Novel Mechanisms Controlling the Expression of the Antidiabetic Hepatokine Fibroblast Growth Factor 21

The incidence of obesity has increased at an alarming rate during the past thirty years. Current therapies for the treatment and prevention of obesity and metabolic syndrome are ineffective and/or produce undesirable side effects. Fibroblast growth factor 21 (FGF21) is a liver derived hormone that mediates adaptive changes in hepatic glucose and lipid metabolism by elevating energy expenditure and reducing lipid storage. Pharmacological administration of FGF21 analogs in both animal and human models induces striking reversals of obesity and type 2 diabetes. However, the activity of FGF21 analogs exhibit short half-lives in blood circulation and are unsuitable for current treatment. Therefore it is imperative that mechanisms mediating the activity of endogenous FGF21 be understood.;FGF21 expression is enhanced during starvation, high-fat low-carbohydrate (HF-LC) ketogenic diet, type 2 diabetes, and obesity. Starvation induces a high glucagon to insulin metabolic hormone ratio in the portal vein. Type 2 diabetics also exhibit chronically high glucagon to insulin ratios in blood circulation. Furthermore, infusions with high glucagon, low insulin hormone concentrations increase the production and secretion of splanchnic FGF21 in man. The mechanisms by which glucagon and insulin work cooperatively to selectively increase hepatic FGF21 expression and secretion is currently unknown.;To understand the molecular mechanisms mediating the induction of FGF21 expression in the liver, we recapitulated the conditions of starvation in primary rat hepatocytes by treating cultures with glucagon and insulin using concentrations similar to that observed in the portal vein during starvation. We then utilized a reverse genetics approach to identify two conserved amino acid response elements (AAREs) on the FGF21 promoter that were critical in mediating the effects of starvation on FGF21 gene expression. We further defined the transcription factor, activating transcription factor 4 (ATF4), as the primary transcription fact responsible in stimulating FGF21 gene transcription during glucagon plus insulin treatment. We then characterized the glucagon and insulin signaling pathways mediating the cooperative actions of glucagon plus insulin on FGF21 gene expression using loss-of-function and gain-of-function techniques, and determined that both the PI3K/Akt/mTORC1 and cAMP/PKA signaling pathways were responsible for inducing FGF21 expression. Lastly, we applied these novel findings to further define the mechanisms other stimuli, such as chenodeoxycholic acid (CDCA), employ to induce FGF21 expression. Understanding the fundamental mechanisms by which these two hormones work together provided the scientific community with in-depth knowledge on pathways mediating the endogenous expression of hepatic FGF21 and also identified additional targets for drug development for the treat of metabolic syndrome

Glucagon and Insulin Cooperatively Stimulate Fibroblast Growth Factor 21 Gene Transcription by Increasing the Expression of Activating Transcription Factor 4

Previous studies have shown that glucagon cooperatively interacts with insulin to stimulate hepatic FGF21 gene expression. Here we investigated the mechanism by which glucagon and insulin increased FGF21 gene transcription in primary hepatocyte cultures. Transfection analyses demonstrated that glucagon plus insulin induction of FGF21 transcription was conferred by two activating transcription factor 4 (ATF4) binding sites in the FGF21 gene. Glucagon plus insulin stimulated a 5-fold increase in ATF4 protein abundance, and knockdown of ATF4 expression suppressed the ability of glucagon plus insulin to increase FGF21 expression. In hepatocytes incubated in the presence of insulin, treatment with a PKA-selective agonist mimicked the ability of glucagon to stimulate ATF4 and FGF21 expression. Inhibition of PKA, PI3K, Akt, and mammalian target of rapamycin complex 1 (mTORC1) suppressed the ability of glucagon plus insulin to stimulate ATF4 and FGF21 expression. Additional analyses demonstrated that chenodeoxycholic acid (CDCA) induced a 6-fold increase in ATF4 expression and that knockdown of ATF4 expression suppressed the ability of CDCA to increase FGF21 gene expression. CDCA increased the phosphorylation of eIF2α, and inhibition of eIF2α signaling activity suppressed CDCA regulation of ATF4 and FGF21 expression. These results demonstrate that glucagon plus insulin increases FGF21 transcription by stimulating ATF4 expression and that activation of cAMP/PKA and PI3K/Akt/mTORC1 mediates the effect of glucagon plus insulin on ATF4 expression. These results also demonstrate that CDCA regulation of FGF21 transcription is mediated at least partially by an eIF2α-dependent increase in ATF4 expression

Perineuronal Net Formation and the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homoeostasis2,3. Because this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, critical-period closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans and rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed in PNN-like structures. In mice, these neurons are densely packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both γ-aminobutyric acid-ergic and leptin-receptor positive, including a majority of Agouti-related-peptide neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the critical period for maturation of Agouti-related-peptide neurons and is dependent on input from circulating leptin, because postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the critical period. We conclude that neurons crucial to metabolic homoeostasis are enmeshed in PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC–median eminence junction, where metabolically relevant humoral signals are sensed

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission

n rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling

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

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

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

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

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