Corticosterone suppresses insulin- and NO-stimulated muscle glucose uptake in broiler chickens (Gallus gallus domesticus)

Comparative Biochemistry and Physiology, Part C 149 (2009) 448–454

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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c

Corticosterone suppresses insulin- and NO-stimulated muscle glucose uptake in broiler chickens (Gallus gallus domesticus) J.P. Zhao, H. Lin ⁎, H.C. Jiao, Z.G. Song Department of Animal Science, Shandong Agricultural University, Taian, Shandong 271018, PR China

a r t i c l e

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Article history: Received 22 July 2008 Received in revised form 14 October 2008 Accepted 17 October 2008 Available online 5 November 2008 Keywords: Corticosterone Glucose Insulin Nitric oxide Chickens

a b s t r a c t We evaluated the effects of stress as mimicked by corticosterone (CORT) administration on the uptake of glucose by skeletal muscles (M. fibularis longus) in broiler chickens (Gallus gallus domesticus). The results showed that both chronic (7 d) and short-term (3 h) CORT administration resulted in hyperglycemia and hyperinsulinemia. Plasma level of nitric oxide (NO) and the activity of NO synthase (NOS) were both suppressed by either chronic or acute stress. In vivo CORT treatment could stimulate the in vitro uptake of 2-deoxy-D-[1,2-3H]-glucose (2-DG). Sodium nitroprusside (SNP) administration improved the in vitro uptake of 2-DG in both CORT and control groups. In CORT treatment, however, the stimulating effect of NO on 2-DG uptake was relatively lower compared to control group, whereas it was restored by insulin. Insulin stimulated muscle in vitro 2-DG uptake in either control or CORT group, with the improvement being significantly higher in control chickens. The results indicated that the reduced circulating and muscle level of NO level via the suppression of NOS by corticosterone treatment was involved in the stress-induced insulin resistance. It appears that CORT could suppress the insulin stimulated glucose uptake in skeletal muscle, inducing insulin resistance in broiler chickens. We conclude that NO could stimulate glucose transport in chicken skeletal muscle and that the reduced circulating and muscle level of NO is involved in the insulin resistance induced by corticosterone treatment. © 2008 Elsevier Inc. All rights reserved.

1. Introduction The principal role of insulin is to control fuel homeostasis, regulating metabolism and growth. Insulin resistance as an impaired biological response to circulating insulin causes a range of metabolic diseases including type 2 diabetes, glucose intolerance, obesity, dyslipidaemia, hypertension and cardiovascular disease in humans (Reaven, 1988). Glucocorticoids (GCs), as the final effectors of the hypothalamic–pituitary–adrenal (HPA) axis, participate in the control of whole body homeostasis and stress response. In mammals, GCs play a key role in the pathogenesis of insulin resistance (reviewed by Reynolds and Walker, 2003). Birds have higher plasma glucose concentrations compared to mammals with similar body weight (reviewed by Braun and Sweazea, 2008). In contrast, the circulating insulin levels are lower. For example, plasma insulin levels of chickens are only about one tenth of those in rats (Dupont et al., 2004). In our previous studies, corticosterone (CORT) administration could induce insulin resistance (hyperglycemia and hyperinsulinemia) in chickens as well (Lin et al., 2006). Moreover,

Abbreviations: CORT, corticosterone; 2-DG, 2-deoxy-D-[1, 2-3H]-glucose; GCs, glucocorticoids; GLUT-4, glucose transporter type 4; INS, insulin; L-NAME, NG-nitroL-Arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; PI 3-kinase, phosphatidylinositol 3-kinase; SNP, sodium nitroprusside. ⁎ Corresponding author. Tel.: +86 538 8249203; fax: +86 538 8241419. E-mail address: [email protected] (H. Lin). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.10.106

CORT treatment resulted in suppressed muscle development and augmented fat deposition (Dong et al., 2007; Yuan et al., 2008). In mammals, skeletal muscle is the most important organ for the disposal of glucose. Both insulin and exercise stimulate glucose transport (Constable et al., 1988). The pathway stimulated by insulin and insulin-like growth factor-1 is wortmannin and phosphatidylinositol 3-kinase (PI 3-kinase) dependent while the contraction or contraction/hypoxia pathway is wortmannin and PI 3-kinase independent (Lund et al., 1995). Moreover, nitric oxide (NO) has been demonstrated to be involved in modulating glucose transport via the pathway of insulin (Roy et al., 1998) or contraction (Roberts et al., 1997) or a mechanism distinct from either (Higaki et al., 2001). Insulin markedly stimulates glucose uptake and utilization in skeletal muscles by activating the translocation of glucose transporter4 (GLUT-4), which is the predominant insulin-responsive transporter in mammalian skeletal muscle (Shepherd and Kahn, 1999). In contrast, birds seem to be deficient in GLUT-4 (Seki et al., 2003; Sweazea and Braun, 2006). The insulin cascade appears to respond normally in liver, but is refractory in muscle (Dupont et al., 2004). Nevertheless, glucose transport across the plasma membrane in vivo in skeletal muscle was increased by insulin administration to chicks, suggesting that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack the GLUT-4 homologous gene (Tokushima et al., 2005). Recently, it was reported that NO is involved in non-insulin mediated glucose transport (Nishiki et al., 2008).

