Effects of resistin on skeletal glucose metabolism

Abstract

Resistin is an adipokine highly related to insulin resistance (IR). The purpose of our research was to investigate how resistin influences skeletal glucose metabolism and explore its mechanisms. We constructed the recombinant plasmid pcDNA3.1 expressing resistin and then transfected it into C2C12 myocytes. The expression of resistin in C2C12 myocytes was detected by Western blotting. Glucose uptake was measured by ³H labeled glucose; glucose oxidation and glycogen synthesis was detected with ¹⁴C-labeled glucose. GLUT4 mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR). We observed that resistin was expressed in transfected myocytes, and resistin decreased insulin induced glucose uptake rate by 28%–31% and inhibited the expression of GLUT4 mRNA. However, there was no significant difference in basal glucose uptake, and glucose oxidation and glycogen synthesis remained unchanged in all groups. It is concluded that resistin inhibits insulin induced glucose uptake in myocytes by downregulating the expression of GLUT4 and it has no effects on glucose oxidation and glycogen synthesis. Our findings may provide a clue to understand the roles of resistin in the pathogenesis of skeletal IR.

Keywords

resistin / insulin resistance / skeletal muscle

Introduction

Resistin, discovered and named by Steppan in 2001, is an adipokine highly related to insulin resistance (IR) [1]. Studies have shown that resistin affects glucose metabolism. For example, blood sugar increases by 28% compared to the control group after intraperitoneal injection of purified recombinant resistin; resistin causes insulin-induced glucose uptake of 3T3-L1 adipocytes to drop 37% [1]. Skeletal muscle is the main tissue where glucose metabolizes; decreases in skeletal sensitivity result in IR [2]. Resistin affects glucose metabolism, but the underlying mechanism in skeletal muscles is unclear. Our study was to investigate the effects of resistin on glucose metabolism and IR in myocytes with recombinant eukaryotic expression vectors.

Materials and methods

Materials

Gibcobrl, pGEM-T Vector system I, was obtained from Promega (USA), pcDNA3.1 from Invitrogen (USA), SuperFect Transfection Reagent from QIAGEN (Germany), HRP-conjugated anti-guinea IgG (heavy and light) from Jingmei Co., Ltd. (China), 3-isobutyl-1-methylxanthine, Cytochalasin B, and benzethonium hydroxide from Sigma (USA), Humulin R from Lilly (USA), ³H-2-deoxyglucose (radioactivity 5.4 Ci/mmol) from China Isotope Corporation, and D-[¹⁴C(U)] glucose (radioactivity 360 mCi/mmol) from NEC (Japan).

Construction of recombinant eukaryotic expression vector pcDNA3.1-resistin

Based on the published mRNA sequence for mouse resistin, primers were designed to amplify the coding region of resistin mRNA. The upstream primer is 5'-CGGAATTCATGAAGAACCTTTCATTTCC-3' (where the underlined part is an EcoR I restriction site) and the downstream primer is 5'-CTAGTCTAGATCAGGAAGCGACCTGCAGCT-3' (where the underlined part is an Xba I restriction site). RNA was extracted using TRIzol reagent from mouse adipose tissue according to the manufacturer’s instructions. Resistin cDNA was subsequently synthesized with the RT-PCR system under optimized conditions. The cDNA was cloned into a pGEM-T vector, double-digested with EcoR I and Xba I, and the resistin cDNA was again cloned to pcDNA3.1 vector. The resultant recombinant eukaryotic expression plasmid was then submitted for sequencing.

Transfection of C2C12 myocytes

According to the instructions included with the SuperFect Transfection Reagent, C2C12 myocytes were transfected with pcDNA3.1-resistin through liposomes. The transfected clones were selected by addition of G418, with a final concentration of 500 μg/mL. Resistin in C2C12 myocytes was assayed by Western blotting.

Grouping of cells

Four groups of cells were used in the following experiments: control (non-transfected) group, transient (transiently transfected pcDNA3.1-resistin) group, vector (transfected real-timely with pcDNA3.1) group, and stable (stably transfected with pcDNA3.1-resistin) group. Three hours after transient transfection, the culture medium of 10% fetal bovine serum (FBS) and high glucose Dulbecco’s modified Eagle’s medium (DMEM) was replaced with horse serum high glucose DMEM culture medium to induce C2C12 myocytes to differentiate into myotubes. The resistin protein expression of the four groups was measured by Western blotting.

Glucose metabolism assay

Each of the four groups was divided into two sub-groups: cells treated with or without 100 nmol/L insulin. All assays were performed six times.

