HNF4α Regulates the Expression of Pancreatic β-Cell Genes Implicated in Glucose ... ·...

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HNF4α Regulates the Expression of Pancreatic β-Cell Genes Implicated in Glucose Metabolism and Nutrient-Induced Insulin Secretion Haiyan Wang, Pierre Maechler, Peter A. Antinozzi, Kerstin A. Hagenfeldt, and Claes B. Wollheim * Running Title: HNF4α Regulated β-Cell Gene Expression and Insulin Secretion * To whom correspondence should be addressed: Claes B. Wollheim Division de Biochimie Clinique Départment de Médecine interne Centre Médical Universitaire CH-1211 Geneva 4 Switzerland Phone: (41 22) 702 5548 1 Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on August 30, 2000 as Manuscript M006612200 by guest on February 26, 2020 http://www.jbc.org/ Downloaded from

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HNF4α Regulates the Expression of Pancreatic β-Cell Genes

Implicated in Glucose Metabolism and Nutrient-Induced Insulin

Secretion

Haiyan Wang, Pierre Maechler, Peter A. Antinozzi, Kerstin A. Hagenfeldt, and Claes

B. Wollheim*

Running Title: HNF4α Regulated β-Cell Gene Expression and Insulin Secretion

*To whom correspondence should be addressed: Claes B. Wollheim

Division de Biochimie Clinique

Départment de Médecine interne

Centre Médical Universitaire

CH-1211 Geneva 4

Switzerland

Phone: (41 22) 702 5548

1

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 30, 2000 as Manuscript M006612200 by guest on February 26, 2020

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Fax: (41 22) 702 5543

E-mail: [email protected]

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Mutations in the HNF4α gene are associated with the subtype 1 of maturity-onset

diabetes of the young (MODY1), which is characterized by impaired insulin secretory

response to glucose in pancreatic β-cells. HNF4α is a transcription factor critical for

liver development and hepatocyte-specific gene expression. However, the role of

HNF4α in the regulation of pancreatic β-cell gene expression and its correlation with

metabolism-secretion coupling has not previously been investigated. The reverse

tetracycline-dependent transactivator system was employed to achieve tightly

controlled and rapidly inducible expression of both wild type (WT) and dominant-

negative mutant (DN) of HNF4α in INS-1 cells. The induction of WT-HNF4α

resulted in a left-shift in glucose-stimulated insulin secretion, whereas DN-HNF4α

selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4α

also caused defective mitochondrial function substantiated by reduced [14C]-

pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane

hyperpolarisation and blunted nutrient-generated cellular ATP production.

Quantitative evaluation of HNF4α-regulated pancreatic β-cell gene expression

revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase,

aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling

protein-2. The patterns of HNF4α regulated gene expression are strikingly similar to

that of its down-stream transcription factor HNF1α. Indeed HNF4α changed the

HNF1α mRNA levels and HNF1α promoter luciferase activity through altered

HNF4α binding. These results demonstrate the importance of HNF4α in β-cell metabolism-

secretion coupling.

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The hepatocyte nuclear factor 4α (HNF4α), a transcription factor of the nuclear

hormone receptor superfamily, is expressed in liver, kidney, gut and pancreatic islets

(1-3). Mutations in the human HNF4α gene lead to maturity onset diabetes of the

young subtype I (MODY I), which is characterized by autosomal dominant

inheritance and impaired glucose-stimulated insulin secretion from pancreatic β-cells

(4-6). These MODY1 mutations located in various domains of the HNF4α protein

result in defective function of the transcription factor (6). The clinical phenotype of

MODY1 patients is indistinguishable from that of MODY3 patients who carry

mutations in the HNF1α gene (5, 6). HNF4α acts upstream of HNF1α in a

transcriptional cascade that drives liver-specific gene expression and hepatocyte

differentiation (7-9). A naturally occurring mutation in the HNF4α binding site of the

HNF1α promoter identified in a MODY3 family (10) suggests that the transcriptional

hierarchy could also be involved in pancreatic β-cell gene expression and function.

