- Email: [email protected]
Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells
European Journal of Pharmacology 649 (2010) 23–28
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells Tatsuo Kobayashi ⁎, Yuko Akiyama, Nobuteru Akiyama, Hideaki Katoh, Sachiko Yamamoto, Kenzo Funatsuki, Toru Yanagimoto, Mitsuru Notoya, Kenji Asakura, Toshihiro Shinosaki, Kohji Hanasaki Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan
a r t i c l e
i n f o
Article history: Received 9 April 2010 Received in revised form 9 July 2010 Accepted 21 August 2010 Available online 6 September 2010 Keywords: Irbesartan Glucose transporter isotype 4 C2C12 cell L6-GLUT4myc cell
a b s t r a c t Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulinstimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Irbesartan is a potent and selective angiotensin II type 1 receptor blocker which is used mainly for treatment of hypertension. It has also been reported to have beneficial effects on glucose and lipid metabolism in clinical trials and animal studies (Henriksen et al., 2001; Sloniger et al., 2005; Bramlage et al., 2004). Adipocyte and skeletal muscle are important tissues which contribute to the development and progression of metabolic disorder. Several recent studies have demonstrated that irbesartan has a partial agonistic activity on the peroxisome proliferator-activated receptor (PPAR)-γ, a member of the nuclear receptor superfamily involved in nutrient metabolism, adipocyte differentiation and insulin signal transduction (Benson et al., 2004; Schupp et al., 2004; Schupp et al., 2005; Janke et al., 2006). In fact, irbesartan enhances adipogenesis and stimulates adiponectin protein expression through PPAR-γ activation in mouse 3T3-L1 (Schupp et al., 2004; Clasen et al., 2005) and human adipocytes (Janke et al., 2006). Also, irbesartan treatment of the
⁎ Corresponding author. Tel.: + 81 6 6331 8081; fax: + 81 6 6332 6385. E-mail address: [email protected] (T. Kobayashi). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.08.037
insulin-resistant obese Zucker rat improves insulin sensitivity and upregulates serum adiponectin (Clasen et al., 2005). Thus, the adipose tissue, at least in part, appears to be an important target organ for irbesartan to elicit an antidiabetic action through PPAR-γ activation. Meanwhile, a previous report showed that irbesartan improves glucose tolerance in obese Zucker rat with the enhancement of glucose uptake in skeletal muscle tissues (Henriksen et al., 2001). However, it is unclear whether in vivo enhancement of glucose uptake in the skeletal muscle is a secondary consequence of improvement in whole-body metabolic functions, or whether irbesartan acts directly on skeletal muscles. The skeletal muscle is the major site of insulinmediated glucose uptake accounting for approximately 75% of insulin-stimulated peripheral glucose disposal, while adipose tissue accounts for less than b1% (Björntorp et al., 1970, 1971; DeFronzo et al., 1981, 1985; Nuutila et al., 1992). Many studies have demonstrated that patients with type 2 diabetes mellitus show impaired glucose uptake, especially in the skeletal muscle, due to insulin resistance (Andréasson et al., 1991; Zierath, 1995; Zierath et al., 1994). Nonetheless, there is a paucity of data on the effects of angiotensin II type 1 receptor blockers on glucose transport in the skeletal muscle. This prompted us to address the question of whether or not irbesartan affects glucose uptake and translocation of the glucose transporter GLUT4 in skeletal muscle cells in vitro.
24
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
2. Materials and methods
differentiated into myotubes by culturing confluent myoblasts in DMEM with 2% heat-inactivated horse serum for 3 days.
2.1. Chemicals Irbesartan was a generous gift from Sanofi-Aventis (Bridgewater, NJ). Pioglitazone is synthesized by a procedure similar to that described by Momose et al. (1991). The structure and purity were confirmed by NMR and elemental analysis. Valsartan was purchased from Apin Chemicals Ltd (Abingdon, UK). 2.2. Animals Six-week-old male genetically obese (fa/fa) Zucker rats and lean (fa/+) rats were obtained from Charles River Laboratories (Yokohama, Japan). The animals were housed in an air-conditioned environment (22 ± 2 °C) with a 12:12-h light–dark cycle (8 AM to 8 PM), and had free access to water and standard laboratory chow (CE2, CLEA Japan, Inc.). Obese Zucker rats were divided into four groups of equal initial average weights: irbesartan 10 mg/kg (n = 8), 30 mg/kg (n = 8), 50 mg/kg (n = 9) groups and vehicle (0.5% methylcellulose) group (n = 9). Lean rats (n = 5) given vehicle treatment served as controls. The rats received a daily oral administration of either irbesartan or vehicle starting from 8 weeks of age for 22 consecutive days. Rats were weighed twice weekly. One day before the end of the 22-day period, blood samples were collected and analyzed for plasma triglyceride. The rats were then subjected to overnight fasting (16 h), blood was collected again, and the plasma was analyzed for glucose and insulin. Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi. 2.3. Plasma determinations Blood was collected from tail veins into capillary tubes and centrifuged (4 °C, 14,000 rpm, 5 min). Plasma glucose (Pureauto GLUR, Daiichi Pure Chemicals, Tokyo, Japan) and triglyceride (Autosera TG-N, Daiichi Pure Chemicals) were analyzed using an Automatic Analyzer (Hitachi 7180, Tokyo, Japan). Plasma insulin was determined using Rat Insulin ELISA KIT (Morinaga, Yokohama, Japan). The Homeostasis Model Assessment Index for Insulin Resistance (HOMA-IR index) was calculated as fasting plasma glucose × fasting plasma insulin, and expressed as relative values normalized by the value for the vehicle group as 1.0. 2.4. 2-Deoxyglucose uptake assay L6-GLUT4myc cells (from Dr. Amira Klip; The Hospital for Sick Children, Toronto, Canada) cultured on 24-well plates were treated with the indicated concentrations of compounds in α-MEM containing 10% FBS for 24 h at 37 °C. After 3 h of serum starvation in α-MEM containing 0.1% BSA, the cells were rinsed twice with Hepes-buffered saline (20 mM Hepes, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2, pH 7.4) and stimulated with 30 or 100 nM insulin (Peptide. Institute, Osaka, Japan) for 30 min at 37 °C. Glucose uptake was determined by the addition of 10 μM 2-deoxygulose containing 0.2 μCi of 2-[3H] deoxyglucose (GE Healthcare, Piscataway, NJ, USA) in Hepes-buffered saline for 10 min. The cells were then placed on ice and immediately washed 3 times with ice-cold 0.9% NaCl. Subsequently, the cells were lysed with 0.25 N NaOH and radioactivity was measured using a liquid scintillation counter. Nonspecific uptake was determined in the presence of 10 μM cytochalasin B and subtracted from the total uptake. The protein content in the cell lysates was measured using a BCA protein assay kit (Thermo Scientific, Inc., Rockford, IL, USA). Glucose uptake in C2C12 cells (DS Pharma Biomedical Co., Ltd., Osaka, Japan) was measured basically by the same method as described earlier except that C2C12 cells were
2.5. Measurement of GLUT4myc translocation L6-GLUT4myc cells cultured on 96-well plates were treated with the indicated concentrations of compounds in α-MEM containing 10% FBS for 24 h at 37 °C. Cells were serum-starved for 3 h in α-MEM containing 0.1% BSA and subsequently incubated with 30 nM insulin for 30 min at 37 °C. Cells were then placed on ice and immediately washed 3 times with ice-cold PBS, and fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. After quenching with 0.1 M glycine in PBS for 10 min, the cells were blocked with PBS containing 5% horse serum for 30 min. To determine the cell surface GLUT4myc content, the cells were then incubated for 1 h with anti-myc antibody 9E10 (Exalpha Biologicals Inc., Watertown, MA, USA) diluted to 0.25 μg/ml in PBS containing 3% horse serum. Thereafter, the cells were treated with a secondary antibody, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling Technology, Beverly, MA, USA) diluted 1:3000 in PBS containing 3% horse serum for 1 h. Following a rinsing step with PBS, TMB substrate (DAKO, Carpinteria, CA, USA) was added and left for over 15 min, and the reaction was stopped by adding 1 N H2SO4. HRP activity was determined by measuring the absorbance at 450 nm with a spectrophotometer. The background absorption, determined using the wells treated similarly in the absence of the primary antibody, was subtracted from each value. 2.6. Akt phosphorylation L6-GLUT4myc cells treated with irbesartan were lysed in lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 2 μg/ml aprotinin, 5 mM EDTA, 4 mM Pefabloc, 1 μM pepstatin, 10 μg/ml leupeptin, 20 mM NaF, 1 mM orthovanadate, 20 mM β-glycerophosphate, 2 mM NaPPi and 1% Triton X-100. Cell lysates were separated in SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dried milk in buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl and 0.05% Tween 20, followed by incubation with primary antibodies against phospho-Akt (Ser473) and Akt (Cell Signaling Technology Inc., Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell signaling Technology Inc, Beverly, MA, USA) was used as secondary antibody. Immunoreactive bands were visualized using ECL system (GE Healthcare, Piscataway, NJ, USA). 2.7. Adiponectin mRNA Total RNA were extracted from the L6-GLUT4myc cells treated with irbesartan or vehicle control using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. RNA concentration was determined by absorbance at 260 nm and RNA quality was checked using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Labeled cRNA were prepared using Quick Amp Labeling Kit, one-color (Agilent Technologies) following recommended procedures. Briefly, 500 ng of total RNA were reverse transcribed with oligo-dT primer containing T7 promoter sequence. Using the resulting cDNA as templates, labeled cRNA were synthesized by in vitro transcription in the presence of cyanine 3-CTP. Cyanine 3-labeled cRNA were applied to an oligonucleotide microarray (Agilent Technologies, Whole Rat Genome), and hybridization was carried out at 65 °C for 17 h according to the manufacturer's instructions. Following hybridization, the slide was washed, dried, and scanned on an Agilent DNA microarray scanner (Agilent Technologies). Fluorescence image was quantified using Feature Extraction software (Agilent Technologies). After