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Hence, we hypothesized that both insulin and NO pathways were involved in the impaired glucose uptake in skeletal muscles by GCs. The objective of the present study was to investigate the effect of CORT on the uptake of glucose in skeletal muscles and the possible pathways involved in glucose regulation, by analyzing the effects of in vivo or in vitro CORT administration on glucose uptake rates. In this study, the M. fibularis longus (FL) muscle, a red muscle functioning in locomotion and jumping, was employed in the measurement of glucose uptake rate. Birds have a high metabolic rate and glucose level, high growth rate and capacity of fat deposition, making broiler muscle an interesting model of insulin resistance. 2. Materials and methods 2.1. Birds and husbandry Male broiler chickens (Arbor Acres, Gallus gallus domesticus) were obtained from a local hatchery (Taiyu Breeder Comp., Shandong, P.R. China) at 1 d of age. Birds were provided a commercial broiler starter diet (12.37 MJ metabolizable energy, ME/kg; 21.5% crude protein, CP) until 21 d of age, after which a commercial grower diet (12.90 MJ ME/kg; 19.48% CP) was provided until the end of the experiment. The brooding temperature was maintained at 35 °C for the first 2 d, and then decreased gradually to 21 °C (45% relative humidity) until 28 d and thereafter maintained at such to 42 d of age. The light regime was 23 Light:1 Dark. All the birds had free access to feed and water during the rearing period. The present study was approved by the University and carried out in accordance with the ‘Guidelines for Experimental Animal’ of the Ministry of Science and Technology. 2.2. Experiment 1 effect of chronic CORT administration on plasma parameters Twenty-four broiler chickens were assigned randomly to two groups of twelve chickens. At 28 d of age, chickens were randomly subjected to one of the two dietary treatments: a basal diet (control) or the basal diet supplemented with 30 mg CORT/kg diet (Sigma-Aldrich Chemical, CORT group; Malheiros et al., 2003; Lin et al., 2004a). A blood sample was obtained from each bird before and at 7 d after CORT treatment, respectively. Blood was drawn from a wing vein using a heparinized syringe within 30 s and collected in iced tubes. Plasma was obtained after centrifugation at 400 g for 10 min at 4 °C and stored at −20 °C for further analysis of glucose, NO, NOS and insulin.

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The right M. fibularis longus (FL) muscle was carefully dissected out and immediately placed in ice-cold 0.9% saline. Thereafter, the muscle was tied to a stainless steel clip to maintain it at its in vivo resting length. FL muscles were incubated initially at 42 °C in incubation flasks for 30 min. The chamber contained 15 mL of preequilibrated incubation medium (Krebs–Henseleit bicarbonate buffer (KHB, pH 7.4) with 8 mM glucose, 32 mM mannitol, 1 mM sodium pyruvate, and 0.1% bovine serum albumin (BSA; fraction V, Sigma Chemical)) to allow for equilibrations. The flasks were continually gassed with 95% O2 + 5% CO2 in a shaking incubator. After the initial 30 min of incubation, FL muscles were transferred to freshly oxygenated flasks containing 15 mL of the same buffer and incubated for 120 min in the presence of 10 mM sodium nitroprusside (SNP, Sigma) or 2 mM NG-nitro-L-arginine methyl ester (LNAME, Sigma) or 100 mU insulin or 100 mU insulin + 10 mM SNP or 100 mU insulin + 2 mM L-NAME. After incubation, muscle samples were rapidly frozen in liquid nitrogen and stored at − 80 °C for the measurement of glycogen, NO and NOS. 2.4.2. 2-deoxyglucose uptake Two groups of six chickens, subjected to either CORT or control treatment, were used and the bilateral FL muscle was carefully dissected out. Glucose uptake in isolated skeletal muscle strips was measured with 2-deoxy-D-[1, 2-3H]-glucose (2-DG), as previously described by Hansen et al. (1994). In brief, the FL muscles were dissected out and rapidly cut into 40- to 60-mg strips. After 30 min pre-equilibration, FL muscles were washed for 20 min at 37 °C in 3 mL of KHB buffer containing 40 mM mannitol, 2 mM sodium pyruvate and 0.1% BSA. Thereafter, muscle strips were incubated for 20 min at 36 °C in 3 mL of KHB buffer containing 8 mM 2-[3H]-DG (1 µCi/mL), 32 mM [14C]-mannitol (0.32 µCi/mL, Amersham Biosciences), 2 mM sodium pyruvate and 0.1% BSA, in the presence of 10 mM SNP or 2 mM L-NAME or 100 mU insulin or 100 mU insulin + 10 mM SNP or 100 mU insulin + 2 mM L-NAME. After the incubation, muscles were rapidly blotted at 4 °C, clamp frozen in liquid nitrogen, and stored at −80 °C. Uptake of 2-[3H]-DG in muscles was detected in perchloric acid extracts. The samples were then counted in a liquid scintillation counter (Wallac1410, Pharmcia). 2-[3H]-DG uptake rate was corrected for extracellular trapping using [14C]-mannitol. 2.5. Experiment 4 effect of in vitro CORT treatment on the in vitro 2-deoxyglucose uptake in FL muscles

