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More and more evidence indicates that chronic, subclinical inflammation may play an important role in the development of type 2 diabetes mellitus (T2DM) and macroangiopathy. The selenoprotein tanis, had been found in Psammomys obesus (Israeli sand rat), which is a T2DM and metabolic syndrome animal model, whose homologue for human is SelS/AD-015.1 Further studies suggested that tanis was regulated by glucose and had a role in insulin resistance. Serum amyloid A (SAA) produced by liver is an acute phase inflammatory response protein and is elevated in patients with T2DM and atherosclerosis.1-3 Yeast two hybrid method suggested that tanis/SelS was a putative receptor of SAA,1 which was also confirmed by Karlsson et al4 who found that skeletal muscle and adipose tissue of tanis/SelS mRNA expression were positively correlated with serum SAA. The interaction between SelS and SAA may contribute to the development of T2DM and atherosclerosis.
In our previous studies,5 it was found that high expression of SelS gene in ECV304 cells could decrease the injury induced by H2O2. In the following experiment, mRNA expression of SelS in omental adipose tissue biopsies from patients with T2DM and age- and weight-matched nondiabetic patients, the relationship of SelS mRNA with Homa-IR and serum SAA level were analyzed.
METHODS
Subjects
Informed consent was received from all patients before participation. Three male and seven female T2DM patients were studied by treating with oral hypoglycemic administration (n=6) or insulin (n=4). The control group consisting of four male and eight female nondiabetic patients was treated similarly. All subjects suffered from gallbladder polypus or cholecystolithiasis without whole or local inflammation, hypertension, coronary heart disease or cerebrovascular disease. Omental adipose tissue biopsies were obtained during the surgery and immediately stored in liquid nitrogen.
Blood chemistry
Two days before surgery, blood samples were taken from all subjects after an overnight fast. Plasma glucose concentration by glucose oxidase method, serum insulin and C-peptide concentrations by radioimmunoassay, serum triglycerides (TG) and total cholesterol (TC) by enzyme method, low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) by endpoint means, high sensitive C-reactive protein (hs-CRP) by immunoturbidimetry, serum SAA level by the human SAA kit (Intermark Inc., SAN JOSE, CA, USA) were determined as the clinical baselines.
Homa-IR
Homa-IR was calculated according to the formula Homa-IR= FINS×FPG/22.5.
RNA extraction and reverse transcription polymerase chain reaction
Omental adipose samples (0.2 g) stored in liquid nitrogen were homogenized in 200 µl Trizol to isolate total RNA. RNA (1 µl) extraction was reversely transcribed and PCR was performed on 2 µl cDNA products using AMV reverse transcriptase and TaKaRa LA Taq® enzyme respectively (TaKaRa Biotechnology Co. Ltd., Dalian, China). Results of SelS gene expression were reported as arbitrary units and β-actin was used as an internal standard to normalize the amount of cDNA. Primer sequences of SelS gene were designed as follows: forward, 5'-ccgctcgagatggaacgccaagaggagtctctg-3', and reverse, 5'-ccatcgatcataaatctccttgaataaagatgc-3'. PCR conditions were 35 cycles of 94°C for 1 minute, 98°C for 10 seconds, 53°C for 1 minute and 72°C for 1 minute followed by 72°C for 10 minutes. The products were separated in 1.0% agarose gel by electrophoresis.
Sequencing
SelS PCR products were linked to pMD18-T vector. DNA sequencing was performed in Dalian Takara Corporation (China) using BcaBWST primers M13-47 and RV-M.
Statistical analysis
Data are presented as mean ± standard deviation (SD). Logarithmic transformation of SAA data had Gaussian distribution. Statistical differences were determined by Student's independent t test. The significance of correlations was determined by Pearson's correlation analysis. P <0.05 was considered statistically significant. All the data were analyzed by SPSSv10.0.
RESULTS
Subject characteristics are reported in Table 1. Age, body mass index (BMI), TG, TC, LDL-C and HDL-C were similar between the diabetic and nondiabetic groups. Fasting plasma glucose, serum insulin, C-peptide, and CRP were significantly higher in the type 2 diabetic subjects.
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Table 1. Basic data in diabetic and control group |
Electrophoresis of PCR products of SelS and β-actin in omental adipose tissue in diabetic and control groups are shown in Figure 1. The sequence of SelS PCR products (54th to the 1155th nucleotides covering the CDs region) agreed 99% with those in GenBank (Figure 2). Moreover, SelS mRNA expression was not correlated with BMI. SelS mRNA expression, Homa-IR, and serum SAA level in diabetic group were compared with control group (Table 2). All were significantly higher in type 2 diabetic group than in control group. Results of SelS mRNA expression are shown as arbitrary units after standardizing SelS expression to β-actin expression.