HNF4α defines the expression of liver-specific genes encoding apolipoproteins,

serum factors, cytochrome P450 isoforms, and proteins involved in the metabolism of

glucose, fatty acids, and amino acids (reviewed in reference 11). However, clinical

characterization of MODY1 subjects reveals that the primary defect is impaired

glucose-stimulated insulin secretion from pancreatic β-cells rather than liver

dysfunction (5, 12-14). Unfortunately, little is known as to how HNF4α regulates β-

cell restricted gene expression and glucose-metabolism, and associated insulin

secretion. Targeted disruption of the hnf4α gene results in defective gastrulation of

mouse embryos due to dysfunction of the visceral endoderm (15). This early

embryonic lethality prevents further analysis of the HNF4α function in pancreatic β-

cells. The precise role of HNF4α in pancreatic β-cells would best be examined by

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conditional β-cell specific deletion of the mouse hnf4α gene. Another alternative is to

up- and down-regulate HNF4α function in pancreatic β-cell lines through gene

manipulation.

In the present study, the wild type HNF4α (WT-HNF4α) and its dominant-

negative mutant (DN-HNF4α) could be induced in INS-1 cells under tight control of

the reverse tetracycline-dependent transactivator (16). DN-HNF4α represents the

epitope myc-tagged truncated HNF4α mutant protein lacking the first 111 amino

acids (myc∆111HNF4α) (17). The HNF4α protein consists of an N-terminal ligand-

independent transactivation domain (amino acids 1-24), a DNA binding domain

containing two zinc fingers (amino acids 51-117), and a large hydrophobic portion

(amino acids 163-368) composed of the dimerization, ligand binding, cofactor

binding, and ligand-dependent transactivation domain (18, 19). DN-HNF4α

therefore suppresses the endogenous WT-HNF4α transcriptional activity by the

formation of heterodimers lacking DNA binding capacity (17). We have investigated

in a quantitative manner the consequences of altered HNF4α function on β-cell

specific expression of genes implicated in glucose metabolism and insulin secretion.

This allowed us to elucidate the molecular basis and HNF4α-target genes responsible

for impaired metabolism-secretion coupling in β-cells deficient in HNF4α function.

EXPERIMENTAL PROCEDURES

Generation of stable cell lines-The rat insulinoma INS-1 cell line-derived stable

clones were cultured in RPMI 1640 in 11 mM glucose, unless indicated otherwise

(20). The establishment of the first-step stable clone INS-r3, which expresses the

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reverse tetracycline-dependent transactivator, was reported previously (21). Plasmids

used in the secondary stable transfection were constructed by subcloning the cDNAs

encoding the rat WT-HNF4α (a generous gift from Dr. Darnell Jr., New York) and

DN-HNF4α into the expression vector PUHD10-3 (a kind gift from Dr. H. Bujard,

University Heidelberg, Germany). DN-HNF4α was PCR amplified from WT-

HNF4α using the following primers: ctaggatccttccgggctggcatgaagaaagaagcc;

ccagaattcctgcagatggttgtcctttag. The PCR fragment was subcloned into pcDNA3.1myc

(Invitrogen, Netherlands) and sequenced. Transfection, clone selection, and screening

procedures were described previously (21).

Immunoblot-Immunoblotting procedures were performed as described previously

using enhanced chemiluminescence (Pierce, Illinois) for detection (22). Dilutions for

antibody against HNF4α, (kindly supplied by Dr. F. M. Sladek, University of

Califernia, Riverside, CA) and anti-myc-tag (9E 10) in myeloma SP2/0 culture

medium were 1:6,000 and 1:10.

Insulin secretion and cellular insulin content-Cells in 24-well dishes were cultured in

2.5 mM glucose medium with or without indicated doses of doxycycline for 14 or 48

h. Insulin secretion was measured over a period of 30 min, in Krebs-Ringer-

Bicarbonate-HEPES buffer (KRBH, 140 mM NaCl, 3.6 mM KCl, 0.5 mM

NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM HEPES, 0.1% BSA)

containing indicated stimulators. Insulin content was determined after extraction with

acid ethanol following the procedures of Asfari et al. (20). Insulin was detected by

radioimmunoassay using rat insulin as standard (22).

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Intracellular ATP-Cells in 6-well dishes were cultured in 2.5 mM glucose medium

with or without 500 ng/ml doxycycline for 48 h. The production of ATP was

measured during 8 min stimulation in KRBH. ATP assay was performed as

previously reported (22).

[14C]-pyruvate oxidation-The production of 14CO2 from [1-14C]-pyruvate or [2-

14C]-pyruvate was measured over 1 h in KRBH containing either 0.05 or 1.0 mM

pyruvate as previously described (23, 24).

