The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 1016-1019
Copyright © 1998 by The Endocrine Society
Mutation/Polymorphism Scanning of Glucose-6-Phosphatase Gene Promoter in Noninsulin-Dependent Diabetes Mellitus Patients1
Issei Yoshiuchi,
Ryosuke Shingu,
Hiromu Nakajima,
Tomoya Hamaguchi,
Yukio Horikawa,
Tomoyuki Yamasaki,
Takanori Oue,
Akira Ono,
Jun-ichiro Miyagawa,
Mitsuyoshi Namba,
Toshiaki Hanafusa and
Yuji Matsuzawa
Second Department of Internal Medicine, Osaka University Medical
School, 2–2 Yamadaoka, Suita, Osaka 565, Japan
Address all correspondence and requests for reprints to: Hiromu Nakajima, M.D., Ph.D., Second Department of Internal Medicine, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan. E-mail: hinakaji{at}imed2.med.osaka-u.ac.jp
 |
Abstract
|
|---|
Glucose-6-phosphatase (G6Pase) catalyzes the rate-limiting step of
gluconeogenesis, and hepatic G6Pase activity is increased in diabetes.
We have cloned and analyzed the human G6Pase gene promoter region and
identified putative regulatory sequences for insulin, cAMP,
glucocorticoid, and hepatocyte nuclear factors. The promoter region of
the G6Pase gene was analyzed in 154 noninsulin-dependent diabetes
mellitus patients and 90 control subjects by PCR-single strand
conformation polymorphism and direct sequencing methods. Polymorphisms
were not found in any subjects. The results suggested that in
noninsulin-dependent diabetic patients, the major cause of the hepatic
glucose overproduction was not attributed to dysregulation of the
G6Pase gene due to mutation/polymorphism of its promoter region.
|
Introduction
|
|---|
NONINSULIN-DEPENDENT diabetes
mellitus (NIDDM) is a genetically heterogeneous disorder caused by
several metabolic defects in pancreatic ß-cells, muscle, adipocytes,
and liver (1). Hepatic glucose production is increased in NIDDM
patients and correlates positively with fasting glucose levels, which
can be attributed to increased gluconeogenesis (2, 3). Thus,
gluconeogenic enzymes are promising candidates for the diabetogenic
genes. Recently, a key enzyme, phosphoenolpyruvate carboxykinase
(PEPCK) gene promoter, has been examined in diabetic patients. However,
no polymorphism was found (4). Glucose-6-phosphatase (G6Pase) also
plays a major role in hepatic glucose production. The activity of
G6Pase is regulated by various hormones, mainly at the transcriptional
level (5, 6, 7). Insulin suppresses the activity by decreasing the amount
of messenger ribonucleic acid of the catalytic subunit (8, 9, 10, 11, 12, 13).
Glucagon, via cAMP, and glucocorticoids increase the activity of this
enzyme. A search for a promoter mutation/polymorphism of G6Pase gene
has not yet been conducted, and it may give some clues to the
elucidation of abnormal hepatic glucose production in NIDDM patients.
In this study, we screened NIDDM patients for the G6Pase promoter
region, including putative insulin-responsive motifs, cAMP response
elements (CREs), glucocorticoid response element (GRE), and hepatocyte
nuclear factor (HNF)-binding sites.
|
Subjects and Methods
|
|---|
Subjects
Unrelated Japanese NIDDM patients (n = 154) and healthy
control subjects (n = 90) participated in the study. NIDDM was
diagnosed using WHO criteria (14). The healthy control subjects
consisted of our department staff and hospital nurses, with normal
hemoglobin A1c values and normal fasting plasma glucose
concentrations (<110 mg/dL). Informed consent was obtained from the
subjects. There were 17 patients with early-onset NIDDM and 137
patients with late-onset NIDDM. Early-onset NIDDM was diagnosed between
14–30 yr of age (mean ± SD, 22.7 ± 5.5; n
= 17), with a body mass index of 22.9 ± 3.3 (range, 16.6–31.6).