Detection of the uptake of glucose

5 × 10⁵ cells were inoculated in a six-well plate, cultured in 10% FBS high glucose DMEM culture medium until the cells reached 60%-70% confluence, and then the C2C12 myocytes were induced to differentiate into myotubes. The medium was replaced with serum-free, antibiotic-free minima essential medium (MEM) containing 5 mmol/L glucose, and the cultures were incubated at 37°C in 5% CO₂ for 30 min. The medium was discarded, and the cells were washed twice with Krebs Ringer phosphate buffer (KRPB). One milliliter of KRPB was added to each well, and the plates were incubated at 37°C with 5% CO₂ for 30 min. In sub-groups, appropriate insulin was added, and the plates were incubated at room temperature for 30 min. To all wells, 1 µCi[3H] deoxyglucose was added at room temperature for 10 min, and then cytochalasin B (to a final concentration of 10 µmol/L) was added to terminate glucose uptake. The cells were washed with cold phosphate buffered saline (PBS) twice, and lysed with 0.2 mL 0.05% NaOH for 20 min. The contents from each well were collected and dotted on Whatman chromatography paper. The chromatography paper was placed into a liquid scintillation vial with a liquid scintillation cocktail and the radioactivity was measured with a liquid scintillation counter. The amount of glucose uptake was calculated as the specific radioactivity of 1 µCi[³H] deoxyglucose and dpm (Pmol/10⁶ cells per 10 min). For example, if a six-well plate contained 2 × 10⁶ cells per well, the glucose uptake would be the measured dpm/ (5.4 Ci/mmol × cell counts).

Detection of glucose oxidation

2 × 10⁵ cells were inoculated in a 12-well plate, cultured in 10% FBS high glucose DMEM culture medium until the cells reached 60%-70% confluence, and then C2C12 myocytes were induced to differentiate into myotubes. The medium was replaced with 1 mL fresh serum-free, antibiotic-free DMEM containing 5.5 mmol/L glucose, and the cells were cultured for 3 h. The medium was then replaced with 1 mL fresh serum-free, antibiotic-free DMEM containing 0.1 µCi/mL D-[U-¹⁴C] and 5.5 mmol/L glucose with or without 100 nmol/L regular insulin (depending on the sub-group). Each well was covered with a hyamine hydrozide-soaked Whatman GF/C chromatography paper, stacked on top of a plastic pad with a cap and was cultured for an additional 2 h. To each well, 0.2 mL 70% perchloric acid was added to terminate the reactions while being gently agitated at 37°C for 1 h. This allowed the chromatography paper to absorb CO₂ generated by oxidization. The paper was dried and placed in scintillation vials to which 3 mL liquid scintillation cocktail was added. Radioactivity was measured using a scintillation counter.

Measuring glycogen synthesis

1 × 10⁵ cells were inoculated in a 24-well plate and cultured in 10% FBS DMEM until 60%-70% cells were confluent; C2C12 myocytes were induced to differentiate into myotubes. The medium was discarded, and the cells were washed twice with PBS. Fresh serum-free, antibiotic-free MEM containing 5 mmol/L glucose was added and the cells were cultured for 18 h. Cells were washed twice with PBS, and then fresh serum-free, antibiotic-free DMEM containing 0.5 µCi/mL D-[U-¹⁴C] and 5.6 mmol/L glucose was added (with or without 100 nmol/L regular insulin, depending on the sub-group) and incubated for 90 min. Cells were washed twice with cold PBS, and 0.1 mL 30% NaOH was added to each well at 95°C and kept for 30 min to lyse the cells. The content of each well was dotted on Whatman filter paper, which were then dried and placed in cold 66% ethanol overnight at 4°C. The paper was washed again with 66% ethanol and dried completely. They were then placed into scintillation vials with 3 mL liquid scintillation cocktail, and the radioactivity was measured using a liquid scintillation counter. Glucose oxidation and glycogen synthesis was also detected by the specific radioactivity of 1 µCi[³H] deoxyglucose and dpm (pmol/10⁶cells·h). dpm/(305mCi/mmol × cell counts) was to stand for its amount.

RT-PCR of GLUT4 mRNA

Total RNA of C2C12 myocytes was extracted by TRIzol reagent; RT-PCR was performed according to the instructions of THERMOSCRIPTTM RT-PCR System under optimal conditions. Products of PCR were measured by gel electrophoresis to calculate GLUT4 mRNA/β-actin mRNA for relative amounts (primers are listed as follows: forward primer for GLUT-4: 5'-GCCCGAAAGAGTCTAAAGCG-3'; reverse primer for GLUT-4: 5'-ACTAAGAGCACCGAGACCAA-3'; forward primer for β-actin: 5'-CTCTTTGATGTCACGCACGATTTC-3'; reverse primer for β-actin: 5'-ATCGTGGGCCGCTCTAGGCACC-3').

