The Gene Expression Of Leptin Receptor In Hepatocyte And Preliminary Study On Its Mechanism

Background and Objective:Leptin, the protein product of the obese (ob) gene, exerts a wide range of physiological functions ranging from regulating appetite, energy homeostasis, neuroendocrine function, cell growth, reproduction and the immune response. By binding to its receptor, leptin inhibits food intake in hypothalamus and increases energy expenditure in peripheral tissues, inhibits insulin release in pancreatic β cells, promotes hepatic glucose production and transportation, increases lipolysis and induces insulin resistance. In humans, persons with congenital leptin deficiency are obese, and treatment with leptin results in dramatic weight loss. Clinical studies demonstrated that disturbances in plasma leptin levels are associated with multiple metabolic disorders resulting in diseases including diabetes, kidney disorder, cardio-vascular disease, non-alcoholic fatty liver disease and multiple kinds of tumors. Therefore, appropriate leptin activity is crucial for maintaining healthy physiological functions. Like all hormores, activity of leptin is under exquisite regulation not only at cellular level, such as expression of leptin and its receptor genes, but also at the level of its stability and clearance in circulation. It is now known that circulating leptin exists in two distinct forms:free or complexed with leptin-binding proteins, with the free form being biologically active. By binding leptin, leptin binding proteins modulate the free/bound ratio (also known as free leptin index, FLI), increasing its stability and decreasing its clearance in circulation, thus modulates its bioactivity. In vitro and in vivo studies indicate that the majority of circulating leptin binding proteins in human plasma is soluble leptin receptor (sOB-R) which is generated by ectodomain shedding of membrane-spanning receptors. However, the relative contribution of each organ in the ectodomain shedding-derived sOB-R in plasma is still unclear. Recent work from rodents indicates that liver leptin receptor may serve as the main source for plasma sOB-R. Studies using tissue distribution of radioactive leptin showed that greater than80%of the radioactivity following i.v. infusion has been retained in the rat liver compartment supporting an important role of the liver in the binding capacity of leptin. Importantly, induction of sOB-R is abolished in mice with selective deletion of leptin receptor from liver using Cre-loxP technology. Moreover, in a cross-sectional study including118healthy people to elucidate the regulation of sOB-R and FLI in humans showed that sOB-R is regulated by adiposity, gender and hormones, and cortisol was significantly correlated with sOB-R (r=0.27, P<0.01). Considering the facts that liver is not only the physiologically important effector organ of glucocoticoids but also the organ with the most abundant expression of leptin receptor, especially the short isoforms, we speculate that glucocoticoids may directly promote hepatocyte leptin receptor gene expression, thus leading to increased plasma sOB-R. We hereby tested this hypothesis in cultured human hepatoma cell line (HepG2) as well as the human normal hepatocyte (L02) using dexamethasone, a chemically synthesized glucocoticoid compound, and determing its effect on leptin receptor gene expression.Meanwhile, recent studies demonstrated that insulin signaling in liver plays an important role in leptin homeostasis. Liver-specific insulin receptor knock-out (LIRKO) mice displayed a10-fold increase in circulating leptin and80-fold increase of sOB-R. Real-time PCR reveals that liver as the source of sOB-R in LIRKO mice with an increase in expression of the short (Ob-Ra), long (Ob-Rb) and soluble (Ob-Re) forms of the leptin receptor. And the increase in leptin receptor expression appears to be a unique feature of the liver, because no alteration is observed for its expression in other tissues of LIRKO mice, or in other mice models, such as fat-, or muscle-specific insulin knockout mice. These res ults indicate that factor(s) downstream of insulin in the liver participate in leptin receptor expression. FOXO1is an important physiological effector downstream of insulin, and is induced by glucocoticoids. We therefore tested whether FOXO1modulates leptin receptor expression by introducing either active FoxO1or its siRNA with lipofectamine-mediated gene transfection into HepG2cells. Reporter gene assay was further included to detect the influence of FOXO1on leptin receptor promoter activity. In aggregate, our studies aim to gain insight into the molecular mechanism of endocrine-regulation of leptin activity. We believe that these studies will gain insight into the sophisticated regulation mechanism of leptin bioactivities, and shed light in the prevention and therapy of leptin dysfunction-related diseases.Materials and Methods:1. HepG2cells or L02cells were incubated and stimulated for48hours in the presence of dexamethsone. Total RNA and proteins were then extracted. The expression of leptin receptor mRNA was determined by RT-PCR and real-time PCR, and normalized with β-actin or GAPDH. Protein levels were determined by Western Blotting.2. In order to study the effect of FOXO1on leptin receptor expression, lipofectamine-mediated gene transfer was used to introduce siRNA into HepG2cells to knockdown endogenous FOXO1level or increase its expression by transfection of a constitutive active form of expression plasmid of FOXO1(FOXO1-TSS).3. A human leptin receptor gene promoter (-3296/+105) which is linked to luciferase reporter gene pGL3.basic plasmid was used. Reporter gene assay was carried out to detect the influence of FOXO1on leptin receptor promoter activity. Luciferase activity was normalized with β-galactase activity.Results:1. Dexamethasone (the chemically synthesized compound of glucocorticoids) induces expression of all isoforms of leptin receptor (also known as OB-Rt) including the long isoform (OB-Rb) in HepG2and L02cells.2. Induction effect by dexamethasone on leptin receptor expression seems to be more potent (more than30fold increase) in hapatoma cell line (HepG2) and functions in a wider range of concentration (50-500nM), whereas its effect was moderate (around3fold) and in a lower dosages (50-100nM) on normal human hepatocyte (L02) leptin receptor expression.3. Dexamethasone increases FOXO1expression both on mRNA and protein levels in HepG2cells.4. Expression of OB-Rb is actively regulated by FOXO1in hepatocyte, and shows that knockdown of FOXOi by siRNA interference decreases OB-Rb expression, in contrast, enforced expression of FOXO1increases OB-Rb.4. Reporter gene assay further showed that leptin receptor promoter activity is stimulated by enforced expression of FOXO1and inhibited by knocking-down of the endogenous FOXO1expression.Conclusion:1. Dexamethasone induces expression of all isoforms of leptin receptor in hepatocytes.2. Hepatic FOXO1is induced by dexamethasone.3. Expression of OB-Rb is actively regulated by FOXO1in hepatocytes, in which OB-Bb decreased by knockdown of FOXO1or enhanced by enforced expression of FOXO1.To the best of knowledge, this is first time showing that FOXO1participates in leptin receptor gene expression. However, the mechanism(s) for this effect is currently unknown. More studies are thus urgently needed to clarify the function of FOXO1via direct binding or indirect induction on other transcriptional factors. In addition, whether FOXO1acts in mediating glucocorticoids on leptin receptor gene expression still wait to be addressed.