Late-onset NIDDM was diagnosed between 31–76 yr of age (46.5 ±
9.2; n = 137), with a body mass index of 22.5 ± 3.2 (range,
16.5–32.7). The 90 healthy control subjects ranged from 21–61 yr of
age (37.5 ± 11.4), with a body mass index of 22.1 ± 2.7
(range, 18.0–28.7).
Isolation of the human G6Pase gene promoter region by inverse
PCR
Primers were prepared according to the published complementary
DNA sequence (15). EcoRI-digested human genomic DNA was
circularized by T4-DNA ligase. By using the primers HG6P12
(5'-CTGGATCCCAAAGTCATGGAGAACATTC) and HG6P22
(5'-CCATGGGCACAGCAGGTGTATACTACG), PCR was performed so as to inversely
amplify the upstream sequence of human G6Pase. An aliquot was subjected
to the nested PCR using the primers HG6P1
(5'-CCTCATTTCCTTGGCACCTCAGGAAG) and HG6P2
(5'-GGTGTATACTACGTGATGGTCACATC). The amplicon was directly sequenced
using the DNA sequencing kit (ABI Prism, Perkin Elmer/Applied
Biosystems Division, Foster City, CA) on a model 310 Genetic Analyzer
(Applied Biosystems, Norwalk, CT). The nucleotide sequence appears in
DDBJ/EMBL/GenBank databases under accession number D87948.
PCR amplification of the human G6Pase gene promoter
We selected three regions to analyze all of the identified
putative functional motifs. Region 1 was located at residues -357 to
-1 containing three insulin response sequences (IRSs), three CREs, a
GRE, and HNF-4-, HNF-3-, and HNF-1-binding sites; region 2 was located
at residues -821 to -450 containing several insulin-responsive motifs
and an HNF-3-binding site. Region 3 was the upstream sequence at
residues -1160 to -838 containing a CRE. The primers used were
designed as follows: for region 1, 5'-AGTGCAGTTGCAGGCATAG and
5'-GAGTCTGTGCCTTGCCCCTG; for region 2, 5'-GACCAGCAAGATGATAGTCCC and
5'-GCTCACACCTGTAATCCCAGC; and for re-gion 3,
5'-TAGCCAGGCATGGTGGCGTGTG and 5'-AGAGTCTAG-GGTCTGCCTCTG.
PCR was performed by denaturation for 1 min at 95 C, annealing for 30
sec at 61 C, and extension for 30 s at 72 C for 35 cycles.
Mutagenized PCR were performed on 50 ng control human genomic DNA in 10
mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 7 mmol/L MgCl2,
0.01% gelatin, 0.2 mmol/L deoxy (d)-ATP and dGTP, 0.25 mmol/L dCTP and
dTTP, 300 nmol/L primers, and 2.5 U AmpliTaq DNA polymerase (Applied
Biosystems). Thermal cycling was performed by denaturation for 1 min at
95 C, annealing for 1 min at 58 C, and extension for 1 min at 72 C, for
35 cycles. The amplicon was subcloned into pUC119 and sequenced. At
least seven clones with one- to three-base substitutions were selected
as mutagenized clones for each region, which were used to optimize the
experimental conditions.
Single strand conformation polymorphism (SSCP) analysis
Samples were heat denatured in 96% formamide at 95 C for
5 min and separated through 5–10% gradient polyacrylamide gels (Ready
Gels-J, Bio-Rad, Richmond, CA) in Tris-glycine buffer (25 mmol/L
Tris-HCl, pH 8.3, and 192 mmol/L glycine). Gel running conditions were
optimized by changing the temperature in increments of 2 or 3° from 7
to 42 C with or without 10% glycerol. The best results were obtained
by electrophoresis with constant power of 20 watts for 75 min at 7 C
and for 60 min at 37 C with 10% glycerol for region 1, for 75 min at 9
C and for 60 min at 30 C with 10% glycerol for region 2, and for 75
min at 7 C and for 60 min at 30 C with 10% glycerol for region 3. Nine
of nine (regions 1 and 2) and six of seven (region 3) mutagenized
clones were distinguished. Gels were stained by SYBR Green I Nucleic
Acid Gel Stains (FMC BioProduct, Rockland, ME) followed by laser
excitation fluorescent image analysis (Fluor Image Analyzer SI and
Image QuaNT, Molecular Dynamics, Sunnyvale, CA).
