Relationship Between Tasl R2, Tasl R3 And TRPM5 Gene Polymorphisms And Type 2 Diabetes Mellitus

Type 2 diabetes is a metabolic disease characterized by hyperglycemia resuiting from defects in insulin secretion in pancreatic beta cells and insulin action on target tissues.Although the mechanism by which type 2 diabetes develops is not well understood,multiple risk factors,including genetic susceptibility and environment factors,are supposed to contribute to the disease(1)Human Tas1r2(taste receptor family 1,member 2)and Tas1r3(taste receptor family 1,member 3)genes,which are clustered in chromosome(chr)1 p36.33,.one of the regions showing potential linkage with type 2 diabetes,encode the proteins of 679 and 843,amino acids respectively,the T1R2 and T1R3. T1R2 and T1R3 are the two G -protein coupled receptors expressed in taste cells of the tongue and palate epithelium.T1R3 recognizes amino acids in combination with T1R1,and functions as a broadly tuned sweet receptor when working with T1R2.Transient receptor potential cation channel,superfamily M,member 5 (TRPM5)is a valtage - modulated and Ca~2+ activated monovalent selective cation channel and the downstream signaling pathway of the taste receptors.TRPM5 gene is located on chr 11p15.5 with 24 exons.The protein it encodes consists 1175 residues.In addition to taste receptor cells,TRPM5 RNA was detected in variety of tissues,including small intestine,liver,lungs,testis and brain.And recently,TRPM5 was found in a rat beta cell line and in human pancreatic islets,suggesting that TRPM5 is an important component of the beta cells.Because sweet -tasting foods have always been considered to elicit the insulin release and it was reported that patients with diabetes had increased preference for sweet food,and because TRPM5 was found in pancreatic beta cell lines,we therefore investigated the role of Tas1r2,Tas1r3 and TRPM5 genes in ObjectiveTo investigate role of the single nucleotide polymorphism(SNP)of the Tas1r2 gene in genetic susceptibility to type 2 diabetes.Subjects and methods1..Subjects1)Type 2 diabetes:We randomly recruited 298 unrelated patients(Male: 154,Female:144,Age:61.4±14.0.)with diabetes from Tohoku University Hospital.Type 2 diabetes was diagnosed according to the World health Organization (WHO)criteria.2)148 elderly subjects meeting the following criteria:older than 60 years of age,no past history of diabetes,HbA1c less than 5.6%and no diabetes in third degree or closer relatives,were enrolled in this study.3)A total of 308 subjects with normal glucose tolerance(NGT)were selected from among people undergoing routine annual health examinations at two hospitals in Japan,Sendai kosei hospital and Shimonoseki Kosei Hospital.Normal glucose tolerance was confirmed by 75 g OGTT according to the WHO criteria.4)The study protocol and genetic analysis of human subjects were reviewed and approved by the Tohoku University Institutional Review Board.Appropriate informed consent was obtained from all subjects examined.2.Methods1)Genomic DNA amplification DNA was isolated from peripheral blood cells using a QIAamp DNA mini kit.2)SNP identification.To amplify the coding region and intron- exon boundaries from genomic DNA,a primer set was developed using the genomic sequence for Tas1r2.PCR was performed and each PCR fragment was directly sequenced in both directions.3)SNP genotyping of Tas1r2 by Direct Sequence.Results1.Polymorphisms in the coding region of Tas1r2 gene.We identified 13 SNPs in the coding region of the Tas1r2 gene,of which 8 with,and 5 without amino acid substitutions.2.An amino acid substitution at position 317,from Arg(949C/950G: wild type)or Gly(949G/950G)to Ala(949G/950C),in the TAS1R2 protein was denoted as 317A.The allele frequency of 950C(317A)was significantly higher in type 2 diabetic patients than in both elderly normal(p = 0.02)and NGT subjects(p = 0. 0003),whereas the allele frequency of other SNPs was essentially identical in these three groups.3.950C(317A)had a significantly higher plasma glucose and insulin levels 30 min,60 min,and 90 min in respond to glucose load on oral glucose tolerance test(p = 0.027,p =0.036,p = 0.022,p = 0.027,p = 0.037,p = 0.02).Furthermore,in the NGT subjects,950C(317A)was associated with a significantly lower insulinogenic index(the ratio of the insulin increment to that of blood glucose 30 min after a glucose load),an indicator of the early insulin response on oral glucose tolerance test(0.52±0.7 vs.0.89±1.32, p = 0.0043),suggesting a reduction in early insulin secretion in response to a glucose load in subjects with 950C(317 A).Conclusions1.There are 13 SNPs in the coding region of the Tas1r2 gene,of which 8 with,5 without amino acid substitutions.2.The G950C(317A)polymorphism in the Tas1r2 gene is associated with the development of type 2 diabetes,via reduced insulin secretion. ObjectiveTo investigate the role of the single nucleotide polymorphism of Tas1r3 gene in genetic susceptibility to type 2 diabetes.Subjects and methods1.Subjects1)Type 2 diabetes:We randomly recruited 363 unrelated patients(Male: 187,Female:176,Age:64.4±11.3)with diabetes from Tohoku University Hospital.Type 2 diabetes was diagnosed according to the World health Organization (WHO)criteria.2)180 elderly subjects meeting the following criteria:older than 60 years of age,no past history of diabetes,HbA1c less than 5.6%and no diabetes in third degree or closer relatives,were enrolled in this study.3)A total of 164 subjects with normal glucose tolerance(NGT)were selected from among people undergoing routine annual health examinations at two hospitals in Japan,Sendai kosei hospital and