TYPE 2 DIABETES MELLITUS is a common, multifactorial

0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(5):2019 2023 Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: 10.1210/jc.2003-031325 The G-250A Promoter Polymorphism of the Hepatic Lipase Gene Predicts the Conversion from Impaired Glucose Tolerance to Type 2 Diabetes Mellitus: The Finnish Diabetes Prevention Study BORYANA TODOROVA, AGATA KUBASZEK, JUSSI PIHLAJAMÄKI, JAANA LINDSTRÖM, JOHAN ERIKSSON, TIMO T. VALLE, HELENA HÄMÄLÄINEN, PIRJO ILANNE-PARIKKA, SIRKKA KEINÄNEN-KIUKAANNIEMI, JAAKKO TUOMILEHTO, MATTI UUSITUPA, AND MARKKU LAAKSO Department of Medicine (B.T., A.K., J.P., M.L.) and Department of Clinical Nutrition (M.U.), University of Kuopio, 70210 Kuopio; Department of Epidemiology and Health Promotion (J.L., J.E., T.T.V., J.T.), Diabetes and Genetic Epidemiology Unit, National Public Health Institute, 00300 Helsinki; Research Department (H.H.), Social Insurance Institution, 20720 Turku; Department of Medicine (P.I.-P.), Finnish Diabetes Association and Tampere University Hospital, 33014 Tampere; Department of Public Health Science and General Practice (S.K.-K.), University of Oulu, Oulu University Hospital and Department of Sport Medicine (S.K.-K.), Oulu Deaconess Institute, 90220 Oulu; and Department of Public Health (J.T.), University of Helsinki, 00300 Helsinki, Finland In population-based studies, dyslipidemia related to insulin resistance (high triglyceride level and low high-density lipoprotein cholesterol level) is a risk factor for type 2 diabetes. Therefore, variants in genes regulating lipid and lipoprotein metabolism are potential candidate genes for diabetes. We investigated whether the G-250A polymorphism of the hepatic lipase gene (LIPC) predicts the conversion from impaired glucose tolerance (IGT) to type 2 diabetes in the Finnish Diabetes Prevention Study. This study randomized subjects to either the intervention group (lifestyle modification aimed at weight loss, such as changes in diet and increased physical exercise) or the control group. Genotyping at position 250 of the LIPC gene was performed with PCR amplification, DraI enzyme digestion, and gel electrophoresis in 490 subjects with IGT whose DNA was available. In the entire study population, the conversion rate to type 2 diabetes was 17.8% among subjects Abbreviations: BMI, Body mass index; DPS, Diabetes Prevention Study; HDL-C, high-density lipoprotein cholesterol; HL, hepatic lipase; IGT, impaired glucose tolerance; LDL, low-density lipoprotein; LIPC, hepatic lipase gene. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. with the G-250G genotype and 10.7% among subjects with the 250A allele (P 0.032). In univariate analysis, the odds ratio for the G-250G genotype to predict the conversion from IGT to type 2 diabetes was 1.80 (95% confidence interval, 1.05 3.10; P 0.034). In multivariate logistic regression analysis, the G-250G genotype predicted the conversion to diabetes independently of the study group (control or intervention), gender, weight, waist circumference at baseline, and change in weight and waist circumference. In the intervention group, 13.0% of subjects with the G-250G genotype and 1.0% of the subjects with the 250A allele converted to diabetes (P 0.001). We conclude that the G-250G genotype of the LIPC gene is a risk factor for type 2 diabetes. Therefore, genes regulating lipid and lipoprotein metabolism may be potential candidate genes for type 2 diabetes. (J Clin Endocrinol Metab 89: 2019 2023, 2004) TYPE 2 DIABETES MELLITUS is a common, multifactorial disease with genetic predisposition that is strongly influenced by environmental and behavioral factors, such as obesity and sedentary lifestyle. Previous studies have indicated that obesity, central obesity, physical inactivity, and a family history of diabetes are major risk factors for type 2 diabetes (1). Furthermore, dyslipidemias related to insulin resistance, i.e. high total triglyceride level, low high-density lipoprotein cholesterol (HDL-C) level, and small dense lowdensity lipoprotein (LDL) particles, are risk factors for diabetes (2, 3). Therefore, variants in genes regulating dyslipidemias are potential risk factors for type 2 diabetes. Hepatic lipase (HL) is an enzyme that regulates the metabolism