Mitochondrial Membrane Potential (∆Ψm)-After a 48 h culture period in 2.5 mM glucose

medium with or without 500 ng/ml doxycycline, cells were trypsinized (0.025%

trypsin, 0.27mM EDTA) and the cell suspension was maintained for 2 h in a spinner

culture with 2.5 mM glucose RPMI1640 plus 1% new born calf serum at 37°C.

Mitochondrial membrane potential (∆Ψm) was measured as described (25). Briefly,

after the spinner culture period, cells were loaded with 10 µg/ml rhodamine-123 (Rh-

123) for 10 min at 37°C. After centrifugation, the cells were resuspended and

transferred to the fluorimeter cuvette at 37°C with gentle stirring in an LS-50B

fluorimeter (Perkin-Elmer, Bucks, England) and fluorescence, excited at 490 nm, was

measured at 530 nm.

Total RNA isolation and Northern blotting-Cells in 10-cm dishes were cultured in

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2.5 mM glucose medium with or without 500 ng/ml doxycycline for 14 or 48 h,

followed by an additional 8 h in culture medium with 2.5, 6, 12 and 24 mM glucose.

Total RNA was extracted and blotted to nylon membranes as described previously (22).

The membrane was prehybridized and then hybridized to 32P-labeled random primer

cDNA probes by the technique of Sambrook et al. (26). To ensure equal RNA loading

and even transfer, all membranes were stripped and re-hybridized with "house-

keeping gene" probes such as β-actin or cyclophilin. cDNA fragments used as probes

for L-pyruvate kinase (L-PK), glucose transporter-2 (GLUT-2), glucokinase,

insulin, PDX1, HNF4α, upstream stimulatory factors (USF), c-Jun, and C/EBPβ

mRNA detection were digested from corresponding expression vectors kindly

provided by, respectively, Drs. A. Kahn, B. Thorens, P. B. Iynedjian, J. Philippe, T.

Edlund, J. E. Darnell Jr., M. Sawadogo, W. Schlegel, and U. Schibler. cDNA probes

for rat aldolase B, glyceraldehyde-3 phosphate dehydrogenase (GAPDH),

dimerization cofactor for HNF1α (DcoH), mitochondrial adenine nucleotide

translocator 1 and 2 (ANT1 & ANT2), mitochondrial uncoupling protein-2 (UCP-2),

mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit, glutamate

dehydrogenase (GDH), Pax4, Pax6, Nkx2.2, Nkx6.1, Isl1, insulin receptor substrate-

2 (IRS2), cyclin-dependent kinase-4 (Cdk4), and cyclophilin were prepared by RT-

PCR and confirmed by sequencing.

Nuclear extract preparation and electrophoretic mobility-shift assay (EMSA)-Cells in

10-cm dishes were grown in culture medium with or without 500 ng/ml doxycycline

for 48 h. The following double-stranded oligonucleotides were used as probes:

5’GGCTGAAGTCCAAAGTTCAGTCCCTTCGC3’ (8). EMSA procedures including

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conditions for nuclear extract preparation, probe labeling, binding reactions,

unlabeled-probe competition, and antibody supershift were performed as previously

reported (22).

Transient transfection and luciferase assay-The HNF1α gene promoter luciferase

reporter plasmids, WT-HNF1αLuc (wild type) and ∆AHNF1αLuc (HNF4α binding

site deleted), were kindly provided by Dr. N. Miura (Akita University, Japan) (27).

Transient transfection experiments and luciferase reporter enzyme assays were carried

out as previously reported (22).

RESULTS

WT-HNF4α or DN-HNF4α protein was induced in INS-1 cells in a dose- and time-

dependent manner-We have obtained 10 and 8 clones positively expressing WT-

HNF4α and DN-HNF4α, respectively. The clones, designated as WT-HNF4α#28

and DN-HNF4α#26 that displayed highest induction levels of transgene proteins,

were chosen for the present study. The time course and dose-response of doxycycline

effect on WT-HNF4α and DN-HNF4α expression was illustrated in Fig. 1A and Fig.