|
Results
|
|---|
The inverse PCR product [1.8 kilobases (kb)] yielded
restriction fragments of 1.4 and 0.4 kb with EcoRI, which
are compatible to the size expected from the reported physical map of
the human G6Pase gene (16). The longer fragment contained 1,214 bp of
the 5'-upstream region of the human G6Pase gene. This sequence had an
insertion of a thymine at -914 compared to that in a previous report
(17). A racial polymorphism may exist, as all Japanese subjects
sequenced (>100 alleles; data not shown) possessed the insertion.
Putative binding sites for transcription factors were identified within
this sequence. Two insulin response elements (IREs) (18), located from
-633 to -626 and from -491 to -484, and two clusters of IRSs
[T(G/A)TTTTG] (19) from -768 to -726 and from -186 to -158
existed. Five potential CREs (20) were also found. A sequence of GRE, a
HNF-1-binding site, a HNF-4 binding site, and two HNF-3-binding sites
were identified using the computer search program Search TRANSFAC
database (http://www.genome.ad.jp/), which is the combination of
TFFACTOR and TFSITE release 3.0 (Fig. 1
).
View larger version (56K):
[in this window]
[in a new window]
|
Figure 1. Nucleotide sequence of the cloned
5'-flanking region of the human glucose-6-phosphatase gene. The
transcription start site is indicated as +1. Putative binding sites for
transcription factors are underlined. A double
underline at -914 shows an insertion of a thymine compared to
a previous report (17). IRS (-), complementary to IRS; HNF-1, -3, and
-4, HNF-1-, -3-, and -4-binding sites; C/EBP, CCAAT/enhancer binding
protein; TATA, TATA box.
|
|
No conformation polymorphism was identified in any subject under
optimal SSCP conditions for the three regions. Moreover, direct
sequencing of regions 1 and 3 failed to reveal any polymorphism in the
100 alleles of the additional 50 NIDDM patients. To test the other CRE
within exon 1, the primer HG6P12 was used to amplify the fragment
between -357 to +120. No polymorphism was found in this CRE by
sequencing 50 patients.
|
Discussion
|
|---|
Promoter mutations have been shown to be pathogenic in patients
with retinoblastoma and ß-thalassemia (21). Recently, promoter
polymorphism of the stromelysin-1 gene has been shown to be closely
associated with atherosclerosis (22). The liver-type and the
ß-cell-type glucokinase promoter variations are associated with
insulin resistance and impaired insulin secretion, respectively (23, 24). In contrast, mutations were clinically silent in the promoters of
insulin, GLUT4, and PEPCK genes (4, 25, 26). The sensitivity of
identifying point mutations with SSCP varies according to the gel
conditions and DNA length and sequence (27, 28). Nonisotopic SSCP in
the minigel format was reported to be capable of detecting over 90% of
point mutations with only one gel condition (29, 30). Our strategy was
to determine the electrophoretic conditions by using mutagenized
clones. At least 96% of the mutagenized sequences were distinguished
by the optimized conditions. We may have missed less than 1 in 154
patients.
