Polymorphisms in the Oxygen-Regulated Protein 150 Gene (ORP150) Are Associated With Insulin Resistance in Pima Indians

The 1,338 subjects are the same subjects analyzed in our previous genomic scan (4). Diabetes status was determined by a 75-g oral glucose tolerance test, and the results were interpreted according to World Health Organization criteria (6). For detailed metabolic testing, nondiabetic, healthy individuals were admitted to our clinical research ward. Oral glucose tolerance was measured after 2–3 days on a weight-maintaining diet. Subjects ingested 75 g glucose, and blood for plasma glucose and insulin measurements was drawn before ingesting the glucose and at 30, 60, 120, and 180 min thereafter. On a different day, subjects also received an intravenous injection of 25 g glucose over 3 min to measure the acute insulin response, as described elsewhere (7). Body composition was estimated by dual-energy X-ray absorptiometry (DPX-1; Lunar Radiation, Madison, WI).

The hyperinsulinemic-euglycemic clamp technique was used to determine basal glucose appearance and insulin-stimulated glucose disappearance (uptake) rates (8). Briefly, insulin was infused to achieve physiologic and maximally stimulating plasma insulin concentrations (137 ± 3 and 2,394 ± 68 μU/ml, respectively) for 100 min for each step. Plasma glucose concentrations were held constant at ∼100 mg/dl by a variable 20% glucose infusion. Tritiated glucose was infused for 2 h before the insulin infusion to calculate rates of postabsorptive glucose appearance and glucose disappearance during the lower dose of insulin infusion. All studies were approved by the Tribal Council and the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases.

Genechip oligonucleotide arrays (Affymetrix, Santa Clara, CA) containing 6,800 genes and 35,000 ESTs were used to compare global gene expression in pools of mRNA extracted from skeletal muscle biopsies of 17 nondiabetic insulin-resistant Pima Indians (five pools) and 18 insulin-sensitive Pima Indians (five pools). Subjects from each pool were matched for age, sex, and percentage body fat. Percutaneous muscle biopsies were obtained from the quadriceps femoris muscle after local anesthesia of skin and fascia with 2% lidocaine. Biopsies were immediately frozen in liquid nitrogen, and RNA was extracted using a ToTally RNA kit (Ambion, Austin, TX).

All 26 exons, including intron/exon splicing sites, the 5′ and 3′ untranslated regions, and the promoter region (900 bp upstream of the first exon) were sequenced in DNA samples from 39 Pima Indians who were not first-degree relatives of the study subjects. Sequencing was performed using the Big Dye Terminator (Applied Biosystems, Foster City, CA) on an automated DNA capillary sequencer (model 3700; Applied Biosystems). Sequence information for oligonucleotide primers is available on the Diabetes website (http://diabetes.diabetesjournals.org/).

Genotyping of single nucleotide polymorphisms (SNPs) in 1,338 subjects was done using the TaqMan assay (Applied Biosystems) for SNPs ORP150-9 and ORP150-12 and pyrosequencing (Pyrosequencing, Uppsala, Sweden) for ORP150-2. The TaqMan genotyping reaction was amplified on a GeneAmp PCR system 9700 (50°C for 2 min; then 95°C for 10 min, 95°C for 15 s, and 62°C for 1 min, for 38 cycles), and fluorescence was detected on an ABI Prism 7700 sequence detector (Applied Biosystems). The pyrosequencing reaction was amplified on a GeneAmp PCR system 9700 (95°C for 10 min; then 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min, for 38 cycles; 72°C for 10 min) and analyzed on PSQ96 sequencer (Pyrosequencing).

Statistical analyses were performed using the statistical analysis system of the SAS Institute (Cary, NC). For continuous variables, the general estimating equation (GEE) procedure was used to adjust for the covariates, including family membership, since some subjects were siblings. Plasma insulin concentrations and rates of glucose disappearance during the low-dose insulin infusion were log-transformed before analyses to approximate a normal distribution. The association of SNP genotypes with diabetes was assessed by analysis of contingency tables.

Genechip oligonucleotide arrays were probed with pooled mRNA extracted from skeletal muscle biopsies of 17 nondiabetic insulin-resistant Pima Indians and 18 insulin-sensitive Pima Indians. The ORP150 gene was more highly expressed in the insulin-resistant pools versus insulin-sensitive pools (signal intensity 433 ± 102 and 233 ± 112, respectively; two-tailed t test, P = 0.02) (Fig. 1).

To determine whether genetic variation in the ORP150 gene itself caused the higher level of its expression in muscle from insulin-resistant Pima Indians, the ORP150 gene was sequenced in 39 Pima Indian DNA samples to detect variation. Twelve SNPs were identified; their positions within the ORP150 gene are shown in Fig. 2. Based on the genotypes from the 39 Pima Indians, ORP150-1, ORP150-4, ORP150-6, ORP150-8, and ORP150-9 are in 100% linkage disequilibrium with each other, whereas ORP150-5, ORP150-7, ORP150-10, ORP150-11, and ORP150-12 are in nearly 100% linkage disequilibrium with each other. In addition, two of the promoter SNPs, ORP150-2 and ORP150-3, are in 100% linkage disequlibrium. Therefore, ORP150-9, ORP150-12, and ORP150-2 were selected as representative SNPs for each unique group of variants and were genotyped in 1,338 Pima Indians. The allele frequencies for these SNPs are given in Table 1. The genotype distributions for all variants were consistent with Hardy-Weinberg equilibrium. The concordance rate between ORP150-2 and ORP150-9 was 81%; between ORP150-2 and ORP150-12, 74%; and between ORP150-9 and ORP150-12, 81%.