of LDL, intermediate-density lipoprotein, and HDL particles (4). High HL activity is associated with low HDL-C level, and HL converts large, triglyceride-rich HDL 2 -C into small, dense HDL 3 -C. HL also catalyzes the hydrolysis of triglyceride and phospholipids in large, buoyant LDL to form more atherogenic small, dense LDL particles. Obesity, and particularly visceral obesity, has been found to be related to high HL activity (4, 5). Consequently, weight loss leading to a reduction of intraabdominal fat results in a decrease of HL activity and an improvement in lipid profile (6, 7). The HL gene (LIPC), located on chromosome 15q21, comprises nine exons and encodes a protein of 449 amino acids. Four different promoter polymorphisms of the LIPC gene have been identified (G-250A, C-514T, T-710C, and A-763G), which are in complete linkage disequilibrium (8, 9). The G-250A substitution in the promoter region of the LIPC gene has been shown to regulate insulin sensitivity (10), but its effect on the risk of type 2 diabetes has not been previously studied. Therefore, we investigated whether the G-250A promoter polymorphism of the LIPC gene predicts the progression from impaired 2019

2020 J Clin Endocrinol Metab, May 2004, 89(5):2019 2023 Todorova et al. Hepatic Lipase and Type 2 Diabetes glucose tolerance (IGT) to diabetes in subjects participating in the Finnish Diabetes Prevention Study (DPS). Subjects and Methods Study population The design of the Finnish DPS and the baseline characteristics of the IGT subjects have been described in details elsewhere (11 13). Briefly, this is a multicenter, longitudinal study performed in five centers in Finland. The aim of the study was to investigate the effect of lifestyle modification on the incidence of diabetes. Altogether, 522 subjects with IGT (defined as fasting plasma glucose concentration 7.8 mmol/liter and 2-h plasma glucose in the range of 7.8 11.0 mmol/liter) were randomly assigned to either the intervention or control group. Their body mass index (BMI) had to be 25 kg/m 2 or greater, and age at baseline had to be between 40 and 65 yr. The mean age was 55 7 yr, and the mean BMI was 31 5 kg/m 2. Criteria for exclusion were the previous diagnosis of diabetes mellitus other than gestational diabetes, any chronic disease making a 6-yr survival improbable or other unbalanced clinical conditions that could interfere with the study, regular vigorous exercise programs, and dietary management and drug treatment that would affect blood glucose level. The subjects in the intervention group were given detailed advice about how to achieve the goals of the intervention, which were a reduction of weight of 5% or more and a reduction in total intake of fat to less than 30% of energy consumed and in intake of saturated fat to less than 10% of energy consumed, an increase in fiber intake to at least 15 g/1000 kcal, and moderate exercise for at least 30 min/d. Every 3 months, 3-d food diaries were completed. Weight was measured at every visit. The subjects in the control group received general oral and written information about diet, physical activity, and weight reduction; additional routine advice was given to them at every annual follow-up visit. Self-reported changes in dietary and exercise habits during the first year of the intervention were significantly greater in the intervention group compared with the control group (decreased consumption of fat 87 vs. 70%, P 0.001; increased consumption of vegetables 72 vs. 62%, P 0.001; decreased consumption of sugar 55 vs. 40%, P 0.001; and increased exercise 36 vs. 16%, P 0.001, respectively) (13). The diagnosis of diabetes was based on the criteria accepted by the World Health Organization in 1985 (14). A new diagnosis of diabetes had to be confirmed by a subsequent oral glucose tolerance tests. All participants provided written informed consent. The Ethics Committee of the National Public Health Institute in Helsinki, Finland, approved the study protocol. Study procedures Medical examinations were done on a yearly basis. In this report, we used measurements at baseline and at 3 yr. Waist circumference was measured midway between the lowest rib and iliac crest, and hip circumference was measured over the great trochanters (both with 0.5-cm precision