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Relationships between polymorphisms of the human serum paraoxonase gene and insulin sensitivity in Japanese patients with Type 2 diabetes
Diabetes Research and Clinical Practice 60 (2003) 79 /85 www.elsevier.com/locate/diabres
Relationships between polymorphisms of the human serum paraoxonase gene and insulin sensitivity in Japanese patients with Type 2 diabetes Yukio Ikeda *, Tadashi Suehiro, Fumiaki Ohsaki, Kaoru Arii, Yoshitaka Kumon, Kozo Hashimoto Second Department of Internal Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan Received 26 August 2002; received in revised form 25 November 2002; accepted 4 December 2002
Abstract Human serum paraoxonase (PON1), which is associated with HDL, is an esterase and has been shown to reduce the susceptibility of LDL to lipid peroxidation. The objective of the study was to determine whether genetic polymorphisms of the PON1 gene are associated with insulin sensitivity. Forty-eight Japanese patients with type 2 diabetes were recruited, and euglycemic hyperinsulinemic clamp was performed to assess insulin sensitivity. The PON1 promoter polymorphism C(/108)T was determined by direct sequencing, and the coding region polymorphism Q192R was determined by polymerase chain reaction and digestion of the amplified fragments. No association was observed between the Q192R polymorphism and the glucose infusion rate (GIR), whereas GIR increased with the following order of genotypes: /108TT B//108CT B/ and /108CC (4.29/1.6, 5.19/2.5, and 6.99/2.5 mg kg 1 min 1, respectively; P B/0.02, ANCOVA). Stepwise regression analysis revealed that the C(/108)T polymorphism significantly contributed to the GIR. It has been reported that oxidative stress attenuates insulin signaling in vitro. The PON1 promoter polymorphism C(/108)T may influence insulin sensitivity by modulating serum antioxidant capacity. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human serum paraoxonase; PON1; Genetic polymorphism; Insulin sensitivity
1. Introduction Human serum paraoxonase (PON1) is associated with apolipoprotein (apo) A-I and apo J in high-density lipoprotein (HDL) [1], and has
* Corresponding author. Tel.: /81-88-880-2343; fax: /8188-880-2344. E-mail address: [email protected] (Y. Ikeda).
been shown to reduce the susceptibility of lowdensity lipoprotein (LDL) to lipid peroxidation [2]. PON1 has two polymorphic sites in the coding region: Leu-Met (L/M) at position 55 of the amino acid sequence, and Gln-Arg (Q/R) at position 192, both of which are involved in serum paraoxonase activity [3,4]. Paraoxonase activity in subjects with the RR genotype is higher than in those with the QQ genotype; such activity in people with the LL
0168-8227/02/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-8227(02)00280-2
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genotype is higher than in those with MM. We identified the C(/108)T polymorphism in the promoter region of the PON1 gene in the Japanese population, and found that this polymorphism affects the transcription and serum concentration of PON1 [5]. Recently, Leviev et al. reported that this PON1 promoter polymorphism is associated with increased serum glucose concentrations in non-diabetic patients [6]. Since plasma glucose concentration is disturbed by insulin resistance in insulin-sensitive tissues and/or by b cell dysfunction, the reported association may suggest that PON1 promoter polymorphism C(/108)T affect insulin sensitivity or b cell function by modulating serum anti-oxidant capacity. We therefore investigated the relationship between the PON1 polymorphism and insulin sensitivity, as determined by a euglycemic hyperinsulinemic clamp in subjects with glucose intolerance.
2. Materials and methods Forty-eight type 2 diabetic patients (30 men and 18 women, mean age9/S.D.; 509/15 years) were recruited among outpatients and inpatients seen at our department. The diagnosis of diabetes mellitus was made according to the criteria set by the World Health Organization (WHO) [7]. Subjects receiving insulin therapy or drugs known to affect insulin sensitivity (e.g. thiazolidine derivates or biguanides), and subjects with cardiovascular, kidney, or liver disease were excluded from the study. All subjects were Japanese and resided in the same area (Kochi prefecture, Japan), and gave their informed consent to participate in the study. All subjects were admitted to the hospital at 08:00 h after a 12-h fast. Twenty of 48 patients were under treatment with sulfonylureas (Glibenclamide or Gliclazide). In those patients, sulfonylureas were taken until the day before the clamp study began. The euglycemic hyperinsulinemic clamp [8] was performed using an artificial pancreas (model STG-22, Nikkiso, Tokyo, Japan). Hyperinsulinemia was established by 15 min of priming with an insulin infusion (Humulin R; Eli Lilly, Indianapolis, IN) and was maintained by constant infusion (1.12 mU kg1 min 1) via a
