ACE Gene Insertion/Deletion Polymorphism Associated With 1998 World Health Organization Definition of Metabolic Syndrome in Chinese Type 2 Diabetic Patients

From February 1998 to February 2001, 711 patients with type 2 diabetes who consecutively attended the diabetes clinic at Pingtung Christian Hospital were studied. The diagnosis of type 2 diabetes was based on the WHO criteria (5). The control subjects were unrelated patients who entered the health examination program of the hospital. Each received a detailed interview about personal disease history and smoking history. All study subjects were of Han Chinese origin, without any known ancestors of other ethnic origin, and were living in the same region at the time of study. This study was approved by the human research ethics committee of our hospital, and informed consent was obtained from each patient. All patients underwent complete physical examinations and routine biochemical analyses of blood and urine as well as an assessment of the presence and extent of macrovascular or microvascular diabetic complications. The anthropometric parameters required to calculate BMI and waist-to-hip ratio (WHR) were measured. Seated blood pressure, plasma biochemical parameters, and urinary microalbumin were measured after overnight fasting. Plasma triglycerides, total, LDL, and HDL cholesterol, uric acid, creatinine, and glucose were determined by standard commercial methods on a parallel-multichannel analyzer (Hitachi 7170A; Hitachi, Tokyo). Urinary albumin concentrations were measured by immunoturbidmetry (Beckman Instruments, Galway, Ireland). The detection limit was 2 mg/l, and the inter- and intra-assay coefficient of variation was <8%.

Metabolic syndrome was defined according to the proposed criteria of the WHO 1998 Consultation on Definition, Diagnosis and Classification of Diabetes Mellitus and Its Complications (5,6), with modification in the definition of hypertension. Metabolic syndrome was defined when a subject with impaired fasting glucose or diabetes had two or more of the following components: 1) raised arterial pressure ≥160/90 mmHg or on antihypertensive treatment; 2) central obesity (WHR >0.9 for men and >0.85 for women and/or BMI >30 kg/m²); 3) microalbuminuria (urinary albumin excretion rate ≥20 μg/min or albumin/creatinine ≥20 mg/g on at least two different occasions) or more advanced nephropathy; and 4) raised plasma triglycerides (≥150 mg/dl) and/or decreased HDL cholesterol (<35 mg/dl for men and <39 mg/dl for women). Because hypertriglyceridemia may be related to hyperglycemia in patients with type 2 diabetes (20), the definition of dyslipidemia in patients with type 2 diabetes was used only when triglycerides or HDL cholesterol were at abnormal levels after a 3-month control of blood glucose.

Genomic DNA was prepared from peripheral blood using standard techniques. For the ACE I/D polymorphism, the primer pairs used and the annealing temperature were as follows: forward 5′-CTGGAGACCACTCCCATCCTTTCT-3′ and reverse 5′-GATGTGGCCATCACATTCGTCAGAT-3′, which amplify the intron 16 region where the I/D fragment is located. PCR amplification products were obtained using 25-μl reactions (0.5 pg genomic DNA, 500 pmol of primers, 0.5 mmol/l each of deoxy-ATP, -GTP, -CTP, and -TTP, 1.5 mmol/l MgCl₂, 0.5 units Tag DNA polymerase [Takara Tag; Takara Shuzo, Otsu Shiga, Japan], 50 mmol/l KCl, 0.001% gelatin, and 10 mmol/l Tris-HCl; pH 8.3) with 4 min denaturation at 94°C, followed by 35 cycles of 15 s at 94°C, 5 s at 67°C, and 30 s at 74°C in a thermal cycler (Gene Amp PCR System 9700; Perkin-Elmer, Foster City, CA). The reaction was terminated at 72°C at 2 min. To avoid ID/DD mistyping of heterozygotes as DD homozygotes (21), all DD genotype samples were confirmed using a pair of primers that produce an amplified product only in the presence of the insertion, which was used to verify the polymorphism: forward 5′-TGGGACCACAGCGCCCGCCACTAC-3′ and reverse 5′-TCGCCAGCCCTCCCATGCCCATAA-3′ (22). The PCR condition was similar to that procedure for I/D detection, except that the annealing temperature was changed to 62°C. All PCR products were visualized after electrophoresis on a 2% agarose gel and ethidium bromide staining. Genotyping was performed in a blinded fashion.

The data are expressed as means ± SD. All statistical analyses were performed using the Statistical Package for Social Science program (SPSS for Windows, version 7.5.1; SPSS, Chicago). The statistical difference in genotype distribution and allele frequencies among the groups was assessed by the Pearson χ² test. Other variables were compared using unpaired t test for normally distributed variables, and ANOVA followed by Scheffe’s test was used to compare the group means. Before statistical testing, fasting plasma glucose and serum triglycerides were logarithmically transformed to achieve a normal distribution.