in standing position). Serum insulin was determined with RIA (Pharmacia, Uppsala, Sweden). Plasma glucose measurements were performed locally and standardized by the central laboratory in Helsinki. Serum lipids were measured by an enzymatic assay. DNA analysis Genotyping at position 250 of the LIPC gene was performed with PCR amplification. The following primers were used: forward, 5 - CCTACCCCGACCTTTGGCAG-3 ; and reverse, 5 -GGGGTCCAG- GCTTTCTTGG-3 (15). DraI enzyme was used for digestion of the amplified product, and electrophoresis was performed on 9% PAGE gel. Statistical analysis We used the SPSS for Windows program (version 10.0; SPSS Inc., Chicago, IL). Data are presented as mean sd. To compare the influence of the G-250A promoter polymorphism of the LIPC gene on continuous variables between the two groups, the t test for independent samples was used. The 2 test was used to compare categorical variables. For the subjects who were diagnosed as diabetic before the 3-yr examination, weight and waist values measured at the visit when diabetes was established were used in statistical analyses. Normality of distribution of variables was tested with the Kolmogorov-Smirnov test, and variables not normally distributed were log transformed for statistical analyses. Logistic regression analysis was performed to evaluate the influence of the genotype and other variables on the conversion from IGT to diabetes. In the logistic regression analysis, the genotypes were coded as follows: G-250A 0, A-250A 0, and G-250G 1; and the study groups were coded as follows: intervention group 0 and control group 1. A P 0.05 was considered statistically significant. Results DNA was available from 490 subjects (161 men and 329 women). The G-250G genotype was found in 274 subjects (55.9%), the G-250A genotype was found in 180 subjects (36.7%), and the A-250A genotype was found in 36 subjects (7.4%). In the intervention group, the G-250G genotype was found in 144 subjects (58.1%), and the 250A allele was found in 104 subjects (41.9%). In the control group, the G-250G genotype was observed in 130 subjects (53.7%), and the 250A allele was observed in 112 subjects (46.3%). The genotype distribution was in Hardy-Weinberg equilibrium. Because the frequency of the A-250A promoter variant was low, we combined it with the G-250A genotype in all statistical analyses. Subjects with and without the 250A allele did not differ with respect to gender, age, BMI, waist to hip ratio, the levels of fasting and 2-h plasma glucose and serum insulin, total cholesterol, HDL-C, and total triglycerides in the entire study population (Table 1) or within the intervention group and control group (data not shown). Of the variables listed in Table 1, only HDL-C changed significantly more in subjects with the 250A allele than in subjects with the G-250G genotype between baseline and 3 yr (G-250G genotype: HDL-C at the 3-yr examination, 1.30 0.32 mmol/liter; and the 250A allele: 1.39 0.32 mmol/liter; P 0.041 for change between the genotypes). Altogether, 69 genotyped subjects developed type 2 diabetes during the 3-yr follow-up period (six men and 13 women in the intervention group, and 21 men and 29 women in the control group). In the entire subset of the genotyped subjects, the conversion rate to type 2 diabetes was 17.8% among subjects with the G-250G genotype and 10.7% among subjects with the 250A allele (P 0.032). In the control group, 23.0% of the subjects with the G-250G genotype and 19.4% of the subjects with the 250A allele converted to TABLE 1. Baseline characteristics of the study subjects according to the G-250A promoter polymorphism of the LIPC gene G-250G (n 274) G-250A/A-250A (n 216) Gender (men/women) 84/190 77/139 Age (yr) 54.8 7.4 55.9 6.6 BMI (kg/m 2 ) 31.4 4.8 31.0 4.3 Waist circumference (cm) 101.6 10.6 101.1 11.4 Waist to hip ratio 0.92 0.07 0.92 0.08 Fasting plasma glucose (mmol/liter) 6.2 0.7 6.1 0.8 2-h plasma glucose (mmol/liter) 9.0 1.5 8.8 1.5 Fasting insulin (pmol/liter) 90 45 88 44 2-h insulin (pmol/liter) 589 428 550 335 Total cholesterol (mmol/liter) 5.60 0.94 5.62 0.89 HDL-C (mmol/liter) 1.19 0.31 1.24 0.27 Total triglycerides (mmol/liter) 1.71 0.77 1.77 0.77 Results are given as mean SD. None of the comparisons was statistically significant.