cubital vein. Blood glucose levels were determined every 5 min during the 120-min clamp study and were maintained at 90 mg dl 1 by a variable-rate infusion of 20% glucose into another cubital vein. The mean glucose infusion rate (GIR) during the last 30-min of the clamping procedure was used as an overall measure of insulin effects on glucose metabolism. Blood samples were obtained before the initiation of the hyperinsulinemic clamping procedure. Genomic DNA was extracted from whole blood using a commercial kit (SMI test; Sumitomo, Tokyo, Japan). All serum and plasma samples were stored at /80 8C and genomic DNA was stored at 4 8C. The PON1 promoter polymorphism C(/108)T was determined by a cycle sequencing method. The DNA fragments were amplified by a polymerase chain reaction (PCR) method using a sense primer (5?-TGGACTAGGCACCTATTCTC-3?) and an antisense primer (5?-GACTGGTGGTTCCTGAAGAG-3?). A PCR fragment separated by electrophoresis in agarose gel was recovered and purified by a commercially available kit (QIAquick PCR Gel Extraction Kit, QIAGEN). The sequence of the PCR fragment was detected using a commercial kit and analyzer (Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit and ABI PRISMTM 310 Genetic Analyzer; PE Applied Biosystems, Foster City, CA), and the same sense or antisense primer of the PCR was used as the sequencing primer. For analysis of the 192Q/R polymorphism, a PCR and digestion of the amplified fragments with Alw I was performed as described previously [4]. Images of the intra-abdominal visceral fat area (VFA) and subcutaneous area (SFA) were obtained by computed tomography at the level of the umbilicus. Values were calculated using imaging software (NIH IMAGE, version 3.1; Wayne Rashand, National Institute of Health, Bethesda, MD). All data are presented as the mean9/S.D. A comparison of variables between two groups was performed using Student’s unpaired t-test. The effect of PON1 genotype on the GIR was estimated by analysis of co-variance (ANCOVA) using genotype as a factor, and age and sex as
Y. Ikeda et al. / Diabetes Research and Clinical Practice 60 (2003) 79 /85
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covariates. Multiple regression analysis with the GIR as the dependent variable was conducted using stepwise forward selection method. The F value for the inclusion and exclusion of variables was set at 4.0. These statistical analyses were performed using STAT VIEW version 5.0 (SAS Institute Inc., Cary, NC) on a personal computer. Statistical significance was defined as P B/0.05.
3. Results The clinical characteristics of the subjects are shown in Table 1. The mean glucose infusion rate (GIR) was 5.59/2.5 mg kg 1 min 1 in the patients, which was significantly decreased, compared with that of sex- and age-matched subjects with normal glucose tolerance (NGT) (8.19/3.2 mg kg1 min1). The frequencies of the Q and R alleles of the Q192R polymorphism were 0.33 and 0.67, respectively, and those of the C and T alleles of the C(/108)T polymorphism were 0.55 and 0.45, respectively. Fig. 1 shows the relationship between the PON1 polymorphisms and the GIR. No association was observed between the Q192R polymorphism and the GIR, whereas the GIR Table 1 Clinical characteristics of patients with type 2 diabetes Characteristics Sex (men/women) Age (years) BMI (kg m 2) FPG (mg dl 1) Fasting IRI (mU ml 1) HbA1C (%) Total cholesterol (mg dl 1) Triglycerides (mg dl 1) Free fatty acid (mEq l 1) HDL-cholesterol LDL-cholesterol Visceral fat area (cm2) Subcutaneous fat area (cm2) Therapy (n ) Diet alone Sulfonylureas
Values 30/18 509/15 25.89/5.5 1289/46 8.69/6.6 7.89/2.2 2039/39 1459/108 0.569/0.24 509/16 1249/38 1139/60 1589/124 28 20
BMI: body mass index, FPG: fasting plasma glucose, IRI: immunoreactive insulin.
Fig. 1. Comparison of the mean (GIR) by coding region Q192R (right) or promoter C(/108)T (left) polymorphisms of the PON1 gene in 48 patients with type 2 diabetes. Values are presented in means9/S.E. Significance of differences in mean GIR values among genotypes was assessed by ANCOVA (using sex and age as covariates), followed by Fisher’s protected least significant difference (PLSD) as a post hoc test. *, P /0.01 vs. TT genotype. $, P B/0.05 vs. CT genotype.
increased in the following genotype order: /108TT B//108CT B/ and /108CC (P B/0.02, ANCOVA). The mean GIR in subjects with CC genotype was significantly higher than in those with TT or CT genotype (P /0.01 and B/0.05, respectively, Fisher’s protected least significant difference). There was also a significant difference in GIR when compared between CC genotype and TT/CT genotypes (mean9/S.D., 4.889/2.30 vs. 6.929/2.49, P B/0.01, unpaired t-test). There were no significant relationships detected between these PON1 polymorphisms and FPG, fasting immunoreactive insulin (F-IRI), serum lipids concentrations, BMI, SFA, or VFA. Table 2 shows the stepwise regression analysis for the GIR. In model 1, the C(/108)T polymorphism was a significant contributor, as well as BMI and HbA1C. Model 2, which included VFA and SFA instead of the BMI as independent variables, also revealed that the C(/108)T polymorphism was a significant contributor to the GIR, together with F-IRI and VFA, both of which are known to be associated with insulin resistance. We also performed the stepwise regression analysis for FPG using the same independent variables including PON1 polymorphisms, but only HbA1C was a significant variable for FPG.
