Effect of Common Genetic Variants of Growth Arrest-Specific 6 Gene on Insulin Resistance, Obesity and Type 2 Diabetes in an Asian Population

<div><p>Objectives</p><p>Growth arrest-specific 6 (Gas6), a vitamin K-dependent protein, has been implicated in systemic inflammation, obesity, and insulin resistance (IR). Data from recent studies suggest that polymorphisms in the <i>Gas6</i> gene are associated with cardiovascular disorders and type 2 diabetes (T2D). However, the association of <i>Gas6</i> gene variants with obesity, IR, and T2D development has not been explored.</p><p>Materials and Methods</p><p>Four common single nucleotide polymorphisms (SNPs) in the <i>Gas6</i> gene were genotyped in 984 participants from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family cohort. An insulin suppression test was performed to determine IR based on steady-state plasma glucose (SSPG). Associations between IR indices and obesity, and SNP genotypes, based on previously-reported data for this cohort (Phase I), were analyzed. In the present follow-up study (Phase II), the effects of gene variants of <i>Gas6</i> on the progression to T2D were explored in individuals who were free of T2D in Phase I. The mean follow-up period for Phase II was 5.7 years.</p><p>Results</p><p>The mean age of the study population in Phase I was 49.5 years and 16.7% of individuals developed T2D during follow-up. After adjusting for covariates, three SNPs (rs8191973, rs8197974, and rs7323932) were found to be associated with SSPG levels (<i>p</i> = 0.007, <i>p</i> = 0.03, and <i>p</i> = 0.011, respectively). This association remained significant after multiple testing and showed a significant interaction with physical activity for SNP rs8191973. However, no other significant correlations were observed between <i>Gas6</i> polymorphisms and other indices of IR or obesity. A specific haplotype, AACG (from rs8191974, rs7323932, rs7331124, and rs8191973), was positively associated with SSPG levels (<i>p</i> = 0.0098). None of the polymorphisms were associated with an increased risk of T2D development.</p><p>Conclusions</p><p>Our results suggest that <i>Gas6</i> gene variants are associated with IR, although their effects on subsequent progression to T2D were minimal in this prospective Asian cohort.</p></div

LD between <i>Gas6</i> SNPs in the study cohort.

<p>Two SNPs, rs7323932 and rs8191973, show moderate LD with <i>r</i>² = 0.34, while other SNP pairs are in linkage equilibrium.</p

Statins stimulate miR-33 expression in RAW264.7 macrophages and BMDMs.

<p>The effect of atorvastatin or pivatostatin on relative miR-33 levels in the cell lysate and medium was investigated in Raw264.7 murine macrophages (A,B) and BMDMs (C,D) by quantitative miRNA real-time PCR as described in the Materials and Methods. The histogram shows the relative fold of miRNA level compared with the control group (set as 1). Results are expressed as mean±SEM of at least three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 versus control group.</p

The levels of ABCA1 protein expression in RAW264.7 macrophages and BMDMs treated with statins.

<p>Raw264.7 murine macrophages were treated with atorvastatin or pitavastatin at the indicated dosages for 24 h (A). The levels of ABCA1 protein expression were determined by western blot analysis as described in the Materials and Methods. BMDMs were treated with atorvastatin or pitavastatin at 1 and 10 μM for 24 h (B). Results represent mean±SEM of at least three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 versus control group.</p

Relative plasma levels of miR-33, miR-103, and miR-155 in subjects before and after treatment with atorvastatin and pitavastatin.

<p>Total RNA was isolated from human plasma and RNA concentrations were determined using a Nanodrop 1000 spectrophotometer. cDNA was generated from the RNA and microRNA assays were performed by real-time PCR using the specific primers for human miR-33a, miR-103, and miR-155. Expression levels of cel-miR-39, RNU6B, and mir-16 were used as the endogenous controls for assays of miR-33, miR-103, and miR-155, respectively. To quantify the relative expression level of each miRNA, threshold cycle (Ct) values were normalized against endogenous reference (ΔCt = Ct (target miR)−Ct (endogenous control)). Expression levels of human plasma miRNAs were calculated by the 2^−ΔCt method. Each microRNA was assayed in triplicates.</p

LD between <i>Gas6</i> SNPs in the study cohort.

