Two Novel Functional Single Nucleotide Polymorphisms of ADRB3 ...

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Two Novel Functional Single Nucleotide Polymorphisms of ADRB3 Are Associated With Type 2 Diabetes in the Chinese Population Qiong Huang, Tian-Lun Yang, Bei-Sha Tang, Xiang Chen, Xi Huang, Xiang-Hang Luo, Yuan-Shan Zhu, Xiao-Ping Chen, Ping-Cheng Hu, Juan Chen, Wei Wei, Hong-Hao Zhou, Ji-Ye Yin, and Zhao-Qian Liu Institute of Clinical Pharmacology (Q.H., Y.-S.Z., X.-P.C., J.C., H.-H.Z., J.-Y.Y., Z.-Q.L.), Hunan Key Laboratory of Pharmacogenetics, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, People’s Republic of China; Institute of Clinical Pharmacology (Q.H., W.W.), Key Laboratory of Antiinflammatory and Immune Medicine, Ministry of Education, Anhui Medical University, Hefei, Anhui 230032, People’s Republic of China; Institutes of Hypertension (T.-L.Y.) and Integrated Traditional Chinese and Western Medicine (X.H.) and Departments of Neurology (B.-S.T.) and Dermatology (X.C.), Xiangya Hospital, Central South University, Changsha, Hunan 410008, People’s Republic of China; Institute of Endocrinology and Metabolism (X.-H.L.), The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, People’s Republic of China; and Department of Epidemiology and Biostatistics (P.-C.H.), School of Public Health, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, People’s Republic of China

Aims: The purpose of this study was to investigate the association of two novel ␤3-adrenergic receptor (ADRB3) gene polymorphisms (Ser165Pro and Ser257Pro) with type 2 diabetes (T2DM) in the Chinese population. Methods: A total of 650 patients with T2DM and 1337 health volunteers were enrolled to conduct the association study. Two candidate polymorphisms were recreated by site-directed mutagenesis and tested for their effect on ADRB3 expression and function in stable transfected human embryonic kidney 293 and Chinese hamster ovary-K1 cells. Real-time PCR, Western blot, confocal microscopy, and cAMP assay were used to determine mRNA, protein expression, trafficking, and ADRB3 function, respectively. Results: We found that both polymorphisms were significantly associated with T2DM (odds ratio ⫽ 2.060 and 95% confidence interval ⫽ 1.303–3.258 for Ser165Pro and odds ratio ⫽ 7.588, 95% confidence interval ⫽ 1.639 –35.138 for Ser257Pro). Patients with T2DM with the Ser165Pro C allele had higher hemoglobin A1c, fasting plasma glucose and postprandial plasma glucose values than those in TT genotypes. We also found that patients with T2DM with the Ser257Pro C allele had lower fasting serum insulin, postprandial serum insulin, and homeostasis model assessment for insulin resistance levels than TT genotype carriers. Further in vitro study indicated that cell lines stably expressing Ser165Pro and Ser257Pro mutants of the ADRB3 gene showed impaired cAMP accumulation activity. However, both polymorphisms had no effect on ADRB3 expression and trafficking. Conclusions: Ser165Pro and Ser257Pro polymorphisms affected ADRB3 function and were significantly associated with susceptibility to and development of T2DM. (J Clin Endocrinol Metab 98: E1272–E1277, 2013)

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received January 16, 2013. Accepted April 15, 2013. First Published Online May 2, 2013

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Abbreviations: CHO, Chinese hamster ovary; CI, confidence interval; ERK, extracellular signal–regulated kinase; FPG, fasting plasma glucose; HEK, human embryonic kidney; PPG, postprandial glucose; SNP, single nucleotide polymorphism.

