Hormonal regulation of visfatin gene in avian Leghorn male hepatoma (LMH) cells

Highlights

• Visfatin is expressed in chicken hepatocytes.

• Hepatocyte visfatin gene is regulated by various hormones.

• TNFα, leptin, T3, and orexin A, but not IL-6 or orexin B modulates visfatin gene expression.

• The hormonal regulation of visfatin is probably mediated via orphan receptors.

Abstract

Visfain has been extensively studied in mammals and has been shown to play an important role in obesity and insulin resistance. However, there is a paucity of information on visfatin regulation in non-mammalian species. After characterization of chicken visfatin gene, we undertook this study to determine its hormonal regulation in avian (non-mammalian) liver cells. Addition of 5 ng/mL TNFα, 100 ng/mL leptin, 1, 3, 10 or 100 ng/mL T3 for 24 h upregulated visfatin gene expression by 1.2, 1.8, 1.95, 1.75, 1.80, and 2.45 folds (P < .05), respectively, compared to untreated LMH cells. Administration of 10 ng/mL of orexin A significantly down regulated visfatin gene expression by 1.35 folds compared to control cells. In contrast, treatment with IL-6 or orexin B for 24 h did not influence visfatin mRNA abundance. These pro-inflammatory cytokines and obesity-related hormones modulate the expression of CRP, INSIG2, and nuclear orphan receptors. Hepatic CRP gene expression was significantly upregulated by IL-6, TNFα, orexin B, and T3 and down regulated by leptin and orexin A. LXR mRNA abundances were increased by orexin A, decreased by orexin B, and T3, and did not affected by IL6, TNFα, or leptin. The expression of FXR gene was induced by IL-6, leptin, and T3, but it was not influenced by TNFα, orexin A or B. CXR gene expression was up regulated by TNFα, leptin, orexin B, and T3, down regulated by 5 ng/mL orexin A, and did not affected by IL-6. INSIG2 mRNA levels were increased by TNFα (5 ng/mL), leptin (100 ng/mL), and T3 (1, 3, 10, and 100 ng/mL), decreased by orexin A, and remained unchanged with IL-6 or orexin B treatment.

Together, this is the first report showing hormonal regulation of visfatin in avian hepatocyte cells and suggesting a potential role of CRP, INSIG2, and nuclear orphan receptor LXR, FXR, and CXR in mediating these hormonal effects.

Introduction

Visfatin or Nampt is an adipokine originally identified as a growth factor for early β cells and thereby called pre-β-cell colony-enhancing factor (PBEF) (Samal et al., 1994; Sethi and Vidal-Puig, 2005). Nampt has an intracellular (iNampt) and extracellular (eNampt) forms. iNampt is a nicotinamide phosphoribosyl transferase (Nampt), a cytosolic enzyme, involved in nicotinamide adenine dinucleotide (NAD) biosynthesis (Rongvaux et al., 2002). It regulates the activity of NAD-consuming enzymes such as sirtuins and affects several metabolic and stress processes (Hognogi and Simiti, 2016). Although the physiological roles of eNampt have been controversial and a matter of debate for decades, several studies have assigned to eNampt a cytokine function and thereby they named it “visfatin” which plays a key role in metabolic disorders (diabetes and obesity) via regulating glucose-stimulated insulin secretion and signaling (Dahl et al., 2007; Revollo et al., 2007; Song et al., 2008; Xie et al., 2007).

Visfatin was found to be expressed in several mammalian cell types and tissues including hepatocytes (Imai and Kiess, 2009) and has been shown to be regulated by various cytokines, hormones, and factors (Hector et al., 2007; MacLaren et al., 2007; Skrzypski et al., 2018; Tan et al., 2009; Zhang et al., 2019). Such studies are currently lacking in avian (non-mammalian) species.

Avian genetic selection for high growth rate and muscle enhancement has resulted in hyperphagic broilers that are prone to obesity. Modern broiler (meat-type) chickens consume over 4 kg of feed to achieve an average slaughter-weight of 2.8 kg in only 42 days. This body weight increase arises mainly from breast (pectoralis) muscle and abdominal fat (Hood, 1982; Scheuermann et al., 2003). Additionally, chickens are hyperglycemic compared to mammals, with their plasma glucose levels averaging three times that found in human (Krzysik-Walker et al., 2008). They require insulin doses greater than four times that required in mammals to achieve hypoglycemia, and hence they are insulin resistant (Akiba et al., 1999; Dupont et al., 2004; Simon et al., 1977). They are also lacking functional brown adipose tissue and glucose transporter GLUT4 (Seki et al., 2003). Interestingly, the majority (>95%) of de novo fatty acid synthesis (lipogenesis) occurs in the liver in chickens (Goodridge and Ball, 1967; Leveille et al., 1975). As a follow up to our previous study where we have shown that visfatin is expressed in chicken liver and is regulated by nutritional status and leptin administration, we undertook this study to determine the effects of IL-6, TNFα, leptin, T3, and orexin A/B on the expression of visfatin gene in chicken hepatocytes (LMH) in culture.