To the best of our knowledge, this report shows the association of rs12617336 and rs17574 DPP4 polymorphisms with HA for the first time. The polymorphisms were also associated with IR (rs12617336 and rs17574), HI (rs12617336), hyperuricemia (rs12617336), high non-HDL-C levels (rs17574) and high apoB levels (rs17574). When compared with non-HA individuals, lower DPP4 levels were observed in the HA group. Differences in DPP4 levels were observed among rs17574 genotypes, with the highest levels in rs17574 AA genotype individuals. Thus, individuals with the G allele presented low levels of DPP4, which agree with the protective effect of this allele.
Adipocytes secret several active proteins called adipokines and it is well-known that the deregulation of their secretion is associated with metabolic diseases. DPP4 is expressed in adipocytes, and in patients with obesity, its expression and activity are elevated. DPP4 plasma levels are related to several markers of obesity, such as BMI, waist circumference, plasma triglyceride levels, leptin concentration, and fat cell volume. In this report, the rs12617336 and rs17574 were associated with a 20–25% risk reduction for the presence of HA, a metabolic abnormality that often coexists with obesity. Moreover, the GTTCG haplotype that contains the rs17574 G protector allele was also related to reduced risk (-25%) of this condition. Neither of the polymorphisms, rs17574 or rs12617336, has been previously associated with HA. Turcot et.al. reported that individuals with rs17574 GG genotype had higher levels of methylation in the promote CpG islands in the visceral adipose tissue. They also showed a positive relationship between methylation levels and HDL-C concentration. Additionally, using informatics tools, important possible functional effects were defined for the two polymorphisms associated with HA. The rs17574 polymorphism, that we report associated with a low risk of HA, is located in exon 2 of the gene; the computer analysis that we carried out establishes that the G allele in this position can affect the efficiency of cutting and splicing, modifying the binding affinity for splicing factors SF2ASF1 and SF2ASF2. This suggests that this polymorphism could be related to the production of DPP4 isoforms with altered activity. Previously, various forms of DPP4 were detected in human and placental plasma[38, 39], as well as in lung cancer tissue and normal tissue[40, 41]. DPP4 isoforms with altered activity could be involved in the development of HA. On the other hand, the rs12617336 G allele produces a binding site for the miRNA-939, miRNA-708, and miRNA-1244, whereas the C allele produces a binding site for the miRNA-1238. It has also been reported that HDLs transport miRNAs[42, 43], some of them regulate crucial pathways for maintaining cardiovascular homeostasis and protect against atherosclerosis by modulating HDL cholesterol efflux capacity and lipid metabolism[45, 46]. These data agree with our results showing that the rs12617336 and rs17574 polymorphisms are associated with a reduced risk for HA. Our findings suggest that rs12617336 C and rs17574 G alleles could be considered as potential genetic markers for HA in our population.
DPP4 enzymatic activity inactivates GLP-1, a hormone that regulates postprandial insulin secretion. Therefore, DPP4 increased level and/or activity may impair insulin sensitivity causing IR and HI. It has been reported that DPP4 serum levels were higher in IR as compared to insulin-sensitive subjects matched for BMI. Moreover, in T2DM it has been shown that increased DPP4 activity is positively associated with IR with a significant increase in IR with rising DPP4 activity quartiles. In the same way, it has been reported that increase DPP4 mRNA levels in the liver of patients with non-alcoholic FL disease correlate with IR.. It has also been reported that DPP4 expression correlates with the amount of VAF, adipocyte size, and inflammation. Macrophages and dendritic cells present in VAF exhibit increased DPP4 expression in response to obese state or inflammation. The best-known non-catalytic function DPP4 exerts is the co-stimulation of T cells through interaction with adenosine deaminase and modulation of the function of antigen-presenting cells. This suggests that DPP4 could have an important participation in the chronic low-grade inflammation present in dyslipidemias, T2DM, IR, obesity, and atherosclerosis. When chronic inflammation is present in liver and adipose tissue, the activated resident macrophages release pro-inflammatory cytokines; by a direct interaction between inflammatory pathways and insulin signaling, these cytokines can cause IR, and in consequently HI. These data support our findings that showed a significant association between the rs17574 and rs12617336 polymorphisms with a low risk for IR and HI (rs12617336). In our study, the carriers of the protective genotype rs17574GG showed the lowest levels of serum DPP4, suggesting that lower DPP4 activity in these subjects would result in higher incretin levels and insulin sensitivity.
