Effects of leptin, an antiobestic factor, on catecholamine synthesis and secretion in bovine adrenal medullary cells

瘦素是最近克隆的肥胖基因的蛋白产物，由脂肪组织分泌，通过能量平衡中枢下丘脑的受体在调节体重和能量消耗方面发挥重要作用。除了下丘脑的作用外，许多证据表明，瘦素直接作用于下丘脑外的一些组织和外周组织，如胰岛和T淋巴细胞。在本研究中，我们研究了瘦素的特异性结合部位，并研究了瘦素对培养的牛肾上腺髓质细胞内儿茶酚胺分泌和合成的影响。1)从牛肾上腺髓质分离的质膜显示一类单一的瘦素特异性结合部位，其表观Kd值为6.6 nm，Bmax为62fmol/mg蛋白。逆转录-聚合酶链式反应检测到瘦素受体a型(Obra)而不表达b型(ObRb)。2)瘦素(3-30 nm)对…有意义对培养的牛肾上腺髓质细胞的儿茶酚胺分泌量无明显影响，但对酪氨酸合成的儿茶酚胺有较强的刺激作用。3)细胞内儿茶酚胺合成的刺激与儿茶酚胺合成的晚期限制酶酪氨酸羟基酶的磷酸化和激活有关。4)细胞与瘦素孵育后，丝裂原活化蛋白激酶(MAPKs)迅速激活。MAPK激酶抑制剂UO126可阻断瘦素对儿茶酚胺合成的刺激作用。5)瘦素增强了乙酰胆碱对儿茶酚胺合成的刺激作用，而瘦素对乙酰胆碱诱导的酪氨酸羟基酶的磷酸化和活化无促进作用。这些结果表明，瘦素通过两种不同的机制来促进儿茶酚胺的合成，一种是依赖于MAPK途径介导的酪氨酸羟基酶的磷酸化，另一种是不依赖于酶的磷酸化。这一信息有助于揭示瘦素作用于代谢稳态的生化基础。较少

Leptin, the protein product of the recently cloned obese gene, is secreted from adipose tissues and plays an important role in regulating body weight and energy expenditure through its receptor in the hypothalamus, the center of energy homeostasis. In addition to its hypothalamic action, several lines of evidence have shown that leptin acts directly on some extrahypothalamic and peripheral tissues such as pancreatic islets and T-lymphocytes.In the present study, we investigated the specific binding sites of leptin and examined the effects of leptin on the secretion and synthesis of catecholamines in cultured bovine adrenal medullary cells. 1) The plasma membranes isolated from bovine adrenal medulla showed a single class of specific binding sites of^<125>I-leptin with an apparent Kd of 6.6nM and Bmax of 62 fmol/mg protein. Leptin receptor type a (ObRa) but not type b (ObRb) mRNA was detected in the tissue by reverse transcriptase-polymerase chain reaction. 2) Leptin (3-30nM) significan … More tly stimulated ^<14>C-catecholamine synthesis from [^<14>C]tyrosine, but not from [^<14>C] DOPA although it had little effect on catecholamine secretion in cultured bovine adrenal medullary cells. 3) The stimulation of catecholamine synthesis in the cells was associated with the phosphorylation and activation of tyrosine hydroxylase, the late-limiting enzyme of catecholamine synthesis. 4) The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, UO126, nullified the stimulatory effect of leptin on the synthesis of ^<14>C-catecholamines. 5) Leptin potentiated the stimulatory effect of acetylcholine on ^<14>C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine.These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent on enzyme phosphorylation. This information helps to unveil the biochemical basis of the mechanism by which leptin acts on metabolic homeostasis. Less