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
25
normalization, the expression value for adioponectin was determined by GeneSpring software version 7.0 (Agilent Technologies). 2.8. Acetyl-CoA carboxylase phosphorylation L6-GLUT4myc cells cultured on 96-well plates were treated with irbesartan. The cells were then placed on ice and immediately washed 2 times with ice-cold PBS, and fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. After washing 3 times with 0.1% Triton X-100 in PBS, the cells were quenched with 0.6% H2O2 in PBS containing 0.1% Triton X-100 for 30 min and blocked with PBS containing 10% fetal bovine serum and 0.1% Triton X-100 for 30 min. To measure the levels of acetyl-CoA carboxylase (ACC) phosphorylation, the cells were then incubated overnight at 4 °C with antiphospho-ACC antibody (1:500 dilutions Cell Signaling Technology Inc.). Thereafter, the cells were treated with a secondary antibody, HRP-conjugated anti-rabbit IgG (1:1000 dilutions Cell Signaling Technology Inc.) for 1 h at room temperature. HRP activity was detected using SuperSignal ELISA Pico substrate (Thermo Scientific, Inc., Rockford, IL, USA). The background chemiluminescence determined, using the wells treated similarly in the absence of the primary antibody, was subtracted from each value. 2.9. Statistical analysis All values are expressed as mean ± S.E.M. The significance of the difference between two groups was tested with Student's t test. Differences between more than two groups were tested using Dunnett's multiple comparison test. Statistical analysis was performed using SAS Ver. 8 (SAS Institute, Cary, NC, USA). A difference of P b 0.05 was considered significant. 3. Results 3.1. Glucose uptake in C2C12 myotubes and L6-GLUT4myc myoblasts To examine the molecular mechanisms of improvement in metabolic parameters by irbesartan, we focused our analysis on its action in the skeletal muscle. First, we examined whether irbesartan could affect glucose uptake in mouse C2C12 myotubes and rat L6 myoblasts stably expressing c-myc epitope-tagged GLUT4 (L6GLUT4myc), two widely used skeletal muscle cell lines. In C2C12 myotubes, insulin (100 nM) treatment alone increased glucose uptake by 17%, and irbesartan significantly increased insulinstimulated glucose uptake by 26% compared with basal uptake (Fig. 1). A comparable effect was also detected with pioglitazone, while valsartan did not affect glucose uptake. Irbesartan significantly increased glucose uptake by 13% even without insulin stimulation. Similarly, pioglitazone increased the uptake by 21% without insulin stimulation. Again, valsartan did not increase glucose uptake from the basal level. In L6-GLUT4myc cells, insulin (30 nM) treatment alone increased glucose uptake by 50%, and irbesartan and pioglitazone significantly increased insulin-stimulated glucose uptake by 72 and 118%, respectively, compared with basal uptake (Fig. 2). Pioglitazone increased glucose uptake without insulin stimulation. Valsartan did not enhance glucose uptake. At the end of the incubation, ATP contents were not different in C2C12 or L6-GLUT4myc cells, showing that cell viability was not affected by any of these treatments (data not shown). 3.2. GLUT4 translocation We examined the effect of irbesartan on the translocation of GLUT4, a key regulatory molecule in insulin-mediated glucose uptake in skeletal muscle. L6-GLUT4myc cells were incubated with irbesartan
Fig. 1. Effects of irbesartan, valsartan and pioglitazone on 2-[3H] deoxyglucose uptake in C2C12 cells in the absence or presence of insulin. Differentiated cells were incubated with irbesartan (50 μM), valsartan (50 μM), pioglitazone (50 μM) or vehicle for 24 h. After washing with HBS, the cells were incubated for 45 min in HBS at 37 °C, followed by insulin stimulation (100 nM) for 15 min. 2-[3H] deoxyglucose uptake was determined as described in Materials and methods Section 2.4. Data are presented as mean ± S.E.M. n = 6; **P b 0.01 vs. vehicle, #P b 0.05 vs. vehicle, ##P b 0.01 vs. vehicle. HBS, Hepes-buffered saline.
for 24 h, and the cell surface level of GLUT4myc was determined. Irbesartan (12.5–100 μM) increased the cell surface GLUT4myc in a concentration-dependent manner under the basal and insulinstimulated (30 nM) conditions (Fig. 3A). The levels were increased by 45 and 79% at the maximal concentration of irbesartan (100 μM) over the basal and insulin-stimulated conditions, respectively. Pioglitazone also enhanced GLUT4 translocation significantly, while valsartan was ineffective (Fig. 3B). To examine whether irbesartan could have an acute effect of enhancing GLUT4 translocation, the cell surface GLUT4myc level was determined after 3 h incubation with irbesartan, valsartan or pioglitazone. Irbesartan had no effect on GLUT4 translocation during this short incubation time. Valsartan also was ineffective. In contrast, pioglitazone enhanced GLUT4 translocation by 45 and 20% over basal and insulin-stimulated conditions, respectively (Fig. 3C).
Fig. 2. Effects of irbesartan, valsartan and pioglitazone on 2-[3H] deoxyglucose uptake in L6-GLUT4myc cells in the absence or presence of insulin. Cells were incubated with irbesartan (50 and 100 μM), valsartan (100 μM), pioglitazone (100 μM) or vehicle for 24 h. After washing with HBS, the cells were incubated for 45 min in HBS at 37 °C, followed by insulin stimulation (30 nM) for 15 min. 2-[3H] deoxyglucose uptake was determined as described in Materials and methods Section 2.4. Data are presented as mean ± S.E.M. n = 3; **P b 0.01 vs. vehicle, #P b 0.05 vs. vehicle, ##P b 0.01 vs. vehicle. HBS, Hepes-buffered saline.
26
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
3.4. Adiponectin mRNA To elucidate molecular pathways for irbesartan-mediated enhancement of glucose transport, the effect of irbesartan on adiponectin mRNA levels was investigated, but no significant change was observed. 3.5. Acetyl-CoA carboxylase phosphorylation We tested whether AMPK activation could be involved in the potentiation of glucose transport by irbesartan. ACC phosphorylation, as a marker of AMPK activation, was measured using cell ELISA technique. We found that the phosphorylation level of ACC was not altered in L6-GLUT4myc cells exposed to irbesartan. 3.6. Body weight and plasma parameters in Zucker fa/fa rats To investigate whether irbesartan could improve insulin resistance, obese fa/fa Zucker rats were used as a model of insulin resistance in this study. Plasma triglyceride, fasting plasma glucose and insulin levels were significantly augmented in obese fa/fa Zucker rats in comparison with lean fa/+ Zucker rats (Table 1; P b 0.01). Obese Zucker rats were treated with vehicle or irbesartan for 3 weeks, after which there was a clear dose-dependent decrease in the fasting plasma insulin level. Significant reductions of 28% (P b 0.05) and 41% (P b 0.01) in plasma insulin were observed at doses of 30 and 50 mg/ kg dose, respectively, compared to the vehicle group. Fasting plasma glucose levels were not different between the groups. The insulin resistance index determined by HOMA was significantly smaller at 50 mg/kg (P b 0.01). The plasma triglyceride tended to be lower in irbesartan groups, but the decrease did not reach statistical significance. Obesity was slightly but significantly attenuated at 50 mg/kg dose (P b 0.05). 4. Discussion
Fig. 3. Effects of irbesartan on GLUT4 translocation in L6-GLUT4myc cells. (A) Concentration-dependency of irbesartan on GLUT4 translocation. Cells were incubated with various concentrations of irbesartan for 24 h. (B) Effect of irbesartan on GLUT4 translocation after 24-h incubation. Cells were incubated with irbesartan (50 and 100 μM), valsartan (100 μM), pioglitazone (100 μM) or vehicle for 24 h. (C) Acute effect of irbesartan on GLUT4 translocation after 3-h incubation. Cells were incubated with irbesartan (50 and 100 μM), valsartan (100 μM), pioglitazone (100 μM) or vehicle in serum-deprived medium (α-MEM 0.1% bovine serum albumin) for 3 h. Cell surface GLUT4 was measured as described in Materials and methods Section 2.5, and expressed as fold increase over the vehicle control without insulin stimulation. Data are presented as mean ± S.E.M. n = 4; *P b 0.05 vs. vehicle, **P b 0.01 vs. vehicle, #P b 0.05 vs. vehicle, ##P b 0.01 vs. vehicle.
