The Functional Q84R Polymorphism of Mammalian Tribbles Homolog TRB3 Is Associated With Insulin Resistance and Related Cardiovascular Risk in Caucasians From Italy

Insulin resistance is pathogenic for dyslipidemia, cardiovascular disease (CVD), and type 2 diabetes (1). Both environmental and genetic factors modulate insulin resistance, with the latter being mostly unknown (2). Akt, a serine/threonine protein kinase, is a key mediator of insulin signaling (3); impaired Akt activity is involved in human and animal models of insulin resistance (4–6). Thus, mediators of Akt activity are candidates for insulin resistance (7). The newly identified TRB3 gene (a mammalian tribbles homolog, also known as TRIB3/NIPK, gene ID 57761) has been reported by most (8,9), although not all (10), studies to affect insulin action by binding to and inhibiting Akt phosphorylation and to play a role in insulin resistance (8,9). TRB3 is located on the 20p13 human chromosome region that has been associated with type 2 diabetes (11,12). TRB3, therefore, is a candidate gene for insulin resistance (7).

By resequencing the coding region of TRB3, five missense and two synonymous variants were identified; four were novel and three already available from the National Center for Biotechnology Information (NCBI) database of single nucleotide polymorphisms (dbSNP). Two additional synonymous variants already available from the NCBI dbSNP were not found (Table 1). Haplotype block partitioning showed that the three polymorphisms with minor allele frequency (MAF) >5% (Q84R, Y111Y, and A323A) were not gathered together into a single block. Strong, although not complete, linkage disequilibrium was observed between Y111Y and A323A (D′ = 0.67; r² = 0.4), leaving an unlinked missense Q84R variant. Because of either their extremely low MAF (four missense) or their unlikely biological significance (two synonymous), only the prevalent missense Q84R variant, which causes the substitution of a polar uncharged amino acid with a charged one, was further tested for association with insulin resistance phenotypes.