<p>Two SNPs, rs7323932 and rs8191973, show moderate LD with <i>r</i>² = 0.34, while other SNP pairs are in linkage equilibrium.</p

Glucose stimulates miR-33 expression in RAW264.7 macrophages and BMDMs.

<p>The effect of glucose on miR-33 levels in the cell lysate and medium was investigated in Raw264.7 murine macrophages (A) and BMDMs (B) by quantitative miRNA real-time PCR. Trizol reagent was used to extract total RNA in cellular lysates and medium from macrophages treated with glucose from 5 to 30 mM. Preparation of microRNA was isolated using the mirVANA microRNA isolation kit and microRNA assays were performed by real-time PCR. The relative miR-33 expression was calculated via the 2^−ΔΔCt method using cel-miR-39 as an endogenous control. The histogram shows the relative fold compared with the control group (set as 1). Results are expressed as mean±SEM of at least three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 versus control group.</p

<i>ABCA1</i> 3'-UTR activity and relative miR-33 expression after statin treatment in RAW264.7 macrophages and BMDMs.

<p>Reporter containing sequence complementary to miR-33a in 3'-UTR of <i>ABCA1</i> was constructed in downstream of the luciferase gene of the pGL3-<i>ABCA1</i> plasmid (A). Macrophages were transfected with the pGL3-<i>ABCA1</i> plasmid and/or statins for 24 h, and then assayed of luciferase activity. A pCMV-β-galactosidase vector encoding β-galactosidase was co-transfected as an internal control. Dose-dependent effects of atorvastatin and pitavastatin on the luciferase activity were analyzed in RAW264.7 macrophages (B). The luciferase activity was normalized to β-galactosidase activity. Data are expressed as ratios relative to the values obtained from the untreated pGL3-<i>ABCA1</i> control group defined as 1.0. The mRNA levels of <i>ABCA1</i> were determined by semi-quantitative RT-PCR (C) and quantitative real-time PCR (D) as described in the Materials and Methods. The histogram shows the relative fold of <i>ABCA1</i> mRNA levels compared with the control group (set as 1). Results are expressed as mean±SEM of at least three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 versus control group.</p

Glucose and statins decrease cholesterol efflux from macrophages.

<p>(A) RAW264.7 macrophages were plated with 0.5 μCi/mL [1,2-³H]cholesterol, 10 μg/mL acetylated low-density lipoprotein, and 30 μg/mL of cholesterol in DMEM containing 0.2% BSA with 25 μg/mL lipid-free apolipoprotein A1 (apoAI) for the indicated time period (n = 4). Cholesterol efflux was expressed as the percentage of radioactivity in the medium relative to the total radioactivity (medium and cell lysate). (B) RAW264.7 macrophages were treated with different concentrations of glucose as indicated for 24 h in present or absent of apoAI. The effect of glucose on cholesterol efflux was measured as described in (A). RAW264.7 macrophages (C) or BMDMs (D) were treated with atorvastatin (ATV) or pitavastatin (PTV) at 1 and 10 μM in the present or absent of lipid-free human apoA1 acceptor for 24 h and the apoA1-mediated cholesterol efflux was calculated as described in (A). Results represent mean±SEM of at least three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 versus control group.</p

Summary of characteristics in Phase I and Phase II studies.

<p>Data presented as mean ± standard deviation unless otherwise indicated. BMI: body mass index, DM: diabetes mellitus, FPG: fasting plasma glucose, FPI: fasting plasma insulin, HOMA-IR: homeostasis model assessment of insulin resistance, SSPG: steady-state plasma glucose, T2D: type 2 diabetes.</p><p>Summary of characteristics in Phase I and Phase II studies.</p