(Hypertension. 1997;30:236-239.)
© 1997 American Heart Association, Inc.
Articles |
Asp905Tyr Polymorphism of Protein Phosphatase 1 G Subunit Gene in Hypertension
From the Department of Geriatric Medicine, Osaka University Medical School; and the Second Department of Internal Medicine, Sapporo Medical University School of Medicine (H.M., K.S.), Japan.
Correspondence to Toshio Ogihara, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.
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Abstract |
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Abstract A possible pathogenic polymorphism in the gene for the G subunit of the glycogen-associated regulatory form of protein phosphatase 1 (PP1 G subunit), causing an Asp-to-Tyr substitution at codon 905 (Asp905Tyr), has been reported to be associated with insulin resistance and hypersecretion of insulin in the white population. Since marked heterogeneity has been reported in the association of mutations of candidate genes with essential hypertension between Japanese and other ethnic groups, we investigated the association of Asp905Tyr with essential hypertension in Japanese subjects. The frequency of the Tyr allele in Japanese control subjects (0.70) was much higher than that in the Danish population (0.10, P<1x10-8), indicating that the Tyr allele, previously reported as a rare variant in white subjects, is a common allele in our population. The genotype distribution in Japanese hypertensive patients (n=109; Asp/Asp=0.09, Asp/Tyr=0.39, Tyr/Tyr=0.52) was not significantly different (
2=0.7, df=2,
P>.6) from that in normotensive control subjects (n=148;
Asp/Asp=0.12, Asp/Tyr=0.36, Tyr/Tyr=0.52). Among subjects with
different PP1 G subunit genotypes, there was no difference in
blood pressure, serum cholesterol, plasma glucose and
insulin levels, and glucose disposal rate estimated by the
euglycemic hyperinsulinemic clamp test.
These data indicate that the Asp905Tyr polymorphism of the PP1 G
subunit is not associated with essential hypertension, nor with
insulin resistance and/or hyperinsulinemia in
Japanese patients with essential hypertension, suggesting that the
polymorphism plays little if any role in susceptibility to insulin
resistance or hypertension.
Key Words: genes • glycogen synthase • hypertension, essential • insulin resistance • genetic predisposition • phosphoprotein phosphatase
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Introduction |
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There is increasing evidence that insulin resistance and/or hyperinsulinemia may play a key role in the pathogenesis of hypertension.1 2 3 4 Nonoxidative glucose disposal (ie, glycogen synthesis) has been found to represent an important site of insulin resistance in essential hypertension.1 5 Several lines of evidence from family studies suggest that genetic factors are involved not only in the development of essential hypertension6 but also in insulin resistance in hypertensive subjects.7 8 9 10 Little is known, however, about the genetic factors responsible for insulin resistance in essential hypertension.11 It is important to elucidate the genetic factors predisposing to insulin resistance in hypertension, because doing so will permit identification of individuals with genetic susceptibility to insulin resistance and hypertension and in turn permit intervention tailored to the underlying abnormalities.
The regulatory G subunit of the glycogen-associated form of protein phosphatase 1 (PP1 G subunit) is a key protein in the stimulation of glycogen synthesis by insulin and thus regulation of nonoxidative glucose disposal.12 13 The PP1 G subunit is expressed in skeletal muscle,14 which is considered a major site of peripheral insulin resistance in essential hypertension.1 15 Both fasting and insulin-stimulated PP1 activities in skeletal muscle have been reported to be reduced in insulin-resistant Pima Indians.16 17 Thus, functional alteration of PP1 may be responsible for impaired insulin-stimulated glycogen synthesis in skeletal muscle, which is characteristic of insulin-resistant individuals, and therefore, the PP1 G subunit is a strong candidate for an inherited trait associated with reduced insulin action in hypertensive subjects. Recently, a polymorphism in the gene for the PP1 G subunit, causing an amino acid substitution at codon 905 of Asp to Tyr (Asp905Tyr), was identified. White subjects who carry the Tyr allele have been reported to exhibit insulin resistance and hypersecretion of insulin.18 Since marked heterogeneity has been reported in the association of candidate genes with glucose intolerance and/or hypertension between Japanese and other ethnic groups,19 20 21 22 we studied the polymorphism of the PP1 G subunit gene in Japanese patients with essential hypertension to investigate the contribution of this locus to genetic susceptibility to essential hypertension and to insulin resistance in hypertension.
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Methods |
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Subjects
One hundred nine unrelated Japanese patients with essential hypertension attending the Osaka University Hospital and an affiliated hospital were investigated. Hypertension was defined on the basis of antihypertensive treatment or according to WHO criteria (arterial blood pressure higher than 160/95 mm Hg). Informed consent was obtained from all the subjects. Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared. An oral glucose tolerance test with 75 g glucose was performed and the insulinogenic index was calculated as the ratio of the increment of insulin (picomoles per liter) to that of plasma glucose (millimoles per liter) 30 minutes after the glucose load.23 Normotensive subjects (n=148) with no family history of essential hypertension served as controls.
