a luciferase promoter plasmid and an
HNF-4α expression vector. We found that overexpression of
HNF-4α in baby hamster kidney (BHK) cells resulted in an increase
of approximately 5-fold in luciferase activity and that this tran-
scriptional activation was abolished when the HNF-4α binding
site sequence was mutated (Figure 6D). Our results demonstrate
that HNF-4α is a transcriptional activator of the Kir6.2 gene.
MODY is a monogenic form of diabetes characterized by early
onset and the progressive loss of insulin secretory capacity (28).
Several recent reports have suggested that the MODY1 subtype
results from the loss of HNF-4α function in the pancreas and
that HNF-4α functions as “master regulator” of multiple tran-
scriptional networks in the islet (11). However, the specific role
for HNF-4α in the maintenance of β cell function has not to our
knowledge been established until now. Here we have attempted to
address this question by genetic means.
We used the Cre-loxP recombination system to delete HNF-4α in
β cells of the adult pancreas. We found that 3- to 5-month-old HNF-
4αloxP/loxP; Ins.Cre mice exhibited elevated plasma insulin levels in
the fasted and fed states but also suffered from impaired glucose
tolerance. Although these results provide evidence that HNF-4α
is required in the β cell for the regulation of insulin secretion, the
overall phenotype of these animals was quite surprising, consider-
ing the hypoinsulinemic hyperglycemia present in humans with
reduced HNF-4α function. Given the relatively late onset of MODY1
in humans (15–25 years) compared with the age of our mice (3–5
months), and given that in MODY1 patients, HNF-4α function
is impaired in the liver in addition to β cells, it appears likely that
long-term and cumulative damage to the β cell contributes to the
more severe phenotype observed in humans. However, consistent
with the observations made in MODY1 patients, HNF-4αloxP/loxP; Ins.
Cre mice failed to secrete adequate amounts of insulin after glucose
stimulation and thus are glucose intolerant. This can be explained
by our finding that isolated islets lacking HNF-4α demonstrated an
abnormal response to glucose in perifusion experiments, including
an attenuated first phase of insulin secretion.
Also surprising is the finding that HNF-1α, which is dependent
on HNF-4α in hepatocytes (26), was not differentially expressed
Glucose-stimulated insulin secretion is dysregulated in isolated islets of
HNF-4αloxP/loxP; Ins.Cre mice. (A) Isolated islets from HNF-4α mutants
(open circles) lack a robust first-phase insulin secretory response to
glucose perifusion compared with that of controls (filled squares),
and fail to rapidly terminate insulin secretion upon switching to 0 mM
glucose (n = 3). (B) ATP levels in isolated islets from HNF-4αloxP/loxP;
Ins.Cre mice (white bars) stimulated with 2, 5, or 10 mM glucose for
60 minutes are virtually indistinguishable from those of control mice
(black bars) (n = 2 per group), indicating that glucose metabolism is not
adversely affected in HNF-4α–deficient β cells. (C) The intracellular
calcium concentration ([Ca2+]i) increases rapidly in response to 16.7
mM glucose (2.9 nM/s), 1 μM glyburide (10.0 nM/s), and 30 mM KCl
in control islets. (D) In contrast, the intracellular calcium concentration
increases at a slower rate in response to glucose (0.7 nM/s) and glybu-
ride (2.0 nM/s) in HNF-4αloxP/loxP; Ins.Cre mice. For all calcium-imaging
experiments, n = 4 per group. These are representative plots.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
at the mRNA or protein level in HNF-4α mutant islets. Given the
reports that HNF-4α binds to the HNF-1α promoter and that a
mutation in the HNF-4α binding site on the HNF-1α promoter
leads to MODY, one would expect HNF-4α to be an essential regula-
tor of HNF-1α expression in the β cell (3, 11). However, as described
by Servitja and colleagues, it is likely that the regulatory relation-
ships between MODY genes vary between tissues and developmen-
tal stages (8). Although our results demonstrate that HNF-4α is
not required for the maintenance of HNF-1α expression in adult β
cells, it remains possible that HNF-4α is required for the initiation of
HNF-1α expression earlier during pancreatic development. Another
possibility is that the regulatory relationship is more important in
the liver than in the β cell. However, the downregulation of HNF-4γ
and PPARα suggests that HNF-4α functions in a transcriptional
regulatory network involving other nuclear receptors.