1B, respectively. WT-HNF4α protein could be induced within a range from 2- to

50-fold above the endogenous protein level (Fig. 1A). Thus, graded overexpression

of WT-HNF4α could be achieved by culturing the WT-HNF4α#28 cells with

varying doses of doxycycline in a defined period of time. Similar induction of DN-

HNF4α protein was detected in the nuclear extracts from DN-HNF4α#26 cells (Fig.

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1B). No leakage of this doxycycline-dependent promoter was observed, since the

expression of DN-HNF4α protein was not detectable in non-induced DN-

HNF4α#26 cells (Fig. 1B). Therefore, the dominant-negative suppression of HNF4α function in

INS-1 cells could be rapidly achieved by culturing the DN-HNF4α#26 cells with a

maximum dose of doxycycline (500 ng/ml).

Effects of WT-HNF4α and DN-HNF4α on insulin secretion-Impaired glucose-

stimulated insulin secretion from pancreatic β-cells is the primary defect causing

hyperglycemia in MODY 1 patients carrying HNF4α mutations. We therefore

examined the consequences of induction of WT-HNF4α and DN-HNF4α on insulin

secretion in INS-1 cells. The graded overexpression of WT-HNF4α led to a left-

shift of glucose-stimulated insulin secretion (Fig. 2A). However, the maximal (above

12 mM) glucose-elicited insulin secretion remained unchanged (Fig. 2A).

Glucose generates ATP and other metabolic coupling factors important for insulin

secretion through glycolysis and mitochondrial oxidation (28). The physiological

insulin secretagogue, leucine, is transported directly into mitochondria to provide

substrates for the tricarboxylic acid (TCA) cycle (28). K+ causes insulin secretion by

depolarisation of the β-cell membrane, resulting in an increase in cytosolic Ca2+

(28). We therefore examined the insulin secretory responses to these three

secretagogues that act at different levels of the signal transduction cascade following

induction of DN-HNF4α. As demonstrated in Fig. 2B, DN-HNF4α selectively

inhibited glucose and leucine stimulated insulin secretion. This could be explained by

defective glucose and leucine metabolism.

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Effects of DN-HNF4α on cellular ATP production and mitochondrial oxidation-To

investigate whether impaired nutrient-evoked insulin secretion is correlated to

defective cellular ATP production, we analysed the impact of DN-HNF4α expression

on the level of ATP generated by glucose and leucine. As shown in Fig. 3A, induction

of DN-HNF4α indeed abolished the ATP generation by glucose and leucine. Since

the mitochondrial substrate leucine failed to generate ATP after induction of DN-

HNF4α, it would seem that HNF4α is required for maintaining normal mitochondrial

function.

To test this hypothesis, we examined the consequences of DN-HNF4α

induction on mitochondrial oxidation of pyruvate. Pyruvate-derived carbons enter

the TCA cycle as either acetyl CoA, catalyzed by pyruvate dehydrogenase, or

oxaloacetate via pyruvate carboxylase. By using pyruvate radiolabled at either the

first or second carbon, the putative defects at various steps in pyruvate metabolism

can be assessed. The radiolabled carbon of [1-14C]pyruvate is lost to CO2 at the

pyruvate dehydrogenase step as pyruvate is converted into acetyl CoA. Alternatively,

if pyruvate enters the TCA cycle via oxaloacetate, the label is lost to CO2 at isocitrate

dehydrogenase within one turn of the cycle. Radiolabled CO2 is generated from [2-

14C] at either OGDH or isocitrate dehydrogenase when pyruvate enters the TCA cycle

as acetyl CoA. Overexpression of DN-HNF4α reduced CO2 formation from [2-

14C] pyruvate by 41 % (Fig. 3B), whereas CO2 formation from [1-14C] pyruvate was

not different between non- and induced conditions (Fig 3C). These results suggest

that the defect in mitochondrial metabolism is not at the point of entry of pyruvate

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into the TCA cycle, rather that the defect appears in reactions within the TCA cycle.

This is in full agreement with the impairment of leucine-stimulation of insulin

secretion since leucine metabolism bypasses pyruvate and enters the TCA cycle solely

as acetyl-CoA. Decreased isocitrate dehydrogenase activity would also be unlikely

since impairment at this step would be observed by both [1-14C] and [2-14C]

pyruvate oxidation. These oxidation experiments suggest that steps following this

reaction beginning with OGDH may be responsible for impaired [2-14C] pyruvate

oxidation.