For studies on insulin sensitivity, a glucose clamp was conducted in 44 patients with essential hypertension randomly selected from the hypertensive group. A euglycemic hyperinsulinemic clamp test was performed according to the method of DeFronzo et al,24 with a modification as reported previously25 ; ie, insulin was infused at 40 mU/m2 per minute and an artificial pancreas, STG-22 (Nikkiso Co Ltd), was used. As an index of insulin sensitivity, the M value (mg/kg per minute) was calculated from the glucose disposal rate during 90 to 120 minutes of the clamp.
Analysis of the PP1 G Subunit Polymorphism
DNA was extracted from peripheral blood leukocytes.
The PP1 G subunit genotypes were determined by polymerase chain
reaction–restriction fragment length polymorphism, according to
the method of Hansen et al.18 The polymerase chain
reaction products were separated on 9% polyacrylamide gel
for genotype determinations.
Statistical Analysis
Results are given as mean±SD. Subjects subdivided by
hypertension status were compared with respect to age, BMI, and blood
pressure by using the unpaired t test. Hardy-Weinberg
equilibrium was checked by 2 test. Difference in
genotype distribution between groups was assessed by using the
2 test. Subjects subdivided by PP1 G subunit
genotypes were compared with respect to clinical variables
by using ANOVA. ANCOVA was used to assess the associations of the
polymorphism with blood pressure, adjusting for age and BMI as
covariates.
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Results |
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PP1 G Subunit Polymorphism and Essential Hypertension
Table 1
summarizes the clinical data
of the hypertensive patients and control subjects, showing that the
hypertensive group was older and had a lower prevalence of males, a
higher BMI, and higher blood pressures than the control group.
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The genotype frequencies of the PP1 G subunit gene, both in the hypertensive patients and control subjects, were in agreement with Hardy-Weinberg equilibrium. The genotype distribution (Asp/Asp=0.12, Asp/Tyr=0.36, Tyr/Tyr=0.52) in Japanese healthy control subjects was significantly different from that in the Danish population (Asp/Asp=0.82, Asp/Tyr=0.17, Tyr/Tyr=0.01; P<1x10-4, df=2).18 The frequency of the Tyr allele was also significantly different between Japanese (0.70) and Danish (0.10; P<1x10-8) healthy subjects.18
The distribution of the genotype was not different between
hypertensive patients and control subjects (Table 2). Among the three groups of Japanese
patients with different PP1 G subunit genotypes, no
statistically significant difference was observed in age, BMI, fasting
plasma glucose, insulin and serum cholesterol levels, and
insulinogenic index (Table 3).
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Since there were significant differences in sex ratio, age, and BMI between the hypertensive group and the control group, ANCOVA was applied to all the subjects, with gender analyzed as a second grouping variable. No significant association was observed between the Asp905Tyr polymorphism and mean blood pressure after controlling for age and BMI (P>.5 and P>.6, respectively).
Insulin Sensitivity and PP1 G Subunit Polymorphism in
Hypertensive Patients
No significant differences were observed in age, BMI, fasting
plasma glucose and insulin levels, and insulinogenic index between the
44 hypertensive patients who underwent the glucose clamp test and the
other hypertensive patients, indicating that the selection was
performed randomly. The genotype distribution of the PP1 G
subunit gene in both groups was in accordance with Hardy-Weinberg
equilibrium. No significant difference in M values was observed among
the subgroups of hypertensive patients with different genotypes
of the PP1 G subunit gene (Table 3
).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study demonstrated marked heterogeneity in the distribution of genotypes and alleles of the PP1 G subunit gene in Japanese and white subjects. The frequency of the Tyr/Tyr genotype or Tyr allele in the Danish population is so low that the Tyr allele is reported as a "variant."18 In contrast, in the Japanese population, the Tyr allele was a major allele, and the Tyr/Tyr genotype frequency was as high as 50%. These data may reflect differences in the genetic backgrounds of the populations and indicate that this polymorphism is not a variant but a polymorphism whose distribution exhibits marked diversity between different ethnic groups. The large number of individuals homozygous for the Tyr allele in our population has allowed us to assess the functional alteration related to the Tyr allele in the homozygous form. Our data demonstrated that the Asp905Tyr polymorphism of the PP1 G subunit was not associated with essential hypertension itself or with insulin resistance in hypertensives. This is the first study to investigate the association of the PP1 G subunit polymorphism with essential hypertension, and it is concluded that this polymorphism is not a major determinant, if it is a determinant at all, of essential hypertension or insulin sensitivity in Japanese patients with essential hypertension.