We genotyped Q84R polymorphism in 178 nondiabetic Caucasian unrelated offspring of type 2 diabetic patients residing in Rome or surrounding areas (Table 2). Insulin sensitivity, as measured by euglycemic-hyperinsulinemic clamp (13), was reduced from Q84Q to Q84R and R84R individuals. Significant changes were also observed in serum insulin, triglycerides, and HDL cholesterol across the three genotypes. Differences in serum insulin and HDL cholesterol were no longer significant when waist circumference (but not BMI) was added into the model, thus suggesting that adipose distribution may modulate TRB3 effects on some insulin resistance–related abnormalities. This is also indicated by the observed interaction between Q84R polymorphism and waist circumference in modulating lean glucose disposal rate. In fact, the expected negative correlation between the two variables was stronger in Q84R and R84R (r = −0.47 and −0.40, respectively) than in Q84Q (r = −0.13) individuals (P for interaction <0.05). We then genotyped Q84R polymorphism in 605 unrelated nondiabetic Caucasians from Gargano (Table 2). Serum insulin level, but not lipid profile, was significantly different across the three genotype groups and higher in R84R individuals, independently of BMI and waist circumference. Worth noting is that individuals from the first sample were offspring of type 2 diabetic patients and, therefore, highly predisposed to insulin resistance because of environmental and/or genetic factors (14). It can be hypothesized that with such a strong unfavorable background, a single copy of the R84 variant (as carried by Q84R heterozygous individuals) is also sufficient to determine insulin resistance. This does not seem to be the case in the second sample, which resembles the general population, where insulin resistance was observed only in homozygous R84R individuals. Insulin resistance may predispose to CVD, particularly among type 2 diabetic patients (15). Accordingly, we studied 716 type 2 diabetic patients (Table 3) from the Gargano area and subgrouped them according to presence of insulin resistance–related cardiovascular risk factors, namely abdominal obesity (waist circumference >88 and >102 cm for woman and men, respectively), dyslipidemia (total cholesterol ≥200 mg/dl and/or HDL cholesterol ≤40 mg/dl in men and 50 mg/dl in woman, and/or triglycerides ≥150 mg/dl or currently on lipid-lowering treatment), and hypertension (systolic blood pressure ≥130 mmHg or diastolic blood pressure ≥85 mmHg or currently on antihypertensive treatment). A total of 356 patients showed the simultaneous presence of three risk factors (very-high-risk subgroup), while 360 patients did not (two or fewer risk factors). The proportion of R84R patients was higher in the very-high-risk group (14 of 356 [3.9%] vs. 7 of 360 [1.9%]; age- and sex-adjusted OR 3.1 [95% CI 1.2–8.2], P = 0.02). A second independent cohort of 100 consecutive type 2 diabetic patients who had suffered myocardial infarction (16) was also genotyped. The age at myocardial ischemia decreased from 58 ± 9 years in Q84Q (n = 79) to 55 ± 10 years in Q84R (n = 18) and to 47 ± 11 years in R84R (n = 3) individuals; P = 0.03 after adjusting for the following three clinical variables that were independently associated with age at myocardial ischemia: sex (P = 0.03), hypertension (P = 0.03), and dyslipidemia (P = 0.05). Insulin resistance may also predispose to type 2 diabetes (1). Accordingly, we compared the distribution of the TRB3 Q84R genotype in the above-described 716 type 2 diabetic patients with that in 330 unrelated nondiabetic control subjects (117 men and 213 women with fasting plasma glucose <6.1 mmol/l, no medications known to affect glucose and lipid metabolism, and absence of systemic diseases) within the same age range (35–76 years) who were recruited in the same region (Gargano and surrounding area) and were part of the previously described sample of 605 individuals. The Q84Q, Q84R, and R84R genotypes, respectively, were carried by 504 (70.4%), 191 (26.7%), and 21 (2.9%) diabetic patients and by 242 (73.4%), 80 (24.2%), and 8 (2.4%) control individuals. The observed tendency of an increased risk of type 2 diabetes for 84R carriers, although compatible with the above-reported association with insulin resistance–related phenotypes, is not significant in the present sample size (P = 0.61). To test the functional role of TRB3 variants on the S⁴⁷³Akt phosporylation, either Q84 or R84 TRB3 full-length cDNAs were transfected in human HepG2 hepatoma cell line. A progressive reduction of insulin-induced S⁴⁷³Akt phosphorylation was observed from untransfected control to Q84- and R84-HepG2 cells, with no change of Akt protein expression (Fig. 1A). Comparable expression levels of the two transfected TRB3 variants were ascertained in cell lysates by immunoprecipitation and blotting with anti-Myc antibody (Fig. 1). On average (n = 3 experiments), insulin effect was significantly (P < 0.05) reduced by 22% in Q84-HepG2 with respect to control cells (Fig. 1B). As compared with Q84-HepG2, a further 23% reduction of Akt phosphorylation was observed in R84-HepG2 cells (Fig. 1B) (P < 0.05 vs. Q84-HepG2 and P < 0.01 vs. control cells). In contrast, in these cell lines, no significant change was observed in the expression of both total Akt and anti-Myc immunoprecipitated TRB3 (P = 0.57 and P = 0.87 by one-way ANOVA, respectively; n = 3 experiments). We here describe for the first time that a functional missense variant of the TRB3 gene (i.e., R84) is associated with several insulin resistance–related abnormalities, including hyperinsulinemia, dyslipidemia, and CVD. This association has been confirmed in several independent samples, making the risk of a false-positive result very unlikely. At variance, at least with the present sample size, no significant association was found with type 2 diabetes. As compared with wild-type Q84, the R84 variant introduces a significant charge change. In addition, R84 variant has a stronger inhibitory activity on insulin-stimulated Akt phosphorylation, a finding that provides a convincing biological explanation for the observed association between the Q84R polymorphism and insulin resistance. Although a comprehensive survey of variations also in the regulatory regions of the gene is certainly needed to deeper understand its allelic structure and several large studies are necessary to better define the specific role of Q84R variant (and possibly other variants) of TRB3 gene, our preliminary findings strongly suggest that the R84 variant contributes to the complex genetic background of insulin resistance and may help identify individuals who are at risk for insulin resistance and related cardiovascular risk at whom preventive programs could be specifically targeted.