3.3. Akt phosphorylation We examined the effect of irbesartan on the phosphorylation status of Akt which plays a primary role in insulin signaling. L6GLUT4myc cells were treated with irbesartan and the Akt phosphorylation was evaluated using Western blot analysis. The phosphorylation level of Akt was not changed by irbesartan treatment.
A number of clinical trials have presented evidence of irbesartan exhibiting favorable effects on glucose and lipid metabolism in patients with essential hypertension or metabolic syndrome (Kyvelou et al., 2006; Negro et al., 2006; Kintscher et al., 2007). Although evidence has accumulated in recent years to show that irbesartan has a direct action in adipocyte that lead to beneficial metabolic effects through PPARγ modulating properties (Schupp et al, 2004; Schupp et al, 2005; Erbe et al, 2006; Pershadsingh 2006), the precise molecular mechanism underlying these effects is not fully understood. The skeletal muscle is a major contributor to glucose metabolism, however, action of angiotensin II type 1 receptor blocker on skeletal muscle has been relatively unexplored. Therefore, we investigated whether or not irbesartan regulates glucose metabolism in skeletal muscles by using well established cell lines. We found that irbesartan enhances glucose uptake in these cell lines, which is likely to be attributed to GLUT4 translocation to the plasma membrane induced by irbesartan. Clearly, these effects are not mediated by angiotensin II receptor blockade, because the cell-based experiments in our study were performed in the absence of angiotensin II. Moreover, the observation that valsartan does not enhance glucose transport or GLUT4 translocation further supports this notion. We showed that chronic administration of irbesartan to the Zucker fatty rat, a widely used disease model of obesity and insulin resistance, elicited significant amelioration of insulin resistance. This observation is in line with what has been previously reported (Henriksen et al., 2001). Plasma concentration of irbesartan in the present study was estimated to be approximately 10 μM. Since, as shown in Fig. 3A, the cell surface GLUT4myc tended to increase at 12.5 μM of irbesartan, we speculate that beneficial metabolic effect of irbesartan observed in
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
27
Table 1 Effect of irbesartan on body weight and plasma parameters in Zucker fa/fa rats.
Vehicle Irbesartan 10 mg/kg Irbesartan 30 mg/kg Irbesartan 50 mg/kg Lean
Body weight (g)
Fasting plasma glucose (mg/dl)
Fasting plasma insulin (ng/ml)
Plasma triglyceride (mg/dl)
Relative HOMA-IR
482.6 ± 6.4 484.0 ± 8.4 483.9 ± 6.0 457.5 ± 7.4a 325.6 ± 6.9c
139.8 ± 7.1 138.3 ± 5.3 139.9 ± 2.8 125.9 ± 1.9 98.6 ± 2.4c
7.59 ± 0.51 7.11 ± 0.84 5.49 ± 0.54a 4.47 ± 0.41b 0.36 ± 0.05c
530.1 ± 58.7 448.1 ± 64.9 391.9 ± 53.2 373.6 ± 62.7 91.6 ± 14.7c
1.00 ± 0.09 0.95 ± 0.14 0.73 ± 0.07 0.53 ± 0.05b 0.03 ± 0.01c
Values are means ± S.E.M. for 5 to 9 animals per group. Relative HOMA-IR is expressed as relative values normalized by the value for vehicle group as 1.0. a Pb0.05 vs. vehicle. b Pb0.01 vs. vehicle. c Pb0.01 vs. vehicle.
the present study may be attributable in part to its direct action on skeletal muscle. Precise molecular pathways for irbesartan to enhance glucose transport in skeletal muscle cells are unknown at present. Since our observation is consistent with an earlier report by Verma et al. (2004), which showed that pioglitazone enhanced glucose uptake in C2C12 cells and that glucose uptake was decreased in C2C12 cells transfected with the antisense construct of PPARγ, one possibility is that irbesartan promotes glucose transport in skeletal muscle through PPARγ modulation. To investigate other potential mechanisms besides PPARγ, we examined three possibilities. First, with the knowledge that activation of the phosphatidylinositol 3-kinase (PI3kinase) pathway affects insulin-mediated stimulation of GLUT4 translocation in the skeletal muscle, we assessed the effect of irbesartan on phosphorylation of Akt, a major downstream target of PI3-kinase in L6-GLUT4myc cells. However, irbesartan did not show a significant effect on Akt phosphorylation under our experimental conditions. Next, we investigated the involvement of adiponectin, adipocytokine derived from adipose tissues with insulin-sensitizing properties. Contrary to the general concept that adiponectin is produced only by the adipose tissue, recent evidence has suggested that skeletal muscle has the potential to produce adiponectin. For instance, rosiglitazone enhanced glucose uptake with increased secretion of adiponectin in L6 cells (Liu et al., 2009). Although we examined the adiponectin mRNA level in L6-GLUT4myc cells exposed to irbesartan, we detected no significant change (data not shown). Third, in order to study the possible involvement of AMPK activation in irbesartan-induced enhancement of glucose transport under our experimental conditions, we evaluated the effects of irbesartan on AMPK activity in L6-GLUT4myc cells by assessing the phosphorylation state of acetyl-CoA carboxylase, a well-known substrate for AMPK. However, AMPK activation was not observed. Since the enhancement of GLUT4 translocation to the plasma membrane did not occur under short-time 3-h treatment, the onset of the increase of glucose uptake induced by irbesartan may be slow and might require the synthesis of new RNAs and proteins. In this regard, a link between irbesartaninduced enhancement of glucose transport and the transcription factors, such as PPARγ, may exist. Further study is needed to identify the molecules involved in enhancement of glucose uptake by irbesartan. Notably, the irbesartan-induced enhancement of whole-body insulin sensitivity was not accompanied by weight gain. Although thiazolidinediones are effective for diabetes treatment, the clinical applications of thiazolidinediones are limited due to undesirable side effects including weight gain, fluid retention, peripheral edema and increased incidence of bone fractures (Rizos et al., 2009). Given that diabetes frequently accompanies hypertension in metabolic syndrome (Geiss et al., 2002), the current findings that chronic irbesartan treatment induces beneficial effects on metabolic parameters in Zucker fatty rats without weight gain indicates a potentially important profile of irbesartan for pharmacological treatment of metabolic syndrome. Recently, several PPARγ partial agonists, which
maintain remarkable insulin-sensitizing efficacy with lesser adverse effects, have been synthesized (Chang et al., 2008; Higgins and Mantzoros, 2008; Larsen et al., 2008; Li et al., 2008; Kim et al., 2009). With respect to irbesartan, there are reports suggesting that irbesartan functions as a partial agonist in cell-based transactivaton assays (Schupp et al., 2004) and in cofactor recruitment assays (Schupp et al., 2005). Thus, the PPARγ partial agonistic activity of irbesartan may explain, at least in part, its beneficial metabolic effect without weight gain. In summary, we provide new evidence that irbesartan exerts a direct effect on skeletal muscle to promote GLUT4 translocation and glucose uptake. This property may be relevant to the beneficial effect of irbesartan in ameliorating the metabolic disorders. Acknowledgment The authors thank Yoko Kajiwara, Hiroyuki Takagi, Michiyo Kanazu and Keiichi Imagawa for technical assistance and helpful discussion. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/ j.ejphar.2010.08.037. References Andréasson, K., Galuska, D., Thörne, A., Sonnenfeld, T., Wallberg-Henriksson, H., 1991. Decreased insulin-stimulated 3-0-methylglucose transport in in vitro incubated muscle strips from type II diabetic subjects. Acta Physiol. Scand. 142, 255–260. Benson, S.C., Pershadsingh, H.A., Ho, C.I., Chittiboyina, A., Desai, P., Pravenec, M., Qi, N., Wang, J., Avery, M.A., Kurtz, T.W., 2004. Identification of telmisartan as a unique angiotensin II receptor antagonist with selective PPARgamma-modulating activity. Hypertension 43, 993–1002. Björntorp, P., Krotkiewski, M., Larsson, B., Somlo-Szücs, Z., 1970. Effects of feeding states on lipid radioactivity in liver, muscle and adipose tissue after injection of labelled glucose in the rat. Acta Physiol. Scand. 80, 29–38. Björntorp, P., Berchtold, P., Holm, J., Larsson, B., 1971. The glucose uptake of human adipose tissue in obesity. Eur. J. Clin. Invest. 