Statistical analysis

Data were expressed as mean±standard deviation (s) and comparisons between groups were performed using analysis of variance (ANOVA). Data were analyzed using SPSS software version 11.0; P<0.05 indicated statistical significance.

Results

Construction of recombinant eukaryotic expression vector pcDNA3.1-resistin

Gel electrophoresis of total RNA of mouse adipose tissue showed no degradation of RNA (Fig. 1). Gel electrophoresis of RT-PCR products showed a band between 250 bp and 500 bp, matching the expected 363 bp resistin cDNA (Fig. 2). The results of the restriction digestion of recombinant vector pGEM-T-resistin demonstrated that the PCR products of resistin cDNA were successfully cloned into pGEM-T (Fig. 3). DNA sequencing confirmed that the sequence of the inserted DNA segment matched the mouse resistin mRNA coding region, indicating successful construction of a recombinant eukaryotic expression vector, pcDNA3.1-resistin, carrying the mouse resistin gene.

Evaluation of cell differentiation

On the sixth day of differentiation of the C2C12 myocytes, 90% of the cells that fused and became filamentous (Fig. 4) and myotubes inside the cells were noted (Fig. 5).

Western blot assay

The transient and stable groups both expressed resistin protein while the control and vector groups had no expression. Thus, pcDNA3.1-resistin was successfully transfected into transient and stable cells, and resistin protein was expressed (Fig. 6).

Measurement of glucose metabolism

There was no significant difference in glucose uptake at baseline conditions between the control, transient, vector, and stable groups (P>0.05; Table 1). A significant difference in insulin-stimulated glucose uptake was noted (P<0.05). Glucose uptake rates were 7.81±0.87, 5.63±1.56, 7.88±1.50, and 5.79±1.26 in the control, transient, vector, and stable groups, respectively. The glucose uptake rates in the transient and stable groups were lower than the control and vector groups by approximately 31%, respectively, indicating that resistin could inhibit insulin-stimulated glucose uptake in myoblasts.

Measurement of glucose oxidization

There was no significant difference in glucose oxidization at baseline conditions among the control, transient, vector, and stable groups (P>0.05; Table 2), indicating that resistin had no observable influences on insulin-stimulated glucose oxidization in C2C12 myocytes under these conditions.

Measurement of glycogen synthesis

There was no significant difference in glycogen synthesis at baseline conditions between the control, transient, vector, and stable groups (P>0.05; Table 3), indicating that resistin had no observable influences on insulin-stimulated glycogen synthesis in C2C12 myocytes under these conditions.

Measurement of GLUT4 mRNA

The relative amount of GLUT4 mRNA was 0.73±0.11, 0.53±0.16, 0.69±0.08 and 0.49±0.09 (P = 0.041, n = 3) in the control, transient-transfected, vector-transfected and stable vector-transfected groups. It showed that resistin inhibited GLUT4 mRNA expression in C2C12 myocytes (Fig. 7, Table 4).

Discussion

Since no murine resistin was commercially available when this study was performed, one was created to investigate its effects on glucose metabolism in C2C12 myocytes. C2C12 myocytes are mouse myoblasts that, when cultured in vitro, can be induced to exit the cell cycle by controlling the ingredients of the medium. Thus, the cells can differentiate into myotubes. This makes them a widely used cell model to study functions of skeletal muscle cells [3]. C2C12 myocytes were successfully differentiated into myotubes using DMEM containing 5% horse serum in this study.

There are two ways of expressing protein post-transfection: transient and stable. In transient transfection, recombinant vectors are introduced into cells to obtain the target gene in temporal but high-level expression. Since it is preferable to measure gene expression one to four days after transfection but C2C12 requires six days of induction to generate myotubes, G418 cells were used to screen stably transfected clones [4]. Transfection was monitored by Western blot, and it was found that the C2C12 cells all expressed resistin, regardless of the type of transfection with pcDNA3.1-resistin.

Both transiently and stably transfected cells had lower glucose uptake than the cells in the control and vector groups, suggesting that resistin repressed insulin-induced glucose uptake in C2C12 myocytes. This is similar to the results reported by Moon [5]. Moon et al. used various amounts of resistin (0-500 nmol/L) to culture L6 rat skeletal muscle cells and found that 100 and 500 nmol/L resistin completely inhibited insulin-induced glucose uptake. Moon et al. also reported that 10 and 50 nmol/L resistin reduced glucose uptake by 10%-14%. Fans [6] had similar findings.