Todorova et al. Hepatic Lipase and Type 2 Diabetes J Clin Endocrinol Metab, May 2004, 89(5):2019 2023 2021 diabetes (P 0.507). In the intervention group, 13.0% of the subjects with the G-250G genotype and 1.0% of the subjects with the 250A allele converted to diabetes (P 0.001; Fig. 1, lower panel). The decrease in the incidence of diabetes was closely associated with weight loss (Fig. 1, upper panel). Subjects with the 250A allele were able to lose more weight than subjects with the G-250G genotype, although the differences between the genotype groups in the control and intervention groups were not statistically significant. We also performed statistical analyses separately for men and women (Fig. 2). Control men with the G-250G genotype had a higher incidence of diabetes than control women with the G-250G genotype (37 vs. 18%, P 0.019; Fig. 2, lower panel). No difference between the genders in the incidence of diabetes with respect to other genotypes was observed in the control group or in the intervention group. Univariate logistic regression analysis showed that subjects with the G-250G genotype had a 1.8 higher risk for the development of diabetes compared with subjects with the 250A allele [95% confidence interval (CI), 1.05 3.10; P 0.034; Table FIG. 2. Three-year weight change (%, upper panel) and the incidence of diabetes (%, lower panel) according to the G-250A polymorphism of the LIPC gene in the control group and in the intervention group by gender (men, black bars; women, white bars). FIG. 1. Three-year weight change (%, upper panel) and the incidence of diabetes (%, lower panel) according to the G-250A polymorphism of the LIPC gene in the control group and in the intervention group. 2]. When the study group, gender, weight at baseline, and weight change were added into the model, the G-250G genotype still predicted the conversion to type 2 diabetes (odds ratio, 1.86; 95% CI, 1.04 3.34; P 0.037). The results remained essentially similar if weight at baseline and weight change were replaced by waist circumference at baseline and waist circumference change during the follow-up (odds ration, 2.89; 95% CI, 0.99 8.46; P 0.053). The interaction term between the genotype and the study group (genotype study group) was statistically significant (P 0.024). All these analyses were also performed separately for men and women. In men, the subjects with the G-250G genotype had a 2.5-fold higher risk for the development of diabetes compared with subjects with the 250A allele (95% CI, 0.97 6.47; P 0.057), whereas in women, the risk was 1.58 (95% CI, 0.75 3.33; P 0.225; model included the following variables: genotype, study group, weight at baseline, and weight change). Thus, no substantial difference in the risk of diabetes was observed between the genders, although the risk was somewhat higher among men. There were 102 women (20.6% of all female DPS study participants) taking

2022 J Clin Endocrinol Metab, May 2004, 89(5):2019 2023 Todorova et al. Hepatic Lipase and Type 2 Diabetes TABLE 2. G-250A promoter polymorphism of the LIPC gene as a predictor for the development of type 2 diabetes (univariate and multivariate logistic regression analysis) Regression coefficient estrogens with or without progestin. The use of estrogens and/or progestins did not modify the risk of diabetes (data not shown). P Odds ratio 95% CI Model 1 Genotype 0.589 0.034 1.80 1.05 3.10 Model 2 Genotype 0.67 0.020 1.95 1.12 3.39 Study group 1.17 0.001 3.23 1.83 5.70 Model 3 Genotype 0.61 0.040 1.84 1.03 3.30 Study group 0.96 0.002 2.60 1.42 4.76 Weight at baseline 0.03 0.001 1.04 1.02 1.05 Weight change 0.10 0.001 1.11 1.05 1.16 Model 4 Genotype 0.62 0.037 1.86 1.04 3.34 Study group 0.96 0.002 2.60 1.42 4.77 Gender 0.14 0.630 0.87 0.49 1.55 Weight at baseline 0.03 0.001 1.03 1.02 1.05 Weight change 0.10 0.001 1.11 1.05 1.16 Model 5 Genotype 1.06 0.053 2.89 0.99 8.46 Study group 1.12 0.038 3.06 1.06 8.82 Gender 0.42 0.394 0.66 0.25 1.73 Waist at baseline 0.077 