1971 (1), 480–485. Bramlage, P., Pittrow, D., Kirch, W., 2004. The effect of irbesartan in reducing cardiovascular risk in hypertensive type 2 diabetic patients: an observational study in 16, 600 patients in primary care. Curr. Med. Res. Opin. 20, 1625–1631. Chang, C.H., McNamara, L.A., Wu, M.S., Muise, E.S., Tan, Y., Wood, H.B., Meinke, P.T., Thompson, J.R., Doebber, T.W., Berger, J.P., McCann, M.E., 2008. A novel selective peroxisome proliferator-activator receptor-gamma modulator-SPPARgammaM5 improves insulin sensitivity with diminished adverse cardiovascular effects. Eur. J. Pharmacol. 14, 192–201. Clasen, R., Schupp, M., Foryst-Ludwig, A., Sprang, C., Clemenz, M., Krikov, M., ThöneReineke, C., Unger, T., Kintscher, U., 2005. PPARgamma-activating angiotensin type1 receptor blockers induce adiponectin. Hypertension 46, 137–143. DeFronzo, R.A., Jacot, E., Jequier, E., Maeder, E., Wahren, J., Felber, J.P., 1981. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30, 1000–1007. DeFronzo, R.A., Gunnarsson, R., Björkman, O., Olsson, M., Wahren, J., 1985. Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus. J. Clin. Invest. 76, 149–155. Erbe, D.V., Gartrell, K., Zhang, Y.L., Suri, V., Kirincich, S.J., Will, S., Perreault, M., Wang, S., Tobin, J.F., 2006. Molecular activation of PPARgamma by angiotensin II type 1receptor antagonists. Vascul Pharmacol. 45, 154–162. Geiss, L.S., Rolka, D.B., Engelgau, M.M., 2002. Elevated blood pressure among U.S. adults with diabetes, 1988–1994. Am. J. Prev. Med. 22, 42–48.
28
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
Henriksen, E.J., Jacob, S., Kinnick, T.R., Teachey, M.K., Krekler, M., 2001. Selective angiotensin II receptor receptor antagonism reduces insulin resistance in obese Zucker rats. Hypertension 38, 884–890. Higgins, L.S., Mantzoros, C.S., 2008. The development of INT131 as a selective PPARgamma modulator: approach to a safer insulin sensitizer. PPAR Res. 936906. Janke, J., Schupp, M., Engeli, S., Gorzelniak, K., Boschmann, M., Sauma, L., Nystrom, F.H., Jordan, J., Luft, F.C., Sharma, A.M., 2006. Angiotensin type 1 receptor antagonists induce human in-vitro adipogenesis through peroxisome proliferator-activated receptor-gamma activation. J. Hypertens. 24, 1809–1816. Kim, M.K., Chae, Y.N., Kim, H.S., Choi, S.H., Son, M.H., Kim, S.H., Kim, J.K., Moon, H.S., Park, S.K., Shin, Y.A., Kim, J.G., Lee, C.H., Lim, J.I., Shin, C.Y., 2009. PAR-1622 is a selective peroxisome proliferator-activated receptor gamma partial activator with preserved antidiabetic efficacy and broader safety profile for fluid retention. Arch. Pharm. Res. 32, 721–727. Kintscher, U., Bramlage, P., Paar, W.D., Thoenes, M., Unger, T., 2007. Irbesartan for the treatment of hypertension in patients with the metabolic syndrome: a sub analysis of the Treat to Target post authorization survey. Prospective observational, two armed study in 14,200 patients. Cardiovasc. Diabetol. 6, 12. Kyvelou, S.M., Vyssoulis, G.P., Karpanou, E.A., Adamopoulos, D.N., Zervoudaki, A.I., Pietri, P.G., Stefanadis, C.I., 2006. Effects of antihypertensive treatment with angiotensin II receptor blockers on lipid profile: an open multi-drug comparison trial. Hellenic J. Cardiol. 47, 21–28. Larsen, P.J., Lykkegaard, K., Larsen, L.K., Fleckner, J., Sauerberg, P., Wassermann, K., Wulff, E.M., 2008. Dissociation of antihyperglycaemic and adverse effects of partial perioxisome proliferator-activated receptor (PPAR-gamma) agonist balaglitazone. Eur. J. Pharmacol. 596, 173–179. Li, Y., Wang, Z., Furukawa, N., Escaron, P., Weiszmann, J., Lee, G., Lindstrom, M., Liu, J., Liu, X., Xu, H., Plotnikova, O., Prasad, V., Walker, N., Learned, R.M., Chen, J.L., 2008. T2384, a novel antidiabetic agent with unique peroxisome proliferator-activated receptor gamma binding properties. J. Biol. Chem. 283, 9168–9176. Liu, Y., Chewchuk, S., Lavigne, C., Brûlé, S., Pilon, G., Houde, V., Xu, A., Marette, A., Sweeney, G., 2009. Functional significance of skeletal muscle adiponectin production, changes in animal models of obesity and diabetes, and regulation by rosiglitazone treatment. Am. J. Physiol. Endocrinol. Metab. 297, E657–E664.
Momose, Y., Meguro, K., Ikeda, H., Hatanaka, C., Oi, S., Sohda, T., 1991. Studies on antidiabetic agents. X. Synthesis and biological activities of pioglitazone and related compounds. Chem. Pharm. Bull. (Tokyo). 39, 1440–1445. Negro, R., Formoso, G., Hassan, H., 2006. The effects of irbesartan and telmisartan on metabolic parameters and blood pressure in obese, insulin resistant, hypertensive patients. J. Endocrinol. Invest. 29, 957–961. Nuutila, P., Koivisto, V.A., Knuuti, J., Ruotsalainen, U., Teräs, M., Haaparanta, M., Bergman, J., Solin, O., Voipio-Pulkki, L.M., Wegelius, U., Yki-Jaryinen, H., 1992. Glucose-free fatty acid cycle operates in human heart and skeletal muscle in vivo. J. Clin. Invest. 89, 1767–1774. Pershadsingh, H.A., 2006. Treating the metabolic syndrome using angiotensin receptor antagonists that selectively modulate peroxisome proliferator-activated receptorgamma. Int. J. Biochem. Cell Biol. 38, 766–781. Rizos, C.V., Elisaf, M.S., Mikhailidis, D.P., Liberopoulos, E.N., 2009. How safe is the use of thiazolidinediones in clinical practice? Expert Opin. Drug Saf. 8, 15–32. Schupp, M., Janke, J., Clasen, R., Unger, T., Kintscher, U., 2004. Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation 109, 2054–2057. Schupp, M., Clemenz, M., Gineste, R., Witt, H., Janke, J., Helleboid, S., Hennuyer, N., Ruiz, P., Unger, T., Staels, B., Kintscher, U., 2005. Molecular characterization of new selective peroxisome proliferator-activated receptor gamma modulators with angiotensin receptor blocking activity. Diabetes 54, 3442–3452. Sloniger, J.A., Saengsirisuwan, V., Diehl, C.J., Kim, J.S., Henriksen, E.J., 2005. Selective angiotensin II receptor antagonism enhances whole-body insulin sensitivity and muscle glucose transport in hypertensive TG (mREN2)27 rats. Metabolism 54, 1659–1668. Verma, N.K., Singh, J., Dey, C.S., 2004. PPAR-gamma expression modulates insulin sensitivity in C2C12 skeletal muscle cells. Br. J. Pharmacol. 143, 1006–1013. Zierath, J.R., 1995. In vitro studies of human skeletal muscle: hormonal and metabolic regulation of glucose transport. Acta Physiol. Scand. Suppl. 626, 1–96. Zierath, J.R., Galuska, D., Nolte, L.A., Thörne, A., Kristensen, J.S., Wallberg-Henriksson, H., 1994. Effects of glycaemia on glucose transport in isolated skeletal muscle from patients with NIDDM: in vitro reversal of muscular insulin resistance. Diabetologia 37, 270–277.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells Tatsuo Kobayashi ⁎, Yuko Akiyama, Nobuteru Akiyama, Hideaki Katoh, Sachiko Yamamoto, Kenzo Funatsuki, Toru Yanagimoto, Mitsuru Notoya, Kenji Asakura, Toshihiro Shinosaki, Kohji Hanasaki Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan
a r t i c l e
i n f o
Article history: Received 9 April 2010 Received in revised form 9 July 2010 Accepted 21 August 2010 Available online 6 September 2010 Keywords: Irbesartan Glucose transporter isotype 4 C2C12 cell L6-GLUT4myc cell
a b s t r a c t Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulinstimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Irbesartan is a potent and selective angiotensin II type 1 receptor blocker which is used mainly for treatment of hypertension. It has also been reported to have beneficial effects on glucose and lipid metabolism in clinical trials and animal studies (Henriksen et al., 2001; Sloniger et al., 2005; Bramlage et al., 2004). Adipocyte and skeletal muscle are important tissues which contribute to the development and progression of metabolic disorder. Several recent studies have demonstrated that irbesartan has a partial agonistic activity on the peroxisome proliferator-activated receptor (PPAR)-γ, a member of the nuclear receptor superfamily involved in nutrient metabolism, adipocyte differentiation and insulin signal transduction (Benson et al., 2004; Schupp et al., 2004; Schupp et al., 2005; Janke et al., 2006). In fact, irbesartan enhances adipogenesis and stimulates adiponectin protein expression through PPAR-γ activation in mouse 3T3-L1 (Schupp et al., 2004; Clasen et al., 2005) and human adipocytes (Janke et al., 2006). Also, irbesartan treatment of the
⁎ Corresponding author. Tel.: + 81 6 6331 8081; fax: + 81 6 6332 6385. E-mail address: [email protected] (T. Kobayashi). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.08.037
