Table 1.
Primers for real-time PCR.
Fig 1.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on: body weight gain (A), food intake (B), serum nitrite + nitrate levels (C), liver COX-2 mRNA (D) and liver TNFα mRNA (E) in control rats or in rats treated with LPS (250 μg/kg i.p.). PF = pair-fed rats. D-Trp(8)-γMSH treatment decreased the inhibitory effect of LPS administration on body weight and food intake as well as the stimulatory effect of LPS on serum concentration of nitrites + nitrates, liver TNFα and COX-2 mRNA (P<0.01). Results are expressed as means ± SE for 8–10 rats per group. *P< 0.05 and **P< 0.01, vs. their respective control group. ++P<0.01 vs. LPS-saline, °P<0.05, °°P<0.01 vs. PF. LSD multiple comparison test, following one-way ANOVA.
Fig 2.
Effect of D-Trp(8)-γMSH (γMSH) (500 μg/kg i.p.) administration on hypothalamic mRNA expression of: IL-1β (A), COX-2 (B) and CRH (C), and on serum concentrations of ACTH (D) and corticosterone (E) in control (C) and in LPS-injected (250 μg/kg) rats. PF = pair-fed rats. Each bar represents the mean ± SE for n = 7–10. mRNA expression was quantified using real-time RT-PCR and is presented as the increase of the mean value in control rats treated with saline. LPS injection increased hypothalamic IL-1β, COX-2 and CRH mRNA levels as well as serum ACTH and corticosterone levels (P<0.01) in rats injected with saline, but not in rats injected with γMSH. *P< 0.05 and **P< 0.01, vs. their respective control group. +P<0.05, ++P<0.01 vs. LPS-saline, °°P<0.01 vs. PF. LSD multiple comparison test, following one-way ANOVA.
Fig 3.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on: IGF-I and IGFBP-3 levels in serum (A and D) and their mRNA in liver (B and E) and gastrocnemius (C and F) in control rats or in rats treated with LPS (250 μg/kg). PF = pair-fed rats. γMSH treatment blocked the inhibitory effect of LPS administration on IGF-I in serum and its mRNA in liver and skeletal muscle. LPS decreased serum IGFBP-3 (P<0.01) and its mRNA in the liver (P<0.05), whereas IGFBP-3 mRNA was increased in muscle by LPS injection (P<0.01). γMSH treatment was unable to modify the effects of LPS on IGFBP-3. mRNA expression was quantified using real-time RT-PCR and is presented as the increase of the mean value in control rats treated with saline. Results are expressed as means ± SE for 6–10 rats per group. *P< 0.05 and **P< 0.01, vs. their respective control group. +P<0.05, ++P<0.01 vs. LPS-saline, °P<0.05, °°P<0.01 vs. PF. LSD multiple comparison test, following one-way ANOVA.
Fig 4.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on; phospho-NF-κB(p65)Ser536 (A), phospho-NF-κB(p65)Ser276 (B), NF-κB(p65) (C), phospho-Akt (D), Akt (E), phospho-mTOR (F) and mTOR (G), in gastrocnemius muscle of control rats and rats treated with LPS (250 μg/kg). PF = pair-fed rats. Proteins were measured by Western blotting with specific antibodies for total and phosphoprotein and expressed as percentage of the control rats treated with saline. Representative Western blots are shown at the middle right. Boxes with immunoblots represent spliced images based on group and treatment order. LPS increased pNF-κB(p65)Ser536 (P<0.01) and pNF-κB(p65)Ser276 (P<0.05), whereas it decreased pAkt (P<0.01) and pmTOR (P<0.05) in rats treated with saline, but not in those treated with γMSH. Data represent means SE (n = 7–10 rats). *P< 0.05 and **P< 0.01, vs. their respective control group. +P<0.05, ++P<0.01 vs. LPS-saline, °P<0.05, °°P<0.01 vs. PF. LSD multiple comparisons test, following one way ANOVA.
Fig 5.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on: phospho-FoxO1 (A), FoxO1 (B), phospho-FoxO3 (C) and FoxO3 (D), in gastrocnemius muscle of control rats and rats treated with LPS (250 μg/kg). PF = pair-fed rats. Proteins were measured by Western blotting with specific antibodies for total and phosphoprotein and expressed as percentage of the control rats treated with saline. Representative Western blots are shown at the right. Boxes with immunoblots represent spliced images based on group and treatment order. Data represent means ± SE (n = 7–10 rats). *P< 0.05 and **P< 0.01, vs. their respective control group. +P<0.05, ++P<0.01 vs. LPS-saline, °°P<0.01 vs. PF. LSD multiple comparisons test, following one way ANOVA.