Given the report by Wang and colleagues that HNF-4α reg-
ulates the expression of genes associated with β cell glucose
metabolism and insulin secretion in insuli-
noma cells (10), we initially hypothesized that
altered expression of these genes and a defect
in glucose metabolism in HNF-4α–deficient β
cells may relate to the observed phenotype of
these mice. However, of all of the targets sug-
gested to be dependent upon HNF-4α in that
study, only L-pyruvate kinase was significantly
reduced in the islets of HNF-4αloxP/loxP; Ins.Cre
mice. This discrepancy may be explained by
the fact that Wang and colleagues used insuli-
noma cells and overexpressed HNF-4α to lev-
els at which the protein will bind promoters
that it will not normally bind in vivo. It is also
likely that several redundant transcriptional
regulatory mechanisms exist in the adult β
cell. Odom and colleagues identified several
cases of “multiple input regulatory circuits”
in the β cell that are controlled by a number
of transcription factors (11). Given the over-
lap of target promoters bound by HNF-1α,
HNF-4α, and other pancreatic transcription
factors, it is possible that these other pro-
teins can compensate for the loss of HNF-4α
to maintain gene expression. Interestingly,
analogous overexpression studies with a dominant negative
HNF-1α in the same insulinoma cell line resulted in a similar
phenotype and gene expression profile, as was the case with
the dominant negative HNF-4α (29). However, when Shih and
colleagues performed a comprehensive gene expression analy-
sis of HNF-1α–deficient islets, they found discrepancies with
the insulinoma studies similar to those we have reported here
(22). Furthermore, it has been shown that downregulation of
L-pyruvate kinase in pancreatic islets has little effect on over-
all pyruvate kinase activity, consistent with the finding that the
M2-pyruvate kinase isoform predominates in β cells (30, 31).
Thus, it is unlikely that changes in L-pyruvate kinase expression
explain the observed phenotype of HNF-4αloxP/loxP; Ins.Cre mice.
In addition, our results, as well as the results reported by Shih
and colleagues, further emphasize the need for animal models
with which to study the functional requirements of transcrip-
tion factors in the regulation of β cell gene expression.
Gene expression analysis in isolated islets of HNF-
4αloxP/loxP; Ins.Cre mice. (A) Levels of mRNA of
MODY genes, as determined by real-time PCR. (B)
Levels of mRNA of pancreatic enriched transcrip-
tion factors, as determined by real-time PCR. (C)
Levels of mRNA of genes involved in glucose and
lipid metabolism, as determined by real-time PCR.
(D) Levels of mRNA of genes involved in stimu-
lus-secretion coupling, as determined by real-time
PCR. *P < 0.05; n = 3–5 for all PCR experiments.
HPRT, hypoxanthine guanine phosphoribosyl
transferase. (E) Western blot analysis of HNF-1α in
isolated islets normalized to α-tubulin protein lev-
els. C, control; M, mutant. (F) Western blot analysis
of Kir6.2 in isolated islets normalized to α-tubulin
protein levels. For all protein quantification, n = 3,
controls, and n = 2, mutants.
1012 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
Using the HNF-4αloxP/loxP; Ins.Cre mouse model, we have revealed
an unexpected role for HNF-4α in the regulation of the KATP chan-
nel–dependent pathway of glucose-stimulated insulin secretion.