insulin-resistant obese Zucker rat improves insulin sensitivity and upregulates serum adiponectin (Clasen et al., 2005). Thus, the adipose tissue, at least in part, appears to be an important target organ for irbesartan to elicit an antidiabetic action through PPAR-γ activation. Meanwhile, a previous report showed that irbesartan improves glucose tolerance in obese Zucker rat with the enhancement of glucose uptake in skeletal muscle tissues (Henriksen et al., 2001). However, it is unclear whether in vivo enhancement of glucose uptake in the skeletal muscle is a secondary consequence of improvement in whole-body metabolic functions, or whether irbesartan acts directly on skeletal muscles. The skeletal muscle is the major site of insulinmediated glucose uptake accounting for approximately 75% of insulin-stimulated peripheral glucose disposal, while adipose tissue accounts for less than b1% (Björntorp et al., 1970, 1971; DeFronzo et al., 1981, 1985; Nuutila et al., 1992). Many studies have demonstrated that patients with type 2 diabetes mellitus show impaired glucose uptake, especially in the skeletal muscle, due to insulin resistance (Andréasson et al., 1991; Zierath, 1995; Zierath et al., 1994). Nonetheless, there is a paucity of data on the effects of angiotensin II type 1 receptor blockers on glucose transport in the skeletal muscle. This prompted us to address the question of whether or not irbesartan affects glucose uptake and translocation of the glucose transporter GLUT4 in skeletal muscle cells in vitro.
24
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
2. Materials and methods
differentiated into myotubes by culturing confluent myoblasts in DMEM with 2% heat-inactivated horse serum for 3 days.
2.1. Chemicals Irbesartan was a generous gift from Sanofi-Aventis (Bridgewater, NJ). Pioglitazone is synthesized by a procedure similar to that described by Momose et al. (1991). The structure and purity were confirmed by NMR and elemental analysis. Valsartan was purchased from Apin Chemicals Ltd (Abingdon, UK). 2.2. Animals Six-week-old male genetically obese (fa/fa) Zucker rats and lean (fa/+) rats were obtained from Charles River Laboratories (Yokohama, Japan). The animals were housed in an air-conditioned environment (22 ± 2 °C) with a 12:12-h light–dark cycle (8 AM to 8 PM), and had free access to water and standard laboratory chow (CE2, CLEA Japan, Inc.). Obese Zucker rats were divided into four groups of equal initial average weights: irbesartan 10 mg/kg (n = 8), 30 mg/kg (n = 8), 50 mg/kg (n = 9) groups and vehicle (0.5% methylcellulose) group (n = 9). Lean rats (n = 5) given vehicle treatment served as controls. The rats received a daily oral administration of either irbesartan or vehicle starting from 8 weeks of age for 22 consecutive days. Rats were weighed twice weekly. One day before the end of the 22-day period, blood samples were collected and analyzed for plasma triglyceride. The rats were then subjected to overnight fasting (16 h), blood was collected again, and the plasma was analyzed for glucose and insulin. Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi. 2.3. Plasma determinations Blood was collected from tail veins into capillary tubes and centrifuged (4 °C, 14,000 rpm, 5 min). Plasma glucose (Pureauto GLUR, Daiichi Pure Chemicals, Tokyo, Japan) and triglyceride (Autosera TG-N, Daiichi Pure Chemicals) were analyzed using an Automatic Analyzer (Hitachi 7180, Tokyo, Japan). Plasma insulin was determined using Rat Insulin ELISA KIT (Morinaga, Yokohama, Japan). The Homeostasis Model Assessment Index for Insulin Resistance (HOMA-IR index) was calculated as fasting plasma glucose × fasting plasma insulin, and expressed as relative values normalized by the value for the vehicle group as 1.0. 2.4. 2-Deoxyglucose uptake assay L6-GLUT4myc cells (from Dr. Amira Klip; The Hospital for Sick Children, Toronto, Canada) cultured on 24-well plates were treated with the indicated concentrations of compounds in α-MEM containing 10% FBS for 24 h at 37 °C. After 3 h of serum starvation in α-MEM containing 0.1% BSA, the cells were rinsed twice with Hepes-buffered saline (20 mM Hepes, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2, pH 7.4) and stimulated with 30 or 100 nM insulin (Peptide. Institute, Osaka, Japan) for 30 min at 37 °C. Glucose uptake was determined by the addition of 10 μM 2-deoxygulose containing 0.2 μCi of 2-[3H] deoxyglucose (GE Healthcare, Piscataway, NJ, USA) in Hepes-buffered saline for 10 min. The cells were then placed on ice and immediately washed 3 times with ice-cold 0.9% NaCl. Subsequently, the cells were lysed with 0.25 N NaOH and radioactivity was measured using a liquid scintillation counter. Nonspecific uptake was determined in the presence of 10 μM cytochalasin B and subtracted from the total uptake. The protein content in the cell lysates was measured using a BCA protein assay kit (Thermo Scientific, Inc., Rockford, IL, USA). Glucose uptake in C2C12 cells (DS Pharma Biomedical Co., Ltd., Osaka, Japan) was measured basically by the same method as described earlier except that C2C12 cells were
2.5. Measurement of GLUT4myc translocation L6-GLUT4myc cells cultured on 96-well plates were treated with the indicated concentrations of compounds in α-MEM containing 10% FBS for 24 h at 37 °C. Cells were serum-starved for 3 h in α-MEM containing 0.1% BSA and subsequently incubated with 30 nM insulin for 30 min at 37 °C. Cells were then placed on ice and immediately washed 3 times with ice-cold PBS, and fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. After quenching with 0.1 M glycine in PBS for 10 min, the cells were blocked with PBS containing 5% horse serum for 30 min. To determine the cell surface GLUT4myc content, the cells were then incubated for 1 h with anti-myc antibody 9E10 (Exalpha Biologicals Inc., Watertown, MA, USA) diluted to 0.25 μg/ml in PBS containing 3% horse serum. Thereafter, the cells were treated with a secondary antibody, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling Technology, Beverly, MA, USA) diluted 1:3000 in PBS containing 3% horse serum for 1 h. Following a rinsing step with PBS, TMB substrate (DAKO, Carpinteria, CA, USA) was added and left for over 15 min, and the reaction was stopped by adding 1 N H2SO4. HRP activity was determined by measuring the absorbance at 450 nm with a spectrophotometer. The background absorption, determined using the wells treated similarly in the absence of the primary antibody, was subtracted from each value. 2.6. Akt phosphorylation L6-GLUT4myc cells treated with irbesartan were lysed in lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 2 μg/ml aprotinin, 5 mM EDTA, 4 mM Pefabloc, 1 μM pepstatin, 10 μg/ml leupeptin, 20 mM NaF, 1 mM orthovanadate, 20 mM β-glycerophosphate, 2 mM NaPPi and 1% Triton X-100. Cell lysates were separated in SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dried milk in buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl and 0.05% Tween 20, followed by incubation with primary antibodies against phospho-Akt (Ser473) and Akt (Cell Signaling Technology Inc., Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell signaling Technology Inc, Beverly, MA, USA) was used as secondary antibody. Immunoreactive bands were visualized using ECL system (GE Healthcare, Piscataway, NJ, USA). 2.7. Adiponectin mRNA Total RNA were extracted from the L6-GLUT4myc cells treated with irbesartan or vehicle control using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. RNA concentration was determined by absorbance at 260 nm and RNA quality was checked using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Labeled cRNA were prepared using Quick Amp Labeling Kit, one-color (Agilent Technologies) following recommended procedures. Briefly, 500 ng of total RNA were reverse transcribed with oligo-dT primer containing T7 promoter sequence. Using the resulting cDNA as templates, labeled cRNA were synthesized by in vitro transcription in the presence of cyanine 3-CTP. Cyanine 3-labeled cRNA were applied to an oligonucleotide microarray (Agilent Technologies, Whole Rat Genome), and hybridization was carried out at 65 °C for 17 h according to the manufacturer's instructions. Following hybridization, the slide was washed, dried, and scanned on an Agilent DNA microarray scanner (Agilent Technologies). Fluorescence image was quantified using Feature Extraction software (Agilent Technologies). After
