Relationship Between Serum Amyloid A Level and Tanis/SelS mRNA Expression in Skeletal Muscle and Adipose Tissue From Healthy and Type 2 Diabetic Subjects

The institutional ethical committee of the Karolinska Institute reviewed and approved the study protocol. Informed consent was received from all subjects before participation. The clinical characteristics of the subjects are presented in Table 1. Ten male type 2 diabetic patients, with mean duration of disease of 4.8 years (range 2–11), were studied. Patients were treated with diet (n = 1); sulfonylureas (n = 5); metformin (n = 1); a combination of sulfonylurea, metformin, acarbose, and insulin (n = 1); a combination of sulfonylurea and metformin (n = 1); or insulin only (n = 1). The control group consisted of 11 healthy male subjects. All healthy subjects underwent an oral glucose tolerance test (8) to exclude diabetes or impaired glucose tolerance. None of the study participants were smokers or were taking any other medication. The subjects were instructed to avoid strenuous physical activity for a period of 72 h before the experiment. On the test day, subjects reported to the laboratory following an overnight fast and, in the case of the type 2 diabetic patients, before administration of any antidiabetic medication.

Skeletal muscle biopsies were obtained under local anesthesia from the vastus lateralis portion of the quadriceps femoris muscle (9,10). Samples were immediately stored in liquid nitrogen.

Insulin-mediated glucose utilization was determined on a separate occasion using the euglycemic-hyperinsulinemic clamp procedure (11,12). (After baseline blood samples were taken, insulin was infused at a rate of 40 mU · m⁻² · min⁻¹ for 180 min.) Adipose tissue biopsies were taken from subcutaneous abdominal adipose tissue at the level of the umbilicus under local anesthesia (mepivacain chloride 5 mg/ml) in the basal state and after 180 min of insulin infusion by needle aspiration (13).

Plasma glucose concentration was determined using a glucose oxidase method (Beckman Instruments, Fullerton, CA). Serum immunoreactive insulin and C-peptide concentrations were determined using a commercially available radioimmunoassay (Pharmacia, Uppsala, Sweden). HbA_1c was determined by specific ion-exchange chromatography, using a kit (Mono S HR 5/5; Pharmacia, Uppsala, Sweden). The normal range for HbA_1c in our laboratory is <5.2%. Plasma free fatty acid level was determined using a microfluorometric method (14). Serum triglycerides, HDL cholesterol, and LDL cholesterol were assessed by reflectance spectrometry using Kodak Ektachem Clinical Chemistry Slides (Eastman Kodak, Rochester, NY). Serum amyloid A levels were measured using the N Latex SAA kit (Dade Behring, Marburg, Germany).

On a separate occasion, maximal oxygen uptake (Vo_2max) was determined on a bicycle ergometer as described (15). Vo_2max was measured continuously with a breath-by-breath data collection technique (Erich Jaeger, Hoechberg, Germany) and calculated at each 20-s interval.

Skeletal muscle biopsies (25–35 mg) and adipose tissue were removed from liquid nitrogen and homogenized using a polytron mixer in 1 ml guanidium thiocyantate-phenol solution (Sigma Tri-Reagent; Sigma, St. Louis, MO), and total RNA was extracted according to the manufacturer’s instructions (RNAeasy mini kit; Qiagen, Crawley, U.K.). RNA extractions were DNase treated before 1 μg mRNA (per 20 μl cDNA) was reverse transcribed (Reverse transcription system; Promega, Southampton, U.K.). Three microliters of cDNA (corresponding to 0.15 μg of total RNA) were amplified using 300 nmol/l of both primers and probe in the Applied Biosystems Taqman Universal PCR mastermix (Branchburg, NJ) in real-time quantitative PCR, using an ABI PRISM 7900 HT (PE Applied Biosystems, Foster City, CA). The nucleotide sequences are reported below. The cDNA specificity of each primer pair was verified by RT-PCR using RNA put through the cDNA protocol with the omission of reverse transcriptase. For normalization of RNA loading, control samples were run using β2-microglobulin housekeeping gene. Expression levels were quantified by generating a five-point serial standard curve.

Expression of Tanis/AD-015 was compared against β2-microglobulin as a housekeeping gene. Primers for β2-microglobulin were purchased from Applied Biosystems (#4310886E Human β2-microglobulin [huB2M] with VIC reporter). For Tanis/AD-015, the primers and probe were designed using the sequence with accession no. AF157317 (Homo sapiens AD-015) in the National Center for Biotechnology Information database. The AD-015 mRNA coding sequence consists of six exons and is 1,209 bp in total length. The probe was designed to overlap the exon 1/exon 2 boundary to selectively detect cDNA from coding sequence mRNA and not DNA from potential contamination of genomic sequences. Probe: 5′-7AC ACC ACG GTG GGC TCC CTG CT-3′; 7 = 6-FAM; reporter: FAM; and quencher: 6-carboxy-tetramethylrhodamine (TAMRA). Forward primer: 5′-CGA GGG GCT GCG CTT C-3′. Reverse primer: 5′-GAT GTA CCA GCC ATA GGT GGC-3′. These primers generate an amplicon of 64 bp.