2.3. Experiment 2 effect of acute CORT administration on plasma parameters Twenty-four 42-day-old broiler chickens were divided into two groups of 12 birds and randomly assigned to one of two treatments: one single subcutaneously injection of CORT (4 mg/kg body mass, BW, dissolved in corn oil, Lin et al., 2004b) or corn oil (control). Feed was withdrawn and chickens had free access to water during the 3-h experimental period. A blood sample was obtained from each chicken immediately before and at 3 h after injection. Plasma was obtained (as in Experiment 1) for further analysis of glucose, NO, NOS and insulin. 2.4. Experiment 3 effect of in vivo CORT treatment on the in vitro 2-deoxyglucose uptake in FL muscles Two groups of 28-d old broiler chickens were employed and randomly subjected to one single subcutaneous injection of CORT (4 mg/kg BW) or corn oil (control). Feed was withdrawn and chickens had free access to water during the 3-h experimental period. Three hours after CORT administration, all birds were killed by intravenous injection of sodium pentobarbitone (100 mg/kg of BW). 2.4.1. Measurement of NO, NOS and glycogen For the measurement of NO, NOS and glycogen, two groups of 36 chickens subjected to either CORT or control treatment were used.

Two groups of thirty six 28-d old broiler chickens were employed and the bilateral fibularis longus (FL) muscles were obtained. The muscle samples from the two groups were randomly subjected to control or CORT treatments. In controls, muscle samples were incubated as in Experiment 3, while the incubation was conducted in the presence of 1 µM CORT in CORT treatments. After incubation, muscle samples were rapidly frozen in liquid nitrogen and stored at −80 °C for the measurement of glycogen, NO and NOS. Another two groups of six chickens were used for the measurement of 2-DG uptake. The bilateral FL muscles were obtained and rapidly cut into 40- to 60-mg strips. In control group, the muscle strips were incubated as mentioned above in Experiment 3. For the CORT treatment, muscle strips were incubated as the control group, except in the presence of 1 µM CORT. After incubation, FL muscles were rapidly frozen with liquid nitrogen and stored at −80 °C for the measurement of glycogen, NO and NOS. 2.6. Measurements 2.6.1. Plasma parameters The plasma levels of glucose (No. F006) and NO (No. A012) and activity of NOS (No. A014-2) were measured spectrophotometrically with commercial colorimetric diagnostic kits (Jiancheng Bioengineering

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Institute, Nanjing, P.R. China). A unit of NOS was defined as 1 mmol NO produced by reacting 1 min in 1 mL of plasma at 37 °C. Plasma insulin was measured by radioimmunoassay (RIA) with a guinea pig anti-porcine insulin serum (3V Bio-engineering Group Co., Weifang, P.R. China). In this measurement, 125I-labeled insulin competes with plasma insulin for sites on insulin-porcine antibody immobilized to the wall of a polypropylene tube. A large crossreaction has been observed between chicken insulin and the porcine anti-sera (Simon et al., 1974). The insulin in this study is referred to as immunoreactive insulin. The sensitivity of the assay was 1 µIU/mL and all samples were included in the same assay to avoid interassay variability. The intraassay coefficient of variation was 6.9%. 2.6.2. Muscle tissue parameters analysis Muscle radioactivity was determined by liquid scintillation counting in a Beckman LS6000 scintillation counter programmed for dualchannel counting. The specific uptake of 2-DG by the muscle strip was calculated by subtracting the 3H activity in the extracellular space from the total 3H activity of each muscle sample. The extracellular trapping of 3H was corrected for using [14C]-mannitol. Muscle glycogen content, level of NO and activity of NOS were measured with commercial colorimetric diagnostic kits (Jiancheng Bioengineering Institute, Nanjing). 2.7. Statistical analysis In Experiment 1 and 2, plasma variables were subjected to Repeated Measurement Analysis (SAS for windows, version 8e, SAS Institute,1998) to evaluate the main effects of treatment and time and their interaction. Paired t-test analysis was used to evaluate the time effect within treatment. In Experiment 3 and 4, the main effect of CORT, insulin and NO treatments as well as their interactions were estimated by three-factor ANOVA. Means were considered significantly different when P b 0.05.