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Table 2 Stepwise regression analysis for glucose infusion rate in patients with type 2 diabetes Variables
Model 1 (n/48) F
b BMI PON1 C(/108)T HbA1c Fasting IRI Age Free fatty acid PON1 Q192R Triglycerides FPG LDL-cholesterol Sex HDL-cholesterol Visceral fat area Subcutaneous Fat area Total R2
Model 2 (n/45)
/0.502 0.383 /0.340
F
b 19.850 11.732 9.112 3.661 3.054 2.365 2.249 2.113 0.943 0.137 0.127 0.115
0.337 /0.320
/0.300 0.501
P B/0.0001
0.428
7.017 3.493 5.775 2.887 3.341 0.581 0.402 0.095 0.620 1.191 0.025 4.845 0.632 P B/0.0001
A stepwise multivariate regression analysis was performed. The F values for the inclusion and exclusion of variables was set at 4.0 at each step. Sex: men /1, women/2; PON1 Q192R: QQ/1, QR/2, RR /3; PON1 C(/108)T: TT/1, CT/2, CC/3. b : partial regression coefficient.
The mean insulin resistance index (IR) assessed by homeostasis model assessment (HOMA) [9] decreased in the following genotype order: /108TT //108CT / and /108CC, although the differences were not significant because of large deviations (mean HOMA-IR9/S.D.; 3.349/ 3.85, 2.539/2.05, and 2.199/1.73, respectively). In a stepwise regression analysis for HOMA-IR using the same independent variables as used in that for GIR (total R2 /0.909, P B/0.0001), the C(/108)T polymorphism was identified as a significant contributor (b//0.104, F /4.517), together with F-IRI (b/0.953, F /329.823), FPG (b/ 0.368, F /56.059), and BMI (b /0.116, F / 4.939).
4. Discussion In this study, we demonstrated that the PON1 promoter polymorphism C(/108)T, but not the coding region polymorphism Q192R, was associated with insulin sensitivity in Japanese patients with type 2 diabetes. The multiple regression
analysis confirmed that the C(/108)T polymorphism was a significant contributor to the GIR, as were other variables known to be related to insulin resistance such as F-IRI, VFA, and BMI. This is the first report suggesting that the PON1 gene polymorphism is an independent factor affecting insulin sensitivity in diabetic patients. No significant difference between the genotypes was observed as regards FPG; this finding contradicted results reported by Leviev et al. [6]. This difference might have been due to differences between study populations (diabetic vs. non-diabetic), or to the influence of sulfonylureas, used for the treatment of our population. Although it remains unclear how this type of polymorphism may affect insulin sensitivity, several possible explanations appear likely. First, by affecting body fat accumulation, the C(/108)T polymorphism may be involved in the development of insulin resistance. However, this polymorphism was not associated with BMI or VFA, suggesting that its effects on insulin sensitivity are not mediated by an influence on body fat mass or composition. Second, the PON1 promoter poly-
Y. Ikeda et al. / Diabetes Research and Clinical Practice 60 (2003) 79 /85
morphism may be in linkage disequilibrium with other genes related to insulin resistance that are also involved in the development of diabetes; this explanation is derived from the results of a genomic scan of Pima Indians [10], which provided evidence for diabetes-susceptibility loci on chromosome 7q, where the gene encoding PON1 is found. However, a genome-wide scan for type 2 diabetes in a Japanese population [11] failed to identify candidate loci on chromosome 7q. In addition, our previous study revealed no significant difference in the prevalence of the C(/108)T polymorphism between subjects with NGT and type 2 diabetic patients [12]. These findings suggest that this polymorphism is not causally related to the development of type 2 diabetes, at least not in the Japanese population. Additionally, it has been shown that oxidative stress is increased in vivo in the diabetic state [13,14], and that such stress can also impair insulin action in vitro [15,16]. It has also been reported that plasma concentrations of partially oxidized LDL were positively correlated to steady-state plasma glucose (SSPG) concentrations [17]. The C(/108)T polymorphism is associated with the transcription and serum concentration of PON1, and the concentration in the subjects with the / 108CC genotype is higher than that with the / 108TT genotype [5]. Among our patients, the GIR increased in the following order, namely, from those with the low- to high-expressor genotype (/ 108TT B//108CT B//108CC). Higher concentrations of circulating PON1 may more strongly prevent oxidative modification in vivo. Therefore, the high-expressor C allele might efficiently prevent the impairment of insulin action caused by oxidative stress in people with diabetes. The difference in the preventive effect associated with the C(/108)T polymorphism might be enhanced in patients suffering from oxidative stress (e.g. diabetic patients). The Q192R polymorphism is closely associated with serum paraoxonase activity [3,4], and the activity increases in the order of the QQ, QR, and RR genotypes. However, Mackness et al. showed in vitro that HDL with the RR genotype, which has high serum paraoxonase activity, had less ability to protect LDL oxidation with time com-