Shimonoseki Kosei Hospital.Normal glucose tolerance was confirmed by 75 g OGTT according to the WHO criteria.4)The study protocol and genetic analysis of human subjects were reviewed and approved by the Tohoku University Institutional Review Board.Appropriate informed consent was obtained from all subjects examined.2.Methods1)Genomic DNA amplification DNA was isolated from peripheral blood cells using a QIAamp DNA mini kit.2)SNP identification.To amplify the coding regions and intron- exon boundaries from genomic DNA,a primer set was developed using the genomic sequence for Tas1r3.PCR was performed and each PCR fragment was directly sequenced in both directions.3)SNP genotyping of Tas1r3,by Direct Sequence.Results1.Polymorphisms in the coding region of Tas1r3 gene.We identified 14 SNPs in the coding region of the Tas1r3 gene,of which 5 with,and 5 without amino acid substitutions.4 are located at the promoter region.2.There were no significant differences in the allele frequency in all the SNPs of the Tas1r3 gene between diabetic subjects and control group.Conclusions1.There are 13 SNPs in the coding region of the Tas1r3 gene,of which 4 are in the promoter,5 with and 5 without amino acid substitutions.2.Tas1r3 gene might not be related to the development of type 2 diabetes.Section A Relationship between TRPM5 Gene Polymorphism and Type 2 DiabetesObjectiveTo investigate the role of the single nucleotide polymorphism of the TRPM5 gene in genetic susceptibility to type 2 diabetes.Subjects and methods1.Subjects1)Type 2 diabetes:We randomly recruited 360 unrelated patients(Male: 184,Female:176,Age:63.4±15.0)with diabetes from Tohoku University Hospital.Type 2 diabetes.was diagnosed according to the World health Organization (WHO)criteria.2)182 elderly subjects meeting the following criteria:older than 60 years of age,no past history of diabetes,HbA1c less than 5.6%and no diabetes in third degree or closer relatives,were enrolled in this study.3)A total of 177 subjects with normal glucose tolerance(NGT)were selected from among people undergoing routine annual health examinations at two hospitals in Japan,Sendai kosei hospital and Shimonoseki Kosei Hospital.Normal glucose tolerance was confirmed by 75 g OGTT according to the WHO criteria.4)The study protocol and genetic analysis of human subjects were reviewed and approved by the Tohoku University Institutional Review Board.Appropriate informed consent was obtained from all subjects examined.2.Methods1)Genomic DNA amplification:DNA was isolated from peripheral blood cells using a QIAamp DNA mini kit.2)SNP identification.To amplify the coding regions and intron- exon boundaries from genomic DNA,a primer set was developed using the genomic sequence for TRPM5.PCR was performed and each PCR fragment was directly sequenced in both directions.3)SNP genotyping of TRPM5 by Direct Sequence.Results1.Polymorphisms in the coding region of TRPM5 gene.We identified 12 SNPs in the coding region of the TRPM5 gene,of which 6 with,6 without amino acid substitutions.2.There was no significant difference in the allele frequency in all the SNPs of the TRPM gene between diabetic subjects and control group.3.An amino acid substitution at position 235,from Ser(704G wild type)to Asn(704A),in the TRPM5 protein was denoted as 235N.In NGT subjects,235N/N had a significantly lower fasting plasma insulin level than N/S,S/S N/S+S/S(p = 0.01,p = 0.016,p = 0.008).Furthermore, in NGT subjects,235N/N was associated with a significantly lower HOMA(R),an indicator of the insulin resistance(p = 0.003,p = 0.0083,p = 0.00000025),suggesting that subjects with 235N/N had lower risk of suffering from insulin resistance.These results were further.confirmed when ISIO composite value,an indicator of insulin sensitivity,was analyzed.Again,the subjects with N/N genotype of the S235N SNP exhibited higher values of ISIO than those with N/S,S/S or N/S + S/S genotypes(p =0.021,0.027 and 0.028 respectively).And the ISIO value in subjects with 235N allele was also higher than that of the 235S allele(p = 0.011),suggesting that 235N is more insulin sensitive than 235S.4.An amino acid substitution at position 578,from Gin(1733G wild type) to Arg(1733A),in the TRPM5 protein was denoted as 578R.In NGT subjects,578R/R had a signifieantly lower fasting plasma insulin level than 578R/Q,578Q/Q及578R/Q + Q/Q(p = 0.027,p = 0.028,p = 0.018.Furthermore,in NGT subjects,235N/N was associated with a significantly higher ISIO composite value.Subjects with 578R/R genotype of the Q578R SNP exhibited higher values of ISIO than those with578R/Q,578Q/Q及. 578R/Q + Q/Q者(p = 0.0036,p = 0.0038,p = 0.0025.And the ISIO value in subjects with 578R allele was also higher than that of the 578Q allele (23.1±15.2 vs.18.9±9.9,p = 0.0135),suggesting that 578R is more insulin sensitive than 578Q.Conclusions1.There are 12 SNPs in the coding region of the TRPM5 gene,of which 6 with,6 without amino acid substitutions.2.There was no significant difference in the allele frequency in all the SNPs of the TRPM5 gene between diabetic subjects and control group.3.235N polymorphism of the TRPM5 gene might be linked to the elevated insulin sensitivity in NGT subjects.4.578 R polymorphism of the TRPM5 gene might,be associated with the elevated insulin sensitivity in NGT subjects. This study firstly investigated the relationship between Tas1r2,Tas1r3 and TRPM5 genes and type 2 diabetes and concluded that:1.Tas1r2 gene might be linked to the development of type 2 diabetes.2.Tas1r3 gene might not be related to the development of type 2 diabetes.3.TRPM5 gene might be associated with the elevated instdin sensitivity in NGT subjects.