Table 2 Effects of subcutaneous administration of corticosterone (CORT, 4 mg/kg BW) on plasma parameters of broiler chickens in experiment 2 CORT

Control

Significance

Glucose, mMol/L 0h 3h

11.4 ± 0.18y 15.8 ± 0.45a,x

11.4 ± 0.22 12.0 ± 0.21b

CORT: P b 0.0001 Time: P b 0.0001 CORT × time: P b 0.0001

Insulin, µIU/mL 0h 3h

9.46 ± 1.01y 46.9 ± 3.3a,x

10.8 ± 1.5 15.5 ± 1.4b

CORT: P b 0.0001 Time: P b 0.0001 CORT × time: P b 0.0001

NO, µmol/L 0h 3h

22.9 ± 1.0x 18.1 ± 1.3b,y

24.1 ± 1.1 23.7 ± 1.2a

CORT: P = 0.0428 Time: P b 0.0001 CORT × time: P = 0.0002

NOS, U/mL 0h 3h

42.7 ± 1.5x 32.2 ± 1.1b,y

42.0 ± 1.0 40.6 ± 1.2a

CORT: P = 0.0099 Time: P b 0.0001 CORT × time: P = 0.0003

NO, nitric oxide; NOS, NO synthase. Values are means ± SEM (n = 12). a,b : means with different superscript within the same time point differ significantly (Pb 0.05). x,y : means with different superscript within the same treatment differ significantly (Pb 0.05).

chickens, plasma levels of glucose and insulin were elevated and NO level was decreased compared to control group after 7-d treatment. Similarly, compared to the initial level before treatment (D 0), plasma levels of glucose and insulin were significantly (P b 0.0001) increased while the level of NO was decreased (P b 0.05) by long-term CORT administration. For the activity of NOS, a significant effect of time as well as its interaction with CORT treatment was detected. Compared to the basal level, NOS level was significantly decreased after 7-d administration in CORT rather than in control treatment.

3. Results 3.1. Chronic effect of CORT administration on plasma concentrations of glucose, insulin and NO and the activity of NOS Plasma levels of glucose, insulin and NO were all significantly influenced by CORT, time and their interaction (Table 1). In CORT

Table 1 Effects of dietary supplementation of corticosterone (CORT, 30 mg/kg diet) on plasma parameters of broiler chickens in experiment 1

Glucose, mmol/L 0d 7d

CORT

Control

Significance

12.2 ± 0.22y 21.2 ± 0.46a,x

12.4 ± 0.18 13.1 ± 0.24b

CORT: P b 0.0001 Time: P b 0.0001 CORT × time: P b 0.0001

Insulin, µIU/mL 0d 7d

9.40 ± 1.02y 88.2 ± 5.9a,x

12.6 ± 1.7 19.4 ± 2.1b

CORT: P b 0.0001 Time: P b 0.0001 CORT × time: P b 0.0001

NO, µmol/L 0d 7d

24.1 ± 1.1x 16.1 ± 1.0b,y

23.4 ± 1.2 22.7 ± 1.0a

CORT: P = 0.0444 Time: P b 0.0001 CORT × time: P = 0.0002

NOS, U/mL 0d 7d

47.2 ± 1.4x 43.2 ± 1.2y

45.1 ± 1.4 44.6 ± 1.2

CORT: NS Time: P = 0.0027 CORT × time: P = 0.0167

NO, nitric oxide; NOS, NO synthase. Values are means ± SEM (n = 12). a,b : Means with different superscript within the same time point differ significantly (P b 0.05). x,y : means with different superscript within the same treatment differ significantly (P b 0.05).

