Upregulation of hepatic melanocortin 4 receptor during rat liver regeneration

Abstract

Background

Melanocortin 4 receptor (MC4R) is predominantly recognized to mediate energy metabolism and anti-inflammation through the central nervous system. However, the expression of MC4R has recently been identified in rat liver and was shown to be upregulated during acute phase response. This study aims to investigate potential roles of MC4R in liver regeneration.

Materials and methods

Rat partial hepatectomy (PH) was performed, and MC4R expression was analyzed at different time points after resection. Sham-operated animals (SH) served as controls. In vitro primary hepatocytes (HCs) were isolated from normal rat liver and stimulated with α-melanocyte–stimulating hormone (MC4R agonist). Real-time polymerase chain reaction, Western blot, and immunofluorescence staining were applied to detect gene expression.

Results

Up to 8 h after PH, hepatic messenger RNA of proinflammatory cytokines interleukin 6 and tumor necrosis factor α reached peak values. Between 8 and 72 h after PH, rat liver regeneration was extremely active as assessed by the regeneration indices labeled by Ki-67. Immunofluorescence staining indicated that MC4R was mostly expressed in hepatocyte nuclear factor 4⁺ cells (HCs) and upregulated during rat liver regeneration. Concurrently, the expression of hepatic MC4R protein was significantly higher in PH than in SH animals, and phosphorylated extracellular signal–regulated kinase 1/2 was remarkably increased in PH compared with SH animals (P < 0.05, respectively). In vitro experiments showed that the expression of proliferating cell nuclear antigen was significantly higher in HCs treated with α-melanocyte–stimulating hormone than in control HCs, which was correlated to the increase of phosphorylated extracellular signal–regulated kinase 1/2 and reduction of phosphorylated signal transducer and activator of transcription 3 (P < 0.05, respectively).

Conclusions

MC4R is predominantly expressed in HCs and upregulated during rat liver regeneration. In vitro stimulation of HC MC4R is associated with a modulation of extracellular signal–regulated kinase and signal transducer and activator of transcription 3 pathways regulating liver regeneration.

Introduction

Melanocortin receptor 4 (MC4R), a G-protein–coupled receptor, plays a pivotal role in the regulation of energy homeostasis and anti-inflammation [1]. MC4R is mechanistically regulated by α-melanocyte-stimulating hormone (α-MSH, MC4R agonist) and agouti-related peptide (AgRP, MC4R inverse agonist), both of which are derived from proopiomelanocortin [1]. MC4R is conventionally considered to be expressed in the central nervous system and exerts its biological effects to peripheral organs [2]. However, recent studies identified the expression of MC4R in liver and adipose tissues as well [3], [4].

Genes involved in direct energy generating processes, such as oxidative phosphorylation, electron transport, and adenosine triphosphate synthesis, were found upregulated in both liver and adipose tissue of α-MSH-treated pigs homozygously expressing missense mutations in MC4R [4]. Interestingly, in mice, central administration of a MC4R antagonist (SHU9119) or AgRP robustly increased feeding behavior, indicating that antagonism of MC4R is an important orexigenic signal [5]. Knockout of MC4R and proopiomelanocortin [6] or overexpression of AgRP [7] led to the same obese phenotype in mice. A similar genetic pattern was also observed in humans with mutations in MC4R [8]. These reports highlighted the crucial effects of MC4R in energy metabolism.

It was reported that α-MSH prevented lipopolysaccharide-induced hepatic inflammation by inhibiting production of chemokines, which then modulated the infiltration of inflammatory cells [9]. Moreover, activation of MC4R attenuated cerebral, myocardial, testicular and renal inflammation and ischemia–reperfusion injury through regulating extracellular signal–related kinase (ERK) 1/2, tumor necrosis factor α (TNF-α) and so forth, consequently trigging repair pathway [10], [11], [12], [13]. Interestingly, gene expression of all MCR subtypes was recently discovered in rat liver, and the level of MC4R was most dramatically increased during acute phase response [3]. However, the precise mechanism of how MC4R exerts anti-inflammatory effects and interacts with other biochemical processes is not well defined yet.

Liver regeneration after partial hepatectomy (PH) is mostly dependent on the replication of hepatocytes (HCs), which are completely differentiated and normally quiescent cells, and do not rely on the activation of a compartment of hepatic stem cells [14]. Tremendous genes are involved in liver regeneration, and the essential circuitry required for the process could be categorized into three networks: metabolic (e.g., Heparin-binding epidermal growth factor and amphiregulin), cytokines (e.g., interleukin 6 and TNF-α), and growth factors (e.g., hepatocyte growth factor, and epidermal growth factor) [15].

Based on the best of present knowledge, MC4R is involved in metabolic and inflammatory processes, so we hypothesized that MC4R could play an important role in the proliferation of liver cells after PH. The present study investigates the MC4R expression pattern in regenerating rat liver after 2/3 PH and its interaction with other signaling pathways being involved in regulation of liver regeneration.