We have found that HNF-4α is required in the β cell for the main-
tenance of normal Kir6.2 mRNA and protein expression and that
HNF-4α is a transcriptional activator of the Kir6.2 gene. Because
Odom and colleagues examined only promoter elements located
–700 bp to +200 bp relative to the transcriptional start site in their
location analysis (11), they failed to identify target genes that are
bound by HNF-4α further upstream, such as Kir6.2. The impor-
tance of Kir6.2 function in glucose homeostasis has been clearly
established, because mutations in the Kir6.2 locus in humans can
lead to persistent hyperinsulinemic hypoglycemia of infancy (32,
33). In addition, several mouse models have demonstrated the
importance of the KATP channel in maintaining β cell function
(34–38). In particular, expression of a dominant negative form
of Kir6.2 or targeted disruption of the Kir6.2 gene in mouse β
cells leads to a reduction in or loss of KATP channel function and,
consequently, elevated basal calcium levels and impaired glucose-
stimulated insulin secretion from isolated islets (34, 36, 37). More
recently, Li and colleagues used a hammerhead ribozyme to reduce
Kir6.2 mRNA levels in RINm5F cells, resulting in a 60% decrease
in KATP channel density, sufficient to diminish glucose-stimulated
insulin release (35). In addition to the observed defects in glucose
and sulfonylurea induced insulin secretion, another hallmark of
KATP channel–deficient mice is the inability of isolated islets to
properly shut off calcium influx and insulin secretion upon glu-
cose withdrawal (39). The insulin secretory defects and calcium
responses we have described here for HNF-4αloxP/loxP; Ins.Cre mice
resemble many of the defects in KATP channel–deficient mice. It
should be noted that because Kir6.2 levels are reduced but not
absent in the HNF-4α mutants, the β cell defect in Kir6.2–/– mice is
more severe than the defect in HNF-4α–deficient β cells. Neverthe-
less, Kir6.2–/– mice paradoxically exhibit an even more mild impair-
ment in glucose tolerance. This is explained by the finding that
Kir6.2–/– mice are hypersensitive to insulin due to the critical role
of Kir6.2 in regulating glucose uptake by adipose tissue and skel-
etal muscle (36, 40). In HNF-4αloxP/loxP; Ins.Cre mice that we have
derived here the expression of HNF-4α is lost only in the pancre-
atic β cells of the islet. Therefore, the skeletal muscle defects seen
in Kir6.2–/– mice are not be present in the HNF-4α mutants. This
was confirmed by the normal insulin sensitivity of HNF-4αloxP/loxP;
Ins.Cre mice (Figure 2G). However, transgenic mouse models of
Kir6.2 have been reported in which a dominant negative form of
Kir6.2 is expressed specifically in β cells (34, 37). Similar to HNF-
4αloxP/loxP; Ins.Cre mice, Kir6.2-transgenic mice are hyperinsulin-
emic and exhibit defects in glucose-stimulated insulin secretion
from isolated islets, including the loss of the first phase of insulin
secretion, and abnormal calcium influx in response to glucose
and sulfonylureas (34). Therefore, we propose that the mainte-
nance of Kir6.2 expression by HNF-4α is necessary for normal
glucose-stimulated insulin secretion and that the downregulation
of Kir6.2 contributes in part to the observed phenotype of the
HNF-4α mutant mice.
Although our data have shown that HNF-4α is required in the
adult β cell for the regulation of insulin secretion, the absence of
HNF 4α from the β cells of 5-month-old HNF-4αloxP/loxP; Ins.Cre
mice is not sufficient to trigger the onset of overt diabetes. Several
possibilities may explain this observation. First, the loss of HNF-4α
from β cells of HNF-4αloxP/loxP; Ins.Cre mice may lead to hypergly-
HNF-4α directly activates the Kir6.2 gene. (A) The consensus binding site for HNF-4α is 13 bp long and was derived from 71 known HNF-4α
binding sequences from the literature (55) using the program Weblogo (http://weblogo.berkeley.edu/). The size of the letters reflects the fre-
quency at which the nucleotide appears at that position in the binding site. (B) Putative HNF-4α binding site in the Kir6.2 promoter identified
using NUBIScan, which uses a transcription factor–binding site–identification algorithm to identify nuclear receptor binding sites. Note that the
site located at position –2,300 matches all determinant nucleotides in the HNF-4α consensus site shown in A. (C) EMSA demonstrates that
HNF-4α binds to the identified binding site in the Kir6.2 gene as well as the HNF-4α consensus site. In supershift experiments using 2 different
antibodies raised against HNF-4α, the identity of the bound protein is confirmed to be HNF-4α. (D) Cotransfection of BHK cells with HNF-4α
and pGL3-Kir6.2, expressing luciferase under the control of the 237-bp region of Kir6.2 containing the binding site, results in a dose-dependent
increase in luciferase activity, indicating that this element serves as an HNF-4α–dependent enhancer. Mutation of this binding site abolishes the
transcriptional activation. Statistical analysis was performed by ANOVA; n = 3 for each transfection condition.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
cemia with increased age, in the presence of genetic modifiers, or
in the presence of environmental factors such as a high-fat diet.