Effect of DN-HNF4a on mitochondrial membrane potential (∆Ψm) in INS-1 cells-

The ∆Ψm was measured in a suspension of INS-1 cells by monitoring rhodamine-123

fluorescence. In control cells (-Dox) addition of 10 mM glucose (12.5 mM final)

potently hyperpolarized ∆Ψm while 1 µM of the protonophore FCCP depolarized

∆Ψm (Fig. 4A). In cells expressing DN-HNF4α (+Dox), the glucose response was

inhibited by 65% (P<0.02). Impaired hyperpolarization of ∆Ψm was also observed

when the glycolysis was bypassed by stimulating cells with the end product of

glycolysis pyruvate (Fig. 4B), indicating mitochondrial dysfunction. Direct activation

with methyl-succinate of the electron transport chain at complex II resulted in a

diminished response in DN-HNF4α induced cells (Fig. 4C). The amplitude of

complete ∆Ψm depolarization by FCCP was also reduced in cells treated with

doxycycline (-43%; P<0.01), suggesting that the mitochondria were partially

uncoupled by suppression of HNF4α function (Fig. 4D).

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Effects of WT-HNF4α and DN-HNF4α on pancreatic β-cell gene expression-The

expression of genes involved in glucose metabolism (Fig. 5A&B) or in pancreatic β-

cell development and differentiation (Fig. 5C&D) was quantitatively evaluated in

WT-HNF4α#28 (Fig. 5A&C) and DN-HNF4α#26 cells (Fig. 5B&D). As shown in

Fig. 5A, WT-HNF4α mRNA could be induced by 2-, 8-, and 50-fold above the

endogenous level. This graded overexpression of WT-HNF4α resulted in a stepwise

increase in the expression of three glucose responsive genes encoding respectively

GLUT-2, L-PK and aldolase B (Fig. 5A). However, the mRNA level of GAPDH,

which is also responsive to glucose, remained unaltered (Fig. 5A). Induction of WT-

HNF4α also caused incremental expression of OGDH E1 subunit transcript (Fig 5A).

Consistently, The mRNA levels of GLUT-2, aldolase B, L-PK, and OGDH E1

subunit were significantly reduced after induction of DN-HNF4α (Fig. 5B). On the

other hand, induction of DN-HNF4α led to increased UCP-2 mRNA expression (Fig.

5B). Therefore, HNF4α regulates the expression of genes involved in both glycolysis

and mitochondrial metabolism. The profile of HNF4α-targeted genes is strikingly

similar to that of HNF1α (29). HNF4α may regulate the expression of genes

implicated in glucose metabolism through HNF1α function as in hepatocytes (7-9).

Since HNF4α is required for liver development and hepatocyte differentiation (9),

we investigated whether HNF4α regulates the expression of genes important for the

pancreatic β-cell phenotype. Induction of WT-HNF4α (Fig. 5C) or DN-HNF4α

(Fig. 5D) did not alter the expression patterns of PDX1, Pax4, Pax6, NKx2.2,

NKx6.1, and Isl-1, which are necessary for normal pancreatic cell development or

differentiation (30). Moreover, HNF4α did not regulate the mRNA levels of USF, c-

Jun, and C/EBPβ (Fig. 5C&D). The expression of these transcription factors appeared

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to be responsive to glucose (Fig. 5C&D). The expression of another glucose-

responsive transcription factor, DcoH, was slightly affected by induction of WT-

HNF4α but not by expression of DN-HNF4α (Fig. 5C&D), suggesting the

involvement of an indirect mechanism. Both Cdk4 and IRS2 are involved in

pancreatic β-cell development (31, 32), but their expression was not regulated by

HNF4α (Fig 5C&D). Induction of DN-HNF4α for 48 h caused 50% reduction in

insulin mRNA levels (Fig. 5D). This may be secondary to decreased HNF1α function,

since HNF1α is required for insulin gene transcription (29).

