Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.

GH regulates several physiological processes in vertebrates, including the promotion of growth, an anabolic process, and the mobilization of stored lipids, a catabolic process. In this study, we used hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) as a model to examine the mechanism of GH action on lipolysis. GH stimulated lipolysis as measured by increased glycerol release in both a time-and a concentration-related manner. The promotion of lipolysis was accompanied by GH-stimulated phosphorylation of the lipolytic enzyme hormone-sensitive lipase (HSL). GH-stimulated lipolysis was also manifested by an increased expression of the two HSL-encoding mRNAs, HSL1 and HSL2. The signaling pathways that underlie GH-stimulated lipolysis were also studied. GH resulted in the activation of phospholipase C (PLC)/protein kinase C (PKC) and the MEK/ERK pathway, whereas JAK-STAT and the PI3K-Akt pathway were deactivated. The blockade of PLC/PKC and the MEK/ERK pathway inhibited GH-stimulated lipolysis and GH-stimulated phosphorylation of HSL as well as GH-stimulated HSL mRNA expression, whereas the blockade of JAK-STAT or the PI3K-Akt pathway had no effect on the activation of lipolysis or the expression of HSL stimulated by GH. These results indicate that GH promotes lipolysis by activating HSL and by enhancing the de novo expression of HSL mRNAs via the activation of PKC and ERK. These findings also suggest molecular mechanisms for activating the lipid catabolic actions of GH while simultaneously deactivating anabolic processes such as antilipolysis and the growth-promoting actions of GH.

Growth hormone (GH) regulates several processes in vertebrates, including two metabolically disparate processes: promotion of growth, an anabolic action, and mobilization of stored lipid, a catabolic action. In this study, we used hepatocytes isolated from continuously fed and long-term (4weeks) fasted rainbow trout (Oncorhynchus mykiss) as a model to investigate the mechanistic basis of the anabolic and catabolic actions of GH. Our hypothesis was that nutritional state modulates the lipolytic responsiveness of cells by adjusting the signal transduction pathways to which GH links. GH stimulated lipolysis as measured by increased glycerol release in both a time- and concentration-related manner from cells of fasted fish but not from cells of fed fish. Expression of mRNAs that encode the lipolytic enzyme hormone-sensitive lipase (HSL), HSL1 and HSL2, also was stimulated by GH in cells from fasted fish and not in cells from fed fish. Activation of the signaling pathways that mediate GH action also was studied. In cells from fed fish, GH activated the JAK-STAT, PI3K-Akt, and ERK pathways, whereas in cells from fasted fish, GH activated the PLC/PKC and ERK pathways. In hepatocytes from fasted fish, blockade of PLC/PKC and of the ERK pathway inhibited GH-stimulated lipolysis and GH-stimulated HSL mRNA expression, whereas blockade of JAK-STAT or of the PI3K-Akt pathway had no effect on lipolysis or HSL expression stimulated by GH. These results indicate that during fasting GH activates the PLC/PKC and ERK pathways resulting in lipolysis but during periods of feeding GH activates a different complement of signal elements that do not promote lipolysis. These findings suggest that the responsiveness of cells to GH depends on the signal pathways to which GH links and helps resolve the growth-promoting and lipid catabolic actions of GH.

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Previously, we reported that extracellular signal-regulated kinase (ERK) and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), mediated somatostatin (SS) inhibition of GH receptor, IGF1, and IGF1 receptor expression. In this study, we used Chinese hamster ovary-K1 cells that stably transfected individually with trout SS receptors (SSTR1A, SSTR1B, and SSTR2) to elucidate receptor-effector pathway linkages. SS induced ERK and Akt activation in a time-and concentration-related manner in all SSTR-expressing cells; however, the PI3K/Akt pathway was activated to a greater extent through SSTR1A than through either SSTR1B or SSTR2, whereas the ERK pathway was activated to a greater extent though SSTR2 than through either SSTR1A or SSTR1B. Although the ERK pathway inhibitor U0126 had no effect on Akt activation, the PI3K inhibitor LY294002 reduced ERK activation to near control levels in all SSTR-expressing cell lines, suggesting some cross talk between the pathways, possibly at the level of c-Raf, the phosphorylation of which also was induced by SS via each SSTR. Pertussis toxin (PTX) completely abolished SS-induced activation of ERK and Akt in SSTR1A-, SSTR1B-, and SSTR2-expressing cells, suggesting that these receptors link to the ERK and PI3K/Akt pathways via PTX-sensitive G-proteins. SS-induced activation of Elk1, Stat3, and C/EBPb also was mediated by each of the trout SSTRs. These findings establish important receptor-effector pathway linkages for fish SSTRs and provide insight into the molecular mechanisms by which SSs may elicit diverse physiological effects in target cells.