DNA sequences that transduce some insulin signals have been
proposed as IRE in glyceraldehyde-3-phosphate dehydrogenase (CCCGCCTC)
(18) and microsomal triglyceride transfer protein (AGCCCACCTACG) (31)
genes. IRS [T(G/A)TTTTG] was also identified in PEPCK (19) and
insulin-like growth factor-binding protein-1 (32) genes. In the rat
G6Pase gene 5'-flanking region, the TAAAACACCA motif complementary to
IRS was identified 1.5 kb upstream from the transcription start site
(33). Very recently, the mouse G6Pase promoter region between -198 and
-159 has been shown to contain an IRS that could confer an inhibitory
transcriptional effect by insulin using the reporter assays (34). Among
several IRS motifs, as determined in this study, the IRS cluster of
-186 to -158 may be the human counterpart, which contributes to the
insulin responsiveness.
Susceptibility to NIDDM can be conferred by multiple and heterogeneous
genetic defects. As partly shown in the experimental animal studies
(13, 35), decreased responsiveness to insulin and/or exaggerated
sensitivity to glucocorticoids and cAMP in the gluconeogenic system can
increase hepatic glucose production. Therefore, gluconeogenic enzyme
gene promoters can be good candidates for diabetogenic genes. We have
extensively searched for the genetic variation of human G6Pase gene
promoter. However, we did not find any polymorphism in the patients or
the control subjects. The frequency of promoter mutation/polymorphism
of G6Pase gene is thus very low. The molecular mechanism of the hepatic
glucose overproduction in NIDDM needs to be further elucidated.
|
Footnotes
|
|---|
1 This work was supported in part by a grant-in-aid from the Ministry
of Education, Science, and Culture, and grants from the Inamori
Foundation, the Senri Lifescience Foundation, the Tanabe Medical
Frontier Conference, the Japan Diabetes Federation, and the Yamanouchi
Foundation for Metabolic Disorders. 
Received August 29, 1997.
Revised October 30, 1997.
Accepted December 2, 1997.
|
References
|
|---|
-
DeFronzo RA, Bonadonna RC, Ferrannini E. 1992 Pathogenesis of NIDDM, a balanced overview. Diabetes Care. 15:318–368.[Abstract]
-
DeFronzo RA. 1988 The triumvirate: ß-cell,
muscle and liver: a collusion responsible for NIDDM. Diabetes. 37:667–687.[Medline]
-
DeFronzo RA, Ferrannini E, Simonsen DC. 1989 Fasting hyperglycemia in non-insulin-dependent diabetes mellitus:
contributions of excessive hepatic glucose production and impaired
tissue glucose uptake. Metabolism. 38:387–395.[CrossRef][Medline]
-
Ludwig DS, Vidal Puig A, O’Brien RM, et al. 1996 Examination of the phosphoenolpyruvate carboxykinase gene
promoter in patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab. 81:503–506.[Abstract]
-
Christ B, Probst I, Jungermann K. 1986 Antagonistic regulation of the glucose/glucose-6-phosphate cycle by
insulin and glucagon in cultured hepatocytes. Biochem J. 238:185–191.[Medline]
-
Arion WJ, Lange AJ, Ballas LM. 1976 Quantitative
aspects of relationship between glucose-6-phosphate transport and
hydrolysis for liver microsomal glucose-6-phosphatase system. J
Biol Chem. 251:6784–6790.[Abstract/Free Full Text]
-
Burchell A, Cain DI. 1985 Rat hepatic microsomal
glucose-6-phosphatase protein levels are increased in
streptozotocin-induced diabetes. Diabetologia. 28:852–856.[CrossRef][Medline]
-
Lange AJ, Argaud D, El-Maghrabi MR, Pan W, Maitra SR,
Pilkis SJ. 1994 Isolation of a cDNA for the catalytic subunit of
rat liver glucose-6-phosphatase: regulation of gene expression in FAO
hepatoma cells by insulin, dexamethazone and cAMP. Biochem Biophys Res
Commun. 201:302–309.[CrossRef][Medline]
-
Liu Z, Barrett EJ, Dalkin AC, Zwart AD, Chou JY. 1994 Effect of acute diabetes on rat hepatic glucose-6-phosphatase
activity and its messenger RNA level. Biochem Biophys Res Commun. 205:680–686.[CrossRef][Medline]
-
Haber BA, Chin S, Chuang E, Buikhuisen W, Naji A, Taub
R. 1995 High levels of glucose-6-phosphatase gene and protein
expression reflect an adaptive response in proliferating liver and
diabetes. J Clin Invest. 95:832–841.
