Regulation of Ghrelin Signaling by a Leptin-induced Gene, Negative Regulatory Element-binding Protein, in the Hypothalamic Neurons

Abstract

Leptin, the product of the ob gene, plays important roles in the regulation of food intake and body weight through its receptor in the hypothalamus. To identify novel transcripts induced by leptin, we performed cDNA subtraction based on selective suppression of the polymerase chain reaction by using mRNA prepared from the forebrain of leptin-injected ob/ob mice. One of the genes isolated was a mouse homolog of human negative regulatory element-binding protein (NREBP). Its expression was markedly increased by leptin in the growth hormone secretagogue-receptor (GHS-R)-positive neurons of the arcuate nucleus and ventromedial hypothalamic nucleus. The promoter region of GHS-R contains one NREBP binding sequence, suggesting that NREBP regulates GHS-R transcription. Luciferase reporter assays showed that NREBP repressed GHS-R promoter activity in a hypothalamic neuronal cell line, GT1-7, and its repressive activity was abolished by the replacement of negative regulatory element in GHS-R promoter. Overexpression of NREBP reduced the protein expression of endogenous GHS-R without affecting the expression of ob-Rb in GT1-7 cells. To determine the functional importance of NREBP in the hypothalamus, we assessed the effects of NREBP on ghrelin action. Although phosphorylation of AMP-activated protein kinase ␣ (AMPK␣) was induced by ghrelin in GT1-7 cells, NREBP repressed ghrelin-induced AMPK␣ phosphorylation. These results suggest that leptin-induced NREBP is an important regulator of GHS-R expression in the hypothalamus and provides a novel molecular link between leptin and ghrelin signaling.Obesity develops when food intake exceeds compensatory increases in energy expenditure, and energy is accumulated as fat (1). Food intake is controlled by the precise coordination between the neural circuitry and peripheral factors that derive from fat, gut, and pancreas. The peripheral factors transduce their signals by binding to the receptors in the hypothalamus and regulate production of the orexigenic and the anorexigenic peptides in the specific subsets of hypothalamic neurons (2). Thus, the hypothalamus is critical in integrating signals from the peripheral factors in the neural circuitry.Leptin, an adipocyte-derived hormone (3), is a key negative regulator of food intake and energy expenditure. Circulating leptin enters the brain through the blood-brain barrier (4) and exerts its effects through binding to the long form of the leptin receptor ob-Rb (5) expressed in the hypothalamus, including the arcuate nucleus, ventromedial hypothalamic nucleus (VMH), 2 dorsomedial hypothalamic nucleus, lateral hypothalamic nucleus, and paraventricular hypothalamic nucleus (6). For example, in the hypothalamic arcuate nucleus, leptin suppresses food intake via decreased expression of the orexigenic peptides, neuropeptide Y, and agouti-related peptide and increased expression of the anorexigenic peptides, proopiomelanocortin, and cocaine-and amphetamine-regulated transcript (6 -9).Although the administration of leptin reverses obesity caused b...

Deficiency of Oncostatin M Receptor β (OSMRβ) Exacerbates High-fat Diet-induced Obesity and Related Metabolic Disorders in Mice

“…Immunohistochemistry-Immunofluorescence staining was performed with some modifications as previously described (25). Briefly, the mice were deeply anesthetized with diethyl ether, and the epididymal adipose tissue was quickly removed.…”

“…Quantitative Real-time PCR-Quantitative real-time PCR was performed with some modifications, as previously described (25). Briefly, total RNA extracted from epididymal adipose tissue, SVF, liver, hypothalamus, skeletal muscle, and pancreas was prepared using TRI reagent (Molecular Research Center).…”

Lack of Oncostatin M Receptor β Leads to Adipose Tissue Inflammation and Insulin Resistance by Switching Macrophage Phenotype

“…Western Blot Analysis-Western blot analysis was performed with some modifications as described previously (25). Lysates were prepared by using radioimmune precipitation assay buffer (Upstate Biotechnology, Lake Placid, NY) containing protease inhibitor mixture (Upstate Biotechnology), 1 mM orthovanadate, 1 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride.…”

“…Cell Culture-Cell culture was performed with some modifications as described previously (25). The mouse macrophage cell line, RAW 264.7, was grown in DMEM (Invitrogen) with 10% FCS, 100 units/ml of penicillin (Invitrogen), and 100 g/ml of streptomycin (Invitrogen).…”

“…Quantitative Real-time PCR-Quantitative real-time PCR was performed with some modifications as described previously (25). Briefly, total RNAs from PEMs were prepared using TRI reagent (Molecular Research Center, Cincinnati, OH).…”