Table 3 Effect of insulin (100 mU/mL), sodium nitroprusside (SNP,10 mM/L) and NG-nitro-L-arginine methyl ester (L-NAME, 2 mM/L) administration during in vitro incubation on 2-deoxyglucose (2-DG) uptake rate, muscle levels of nitric oxide (NO) and glycogen and the activity of NO synthase (NOS) in M. fibularis longus muscles isolated from broiler chickens subjected to one single subcutaneous injection of corticosterone (4 mg/kg body mass) in experiment 3

2-DG, nmol/g/min CORT Insulin NO

No treatment

Treatment (+)

42.17x 26.72y 37.40y

38.85y 54.30x 47.15x (SNP)

CORT⁎Insulin Significance b 0.0001 Glycogen, mg/g wet tissue CORT 1.43 Insulin 1.34y NO 1.40y

CORT⁎NO NS

CORT⁎Insulin 0.0041

CORT⁎NO NS

2.96x 2.22y 1.11y CORT⁎Insulin NS

2.24y 2.98x 5.62x (SNP) CORT⁎NO b 0.0001

1.24x 0.99y 1.21x

1.09y 1.34x 1.23x (SNP)

CORT⁎Insulin NS

CORT⁎NO NS

Significance NO, μmol/g protein CORT Insulin NO Significance NOS, U/mg protein CORT Insulin NO

Significance

1.44 1.53x 1.52x (SNP)

Treatment (−)

36.98y (L-NAME) Insulin⁎NO 0.0382

1.40y (L-NAME) Insulin⁎NO NS

1.07y (L) Insulin⁎NO 0.1056

1.05y (L-NAME) Insulin⁎NO NS

Significance 0.0003 b0.0001 b0.0001 CORT⁎Insulin⁎NO NS NS b0.0001 b0.0001 CORT⁎Insulin⁎NO NS b0.0001 b0.0001 b0.0001 CORT⁎Insulin⁎NO NS b0.0001 b0.0001 b0.0001 CORT⁎Insulin⁎NO NS

Values are means ± SEM (n = 36). x,y : Means with different superscript within the same row differ significantly (P b 0.05).

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3.2. Acute effect of CORT administration on plasma concentrations of glucose, insulin and NO and the activity of NOS Compared to controls, plasma glucose and insulin concentrations were elevated while NO level and NOS activity were decreased by acute CORT treatment at 3-h after CORT administration (Table 2). For all the plasma variables, there was a significant time effect as well, as the interaction of time and CORT treatment. In CORT chickens, compared to the initial levels, glucose and insulin levels were increased while the NO level and NOS activity were reduced at 3-h post injection. In contrast, there was no significant time effect in control treatment. 3.3. Effect of in vivo CORT administration on 2-DG uptake rate and muscle levels of NO, NOS and glycogen The in vitro uptake of 2-DG was significantly (P b 0.001) suppressed by in vivo CORT treatment, whereas it was significantly (P b 0.0001) improved by in vitro INS or SNP treatments (Table 3, Fig. 1). In contrast, L-NAME treatment had no detectable effect on 2-DG uptake. There were significant interactions between INS and CORT and NO treatments. In basal group, the uptake rate of 2-DG was significantly higher in CORT treatment compared to control treatment, whereas the inverse was true when INS was present (Fig. 1). In the absence of insulin, the improvement of 2-DG uptake rate by SNP was greater compared to CORT chickens (Control, 59.7% vs. CORT, 16.4%), resulting the diminished difference between CORT and control treatment. In contrast, when insulin was present, the stimulating effect of SNP on 2-DG uptake was similar in the two groups of chickens (Control, 22.1% vs. CORT, 21.3%).

Fig. 2. Effect of insulin (100 mU/mL), sodium nitroprusside (SNP, 10 mM/L) and NGnitro-L-arginine methyl ester (L-NAME, 2 mM/L) administration on muscle level of nitric oxide (NO, A) and NO synthase activity (NOS, B) in M. fibularis longus muscles isolated from broiler chickens subjected to one single subcutaneous injection of corticosterone (CORT, 4 mg/kg body mass) in experiment 3. Values are means ± SEM (n = 6); a–d: Means with different superscript in CORT group differ significantly (P b 0.05); w–z: Means with different superscript in control group differ significantly (P b 0.05); ⁎: Means within the same in vitro treatment differ significantly (P b 0.05).

Muscle glycogen was not affected by CORT treatment, but it was increased (P b 0.0001) by INS treatment (Table 3, Fig. 1). NO treatment influenced (P b 0.0001) muscle glycogen concentration, which was improved by SNP. There was a significant (P b 0.01) interaction of CORT and INS. In basal group, CORT treatment had higher glycogen store compared to control group, whereas the inverse was true in the presence of INS. Muscle levels of NO and NOS were decreased by CORT treatment, but increased by INS treatment (P b 0.0001; Table 3, Fig. 2). Muscle NO level was increased by SNP treatment (P b 0.0001), regardless of CORT or insulin treatment, whereas L-NAME treatment had no significant influence (P N 0.05). There was an obvious interaction of CORT and NO treatments and the improvement by SNP was lower in CORT treatment compared to control group (CORT, 3.74 fold vs. Control, 4.30 fold). In contrast, the activity of NOS was suppressed (P b 0.0001) by L-NAME treatment and was not altered by SNP treatment. 3.4. Effect of in vitro CORT administration on 2-DG uptake rate and muscle levels of NO, NOS and glycogen