A total of 750 sex- and age-matched subjects from 1,309 control subjects were used as the control group. Among these control subjects, 43.2% were found to be obese, 29.5% had dyslipidemia, 23.6% had hypertension, 12.3% had impaired fasting glucose or diabetes, and 10.3% had microalbuminuria or more advanced nephropathy. Eighty-two (10.9%) control subjects were found to fit the criteria of metabolic syndrome, and 229 subjects were free of any abnormality of components of metabolic syndrome. Table 1 presents the clinical characteristics of control and type 2 diabetic subjects. Diabetic subjects had significantly higher levels of BMI, WHR, fasting glucose, blood pressure, serum total cholesterol, and triglycerides than those of the control group. Of 711 type 2 diabetic subjects, 534 (75.1%) fulfilled the WHO criteria for metabolic syndrome. Diabetic subjects with metabolic syndrome were older and had significantly higher blood glucose, HbA_1c, blood pressure, serum triglyceride, total cholesterol, creatinine, and uric acid levels as well as higher urinary albumin/creatinine ratio and lower HDL cholesterol levels than those without metabolic syndrome (Table 2).

The ACE gene I/D genotype distributions within the study groups are presented in Table 1. In each study group, the genotype frequency distributions of this polymorphism were in Hardy-Weinberg equilibrium. No differences in ACE genotype distribution and allele frequencies were found between patients with type 2 diabetes and control subjects. Table 3 shows the prevalence of the different components of metabolic syndrome in control and diabetic subjects with different ACE genotypes. The highest prevalence of metabolic derangement was obesity, and the most common combination was obesity plus dyslipidemia. The prevalence of metabolic syndrome in control subjects with II, ID, and DD genotype was 9.4, 11.5, and 15.4%, respectively, and in patients with type 2 diabetes, it was 68.6, 79.2, and 86.1%, respectively. The ACE I/D polymorphism was significantly associated with the syndrome (P = 0.001) (Table 3) in patients with type 2 diabetes. When pooling the control with diabetic subjects, the prevalence of metabolic syndrome in the whole study group with II, ID, and DD genotype was 37.9, 44.5, and 51.0%, respectively, and ACE I/D polymorphism was still significantly associated with metabolic syndrome (P = 0.003). Diabetic patients with DD genotype were also found to have a higher prevalence of dyslipidemia (II/ID/DD = 43.1/53.1/65.8%, P < 0.001) and albuminuria (36.0/44.6/50.6%, P = 0.018) and to have higher serum triglyceride levels (II, ID, and DD = 155 ± 114, 170 ± 140, and 199 ± 132 mg/dl, respectively, P < 0.05) (Table 1). Control subjects with DD genotype were also found to have a higher prevalence of albuminuria or more advanced nephropathy (II/ID/DD = 5.7/14.0/15.4%, P = 0.001). When pooling the control with diabetic subjects, ACE genotype could still be significantly associated with dyslipidemia (II/ID/DD = 34.7/41.3/52.2%, P < 0.001) and albuminuria or more advanced nephropathy (20.3/28.9/33.1%, P < 0.001). There were no statistically significant differences in the variables found among the control subjects with different ACE genotypes (Table 1).

Our results demonstrate a high prevalence (75.1%) of metabolic syndrome in Chinese patients with type 2 diabetes, a finding comparable with a Scandinavian report (7,8). The current study gives the actual prevalence for the syndrome in type 2 diabetic patients, which could only be made once the criteria for the syndrome was made available in 1998 (5). This finding suggests that the etiology of diabetes, obesity, dyslipidemia, hypertension, and nephropathy may have a common factor(s), and it also provides clues to the high incidence of macro- or microvascular complications in patients with type 2 diabetes. An elevated mean serum uric acid level was also noticed in our diabetic patients with metabolic syndrome, which is in accordance with the finding of elevated uric acid often found concurrently in obesity, dyslipidemia, and essential hypertension (23).

The current study reveals no association between ACE genotype and type 2 diabetes in the Chinese population, although Chinese diabetic patients with ACE DD genotype have a higher prevalence of metabolic syndrome. To our knowledge, this is the first report showing the association of ACE gene polymorphism with metabolic syndrome using the new WHO criteria. This fact may provide genetic evidence for the clustering of metabolic syndrome or insulin resistance syndrome.

