Glucagon stimulates hepatic FGF21 secretion through a PKA- and EPAC-dependent posttranscriptional mechanism.

Previous studies have shown that whole body deletion of the glucagon receptor suppresses the ability of starvation to increase hepatic fibroblast growth factor 21 (FGF21) expression and plasma FGF21 concentration. Here, we investigate the mechanism by which glucagon receptor activation increases hepatic FGF21 production. Incubating primary rat hepatocyte cultures with glucagon, dibutyryl cAMP or forskolin stimulated a 3-4-fold increase in FGF21 secretion. The effect of these agents on FGF21 secretion was not associated with an increase in FGF21 mRNA abundance. Glucagon induction of FGF21 secretion was additive with the stimulatory effect of a PPARα activator (GW7647) on FGF21 secretion. Inhibition of protein kinase A (PKA) and downstream components of the PKA pathway [i.e. AMP-activated protein kinase and p38 MAPK] suppressed glucagon activation of FGF21 secretion. Incubating hepatocytes with an exchange protein directly activated by cAMP (EPAC)-selective cAMP analog [i.e. 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME)], stimulated a 3.9-fold increase FGF21 secretion, whereas inhibition of the EPAC effector, Rap1, suppressed glucagon activation of FGF21 secretion. Treatment of hepatocytes with insulin also increased FGF21 secretion. In contrast to glucagon, insulin activation of FGF21 secretion was associated with an increase in FGF21 mRNA abundance. Glucagon synergistically interacted with insulin to stimulate a further increase in FGF21 secretion and FGF21 mRNA abundance. These results demonstrate that glucagon increases hepatic FGF21 secretion via a posttranscriptional mechanism and provide evidence that both the PKA branch and EPAC branch of the cAMP pathway play a role in mediating this effect. These results also identify a novel synergistic interaction between glucagon and insulin in the regulation of FGF21 secretion and FGF21 mRNA abundance. We propose that this insulin/glucagon synergism plays a role in mediating the elevation in FGF21 production during starvation and conditions related to metabolic syndrome

Glucagon synergistically interacts with insulin to induce FGF21 secretion and FGF21 mRNA abundance.

<p>A: effect of insulin on FGF21 secretion, FGF21 mRNA abundance, and albumin secretion in primary rat hepatocytes incubated in the absence and presence of glucagon. Cells were incubated with or without insulin (50 nM), glucagon (25 nM), or insulin plus glucagon for 6 h. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of cells incubated no additions were set to 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). B: effect of different concentrations of insulin on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated concentrations of insulin and glucagon for 6 h. The abundance of FGF21 mRNA in cells incubated with no hormones was set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 3). * Significantly different (<i>P</i><0.05) from that of cells incubated with no additions. ^# Significantly different (<i>P</i><0.05) from that of cells incubated with glucagon or insulin alone.</p

Glucagon increases FGF21 secretion via a mechanism not involving changes in FGF21 mRNA abundance.

<p>A: effect of different concentrations of glucagon on FGF21 secretion, FGF21 mRNA abundance, and albumin secretion in primary rat hepatocyte cultures. Cells were incubated with the indicated concentrations of glucagon in serum-free Medium 199 for 6 h. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of cells incubated with 0 nM glucagon (Gln) were set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 3). B: time course of the effect glucagon on FGF21 secretion, FGF21 mRNA abundance, and albumin secretion. Rat hepatocytes were incubated with or without glucagon (25 nM) for the indicated time periods. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of cells incubated with no additions (NA) for 2 h were set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). C: the effect of glucagon on FGF21 secretion is reversible. Rat hepatocytes were initially incubated with or without glucagon for 18 h. The culture medium was then replaced with one containing glucagon or NA, and the incubation was continued for 6 h. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of cells incubated with NA for both treatment periods were set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). D: interaction between glucagon and PPARα in the regulation of FGF21 secretion and FGF21 mRNA abundance. Rat hepatocytes were incubated with or without glucagon (25 nM), GW7647 (1 µM), or glucagon plus GW7647 for 6 h. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of cells incubated no additions were set to 1, and the other values were adjusted proportionately. Values are means ± SE (n = 3). * Significantly different (<i>P</i><0.05) from that of cells incubated with no additions. ^# Significantly different (<i>P</i><0.05) from that of cells incubated with glucagon or GW7647 alone.</p

Activation of EPAC induces FGF21 secretion.

<p>Primary rat hepatocytes (A), rat H4IIE hepatoma cells (B) and human HepG2 hepatoma cells (C) were incubated with cpTOME (5 µM) for 6 and 12 h. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of cells incubated with no treatments for 6 h were set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). * Significantly different (<i>P</i><0.05) from that of cells incubated without cpTOME for the same time period.</p

Inhibition of AMPK suppresses the stimulatory effect of EPAC activation on FGF21 secretion.