Of the five DPP4 polymorphisms included in the present study, three of them have previously been associated with serum lipid levels[51, 52] and apoB levels. Among T2DM individuals, dyslipidemia is a major risk factor for CVD. ApoB plasma levels, the principal protein component of very-low-density lipoprotein (VLDL) remanent, VLDL, and LDL particles, represent the total atherogenic lipoprotein particles in circulation and correlates with the concentration of non-HDL-C. In South Asians, Bayle et al identified and replicated the association between variation at the rs4664443 DPP4 polymorphism and apoB levels. They also report an interaction between the SNP and BMI for plasma apoB levels in Europeans. On the other hand, the DPP4 rs1558957 has been previously associated with high total cholesterol, HDL-C, LDL-C, and triglycerides plasma concentrations in a study of obese individuals of European ancestry, although these results were inconsistent. These previous reports support our findings. Here, we show an association between rs17574 and the presence of high apoB and non-HDL-C levels. Consistent with the observed association between polymorphisms in the DPP4 gene with apoB and non-HDL-C, pharmacological inhibition of DPP4, has been found associated with lower total cholesterol, apoB, triglycerides, non-HDL-C and VLDL levels[55–57].
Uric acid, the product of purine catabolism, is a natural antioxidant. However, it is well recognized that hyperuricemia is associated with metabolic diseases and endothelial dysfunction. Consistent with our observed association between variation in DPP4 polymorphisms and hyperuricemia, Mohandas et al. demonstrated that uric acid inhibits DPP4 activity when it is anchored to the membrane and that the inhibitory effect depends on the redox state of cells and formation of intracellular triuret.
DPP4 is ubiquitously expressed on the surface of several cell types, however, it can also be found in circulation after it is shed from the membrane by proteolytic cleavage. Increased DPP4 circulatory levels, in metabolic diseases, could be explained by an aberrant DPP4 shedding. It has been reported that in smooth muscle cells and adipocytes, hypoxia increased DPP4 shedding by matrix metalloproteases. The constitutive shedding mechanism of DPP4 is varied with cell specificity as well as cells and tissues circumstances and occurs due to a complex interplay between different proteases in cell type-specific manner. Our results showed that non-HA subjects have significantly higher DPP4 concentrations. These findings appear to be contradictory to those expected. However, epigenetic modifications and changes at the DNA level are part of the complex mechanisms that modulate the production of several molecules, including DPP4. As previously mentioned, Turcot et al. in 2011 reported in premenopausal obese nondiabetic women that carriers of the rs17574 GG genotype have higher levels of methylation. Our findings show that carriers of the rs17574 GG genotype have significantly lower concentrations compared to AG and AA genotypes. When stratified by the presence of HA, the statistically significant difference was maintained in the subjects with HA. Individuals with HA, have a higher amount of VAF in comparison with non-HA (159 cm2 vs 134 cm2, respectively, p < 0.001). The aforementioned suggests that in this group, the presence of higher VAF could be associated with an increase in the percentage of methylation in carriers of the rs17574 GG genotype, and thus, lead to lower expression of the DPP4 gene and, therefore, to lower levels in protein plasma.
The main strength of the present work is the inclusion of a large cohort of Mexican individuals, with and without HA, who were broadly characterized from a tomographic, clinical, and biochemical point of view using standardized methods, which allowed adjust the results by an important number of potential confounders. Although the selection of the participants was not random, the studied subjects were not aware of being carriers of the variants of the DPP4 gene polymorphisms studied, given this, it could be expected that the observed associations were similar to those of a sample random and therefore be applied to the general population. Despite these strengths, our study has some limitations. First, due to the cross-sectional nature of the study, we cannot make causal conclusions. Second, our results were not replicated in the second cohort of individuals with and without HA. Thirdly, IR was not determined by the gold standard: the euglycemic clamp; nevertheless, insulin sensitivity can be accurately estimated by the homeostasis model assessment of insulin resistance (HOMA-IR) index that has proven to be a reliable measure. Fourthly, our data may not apply to other ethnicities, considering that the GEA participants are exclusively Mexican-Mestizo subjects. The DPP4 polymorphism associations detected in our study should be investigated in other populations to establish if they are shared with other ethnic groups or are specific for the Mexican population.