Fig. 1. Effect of insulin (100 mU/mL), sodium nitroprusside (SNP, 10 mM/L) and NGnitro-L-arginine methyl ester (L-NAME, 2 mM/L) administration on in vitro 2deoxyglucose (2-DG) uptake rate (A) and glycogen stores (B) in M. fibularis longus muscles isolated from broiler chickens subjected to one single subcutaneous injection of corticosterone (CORT, 4 mg/kg body mass) in experiment 3. Values are means ± SEM (n = 6); a–d: Means with different superscript in CORT group differ significantly (P b 0.05); w–z : Means with different superscript in control group differ significantly (P b 0.05); ⁎: Means within the same in vitro treatment differ significantly (P b 0.05).

The uptake rate of 2-DG was not affected (P N 0.05) by either CORT or L-NAME treatment (Table 4, Fig. 3). In contrast, both INS and SNP treatments significantly increased the uptake rate of 2-DG. There was a significant (P b 0.01) interaction of CORT and INS for the uptake of 2-DG and the effect of CORT was reversed in the presence of insulin. CORT treatment had no detectable effect (P N 0.05) on muscle glycogen store whereas it was increased (P b 0.0001) by INS treatment. Glycogen store was significantly increased by SNP treatment rather than L-NAME treatment.

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Table 4 Effect of corticosterone (CORT, 1μM/L), insulin (100 mU/mL), sodium nitroprusside (SNP, 10 mM/L) and NG-nitro-L-arginine methyl ester (L-NAME, 2 mM/L) administration during in vitro incubation on 2-deoxyglucose (2-DG) uptake rate, muscle levels of nitric oxide (NO) and glycogen and the activity of NO synthase (NOS) in M. fibularis longus muscles isolated from normal chickens in experiment 4

2-DG, nmol/g/min CORT Insulin NO

No treatment

Treatment (+)

69.48 50.40y 56.82y

61.74 80.82x 82.89x (SNP)

CORT⁎Insulin Significance 0.0051 Glycogen, mg/g wet tissue CORT 1.54 Insulin 1.42y NO 1.54y

Significance NO, μmol/g protein CORT Insulin NO

Significance NOS, U/mg protein CORT Insulin NO

Significance

CORT⁎NO NS 1.56 1.68x 1.63x (SNP)

CORT⁎Insulin NS

CORT⁎NO NS

4.17 3.56y 1.80y

4.40 5.01x 9.28x (SNP)

CORT⁎Insulin NS

CORT⁎NO NS

2.12 1.88y 2.29x

2.11 2.35x 2.28x (SNP)

CORT⁎Insulin NS

CORT⁎NO NS

Treatment (−)

57.13y (L-NAME) Insulin⁎NO NS

1.48z (L-NAME) Insulin⁎NO NS

1.77y (L-NAME) Insulin⁎NO NS

1.77y (L-NAME) Insulin⁎NO NS

Significance CORT: NS Insulin: b 0.0001 NO: b 0.0001 CORT⁎Insulin⁎NO NS CORT: NS Insulin: b 0.0001 NO: b 0.0001 CORT⁎Insulin⁎NO NS CORT: NS Insulin: 0.0014 NO: b 0.0001 CORT⁎Insulin⁎NO NS CORT: NS Insulin: b 0.0001 NO: b 0.0001 CORT⁎Insulin⁎NO NS

Values are means ± SEM (n = 36). x,y : Means with different superscript within the same row differ significantly (P b 0.05).