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
25
normalization, the expression value for adioponectin was determined by GeneSpring software version 7.0 (Agilent Technologies). 2.8. Acetyl-CoA carboxylase phosphorylation L6-GLUT4myc cells cultured on 96-well plates were treated with irbesartan. The cells were then placed on ice and immediately washed 2 times with ice-cold PBS, and fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. After washing 3 times with 0.1% Triton X-100 in PBS, the cells were quenched with 0.6% H2O2 in PBS containing 0.1% Triton X-100 for 30 min and blocked with PBS containing 10% fetal bovine serum and 0.1% Triton X-100 for 30 min. To measure the levels of acetyl-CoA carboxylase (ACC) phosphorylation, the cells were then incubated overnight at 4 °C with antiphospho-ACC antibody (1:500 dilutions Cell Signaling Technology Inc.). Thereafter, the cells were treated with a secondary antibody, HRP-conjugated anti-rabbit IgG (1:1000 dilutions Cell Signaling Technology Inc.) for 1 h at room temperature. HRP activity was detected using SuperSignal ELISA Pico substrate (Thermo Scientific, Inc., Rockford, IL, USA). The background chemiluminescence determined, using the wells treated similarly in the absence of the primary antibody, was subtracted from each value. 2.9. Statistical analysis All values are expressed as mean ± S.E.M. The significance of the difference between two groups was tested with Student's t test. Differences between more than two groups were tested using Dunnett's multiple comparison test. Statistical analysis was performed using SAS Ver. 8 (SAS Institute, Cary, NC, USA). A difference of P b 0.05 was considered significant. 3. Results 3.1. Glucose uptake in C2C12 myotubes and L6-GLUT4myc myoblasts To examine the molecular mechanisms of improvement in metabolic parameters by irbesartan, we focused our analysis on its action in the skeletal muscle. First, we examined whether irbesartan could affect glucose uptake in mouse C2C12 myotubes and rat L6 myoblasts stably expressing c-myc epitope-tagged GLUT4 (L6GLUT4myc), two widely used skeletal muscle cell lines. In C2C12 myotubes, insulin (100 nM) treatment alone increased glucose uptake by 17%, and irbesartan significantly increased insulinstimulated glucose uptake by 26% compared with basal uptake (Fig. 1). A comparable effect was also detected with pioglitazone, while valsartan did not affect glucose uptake. Irbesartan significantly increased glucose uptake by 13% even without insulin stimulation. Similarly, pioglitazone increased the uptake by 21% without insulin stimulation. Again, valsartan did not increase glucose uptake from the basal level. In L6-GLUT4myc cells, insulin (30 nM) treatment alone increased glucose uptake by 50%, and irbesartan and pioglitazone significantly increased insulin-stimulated glucose uptake by 72 and 118%, respectively, compared with basal uptake (Fig. 2). Pioglitazone increased glucose uptake without insulin stimulation. Valsartan did not enhance glucose uptake. At the end of the incubation, ATP contents were not different in C2C12 or L6-GLUT4myc cells, showing that cell viability was not affected by any of these treatments (data not shown). 3.2. GLUT4 translocation We examined the effect of irbesartan on the translocation of GLUT4, a key regulatory molecule in insulin-mediated glucose uptake in skeletal muscle. L6-GLUT4myc cells were incubated with irbesartan
Fig. 1. Effects of irbesartan, valsartan and pioglitazone on 2-[3H] deoxyglucose uptake in C2C12 cells in the absence or presence of insulin. Differentiated cells were incubated with irbesartan (50 μM), valsartan (50 μM), pioglitazone (50 μM) or vehicle for 24 h. After washing with HBS, the cells were incubated for 45 min in HBS at 37 °C, followed by insulin stimulation (100 nM) for 15 min. 2-[3H] deoxyglucose uptake was determined as described in Materials and methods Section 2.4. Data are presented as mean ± S.E.M. n = 6; **P b 0.01 vs. vehicle, #P b 0.05 vs. vehicle, ##P b 0.01 vs. vehicle. HBS, Hepes-buffered saline.
for 24 h, and the cell surface level of GLUT4myc was determined. Irbesartan (12.5–100 μM) increased the cell surface GLUT4myc in a concentration-dependent manner under the basal and insulinstimulated (30 nM) conditions (Fig. 3A). The levels were increased by 45 and 79% at the maximal concentration of irbesartan (100 μM) over the basal and insulin-stimulated conditions, respectively. Pioglitazone also enhanced GLUT4 translocation significantly, while valsartan was ineffective (Fig. 3B). To examine whether irbesartan could have an acute effect of enhancing GLUT4 translocation, the cell surface GLUT4myc level was determined after 3 h incubation with irbesartan, valsartan or pioglitazone. Irbesartan had no effect on GLUT4 translocation during this short incubation time. Valsartan also was ineffective. In contrast, pioglitazone enhanced GLUT4 translocation by 45 and 20% over basal and insulin-stimulated conditions, respectively (Fig. 3C).
Fig. 2. Effects of irbesartan, valsartan and pioglitazone on 2-[3H] deoxyglucose uptake in L6-GLUT4myc cells in the absence or presence of insulin. Cells were incubated with irbesartan (50 and 100 μM), valsartan (100 μM), pioglitazone (100 μM) or vehicle for 24 h. After washing with HBS, the cells were incubated for 45 min in HBS at 37 °C, followed by insulin stimulation (30 nM) for 15 min. 2-[3H] deoxyglucose uptake was determined as described in Materials and methods Section 2.4. Data are presented as mean ± S.E.M. n = 3; **P b 0.01 vs. vehicle, #P b 0.05 vs. vehicle, ##P b 0.01 vs. vehicle. HBS, Hepes-buffered saline.
26
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
3.4. Adiponectin mRNA To elucidate molecular pathways for irbesartan-mediated enhancement of glucose transport, the effect of irbesartan on adiponectin mRNA levels was investigated, but no significant change was observed. 3.5. Acetyl-CoA carboxylase phosphorylation We tested whether AMPK activation could be involved in the potentiation of glucose transport by irbesartan. ACC phosphorylation, as a marker of AMPK activation, was measured using cell ELISA technique. We found that the phosphorylation level of ACC was not altered in L6-GLUT4myc cells exposed to irbesartan. 3.6. Body weight and plasma parameters in Zucker fa/fa rats To investigate whether irbesartan could improve insulin resistance, obese fa/fa Zucker rats were used as a model of insulin resistance in this study. Plasma triglyceride, fasting plasma glucose and insulin levels were significantly augmented in obese fa/fa Zucker rats in comparison with lean fa/+ Zucker rats (Table 1; P b 0.01). Obese Zucker rats were treated with vehicle or irbesartan for 3 weeks, after which there was a clear dose-dependent decrease in the fasting plasma insulin level. Significant reductions of 28% (P b 0.05) and 41% (P b 0.01) in plasma insulin were observed at doses of 30 and 50 mg/ kg dose, respectively, compared to the vehicle group. Fasting plasma glucose levels were not different between the groups. The insulin resistance index determined by HOMA was significantly smaller at 50 mg/kg (P b 0.01). The plasma triglyceride tended to be lower in irbesartan groups, but the decrease did not reach statistical significance. Obesity was slightly but significantly attenuated at 50 mg/kg dose (P b 0.05). 4. Discussion
Fig. 3. Effects of irbesartan on GLUT4 translocation in L6-GLUT4myc cells. (A) Concentration-dependency of irbesartan on GLUT4 translocation. Cells were incubated with various concentrations of irbesartan for 24 h. (B) Effect of irbesartan on GLUT4 translocation after 24-h incubation. Cells were incubated with irbesartan (50 and 100 μM), valsartan (100 μM), pioglitazone (100 μM) or vehicle for 24 h. (C) Acute effect of irbesartan on GLUT4 translocation after 3-h incubation. Cells were incubated with irbesartan (50 and 100 μM), valsartan (100 μM), pioglitazone (100 μM) or vehicle in serum-deprived medium (α-MEM 0.1% bovine serum albumin) for 3 h. Cell surface GLUT4 was measured as described in Materials and methods Section 2.5, and expressed as fold increase over the vehicle control without insulin stimulation. Data are presented as mean ± S.E.M. n = 4; *P b 0.05 vs. vehicle, **P b 0.01 vs. vehicle, #P b 0.05 vs. vehicle, ##P b 0.01 vs. vehicle.