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pared with that of the QR or QQ genotype [18]. Serum paraoxonase activity reveals an enzymatic function with the use of paraoxon, which is never detected in the human body, as a substrate. The actual in vivo substrate of PON1 is not yet known, and the site of PON1 against the oxidization of LDL has not yet been established. However, the cystein at 284 of the amino acid sequence has been highly suspected to be an active site for antioxidization [19], which appears to be different from the enzyme-active site [20]. Therefore, the C(/108)T polymorphism, which is involved in serum PON1 concentration, may be more important in the protection from oxidative stress rather than the Q192R polymorphism. Serum paraoxonase concentrations or activity can also predict susceptibility to insulin resistance. However, we and other investigators have shown that serum paraoxonase activity was decreased in diabetic patients, and was further decreased in those with diabetic complications, independent of the PON1 gene polymorphism [21,22]. We have also shown that not only enzyme activity, but also enzyme concentrations were lower in uremic patients, who often suffer severe oxidative stress [23,24]. It has been shown that treatment of cultured HepG2 cells with mildly oxidized LDL or oxidized phospholipids decreased PON1 mRNA levels, and that injection of oxidized phospholipids into C57BL/6J mice resulted in a marked reduction in serum paraoxonase activity [25,26]. Yamada et al. reported that paraoxonase activity unexpectedly increased in non-diabetic subjects with insulin resistance, as assessed by HOMA [27]. Thus, serum enzyme activity or the concentration of PON1 can be altered secondarily to various diseases or pathological conditions, regardless of insulin resistance. In fact, we measured serum PON1 concentrations in 36 patients of this study subjects, using a competitive enzyme immunoassay [12], and found no significant association between PON1 concentrations and GIR in a stepwise regression analysis (data not shown). Therefore, in subjects with diabetes or glucose intolerance, the genetic polymorphism C(/108)T could be a better predictor of susceptibility to insulin resistance than either PON1 enzyme concentration or activity.
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In conclusion, our data suggests that the PON1 gene polymorphism in the promoter region exerts an influence on insulin sensitivity in diabetic patients who might be particularly susceptible to oxidative stress. However, the number of subjects participating in this study was too small to arrive at definitive conclusions. Therefore, further study is required to establish a link between the PON1 polymorphism and the development of insulin resistance.
References [1] M.-C. Blatter, R.W. James, S. Messmer, F. Barja, D. Pometta, Identification of a distinct human high-density lipoprotein subspecies defined by a lipoprotein- associated protein, K-45. Identity of K-45 with paraoxonase, Eur. J. Biochem. 211 (1993) 871 /879. [2] M.I. Mackness, S. Arrol, C. Abbott, P.N. Durrington, Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase, Atherosclerosis 104 (1993) 129 /135. [3] S. Adkins, K.N. Gan, M. Mody, B.N. La Du, Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes, Am. J. Hum. Genet. 52 (1993) 598 /608. [4] R. Humbert, D.A. Adler, C.M. Disteche, C. Hassett, C.J. Omiecinski, C.E. Furlong, The molecular basis of the human serum paraoxonase activity polymorphism, Nat. Genet. 3 (1993) 73 /76. [5] T. Suehiro, T. Nakamura, M. Inoue, et al., A polymorphism upstream from the human paraoxonase (PON1) gene and its association with PON1 expression, Atherosclerosis 150 (2000) 295 /298. [6] I. Leviev, B. Kalix, M.-C. Brulhart Meynet, R.W. James, The paraoxonase PON1 promoter polymorphism C(/ 107)T is associated with increased serum glucose concentrations in non-diabetic patients, Diabetologia 44 (2001) 1177 /1183. [7] World Health Organization, Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications. Part 1: Diagnosis and Classification of diabetes mellitus. Report of a WHO Consultation, Geneva, World Health Organization, 1999. [8] R.A. DeFronzo, J.D. Tobin, R. Andres, Glucose clamp technique: a method for quantifying insulin secretion and resistance, Am. J. Physiol. 237 (1979) E214 /E223. [9] D.R. Matthews, J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F. Treacher, R.C. Turner, Homeostasis model assessment: insulin resistance and b-cell function from fasting plasma glucose and insulin concentrations in man, Diabetologia 28 (1985) 412 /419.