J Clin Endocrinol Metab, July 2013, 98(7):E1272–E1277

doi: 10.1210/jc.2013-1137

doi: 10.1210/jc.2013-1137

DRB3 (␤3-adrenergic receptor) belongs to the G protein– coupled receptor superfamily and has 7 hydrophobic stretch transmembrane domains. It plays an important role in energy metabolism, which includes enhancing lipolysis, mobilization of free fatty acid into the circulation in white adipose tissue, and thermogenesis in brown adipose tissue (1). It can be activated by ␤-adrenergic agonists and results in adenylate cyclase activation, which then increases the amount of intracellular cAMP (2). In an animal study, ADRB3 agonists induce increases in lipolysis, fatty acid oxidation, and energy expenditure and decreases in blood glucose; thus, the ADRB3 is a potential target for antiobesity and antidiabetes therapy (3). To date, several polymorphisms have been identified in the ADRB3 gene, including Trp64Arg and Ile62Met (4, 5). A previous study showed that compared with the wild type and heterozygotes, Trp64Arg Pima Indian homozygote carriers had an earlier onset of T2DM and a lower resting metabolic rate (6). Furthermore, Trp64Arg polymorphism was also associated with insulin resistance and lower insulin secretory activity (7, 8). However, some studies failed to find an association between ADRB3 Trp64Arg polymorphism and T2DM. For example, a study in Japanese suggested that Trp64Arg polymorphism was not associated with obesity and hyperinsulinemia (9), another study also showed negative results for their association in members of families with a strong predisposition to T2DM (10), and Urhammer et al (11) reported that only the homozygous genotype of the Trp64Arg polymorphism may be associated with obesity in young healthy Danes. Although controversy exists, ADRB3 polymorphisms may affect its function, which in turn affect susceptibility for T2DM. In the present study, we identified two novel polymorphisms (Ser165Pro and Ser257Pro) of ADRB3 and found that these two polymorphisms were implicated in the susceptibility of T2DM in Chinese population. We further conducted a functional study for these two polymorphisms in vitro and found that mutated cell lines showed impaired cAMP accumulation activity.

A

Subjects and Methods Participants and genotyping Distribution of the polymorphisms was determined in 1337 healthy control subjects and 650 unrelated patients with T2DM. All subjects were randomly recruited from the Department of Endocrinology (patients) and the Health Screening Center (healthy control subjects), Xiangya Hospital of Central South University (Changsha, Hunan, China). The clinical characteris-


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tics of all subjects are summarized in Supplemental Table 1 published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org. T2DM was diagnosed according to a fasting plasma glucose (FPG) level of ⱖ7.0 mmol/L or a postprandial plasma glucose (PPG) test result of ⱖ11.1 mmol/L (12). Patients who were receiving insulin treatment, pregnant or lactating women, or women with serious diseases such as acute myocardial infarction, cerebrovascular accident, trauma, hepatic, kidney, or liver disease were excluded from this study. The study protocol was approved by the Ethics Committee of Xiangya School of Medicine, Central South University. Genomic DNA were extracted from leukocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin) and stored at 4°C until use. The genotypes of two polymorphisms were determined by pyrosequence assay. (The details are described in the Supplemental Materials and Methods.)

Construction of plasmids and site-directed mutagenesis A human ADRB3 wild-type expression plasmid pcDNA3.1(⫹)ADRB3WT was used to generate ADRB3Trp64Arg (Trp64Arg mutation), ADRB3Ser165Pro (Ser165Pro mutation), and ADRB3Ser257Pro (Ser257Pro mutation) mutant plasmids by site-directed mutagenesis. The details are provided in the Supplemental Materials and Methods.

Cell culture and transfections Human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO)-K1 cells were grown in Dulbecco’s modified Eagle medium and Ham’s F12/Dulbecco’s modified Eagle medium containing 10% (v/v) calf serum. The details of transfection are provided in the Supplemental Materials and Methods.

Sample preparation, real-time PCR, Western blot, confocal immunofluorescence, and cAMP assay mRNA, protein expression, and protein trafficking were detected by real-time PCR, Western blot, and confocal microscopy, respectively. The cAMP assay was performed by a cAMP-Glo Assay Kit (Promega). The details of these methods are provided in the Supplemental Materials and Methods.