HNF4α regulates pancreatic β-cell gene expression through HNF1α function-We

performed EMSA for studying HNF4α binding activity to HNF1α promoter,

luciferase reporter enzyme assay for HNF1α promoter activity, and Northern blotting

for the HNF1α mRNA expression. Nuclear extracts were prepared from WT-

HNF4α#28 and DN-HNF4α#26 cells cultured for 48 h in the presence or absence of 500

ng/ml doxycycline. The murine HNF1α promoter segment, which contains the

HNF4α binding site, was used as probe (8). Induction of WT-HNF4α resulted in a dramatic

increase in the signal density of HNF4α binding (Fig. 6A). On the other hand,

induction of DN-HNF4α almost completely abolished the binding activity of

endogenous HNF4α to the HNF1α promoter (Fig. 6A). DN-HNF4α exerts its

dominant-negative function by forming DN-HNF4α/WT-HNF4α heterodimers that

lack DNA binding capacity (11). The retarded DNA binding complexes

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corresponding to endogenous WT-HNF4α and/or induced transgene WT-HNF4α

homodimers were supershifted by a specific antibody against HNF4α (Fig. 6A).

Consistently, induction of WT-HNF4α resulted in a 2-fold increase in

endogenous HNF1α mRNA level, while DN-HNF4α completely eliminated the

HNF1α expression (Fig. 6B). To confirm that HNF4α directly regulates HNF1α

transcription, we transiently transfected WT-HNF4α#28 and DN-HNF4α#26 cells

with a luciferase reporter construct containing either the wild type HNF1α gene

promoter (HNF1αLuc) or a promoter that lacks a functional HNF4α-binding site

(∆AHNF1αLuc). As demonstrated in Fig. 6C, ovexpression of WT-HNF4α caused a 2.5-

fold increase in the luciferase reporter enzyme activity in WT-HNF4α#28 cells

transfected with HNF1αLuc. Deletion of the HNF4α-binding site in the HNF1α

promoter (∆AHNF1α) abolished the activation induced by WT-HNF4α (Fig. 6C). In

contrast, induction of DN-HNF4α caused a 71% reduction in wild-type HNF1α

promoter activity (Fig. 6C). The inhibitory effect of DN-HNF4α was no-longer

present in DN-HNF4α#26 cells transfected with ∆AHNF1αLuc (Fig. 6C). Therefore,

HNF4α directly controls HNF1α gene expression in pancreatic β-cells as it does in

hepatocytes.

DISCUSSION

It has been demonstrated that HNF4α controls the expression of a large array of liver-

specific genes encoding several apolipoproteins, metabolic proteins, and serum factors

which are essential for hepatocyte differentiation and liver development (9). HNF4α

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is also required for HNF1α expression in hepatocytes (7-9). Another study in

embryonic stem cell differentiated embryoid bodies (33) shows that the absence of

HNF4α affects the expression of genes encoding GLUT-2, aldolase B, and L-PK,

which are involved in glucose transport and glycolysis. However, little is known as to

how HNF4α regulates pancreatic β-cell gene expression. The primary cause of the

MODY1 phenotype is impaired glucose stimulated insulin secretion from pancreatic

β-cells (5). The present study was therefore designed to investigate the role of HNF4α

in the regulation of the expression of β-cell genes implicated in glucose metabolism,

and associated insulin secretion.

We found that overexpression of WT-HNF4α caused a left-shift of glucose-

stimulated insulin secretion while dominant-negative suppression of HNF4α

selectively blunted the insulin release induced by glucose and leucine, but not by K+

depolarization. The diminished nutrient-evoked insulin secretion is associated with

reduced ATP production in DN-HNF4α expressing cells. The physiological insulin

secretagogue leucine raises the cytosolic and mitochondrial Ca2+ concentrations

through mitochondrial metabolism down-stream of glycolysis (28, 34). We therefore

suggest that loss of HNF4α function leads to defective mitochondrial metabolism and,

as a consequence, impaired insulin secretion. The reduced mitochondrial oxidation of

[2-14C]-pyruvate and the abrogation of mitochondrial membrane hyperpolarisation

elicited by glucose, pyruvate, and methyl-succinate indicate impaired mitochondrial

TCA-cycle enzyme activity and partial uncoupling of the mitochondrial respiratory

chain.