[10] R.L. Hanson, M.G. Ehm, D.J. Pettitt, et al., An autosomal genomic scan for loci linked to type II diabetes mellitus and body-mass index in Pima Indians, Am. J. Hum. Genet. 63 (1998) 1130 /1138. [11] Y. Mori, S. Otabe, C. Dina, et al., Genome-wide search for type 2 diabetes in Japanese affected sib-pairs confirms susceptibility genes on 3q, 15q, and 20q and identifies two new candidate loci on 7p and 11p, Diabetes 51 (2002) 1247 /1255. [12] M. Inoue, T. Suehiro, T. Nakamura, Y. Ikeda, Y. Kumon, K. Hashimoto, Serum arylesterase/diazoxonase activity and genetic polymorphisms in patients with type 2 diabetes, Metabolism 49 (2000) 1400 /1405. [13] S.P. Wolff, Z.Y. Jiang, J.V. Hunt, Protein glycation and oxidative stress in diabetes mellitus and aging, Free Radic. Biol. Med. 10 (1991) 339 /352. [14] J. Nourooz-Zadeh, J. Tajaddini-Sarmadi, S. McCarthy, D.J. Betteridge, S.P. Wolff, Elevated levels of authentic plasma hydroperoxides in NIDDM, Diabetes 44 (1995) 1054 /1058. [15] A. Rudich, N. Kozlovsky, R. Potashnik, N. Bashan, Oxidant stress reduces insulin responsiveness in 3T3-L1 adipocytes, Am. J. Physiol. 272 (1997) E935 /E940. [16] A. Tirosh, R. Potashnik, N. Bashan, A. Rudich, Oxidative stress disrupts insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3kinase in 3T3-L1 adipocytes, J. Biol. Chem. 274 (1999) 10595 /10602. [17] M. Carantoni, F. Abbasi, F. Warmerdam, et al., Relationship between insulin resistance and partially oxidized LDL particles in healthy, nondiabetic volunteers, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 762 /767. [18] M.I. Mackness, S. Arrol, B. Mackness, P.N. Durrington, Alloenzymes of paraoxonase and effectiveness of highdensity lipoproteins in protecting low-density lipoprotein against lipid peroxidation, Lancet 349 (1997) 851 /852. [19] M. Aviram, S. Billecke, R. Sorenson, et al., Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 1617 /1624. [20] H. Cao, A. Girard-Globa, F. Berthezene, P. Moulin, Paraoxonase protection of LDL against peroxidation is independent of its esterase activity towards paraoxon and is unaffected by the Q0/R genetic polymorphism, J. Lipid Res. 40 (1999) 133 /139. [21] Y. Ikeda, T. Suehiro, M. Inoue, et al., Serum paraoxonase activity and its relationship to diabetic complications in patients with non-insulin-dependent diabetes mellitus, Matabolism 47 (1998) 598 /602. [22] C.A. Abbott, M.I. Mackness, S. Kumar, A.J. Boulton, P.N. Durrington, Serum paraoxonase activity, concentration, and phenotype distribution in diabetes mellitus and its relationship to serum lipids and lipoproteins, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 1812 /1818.
Y. Ikeda et al. / Diabetes Research and Clinical Practice 60 (2003) 79 /85 [23] T. Itahara, T. Suehiro, Y. Ikeda, et al., Serum paraoxonase and arylesterase activities in hemodialysis patients, J. Atheroscler. Thromb. 7 (2000) 152 /158. [24] T. Suehiro, Y. Ikeda, T. Shiinoki, et al., Serum paraoxonase (PON1) concentration in patients undergoing hemodialysis, J. Atheroscler. Thromb. 9 (2002) 133 /138. [25] M. Navab, S. Hama-Levy, B.J. Van Lenten, et al., Mildly oxidized LDL induces an increased apolipoprotein J/ paraoxonase ratio, J. Clin. Invest. 99 (1997) 2005 /2019.
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[26] B.J. Van Lenten, A.C. Wagner, M. Navab, A.M. Fogelman, Oxidized phospholipids induce changes in hepatic paraoxonase and apoJ but not monocyte chemoattractant protein-1 via interleukin-6, J. Biol. Chem. 276 (2001) 1923 /1929. [27] A. Yamada, T. Shoji, H. Tahara, M. Emoto, Y. Nishizawa, Effect of insulin resistance on serum paraoxonase activity in a nondiabetic population, Metabolism 50 (2001) 805 /811.
Relationships between polymorphisms of the human serum paraoxonase gene and insulin sensitivity in Japanese patients with Type 2 diabetes Yukio Ikeda *, Tadashi Suehiro, Fumiaki Ohsaki, Kaoru Arii, Yoshitaka Kumon, Kozo Hashimoto Second Department of Internal Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan Received 26 August 2002; received in revised form 25 November 2002; accepted 4 December 2002
Abstract Human serum paraoxonase (PON1), which is associated with HDL, is an esterase and has been shown to reduce the susceptibility of LDL to lipid peroxidation. The objective of the study was to determine whether genetic polymorphisms of the PON1 gene are associated with insulin sensitivity. Forty-eight Japanese patients with type 2 diabetes were recruited, and euglycemic hyperinsulinemic clamp was performed to assess insulin sensitivity. The PON1 promoter polymorphism C(/108)T was determined by direct sequencing, and the coding region polymorphism Q192R was determined by polymerase chain reaction and digestion of the amplified fragments. No association was observed between the Q192R polymorphism and the glucose infusion rate (GIR), whereas GIR increased with the following order of genotypes: /108TT B//108CT B/ and /108CC (4.29/1.6, 5.19/2.5, and 6.99/2.5 mg kg 1 min 1, respectively; P B/0.02, ANCOVA). Stepwise regression analysis revealed that the C(/108)T polymorphism significantly contributed to the GIR. It has been reported that oxidative stress attenuates insulin signaling in vitro. The PON1 promoter polymorphism C(/108)T may influence insulin sensitivity by modulating serum antioxidant capacity. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human serum paraoxonase; PON1; Genetic polymorphism; Insulin sensitivity
1. Introduction Human serum paraoxonase (PON1) is associated with apolipoprotein (apo) A-I and apo J in high-density lipoprotein (HDL) [1], and has
* Corresponding author. Tel.: /81-88-880-2343; fax: /8188-880-2344. E-mail address: [email protected] (Y. Ikeda).