Statistical analysis Statistical analyses were performed in SPSS software (version 18.0 for Windows; SPSS Inc, Chicago, Illinois). Details of the statistical analyses are provided in the Supplemental Materials and Methods.

Results ADRB3 gene Ser165Pro and Ser257Pro polymorphisms increased T2DM risk and were associated with T2DM development in the Chinese population In our previous study, we cloned the ADRB3 gene from the perinephric fat tissue from a 35-year-old male Chinese patient with renal lithiasis (13). While sequencing the coding region of ADRB3, we identified two novel polymorphisms: Ser165Pro and Ser257Pro. These two single nu-

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Table 1.

ADRB3 Novel SNPs and T2DM


Clinical Characteristics of Patients With T2DM With Different ADRB3 Genotypes Ser165Pro

No. (men/ women) Age, y BMI, kg/m2 WHR SBP, mm Hg DBP, mm Hg HbA1c, % FPG, mmol/L PPG, mmol/L FINS, mU/L PINS, mU/L HOMA-IR

Ser257Pro

TT

TC ⴙ CC

P Values

TT

TC ⴙ CC

P Values

543 (309/234)

107 (51/56)

.079

473 (257/216)

26 (17/9)

.270

49.73 ⫾ 10.93 (48.80 –50.65) 25.58 ⫾ 3.79 (25.25–25.91) 0.91 ⫾ 0.06 (0.90 – 0.91) 133.70 ⫾ 20.86 (131.85–135.55) 84.73 ⫾ 13.30

51.53 ⫾ 11.24 (49.38 –53.69) 25.64 ⫾ 3.43 (24.97–26.31) 0.90 ⫾ 0.06 (0.89 – 0.91) 131.17 ⫾ 21.99 (126.85–135.48) 81.76 ⫾ 13.63

1.000

9.44 ⫾ 2.58 (8.94 –9.94) 8.92 ⫾ 2.93 (8.36 –9.47) 15.76 ⫾ 4.82 (14.75–16.78) 10.34 ⫾ 5.65 (9.00 –11.69) 42.43 ⫾ 35.08 (33.66 –51.19) 4.22 ⫾ 2.56 (3.61– 4.83)

50.04 ⫾ 9.98 (46.01–54.07) 25.65 ⫾ 3.59 (24.17–27.13) 0.90 ⫾ 0.06 (0.87– 0.92) 127.54 ⫾ 16.20 (121.00 –134.08) 80.19 ⫾ 9.35 (76.42– 83.97) 8.86 ⫾ 2.55 (7.59 –10.12) 9.27 ⫾ 3.45 (7.88 –10.67) 15.80 ⫾ 5.97 (13.39 –18.21) 8.06 ⫾ 3.41 (6.69 –9.44) 30.24 ⫾ 14.90 (24.22–36.26) 3.08 ⫾ 1.27 (2.56 –3.59)

1.000

8.33 ⫾ 2.75 (8.01– 8.66) 7.81 ⫾ 3.38 (7.52– 8.09) 13.51 ⫾ 6.45 (12.85–14.17) 10.42 ⫾ 7.22 (9.58 –11.25) 52.48 ⫾ 44.20 (47.49 –57.46) 3.88 ⫾ 2.76 (3.56 – 4.20)

49.58 ⫾ 10.99 (48.58 –50.57) 25.69 ⫾ 3.42 (25.37–26.01) 0.90 ⫾ 0.06 (0.90 – 0.92) 127.71 ⫾ 17.67 (126.03–129.39) 80.61 ⫾ 10.57 (79.61– 81.62) 8.80 ⫾ 2.57 (8.52–9.08) 8.67 ⫾ 3.38 (8.37– 8.98) 14.63 ⫾ 6.13 (14.01–15.25) 10.60 ⫾ 7.12 (9.83–11.37) 52.31 ⫾ 43.97 (47.63–57.00) 4.02 ⫾ 2.79 (3.71– 4.32)

