Polymorphism of the Glycogen Synthase Gene in Hypertensive and Normotensive Subjects
Abstract Hypertension and non–insulin-dependent diabetes mellitus (NIDDM) are characterized by a strong genetic component and impaired ability to store glucose as glycogen in skeletal muscle. Impaired insulin activation and altered genetic control of muscle glycogen synthase, the rate-limiting enzyme for glucose storage in skeletal muscle, could provide an explanation for this insulin resistance. We examined whether there is an association between the glycogen synthase gene (Xba I polymorphism) and hypertension in 304 nondiabetic subjects. We examined glucose tolerance with an oral glucose tolerance test and glucose storage in skeletal muscle with the euglycemic insulin clamp technique in combination with indirect calorimetry. The Xba I A2 allele of the glycogen synthase gene was enriched in subjects with hypertension and a family history of NIDDM (48%) compared with normotensive subjects without a family history of NIDDM (6%, P<.0001). The presence of the A2 versus the A1 allele was associated with decreased rates of insulin-stimulated glucose storage in hypertensive subjects (11.2±2.3 versus 16.9±2.6 μmol/kg lean body mass per minute, P=.029) but not in normotensive subjects (28.0±4.6 versus 29.6±3.7 μmol/kg lean body mass per minute). In conclusion, Xba I polymorphism of the glycogen synthase gene identifies a subgroup of hypertensive subjects with a family history of NIDDM. The data suggest that a locus in the glycogen synthase gene region on chromosome 19 may serve as a “thrifty gene,” increasing susceptibility for insulin resistance when exposed to other environmental or genetic factors.
- glycogen synthase
- polymorphism, genetics
- insulin resistance
- blood pressure
- non–insulin-dependent diabetes mellitus
Essential hypertension and NIDDM are both characterized by insulin resistance, manifested as an impaired ability to store glucose as glycogen in skeletal muscle.1 2 3 4 Both conditions have a strong genetic component, and defects in carbohydrate metabolism can be demonstrated already in first-degree relatives of subjects with NIDDM or hypertension.5 6 7 8 9 The key regulatory enzyme for glycogen synthesis in skeletal muscle is GS.10 Decreased rates of insulin-stimulated glucose storage and impaired activation of muscle GS activity by insulin are early events in the pathogenesis of NIDDM.6 7 In nondiabetic subjects increased blood pressure is related to both decreased rates of glucose storage and impaired GS activity.11 The GS gene has been considered a candidate gene in the pathogenesis of NIDDM. Evidence for an association between the GS gene and NIDDM comes from studies in Finnish,12 French,13 and Japanese14 patients as well as Pima Indians.15 Interestingly, both the Finnish and French NIDDM patients identified by Xba I polymorphism of the GS gene had a high prevalence of hypertension.12 13 It is not known whether there is an association between polymorphism of the GS gene and hypertension in nondiabetic subjects. To address this question, we examined 304 nondiabetic subjects, 58 of whom had essential hypertension, for a possible association with the GS gene (Xba I polymorphism). We studied the metabolic consequences of this polymorphism with euglycemic insulin clamp combined with indirect calorimetry.
All subjects participating in the study were European and came from the Helsinki area in southern Finland. Liver, kidney, and thyroid function tests were normal in all subjects. Informed consent was obtained from all subjects, and the protocol was approved by the local ethics committee.
All laboratory specimens were taken after a 12-hour fast. Hypertension was defined as blood pressure above 160/95 mm Hg documented on at least three occasions during the previous 3 months or known treatment for hypertension. Blood pressure was measured with a mercury sphygmomanometer with the subject in the sitting position after a 15-minute rest. Blood pressure values are given as the average of three measurements. Subjects with secondary forms of hypertension were excluded from the study. FHx of NIDDM was regarded as positive if one of the parents or siblings had NIDDM. Table 1⇓ shows the characteristics of the study subjects.
Oral Glucose Tolerance Test
Glucose tolerance was examined in 172 subjects, 136 of whom were normotensive (115 with A1 allele; 21 with A2 allele) and 36 hypertensive (25 with A1 allele; 11 with A2 allele) according to World Health Organization criteria.16 Diagnostic values for IGT were fasting plasma glucose less than 7.8 mmol/L and 2-hour glucose of 7.8 to 11.1 mmol/L.16 Insulin and glucose concentrations were measured before and at 30, 60, 90, and 120 minutes after a 75-g oral glucose load. The area under the curve was calculated for both insulin and glucose.