The same factors have been shown to play a role in the eventual
development of hyperglycemia observed in older Kir6.2-deficient
mice. Older mice transgenic for a dominant negative mutant of
Kir6.2 develop hyperglycemia and glucose intolerance (37). The
progression to hyperglycemia is accelerated when these mice are
fed a high-fat diet (41). Second, several mechanisms for nutrient-
stimulated insulin secretion exist in the β cell. The mild impair-
ment in glucose tolerance exhibited by KATP channel–deficient
mice has been explained in part by the increased activity of KATP
channel–independent pathways leading to insulin secretion (42,
43). Thus, while HNF-4α is essential for KATP channel–dependent
insulin secretion, it is possible that other pathways that potenti-
ate nutrient-stimulated insulin secretion can compensate in the
short term to prevent more-severe impairments in glucose homeo-
stasis. One possible alternate pathway is suggested by our find-
ing that expression of the nuclear receptor PPARα was reduced
in HNF-4α–deficient β cells. PPARα has been shown to activate
genes encoding enzymes of the β-oxidation pathway of fatty acids.
A decrease in β-oxidation is suggested to result in the accumula-
tion of lipids in the cytoplasm, resulting ultimately in increased
insulin secretion (44, 45). Given the postulated role of PPARα in
the regulation of β cell lipid metabolism, it is possible that the
lower level of PPARα in the HNF-4α mutants partially contrib-
utes to the elevated basal insulin levels (27). Third, as described
above, it is possible that HNF-4α also plays an important role in
the developing pancreas prior to excision of the floxed HNF-4α
allele by the Ins.Cre transgene. Like many other pancreatic tran-
scription factors such as Pdx-1 (MODY4) or Foxa2, HNF-4α may
be required at multiple stages of pancreatic development. Finally,
it is also likely that in mice and humans, contributions of other
HNF-4α–deficient organs are necessary for the progression to
type 2 diabetes. MODY 1 diabetics exhibit impairments in lipid
homeostasis prior to the onset of hyperglycemia, indicating a pri-
mary hepatic lesion in these patients (46). Targeted disruption of
HNF-4α in the adult liver also results in impaired lipid homeo-
stasis, abnormal glycogen deposition, and hepatic hypertrophy
(26). More recently, Parviz and colleagues have demonstrated the
importance of HNF-4α in the formation of a hepatic epithelium
during liver development (47). Thus, this raises the possibility
that haploinsufficiency for HNF-4α in the liver also contributes
to the progression of hyperglycemia in MODY1. Supporting this
hypothesis are studies of MODY5. Patients with mutations in the
MODY5 gene HNF-1β suffer diabetes and renal dysfunction (48).
However, mice with a targeted deletion of HNF-1β in β cells are
glucose intolerant due to impaired insulin secretion but do not
exhibit hyperglycemia, providing another example of how the loss
of a MODY gene only in the β cell may not be sufficient to trigger
overt diabetes (49). Nevertheless, the model of HNF-1β and the
model of HNF-4α reported in this article provide excellent tools to
elucidate the mechanisms by which these MODY genes contribute
to the maintenance of glucose homeostasis by the β cell.
In summary, our data provide genetic evidence that HNF-4α is
required in the adult β cell for the regulation of β cell function.
Our data also reveal an unexpected role for HNF-4α in the regula-
tion of the KATP channel–dependent pathway of insulin secretion.
The model derived here will serve as a useful tool for identifying
additional genes and pathways dependent on HNF-4α activity and
may lead to novel treatment regimens for type 2 diabetes.
Animals and genotype analysis. The derivation of both HNF-4aloxP/loxP and Ins.