1.000 1.000 1.000 .672 .000b .016a .000b 1.000 1.000 1.000

1.000 1.000 1.000 1.000 1.000 1.000 1.000 .032a .000b .032a

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; FINS, fasting serum insulin; HOMA-IR, homeostasis model assessment for insulin resistance; PINS, postprandial serum insulin; SBP, systolic blood pressure; WHR, waist-to-hip ratio. Data are given as means ⫾ SD (95% CI). P values represent the statistical difference between TT and TC ⫹ CC groups. a

P ⬍ 0.05.

b

P ⬍ 0.01.

cleotide polymorphism (SNPs) were then submitted to the dbSNP database (http://www.ncbi.nlm.nih.gov/snp) and registered as rs72655364 (Ser165Pro) and rs72655365 (Ser257Pro), respectively. We then detected whether these two polymorphisms were associated with T2DM in a casecontrol study. A total of 1337 healthy control subjects and 650 unrelated patients with T2DM were genotyped. The C allelic and CC genotypic frequencies of Ser165Pro polymorphism in patients with T2DM were significantly higher than those in healthy control subjects (P ⬍ .001 and P ⫽ .001, respectively). Considering that the CC homozygous genotype was limited, we thus applied a recessive model to detect the association of Ser165Pro polymorphism with T2DM and found that C allele carriers had a relative risk of 1.639 (95% confidence interval [CI], 1.223–2.198). Similarly, the C allelic and CC genotypic frequencies of Ser257Pro polymorphism in the T2DM group were also significantly higher than those in healthy control subjects (P ⬍ .001 and P ⬍ .001, respectively). The recessive model showed that the Ser257Pro genotype was significantly associated with T2DM, and C allele carriers had a relative risk of 5.371 (95% CI, 2.312–12.476). After adjustment by age, sex, body mass index, waist-to-hip

ratio and diastolic blood pressure, both polymorphisms showed to be correlated with T2DM (odds ratio ⫽ 2.060 and 95% CI ⫽ 1.303–3.258 for Ser165Pro and odds ratio ⫽ 7.588 and 95% CI ⫽ 1.639 –35.138 for Ser257Pro). Table 1 summarizes the clinical characteristics of different genotypes of ADRB3 Ser165Pro and Ser257Pro in all patients. We found that patients with T2DM who had the C allele of Ser165Pro polymorphism had higher hemoglobin A1c (percentage), FPG (millimoles per liter), and PPG values than those in TT genotype carriers. For Ser257Pro, we found that patients with T2DM with the C allele had lower fasting serum insulin (milliunits per liter), postprandial serum insulin (milliunits per liter), and homeostasis model assessment for insulin resistance levels compared with TT genotype carriers. Therefore, patients with mutated ADRB3 gene Ser165Pro or Ser257Pro polymorphisms showed more serious metabolic disorder. Ser165Pro and Ser257Pro did not affect ADRB3 expression and subcellular localization To further determine whether Ser165Pro and Ser257Pro polymorphisms affected ADRB3 expression, two mutations and their combined mutation were