Euglycemic Insulin Clamp
Insulin sensitivity was measured with the euglycemic, hyperinsulinemic clamp technique in combination with indirect calorimetry and infusion of [3-3H]glucose for determination of hepatic glucose production as previously described.6 After three baseline samples had been taken for measurement of glucose and insulin concentrations, a primed constant infusion of short-acting human insulin (Actrapid, Novo-Nordisk) was administered at a rate of 45 mU/m2 per minute (340 pmol/m2 per minute) for 2 hours. Plasma glucose concentration was determined at 5-minute intervals, and 20% glucose was infused for maintenance of a constant plasma glucose concentration.
Indirect calorimetry was used during 60 minutes in the basal state and during the last 60 minutes of the insulin clamp for estimation of glucose oxidation rates.17 A computerized, open-circuit system was used for measurement of gas exchange through a transparent plastic canopy (Deltatrac, Datex Inc). Hepatic glucose production was measured by the isotope-dilution technique with [3-3H]glucose (Amersham) administered as a primed (25 μCi) constant (0.25 μCi/min) infusion for 150 minutes before the insulin clamp was started and was continued throughout the study. Total body glucose metabolism equals the mean rate of glucose infusion during the last 60 minutes of the clamp, provided there is no entry of glucose from the liver. Nonoxidative glucose metabolism, ie, glucose storage in skeletal muscle, was calculated as the difference between total body glucose metabolism and glucose oxidation as determined by indirect calorimetry. Lean body mass was determined with bioelectrical impedance.18
Plasma glucose was measured with the glucose oxidase method adapted for the Beckman Glucose Analyzer II. Serum-free insulin concentrations were measured by double-antibody radioimmunoassay (Pharmacia). Serum cholesterol, high-density lipoprotein cholesterol, and triglycerides were measured by specific enzymatic assays.
GS Xba I Polymorphism
A nonradioactive polymerase chain reaction method was developed for the determination of Xba I polymorphism of the GS gene. A genomic clone was isolated from a human genomic library constructed with Lambda dash vector (Stratagene) with the use of [α-32P]dCTP–labeled human GS cDNA as a probe.19 The hybridizations were carried out at 42°C with 50% formamide. The positive clones were confirmed by Southern blotting and sequenced with Sequenase 2.0 (USB) single-stranded DNA of the subcloned lambda DNA (Bluescript SK, Stratagene). Genomic lambda clone GST11 was isolated and subcloned. The polymorphic Xba I site was in an intron flanked by the exons coding bases 1806 to 1969 and 1970 to 2050 of the cDNA. Two oligonucleotides were synthesized: 5′-CTCCTTCCTCTACAGTTTCTG-3′, located upstream, and 5′-GTGAGTCTCCTCTTTGGCCA-3′, located downstream of the polymorphic Xba I site. Genomic DNA was extracted from peripheral blood leukocytes by the standard method. Genomic DNA (100 ng) was amplified with polymerase chain reaction (Perkin-Elmer Cetus) in a reaction mixture containing 10 pmol of both primers, 1.25 mmol/L MgCl2, 0.1 U Taq polymerase (Promega) and 1× Taq polymerase reaction buffer (Promega), in a total volume of 20 μL. The reaction was carried out at 96°C for 3 minutes, followed by 35 cycles at 96°C for 1 minute, at 61°C for 1 minute, and at 72°C for 1 minute and final extension for 10 minutes at 72°C. After amplification, 2 U Xba I restriction enzyme (Promega), 2 μL of 10× restriction enzyme buffer (Promega), and distilled H2O were added to a final volume of 40 μL and incubated for 1 hour at 37°C. The resulting fragments were separated and analyzed on ethidium bromide–stained 1% agarose gel in 1× TBE buffer (90 mmol/L Tris-borate, 2 mmol/L EDTA) at a constant voltage of 4 V/cm for 2 hours.
Data are expressed as mean±SEM. Statistical analyses were performed with a BMDP computer program. The significance of differences between group means was tested with the Mann-Whitney rank sum test (nonparametric). In addition, ANCOVA was performed with BMI and age (OGTT data) and BMI, age, and fasting plasma-glucose (clamp data) as covariates to adjust for differences in these parameters between the groups studied. The significance of the frequency difference of GS Xba I alleles was tested by χ2 analysis with Yates’ correction.
