Abstract
substitution and insulin resistance in subjects with normal insulin secretory capacity, since it has been reported that PPAR may affect not only insulin resistance but also insulin secretion. We examined 81 Japanese male patients with untreated essential hypertension using the glucose clamp technique. We found 77 subjects with Pro/Pro and 4 subjects with Pro/Ala genotype, and the glucose disposal rate was not signifi- 
Introduction
The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor superfamily and plays an important role in the regulation of adipocyte differentiation (1) . A new class of antidiabetic drugs, thiazolidinediones, are known to be ligands for PPARγ and to improve insulin sensitivity. On the other hand, the results of a study using PPARγ knockout mouse have suggested that PPARγ-deficient mice have higher insulin sensitivity (2) . The heterozygous PPARγ-deficient mouse has been shown to be protected from the detution was associated with lower body mass index (BMI) and improved insulin sensitivity in middle-aged and elderly Finns. On the other hand, other reports have shown that PPARγ 2 substitution was not associated with insulin sensitivity (5, 6) . Further investigation on PPARγ 2 substitution and insulin resistance may help us identify the role of PPARγ in insulin resistance.
A study on a large number of Japanese subjects showed that the frequency of the PPARγ Ala allele was significantly lower in type 2 diabetic patients than in nondiabetic control subjects (7) . However, this result does not necessarily mean that the Ala allele in PPARγ is associated with better insulin sensitivity and hence lower prevalence of diabetes, because lowered insulin secretion is thought to be a major determinant in the development of diabetes in Oriental population. We therefore investigated the relationship between Pro12Ala substitution and insulin resistance in nondiabetic hypertensive subjects with normal insulin secretory capacity.
β3 adrenergic receptor (β 3 AR) is expressed in adipose tissue, and is considered responsible for increases in lipolysis. The β3 AR Trp64Arg polymorphism has been considered to have an association with obesity (8) . And a previous study using the glucose clamp method reported that this mutation was associated with decreased insulin sensitivity in type 2 diabetic Finns (9) . Moreover, the allele frequency of this mutation is about 0.20 i.e., is relatively common in Japanese. We therefore investigated the β3 AR Trp64Arg polymorphism in addition to PPARγ Pro12Ala substitution.
Methods

Subjects
Subjects were 81 Japanese male patients with untreated essential hypertension. All the patients were diagnosed as hypertension according to the WHO criteria. They were in good health, except for having hypertension, as determined by case history, physical examination, and routine laboratory screening. Aside from having hypertension, all subjects were in good health, as determined by case history, physical examination, and routine laboratory screening. None were diabetic as assessed by 75 g oral glucose tolerance test (OGTT) according to the WHO criteria. The study was approved by the Medical Research Review Board of Jikei University and written informed consent was obtained from each subject.
DNA Analysis
Genomic DNA was extracted from peripheral leukocytes by using a blood DNA kit (QIAGEN, Hilden, Germany). Presence of the PPARγ Pro12Ala polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis (10) . The flanking primer pair was 5 -TCTGGGAGATTCTCCTATTGGC-3 and 5 -CTGGAAGACAACTACAAGAG-3 . A 50 µl reaction mixture containing 50 -500 ng of genomic DNA, 0.2 µM of the primers, 2.5 mM of KCl, and 1 U of Ex. Taq (Takara, Ohtsu, Japan) was used for PCR. The reaction mixture was incubated at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s. The PCR products were digested with BstU-I (New England BioLabs, Beverly, USA) at 60°C for 2 h, electrophoresed on 4% agarose gel, and stained with ethidium bromide. The Pro12 allele gives one 154-bp fragment whereas the Ala12 allele gives 132-bp and 22-bp fragments. The β3 adrenergic receptor polymorphism was analyzed by the PCR-RFLP method (10) . The forward primer was 5 -CGCCCAATACCGCCAACAC-3 and the reverse primer was 5 -CCACCAGGAGTCCCATCACC-3 . The PCR products were digested with Bst-O1 (Promega, Madison, USA) at 60°C overnight, and electrophoresed on 10 Ready Gel J (Bio-Rad, Hercules, USA) and stained with ethidium bromide.