3.3. Akt phosphorylation We examined the effect of irbesartan on the phosphorylation status of Akt which plays a primary role in insulin signaling. L6GLUT4myc cells were treated with irbesartan and the Akt phosphorylation was evaluated using Western blot analysis. The phosphorylation level of Akt was not changed by irbesartan treatment.
A number of clinical trials have presented evidence of irbesartan exhibiting favorable effects on glucose and lipid metabolism in patients with essential hypertension or metabolic syndrome (Kyvelou et al., 2006; Negro et al., 2006; Kintscher et al., 2007). Although evidence has accumulated in recent years to show that irbesartan has a direct action in adipocyte that lead to beneficial metabolic effects through PPARγ modulating properties (Schupp et al, 2004; Schupp et al, 2005; Erbe et al, 2006; Pershadsingh 2006), the precise molecular mechanism underlying these effects is not fully understood. The skeletal muscle is a major contributor to glucose metabolism, however, action of angiotensin II type 1 receptor blocker on skeletal muscle has been relatively unexplored. Therefore, we investigated whether or not irbesartan regulates glucose metabolism in skeletal muscles by using well established cell lines. We found that irbesartan enhances glucose uptake in these cell lines, which is likely to be attributed to GLUT4 translocation to the plasma membrane induced by irbesartan. Clearly, these effects are not mediated by angiotensin II receptor blockade, because the cell-based experiments in our study were performed in the absence of angiotensin II. Moreover, the observation that valsartan does not enhance glucose transport or GLUT4 translocation further supports this notion. We showed that chronic administration of irbesartan to the Zucker fatty rat, a widely used disease model of obesity and insulin resistance, elicited significant amelioration of insulin resistance. This observation is in line with what has been previously reported (Henriksen et al., 2001). Plasma concentration of irbesartan in the present study was estimated to be approximately 10 μM. Since, as shown in Fig. 3A, the cell surface GLUT4myc tended to increase at 12.5 μM of irbesartan, we speculate that beneficial metabolic effect of irbesartan observed in
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
27
Table 1 Effect of irbesartan on body weight and plasma parameters in Zucker fa/fa rats.
Vehicle Irbesartan 10 mg/kg Irbesartan 30 mg/kg Irbesartan 50 mg/kg Lean
Body weight (g)
Fasting plasma glucose (mg/dl)
Fasting plasma insulin (ng/ml)
Plasma triglyceride (mg/dl)
Relative HOMA-IR
482.6 ± 6.4 484.0 ± 8.4 483.9 ± 6.0 457.5 ± 7.4a 325.6 ± 6.9c
139.8 ± 7.1 138.3 ± 5.3 139.9 ± 2.8 125.9 ± 1.9 98.6 ± 2.4c
7.59 ± 0.51 7.11 ± 0.84 5.49 ± 0.54a 4.47 ± 0.41b 0.36 ± 0.05c
530.1 ± 58.7 448.1 ± 64.9 391.9 ± 53.2 373.6 ± 62.7 91.6 ± 14.7c
1.00 ± 0.09 0.95 ± 0.14 0.73 ± 0.07 0.53 ± 0.05b 0.03 ± 0.01c
Values are means ± S.E.M. for 5 to 9 animals per group. Relative HOMA-IR is expressed as relative values normalized by the value for vehicle group as 1.0. a Pb0.05 vs. vehicle. b Pb0.01 vs. vehicle. c Pb0.01 vs. vehicle.
the present study may be attributable in part to its direct action on skeletal muscle. Precise molecular pathways for irbesartan to enhance glucose transport in skeletal muscle cells are unknown at present. Since our observation is consistent with an earlier report by Verma et al. (2004), which showed that pioglitazone enhanced glucose uptake in C2C12 cells and that glucose uptake was decreased in C2C12 cells transfected with the antisense construct of PPARγ, one possibility is that irbesartan promotes glucose transport in skeletal muscle through PPARγ modulation. To investigate other potential mechanisms besides PPARγ, we examined three possibilities. First, with the knowledge that activation of the phosphatidylinositol 3-kinase (PI3kinase) pathway affects insulin-mediated stimulation of GLUT4 translocation in the skeletal muscle, we assessed the effect of irbesartan on phosphorylation of Akt, a major downstream target of PI3-kinase in L6-GLUT4myc cells. However, irbesartan did not show a significant effect on Akt phosphorylation under our experimental conditions. Next, we investigated the involvement of adiponectin, adipocytokine derived from adipose tissues with insulin-sensitizing properties. Contrary to the general concept that adiponectin is produced only by the adipose tissue, recent evidence has suggested that skeletal muscle has the potential to produce adiponectin. For instance, rosiglitazone enhanced glucose uptake with increased secretion of adiponectin in L6 cells (Liu et al., 2009). Although we examined the adiponectin mRNA level in L6-GLUT4myc cells exposed to irbesartan, we detected no significant change (data not shown). Third, in order to study the possible involvement of AMPK activation in irbesartan-induced enhancement of glucose transport under our experimental conditions, we evaluated the effects of irbesartan on AMPK activity in L6-GLUT4myc cells by assessing the phosphorylation state of acetyl-CoA carboxylase, a well-known substrate for AMPK. However, AMPK activation was not observed. Since the enhancement of GLUT4 translocation to the plasma membrane did not occur under short-time 3-h treatment, the onset of the increase of glucose uptake induced by irbesartan may be slow and might require the synthesis of new RNAs and proteins. In this regard, a link between irbesartaninduced enhancement of glucose transport and the transcription factors, such as PPARγ, may exist. Further study is needed to identify the molecules involved in enhancement of glucose uptake by irbesartan. Notably, the irbesartan-induced enhancement of whole-body insulin sensitivity was not accompanied by weight gain. Although thiazolidinediones are effective for diabetes treatment, the clinical applications of thiazolidinediones are limited due to undesirable side effects including weight gain, fluid retention, peripheral edema and increased incidence of bone fractures (Rizos et al., 2009). Given that diabetes frequently accompanies hypertension in metabolic syndrome (Geiss et al., 2002), the current findings that chronic irbesartan treatment induces beneficial effects on metabolic parameters in Zucker fatty rats without weight gain indicates a potentially important profile of irbesartan for pharmacological treatment of metabolic syndrome. Recently, several PPARγ partial agonists, which
maintain remarkable insulin-sensitizing efficacy with lesser adverse effects, have been synthesized (Chang et al., 2008; Higgins and Mantzoros, 2008; Larsen et al., 2008; Li et al., 2008; Kim et al., 2009). With respect to irbesartan, there are reports suggesting that irbesartan functions as a partial agonist in cell-based transactivaton assays (Schupp et al., 2004) and in cofactor recruitment assays (Schupp et al., 2005). Thus, the PPARγ partial agonistic activity of irbesartan may explain, at least in part, its beneficial metabolic effect without weight gain. In summary, we provide new evidence that irbesartan exerts a direct effect on skeletal muscle to promote GLUT4 translocation and glucose uptake. This property may be relevant to the beneficial effect of irbesartan in ameliorating the metabolic disorders. Acknowledgment The authors thank Yoko Kajiwara, Hiroyuki Takagi, Michiyo Kanazu and Keiichi Imagawa for technical assistance and helpful discussion. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/ j.ejphar.2010.08.037. References Andréasson, K., Galuska, D., Thörne, A., Sonnenfeld, T., Wallberg-Henriksson, H., 1991. Decreased insulin-stimulated 3-0-methylglucose transport in in vitro incubated muscle strips from type II diabetic subjects. Acta Physiol. Scand. 142, 255–260. Benson, S.C., Pershadsingh, H.A., Ho, C.I., Chittiboyina, A., Desai, P., Pravenec, M., Qi, N., Wang, J., Avery, M.A., Kurtz, T.W., 2004. Identification of telmisartan as a unique angiotensin II receptor antagonist with selective PPARgamma-modulating activity. Hypertension 43, 993–1002. Björntorp, P., Krotkiewski, M., Larsson, B., Somlo-Szücs, Z., 1970. Effects of feeding states on lipid radioactivity in liver, muscle and adipose tissue after injection of labelled glucose in the rat. Acta Physiol. Scand. 80, 29–38. Björntorp, P., Berchtold, P., Holm, J., Larsson, B., 1971. The glucose uptake of human adipose tissue in obesity. Eur. J. Clin. Invest. 1971 (1), 480–485. Bramlage, P., Pittrow, D., Kirch, W., 2004. The effect of irbesartan in reducing cardiovascular risk in hypertensive type 2 diabetic patients: an observational study in 16, 600 patients in primary care. Curr. Med. Res. Opin. 20, 1625–1631. Chang, C.H., McNamara, L.A., Wu, M.S., Muise, E.S., Tan, Y., Wood, H.B., Meinke, P.T., Thompson, J.R., Doebber, T.W., Berger, J.P., McCann, M.E., 2008. A novel selective peroxisome proliferator-activator receptor-gamma modulator-SPPARgammaM5 improves insulin sensitivity with diminished adverse cardiovascular effects. Eur. J. Pharmacol. 14, 192–201. Clasen, R., Schupp, M., Foryst-Ludwig, A., Sprang, C., Clemenz, M., Krikov, M., ThöneReineke, C., Unger, T., Kintscher, U., 2005. PPARgamma-activating angiotensin type1 receptor blockers induce adiponectin. Hypertension 46, 137–143. DeFronzo, R.A., Jacot, E., Jequier, E., Maeder, E., Wahren, J., Felber, J.P., 1981. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30, 1000–1007. DeFronzo, R.A., Gunnarsson, R., Björkman, O., Olsson, M., Wahren, J., 1985. Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus. J. Clin. Invest. 76, 149–155. Erbe, D.V., Gartrell, K., Zhang, Y.L., Suri, V., Kirincich, S.J., Will, S., Perreault, M., Wang, S., Tobin, J.F., 2006. Molecular activation of PPARgamma by angiotensin II type 1receptor antagonists. Vascul Pharmacol. 45, 154–162. Geiss, L.S., Rolka, D.B., Engelgau, M.M., 2002. Elevated blood pressure among U.S. adults with diabetes, 1988–1994. Am. J. Prev. Med. 22, 42–48.