Xba I Polymorphism of the GS Gene
With the use of the restriction enzyme Xba I, two polymorphic patterns can be demonstrated in the GS gene. The A1 allele lacks the Xba I site and gives a fragment of approximately 600 bp in size, whereas the A2 allele is cleaved into two fragments of 500 and 100 bp in size.
Of 304 unrelated, nondiabetic subjects, 253 (83%) had the genotype A1A1 and 51 (17%) the A2 allele. Most subjects with the A2 allele (n=48) were heterozygous, whereas homozygosity for the A2 allele (A2A2) could be demonstrated in only 3 subjects.
The characteristics of study subjects are given in Table 1⇑. Hypertensive subjects were characterized by significantly higher fasting insulin values, higher total cholesterol and triglyceride concentrations, lower high-density lipoprotein cholesterol concentrations, and increased BMI and age compared with normotensive subjects (Table 1⇑).
The A2 allele was twice as frequent among the hypertensive as among the normotensive subjects (28% versus 14%, P=.024) (Fig 1⇓). The highest frequency of the A2 allele (48%) was observed among hypertensive subjects with FHx of NIDDM and the lowest in normotensive subjects without FHx of NIDDM (6%); the frequency in subjects with isolated FHx of NIDDM (26%) or hypertension (16%) was intermediary (χ2=33.7, 3 df, P<.0001) (Fig 2⇓).
Characteristics of Subjects With the A1 or A2 Allele
Characteristics of normotensive and hypertensive subjects with the A1 or A2 allele are given in Table 2⇓. A reliable FHx of hypertension was obtained for 103 subjects. Of these subjects, 57% of those with the A2 allele and 46% with the A1 allele had FHx of hypertension (P=NS). Sixteen percent of subjects with the A2 allele and 13% of those with the A1 allele were taking antihypertensive drugs.
Normotensive subjects with the A2 allele were younger than normotensive subjects with the A1 allele (39.0±2 versus 45±2 years, P=.047). Glucose tolerance was similar in normotensive subjects with the A1 or A2 allele (P=NS). The areas under the curve for glucose (27.3±0.5 versus 25.3±1.6 mmol/L×120 minutes, P=.40) and insulin (1441±87 versus 989±118 pmol/L×120 minutes, P=.10) did not differ significantly between subjects with the A1 or A2 allele, respectively (adjusted for age and BMI).
In hypertensive subjects, the prevalence of IGT was significantly higher in subjects with the A2 compared with those with the A1 allele (36% versus 4%, P=.04). The areas under the curve for glucose (32.4±2.1 versus 29.8±0.9 mmol/L×120 minutes, respectively; P=.57) and insulin (1446±240 pmol/L versus 1598±190×120 minutes, respectively; P=.68) did not differ significantly between the two groups (adjusted for age and BMI).
Rates of Insulin-Stimulated Glucose Storage
To examine whether the A2 allele was associated with decreased rates of glucose storage, we performed euglycemic insulin clamp studies in combination with indirect calorimetry (Table 3⇓) in matched subgroups of normotensive subjects (A1 allele: 4 women/4 men; mean age, 39±2 years; BMI, 25.0±1.4 kg/m2; A2 allele: 4 women/4 men; mean age, 38±2 years; BMI, 24.6±0.8 kg/m2) and hypertensive subjects (A1 allele: 1 woman/6 men; mean age, 49±5 years; BMI, 27.8±1.6 kg/m2; A2 allele: 7 men; mean age, 53±4 years; BMI, 28.3±1.4 kg/m2). Insulin sensitivity and glucose storage rates did not differ between normotensive subjects with the A1 or A2 allele (Table 3⇓). Among hypertensive subjects, subjects with the A2 allele had lower rates of insulin-stimulated glucose storage (P=.029) compared with subjects with the A1 allele (Table 3⇓).
