DOI: https://doi.org/10.21203/rs.3.rs-2079095/v1
Gastric inhibitory polypeptide receptor (GIPR) encodes a G-protein coupled receptor for gastric inhibitory polypeptide (GIP), which was demonstrated to stimulate insulin secretion. Relation of GIPR gene variation to type 2 diabetes mellitus has been suggested in previous studies. The aim of the study was to investigate single nucleotide polymorphisms (SNPs) in the promoter and coding regions of GIPR in type 2 diabetes mellitus (T2DM).
Two hundred subjects including 100 healthy and 100 T2DM patients were recruited in the study. Genotypes and allele frequency of rs34125392, rs4380143 and rs1800437 in the promoter, 5ʹ UTR and coding region of GIPR were investigated by RFLP-PCR and Nested-PCR.
Our finding indicated that rs34125392 genotype distribution was statistically different between T2DM and healthy groups (P = 0.043). In addition, distribution of T/- + -/- versus TT was significantly different between the both groups (P = 0.021). Moreover, rs34125392 T/- genotype increased the risk of T2DM (OR = 2.68, 95%CI = 1.203–5.653, P = 0.015). However, allele frequency and genotype distribution of rs4380143 and rs1800437 were not statistically different between the groups (P > 0.05).
We concluded that GIPR gene polymorphism is associated with T2DM.In addition; rs34125392 heterozygote genotype may increase the risk of T2DM. More studies with large sample size in other populations are recommended to show the ethnical relation of these polymorphisms to T2DM.
Global studies indicated that 415 million people lived with diabetes mellitus (DM) until 2017. Estimations suggest that 608 million people are suffered from DM that 32% of them are undiagnosed. More than 90% of DM patients accounts for type 2 diabetes mellitus (T2DM) (1). T2DM is a common metabolic disorders mainly caused by two mechanisms: reduced insulin secretion by pancreatic B cells or resistance of target tissues to insulin (2). Any defect in one of these mechanisms could trigger a T2DM phenotype. The most popular risk factors for T2DM are combinations of genetic, environmental and metabolic factors (3, 4) Contribution of genes to T2DM is well studied previously and large-scale genotyping using the Metabochip indicated that some loci increase the susceptibility to T2DM (5) Now the question is that whether the risk loci are confined to the ones that have been identified in previous studies.
Gastric inhibitory polypeptide receptor (GIPR, Gene ID: 2696) or glucose-dependent insulinotropic polypeptide receptor (https://www.uniprot.org/uniprotkb/P48546/entry) has several isoforms and encodes a G-protein coupled receptor for gastric inhibitory polypeptide (GIP), which was originally identified as a hormone that acts in gut extracts (https://www.ncbi.nlm.nih.gov/gene/2696) and inhibits the releasing of gastrin and subsequently gastric acid. Now it has been demonstrated that GIP is secreted in response to oral glucose and stimulates insulin release (6). GIPRs are located in pancreas and insulin-sensitive tissues such as adipocytes (7). GIPR, through interaction with GIP, increases lipoprotein lipase activity (LPL), fatty acid and glucose uptake, induces fatty acid adsorption into adipocytes and inhibits lipolysis (8). Recent studies have indicated that GIP induces resistance to insulin in adipose tissues (9–11) and the plasma level of GIP is increased in T2DM (12, 13) .
GIPR gene located on 19q13.32 is present in β cells of Langerhans islands. Despite differential expression in extra pancreatic tissues, its expression in adipose tissues is relatively high (7). Previous findings suggest that pharmacological activation of GIPR may have a therapeutic benefit on peripheral energy metabolism (14).
As indicated by recent studies, single nucleotide polymorphism in GIPR gene are related to changes in the secretion of hormones and adipokines in obese type 2 diabetic patients (15). GIPR gene polymorphisms have been studied in other diseases including schizophrenia (16, 17), pre-diabetic and diabetic patients (18) metabolic syndrome (19) and obesity (20). The aim of the present study was to investigate the association of GIPR gene polymorphism with type 2 diabetes mellitus in Iranian patients.