There was no association between the genotypes of ORP150-9, ORP150-12, or ORP150-2 with either type 2 diabetes or BMI in the 1,338 Pima Indians. However, among 227 Pima Indians from this group who had normal glucose tolerance and had undergone a hyperinsulinemic- euglycemic clamp study to assess insulin resistance, both ORP150-2 and ORP150-9 were significantly associated with insulin action at physiologic plasma insulin concentrations (Table 2). In addition, both SNPs were significantly associated with insulin action at maximally stimulating insulin concentrations.

Cellular responses to environmental stresses, such as high temperature and glucose or oxygen deprivation, include induction of de novo protein synthesis of stress-associated proteins such as heat shock proteins, glucose-regulated proteins (GRPs), and oxygen-regulated proteins (ORPs). The rat ORP150 has been purified and characterized (9), and the human cDNA encoding ORP150 has been cloned from a hypoxia-treated astrocytoma library (10). Based on its remarkable sequence homology and shared tissue specificity with known GRPs, ORP150 was proposed to be an endoplasmic reticulum (ER)-associated chaperone involved in protein folding. ORP150 has subsequently been shown to be involved in protection against hypoxia/ischemia-induced neuronal death, regulation of tumor angiogenesis, and acceleration of wound healing by modulated intracellular vascular endothelial growth factor (VEGF) transport (11–13).

ORP150, unlike glucose-regulated proteins GRP78 and GRP94, is markedly expressed in MIN6 β-cells under normal oxygen conditions, suggesting that ORP150 plays a role as an ER chaperone in β-cells (5). It has been proposed that ORP150 may mediate the insulin-synthesizing process by assisting the folding of insulin, prohormone convertase, or other proteins entering secretory granules. Furthermore, decreased serum glucose concentrations severely suppress ORP150 expression in the pancreas, which is consistent with a decrease of serum insulin levels (5). This suggests a tight correlation between ORP150 expression and insulin secretion and indicates that abnormalities of ORP150 could potentially contribute to the genetic predisposition for diabetes and its underlying pathophysiological mechanisms.

The human ORP150 gene maps to a region on chromosome 11q23–24 previously found to be linked to type 2 diabetes and BMI (4). In the present study, we report that ORP150 is more highly expressed in skeletal muscle mRNA from insulin-resistant subjects compared with insulin-sensitive subjects. Although insulin resistance is a strong predictor of type 2 diabetes, our association analysis of three SNPs in ORP150 suggest that this gene, by itself, does not appear to be a major susceptibility gene for type 2 diabetes. Nor was the gene strongly associated with insulin secretion, as measured by the acute insulin response to intravenous glucose (P = 0.07 for ORP150–2), despite findings that MIN6 cells, which constitutively secrete insulin, express very high levels of ORP150 and that ORP150 expression and insulin content are markedly reduced in islets of fasting mice (5).

However, among 227 normal glucose-tolerant Pima subjects who were genotyped, ORP150-2 and ORP150-9 were significantly associated with insulin action at physiologic as well as maximally stimulating insulin levels. ORP150-9 is positioned within exon 19 but does not predict an amino acid substitution; therefore, we do not believe this SNP is a functional variant within this gene, but rather assume that it is in high linkage disequilibrium with a nearby functional variant.

ORP150-2 is positioned within the putative promoter region. Our microarray studies indicated that ORP150 is more highly expressed in muscle from insulin-resistant subjects; therefore, it is possible that a variation in the ORP150 promoter is responsible for the altered expression. Three distinct mRNAs are transcribed from the ORP150 gene by use of alternative promoters (14). The three messages start with exon 1A, exon 1B, and exon 2. For each transcript, regulatory sequences just upstream of the start site were essential for transcription. ORP150-2 is located at nucleotide position −429 from exon 1B. Although most transcriptional activity has been proposed to be downstream of nucleotide −332, it has been shown that the region upstream of −332 might carry additional transcriptional regulatory sequences (14). However, the MatInspector V2.2 based on TRANSFAC database (http://transfac.gbf.de/TRANSFAC/) did not reveal any known transcriptional factor binding site in the sequence surrounding ORP150-2. In contrast, the region surrounding ORP150-1 matches a transcriptional binding site for Ik2, a potent transcriptional stimulator (15), and ORP150-4 is positioned within one of several putative Sp1 binding sites. Because ORP150-1 and ORP150-4 are in complete linkage disequilibrium with ORP150-9 (and are therefore associated with insulin resistance), it would be worthwhile to conduct further analyses to explore a possible role of these promoter variants on ORP150 expression.

ORP150-5, which is in 100% linkage disequilibrium with OPR150-12, is positioned within a hypoxia-responsive element. This cis-acting element is responsible for selective induction of transcription from exon 1B upon hypoxia or tunicamycin treatment (14). Despite its seemingly functionally important location, ORP150-5 was not associated with diabetes or insulin action in our study.

Based on these data, we suggest that the human ORP150 gene, through its potential role in influencing insulin action, may have a minor contribution in determining, or modifying, type 2 diabetes in Pima Indians. A mechanism by which altered levels of ORP150 could result in insulin resistance, and therefore increase risk for type 2 diabetes, is unknown. The high sequence homology between ORP150 and GRPs may suggest that ORP150 has some role in responding to glucose levels by altering muscle glucose uptake. However, further genetic studies in other populations, as well as functional studies, are needed to better define the relative importance of these genetic variants. It is our hope that once major susceptibility genes for type 2 diabetes are identified, the roles and mechanisms of minor, modifier genes, such as ORP150, will become clearer for this polygenic disease.