Cre mice has been reported previously (15, 50). All mice were maintained
on the CD1 background. Genotyping was performed by PCR analysis using
genomic DNA isolated from the tail tips of newborn mice. All of the experi-
ments described here focused on female mice, because female HNF-4αloxP/loxP;
Ins.Cre mice showed larger impairments in glucose homeostasis compared
with age- and sex-matched control mice on a standard diet than did male
mice (Figure 2). Littermate HNF-4αloxP/loxP and HNF-4αloxP/+ female mice were
used as controls. All procedures involving mice were approved by the Uni-
versity of Pennsylvania Institutional Animal Care and Use Committee.
Immunofluorescence and immunohistochemistry. Indirect immuno-
fluorescence was performed as described previously (18) and was exam-
ined using confocal microscopy (Leica Microsystems Inc.). The follow-
ing antibodies were used: guinea pig anti-insulin (1:800 dilution; Linco
Research Inc.), rabbit anti-glucagon (undiluted; Zymed Laboratories
Inc.), rabbit anti-somatostatin (1:50 dilution; Zymed Laboratories Inc.),
indocarbocyanine-conjugated donkey anti-rabbit IgG (1:750 dilution;
Jackson ImmunoResearch Laboratories Inc.), and carbocyanine-con-
jugated donkey anti–guinea pig IgG (1:200 dilution; Jackson Immuno-
Research Laboratories Inc.).
For immunohistochemistry, slides were blocked with avidin D and
biotin blocking reagents (Vector Laboratories) for 15 minutes at room
temperature with a quick rinse in PBS in between. All slides were blocked
with protein-blocking reagent (Immunotech) for 20 minutes at room
temperature. Anti–HNF-4α (SC-6556; Santa Cruz Biotechnology) was
diluted in phosphate buffered saline plus tween (PBT) and was incubated
with tissue overnight at 4°C. Slides were washed in PBS and were incu-
bated with biotinylated anti-goat. HRP-conjugated avidin–biotinylated
enzyme complex reagent was used following the manufacturer’s proto-
col (Vector Laboratories). Signals were developed using 3,3-diaminoben-
zidine tetrahydrochloride as substrate. For β cell mass determination,
pancreata were laid flat during the paraffin-embedding process. The
section with the largest tissue surface area was stained for insulin by
immunohistochemistry as outlined above. Quantification of β cell mass
was performed as described previously (16).
Glucose and insulin tolerance tests. For glucose tolerance tests, animals that
had fasted overnight (16 hours) were injected intraperitoneally with 2
grams of glucose (Sigma-Aldrich) per kilogram of body weight. Glucose
levels were measured at 0, 15, 30, 60, 90, and 120 minutes with Glucometer
Elite (Bayer Corporation). For determination of plasma insulin concen-
trations during glucose tolerance tests, animals that had fasted overnight
were injected with 3 grams of glucose per kilogram of body weight and
blood was collected from the tail vein at 0, 2, 5, 15, and 30 minutes after
injection. Plasma insulin measurements were performed by ELISA (Crystal
Chem Inc.) For insulin tolerance tests, mice that had fasted for 4 hours
were injected intraperitoneally with 0.75 units of insulin per kilogram of
body weight. Glucose levels were measured at 0, 15, 30, 60, 90, and 120
minutes as described above.
Islet perifusions. For each experiment, 100 islets were isolated from 3- to
5-month-old mutants and controls using standard collagenase digestion
followed by purification through a Ficoll gradient (51). One hundred
islets were “hand-picked” under a light microscope and were placed into a
perifusion chamber (Millipore). A computer-controlled fast-performance
HPLC system (625 LC System; Waters Corporation) allowed for program-
mable rates of flow and concentration of the appropriate solutions held at
37°C in a water bath. Islets were perifused with Krebs bicarbonate buffer
(2.2 mM Ca2+, 0.25% bovine serum albumin, 10 mM HEPES [acid], and 95%
O2 and 5% CO2 equilibration, pH 7.4) to reach baseline hormone secretion
values before the addition of the appropriate secretagogues. Samples were
1014 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
collected at regular intervals with a fraction collector (Waters Corporation)
and insulin content was determined using a radioimmune assay (Univer-
sity of Pennsylvania Diabetes Center).
