Ghrelin ameliorates tumor-induced adipose tissue atrophy and inflammation via Ghrelin receptor-dependent and -independent pathways

Figures

• 

  Figure 1. Effects of ghrelin on body weight, fat mass, and food intake in LLC-induced cachexia. HK+V: heat-killed + vehicle; T+V: tumor + vehicle; T+G: tumor + ghrelin. Changes in (A) body weight (carcass weight, body weight at endpoint without tumor, n = 8–10) expressed as % change from baseline body weight (before tumor implantation) and (B) fat body mass by NMR expressed as % change from baseline (fat mass by NMR before tumor implantation, n = 8–10). (C) Fat pad mass normalized to baseline NMR fat mass (mg/g, n = 4–6). (D) Average cumulative food intake (FI) normalized to baseline daily FI [kcal/kcal, 72 h cumulative FI during CLAMS before sacrificing/baseline daily FI (before LLC implantation), n = 4–6). Two-way ANOVA was performed to detect genotype and treatment differences. * p < 0.05 compared to HK+V within the same genotype. # p < 0.05 compared to T+V within the same genotype. Genotype effects are shown in p-values above the corresponding figures (p < 0.05). Data are shown as mean ± SE.

• 

  Figure 2. Effects of ghrelin on LLC-induced protein-level changes in lipases in iWAT. HK+V: heat-killed + vehicle; T+V: tumor + vehicle; T+G: tumor + ghrelin; T+G+P: tumor + ghrelin + pair-fed (A) Representative Western blots for ATGL, HSL, and GAPDH. (B) ATGL expression in each group. (C) HSL expression in each group. Western blots were quantified by densitometry and normalized to GAPDH. Results are presented as fold change of Ghsr+/+ HK+V. Two-way ANOVA was performed to detect genotype and treatment differences. * p < 0.05 compared to HK+V within the same genotype. # p < 0.05 compared to T+V within the same genotype. No genotype difference was detected. Data are shown as mean ± SE. n = 4/group.

• 

  Figure 3. Effects of ghrelin on LLC-induced changes in inflammation and macrophages in iWAT. HK+V: heat-killed + vehicle; T+V: tumor + vehicle; T+G: tumor + ghrelin. Protein levels of inflammatory markers (A) IL-1β, (B) IL-6, and (C) TNF; and (D) macrophage marker MCP-1 in iWAT (pg/mg). Two-way ANOVA was performed to detect genotype and treatment differences. * p < 0.05; ** p < 0.01 compared to HK+V within the same genotype. # p < 0.05 compared to T+V within the same genotype. No genotype difference was detected. Data are shown as mean ± SE. n = 6–7/group. (E and F) Colocalization of inflammation and macrophages in iWAT. (E) Representative images of colocalization of inflammatory marker IL-6 and macrophage marker F4/80 in iWAT (IL-6 in Texas red; F4/80 in FITC green; nuclei in DAPI blue). (F) Representative images of colocalization of inflammatory marker TNF and macrophage marker F4/80 in iWAT (TNF in FITC green; F4/80 in Texas red; nuclei in DAPI blue). Positively stained inflammatory markers and colocalizations with macrophages are indicated by white arrows. Scale bars, 50 μm.

• 

  Figure 4. Effects of ghrelin on LLC-induced changes in inflammation and macrophages in BAT. HK+V: heat-killed + vehicle; T+V: tumor + vehicle; T+G: tumor + ghrelin. Protein levels of inflammatory markers (A) IL-1β, (B) IL-6, and (C) TNF; and (D) macrophage marker MCP-1 in BAT (pg/mg). Two-way ANOVA was performed to detect genotype and treatment differences. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to HK+V within the same genotype. # p < 0.05; ### p < 0.001 compared to T+V within the same genotype. No genotype difference was detected. Data are shown as mean ± SE. n = 6–7/group. (E and F) Colocalization of inflammation and macrophages in BAT. (E) Representative images of colocalization of inflammatory marker IL-6 and macrophage marker F4/80 in BAT (IL-6 in Texas red; F4/80 in FITC green; nuclei in DAPI blue). (F) Representative images of colocalization of inflammatory marker TNF and macrophage marker F4/80 in BAT (TNF in FITC green; F4/80 in Texas red; nuclei in DAPI blue). Positively stained inflammatory markers and colocalizations with macrophages are indicated by white arrows. Scale bars, 50 μm.

• 

  Figure 5. Expression of UCP-1 in iWAT and BAT. HK+V: heat-killed + vehicle; T+V: tumor + vehicle; T+G: tumor + ghrelin. (A) Representative H&E and IHC images of UCP-1 in iWAT. (B) UCP-1 positive area is expressed as % of the total analyzed area in iWAT (n = 4–6). (C) Representative H&E and IHC images of UCP-1 in BAT. (D) UCP-1 positive area is expressed as % of the total analyzed area in BAT (n = 4–6). Two-way ANOVA was performed to detect genotype and treatment differences. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to HK+V within the same genotype. Genotype effects are shown as p-values above the corresponding figures (p < .05). Data are shown as mean ± SE. Scale bars, 50 μm.

• 

  Figure 6. Indirect calorimetry measurements by CLAMS. HK+V: heat-killed + vehicle; T+V: tumor + vehicle; T+G: tumor + ghrelin. (A–B) Energy expenditure (EE) adjusted by LBM (without tumor) is expressed as kcal/h/kg (A) during 72 hours before sacrificing (2 weeks of ghrelin treatment); (B) average daily EE over the 72 hours before sacrificing. (C–D) Ambulatory activity (C) during 72 hours before sacrificing (counts/6 h). (D) Average daily ambulatory activity over the 72 hours before sacrificing (counts/day). (E–F) Respiratory Quotient (RQ) is calculated by VCO2/VO2 (E) during 72 hours before sacrificing (RQ/6 h); (F) average daily RQ over the 72 hours before sacrificing (RQ/day). Two-way ANOVA was performed to detect genotype and treatment differences. * p < 0.05 compared to HK+V within the same genotype. Genotype effects are shown in p-values above the corresponding figures (p < 0.05). N = 4 for HK+V groups and N = 6 for the rest of the groups. Data are shown as mean ± SE.