Two hundred subjects referred to Peymanieh hospital (Jahrom city, Iran) including 100 healthy individuals and 100 type 2 diabetes mellitus patients were recruited in the study. Patients had a fasting blood glucose (FBG) > 125 mg/dl. However, subjects with underlying disease including cancer, liver and kidney disease, gastrointestinal tract disease were excluded from the study. Low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triacylglycerol (TG) and FBG were measured by routine biochemical assays.
Venous blood samples were drawn in an EDTA-containing tube and stored in -80 C until the DNA extraction. Salting out technique was used to extract the genomic DNA (21) and stored rapidly in -20. Three single nucleotide polymorphisms were investigated in the study. On the other hand, rs4380143 T > C with minor allele frequency (MAF) 0.3 was located on upstream promoter region, rs34125392 T>- with MAF 0.26 was located on 5ʹUTR region, and rs1800437 G > C (Glu to Gln) with MAF 0.2 was located on coding region. RFLP-PCR was applied to genotyping and allele identification. The sequences of primers used in the study are summarized in Table 1.
Primers |
Sequence |
---|---|
GIPR5392. R |
5'-GGTGGGACAGCATGAGAGATTGTA − 3' |
GIPR5392.F |
5'-GTTATCTAGCAGCTAACCAGAGATGGA-3' |
GIPR0143. R |
5'-CCAAGAGTTGGAGACCAGCATGG -3' |
GIPR0143.F |
5'-CAGTTCCAACAACACTGTCAATCACC-3' |
GIPR0143.Nes.R |
5'-GTTCCAGTGCACTCCACTCTCAT − 3' |
GIPR0143.Nes.F |
5'-CAGGCTGGTCTCAAACTCCTG-3' |
GIPR00437. R |
5'-GCATTCTTGGCATTCTCCTGTCC − 3' |
GIPR00437.F |
5'-GAAGGAGCTGAGGAAGATCTCAAAGC-3' |
F: forward primer, R: Reverse primer, Nes: Nested-PCR |
Reactions were performed in a micro-tube with final volume of 25 µl containing 0.2 µg genomic DNA, 0.8 U Hot start Taq DNA polymerase and 1.5 mM MgCl2. The temperature cycles for rs34125392 were 940C for 30 seconds, 630C for 45 seconds and 720C for 35 seconds for 30 cycles. The temperature cycles for rs4380143 were 940C for 30 seconds, 650C for 45 seconds and 720C for 50 seconds for 30 cycles. The product of this step was the template for Nested-PCR with new pair of primers (Table 1) to detect rs43800143. The temperature cycles for Nested-PCR were 940C for 30 seconds, 620C for 25 seconds and 720C for 35 seconds for 25 cycles. The temperature cycles for rs1800437 were 940C for 30 seconds, 650C for 45 seconds and 720C for 20 seconds for 30 cycles. Initial incubation period was 5 minutes at 940C and a final extension incubation step was set at 720C for 5 minutes for all reactions.
Then, the PCR products of rs34125392, rs4380143 and rs1800437 were subjected to digestion with BtsI, FatI and BssSI, respectively (Newengland Biolab; 10U, overnight). The digested PCR products were run on 3% agarose gel and visualized by UV transillumination after DNA green viewer staining.
SPSS v.18 (Chicago) was used for statistical analysis. Normality of the data was checked by Kolmogorov-Smirnov test. Hardy-Weinberg equilibrium was performed to survey allele distribution. Logistic regression was used to survey odds ratios. The numeric data were reported as mean ± Standard Error (SE). Student t and Chi square tests were applied to investigate the differences between groups, genotype and allele variations. P value less than 0.05 was considered to be significant.
The dataset generated and/or analysed during the current study are available as Supplementary file 1. Two hundred subjects including 100 healthy individuals and 100 T2DM patients were recruited in the study. Characteristics of study population are summarized in Table 2. Healthy subjects and T2DM patients had a different age (P = 0.29). Distribution of the sex between the both group was not significantly different (P > 0.05). FBS, LDL-C, TC and TG were statistically different between the groups (P < 0.001). High density lipoprotein cholesterol was not different between T2DM and healthy subjects (P > 0.05).