28
T. Kobayashi et al. / European Journal of Pharmacology 649 (2010) 23–28
Henriksen, E.J., Jacob, S., Kinnick, T.R., Teachey, M.K., Krekler, M., 2001. Selective angiotensin II receptor receptor antagonism reduces insulin resistance in obese Zucker rats. Hypertension 38, 884–890. Higgins, L.S., Mantzoros, C.S., 2008. The development of INT131 as a selective PPARgamma modulator: approach to a safer insulin sensitizer. PPAR Res. 936906. Janke, J., Schupp, M., Engeli, S., Gorzelniak, K., Boschmann, M., Sauma, L., Nystrom, F.H., Jordan, J., Luft, F.C., Sharma, A.M., 2006. Angiotensin type 1 receptor antagonists induce human in-vitro adipogenesis through peroxisome proliferator-activated receptor-gamma activation. J. Hypertens. 24, 1809–1816. Kim, M.K., Chae, Y.N., Kim, H.S., Choi, S.H., Son, M.H., Kim, S.H., Kim, J.K., Moon, H.S., Park, S.K., Shin, Y.A., Kim, J.G., Lee, C.H., Lim, J.I., Shin, C.Y., 2009. PAR-1622 is a selective peroxisome proliferator-activated receptor gamma partial activator with preserved antidiabetic efficacy and broader safety profile for fluid retention. Arch. Pharm. Res. 32, 721–727. Kintscher, U., Bramlage, P., Paar, W.D., Thoenes, M., Unger, T., 2007. Irbesartan for the treatment of hypertension in patients with the metabolic syndrome: a sub analysis of the Treat to Target post authorization survey. Prospective observational, two armed study in 14,200 patients. Cardiovasc. Diabetol. 6, 12. Kyvelou, S.M., Vyssoulis, G.P., Karpanou, E.A., Adamopoulos, D.N., Zervoudaki, A.I., Pietri, P.G., Stefanadis, C.I., 2006. Effects of antihypertensive treatment with angiotensin II receptor blockers on lipid profile: an open multi-drug comparison trial. Hellenic J. Cardiol. 47, 21–28. Larsen, P.J., Lykkegaard, K., Larsen, L.K., Fleckner, J., Sauerberg, P., Wassermann, K., Wulff, E.M., 2008. Dissociation of antihyperglycaemic and adverse effects of partial perioxisome proliferator-activated receptor (PPAR-gamma) agonist balaglitazone. Eur. J. Pharmacol. 596, 173–179. Li, Y., Wang, Z., Furukawa, N., Escaron, P., Weiszmann, J., Lee, G., Lindstrom, M., Liu, J., Liu, X., Xu, H., Plotnikova, O., Prasad, V., Walker, N., Learned, R.M., Chen, J.L., 2008. T2384, a novel antidiabetic agent with unique peroxisome proliferator-activated receptor gamma binding properties. J. Biol. Chem. 283, 9168–9176. Liu, Y., Chewchuk, S., Lavigne, C., Brûlé, S., Pilon, G., Houde, V., Xu, A., Marette, A., Sweeney, G., 2009. Functional significance of skeletal muscle adiponectin production, changes in animal models of obesity and diabetes, and regulation by rosiglitazone treatment. Am. J. Physiol. Endocrinol. Metab. 297, E657–E664.
Momose, Y., Meguro, K., Ikeda, H., Hatanaka, C., Oi, S., Sohda, T., 1991. Studies on antidiabetic agents. X. Synthesis and biological activities of pioglitazone and related compounds. Chem. Pharm. Bull. (Tokyo). 39, 1440–1445. Negro, R., Formoso, G., Hassan, H., 2006. The effects of irbesartan and telmisartan on metabolic parameters and blood pressure in obese, insulin resistant, hypertensive patients. J. Endocrinol. Invest. 29, 957–961. Nuutila, P., Koivisto, V.A., Knuuti, J., Ruotsalainen, U., Teräs, M., Haaparanta, M., Bergman, J., Solin, O., Voipio-Pulkki, L.M., Wegelius, U., Yki-Jaryinen, H., 1992. Glucose-free fatty acid cycle operates in human heart and skeletal muscle in vivo. J. Clin. Invest. 89, 1767–1774. Pershadsingh, H.A., 2006. Treating the metabolic syndrome using angiotensin receptor antagonists that selectively modulate peroxisome proliferator-activated receptorgamma. Int. J. Biochem. Cell Biol. 38, 766–781. Rizos, C.V., Elisaf, M.S., Mikhailidis, D.P., Liberopoulos, E.N., 2009. How safe is the use of thiazolidinediones in clinical practice? Expert Opin. Drug Saf. 8, 15–32. Schupp, M., Janke, J., Clasen, R., Unger, T., Kintscher, U., 2004. Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation 109, 2054–2057. Schupp, M., Clemenz, M., Gineste, R., Witt, H., Janke, J., Helleboid, S., Hennuyer, N., Ruiz, P., Unger, T., Staels, B., Kintscher, U., 2005. Molecular characterization of new selective peroxisome proliferator-activated receptor gamma modulators with angiotensin receptor blocking activity. Diabetes 54, 3442–3452. Sloniger, J.A., Saengsirisuwan, V., Diehl, C.J., Kim, J.S., Henriksen, E.J., 2005. Selective angiotensin II receptor antagonism enhances whole-body insulin sensitivity and muscle glucose transport in hypertensive TG (mREN2)27 rats. Metabolism 54, 1659–1668. Verma, N.K., Singh, J., Dey, C.S., 2004. PPAR-gamma expression modulates insulin sensitivity in C2C12 skeletal muscle cells. Br. J. Pharmacol. 143, 1006–1013. Zierath, J.R., 1995. In vitro studies of human skeletal muscle: hormonal and metabolic regulation of glucose transport. Acta Physiol. Scand. Suppl. 626, 1–96. Zierath, J.R., Galuska, D., Nolte, L.A., Thörne, A., Kristensen, J.S., Wallberg-Henriksson, H., 1994. Effects of glycaemia on glucose transport in isolated skeletal muscle from patients with NIDDM: in vitro reversal of muscular insulin resistance. Diabetologia 37, 270–277.