ATP assays. Isolated islets were cultured at 37°C and 5% CO2 for 3 days
in RPMI 1640 medium (glucose-free; Sigma-Aldrich) supplemented with
10% FBS, 2 mM glutamine, 100 units/ml penicillin, 50 μg/ml streptomy-
cin, and 10 mM glucose. Then, 50 islets per condition were preincubated
for 90 minutes at 37°C and 5% CO2 in glucose-free Krebs bicarbonate
buffer and then were incubated for 60 minutes in buffer containing the
indicated amounts of glucose (Figure 4B). ATP was extracted from islets
and assayed as described previously (52).
Calcium-imaging. Isolated islets were cultured for 3 days in 10 mM glucose
and were pretreated at 37°C for 40 minutes in Krebs bicarbonate buffer
supplemented with 1 mM fura-2 acetoxymethylester (fura-2AM; Invitrogen
Corp.). The fura-2AM–loaded islets were transferred to a perifusion cham-
ber and were placed on the homeothermic platform of an inverted Zeiss
microscope for visualization with a 40× oil-immersion objective (Carl Zeiss
MicroImaging Inc.). Islets were perifused with Krebs bicarbonate buffer at
37°C at a flow rate of 2 ml/min while various treatments were applied. The
intracellular calcium concentration was determined by the ratio of the exci-
tation of fura-2AM at 334 nm to that at 380 nm. Emission was measured at
520 nm with an Attofluor charge-coupled device camera and was calibrated
using Attofluor Ratio Vision Software (BD Biosciences). The rate of change
in intracellular calcium was calculated from the first 6 data points starting
with the data point prior to the first observed increase in calcium.
Real-time PCR and Western blot analysis. Islets from 3- to 5-month-old mice
were isolated using the standard collagenase procedure as described above.
Total RNA from islets was isolated in Trizol (Invitrogen Corp.) according to
the manufacturer’s instructions. Islet RNA was reverse-transcribed using 1
μg oligo(dT) primer, Superscript II Reverse Transcriptase, and accompany-
ing reagents (Invitrogen Corp.). PCR reaction mixes were assembled using
the Brilliant SYBR Green QPCR Master Mix (Stratagene). Reactions were
performed using the SYBR Green (with Dissociation Curve) program on
the Mx4000 Multiplex Quantitative PCR System (Stratagene). All reactions
were performed in triplicate with reference dye normalization, and median
cycling threshold values were used for analysis. Primer sequences are avail-
able upon request. Islet purity was assessed as previous described (25). For
Western blots, islet extracts were prepared as described previously (53),
were separated by SDS-PAGE, were transferred to immobilin P membranes
(Millipore), and were probed with rabbit polyclonal anti-Kir6.2 (Chemicon
International Inc.), rabbit anti–HNF-1α (Santa Cruz Biotechnology Inc.),
and monoclonal anti–α-tubulin (Sigma-Aldrich). The ECL Plus detection
system was used to detect the signal (Amersham Pharmacia). Band intensi-
ties were quantified using the QuantityOne 4.3.1 program (Biorad Labora-
tories). Intensities were normalized to those obtained for α-tubulin.
Computational identification of HNF-4α binding sites in the Kir6.2 promoter.
For the identification of potential HNF-4α binding sites in the Kir6.2
promoter, 4.5 kilobases of promoter sequence upstream of the transcrip-
tional start site was retrieved from the University of California Santa Cruz
Genome Browser (http://www.genome.ucsc.edu) and was uploaded into
the NUBIScan website (http://www.nubiscan.unibas.ch), which uses an
in silico approach for predicting nuclear receptor response elements (54).