0.001 1.08 1.04 1.13 Waist change 0.073 0.080 1.08 0.99 1.17 CI, Confidence interval. Study groups were coded as 0 intervention group and 1 control group, and genotypes were coded as 0 G-250A, 0 A-250A, 1 G-250G. Weight at baseline was measured in kilograms, and waist circumference was measured in centimeters. Weight change was calculated as [weight (kg) at 3 yr weight (kg) at baseline]/[weight (kg) at baseline] 100%; waist circumference change was calculated as [waist (cm) at 3 years waist (cm) at baseline]/[waist (cm) at baseline] 100%. Discussion This is the first study to demonstrate that a gene regulating lipoprotein metabolism, specifically the G-250A promoter polymorphism of the LIPC gene, predicts the progression from IGT to diabetes. The G-250G genotype was associated with an approximately 2-fold higher risk of developing type 2 diabetes than the 250A allele, independent of study group, gender, weight at baseline, and weight change. Dyslipidemia associated with insulin resistance includes low HDL-C, high triglycerides, small and dense LDL, and postprandial lipemia. Previous population-based observational studies have shown that these lipid and lipoprotein abnormalities are predictors of type 2 diabetes (2, 3). HL plays a central role in dyslipidemia of insulin resistance. Zambon et al. (15) have shown that the G3A substitution at the 250 locus of the LIPC gene leads to decreased HL activity and, therefore, to buoyant LDL particles and high HDL-C. There are also other studies suggesting that the rare haplotype correlates with lower HL activity compared with the common haplotype (16). Obesity, and particularly upper body obesity, is associated with dyslipidemia as well as with high HL level (4). In vitro analysis has shown that the C514T substitution, which is in complete linkage dysequilibrium with the G-250A variant, reduces transcriptional activity of the LIPC promoter (17). Intraabdominal fat leads to high HL activity, and the maximum activity level of HL is 58% lower in the presence of the 250A allele than in the presence of the G-250G genotype (18). Both intraabdominal fat and the 250A allele are independently associated with HL activity when included in the same model, and the effect of intraabdominal fat on HDL-C level is mediated by HL (19). In previous studies and also in our study, HDL-C at baseline tended to be higher in subjects with the 250A allele than in subjects with the G-250G genotype, and the change in HDL-C level from baseline to 3 yr was greater in subjects with the 250A allele than in subjects with the G-250G genotype (P 0.041). Modification of lifestyle leads to reduction of body weight, decrease in HL activity, increase in HDL-C, and insulin sensitivity (6, 7). The most marked weight decrease ( 4%) in our study was observed in subjects with the 250A allele in the intervention group. Subjects with the G-250G genotype in the control group had the least reduction in weight ( 0.5%), which was significantly less (P 0.001) than in subjects with the 250A allele in the intervention group. This resulted in a 23% incidence of diabetes among subjects with the G-250G genotype in the control group, whereas only one subject (1%) with the 250A allele in the intervention group developed diabetes (Fig. 1). In the control group, weight reduction was closely associated with the decrease in the incidence of diabetes, whereas in the intervention group, the decrease in the incidence of diabetes was even greater (13 vs. 1%, P 0.001) between the subjects with the G-250G genotype and the 250A allele than could be expected on the basis of weight loss alone. Therefore, other changes in lifestyle in addition to weight loss, such as composition of diet (20) or increased physical activity, or other factors independent of lifestyle changes, could have contributed to the highly beneficial protective effect in subjects having the 250A allele. Gender has a significant influence on HL activity. Compared with women, men have about 30% higher