<p>Primary rat hepatocytes were isolated and incubated in serum-free Medium 199. At 66 h of incubation, the medium was replaced with one of the same composition containing compound C (5 µM), SB203580 (5 µM) or vehicle (DMSO) with or without cpTOME (5 µM), and the incubation was continued for 6 h. A: the level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA were measured. Values for cells incubated with vehicle in the absence of glucagon were set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). * Significantly different (<i>P</i><0.05) from that of cells incubated with vehicle. B: the abundance of phosphorylated ACC1 (Ser⁷⁹), phosphorylated ACC2 (Ser²¹²), total ACC1, and total ACC2 in total cell lysates were measured by Western analysis. The ratio of phosphorylated ACC1 and ACC2 to total ACC1 and ACC2 of cells treated with no additions was set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). ^# Significantly different (<i>P</i><0.05) from any other treatment group. C: the abundance of phosphorylated p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) and total p38 MAPK in cells treated with cpTOME for 6 h.</p

The glucagon-induced increase in FGF21 secretion is mediated by Rap1 activation.

<p>Primary rat hepatocytes were transduced with a Rap1GAP expressing adenovirus (AV-Rap1GAP) or a control adenovirus (AV-1LacZ). After 24 h, cells were incubated with or without glucagon for 24 h. The level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA of AV-LacZ-infected cells incubated with no additions (NA) were set at 1, and the other values were adjusted proportionately. The effect of glucagon was calculated as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094996#pone-0094996-g003" target="_blank">Fig. 3</a>. Values are means ± SE (n = 4). * Significantly different (<i>P</i><0.05) from that of cells infected with AV-LacZ.</p

Inhibition of AMPK and p38 MAPK suppresses the stimulatory effect of glucagon on FGF21 secretion.

<p>Primary rat hepatocytes were isolated and incubated in serum-free Medium 199. At 66 h of incubation, the medium was replaced with one of the same composition containing compound C (5 µM), SB203580 (5 µM) or vehicle (DMSO) with or without glucagon, and the incubation was continued for 6 h. A: the level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA were measured. Values for cells incubated with vehicle in the absence of glucagon were set at 1, and the other values were adjusted proportionately. The effect of glucagon was calculated as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094996#pone-0094996-g003" target="_blank">Fig. 3</a>. Values are means ± SE (n = 4). * Significantly different (<i>P</i><0.05) from that of cells incubated with vehicle. B: the abundance of phosphorylated ACC1 (Ser⁷⁹), phosphorylated ACC2 (Ser²¹²), total ACC1, and total ACC2 in total cell lysates were measured by Western analysis. The ratio of phosphorylated ACC1 and ACC2 to total ACC1 and ACC2 of cells treated with no additions was set at 1, and the other values were adjusted proportionately. Values are means ± SE (n = 4). ^# Significantly different (<i>P</i><0.05) from any other treatment group. C: the abundance of PEPCK mRNA in total RNA was measured.</p

Proposed model for how starvation increases hepatic FGF21 gene expression and secretion.

<p>Starvation increases the secretion of glucagon into the portal circulation. At the liver, glucagon binds to the glucagon receptor (GR) triggering a signaling cascade resulting in the activation of the PKA and EPAC branches of the cAMP pathway. Activation of PKA and EPAC stimulates FGF21 secretion via a translational and/or posttranslational mechanism. Additional components of the glucagon pathway regulating FGF21 secretion include P38 MAPK and AMPK; these proteins function downstream of PKA and/or EPAC. Glucagon also increases FGF21 secretion via a pretranslational mechanism, and this effect is unmasked by the presence of insulin. Activation of Akt may play a role in mediating the effect of glucagon and insulin on FGF21 gene expression. The activation of PPARα also contributes to the starvation-induced increase in FGF21 gene expression. IR: insulin receptor.</p

Inhibition of PKA suppresses the stimulatory effect of glucagon on FGF21 secretion.

<p>Primary rat hepatocytes were isolated and incubated in serum-free Medium 199. At 66 h of incubation, the medium was replaced with one of the same composition containing H89 (10 µM) or vehicle (DMSO) with or without glucagon, and the incubation was continued for 6 h. A: the level of FGF21 and albumin in the culture medium and the abundance of FGF21 mRNA in total RNA were measured. B: the abundance of PEPCK mRNA was measured. C: the abundance of SOCS3 mRNA was measured. Values for cells incubated with vehicle in the absence of glucagon were set at 1, and the other values were adjusted proportionately. The effect of glucagon is the level of FGF21 secretion, albumin secretion, FGF21 mRNA, PEPCK mRNA, or SOCS3 mRNA in cells treated with glucagon expressed as a percentage of the level in cells treated with NA. The effect of glucagon was calculated for individual experiments and then averaged. Values are means ± SE (n = 5). * Significantly different (<i>P</i><0.05) from that of cells incubated with vehicle.</p