Measurement of Insulin Sensitivity
The hyperinsulinemic euglycemic clamp was performed to assess insulin sensitivity. Insulin (300 mU/ml) was infused at a rate of 40 mU/m 2 body surface area per min with a syringe pump (Syringe Pump 1235; Atom, Tokyo, Japan) as described elsewhere (11) . Blood glucose was maintained at the fasting level by infusing glucose. The glucose clamp was continued for 120 min. Glucose infusion rate during the last 30 min of the glucose clamp was used as an index of insulin sensitivity. Insulin sensitivity was determined using Biostator, an artificial endocrine pancreas (Life Science Instrument, Elkhart, USA) in 39 cases (12) . In the remaining 42 cases, insulin sensitivity was determined according to the method of DeFronzo because the artificial endocrine pancreas broke down (13) . Blood glucose was determined every 5 min with a glucose meter (Antsense II; Daikin, Osaka, Japan) and 10% glucose was infused to maintain the fasting blood glucose level. As insulin was infused in exactly the same way using the same insulin infusate and the same infusion pump, data obtained by the two glucose clamp methods were treated together in the results as previously (14) . In 4 of the cases, the glucose infusion rate had increased to 8 mg/kg min by 30 min after the start of the glucose clamp. Because it was anticipated that, at 90 and 120 min, the glucose infusion rate would increase to a level beyond the capacity of the pump, glucose clamping was cancelled midway and the highest value yet obtained at that point (11.2 mg/kg min) was taken as the infusion rate for these 4 cases.
Fasting plasma glucose, fasting immunoreactive insulin (IRI), total cholesterol, HDL cholesterol, and triglyceride were measured. The 75 g OGTT was performed on another day after an overnight fast. Area under the curve (AUC) for plasma glucose (PG) and for IRI were calculated by trapezoidal rule. HOMA-R (homeostasis model assessment of insulin resistance) values were calculated as described else-where (15) according to the formula:
HOMA-R fasting insulin (µU/ml) glucose (mmol/l)/22.5
Statistical Analysis
Results are expressed as mean SEM. The unpaired Student's t-test was used for comparison of the obtained data. Values of p<0.05 were considered to indicate statistical significance.
Results
The PPARγ 2 Pro12Ala distribution revealed that 77 subjects had the Pro/Pro, 4 subjects the Pro/Ala, and no subjects the Ala/Ala genotype. The allele frequency of the Ala genotype was 0.025. The genotype distribution was in Hardy-Weinberg equilibrium (Pro/Pro, 95.1%; Pro/Ala, 4.9%; Ala/Ala, 0.06%). Age, BMI, and blood pressure were not significantly different between the Pro/Pro group and Pro/Ala group ( Table 1) . The glucose disposal rate (GDR) was 6.02 0.27 mg/kg min (mean SEM) in the Pro/Pro group and 6.39 1.21 mg/kg min in the Pro/Ala group, and there was no significant difference between the two groups (Fig. 1) . Levels of fasting plasma glucose, fasting IRI, total cholesterol, HDL cholesterol, and triglyceride were not significantly different between the two groups. There were also no significant differences between groups in HOMA-R values, AUC for PG, or AUC for IRI in 75 g OGTT ( Table 2) .
The β3 adrenergic receptor Trp64Arg polymorphism distribution showed 56 subjects with the Trp/Trp, 24 subjects with the Trp/Arg, and 1 subject with the Arg/Arg. The allele frequency of the Arg genotype was 0.16. The genotype distribution was in Hardy-Weinberg equilibrium (Trp/Trp, 2.6%; Trp/Arg, 26.9%; Arg/Arg, 70.6%). There were no differences in BMI between the Trp/Trp group and Trp/Arg Arg/Arg group. GDR was 6.25 0.34 mg/kg min (mean SEM) in members of the Trp/Trp group and 5.55 0.33 mg/kg min in those of the Trp/Arg Arg/Arg group. GDR was not statistically analyzed according to the two gene polymorphisms (Table 3) due to the small numbers in each group.
