Mechanism of the impairment of the glucagon-stimulated phosphoenolpyruvate carboxykinase gene expression by interleukin-6 in rat hepatocytes: Inhibition of the increase in cyclic 3?,5? adenosine monophosphate and the downstream cyclic 3?,5? adenosine monophosphate action

Abstract

ferase reporter gene construct under the control of a PCK In cultured rat hepatocytes, the gluconeogenic key enzyme, phosphoenolpyruvate carboxykinase (PCK), is induced by gene promoter fragment (base 0979 to base /32). Luciferase activity was determined after stimulation of the cells with glucagon via elevation of cyclic 3 ,5 adenosine monophosphate (cAMP). The proinflammatory cytokine, interleukin-6 CPT-cAMP in the absence or presence of IL-6. CPT-cAMP increased luciferase activity by 1.7-fold, which was inhibited (IL-6), which in the liver together with IL-1b and tumor necrosis factor a triggers the acute-phase response, had been shown in the presence of IL-6. It is concluded that IL-6 had a dual inhibitory effect on the stimulation of PCK gene expression to attenuate the glucagon-induced increase in PCK gene transcription, messenger (mRNA) levels, and enzyme activity. The by glucagon. It inhibited the increase in cAMP at a site before cAMP formation by adenylate cyclase and at a site after cAMP molecular mechanism of this inhibition was investigated in the present study. Glucagon increased cyclic cAMP and PCK formation, the activation of the PCK gene promoter by cAMP.(HEPATOLOGY 1997;26:73-80.) mRNA levels to a transient maximum twofold and fivefold, respectively. The increases were attenuated by IL-6. Forskolin, which stimulates adenylate cyclase activity, increasedIn the liver, the proinflammatory cytokine, interleukin-6 cAMP and PCK mRNA levels 1.6-fold and fivefold, respec-(IL-6), triggers the acute-phase response, which is mainly tively. However, IL-6 attenuated the forskolin-stimulated in-characterized by the increased synthesis and secretion of poscrease in PCK mRNA but not the increase in cAMP. This itive acute-phase plasma proteins and the decreased synthesis showed that IL-6 inhibited PCK mRNA increase in part by and secretion of negative acute-phase plasma proteins (for the attenuation of cAMP increase, but also beyond cAMP review, see Heinrich et al. 1 and Akira et al. 2 ). IL-6 binds to formation. This was confirmed in experiments in which PCK its 80-kd receptor at the cell surface, which associates with mRNA levels were increased by the nonhydrolyzable cAMP-a dimer of the signal-transducing 130-kd glycoprotein, analogue, chlorophenylthio (CPT)-cAMP. The increase in PCK gp130. 3,4 After the binding of IL-6 to its receptor, signal mRNA was again attenuated by IL-6. In pertussis toxin-and transduction is accomplished by two different routes in the in isobutylmethylxanthine-treated hepatocytes, IL-6 still in-cell. Phosphorylation on threonine of the basic leucine zipper hibited the glucagon-stimulated increase in cAMP, indicating transcription factor, C/EBPb (CAAT-box enhancer binding that IL-6 did not activate an inhibitory G-protein or phospho-protein b) (also termed NF-IL-6, IL-6-DBP, or LAP), leads diesterase, which could cause the impairment of cAMP in-to transcriptional activation of C/EBPb, which binds to C/ crease. To demonstrate whether the inhibition of PCK gene EBPb-binding sites in the ...

Regulation by glucagon (cAMP) and insulin of the promoter of the human phosphoenolpyruvate carboxykinase gene (cytosolic) in cultured rat hepatocytes and in human hepatoblastoma cells

“…Insulin, the physiological antagonist of glucagon action, decreases the glucagon-stimulated increase in cAMP by the activation of phosphodiesterase, thereby attenuating the stimulation by glucagon of PCK gene expression [14,15,22]. In the present study insulin inhibited the stimulation by glucagon or cAMP of luciferase expression from the human as well as from the rat cytosolic PCK gene promoter, indicating that insulin was antagonistic through both promoters.…”

“…In the present study insulin inhibited the stimulation by glucagon or cAMP of luciferase expression from the human as well as from the rat cytosolic PCK gene promoter, indicating that insulin was antagonistic through both promoters. However, because insulin is also inhibitory when the non-hydrolysable cAMP analogue CPT-cAMP is used, the effect must occur downstream from cAMP hydrolysis, as has been suggested previously [22,25].…”

“…In the present study a 48 h culture period was employed, because after that time the hepatocytes had completely recovered from the isolation procedure. After 48 h of culture the expression of the transgene was stimulated with glucagon or CPT-cAMP for 8 h. During this stimulation period the expression of the luciferase gene increased linearly with time [22][23][24].…”

Inhibition of Glucagon-Signaling and Downstream Actions by Interleukin 1β and Tumor Necrosis Factor α in Cultured Primary Rat Hepatocytes

“…Hence, at high cAMP concentrations the inhibitory effect of the cytokines on the increase in PCK1 mRNA could be overridden ( Table 1 ). Under the same conditions, it has been shown that the inhibition by insulin of the glucagon-stimulated increase in cAMP levels could be prevented in the presence of IBMX [9] . This was expected, because insulin is known to counteract the increase in cAMP by the activation of phosphodiesterase [14] .…”

“…PCK1 mRNA and cAMP levels were determined after 2 hours, and 3 minutes, respectively, at which points in time, levels were increased transiently to a maximum after glucagon addition as shown in previous studies. This increase in mRNA and cAMP at 2 hours, and 3 minutes, respectively, after glucagon addition, was set equal to 100 % in each single experiment, relative to other values [9,10] . Under these conditions, the cytokines still inhibited the glucagon-stimulated increase in cAMP and in PCK1 mRNA indicating that this inhibition was not mediated by the activation of an inhibitory G-protein ( Table 1 ).…”