Quantitative Northern blot analysis allows us to identify HNF4α-target genes

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responsible for defective metabolism-secretion coupling. HNF4α indeed regulates the

expression of genes encoding GLUT-2, aldolase B and L-PK in pancreatic β-cells

(Fig. 5), as inferred from previous studies in hepatocytes and embryonic stem cell

differentiated embryoid bodies (8, 9). Most importantly, we demonstrate that HNF4α

alters the mRNA expression of mitochondrial OGDH E1 subunit and UCP-2 (Fig. 5),

which may indeed contribute more significantly to the impaired metabolism-secretion

coupling. The phenotype and gene expression patterns in DN-HNF4α expressing

cells are strikingly similar to those of DN-HNF1α expressing cells (22, 29). This

prompted us to investigate whether HNF4α regulates β-cell expression through

HNF1α function, as in hepatocytes (9). We provide unprecedented evidence that

HNF4α is required for HNF1α expression in pancreatic β-cells.

This conclusion is based on the use of an artificial dominant-negative hnf4α

mutation. The naturally occurring human mutations of HNF4α do not function in a

dominant-negative manner (6, 35). It is to be expected that a mutation with such

repressive action on the endogenous HNF4α function would cause embryonic

lethality, as is the case in the hnf4α knock out mouse (15). Haploinsufficiency or

reduced gene dosage of HNF4α may thus explain the mechanism leading to the

MODY1 phenotype (33). The INS-1 cell line expressing DN-HNF4α provides a

convenient model to explore the impact of impaired HNF4α function on β-cell gene

expression and metabolism-secretion coupling. This goal can not be achieved by the

introduction of one of the human HNF4α mutations into β-cell lines. In fact, the

induction of a nonsense mutation HNF4αQ268X to a level similar to DN-HNF4α

had no detectable consequences on β-cell gene expression and metabolism-secretion

coupling (unpublished results).

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MODY1 patients display secretory defects not only in β-cells but also in the

glucagon secreting α-cells and the pancreatic polypeptide secreting cells (36, 37).

This general effect on islet hormone release does however not seem due to an effect

on the development and differentiation of the endocrine pancreas, since altered

HNF4α function did not affect the expression of PDX1 and other transcription factors

determining pancreatic phenotype. On the other hand, loss of HNF4α function may

cause reduced β-cell insulin content secondary to defective HNF1α function (22, 29).

ACKNOWLEDGEMENTS

We are grateful to D. Harry, G. Chaffard, C. Bartley, and E.-J. Sarret for expert

technical assistance. We are indebted to Drs F. M. Sladek (HNF4α antibody), J. E.

Darnell Jr. (HNF4α cDNA), W. Schlegel (c-Jun cDNA), P.B. Iynedjian (glucokinase

cDNA), U. Schibler (C/EBPβ cDNA), T. Edlund (PDX1 cDNA), M. Sawadogo (USF

cDNA), A. Kahn (L-PK cDNA), B. Thorens (GLUT-2 cDNA), J. Philippe (insulin I

cDNA), H. Bujard (PUHD 10-3 plasmid), and N. Quintrell (pTKhygro plasmid). This

work was supported by the Swiss National Science Foundation (grant no. 32-

49755.96), and by a European Union Network Grant (through the Swiss Federal

Office for Education and Science), and by a research grant from the Eli-Lilly

Company, Indianapolis, IN.

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FIGURE LEGENDS

FIG. 1. Dose-response and time-course of doxycycline effect on WT-HNF4α (A)

and DN-HNF4α (B) expression. For studying dose-response, cells were cultured

with the indicated doses of doxycycline (Dox) for 48 h. For studying time-course,

cells were cultured in medium containing 500 ng/ml doxycycline and harvested for

nuclear extracts at the indicated times. Nuclear extracts from WT-HNF4α#28 (50

µg/lane) (A) and DN-HNF4α#26 (10 µg/lane) (B) were resolved in 9% SDS-PAGE,

transferred to nitrocellulose, and immunoblotted with antibodies against HNF4α (A)

and the Myc-tag (B) respectively.

FIG. 2. HNF4α regulates nutrient-evoked insulin secretion in INS-1 cells. Insulin

secretion was quantified as described in the Experimental Procedures and normalized

by cellular DNA content. (A) Glucose-stimulated insulin secretion in WT-

HNF4α#28 cells induced with indicated doses of doxycycline for 14 h. Data represent the mean

+ SEM of six independent experiments. Statistical significance between doxycycline-

induced and non-induced cells was obtained at 2.5 and 6 mM glucose (p<0.001,

unpaired Student’s t-test). (B) Glucose-, leucine-, and K+-elicited insulin secretion

in DN-HNF4α#26 cells induced with 500 ng/ml doxycycline for 48 h. Insulin

secretion was measured during 30 min incubation with 2.5 mM (Basal) and 24 mM

glucose in KRBH, or with 20 mM leucine and 20 mM KCl added in KRBH

containing 2.5 mM glucose. Data are the mean + SEM of six separate experiments.

