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，并通过其在能量稳态中心下丘脑的受体在调节体重和能量消耗方面发挥重要作用。除了它的下丘脑作用，一些证据表明瘦素直接作用于一些下丘脑外和周围组织，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位点，并检测了瘦素对培养的牛肾上腺髓细胞分泌和合成儿茶酚胺的影响。1)从牛肾上腺髓质分离的质膜上发现了一类特异性结合位点^<125>I-leptin，表观Kd为6.6nM， Bmax为62 fmol/mg蛋白。逆转录聚合酶链反应检测到瘦素受体a型（ObRa） mRNA，未检测到b型（ObRb） mRNA。2)瘦素（3-30nM）对[^<14>C]酪氨酸合成^<14>C-儿茶酚胺的刺激作用更大，但对[^<14>C]多巴合成^<14>C-儿茶酚胺的影响较小。3)细胞对儿茶酚胺合成的刺激与儿茶酚胺合成的后期限制酶酪氨酸羟化酶的磷酸化和活化有关。4)用瘦素孵育细胞导致有丝分裂原活化蛋白激酶（MAPKs）的快速激活。MAPK激酶抑制剂UO126抵消了瘦素对^<14> c -儿茶酚胺合成的刺激作用。5)瘦素增强了乙酰胆碱对^<14> c -儿茶酚胺合成的刺激作用，而对乙酰胆碱诱导的酪氨酸羟化酶的磷酸化和活化作用没有增强作用。这些发现表明，瘦素通过激活酪氨酸羟化酶刺激儿茶酚胺的合成有两种不同的机制，即一种依赖于MAPK途径介导的酪氨酸羟化酶磷酸化，另一种不依赖于酶磷酸化。这一信息有助于揭示瘦素作用于代谢稳态机制的生化基础。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