been shown to reduce the susceptibility of lowdensity lipoprotein (LDL) to lipid peroxidation [2]. PON1 has two polymorphic sites in the coding region: Leu-Met (L/M) at position 55 of the amino acid sequence, and Gln-Arg (Q/R) at position 192, both of which are involved in serum paraoxonase activity [3,4]. Paraoxonase activity in subjects with the RR genotype is higher than in those with the QQ genotype; such activity in people with the LL
0168-8227/02/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-8227(02)00280-2
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Y. Ikeda et al. / Diabetes Research and Clinical Practice 60 (2003) 79 /85
genotype is higher than in those with MM. We identified the C(/108)T polymorphism in the promoter region of the PON1 gene in the Japanese population, and found that this polymorphism affects the transcription and serum concentration of PON1 [5]. Recently, Leviev et al. reported that this PON1 promoter polymorphism is associated with increased serum glucose concentrations in non-diabetic patients [6]. Since plasma glucose concentration is disturbed by insulin resistance in insulin-sensitive tissues and/or by b cell dysfunction, the reported association may suggest that PON1 promoter polymorphism C(/108)T affect insulin sensitivity or b cell function by modulating serum anti-oxidant capacity. We therefore investigated the relationship between the PON1 polymorphism and insulin sensitivity, as determined by a euglycemic hyperinsulinemic clamp in subjects with glucose intolerance.
2. Materials and methods Forty-eight type 2 diabetic patients (30 men and 18 women, mean age9/S.D.; 509/15 years) were recruited among outpatients and inpatients seen at our department. The diagnosis of diabetes mellitus was made according to the criteria set by the World Health Organization (WHO) [7]. Subjects receiving insulin therapy or drugs known to affect insulin sensitivity (e.g. thiazolidine derivates or biguanides), and subjects with cardiovascular, kidney, or liver disease were excluded from the study. All subjects were Japanese and resided in the same area (Kochi prefecture, Japan), and gave their informed consent to participate in the study. All subjects were admitted to the hospital at 08:00 h after a 12-h fast. Twenty of 48 patients were under treatment with sulfonylureas (Glibenclamide or Gliclazide). In those patients, sulfonylureas were taken until the day before the clamp study began. The euglycemic hyperinsulinemic clamp [8] was performed using an artificial pancreas (model STG-22, Nikkiso, Tokyo, Japan). Hyperinsulinemia was established by 15 min of priming with an insulin infusion (Humulin R; Eli Lilly, Indianapolis, IN) and was maintained by constant infusion (1.12 mU kg1 min 1) via a
cubital vein. Blood glucose levels were determined every 5 min during the 120-min clamp study and were maintained at 90 mg dl 1 by a variable-rate infusion of 20% glucose into another cubital vein. The mean glucose infusion rate (GIR) during the last 30-min of the clamping procedure was used as an overall measure of insulin effects on glucose metabolism. Blood samples were obtained before the initiation of the hyperinsulinemic clamping procedure. Genomic DNA was extracted from whole blood using a commercial kit (SMI test; Sumitomo, Tokyo, Japan). All serum and plasma samples were stored at /80 8C and genomic DNA was stored at 4 8C. The PON1 promoter polymorphism C(/108)T was determined by a cycle sequencing method. The DNA fragments were amplified by a polymerase chain reaction (PCR) method using a sense primer (5?-TGGACTAGGCACCTATTCTC-3?) and an antisense primer (5?-GACTGGTGGTTCCTGAAGAG-3?). A PCR fragment separated by electrophoresis in agarose gel was recovered and purified by a commercially available kit (QIAquick PCR Gel Extraction Kit, QIAGEN). The sequence of the PCR fragment was detected using a commercial kit and analyzer (Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit and ABI PRISMTM 310 Genetic Analyzer; PE Applied Biosystems, Foster City, CA), and the same sense or antisense primer of the PCR was used as the sequencing primer. For analysis of the 192Q/R polymorphism, a PCR and digestion of the amplified fragments with Alw I was performed as described previously [4]. Images of the intra-abdominal visceral fat area (VFA) and subcutaneous area (SFA) were obtained by computed tomography at the level of the umbilicus. Values were calculated using imaging software (NIH IMAGE, version 3.1; Wayne Rashand, National Institute of Health, Bethesda, MD). All data are presented as the mean9/S.D. A comparison of variables between two groups was performed using Student’s unpaired t-test. The effect of PON1 genotype on the GIR was estimated by analysis of co-variance (ANCOVA) using genotype as a factor, and age and sex as
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covariates. Multiple regression analysis with the GIR as the dependent variable was conducted using stepwise forward selection method. The F value for the inclusion and exclusion of variables was set at 4.0. These statistical analyses were performed using STAT VIEW version 5.0 (SAS Institute Inc., Cary, NC) on a personal computer. Statistical significance was defined as P B/0.05.
