Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response

Abstract

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1β and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.

Fig. 1

qRT-PCR analysis of total RNA from rat liver during acute-phase reaction. a Fold change in mRNA expression of MC1R, MC2R, MC3R, MC4R and MC5R; the TO-treated liver at different time points (1–48 h) related to saline-treated controls for each time point. qRT-PCR was normalized by using two housekeeping genes: β-actin and ubiquitin C. Results represent mean value ± SEM of three animals, *p < 0.05. b PCR analysis of total RNA extracted from rat brain and liver tissue. The PCR product was analyzed by agarose (1.5%) gel electrophoresis (UV light picture)

Fig. 2

Immunofluorescence staining of liver sections with polyclonal antibody directed against MC4R (red), followed by fluorescent immunodetection in rat liver sections during acute-phase response after TO treatment. Left control liver, right 6 h after TO treatment. Upper MC4R and DAPI staining; middle MC4R staining; lower DAPI staining. Inset shows higher magnification. Results show the representative picture of three animals and six slides per time point (original magnification, ×200, scale bar 20 μm)

Fig. 3

Immunofluorescence staining in rat liver sections with monoclonal antibody against ED1 (upper left, green), polyclonal antibody directed against MC4R (upper middle, red) and merged ED1 and MC4R (upper right); middle staining with monoclonal antibody against CK-19 (middle left, green), polyclonal antibody directed against MC4R (middle, red) and merged CK-19 and MC4R (middle right); lower staining with monoclonal antibody against SMA (lower left, green), polyclonal antibody directed against MC4R (middle, red) and merged SMA and MC4R (lower right) followed by fluorescent immunodetection. Inset shows higher magnification. Results show the representative picture of three animals and six slides per time point (original magnification, ×100, scale bar 20 μm)

Fig. 4

Immunofluorescence staining of brain sections with polyclonal antibody directed against MC4R (green) followed by fluorescent immunodetection in sections of rat brain during APR. a Control brain section and b 6 h after TO treatment. Results show the representative picture of three animals and six slides (original magnification, ×100, scale bar 10 μm)

Fig. 5

Immunofluorescence staining of liver and brain sections during APR. Immunofluorescence staining with polyclonal antibody directed against MC3R a in liver sections (green) and b brain section (red) followed by fluorescent immunodetection during APR. Inset shows higher magnification. Results show the representative picture of three animals and six slides (original magnification, ×100, scale bar 10 μm)

Fig. 6

Detection of MC4R and MC3R by Western blot analysis of rat liver and brain. a Western blot analysis revealed two immunoreactive bands: one at 37 kDa and a second at 55 kDa. Lane (L)1 contains rat liver total lysate, L2 liver cytosolic extraction, L3 liver nuclear extracts and L4 total protein of cultured rat hepatocytes. b Top rat liver total lysates with immunoreactive bands at 37 and 55 kDa, middle after neutralizing the anti-MC4R antibody with a specific immunizing peptide. Bottom reprobing of the same membrane with the antibody against MC4R without immunizing peptide. c Changes in MC4R protein level during APR. Top contains rat liver total lysate, middle liver cytosolic extraction, bottom liver nuclear extracts. d Identification and changes in MC4R level in rat brain total lysate during APR, e detection and changes of MC3R level in rat liver total lysate. f Changes in serum concentration of α-MSH and leptin during APR. α-MSH (top) and leptin (bottom) levels were measured with enzyme-linked immunosorbent assay (ELISA). Results represent mean value ± SEM (*P < 0.05 analyzed by one-way ANOVA; n = 3). g Detection of MC4R in the liver of wild-type and IL-6-ko mice during APR. Total protein lysate of mouse liver after TO administration; h total protein lysate of mice liver after LPS treatment. β-actin (43 kDa) was used as equal loading control in Western blot analysis. Results show the representative picture of three animals

Fig. 7

Double immunofluorescence staining with polyclonal antibody directed against MC4R (red) and F4/80 (green) followed by fluorescent immunodetection in sections of mouse liver during APR. Upper control liver, lower 6 h after TO treatment. Inset shows higher magnification. White arrow indicates the hepatocytes positive for MC4R and white arrowhead indicates the Kupffer cells positive for MC4R and F4/80. Results show the representative picture of three animals and six slides (original magnification, ×200, scale bar 20 μm)

Fig. 8

Immunofluorescence detection of MC4R in isolated rat a Kupffer cells, b hepatocytes and c human hepatoma cell line (HepG2). Results are representative of three experiments for each cell type (original magnification ×400, scale bar 40 μm)

Fig. 9

RT-PCR analysis of total RNA from rat isolated Kupffer cells treated with α-MSH and THIQ. Data shown as fold changes in mRNA expression of MC1R, MC2R, MC3R, MC4R and MC5R at different time points related to untreated controls for each time point. qRT-PCR was normalized by using two housekeeping genes: β-actin and ubiquitin C. Results represent mean value ± SEM of three experiments, *p < 0.05

Fig. 10

a RT-PCR analysis of total RNA from rat isolated Kupffer cells treated with LPS in the presence and absence of α-MSH and THIQ and b isolated hepatocytes treated only with α-MSH and THIQ. Data shown as fold changes in mRNA expression of TNF-α, IL-6, IL-1β, CCL2 and CXCL1 at different time points related to untreated controls for each time point. qRT-PCR was normalized by using two housekeeping genes: β-actin and ubiquitin C. Results represent mean value ± SEM of three experiments, *p < 0.05. L LPS, M α-MSH and T THIQ