Meta-analyses assessing the influence of the ACE gene polymorphism on disease susceptibility have demonstrated significant odds ratios in individuals with the DD genotype for both diabetic and nondiabetic renal disease (17), hypertension (14), and coronary artery diseases (15,16). Most evidence appears to show that the D allele is a risk factor for development and progression of micro- or macrovascular diabetic complications. It has been found that an I/D polymorphism of ACE gene affects the serum ACE level (24), and ACE gene polymorphism has been known to contribute to the ethnic differences in response to ACE inhibitor treatment (25). In Caucasians, 44% of the variance in circulating ACE activity is accounted for by genetic polymorphism (26), and plasma ACE activity has also been found to be genetically determined in the Chinese population (27). Activated angiotensin systems may lead to organ damage by enhancement of cellular hypertrophy and proliferation and disruption of the extracellular matrix, and induction of cytokine or growth factor secretion further exacerbates the injury (12). Patients with the DD genotype have activated angiotensin systems (24) and, thus, may be prone to vascular injury. Our results are similar to many reports (13–17) that reveal that type 2 diabetic patients with ACE DD genotype have a higher prevalence of albuminuria. Combined, these results support the finding that the ACE genotype is associated with the metabolic syndrome and give evidence to support the hypothesis that the D allele is a risk factor for micro- and macrovascular diseases (13–17).

The HOPE (Heart Outcomes Prevention Evaluation) and MICRO-HOPE studies demonstrated that when ACE inhibitor treatment was administered to patients with high risk for cardiovascular disease and diabetes, cardiovascular events and overt nephropathy were significantly decreased (28,29). These studies also revealed that the cardiovascular benefit was greater than that attributable to the decrease in blood pressure and concluded that ACE inhibition represents a vasculoprotective and renoprotective effect for people with high cardiovascular risk and diabetes. These facts indeed suggest that the angiotensin system plays a role in the pathogenesis of cardiovascular and renovascular complications.

Patients with type 2 diabetes with DD genotype were found in this study to have a significantly higher prevalence of dyslipidemia and higher serum triglyceride levels than those with II genotype; the difference was also present when pooling the control subjects with diabetic patients. This result may suggest that the angiotensin system plays some role in lipid metabolism. All components of RAS genes are found to be expressed in adipose tissue (30). The adipose tissue RAS may have multiple functions, including prostaglandin synthesis and adipose tissue lipolysis, and may play a role in adipocyte metabolic function and obesity (31,32). Nagi et al. (18) found that plasma ACE levels, but not ACE genotype, were associated with plasma triglyceride and total cholesterol levels in Pima Indians. At the same time, ACE genotypes were found to be significantly correlated with plasma ACE levels in both diabetic and nondiabetic groups, leading them to conclude that ACE genotype is not a major determinant of circulating plasma ACE levels in Pima Indians.

Our study revealed that ACE genotype was significantly associated with serum triglyceride levels, not total cholesterol levels, in Chinese subjects with type 2 diabetes. The discrepancy between the results of our study and that of Nagi et al. in Pima Indians may be due to the larger number of case subjects in our study (1,461 vs. 305). The prevalence of ACE DD genotype has been reported to be lower among Chinese compared with Caucasians but comparable with that of Pima Indians (18,25,27). To avoid the confounding effect of glucose level on serum triglyceride concentrations (20), the serum triglyceride level used for classification was the level measured after a 3-month control of blood glucose. Table 1 shows that the plasma sugar and HbA_1c levels were not significantly different among the diabetic subgroups. Thus, our results suggest that RAS may play some role in triglyceride metabolism.

Angiotensin II has been found to be a modulator of insulin sensitivity in both diabetic and nondiabetic subjects (33,34). Our results show no difference in ACE genotype distribution and allele frequencies between patients with type 2 diabetes and control subjects and imply that the ACE genotype may not be associated with the pathogenesis of insulin sensitivity or type 2 diabetes. Although the angiotensin system may not, in general, be a causative factor for diabetes, according to our studies and the studies of others, angiotensin II may have certain detrimental effects on metabolic syndrome.

Our study also indicates that ACE I/D polymorphism is not associated with hypertension in patients with type 2 diabetes. This result is in accordance with a report by Staesen et al. (35) in which a meta-analysis of other reports showed no association of hypertension with ACE polymorphism. The correlation between angiotensin system and obesity is interesting. Plasma angiotensin level, plasma renin activity, and plasma ACE activity were found to be correlated with BMI in obese human subjects of different human populations (36,37), and the linkage between obesity and an angiotensin system gene polymorphism has been demonstrated in a genetically isolated population (38). In this study, we did not find any anthropometric parameters associated with the ACE gene genotypes, and thus, we believe this association needs clarification by further observations in the Chinese population.

In summary, we have found an association of the ACE genotype polymorphism with metabolic syndrome in Chinese patients with type 2 diabetes. This result may provide genetic evidence to explain the clustering of metabolic syndrome, and suggests that RAS is involved in the pathophysiology of metabolic derangement in patients with type 2 diabetes.

We are grateful to Yi-Su Chung for her excellent technical assistance and to the staff of the clinical and nutrition diabetes section for their assistance in various measurements and other organizational aspects of this study.