Association of the Diabetes Gene Calpain-10 With Subclinical Atherosclerosis : The Mexican-American Coronary Artery Disease Study

The clinical characteristics of the phenotyped offspring generation are shown in Table 1. Weight and insulin-to-glucose ratio at 30 min (IGR30) were significantly different between the men and women but not when standardized to BMI and disposition index (DI)-1, respectively. Carotid intima-media thickness (IMT) was significantly greater in the men.

The overall mean carotid IMT was 0.66 ± 0.12 mm. Haplotype 1221 was significantly associated with decreased carotid IMT, with and without covariate adjustment (Fig. 1A). Haplotype 1112 showed a significant association with increased carotid IMT and marginal significance when age, sex, and BMI were analyzed as covariates.

Given previous reports of the association of the 43-19-63 haplotype combination 112/121 with diabetes (3,4) and diabetes-related traits (5), we specifically analyzed the equivalent 44-43-56-63 haplotype combination 1112/1121 for phenotype associations. Haplotype combination 1112/1121 was significantly associated with increased IMT (P = 0.012). A total of 34 subjects with 1112/1121 had a mean IMT of 0.72 ± 0.16 mm, whereas 275 subjects with any other haplotype combination had a mean IMT of 0.65 ± 0.11 mm (Fig. 1A).

The robust associations of haplotype 1221 and the haplogenotype 1112/1121 with carotid IMT remained significant even when blood pressure or lipid parameters were included as covariates (data not shown).

Haplotype 1112 demonstrated association with sensitivity index (SI) (Table 2). Mean levels indicated that 1112 was associated with significantly reduced insulin sensitivity (Fig. 1B). This association with insulin sensitivity remained significant even when IGR30 or insulin at 30 min–to–insulin at baseline ratio (INSR30) were included as covariates (data not shown).

Haplotype 1112 was associated with IGR30, DI1, and DI2 (P = 0.029, P = 0.0046, and P = 0.0066, respectively), and haplotype 1221 was associated with INSR30 (P = 0.029) (Table 2). Figure 1C illustrates that haplotype 1112 was associated with lower insulin secretion and that 1221 was associated with higher insulin secretion. To further assess CAPN10 association with insulin secretion indexes correcting for insulin sensitivity, besides using DI, we also evaluated the association of CAPN10 with IGR30 and INSR30 by including SI as a covariate. In these models, the CAPN10 associations with IGR30 and INSR30 remained significant (Table 2).

Because diabetes itself and its preclinical abnormalities may predispose to atherosclerosis, we sought to determine whether the association of variation in CAPN10 with carotid IMT was independent of diabetes-related phenotypes. Table 3 shows that the association of haplotype 1221 with carotid IMT was still significant when SI, IGR30, and INSR30, alone or in combination, were analyzed as covariates. The association of the haplotype combination 1112/1121 with carotid IMT was consistently significant under all covariate models (Table 3).

In a nondiabetic population at risk for CAD, we demonstrated an association of variation in the CAPN10 gene with carotid IMT, a measure of subclinical atherosclerosis, and also with insulin sensitivity and insulin secretion, the critical factors contributing to diabetes. Adjusting each haplotype-phenotype association for the other phenotypes showed that CAPN10 was independently associated with each trait. Haplotype 1112 was associated with increased common carotid IMT, insulin resistance, and impaired insulin secretion, whereas haplotype 1221 was associated with favorable levels of these phenotypes. Of note, the haplotype combination 112/121, found by others to increase risk of diabetes and diabetes-related traits (3–5), was also associated with the greatest degree of subclinical atherosclerosis in our population.

Our results provide an additional genetic link between atherosclerosis and diabetes. To date, there has been little evidence for such common genetic determinants identified in the same study population (2). Single nucleotide polymorphisms (SNPs) in the genes for IGF-I, methylenetetrahydrofolate reductase, adiponectin, glucokinase, and the platelet-surface integrin GPIIB-IIIa have been found to be associated with type 2 diabetes and CAD (6–9). In our Mexican-American population, we previously demonstrated an association of LPL haplotypes with CAD and insulin sensitivity (10,11). The present study is unique in that it identifies in healthy adults a gene underlying both subclinical atherosclerosis and pre-diabetic phenotypes.