-
Massillon D, Barzilai N, Chen W, Hu M, Rossetti L. 1996 Glucose regulates in vivo glucose-6-phosphatase gene
expression in the liver of diabetic rats. J Biol Chem. 271:9871–9874.[Abstract/Free Full Text]
-
Mithieux G, Vidal H, Zitoun C, Bruni N, Daniele N,
Minassian C. 1996 Glucose-6-phosphatase mRNA and activity are
increased to the same extent in kidney and liver of diabetic rats. Diabetes. 45:891–896.[Abstract]
-
Shingu R, Nakajima H, Horikawa Y, et al. 1996 Expression and distribution of glucose-6-phosphatase catalytic subunit
messenger RNA and its changes in the diabetic state. Res Commun Mol
Pathol Pharmacol. 93:13–24.[Medline]
-
Bennett PH. 1994 Definition, diagnosis, and
classification of diabetes mellitus and impaired glucose tolerance. In:
Kahn CR, Weir GC, eds. Joslin’s diabetes mellitus, 13th ed.
Philadelphia: Lea and Febiger; 193–200.
-
Lei KJ, Pan CJ, Shelly LL, Liu JL, Chou JY. 1994 Identification of mutations in the gene for glucose-6-phosphatase, the
enzyme deficient in glycogen storage disease type 1A. J Clin
Invest. 93:1994–1999.
-
Lei KJ, Shelly LL, Pan CJ, Sidbury JB, Chou JY. 1993 Mutations in the glucose-6-phosphatase gene that cause glycogen
storage disease type 1a. Science. 262:580–583.[Abstract/Free Full Text]
-
Schmoll D, Allan BB, Burchell A. 1996 Cloning and
sequencing of 5' region of the human glucose-6-phosphatase gene:
transcriptional regulation by cAMP, insulin and glucocorticoids in
H4IIE hepatoma cells. FEBS Lett. 383:63–66.[CrossRef][Medline]
-
Nasrin N, Ercolani L, Denaro M, Kong XF, Kang I,
Alexander M. 1990 An insulin response element in the
glyceraldehyde 3-phophate dehydrogenase gene binds a nuclear protein
induced by insulin in cultured cells and by nutritional manipulations
in vivo. Proc Natl Acad Sci USA. 87:5273–5277.[Abstract/Free Full Text]
-
O’Brien RM, Bonovich MT, Forest CD, et al. 1991 Signal transduction convergence: phorbol esters and insulin inhibit
phosphoenol pyruvate carboxykinase gene transcription
through the same 10-base-pair sequence. Proc Natl Acad Sci USA. 88:6580–6584.[Abstract/Free Full Text]
-
Roesler WJ, Vandenbark GR, Hanson RW. 1988 Cyclic
AMP and the induction of eukaryotic gene transcription. J Biol
Chem. 263:9063–9066.[Free Full Text]
-
Cooper D. 1992 Regulatory mutations and human
genetic disease. Ann Med. 24:427–437.[Medline]
-
Ye S, Eriksson P, Hamsten A, Kurkinen M, Humphries SE,
Henney AM. 1996 Progression of coronary atherosclerosis is
associated with a common genetic variant of the human stromelysin-1
promoter which results in reduced gene expression. J Biol Chem. 271:13055–13060.[Abstract/Free Full Text]
-
Chiu KC, McCarthy JE. 1996 Promoter variation in
the liver glucokinase is a risk factor for Non-insulin-dependent
diabetes mellitus. Biochem Biophys Res Commun. 221:614–618.[CrossRef][Medline]
-
Stone LM, Kahn SE, Fujimoto WY, Deeb SS, Porte Jr
D. 1996 A variation at position -30 of the ß-cell glucokinase
gene promoter is associated with reduced ß-cell function in
middle-aged Japanese-American men. Diabetes. 45:422–428.[Abstract]
-
Olansky L, Welling C, Giddings S, et al. 1992 A
variant insulin promoter in non-insulin-dependent diabetes mellitus. J Clin Invest. 89:1596–1602.