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Figure 1. Electrophoresis of SelS and β-actin PCR products in omental adipose tissue in each group. Lines 1 to 10 were PCR products in diabetic group (A). Lines 11 to 22 were PCR products in control group (B). M: DNA marker DL2000. |
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Figure 2. Comparison of sequence of SelS PCR product with NM_018445.4 in GenBank, 99% identities were found from the 54th to the 1155th nucleotides, which covered the CDs region. A was the sequence of SelS PCR product, and B was the sequence of NM_018445.4. SELS transcript variant 2 in GenBank. |
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Table 2. Levels of SelS mRNA, Homa-IR and SAA in different groups |
It was found that SelS mRNA expression was significantly and positively correlated with Homa-IR (Figure 3) similarly for SelS mRNA expression with SAA in each group (Figure 4).
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Figure 3. Relationship between SelS mRNA and Homa-IR in omental adipose tissue. |
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Figure 4. Relationship between SelS mRNA and SAA in diabetic (A) and control groups (B). SAA: serum amyloid A. |
DISCUSSION
Insulin deficiency and insulin resistance are the two key indicators in pathogenesis of T2DM. However, macroangiopathy is the main cause of death in patients with T2DM. Chronic subclinical inflammation is a part of the insulin resistance syndrome6 and diabetes and atherosclerosis may share a common inflammatory basis.7 Tanis may be the link between T2DM and inflammation. Tanis is a cDNA product of 1155 bp composed of six exons and five introns.1 The human homologue for tanis is AD-015/SelS, which encodes a 204 amino acid protein.1 Tanis is localized in plasma and microsomal membranes and highly expressed in three primary insulin target tissues: liver, adipose, and skeletal muscles.1,8 In vitro studies reveal that tanis overexpression could reduce glucose uptake, basal and insulin stimulated glycogen synthesis and attenuate the suppression of PEPCK gene expression by insulin.8 These studies suggested that tanis had a role in insulin resistance. mRNA expression of SelS in omental adipose tissues and Homa-IR were found higher in diabetic subjects than in age- and weight-matched nondiabetic control subjects. Meanwhile, SelS mRNA expression was positively correlated with Homa-IR. The increased expression of SelS mRNA in T2MD subjects might share the same mechanism with the in vitro study and thus contribute to insulin resistance.
SAA is an inflammatory mediator produced by the liver and by adipose tissues.9 Numerous studies suggest that SAA have an important role in the development of atherosclerosis.10-16 Expression of SAA mRNA has been detected in many cells such as smooth muscle cells and vascular endothelial cells in human atherosclerotic lesions.17 Additionally, SAA has a role in preventing inflammation. For example, SAA has been reported to bind to neutrophils and inhibit the oxidative burst, suggesting that it may help prevent oxidative tissue damage during inflammation, but this effect was restricted to lower concentrations of SAA.11
Walder et al1 identified the binding of SAA by tanis using yeast two hybrid screening and suggested that tanis was the receptor for SAA. Karlsson et al4 revealed that tanis/SelS mRNA expression in skeletal muscles and subcutaneous adipocyte was positively correlated with plasma SAA in humans, further confirming that tanis/SelS was the putative receptor for SAA. In our study, the positive correlation between SAA and SelS mRNA expression in human omental cells supported this hypothesis.
SelS is one of 25 selenoproteins identified in the human genome and one of only two membrane selenoproteins that have been described.4 Overexpression of SelS gene in Min6 pancreatic β-cells and ECV304 cells could significantly increase their tolerance to oxidative stress.5,18 SAA had pro- and anti-inflammatory roles. All these imply that tanis and its interaction with SAA might link type 2 diabetes, inflammation and cardiovascular disease.
Adipose tissue is not only a terminal differentiated organ to store and provide energy but also an endocrine and secretory organ to secrete many adipose cytokines and proteins. Among them, leptin and adiponectin are the cytokines expressed specifically in adipose tissues, but IL-6, TNF-α and SelS are not cytokines specifically in adipose tissues.19,20 SAA, insulin and SelS all occur in adipose tissue, so the interaction of SAA with SelS may participate in the inflammatory process of insulin resistance, T2DM and atherosclerosis. However, the mechanism is not clear and further studies are needed.
In summary, SelS gene was detected in human omental adipose tissue by reverse transcription polymerase chain reaction. SelS may also have a role of insulin resistance in Chinese with T2DM. SelS reacted with SAA plays an important role in the development of T2DM and atherosclerosis. Further studies are warranted to study other functions and mechanism of SelS in T2DM.
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