Statistical significance between doxycycline-induced and non-induced cells was

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observed at 24 mM glucose and 20 mM leucine stimulated conditions (p<0.001).

Insulin content was reduced by 30 + 8.2% after induction of DN-HNF4α.

FIG. 3. Induction of DN-HNF4α impairs cellular ATP production and mitochondrial

oxidation. (A) Cellular ATP levels in DN-HNF4α#26 cells were measured after 8

min incubation with 2.5 mM (Basal) and 24 mM glucose in KRBH or 20 mM leucine

and 20 mM KCl added in KRBH containing 2.5 mM glucose. Data represent mean +

SEM of three independent experiments. Glucose and leucine stimulated ATP

production was significantly inhibited after treatment with 500 ng/ml doxycycline for

48 h (P<0.005 and P<0.001, respectively). (B) [2-14C] Pyruvate oxidation was

measured during 1 h incubation in KRBH containing 0.05 or 1 mM pyruvate. Data

represent the mean ± SE performed in triplicate from one of four similar experiments.

*P<0.02. (C) [1-14C] Pyruvate oxidation was measured with identical conditions in

the same preparation of cells as in Figure 3B. Data represent the mean ± SE

performed in triplicate from one of three similar experiments.

FIG. 4. Effect of DN-HNF4α on mitochondrial membrane potential (∆Ψm) in INS-1

cells. The ∆Ψm was measured in a suspension of 2x106 INS-1 cells per 2 ml KRBH

using rhodamine-123 (Rh-123) fluorescence after a spinner culture period. In panel

A, glucose-induced (12.5mM final) hyperpolarization of ∆Ψm was tested followed by

the complete depolarization of ∆Ψm using 1µM of the uncoupler FCCP. In panel B,

the end product of glycolysis pyruvate (10mM) was added 10 min before FCCP. In

panel C, the mitochondrial substrate methyl-succinate (10 mM) was tested. The

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effects of these various substrates (5 min after addition) as well as that of FCCP are

summarized with statistics in panel D. *P<0.05; **P<0.01. Each trace (A-C) is

representative of 4-8 independent experiments.

FIG. 5. Effects of WT-HNF1α and DN-HNF1α on pancreatic β-cell gene

expression. Northern blotting was used to quantify the gene expression in WT-

HNF4α#28 (A&C) and DN-HNF4α#26 (B&D) cells induced with indicated doses of

doxycycline and cultured at given concentrations of glucose (detailed in Experimental

Procedures). RNA samples were analysed by hybridization with the indicated cDNA

probes.

FIG. 6. Induction of WT-HNF1α and DN-HNF1α regulates the HNF1α mRNA

expression and HNF1α promoter luciferase activity through altered HNF4α binding.

EMSA (A), Northern blotting (B), and luciferase enzyme reporter activity (C) assays

were performed in WT-HNF4α#28 and DN-HNF4α#26 cells cultured in the

presence or absence of 500 ng/ml doxycycline for 48 h. (A). For EMSA, the

oligonucleotide duplex corresponding to the murine HNF1α promoter fragment

containing HNF4α binding site was used as probe. (B). For Northern blot analysis,

cells were cultured in 2.5 mM glucose medium for 48 h, and continued for 8 h with

indicated glucose concentrations. RNA samples from WT-HNF4α#28 (upper panel)

and DN-HNF4α#26 (lower panel) cells were hybridized with HNF1α cDNA probe.

(C). Cells were transiently transfected with HNF1αLuc or ∆AHNF1αLuc by calcium

phosphate-DNA co-precipitation. Luciferase activity measured in non-induced cells

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was defined as 100%. Data are the mean + SEM of six separate experiments.

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WollheimHaiyan Wang, Pierre Maechler, Peter A. Antinozzi, Kerstin A. Hagenfeldt and Claes B.

metabolism and nutrient-induced insulin secretionHNF4alpha regulates the expression of pancreatic beta-cell genes implicated in glucose

published online August 30, 2000J. Biol. Chem. 

  10.1074/jbc.M006612200Access the most updated version of this article at doi:

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