glycogen store in muscle of CORT chickens. Similarly, in vitro CORT administration tended to increase the 2-DG uptake in FL muscle from normal chickens (Experiment 4). Hence, the result may suggest that CORT solely is favourable, rather than disadvantageous, for the uptake of glucose. As birds are proved to be GLUT-4 deficient (Seki et al., 2003; Sweazea and Braun, 2006), the underlying mechanism needs to be investigated further. 4.2. CORT suppressed insulin-dependent glucose uptake Insulin is a potent stimulator of glucose transport in skeletal muscle. In line with previous works in mammals (Roy et al., 1998; Higaki et al., 2001) and chickens (Tokushima et al., 2005), INS treatment stimulated the in vitro uptake of 2-DG in skeletal muscles. In mammals, the elevated glucose uptake by insulin administration could be due to its direct stimulation of the intrinsic or functional activity of GLUT-4 (Garvey et al., 1991), which positively alters the recruitment of GLUT-4 from an intracellar pool to plasma membrane (Bao et al., 1995). As birds intrinsically lack GLUT-4 homologous gene (Seki et al., 2003; Tokushima et al., 2005; Sweazea and Braun, 2006), the result suggests that an insulin-responsive glucose transport mechanism is present in chickens as well, even though they intrinsically lack GLUT-4 homologous gene. The insulin-stimulated glucose uptake, however, was arrested partially in CORT chickens. Compared to control group, insulin-induced glucose uptake was suppressed by 16.3% in CORT-chickens. This result was in accordance with the result of glycogen content that the insulinstimulated glycogen synthesis was reduced by 7.1% in CORT treatment. In mammals, dexamethasone treatment reduced insulin-stimulated glucose uptake and glycogen synthesis by 30–70% in the epitrochlearis and soleus (Burén et al., 2008). Hence, it could be concluded that CORT could suppress the insulin stimulated muscle glucose uptake in chickens as well.

Neither muscle NO level nor NOS activity was significantly influenced by CORT treatment (P N 0.05, Table 4, Fig. 4). In contrast, both NO (P b 0.01) and NOS (P b 0.0001) levels were significantly increased by INS treatment. SNP treatment significantly (P b 0.0001) increased the level of NO but had no detectable influence (P N 0.05) on the activity of NOS. Inversely, L-NAME treatment significantly (P b 0.0001) suppressed NOS activity and had no significant (P N 0.05) effect on muscle NO level. No significant (P N 0.05) interaction of CORT, insulin and NO treatment was observed for either NO or NOS. 4. Discussion In stressed animals, the increased circulating levels of GCs are important for coping with a stressor. In our previous work, dietary CORT supplementation (30 mg/kg diet) or one single subcutaneous injection of CORT at a dose of 4 mg/kg BW increased circulating CORT within reasonable physiological limits (Lin et al., 2004a,b). In Experiment 1 and 2 of the present study, the hyperglycemia and hyperinsulinemia induced by either chronic or acute CORT administration indicated the induction of insulin resistance in broiler chickens. 4.1. In vivo CORT administration stimulated in vitro muscle glucose uptake Glucocorticoids initiate whole body insulin resistance in mammals. The present result showed that in vitro 2-DG uptake and glycogen stores were enhanced in muscle from CORT-challenged chickens (basal group), indicating that CORT administration could stimulate glucose uptake. This result was in line with the work of Ewart et al. (1998), who reported that dexamethasone treatment elevated GLUT1 and GLUT4 proteins by 68% and 94%, respectively, in L6 skeletal muscle cells. The result was supported by the observation of augmented

Fig. 3. Effect of corticosterone (CORT, 1 µM/L), insulin (100 mU/mL), sodium nitroprusside (SNP, 10 mM/L) and NG-nitro-L-arginine methyl ester (L-NAME, 2 mM/ L) administration on in vitro 2-deoxyglucose (2-DG) uptake rate (A) and glycogen stores (B) in M. fibularis longus muscles isolated from normal broiler chickens in experiment 4. Values are means ± SEM (n = 6); a–d: Means with different superscript in CORT group differ significantly (P b 0.05); w–z: Means with different superscript in control group differ significantly (P b 0.05).

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Fig. 4. Effect of corticosterone (CORT, 1 µM/L), insulin (100 mU/mL), sodium nitroprusside (SNP, 10 mM/L) and NG-nitro-L-arginine methyl ester (L-NAME, 2 mM/ L) administration on muscle level of nitric oxide (NO, A) and NO synthase (NOS, B) in M. fibularis longus muscles isolated from normal broiler chickens in experiment 4. Values are means ± SEM (n = 6); a–c: Means with different superscript in CORT group differ significantly (P b 0.05); w–z: Means with different superscript in control group differ significantly (P b 0.05).