To our knowledge this is the first report associating CAPN10 with atherosclerosis. Demonstration of an effect of CAPN10 haplotypes on development of overt atherosclerosis in a longitudinal study would provide convincing support to our cross-sectional results. Our results may not apply to other ethnic groups, especially those with a low frequency of haplotype 1112 or 1221. Replication in other populations will be necessary to substantiate CAPN10 as a common genetic determinant of diabetes and atherosclerosis. Our data suggest that CAPN10 affects these conditions independently. CAPN10 haplotypes, in particular 1221 but also 1112, were associated with carotid IMT. These associations persisted even after adjustment for insulin sensitivity and insulin secretion, showing that at least some portion of the CAPN10 effect on carotid IMT is independent of diabetes-related phenotypes. The observed pleiotropic effect of CAPN10 on atherosclerosis and diabetes-related phenotypes is an important observation because diabetes itself would be expected to increase atherosclerosis. Therefore, CAPN10 may affect atherosclerosis both directly and indirectly via diabetes.

Physiological studies using calpain inhibitors suggest that calpains may affect atherosclerosis. Experimental hyperglycemia in rats increased adhesion molecule expression, increased leukocyte adherence to vascular endothelium, and decreased nitric oxide (NO) production; calpain inhibition prevented all these effects and stabilized endothelial NO synthase (12). Calpains also play a role in vascular smooth muscle cell proliferation and migration (13), as well as platelet aggregation, degranulation, and spreading (14).

Experimental work also supports a role for calpains in insulin sensitivity and secretion. Specific inhibition of CAPN10 with antisense expression reduced insulin-stimulated glucose uptake and glucose transporter GLUT4 translocation in adipocytes (15); calpain inhibitors also reduced insulin-mediated glucose transport and glycogen synthesis in mouse muscle strips (16). In mouse pancreatic islet cells, effects of calpain inhibition on insulin secretion depended on duration of exposure, with acute inhibition increasing insulin exocytosis and chronic inhibition decreasing insulin secretion (16,17).

In summary, we demonstrate that variation in CAPN10 is independently associated with subclinical atherosclerosis and physiological determinants of type 2 diabetes. This report of a common genetic determinant of both atherosclerosis and diabetes has the support of compelling functional studies.

The University of California at Los Angeles (UCLA)/Cedars-Sinai Mexican-American Coronary Artery Disease (MACAD) Study enrolled families ascertained through a proband (parent) with documented CAD (11). Two generations were enrolled: 1) the proband and proband spouses (parental generation) and 2) their adult (aged 18 years or older) offspring and the spouses of those offspring (offspring generation). All subjects were genotyped, and only the offspring generation was phenotyped. By design, the offspring were free of diabetes and clinically manifest cardiovascular disease, thus avoiding secondary changes in phenotype caused by overt disease.

All studies were approved by the human subject protection institutional review boards at UCLA and Cedars-Sinai Medical Center. All subjects gave informed consent before participation.

We genotyped SNPs 44, 43, 56, and 63 of CAPN10. SNP-56 is in near-perfect linkage disequilibrium with Indel-19 and thus served as a surrogate for Indel-19 that was compatible with our genotyping technology (3,18). To maintain consistency with the literature in terms of haplotype designations, we represented SNP-56 A as allele 1 and SNP-56 G as allele 2; for the other SNPs, 1 = major allele and 2 = minor allele. Genotyping in 486 subjects from 85 MACAD families was performed using the 5′-exonuclease (Taqman MGB) assay (11,19). PCR primer and oligonucleotide probe sequences are available from the authors. The genotype frequencies for all four markers were in Hardy-Weinberg equilibrium. Linkage disequilibrium among the four markers (D′) ranged from 0.56 to 0.85 (average pairwise D′ of 0.74).