Data are presented as mean ± SE. Statistical differences were determined using Student’s unpaired t test or the Wilcoxon’s matched-pairs signed-rank test, as appropriate. The significance of correlations was determined using Pearson’s correlation analysis. P < 0.05 was considered statistically significant.

Subject characteristics are reported in Table 1. Age and BMI were similar between the type 2 diabetic and healthy subjects. Physical fitness as assessed by Vo_2max was not different between the two groups. Insulin-mediated peripheral glucose utilization, achieved during steady-state hyperinsulinemia, was reduced by 50% in type 2 diabetic subjects (P < 0.05 vs. healthy subjects). Serum and LDL cholesterol were lower in the type 2 diabetic subjects (P < 0.05 vs. healthy subjects). The metabolic control of the type 2 diabetic subjects was good (HbA_1c 6.0 ± 0.3%).

A search of the National Center for Biotechnology Information database revealed AD-015 to be the human homologue of Tanis. PCR primers were designed to determine mRNA expression of human Tanis/AD-015. Tanis/SelS mRNA expression in skeletal muscle was similar between healthy and type 2 diabetic subjects (Fig. 1). Furthermore, Tanis/SelS mRNA expression in adipose tissue biopsies was similar between healthy and type 2 diabetic subjects.

Insulin action on Tanis/SelS mRNA expression was determined. To correct the hyperglycemia in type 2 diabetic patients, insulin was infused for ∼60 min before initiating the glucose infusion. Plasma free insulin concentrations during the insulin infusion were comparable between type 2 diabetic and healthy subjects (∼60 mU/l, respectively). Steady-state plasma glucose concentration was maintained at 5.5 mmol/l in type 2 diabetic and healthy subjects. Following insulin infusion, Tanis/SelS mRNA increased in adipose tissue from type 2 diabetic subjects (P = 0.052) (Fig. 2), whereas mRNA levels in healthy subjects were unaltered.

Tanis has been proposed (5) to be a receptor for serum amyloid A. We assessed serum amyloid A concentration in healthy and type 2 diabetic subjects. When all subjects were analyzed collectively, plasma serum amyloid A levels were positively correlated with Tanis/SelS mRNA expression in skeletal muscle (r = 0.51, P < 0.05) (Fig. 3A) and adipose tissue (r = 0.60, P < 0.05) (Fig. 3B). Neither skeletal muscle nor adipose Tanis/SelS mRNA was correlated with glucose, insulin, blood lipid levels, or whole-body glucose utilization.

Tanis was identified (5) as a novel diabetes-associated gene that is downregulated in the liver and adipose tissue of the Israeli sand rat (Psammomys obesus). Tanis encodes a 189–amino acid protein and contains a putative transmembrane domain. Tanis is primarily localized to plasma and microsomal membranes (6). Tanis is highly expressed in three primary insulin target tissues: liver, adipose, and skeletal muscle (5), suggesting a putative role in modulating insulin action and/or glucose metabolism. In diabetic rodents (Psammomys obesus) fasting (24 h) increased hepatic Tanis expression approximately 3-fold, whereas in control animals, only a marginal (1.5-fold) increase was noted (5). Functional studies (6) reveal that adenoviral-mediated overexpression of Tanis in hepatoma cells is associated with decreased insulin-stimulated glucose uptake and glycogen synthesis. The mechanism by which Tanis interferes with insulin action remains to be determined, as no effect on insulin receptor phosphorylation has been reported (6). Given that Tanis is associated with insulin resistance, we have determined Tanis/SelS mRNA expression in skeletal muscle and adipose tissue from type 2 diabetic and age- and weight-matched healthy subjects. Tanis/SelS mRNA expression in skeletal muscle and adipose tissue was similar between type 2 diabetic and healthy subjects. Moreover, plasma serum amyloid A was positively correlated with Tanis/SelS mRNA expression in both muscle and adipose tissue.

Tanis/SelS is one of 25 selenoproteins identified in the human genome and one of only two membrane selenoproteins that has been described (7). Selenoproteins are characterized by the presence of the amino acid selonocysteine, which in several cases has been found in the active sites of the enzyme. Selenoproteins include several glutathione peroxidases and thyroid hormone deiodinases (16). Selenoproteins are thought to be responsible for the majority of the biomedical effects of dietary selenium.