A directed search for DR1 (direct repeat with single nucleotide spacing)
half-sites using the HNF-4α matrix was performed to identify the putative
binding sites. The site with the highest score was analyzed in gel-shift and
EMSA. Oligonucleotides were synthesized corresponding to the HNF-4α
consensus site (sc-2599; Santa Cruz Biotechnology Inc.) and to the HNF-4α
inding site in the Kir6.2 gene located 2.3 kilobases upstream of the tran-
scriptional start site. Radiolabeled probes were generated by the incuba-
tion of 250 ng of annealed oligonucleotides for 15 minutes at 37°C with
20 μCi [32P]dCTP in the presence of Klenow DNA polymerase (Roche
Applied Science). Radiolabeled probes were subsequently separated from
free nucleotide using G-50 column purification (Amersham Biosciences).
Liver nuclear extract (a kind gift from L.E. Greenbaum, University of Penn-
sylvania, Philadelphia, Pennsylvania, USA) was then incubated at room
temperature for 15 minutes with radiolabeled probe at 100,000 dpm and
1 μg poly(dI-dC) in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT,
1 mM EDTA, and 5% glycerol. Binding reactions were then incubated with
the specified Santa Cruz HNF-4α antisera for 30 minutes at room tem-
perature. Samples were resolved by 5% polyacrylamide gel electrophore-
sis in 0.5% Tris-buffered EDTA at 300 V for 2 hours. The dried gels were
exposed to a phosphorimager cassette (Amersham Biosciences) and were
analyzed with Storm840 software (Amersham Biosciences). Oligonucle-
otide sequences for the HNF-4α site in the Kir6.2 gene were as follows:
forward, 5′-GGGGAAGGCCAGACCAAAGGGCAGACCCT-3′; reverse,
Transient transfections and luciferase reporter assays. A 237-bp fragment of
the Kir6.2 promoter containing the putative HNF-4α binding site was
cloned by PCR to contain KpnI and BglII restriction enzyme sites on the 5′
and 3′ ends, respectively. A mutated version of this element was generated
by overlap PCR (Figure 6D). The wild-type and mutant PCR fragments
were then cloned into the KpnI and BglII sites of the pGL3 promoter
luciferase vector (Promega Corp.). The pCMVβ-HNF 4α plasmid (a kind
gift from M. Stoffel, Rockefeller University, New York, New York, USA)
was used to expresses FLAG-tagged HNF-4α under control of the cyto-
megalovirus promoter. BHK cells (5 × 105) were seeded 16 hours prior to
transfection in 60-mm dishes and were cultured in DMEM supplemented
with 10% FBS, L-glutamine, penicillin, and streptomycin. Transient trans-
fections were performed using the Effectine transfection reagent (QIA-
GEN) according to the manufacturer’s instructions. At 24 hours after
transfection, cells were harvested and luciferase activity was measured
using the Dual Luciferase Reporter Assay (Promega Corp.). Luciferase
activity was normalized for transfection efficiency by the corresponding
Renilla luciferase activity.
The authors are grateful to M.A. Lazar, D.A. Stoffers, and J.R.
Friedman for critical reading of the manuscript. The authors
thank Kristen A. Lantz, Sara D. Sackett, Regina K. Gorski, Maria
Golson, John E. Brestelli, and Peter White for contributions to
this work, and Shamina Rangwala and Phillip P. Le for valuable
discussions. The authors are also grateful to Heather Collins for
performing radioimmune assays, Nicolai Doliba for help with cal-
cium-imaging experiments, and Harshani Peiris and Linda Green-
baum for liver nuclear extracts. Our studies were assisted by the
University of Pennsylvania Diabetes Center (P30DK19525) and the
Penn Center for Molecular Studies in Digestive and Liver Disease
(P30DK50306). This work was supported by the National Institute
of Diabetes and Digestive and Kidney Diseases (grants DK55342
and DK56947 to K.H. Kaestner; DK55743 and DK60064 to S.A.
Duncan; and DK61226 to C.S. Lee).
Received for publication June 7, 2004, and accepted in revised form
January 18, 2005.
Address correspondence to: Klaus H. Kaestner, Department of Genet-
ics, University of Pennsylvania School of Medicine, 415 Curie Boule-
vard, Philadelphia, Pennsylvania 19104-6145, USA. Phone: (215) 898-
8759; Fax: (215) 573-5892; E-mail:email@example.com.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 4 April 2005
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