The development of essential hypertension is suggested to be at least
in part genetically determined,6 and the
angiotensinogen I gene on chromosome 1 has been reported to
contribute to the development of essential hypertension in different
ethnic groups.20 26 Insulin resistance in hypertension has
been suggested to be genetically determined7 8 9 10 11 and
appears to precede the development of essential
hypertension.27 The recently identified Asp905Tyr
polymorphism of the PP1 G subunit was reported to be associated
with insulin resistance in Danish subjects, suggesting that this
genetic variation may well confer susceptibility to hypertension as
well as to insulin resistance. Since insulin sensitivity was reported
to be decreased in heterozygous Danish subjects,18 one
might have expected individuals with the Tyr/Tyr genotype to be
markedly resistant to insulin. This, however, was not the case
in hypertensive subjects (Table 3). Furthermore, the patients
homozygous for the normal "Asp" allele even showed a tendency
to have a lower M value and higher insulinogenic index than those with
other genotypes (Table 3), suggesting that the Asp allele
rather than the Tyr allele is associated with insulin resistance.
These observations suggest one plausible explanation, ie, an unknown
etiologic mutation is in linkage disequilibrium with the Tyr allele
in the Danish and with the Asp allele in the Japanese.
Alternatively, since our data could not show a significant relation,
there appear to be other possibilities to explain the observations. The
first possibility is, on the basis of a racial difference, that the
Japanese population lacks other putative genetic and/or environmental
factors that would need to coexist to uncover the effect of the Tyr
allele on insulin sensitivity. Second, the effect of this mutation
on insulin sensitivity is not so strong that the effect recognized
among normotensive subjects is weakened under the pathological
condition of hypertension. In either case, the Tyr allele of the
PP1 G subunit gene per se is unlikely to contribute primarily to
susceptibility to insulin resistance in hypertension.
With respect to insulin secretion, Danish obese carriers of the Tyr allele showed reduced early-phase insulin secretion evaluated by an intravenous glucose tolerance test.18 Although it may be possible that ordinary hyperinsulinemia resulting from peripheral insulin resistance could affect beta cell function, it seems unlikely that an amino acid substitution of the PP1 G subunit, mainly expressed in muscle,14 directly affects the early insulin response of beta cells to glucose. We previously reported that the insulinogenic index showed a good correlation with early-phase insulin response.23 In addition, orally loaded glucose is more physiological than that loaded intravenously. Thus, the lack of association of the polymorphism with insulinogenic index in this study suggests that the PP1 G subunit locus plays little role in glucose-stimulated insulin secretion, at least under physiological conditions.
Although we could not find any association of this polymorphism with essential hypertension and/or insulin sensitivity, one cannot exclude the possibility of the contribution of this locus to disease pathogenesis. First, a polymorphism (other than the Asp905Tyr polymorphism) that we did not study could possibly be associated with the phenotypes. Second, because of the relatively weak contribution of this polymorphism to the genetic predisposition to hypertension, it is possible that this sample size was not sufficient to clarify its effect. Third, because insulin resistance is associated with atherosclerotic complications, this locus might affect morbidity and mortality of cardiovascular and cerebrovascular disease, which were not evaluated in this study. To understand the biological effect of the Asp905Tyr substitution of the PP1 G subunit on its function, further studies, including in vitro experiments with cultured cells expressing different subtypes of the PP1 G subunit, are needed.
Regarding the statistical power of this study, the estimates were
derived with modeling and simulation. The probability to detect a 5%
difference in allele frequency by 2 test (2
df) between a normotensive group (frequency of Asp
allele=0.3, as observed) and a hypertensive group (frequency of Asp
allele=0.25 or 0.35) was estimated as 78% or 76%, respectively,
given a 2 statistic greater than 0.74, which
corresponded to our observation. Thus, even with our sample numbers, it
seems possible to detect a difference with relatively high power if the
effect of the polymorphism is marked.
In conclusion, we have demonstrated that (1) the frequency of the Tyr allele in Japanese subjects was significantly higher than that in Danes and (2) the Asp905Tyr polymorphism of the PP1 G subunit gene was not associated with essential hypertension or with insulin resistance in Japanese patients with essential hypertension. These data suggest that the previously reported Tyr allele itself plays little if any role in susceptibility to insulin resistance or hypertension.
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Acknowledgments |
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This work was supported in part by a grant for diabetes research from the Ministry of Health and Welfare, a grant-in-aid from the Ministry of Education, Science, and Culture, a grant-in-aid from the Japan Medical Association, a grant from Otsuka Pharmaceutical Co, a grant from the SANDOZ Foundation for Gerontological Research, and a Mitsukoshi grant-in-aid. We thank Yumiko Ueno for her skillful technical support. We also thank Dr Yoshihiko Kawaguchi for his comments on the manuscript.
Received July 1, 1996; first decision August 15, 1996; accepted January 24, 1997.
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