In contrast to the reduction in hepatic Tanis mRNA expression noted in fed diabetic Psammomys obesus rodents, Tanis/SelS mRNA expression was unaltered in skeletal muscle and adipose tissue from overnight-fasted type 2 diabetic patients as compared with control subjects. This could reflect tissue, nutritional, and/or species-specific differences. However, in Psammomys obesus rodents, fasting increased liver expression of Tanis in diabetic, but not in control, animals, such that the net effect was reflected as a similar level of Tanis expression between diabetic and healthy rodents under fasting conditions (5). Because skeletal muscle and adipose tissue biopsies in the present study were obtained from fasted subjects, a putative difference under fed conditions may have been masked.

We determined effect of short-term hyperinsulinemia on Tanis/SelS mRNA expression in adipose tissue obtained during a euglycemic-hyperinsulinemic clamp. During the euglycemic-hyperinsulinemic clamp procedure, fasting hyperglycemia in the type 2 diabetic patients was corrected by an insulin infusion for 60 min before commencing the glucose infusion (11). Thus, all subjects were studied under normoglycemic conditions (5.5 mmol/l). Tanis/SelS mRNA expression was increased under insulin-stimulated conditions in type 2 diabetic subjects, whereas levels were unchanged in control subjects. In vitro studies (5) in cultured cells have provided evidence that insulin and glucose suppress Tanis mRNA. However, these results could be misleading because results from these cell-based experiments were performed in cells incubated in insulin- and glucose-free media, a situation that is impossible to mimic in humans in vivo. In fact, apart from a baseline permissive effect, glucose and insulin do not suppress Tanis/SelS mRNA in adipose cells (5). Rather, our data provide evidence to suggest that in the context of type 2 diabetes, hyperinsulinemia may increase Tanis/SelS mRNA expression. Overexpression of Tanis in hepatoma cells leads to reduced insulin action on glucose uptake and glycogen synthesis (6). Whether increased Tanis/SelS mRNA in adipose cells in human type 2 diabetic subjects contributes to reduced insulin action remains to be determined.

Emerging evidence (17–19) suggests that type 2 diabetes is an inflammatory disorder with elevated circulating concentrations of several acute-phase reactants. Similarly, adiponectin, an adipocyte-derived hormone that has been shown to play important roles in the regulation of glucose and lipid metabolism, is structurally related to the complement 1q family (20), well-known players in the inflammatory processes. The relationship between Tanis mRNA and the acute-phase protein serum amyloid A suggests an interaction between innate immune system responses and Tanis expression in muscle and adipose tissue. Moreover, yeast 2 hybrid studies (5) reveal that Tanis binds serum amyloid A, suggesting that Tanis is the receptor for serum amyloid A. The positive correlation between plasma serum amyloid A and Tanis/SelS mRNA expression identified in the present study further supports this hypothesis. Thus, the serum amyloid A level is a surrogate marker for Tanis/SelS gene expression.

Selenium as a nutrient is related to immune system function (21). Selenium deficiency in AIDS patients is associated with increased mortality (16). Whether Tanis/SelS plays a role in selenium-mediated effects on the immune systems and inflammation in type 2 diabetes is an exciting hypothesis that requires further study.

In summary, Tanis/SelS mRNA is expressed to a similar level in both skeletal muscle and adipose tissue from type 2 diabetic patients and age- and weight-matched healthy subjects. Expression of Tanis/SelS mRNA is positively correlated with serum amyloid A in type 2 diabetic and control subjects, supporting the hypothesis that Tanis/SelS may be the cell-surface receptor for serum amyloid A. Thus, serum amyloid A levels are also a good candidate surrogate marker for Tanis/SelS gene expression. Future studies are warranted to ascertain the functional role of Tanis/SelS in the regulation of glucose homeostasis in humans.

This study was supported by grants from the Swedish Research Council, the Thurings Foundation, the Swedish Medical Association, Tore Nilsons Stiftelse, the Novo-Nordisk Foundation, Harald and Greta Jeanssons Stiftelse, the Swedish Diabetes Association, the Markus and Amalia Wallenberg foundation, and the Swedish Fund for Research without Animal Experiments. H.A.K. was supported by fellowships from the Emil Aaltonen Foundation, the Finnish Academy of Science (Grant 52841), the Finnish Diabetes Research Foundation, the Finnish Medical Foundation, and the governmental subsidy for research of Helsinki University Central Hospital (EVO).

The authors would like to thank Professor Juleen R. Zierath for critical reading of the manuscript.