-
Bjorbaek C, Echwald SM, Hubricht P, et al. 1994 Genetic variants in promoters and coding regions of the muscle glycogen
synthase and the insulin-responsive GLUT4 genes in NIDDM. Diabetes. 43:976–983.[Abstract]
-
Orita M, Suzuki Y, Sekiya T, Hayashi K. 1989 Rapid
and sensitive detection of point mutations and DNA polymorphisms using
the polymerase chain reaction. Genomics. 5:874–879.[CrossRef][Medline]
-
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya
T. 1989 Detection of polymorphisms of human DNA by gel
electrophoresis as single-strand conformation polymorphisms. Proc Natl
Acad Sci USA. 86:2766–2770.[Abstract/Free Full Text]
-
Vidal-Puig A, Moller DE. 1994 Comparative
sensitivity of altenative single strand conformation polymorphism
(SSCP) methods. Biotechnology. 17:490–496.
-
Hongyo T, Buzard GS, Calvert RJ, Weghorst M. 1993 ‘Cold SSCP:’ a simple, rapid and non-radioactive method for optimized
single-strand conformation polymorphism analyses. Nucleic Acids Res. 21:3637–3642.[Abstract/Free Full Text]
-
Hagen DL, Kienzle B, Jamil H, Hariharan N. 1994 Transcriptional regulation of human and hamstar microsomal triglyceride
transfer protein genes. J Biol Chem. 269:28737–28744.[Abstract/Free Full Text]
-
O’Brien RM, Noisin EL, Suwanichkul A, et al. 1995 Hepatic nuclear factor 3- and hormone-regulated expression of the
phosphoenolpyruvate carboxykinase and insulin-like growth
factor-binding protein 1 genes. Mol Cell Biol. 15:1747–1758.[Abstract]
-
Argaud D, Zhang Q, Pan W, Maitra S, Pilkis SJ, Lange
AJ. 1996 Regulation of rat liver glucose-6-phosphatase gene
expression in different nutritional and hormonal states: gene structure
and 5'-flanking sequence. Diabetes. 45:1563–1571.[Abstract]
-
Streeper RS, Svitek CA, Chapman S, Greenbaum LE, Taub R,
O’Brien RM. 1997 A multicomponent insulin response sequence
mediates a strong repression of mouse glucose-6-phosphatase gene
transcription by insulin. J Biol Chem. 272:11698–11701.[Abstract/Free Full Text]
-
Hotta K, Kuwajima M, Ono A, et al. 1996 Disordered
expression of hepatic glycolytic and gluconeogenic enzymes in Otsuka
Long-Evans Tokushima fatty rats with spontaneous long-term
hyperglycemia. Biochim Biophys Acta. 1289:145–149.[Medline]
This article has been cited by other articles:
|
|
|
|
 
C. Bouche, S. Serdy, C. R. Kahn, and A. B. Goldfine
The Cellular Fate of Glucose and Its Relevance in Type 2 Diabetes
Endocr. Rev.,
October 1, 2004;
25(5):
807 - 830.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Cai, J. Xie, J.-X. She, and A. L. Notkins
Analysis of the Coding and Promoter Regions of the Autoantigen IA-2 in Subjects With and Without Autoantibodies to IA-2
Diabetes,
October 1, 2001;
50(10):
2406 - 2409.
[Abstract]
[Full Text]
|
|
|
Top
Abstract
Introduction