3. Results The clinical characteristics of the subjects are shown in Table 1. The mean glucose infusion rate (GIR) was 5.59/2.5 mg kg 1 min 1 in the patients, which was significantly decreased, compared with that of sex- and age-matched subjects with normal glucose tolerance (NGT) (8.19/3.2 mg kg1 min1). The frequencies of the Q and R alleles of the Q192R polymorphism were 0.33 and 0.67, respectively, and those of the C and T alleles of the C(/108)T polymorphism were 0.55 and 0.45, respectively. Fig. 1 shows the relationship between the PON1 polymorphisms and the GIR. No association was observed between the Q192R polymorphism and the GIR, whereas the GIR Table 1 Clinical characteristics of patients with type 2 diabetes Characteristics Sex (men/women) Age (years) BMI (kg m 2) FPG (mg dl 1) Fasting IRI (mU ml 1) HbA1C (%) Total cholesterol (mg dl 1) Triglycerides (mg dl 1) Free fatty acid (mEq l 1) HDL-cholesterol LDL-cholesterol Visceral fat area (cm2) Subcutaneous fat area (cm2) Therapy (n ) Diet alone Sulfonylureas
Values 30/18 509/15 25.89/5.5 1289/46 8.69/6.6 7.89/2.2 2039/39 1459/108 0.569/0.24 509/16 1249/38 1139/60 1589/124 28 20
BMI: body mass index, FPG: fasting plasma glucose, IRI: immunoreactive insulin.
Fig. 1. Comparison of the mean (GIR) by coding region Q192R (right) or promoter C(/108)T (left) polymorphisms of the PON1 gene in 48 patients with type 2 diabetes. Values are presented in means9/S.E. Significance of differences in mean GIR values among genotypes was assessed by ANCOVA (using sex and age as covariates), followed by Fisher’s protected least significant difference (PLSD) as a post hoc test. *, P /0.01 vs. TT genotype. $, P B/0.05 vs. CT genotype.
increased in the following genotype order: /108TT B//108CT B/ and /108CC (P B/0.02, ANCOVA). The mean GIR in subjects with CC genotype was significantly higher than in those with TT or CT genotype (P /0.01 and B/0.05, respectively, Fisher’s protected least significant difference). There was also a significant difference in GIR when compared between CC genotype and TT/CT genotypes (mean9/S.D., 4.889/2.30 vs. 6.929/2.49, P B/0.01, unpaired t-test). There were no significant relationships detected between these PON1 polymorphisms and FPG, fasting immunoreactive insulin (F-IRI), serum lipids concentrations, BMI, SFA, or VFA. Table 2 shows the stepwise regression analysis for the GIR. In model 1, the C(/108)T polymorphism was a significant contributor, as well as BMI and HbA1C. Model 2, which included VFA and SFA instead of the BMI as independent variables, also revealed that the C(/108)T polymorphism was a significant contributor to the GIR, together with F-IRI and VFA, both of which are known to be associated with insulin resistance. We also performed the stepwise regression analysis for FPG using the same independent variables including PON1 polymorphisms, but only HbA1C was a significant variable for FPG.
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Table 2 Stepwise regression analysis for glucose infusion rate in patients with type 2 diabetes Variables
Model 1 (n/48) F
b BMI PON1 C(/108)T HbA1c Fasting IRI Age Free fatty acid PON1 Q192R Triglycerides FPG LDL-cholesterol Sex HDL-cholesterol Visceral fat area Subcutaneous Fat area Total R2
Model 2 (n/45)
/0.502 0.383 /0.340
F
b 19.850 11.732 9.112 3.661 3.054 2.365 2.249 2.113 0.943 0.137 0.127 0.115
0.337 /0.320
/0.300 0.501
P B/0.0001
0.428
7.017 3.493 5.775 2.887 3.341 0.581 0.402 0.095 0.620 1.191 0.025 4.845 0.632 P B/0.0001
A stepwise multivariate regression analysis was performed. The F values for the inclusion and exclusion of variables was set at 4.0 at each step. Sex: men /1, women/2; PON1 Q192R: QQ/1, QR/2, RR /3; PON1 C(/108)T: TT/1, CT/2, CC/3. b : partial regression coefficient.
The mean insulin resistance index (IR) assessed by homeostasis model assessment (HOMA) [9] decreased in the following genotype order: /108TT //108CT / and /108CC, although the differences were not significant because of large deviations (mean HOMA-IR9/S.D.; 3.349/ 3.85, 2.539/2.05, and 2.199/1.73, respectively). In a stepwise regression analysis for HOMA-IR using the same independent variables as used in that for GIR (total R2 /0.909, P B/0.0001), the C(/108)T polymorphism was identified as a significant contributor (b//0.104, F /4.517), together with F-IRI (b/0.953, F /329.823), FPG (b/ 0.368, F /56.059), and BMI (b /0.116, F / 4.939).