Parameters |
Control N = 100 |
T2DM N = 100 |
P value |
---|---|---|---|
Age(year) |
54 ± 1.23 |
57.3 ± 0.9 |
P = 0.029 |
HDL-C(mg/dl) |
46.6 ± 2 |
45.2 ± 3 |
P > 0.05 |
LDL-C(mg/dl) |
90.2 ± 3.02 |
110.9 ± 4.34 |
P < 0.001 |
TG(mg/dl) |
104.6 ± 3.64 |
159.6 ± 9.83 |
P < 0.001 |
TC(mg/dl) |
149.9 ± 3.3 |
186.6 ± 5.33 |
P < 0.001 |
FBS(mg/dl) |
84.7 ± 2.42 |
166.58 ± 7.09 |
P < 0.001 |
Sex(male/female) |
28/72 |
40/60 |
P > 0.05 |
FBS: fasting blood sugar; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; TC: total cholesterol; T2DM: type 2 diabetes mellitus: TG: triacylglycerol |
Distribution of genotypes and allele frequency of rs34125392, rs4380143 and rs1800437 are summarized in Table 3. rs34125392 genotype distribution was statistically different between T2DM and healthy groups(P = 0.043). In addition, distribution of T/- + -/- versus TT was significantly different between the both groups (P = 0.021). Moreover, rs34125392 T/- genotype increased the risk of T2DM (OR = 2.68, 95%CI = 1.203–5.653, P = 0.015). However, allele frequency and distribution of rs34125392 genotype in men and women was not significantly different between the groups (P > 0.05).
Allele/Genotype |
Control (n = 100) |
T2DM (n = 100) |
P value |
|
---|---|---|---|---|
rs34125392 |
||||
Allele |
T |
97(48.5%) |
80(40%) |
NS |
- |
103(51.5%) |
120(60%) |
||
Genotype |
T/T |
27(27%) |
13(13%) |
0.043 |
T/- |
43(43%) |
54(54.9%) |
||
-/- |
30(30%) |
33(33%) |
||
T/- + -/- |
73 |
87 |
0.021 |
|
rs4380143 |
||||
Allele |
T |
110(55%) |
102(51%) |
NS |
C |
90(45%) |
98(49%) |
||
Genotype |
T/T |
23(23%) |
16(16%) |
NS |
T/C |
65(65%) |
70(70%) |
||
C/C |
12(12%) |
14(14%) |
||
T/C + C/C |
77 |
84 |
NS |
|
rs1800437 |
||||
Allele |
G |
154(23%) |
150(75%) |
NS |
C |
46(77%) |
50(25%) |
||
Genotype |
G/G |
7(7%) |
5(5%) |
NS |
G/C |
32(32% |
40(40%) |
||
C/C |
61(61%) |
55(55%) |
||
G/C + C/C |
93 |
95 |
NS |
On the other hand, allele frequency and genotype distribution of rs4380143 was not significantly different between healthy and T2DM subjects (P > 0.05). T/C + C/C versus TT were not different between the groups (P > 0.05). In addition, allele frequency and genotypes distribution of rs4380143 was the same in men and women between the groups (P > 0.05). There was no increased risk of rs4380143 for T2DM (P > 0.05).
Moreover, rs1800437 genotype distribution and Allele frequency was the same in the both groups (P > 0.05). G/C + C/C versus GG were not different between the groups (P.0.05). We found no increased risk of genotypes and Alleles for T2DM (P > 0.05).
The main finding of our study was that rs34125392 in GIPR gene is associated with T2DM and this polymorphism increased the risk of the disease. T2DM is a multifactorial disease which affects many people worldwide (22) and virtually no physician is found that has no patient with T2DM. Gene variations have a strong role in T2DM and the list of the genes that involved in the pathogenesis of the disease is increasing to date (23).
