The Effect Of Leptin On The Secretion Of Growth Hormone And Its Mechanism

Growth hormone (GH) is an important hormone that regulates body growth, development, metabolism and immune function. Its parasecretion will result in many diseases, such as malnutrition, obese, and diabetes. There are many factors to influence the GH synthesis and secretion, such as GH-releasing hormone (GHRH) and somatostatin (SS) from hypothalamus, as well as synthetical GH-releasing peptides (GHRP) and endogenic GHRP, ghrelin. Besides, many neurotransmitters, cytokines and some peripheral materials also exert regulating effects on synthesis and secretion of GH. Information in existence indicates that there are many bioactive peptides produced in local pituitary and peripheral tissues. Through the role of endocrine, paracrine or autocrine, these peptides regulate not only cell differentiation, development, and maturity of anterior pituitary (AP), but also cell function. They form an information network to conform all information from central and peripheral nervous systems to regulate GHsynthesis and secretion at pituitary level.Leptin, a protein composed of 167 amino acids, encoded by the obese gene, and mainly produced in the adipose tissue, reduces food intake, increases energy expenditure, and results in weight loss in mammals. Studies in recent years have shown that leptin is an important metabolic signal to regulate GH synthesis and secretion. Leptin receptor is highly expressed in the hypothalamus. Leptin affects GH secretion from pituitary through regulating the expression of GHRH and SS gene in hypothalamus. So it is indicated that leptin has an important role in neuroendocrine regulation of AP function. To date, many studies have proven that there are expressions of leptin and its receptor in anterior lobe of pituitary, whose subtypes are different between animals, suggesting that leptin may affect the secretion of many pituitary hormones. These data imply a cell-specific regulatory role of leptin in the endocrine and paracrine regulation of AP cell function. The GH cell may be a key site for leptin to regulate GH secretion directly and indirectly.There are still many questions that remain unclear. Such as which subtype is expressed in AP and GH3 cells? How is the effect of leptin on GH synthesis and secretion? In signal transduction pathway, which molecule is concerned with leptin action? These questions are aims of our present study.In this study, we firstly used RT-PCR to confirm the expression of leptin receptors, OB-R mRNA, in rat anterior pituitary (AP) and GH3 cells, growth hormone-secreting rat pituitary adenomas, and observed the effect of leptin on the expression of OB-R mRNA. Then, we used Percoll gradient centifugation to isolate the rat somatotrope and observed the effect of leptin on the cell proliferation. Thirdly, MTT, flow cytometery (FCM) and Annexin V-EGFP Apoptosis Detection Kit were employed to investigate the influence of leptin on the proliferation, metabolism and apoptosis of GH3 cells. Fourthly, we used ELISA to study the effect of leptin on GH secretion of GH3 cells. Andfinally, laser scanning confocal microscope and RIA were employed to assay the influence of leptin on cytoplasm free calcium level ([Ca2+]0 and cAMP/cGMP of GH3 cells. The main results were as follows:1. With the method of semi-quantity RT-PCR, we found there were expression of leptin receptors in both normal male rat AP tissue and cultured GH3 cells, including OB-R (common form), OB-Ra (short form) and OB-Rb (long form). Leptin (10'8 mol/L) treatment for 48 h up-regulated the expression of OB-Ra and OB-Rb in AP tissue and GH3 cells.2. We used ELISA method to study the effect of leptin on GH secretion of GH3 cells. The results indicated that leptin at 10~9~10"7 mol/L inhibited the basal growth hormone secretion of GH3 cells in a concentrationQdependent manner. And short-term leptin treatment (10" mol/L) for 30 min, 1 and 3 h did not affect basal GH secretion. However, treatment of the GH3 cells with leptin (10" mol/L) for 1 d or longer time resulted in an inhibition of GH secretion, indicating that leptin may regulate secretory function of pituitary through its specific receptor in pituitary.3. The results of MTT and Quick Cell Proliferation Assay showed that leptin inhibited significantly the proliferation of rat AP cells, growth hormone cells and GH3 cells in a concentration-dependent manner (P<0.05).4. We used flow cytometery (FCM) to study the effect of leptin on the cell cycle of rat AP and GH3 cells. The results showed that, after treatment with leptin (10"8 mol/L) for 48 h, the proportion of GH3 cells in S phase decreased from (15.3±4.5)% to (3.6 + 2.3)%, G2 phase from (4.1 ±1.5)% to (3.7+1.7) % and the proportion cells in Gi phase increased from (80.6 ±4.9)% to (92.7 + 3.9)%. The proportion of AP cells in S phase decreased from (11.4±3.7)% to (5.4 + 3.1)%, G2 phase from (6.1 ±2.5)% to (4.2±1.3)% and the proportion cells in Gt phase increased from (82.5±3.9)% to (90.4 + 2.7)%. These results indicated that leptin (10~8 mol/L) decreased the proportion of cells in S and G2 phase, but increased the proportion of cells in Gi phase (PO.05). So leptin inhibited the proliferation of AP and GH3 cells. These results suggested that the regulatory effect of leptin on the proliferation and differentiation of GH cells might be due to the inhibition of DNA synthesis of cells.5. By flow cytometry and Annexin V-EGFP Apoptosis Detection, it was founded that leptin induced the apoptosis of GH3 cells. After treatment with 10"8 and 10~7 mol/L leptin for 48 h, the proportion of cells in 2 and 4 phases increased. The apoptosis index increased from (4.3 + 1.7)% to (8.1 ±2.1)% and (12.2+2.6)% respectively. These results indicated that leptin induced apoptosis of (?H3 cells, and it might play a role in inhibiting synthesis of GH3 cells.6. The laser scanning confocal microscopy revealed that intracellular free Ca2+ level of cultured male rat GH cells and GH3 cells decreased rapidly upon the addition of leptin (10'8 mol/L).7. RIA results demonstrated that, after leptin (10'8 mol/L) treatment for 15 min, the amount of cAMP in the cytoplasm of GH3 cells increased from 4.1 ±0.31 nmol/Lto 4.75 + 1.02 nmol/L, 5.55 +1.21 nmol/L and 6.58 + 1.57 nmol/L respectively, and the effect displayed in a concentration dependent manner. Leptin had no influence on the cGMP concentration of GH3 cells.We firstly reported that there is expression of leptin receptor in rat AP and GH3 cells with both long form and short form, and leptin regulates their expression in these tissues. Leptin inhibits the basal GH secretion of GH3 cells, which may be due to the inhibition of synthesis and advanced apoptosis...