Association of Neuropeptide-Y (NPY) and Interleukin-1beta (IL1B), Genotype-Phenotype Correlation and Plasma Lipids with Type-II Diabetes

Abstract

Background: Neuropeptide Y (NPY) is known to play a role in the regulation of satiety, energy balance, body weight, and insulin release. Interleukin-1beta (IL1B) has been associated with loss of beta-cell mass in type-II diabetes (TIID).

Objectives: The present study attempts to investigate the association of NPY exon2 +1128 T/C (Leu7Pro; rs16139), NPY promoter -399 T/C (rs16147) and IL1B -511 C/T (rs16944) polymorphisms with TIID and their correlation with plasma lipid levels, BMI, and IL1B transcript levels.

Methods: PCR-RFLP was used for genotyping these polymorphisms in a case-control study involving 558 TIID patients and 1085 healthy age-matched controls from Gujarat. Linkage disequilibrium and haplotype analysis of the NPY polymorphic sites were performed to assess their association with TIID. IL1B transcript levels in PBMCs were also assessed in 108 controls and 101 patients using real-time PCR.

Results: Our results show significant association of both structural and promoter polymorphisms of NPY (p<0.0001 and p<0.0001 respectively) in patients with TIID. However, the IL1B C/T polymorphism did not show any association (p = 0.3797) with TIID patients. Haplotype analysis revealed more frequent association of CC and CT haplotypes (p = 3.34 x 10-5, p = 6.04 x 10-9) in diabetics compared to controls and increased the risk of diabetes by 3.02 and 2.088 respectively. Transcript levels of IL1B were significantly higher (p<0.0001) in patients as compared to controls. Genotype-phenotype correlation of IL1B polymorphism did not show any association with its higher transcript levels. In addition, NPY +1128 T/C polymorphism was found to be associated with increased plasma LDL levels (p = 0.01).

Conclusion: The present study provides an evidence for a strong correlation between structural and promoter polymorphisms of NPY gene and upregulation of IL1B transcript levels with susceptibility to TIID and altering the lipid metabolism in Gujarat population.

Fig 1. PCR-RFLP analysis of NPY exon 2 (+1128; T/C), NPY promoter (-399; T/C) and IL1B promoter (-511; C/T) polymorphisms.

(A) PCR-RFLP analysis of NPY exon 2 (+1128; T/C) polymorphism on 2.0% agarose gel: lanes: 1 and 5 show homozygous (TT) genotypes; lane: 2 and 6 show heterozygous (TC) genotypes; lane: 3 shows homozygous (CC) genotype; lane: 4 shows 100 bp DNA ladder. (B) PCR-RFLP analysis of NPY promoter (-399; T/C) polymorphism on 3.5% polyacrylamide gel: lanes: 1, 3 and 4 show heterozygous (TC) genotypes; lanes: 2, 5 and 6 show homozygous (TT) genotypes; lane: 3 shows homozygous (TT) genotype; lane: 7 shows 100 bp DNA ladder. (C) PCR-RFLP analysis of IL1B promoter (-511; C/T) polymorphism on 2.0% agarose gel: lanes: 1 and 6 show homozygous (CC) genotypes; lanes: 2, 3 and 5 show heterozygous (CT) genotypes; lane: 4 shows homozygous (TT) genotype.

Fig 2. Relative gene expression of IL1B in controls and TIID patients.

(A) Expression of IL1B transcripts in 108 controls, 101 TIID patients, as suggested by Mean ΔCp. TIID patients showed significantly increased mRNA levels of IL1B as compared to controls (Mean ΔCp ± SEM: 2.197 ± 0.2777 vs 0.2286 ± 0.3209; p<0.0001). (B) Expression fold change of IL1B transcripts in 108 controls and 101 TIID patients showed 3.92 fold change as determined by 2^-ΔΔCp method. (C) Expression fold change of IL1B transcripts with respect to genotypes of IL1B C/T (rs16944) promoter polymorphism in individuals having n = 28 CC, n = 95 CT, and n = 49 TT. There was no significant difference between CC vs CT genotype, CC vs TT genotype and CC vs CT vs TT genotype (p = 0.8043, p = 0.8403 and p = 0.9585 respectively) [ns = non-significant].

Fig 3. Analysis of fasting blood sugar, plasma lipid levels and BMI in TIID patients and controls.

(A) Correlation of FBS, total cholesterol, triglycerides, HDL, and LDL levels between controls and TIID patients (p<0.0001, p = 0.4104, p<0.0001, p<0.0001, p = 0.0005). (B) Correlation of BMI between controls and TIID patients (p = 0.0011).

Fig 4. Correlation of NPY +1128 T/C, NPY -399 T/C, and IL1B -511 C/T polymorphisms with plasma lipid concentration and BMI.

(A) Genotype-phenotype correlation of NPY +1128 T/C, NPY -399 T/C, and IL1B -511 C/T polymorphisms with total cholesterol (p = 0.6798, p = 0.6704, p = 0.8418 respectively). (B) Genotype-phenotype correlation of NPY +1128 T/C, NPY -399 T/C, and IL1B -511 C/T polymorphisms with triglycerides (p = 0.8648, p = 0.7037, p = 0.4278 respectively). (C) Genotype-phenotype correlation of NPY +1128 T/C, NPY -399 T/C, and IL1B -511 C/T polymorphisms with HDL (p = 0.5064, p = 0.05, p = 0.8936 respectively). (D) Genotype-phenotype correlation of NPY +1128 T/C, NPY -399 T/C, and IL1B -511 C/T polymorphisms with LDL (p = 0.01, p = 0.9289, p = 0.6244 respectively). (E) Genotype-phenotype correlation of NPY +1128 T/C, NPY -399 T/C, and IL1B -511 C/T polymorphisms with BMI (p = 0.7783, p = 0.2092, p = 0.8016 respectively).

Fig 5. Role of NPY -399 T/C, exon 2 T/C and IL1B -511 C/T polymorphisms and IL1B in Type-II diabetes.

NPY -399 T/C, exon 2 T/C SNPs lead to elevated levels of NPY which results in inhibition of glucose-stimulated insulin secretion. Increase in IL1B levels is involved in apoptosis of β cells and impaired insulin secretion and further increase the levels of NPY. Increased blood glucose level causes glucotoxicity which further stimulates macrophages to secrete proinflammatory cytokine IL1B leading to the destruction of β-cells and thereby causing TIID. In Type-II diabetes, chronic hyperglycemia further worsens the condition.