Insulin-induced glucose uptake occurs via binding of insulin to the α sub-functional group resulting in self-phosphorylation of the tyrosine group of the β subgroup. In turn, multiple tyrosine groups of the insulin receptor substrates are phosphorylated and PI3-K and mitogen-activated protein kinase (MAPK) are activated, and then glucose transporters translocate to the plasma membrane and induce glucose to enter cells. The exact pathway by which resistin inhibits glucose uptake in C2C12 myocytes is unclear. Nonetheless, our study found that resistin inhibited insulin-induced glucose uptake of C2C12 myocytes, indicating that resistin might reduce GLUT4 or weaken its function by affecting some links in an insulin-regulated pathway. Resistin may reduce GLUT4 by controlling the activity of PI3-K in C2C12 myocytes to lower insulin-induced glucose uptake. Moon [5] reported that resistin inhibited glucose uptake of L6 cells neither by influencing translocation of GLUT4 to plasma membrane nor by affecting phosphorylation of IRS-1 tyrosine, but rather by affecting the activity of the p85 subgroup of PI3-K so that the function(s) of translocacted GLUT4 was/were attenuated. Palanivel [7] reported that resistin reduced the translocation of GLUT4 to the cell surface of L6 rat skeletal cells and consequently inhibited insulin-induced glucose uptake by regulating IRS-1 and protein kinase B (PKB/Akt).

Although our study found that resistin can inhibit insulin-induced glucose uptake in C2C12 myocytes, resistin had no observable impact on either cell basal or insulin-induced glucose oxidization or glycogen synthesis. One reason for it could be that glucose uptake and glucose oxidization occurred or glycogen synthesis belonged to different glucose metabolism processes. Glucose uptake relies on the amount and activity of GLUT4 whereas glucose oxidization and glycogen synthesis hinge on restricting enzymes and their activity. Resistin can reduce glucose oxidization by inhibiting the Krebs citric acid cycle, and resistin can also reduce insulin-induced glycogen synthesis by inhibiting glycogen synthesis kinase-3 beta (GSK-3β) [8].

There are various conclusions from previous studies regarding the impact of resistin on cellular glucose metabolism. Dietzed [9] co-cultured human skeletal muscle cells with human adipose cells and found that cell factors secreted by adipocytes caused IR in skeletal muscle cells. Resistin is adipose-specific in rodents [1], but in humans it is predominantly expressed in monocytes/macrophages, and its expression is very low on the surface of skeletal muscle cells [10-12]. Resistin is implied to have involvement in the pathogenesis of IR in skeletal muscle cells.

The effects of resistin on cellular glucose metabolism are very complicated, and the findings are not consistent [13]. Enzyme-facilitated conversion of intracellular glucose to glycogen is one of the major limiting steps of glucose metabolism [14]; however, the present study indicated that resistin only inhibits insulin-induced glucose uptake in C2C12 myocytes, but has no effect on glucose oxidization or glycogen synthesis. Hence, it is inferred that resistin regulates glucose metabolism not via skeletal muscle cells, but by other tissues. Studies by Banerjee [15], Evan [16] and Satoh [17] support our hypothesis. Resistin-induced malfunction of glucose metabolism is mainly due to increase of hepatic glucose output, which is in accord with our further study [18]. Resistin inhibits phosphorylation of AMP-activated protein kinase (AMPK) in liver cells, and therefore, the expression of the key enzymes of gluconeogenesis, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), was upregulated, consequently leading to intensification of gluconeogenesis and the synthesis of glycogen.

In humans, the resistin expression is high in monocytes, which increases during differentiation to macrophages, and resistin in adipose tissue is also derived from infiltrating macrophages, both of which are inflammatory cells, suggesting that resistin might be associated with inflammation. Many studies have shown that hyper-resistinemia is an inflammatory state; it was positively correlated with inflammatory cytokines such as C-reactive protein and tumor necrosis factor-α [19-21]. In human primary monocyte-derived macrophages, resistin induced release of inflammatory cytokines via activated nuclear factor-κB [22,23], and consequently promoted inflammation, which could induce IR and inhibit insulin-induced glucose uptake.

Our study showed that the only effect of resistin on skeletal glucose metabolism was to inhibit insulin-induced glucose uptake by downregulating the expression of GLUT4. As for glucose oxidization or glycogen synthesis, which plays pivotal roles in skeletal glucose metabolism, no positive effects of resistin were found. Therefore, in this perspective the roles of resistin were not underestimated. The functions and mechanisms of resistin on IR require further investigation.

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