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Figure 1. Effects of ADRB3 Ser165Pro, Ser257Pro, and Trp64Arg polymorphisms on the expression, trafficking, and function of ADRB3. The vector control, wild-type, Trp64Arg, Ser165Pro, Ser257Pro, and the combined mutation of the ADRB3 gene (Ser165Pro/Ser257Pro) were transiently transfected to HEK293 and CHO-K1 cells. A and E, Expression levels of ADRB3 mRNA were determined in HEK293 (A) and CHO-K1 cells (E) using real-time PCR. B and F, Expression levels of ADRB3 protein were measured in HEK293 (B) and CHO-K1 cells (F) using Western blot. mRNA and protein level shown are relative to actin. The mean ⫾ SD is the average of at least three independent experiments. ␤-Actin was used as a control for the real-time PCR and Western blot analysis. C and G, Subcellular localization of wild-type and mutant ADRB3 in HEK293 (C) and CHOK1 cells (G). Cells were immunostained by use of ADRB3 antibody and fluorescein isothiocyanate– conjugated secondary antibody. Nuclei were counterstained with 4⬘,6⬘-diamidino-2-phenylindole (DAPI). D and H, Comparisons of cAMP concentrations in cell lines transfected with different genotypic ADRB3. The wild-type and Ser165Pro and Ser257Pro mutants were stably expressed to equivalent levels in HEK293 (D) and CHO-K1 (H). Then cells were serum starved in Krebs buffer containing 2% fatty acid–free bovine serum albumin for 3 hours before addition of various concentrations of CL316,243 as a selective agonist of ADRB3. After cells were treated with 100 nM, 1 ␮M, and 10 ␮M CL316,243, the concentrations of cAMP in cells were detected. Both HEK293 (D) and CHO-K1 (H) cells transfected with Ser165Pro and Ser257Pro and their combination mutant (Ser165Pro/Ser257Pro) had significantly lower cAMP concentrations than wild-type cells. Results are from at least three independent experiments. *, P ⬍ .05 compared with wild-type cells.

recreated and transiently transfected into HEK293 and CHO-K1 cells. As shown in Figure 1, A and B, there were no significant differences in the expression levels of ADRB3 mRNA and protein between the wild-type and mutant HEK293 cells. To ensure that the above finding is not cell line–specific, we performed the same experiments using CHO-K1 cells. Figure 1, E and F, showed similar results, and no significant differences were detected. We next determined whether ADRB3 Ser165Pro and Ser257Pro and their combined mutation influenced the trafficking of ADRB3 protein to the cell membrane through the process of immunostaining. As shown in Figure 1C, ADRB3 was not observed in cells transfected with empty vector, whereas cells transfected with wild-type ADRB3 showed strong plasma membrane staining as expected. Plasma membrane staining was also observed in the mutant cells. To further determine whether membrane ADRB3 expression was affected by the polymorphisms, fluorescence intensity on the cell peripherals was quantitated. As shown in Supplemental Figure 1A, no significant

difference was detected between wild-type and mutant cells. To validate this result, we next conducted Western blotting using membrane fractions of different genotype cells. As shown in Supplemental Figure 2A, both wild-type and mutant cells expressed similar ADRB3. We also performed similar experiments using CHO-K1 cells. As shown in Figure 1G and Supplemental Figures 1B and 2B, similar results were obtained. Thus, we concluded that Ser165Pro and Ser257Pro did not affect ADRB3 expression and trafficking. As a control, we also used the method above to detect the effect of polymorphism Trp64Arg on ADRB3 expression and trafficking. Similarly, Trp64Arg also did not affect trafficking as revealed by the cytoplasm staining of ADRB3. Functional study of ADRB3 gene Ser165Pro and Ser257Pro polymorphisms Next, we investigated the possible functional implication of two polymorphisms to cAMP and extracellular signal–regulated kinase (ERK) pathways. We first stably expressed the wild-type and mutant ADRB3 in CHO-K1

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ADRB3 Novel SNPs and T2DM

and HEK-293 cells. After cells were treated with 100 nM, 1 ␮M, and 1 mM of CL316,243, cAMP accumulation was detected. As shown in Figure 1D, the concentrations of cAMP in HEK293 cells transfected with ADRB3 gene Ser165Pro and Ser257Pro mutants and their combination mutant (Ser165Pro/Ser257Pro) were significantly lower than that in wild-type cells. Similar results were also observed in CHO-K1 cells (Figure 1H). We next detected the phosphorylation level of ERK1/2 in response to ADRB3 activation in HEK293 cells. In both wild-type and mutant cells, the expression level of phosphorylated ERK1/2 were increased with increasing concentrations of CL316,243. However, for each concentration, there were no significant differences in the phosphorylation of ERK1/2 between wild-type and mutant cells (Supplemental Figure 3).