To the best of our knowledge this the first study that investigated rs34125392 and rs4380143 polymorphisms in T2DM. We searched PubMed, Google and dbSNP databases and found no study that included these polymorphisms in their studies. Our findings showed that the distribution of rs34125392 genotypes is different between the healthy and patient group. Also our finding indicated that rs34125392 T/- genotype may increase the risk of T2DM. Taking together, since this polymorphism is located on 5ʹUTR region of GIPR gene and we didn’t measure the expression level of GIPR gene, so the main question is that whether this gene variation could alter gene expression. Skuratovskaia et al (15) investigated the association of GIPR gene polymorphism with plasma level of mediators which have a role in the regulation of carbohydrate metabolism in obese T2DM patients. They found that GIPR gene expression in adipose tissue of the small intestine mesentery in patients bearing rs2302382 CC and rs8111428 AA genotypes was decreased and this was in relation to increase level of leptin. They claimed that during normal expression plasma concentration of insulin and GIP in subjects bearing rs2302382 polymorphism and rs8111428 AG genotypes were increased. However, we found no increased risk of rs4380143 genotypes in T2DM and the distribution of alleles and genotypes were the same in patients and healthy groups.
Shalaby group (24) studied rs1800437 polymorphism in Egyptian T2DM patients. Their findings indicated that the distribution of C haplotype is statistically higher in patients than controls. They concluded that there is no association between this polymorphism and the risk of T2DM. In contrast to Shalaby group our finding showed that the distribution of rs1800437 genotypes and allele frequency in T2DM patients and healthy subjects were not different. This inconsistency may be related to different races. However, we found that there is no association between rs1800437 genotypes and alleles with T2DM. Jeannine group (25) investigated the association of variants in GIPR gene with impaired glucose homeostasis in obese children and adolescents from Berlin. They found an association between rs1800437 C allele and elevated homeostasis model of insulin resistance values. They concluded that GIPR gene variations are not related to childhood obesity but rs1800437 may have a potential role in glucose homeostasis. Recent studies are focused on the GIPR agonist to improve the extra pancreatic effects of GIP and its role in secretion of insulin. Nicholas group (26) performed a study to clarify whether the increased risk of coronary artery disease (CAD) is mediated via GIPR or is instead the result of linkage disequilibrium (LD) confounding between variants at the GIPR locus. They found that rs1800437 G allele is common among the fasting GIP levels, glycemic traits, and adiposity-related traits and it is independent of CAD and lipid traits.
our findings indicated that GIPR gene polymorphism is associated with T2DM in Iranian patients. In addition, the results showed that T/- genotype may increase the risk of T2DM. Since this polymorphism is located on 5ʹUTR region of GIPR gene, further studies are needed to show that whether genotype variation could alter GIPR phenotype. Future studies with large sample size in other populations are recommended to show the ethnical relation of these polymorphisms to T2DM. Our sample size was relatively small so our findings should interpret with caution.
CAD: Coronary artery disease
DM: Diabetes mellitus
FBG: Fasting blood glucose
GIPR: Gastric inhibitory polypeptide receptor
GIP: Gastric inhibitory polypeptide
HDL-C: High density lipoprotein cholesterol
LD: Linkage disequilibrium
LDL-C: Low density lipoprotein cholesterol
LPL: Lipoprotein lipase
MAF: Minor allele frequency
TC: Total cholesterol
T2DM: Type 2 diabetes mellitus
TG: Triacylglycerol
Ethical approval and consent to participate: Ethics committee of Jahrom University of Medical sciences approved the study. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardian(s).
Availability of data and materials: The dataset generated and/or analyzed during the current study are available as Supplementary file 1.
Competing interests: The authors declare that they have no competing interests
Consent to publish: Not applicable
Funding: The study was founded by Jahrom University of medical sciences
Authors contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Saiedeh Erfanian and Abazar Roustazadeh. The first draft of the manuscript was written by Saiedeh Erfanian, Abazar Roustazadeh and Hamed Mir and all authors commented on previous versions of the manuscript. Saiedeh Erfanian and Abazar Roustazadeh assisted in laboratory measurements. All authors read and approved the final manuscript.
Acknowledgment: This study was supported by vice chancellery of research affairs of Jahrom University of Medical Sciences.