Discussion
In an obese rat model, thiazolidinediones have been shown to improve insulin sensitivity through activation of PPARγ, to increase the population of small adipocytes, and to decrease the population of large adipocytes (1) . And a recent study The PPARγ 2 Ala allele has been shown to demonstrate decreased binding affinity to the promoter element and reduced ability to transactivate responsive promoters in vitro (4) . In the same study, this allele was also associated with lower body mass index and lower fasting insulin level in middle-aged and elderly Finns (4). These results have suggested that the Ala allele may be associated with better insulin sensitivity. Whereas the Ala allele has also been associated with higher BMI in Caucasian obese subjects (17) , reports in Japanese healthy subjects and impaired glucose tolerant (IGT) subjects have shown no significant relationship between PPARγ 2 substitution and insulin sensitivity as assessed by 75 g OGTT, the HOMA model, and fat distribution (5) . In a recent study on a large number of Japanese type 2 diabetic patients and control subjects, frequency of the Ala allele was significantly lower in the type 2 diabetic group than in controls (7). However, impaired insulin secretion is observed more frequently and is thought to play a more important role in the development of type 2 diabetes in the Japanese population. Moreover, HOMA-β was significantly reduced in the Ala allele group (7), suggesting that impaired insulin secretion may be involved. Another study found that PPARγ-deficient mice had lower capacity for insulin secretion than wild-type mice, as assessed by an oral glucose tolerance test (2) . We therefore investigated the relationship between insulin resistance and PPARγ 2 substitution in nondiabetic hypertensive subjects with normal insulin secretion using the glucose clamp technique.
We found no significant relationship between PPARγ 2 polymorphism and insulin resistance in our cohort of Japanese essential hypertensive patients. However, previous studies have reported racial differences in the frequency of PPARγ 2 substitution. The Ala allele frequency is 12 -20% in Caucasians, and only 2 -3% in Japanese. In the present study, we found only 4 cases of Pro12Ala heterozygosity, and no cases of homozygous substitution.
In a previous study using the glucose clamp method, no significant relationship was observed between PPARγ 2 substitution and insulin sensitivity in nondiabetic Caucasian subjects who had a history of type 2 diabetes in a first-degree relative (18) . However, the Ala allele was associated with significantly higher insulin sensitivity only in severely obese subjects with BMI more than 30 kg/m 2 , showing that the influence of PPARγ 2 substitution on insulin sensitivity was limited to obesity-associated insulin resistance. Another study has shown that heterozygous PPARγ -deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet (2) . PPARγ is expressed mainly in the brown and white adipose tissue. Thiazolidinediones have been shown to suppress resistin in adipocytes leading to improvement of insulin sensitivity. PPARγ is also expressed in the skeletal muscle and the liver, although PPARγ levels are 10 -30 times higher in fat than in the latter tissues (19) . Pharmacological doses of thiazolidinediones may stimulate PPARγ not only in the adipose tissue, but also in the skeletal muscle and the liver (19) . Therefore, another possible mechanism is that thiazolidinediones act directly on PPARγ in the skeletal muscle. A report on the improvement of insulin sensitivity by troglitazone in the lipodystrophic mouse has suggested that regulation of PPARγ can also occur in the near absence of fat (20) .
In order to exclude the confounding factors of severe obesity and impaired insulin secretion, we therefore examined insulin sensitivity in subjects who were not severely obese. In our study, subjects examined by the glucose clamp method were patients with essential hypertension, and their mean BMI was 24.7 kg/m 2 . There was no significant correlation between PPARγ 2 mutation and insulin resistance in terms of glucose disposal rate.
As insulin sensitivity would probably be determined by polygenic factors, we also determined β3 AR Trp64Arg polymorphism as a possible determinant of insulin resistance. GDR was not statistically analyzed according to PPARγ 2 and β3 AR polymorphisms because of the small numbers in each group.
In conclusion, we found no significant association between PPARγ 2 substitution and insulin sensitivity in our cohort of Japanese hypertensive patients. However, further investigations will be needed to determine the relationship between PPARγ 2 and insulin sensitivity in a larger number of subjects.

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