Measurement of carotid IMT using B-mode ultrasound, an oral glucose tolerance test (OGTT), and a hyperinsulinemic-euglycemic clamp were performed on separate days. Of the 365 subjects from the offspring generation, 327 underwent the OGTT, 309 underwent carotid IMT, and 294 underwent the euglycemic clamp at the time of analyses reported herein.

B-mode carotid artery images of the right distal common carotid artery were obtained using a standardized technique (20). The distance between the blood-intima and the media-adventitia echoes, measured by automated computerized edge detection algorithm (Prowin, patent pending), was taken as the IMT measure. The measure of distal common carotid IMT represented the average of ∼80–100 IMT measurements made over 1 cm.

The hyperinsulinemic-euglycemic clamp (21) was performed as previously described with an insulin infusion of 60 mU · m⁻² · min⁻¹ (10). The clamp-derived insulin SI (mg/m² · ml · min⁻¹ · μIU⁻¹) is the mean steady-state glucose infusion rate divided by the body surface area and steady-state insulin level.

The OGTT consisted of baseline glucose and insulin measurements followed by administration of 75-g oral glucose with blood drawn at 30, 60, 90, 120, and 180 min. Insulin secretion indexes were obtained from the OGTT 30-min time-point glucose and insulin measurements to estimate early insulin secretion. The insulin-to-glucose ratio (IGR30, μIU · dL · mg⁻¹ · ml⁻¹) is the 30-min insulin value divided by the 30-min glucose value, and the insulin ratio (INSR30) is the insulin value at 30 min divided by the baseline insulin value (22). We also used the DI, defined as the product of an insulin secretion index with an insulin SI, a measure of β-cell compensation for insulin resistance, based on the demonstrated hyperbolic relationship between insulin sensitivity and insulin secretion (23). We defined DI1 as IGR30 × SI and DI2 as INSR30 × SI.

Haplotypes were determined by the maximum likelihood method, using the simulated annealing algorithm implemented in the program Simwalk2 (24). Five common haplotypes were observed; these were analyzed for genotype-phenotype associations. Haplotype (comprised of SNPs 44, 43, 56, and 63) frequencies were 34.6% for 1121, 31.7% for 1221, 16.7% for 1112, 9.7% for 1111, and 7.2% for 2111.

Log-transformed or square-root–transformed trait values were used to reduce skewness for all analyses. T tests were used to compare trait values between men and women. Mean trait values are given as arithmetical mean ± SD.

Because family data are correlated, we evaluated association using a robust variance estimation approach using the generalized estimating equation (GEE1 ref. 25) to test hypothesized associations between phenotypes and haplotypes, while accounting for familial correlations present in the data. The PROC GENMOD procedure in SAS (version 8.0; SAS Institute, Cary, NC) was used for the analysis using the GEE1 model. Family was taken as the cluster factor, i.e., members from the same family were assumed to be correlated. Analyses were conducted using age, sex, and BMI as covariates unless otherwise specified. Because insulin secretion is influenced by insulin sensitivity, we further assessed the association of CAPN10 with insulin secretion in two ways. First, we evaluated the association of CAPN10 with the DIs. Second, we evaluated CAPN10 association with the insulin secretion indexes including insulin SIs as covariates. For CAPN10 association with carotid IMT, we included SI, IGR30, INSR30, and their combination or products (DI1 or DI2) as covariates.

Given the focus on haplotypes in the literature on CAPN10, we reported the results of haplotype-phenotype associations. Statistical correction for multiple testing was not used because our primary goal was to test the specific hypothesis that diabetogenic CAPN10 haplotypes were associated with carotid IMT. Subsequent analyses focused on elucidating the relationship of these haplotypes with diabetes-related traits and carotid IMT adjusted for these traits.

The Mexican-American Coronary Artery Disease (MACAD) project is supported by National Institutes of Health Program Project Grant HL-60030. Further support was from the Cedars-Sinai Board of Governors’ Chair in Medical Genetics (to J.I.R.) and the Cedars-Sinai General Clinical Research Center Grant RR000425. M.O.G. was supported by National Research Service Award Grant 5 T32 GM08243-16 and the UCLA Specialty Training and Advanced Research Program.