HL activity, independently of the genotype, and lower HDL-C (21). Intraabdominal fat accounts for only a portion of this gender difference. Also in our study, HDL-C was lower in men than in women in each genotype group (data not shown). Furthermore, the conversion to diabetes tended to be higher among male subjects (17.3%) than among female subjects (13.4%), and in the control group, the difference in the incidence of diabetes between men and women was statistically significant among subjects with the G-250G genotype (37 vs. 18%, P 0.019). This difference is partly explained by a greater weight loss among women than among men with the G-250G genotype, but high absolute HL level could also contribute to high incidence of diabetes among men with the G-250G genotype. Moreover, none of the women with the 250A allele in the intervention group developed diabetes during the 3-yr follow-up. Estrogens have been found to decrease postheparin HL activity (22, 23). Estrogen levels are positively associated with HDL-C levels, and premenopausal women have lower HL activity compared with postmenopausal women. In our study, 102 women (20.8% from the study population) had hormone replacement therapy. However, we did not find a significant influence of the hormone replacement therapy on the conversion rate from IGT to type 2 diabetes among women.

Todorova et al. Hepatic Lipase and Type 2 Diabetes J Clin Endocrinol Metab, May 2004, 89(5):2019 2023 2023 Two mechanisms can be considered to explain why the G-250A polymorphism of the LIPC gene regulates the conversion from IGT to diabetes. First, subjects with the G-250G genotype could be more prone to accumulate visceral fat, which could lead to high HL levels and dyslipidemia and, finally, to frank hyperglycemia. BMI, waist circumference, and waist to hip ratio did not differ at baseline between the groups (Table 1), but subjects with the G-250G genotype tended to loose less weight (and probably also less intraabdominal fat) than subjects with the 250A allele both in the control group and in the intervention group. However, the G-250G genotype of the LIPC gene predicted the conversion to diabetes independently of weight at baseline and weight change, as well as independently of waist circumference and its change during the follow-up. Therefore, it is not very likely that differences in the amount of visceral fat per se could explain differences in the incidence of diabetes between the subjects with the G-250G genotype and the 250A allele. Second, the driving force for the high incidence of diabetes could have been high HL activity itself. Although we did not measure HL activity, indirect evidence can be extrapolated from changes in HDL-C concentration. During the 3-yr follow-up, HDL-C level increased significantly more among subjects with the 250A allele than among subjects with the G-250G genotype. Therefore, a hypothesis could be presented that high HL activity itself, due to the G-250G genotype and independent of visceral obesity, is determining the conversion from IGT to diabetes. In summary, our findings show that the G-250G genotype of the LIPC gene is associated with the risk of diabetes among subjects with IGT. These results imply that genes regulating characteristic lipid and lipoprotein abnormalities related to insulin resistance (low HDL cholesterol, high triglycerides, and small dense LDL) are also potential candidate genes for type 2 diabetes. However, our study population included only Finnish and high-risk individuals with respect to the conversion to diabetes. Therefore, our findings should be replicated in other populations and in subjects who are at lower risk of developing type 2 diabetes. Acknowledgments Received July 31, 2003. Accepted January 8, 2004. Address all correspondence and requests for reprints to: Markku Laakso, M.D., Department of Medicine, University of Kuopio, 70210 Kuopio, Finland. E-mail: markku.laakso@kuh.fi. 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