4. Discussion In this study, we demonstrated that the PON1 promoter polymorphism C(/108)T, but not the coding region polymorphism Q192R, was associated with insulin sensitivity in Japanese patients with type 2 diabetes. The multiple regression
analysis confirmed that the C(/108)T polymorphism was a significant contributor to the GIR, as were other variables known to be related to insulin resistance such as F-IRI, VFA, and BMI. This is the first report suggesting that the PON1 gene polymorphism is an independent factor affecting insulin sensitivity in diabetic patients. No significant difference between the genotypes was observed as regards FPG; this finding contradicted results reported by Leviev et al. [6]. This difference might have been due to differences between study populations (diabetic vs. non-diabetic), or to the influence of sulfonylureas, used for the treatment of our population. Although it remains unclear how this type of polymorphism may affect insulin sensitivity, several possible explanations appear likely. First, by affecting body fat accumulation, the C(/108)T polymorphism may be involved in the development of insulin resistance. However, this polymorphism was not associated with BMI or VFA, suggesting that its effects on insulin sensitivity are not mediated by an influence on body fat mass or composition. Second, the PON1 promoter poly-
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morphism may be in linkage disequilibrium with other genes related to insulin resistance that are also involved in the development of diabetes; this explanation is derived from the results of a genomic scan of Pima Indians [10], which provided evidence for diabetes-susceptibility loci on chromosome 7q, where the gene encoding PON1 is found. However, a genome-wide scan for type 2 diabetes in a Japanese population [11] failed to identify candidate loci on chromosome 7q. In addition, our previous study revealed no significant difference in the prevalence of the C(/108)T polymorphism between subjects with NGT and type 2 diabetic patients [12]. These findings suggest that this polymorphism is not causally related to the development of type 2 diabetes, at least not in the Japanese population. Additionally, it has been shown that oxidative stress is increased in vivo in the diabetic state [13,14], and that such stress can also impair insulin action in vitro [15,16]. It has also been reported that plasma concentrations of partially oxidized LDL were positively correlated to steady-state plasma glucose (SSPG) concentrations [17]. The C(/108)T polymorphism is associated with the transcription and serum concentration of PON1, and the concentration in the subjects with the / 108CC genotype is higher than that with the / 108TT genotype [5]. Among our patients, the GIR increased in the following order, namely, from those with the low- to high-expressor genotype (/ 108TT B//108CT B//108CC). Higher concentrations of circulating PON1 may more strongly prevent oxidative modification in vivo. Therefore, the high-expressor C allele might efficiently prevent the impairment of insulin action caused by oxidative stress in people with diabetes. The difference in the preventive effect associated with the C(/108)T polymorphism might be enhanced in patients suffering from oxidative stress (e.g. diabetic patients). The Q192R polymorphism is closely associated with serum paraoxonase activity [3,4], and the activity increases in the order of the QQ, QR, and RR genotypes. However, Mackness et al. showed in vitro that HDL with the RR genotype, which has high serum paraoxonase activity, had less ability to protect LDL oxidation with time com-
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pared with that of the QR or QQ genotype [18]. Serum paraoxonase activity reveals an enzymatic function with the use of paraoxon, which is never detected in the human body, as a substrate. The actual in vivo substrate of PON1 is not yet known, and the site of PON1 against the oxidization of LDL has not yet been established. However, the cystein at 284 of the amino acid sequence has been highly suspected to be an active site for antioxidization [19], which appears to be different from the enzyme-active site [20]. Therefore, the C(/108)T polymorphism, which is involved in serum PON1 concentration, may be more important in the protection from oxidative stress rather than the Q192R polymorphism. Serum paraoxonase concentrations or activity can also predict susceptibility to insulin resistance. However, we and other investigators have shown that serum paraoxonase activity was decreased in diabetic patients, and was further decreased in those with diabetic complications, independent of the PON1 gene polymorphism [21,22]. We have also shown that not only enzyme activity, but also enzyme concentrations were lower in uremic patients, who often suffer severe oxidative stress [23,24]. It has been shown that treatment of cultured HepG2 cells with mildly oxidized LDL or oxidized phospholipids decreased PON1 mRNA levels, and that injection of oxidized phospholipids into C57BL/6J mice resulted in a marked reduction in serum paraoxonase activity [25,26]. Yamada et al. reported that paraoxonase activity unexpectedly increased in non-diabetic subjects with insulin resistance, as assessed by HOMA [27]. Thus, serum enzyme activity or the concentration of PON1 can be altered secondarily to various diseases or pathological conditions, regardless of insulin resistance. In fact, we measured serum PON1 concentrations in 36 patients of this study subjects, using a competitive enzyme immunoassay [12], and found no significant association between PON1 concentrations and GIR in a stepwise regression analysis (data not shown). Therefore, in subjects with diabetes or glucose intolerance, the genetic polymorphism C(/108)T could be a better predictor of susceptibility to insulin resistance than either PON1 enzyme concentration or activity.
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In conclusion, our data suggests that the PON1 gene polymorphism in the promoter region exerts an influence on insulin sensitivity in diabetic patients who might be particularly susceptible to oxidative stress. However, the number of subjects participating in this study was too small to arrive at definitive conclusions. Therefore, further study is required to establish a link between the PON1 polymorphism and the development of insulin resistance.
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