Fig 6.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on; autophagy-related marker LC3b mRNA (A), Bnip-3 (B) and Gabarap1 mRNA (C), on LC3a/b protein I and II (D and E), in gastrocnemius muscle of control rats and rats treated with LPS (250 μg/kg). PF = pair-fed rats. mRNA expression was quantified using real-time RT-PCR and is presented as increase of the mean value in control rats treated with saline. Proteins were measured by Western blotting and expressed as percentage of control rats treated with saline. Representative Western blots are shown at the bottom right. Boxes with immunoblots represent spliced images based on group and treatment order. LPS increased levels of LC3b, Bnip-3 and Gabarap1 mRNA and LC3a/b II protein (P<0.01) in rats treated with saline, but not in the group that received D-Trp(8)-γMSH. Results are expressed as means ± SE for 7–10 rats per group. **P< 0.01, vs. their respective control group. +P<0.05, ++P<0.01 vs. LPS-saline, °P<0.05, °°P<0.01 vs. PF. LSD multiple comparisons test, following one way ANOVA.
Fig 7.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on; MuRF1 mRNA and protein (A and C), and on atrogin-1 mRNA and protein (B and D), in gastrocnemius muscle of control rats and rats treated with LPS (250 μg/kg). PF = pair-fed rats. mRNA expression was quantified using real-time RT-PCR and is presented as increase of the mean value in control rats treated with saline. Proteins were measured by Western blotting and expressed as percentage of the control rats treated with saline. Representative Western blots are shown at the bottom right. Boxes with immunoblots represent spliced images based on group and treatment order. LPS increased levels MuRF1 and atrogin-1 proteins (P<0.01) in the rats treated with saline, but not in the group that received D-Trp(8)-γMSH. MuRF1 and atrogin-1 mRNAs were increased by LPS injection (P<0.01), and D-Trp(8)-γMSH attenuated these increases (P<0.01). Results are expressed as means ± SE for 7–10 rats per group. *P< 0.05 and **P< 0.01, vs. their respective control group. ++P<0.01 vs. LPS-saline, °°P<0.01 vs. PF. LSD multiple comparisons test, following one way ANOVA.
Fig 8.
Effect of D-Trp(8)-γMSH (γMSH) treatment (500 μg/kg i.p.) on; MCH I (A) and MHC IIa mRNA (B), in gastrocnemius muscle of control rats and rats treated with LPS (250 μg/kg). PF = pair-fed rats. mRNA expression was quantified using real-time RT-PCR and is presented as the increase of the mean value in control rats treated with saline. LPS decreased gastrocnemius MCH I (P<0.05) and MCH II mRNA (P<0.01). The rats treated with D-Trp(8)-γMSH and LPS had MCH I and MCH II mRNA levels between those of control rats treated with D-Trp(8)-γMSH and rats treated with LPS alone. Data represent means ± SE (n = 7–9). *P< 0.05 and **P< 0.01, vs. their respective control group. °P<0.05 vs. PF. LSD multiple comparisons test, following one way ANOVA.
Fig 9.
Effect of D-Trp(8)-γMSH (0, 50 or 200 nM) on; phospho-NF-κB(p65)Ser276 (A), NF-κB(p65) (B), phospho-Akt (C) Akt (D), IGF-I mRNA (E) and MHC I mRNA (F) in L6 myotubes cell cultures incubated with TNFα (10 μg/ml) or DMEM. Representative Western blots are shown at the middle right. Boxes with immunoblots represent spliced images based on group and treatment order. TNFα increased NF-κB(p65) phosphorylation (P<0.01) and decreased Akt phosphorylation (P<0.05), whereas D-Trp(8)-γMSH prevented those effects. IGF-I mRNA was decreased by TNFα (P<0.05), but not in the cells cultures with TNFα and D-Trp(8)-γMSH. TNFα also decreased MHC I mRNA (P<0.01) and D-Trp(8)-γMSH attenuated this effect. Data are expressed as mean ± SE for n = 6–8 wells per group, *P<0.05, **P<0.01 vs their respective myotube group incubated without TNFα, °P<0.05, °°P<0.01 vs their respective myotube group incubated without D-Trp(8)-γMSH. LSD multiple comparisons test, following one way ANOVA.