Hypertension is already observed in about half of NIDDM patients at diagnosis. Both hypertension and NIDDM are characterized by insulin resistance manifested as a decreased ability to store glucose as glycogen in skeletal muscle.2 4 We12 and others13 have previously demonstrated an association between polymorphism of the GS gene and NIDDM. It is not known whether hypertension in these subjects represents a secondary phenomenon or predisposes to the development of NIDDM. The present study examined GS Xba I polymorphism in a nondiabetic population, and the A2 allele of this polymorphism was highly enriched in subjects having overt hypertension and FHx of NIDDM. On the basis of results from twin and adoption studies and statistical analyses of blood pressure in various pedigrees, approximately 20% to 60% of the population variability in blood pressure is estimated to be genetically determined.20 21 GS Xba I polymorphism seems to identify a subgroup of subjects with a genetic background involving both hypertension and NIDDM. High blood pressure frequently associates with IGT, indicating, at least in part, a common genetic background for these disorders.22 23 24 25 The significant association with the GS gene in subjects having both hypertension and FHx of NIDDM implies that this may represent a distinct disease entity, the susceptibility of which is increased by some genetic defect or defects within or in the vicinity of the GS gene.
IGT was more frequent in hypertensive subjects with the A2 than with the A1 allele. No such difference was observed in normotensive subjects. However, caution is warranted in the interpretation of these data because normotensive subjects with the A2 allele were younger than those with the A1 allele. Therefore, this could have masked an expected age-dependent increase in the frequency of IGT in these subjects.
In subjects with NIDDM the presence of the A2 allele was associated with impaired insulin-stimulated glucose storage.12 In keeping with this finding we observed a significant decrease in the rate of insulin-stimulated glucose storage also in hypertensive subjects with the A2 allele. However, this difference was not seen in the normotensive subjects. In support of this, there was no significant difference in insulin responses during the OGTT in normotensive subjects. Insulin responses during the OGTT were similar also in hypertensive subjects. If anything, insulin responses tended to be diminished in the subjects with the A2 allele. How could these data be reconciled? First, changes in insulin sensitivity explain only 30% of the variance in insulin concentrations during the OGTT.26 Second, the defect was confined to the glucose storage pathway of glucose metabolism, whereas glucose oxidation was normal. These changes in the intracellular partitioning of glucose may be too subtle to be reflected by changes in insulin concentration during the OGTT. Third, impaired insulin secretion was recently reported in hypertensive subjects with IGT independent of insulin resistance.27 This could explain why the hypertensive subjects with the A2 allele did not show an increase in the insulin response during the OGTT.
Why would the presence of the A2 allele be associated with impaired skeletal muscle glycogen synthesis in patients with NIDDM and hypertension but not in lean, normoglycemic individuals? A recent animal study may shed some light on this discrepancy. The trait of developing diabetes during fat feeding was recently linked to the GS gene locus on chromosome 7 in the C57BL/6J mouse.28 The diabetes-prone mouse was further characterized by impaired GS activity in skeletal muscle and elevated fasting insulin concentrations (as a measure of insulin resistance). When kept on a normal diet, the mice maintained normal weight and normal glucose and insulin concentrations.28 These data suggest that no metabolic abnormalities could be discerned until the “thrifty gene” was exposed to an environment of high energy intake. If the same applies for Xba I polymorphism of the GS gene in humans, the presence of the genetic marker in an otherwise lean and healthy person should not be associated with the metabolic abnormalities of insulin resistance. However, we do not know what the pathogenetic link is between this polymorphism and insulin resistance. Since insulin resistance was observed mainly in obese (NIDDM12 or hypertensive) subjects with the A2 allele, obesity may be the common denominator predisposing to the other conditions.
In conclusion, Xba I polymorphism of the GS gene identifies a subgroup of subjects with both hypertension and FHx of NIDDM. Since this polymorphism is associated with insulin resistance only in obese subjects with hypertension and/or NIDDM, it may represent a “thrifty gene,” which has to be exposed to an affluent environment before the associated metabolic abnormalities are unmasked.
Selected Abbreviations and Acronyms
|BMI||=||body mass index|
|IGT||=||impaired glucose tolerance|
|NIDDM||=||non–insulin-dependent diabetes mellitus|
|OGTT||=||oral glucose tolerance test|
This work was supported by grants from the Sigrid Jusélius Foundation, the Finnish Diabetes Research Foundation, Finska Läkaresällskapet, the Perklén Foundation, and Svenska Kulturfonden. We are grateful to Maija Parkkonen, RT, and Esa Laurila, RT, for technical assistance.
Reprint requests to C. Schalin-Jäntti, MD, IV Department of Medicine, Helsinki University Hospital, Unioninkatu 38, FIN-00170, Helsinki, Finland.
- Received April 17, 1995.
- Revision received July 25, 1995.
- Accepted September 6, 1995.
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