In mammals, insulin exerts most of its actions through the insulin receptor substrate-1 and -2 (IRS-1 and -2), leading to glucose transport, glycogen synthesis and protein synthesis (Sun et al., 1991). There is evidence that GCs are involved in the early event of insulin-induced signalling pathways in mammals. Dexamethasone down-regulates insulin receptor substrate-1 protein content and insulin-induced tyrosine phosphorylation of IRS-1 (Ewart et al., 1998), and results in a reduction of insulin-stimulated IRS-1-associated PI 3-kinase (Saad et al., 1993), suggesting the depression of intracellular insulin-induced signalling. Glucocorticoids decrease the expression of protein kinase B (PKB) and insulin-stimulated phosphorylation, and increase glycogen synthase phosphorylation (Ruzzin et al., 2005; Burén et al., 2008). Hence, in mammals, the GC induced insulin resistance should be related to the depressed insulin signalling pathways such as the PKB/AKT and PI 3-Kinase/mTOR pathways (Wang et al., 1999; Müssig et al., 2005). In chickens, it was reported that the expression of IRS and IR-associated PI 3-kinase activity were all decreased by CORT treatment (Dupont et al., 1999). In chickens, however, the early steps of insulin signaling pathway are different from that of mammals. Recently, it was found that the basal levels of tyrosine phosphorylation of IR and of PI 3-kinase activity were much higher in chickens than in rats (Dupont et al., 2004). Moreover, in contrary to the strong activation for all components of the cascade by insulin in rats, no activation was observed in chickens (Dupont et al., 2004). The associated signalling pathways in stress-challenged chickens need to be investigated further. 4.3. NO involved in CORT-induced insulin resistance In mammals, NO has been proven to be an important contributor to mediate glucose transport. In mammals, part of the mechanism by which insulin increases glucose transport in vivo involves enhanced

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blood flow and glucose delivery to the muscle, a process mediated by the release of NO from the endothelium (Roy et al., 1998). Furthermore, there is evidence to show that NO is involved in the enhanced glucose transport by contraction as well (Roberts et al., 1997; Balon and Nadler, 1997). Recently, it is found that NO could mediate skeletal muscle glucose uptake through a mechanism distinct from both the insulin and contraction signaling pathways (Higaki et al., 2001). NO is produced in a variety of tissues through the activation of NO synthase (NOS). Of the three isoforms of NOS, both the type I (neuronal) and type III (endothelial) isoforms of NOS are expressed in skeletal muscle (Kobzik et al., 1994, 1995). In line with the previous work in mammals (Balon and Nadler, 1997; Etgen et al., 1997; Young et al., 1997) and in chickens (Nishiki et al., 2008), NO administration improved glucose uptake in skeletal muscle. In chickens, it is recently reported that NO is involved in the modulation of non-insulin mediated glucose transport in skeletal muscles as well (Nishiki et al., 2008). In the present study, the result showed that there was an additive effect on glucose uptake between SNP and insulin treatments, indicating that NO could solely improve glucose uptake in skeletal muscle. Compared to mammals, the NO may play a lesser role in the modulation of glucose transport in chickens (Nishiki et al., 2008). Moreover, the stimulating effect of NO on glucose uptake rate was less than that of insulin (Experiment 3: insulin, 103.2% vs. SNP, 26.1%; Experiment 4: insulin, 60.4% vs. SNP, 45.9%), suggesting that NO may play a less important role in the regulation of glucose transport in chicken skeletal muscle than insulin. In the present study, the circulating level of NO was decreased by either chronic or acute upregulation of CORT. The simultaneously suppressed NOS activity indicated that CORT administration reduces circulating level of NO via suppressed NOS activity. The result implied that the NO-related activation of glucose transport might be reduced in CORT chickens. Furthermore, the stimulating effect of NO on glucose uptake seemed to be suppressed by CORT. Without the presence of insulin, the activation of glucose uptake by SNP was higher in control group compared to CORT treatment (Experiment 3: control, 59.7% vs. CORT, 16.4%; Experiment 4: control, 63.3% vs. CORT, 29.8%). In the presence of insulin, however, the stimulating effect of NO in CORT chickens seemed to be restored in the presence of insulin as the increment of 2-DG uptake rate was similar in the two corresponding treatments (Experiment 3: control, 22.1% vs. CORT, 21.3%; Experiment 4: control, 48.7% vs. CORT, 42.9%). The result implies that the suppression effect of CORT on the stimulating effect of NO on glucose uptake is insulin-dependent. Nevertheless, it could be concluded that the depressed NO production is involved in the CORT-induced insulin resistance in stressed-chickens. Skeletal muscle is the main tissue responsible for the insulinstimulated glucose disposal. In our previous work, it has been proven that CORT treatment significantly suppressed the development of skeletal muscle (Lin et al., 2006; Dong et al., 2007; Yuan et al., 2008). Hence, the reduced skeletal muscle mass should be responsible at least partially for the stress-induced insulin resistance as well. In conclusion, CORT could suppress the insulin stimulated glucose uptake in skeletal muscle, inducing insulin resistance in broiler chickens. The results suggest that NO could stimulate glucose transport in skeletal muscle of chickens and the reduced circulating and muscle level of NO is involved in the insulin resistance induced by corticosterone treatment. Acknowledgements This work was supported by grants from the National Basic Research Program of China (2004CB117507), National Natural Science Foundation of China (No.30771573), Program for New Century Excellent Talents in University and the research fund for the Doctoral Program of Higher Education (RFDP).

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