This study has some limitations. Although the in vitro study showed that these two amino acid substitutions affected ADRB3 function, a ligand-binding study was lacking. Therefore, we cannot determine whether the function impairment was due to a decreased number of receptors on the cell surface or a binding affinity change. In the present study, fat mass in subjects was not direct detected by dualenergy X- ray absorptiometry, which was a more accurate method. In addition, linkage disequilibrium analysis between these two and other genetic polymorphisms of ADRB3 was lacking in this study. We are trying to clarify these limitations. Finally, we conducted the association study in just the Chinese population; thus, our results need to be verified in other ethnicities.

Acknowledgments Discussion In the present study, we identified two novel nonsynonymous polymorphisms, Ser165Pro and Ser257Pro, of the ADRB3 gene. These two amino acid substitutions affected ADRB3 function and were associated with T2DM susceptibility and development. ADRB3 binds endogenous ligands epinephrine and norepinephrine to regulate the intracellular cAMP level (14). When ADRB3 was activated by agonists, it coupled with G␣s protein to stimulate cAMP production and protein kinase A2 activity. In the present study, cAMP levels were determined in wild-type and mutant cells after addition of different concentrations of CL316,243. In the CL316,243 100 nM, 1 ␮M, and 10 ␮M groups, we found that the cAMP levels in mutated genotype cells were significantly lower than that in wild-type cells. This result suggested that the mutated ADRB3 may have less coupled efficiently to the G␣s protein than the wild-type receptor. This result was consistent with a previous study (15). We thus proposed that these two polymorphisms impaired ADRB3-mediated cAMP production through decreasing the binding capacity to G␣s protein. ADRB3 also couples with G␣i protein to stimulate mitogen-activated protein kinase production and ERK activity (16, 17). However, in the present study, we found that phosphorylation ratios of ERK1/2 were not changed in mutant ADRB3 cells compared with wild-type cells. Currently, it is not know why these two polymorphisms did not affect ERK activation. One hypothesis was that these two polymorphisms impaired binding capacity of ADRB3 to G␣s, but did not affect G␣i binding. We thus speculated that Ser165Pro and Ser257Pro may only affect the binding of G␣s. We will address this hypothesis in future studies.

Address all correspondence and requests for reprints to: Professor Zhao-Qian Liu, Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha, Hunan 410078, People’s Republic of China. E-mail: [email protected]; or Dr. Ji-Ye Yin, Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha, Hunan 410078, People’s Republic of China. E-mail: [email protected]. This work was supported by the National High-Tech R&D Program of China (863 Program; 2012AA02A517), National Natural Science Foundation of China (81202596, 81173129, and 81202595), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT0946), Special Scientific Research Foundation of Doctor Disciplines in University of Ministry of Education of China (20110162110034), Natural Science Innovation Group Foundation of Hunan Province (12JJ7006), Specialized Research Fund for the Doctoral Program of Higher Education (20113420120006), Grants for Scientific Research of BSKY (XJ201021), and Young Top-Notch Talent Support Programs from Anhui Medical University. Q.H., J.-Y.Y. and Z.-Q.L. conceived and designed the experiments. Q.H. and J.-Y.Y. performed the experiments. Q.H. and J.-Y.Y. analyzed the data. Q.H., T.-L.Y., B.-S.T., X.C., X.H., X.-H.L., Y.-S.Z., X.-P.C., P.-C.H., and J.C contributed clinical samples and data. Q.H. wrote the manuscript. Q.H., J.-Y.Y., Z.-Q.L., W.W., and H.-H.Z. reviewed/edited the manuscript. This study was registered with clinical trial registration number ChiCTR-CCC00000406. Disclosure Summary: The authors have nothing to disclose.

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