Elsevier

Cellular Signalling

Volume 84, August 2021, 110010
Cellular Signalling

Research Article
Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis

https://doi.org/10.1016/j.cellsig.2021.110010Get rights and content

Highlights

  • Glucagon elicited a biphasic mTORC1 signaling response.

  • Transient activation of mTORC1 by glucagon required PI3K/Akt and EPAC.

  • Glucagon promoted Rap1-GTP loading.

  • Glucagon acutely stimulated EPAC-dependent protein synthesis.

  • Latent repression of mTORC1 by glucagon was consistent with PKA activation.

Abstract

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.

Introduction

The master kinase known as mechanistic target of rapamycin (mTOR) exists in two independent protein complexes that act to govern cellular proliferation and metabolism. Signaling through mTOR complex 1 (mTORC1) stimulates protein synthesis through multiple mechanisms including phosphorylation of the 70 kDa ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). During fasting, hepatic mTORC1 activity is repressed, whereas a meal rapidly activates the complex [1]. Coordinate regulation of mTORC1 in the liver is mediated by levels of nutrients, such as amino acids, and hormones, such as insulin and glucagon [[2], [3], [4]].

Insulin-stimulated mTORC1 activation in liver occurs primarily through the PI3K/Akt signaling pathway [4,5]. Phosphatidylinositol 3,4,5-triphosphate (PIP3) binds to the pleckstrin homology domain of Akt, to promote Akt translocation to the plasma membrane, where it is then phosphorylated by PDK1 and mTOR complex 2 (mTORC2) [5]. Evidence supports direct activation of mTORC2 by PIP3 [6]. Active mTORC2 phosphorylates the hydrophobic motif of Akt at Ser473 [5]. In turn, Akt phosphorylates tuberous sclerosis complex 2 (TSC2) to inhibit GTPase activator protein (GAP) activity of the TSC complex toward the small GTPase Ras homolog enriched in brain (Rheb) [7]. Binding of Rheb-GTP, but not Rheb-GDP, activates mTORC1 [8]. On the other hand, amino acid sufficiency signals through the heterodimeric Rag GTPases to promote mTORC1 translocation to lysosomal membranes, where the complex interacts with Rheb [9].

The peptide hormone glucagon (GCG) is secreted by alpha cells of the pancreas in response to hypoglycemia and acts on a limited number of target tissues that express the glucagon receptor (GCG-R). GCG action on hepatic carbohydrate metabolism has been studied extensively, and the hormone is conventionally viewed as counter-regulatory to insulin [[10], [11], [12], [13]]. GCG increases hepatic glucose production by activating signaling pathways that stimulate gluconeogenesis [14,15] and glycogenolysis [12,16]. Reports from our laboratory [2,3,17] and others [18] provide evidence that GCG acts to negatively regulate signaling through mTORC1. Specifically, the suppressive effects of GCG on mTORC1 are associated with the activation of protein kinase A (PKA) and enhanced phosphorylation of AMP-activated protein kinase (AMPK) by LKB1 [2]. AMPK negatively regulates mTORC1 both directly through phosphorylation of raptor [19] and indirectly by phosphorylating TSC2 to promote GAP activity of the TSC complex [20].

Recently, a surprising role for GCG in energy hemostasis was suggested by reports that the hormone may also act to increase energy expenditure [21]. In fact, evidence supports that the impact of GCG signaling may differ between fasting and post-prandial conditions [22]. For example, acute agonism of the GCG-R synergistically enhances hepatic insulin action and facilitates glucose disposal [23]. Elevated plasma GCG levels are conventionally associated with fasting, but the secretion of both glucagon and insulin are rapidly stimulated by consumption of a protein meal [24]. This increase is due to the glucogonotrophic effects of amino acids like arginine, glutamine, and alanine on the pancreas. Notably, the increase in GCG secretion from isolated pancreatic islet cells upon exposure to amino acids is many times greater than what is seen with hypoglycemia [25].

A post-prandial increase in plasma GCG would theoretically suppress protein synthesis in the liver by countering amino acid- and insulin-stimulated mTORC1 activation. However, the rates of translation initiation and protein synthesis are higher in the liver shortly after ingestion of a protein-rich diet compared to fasted animals [26]. Evidence from cultured hepatocytes exposed to GCG also supports an acute increase in protein synthesis by the hormone [27]. Based on these confounding observations, we investigated the impact of insulin and GCG on mTORC1 activation in rat H4IIE and human HepG2 cell cultures. Surprisingly, the effect of GCG on mTORC1 signaling was biphasic, wherein there was an acute increase in phosphorylation of mTORC1 substrates over the first hour of exposure, followed by latent suppression. GCG-induced mTORC1 activation was associated with enhanced cAMP levels and required activation of exchange protein activated by cAMP (EPAC). Overall, the findings support that glucagon elicits acute activation of hepatic mTORC1/2 by an EPAC-dependent increase in Rap1-GTP, which is followed by mTORC1 repression.

Section snippets

Cell culture

Rat H4IIE (CRL-1548™) and Human HepG2 (HB-8065™) hepatoma cultures, and human embryonic kidney HEK293 (CRL-1573™) cells were obtained from American Type Cell Culture (ATCC®). All cells were cultured at 37 °C, 5% CO2 on CellBIND plates (Corning). Cells were cultured in EMEM (Invitrogen) or DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. In specific studies, cells were serum deprived for 16 h before culture medium was supplemented with 25 nM GCG (Sigma-Aldrich), PKA agonist

Glucagon elicits a biphasic response on mTORC1 signaling in hepatocyte cultures

To evaluate the coordinate effects of insulin and GCG on mTORC1 signaling, serum-deprived H4IIE cells were exposed to culture medium supplemented with insulin and/or GCG. As expected, insulin enhanced S6K1 phosphorylation (Fig. 1A). Surprisingly, GCG addition to culture medium also promoted an acute increase in S6K1 phosphorylation (Fig. 1A and Fig. S1A–C). Insulin-stimulated S6K1 phosphorylation was greater than that observed with GCG, and cells exposed to both insulin and GCG did not exhibit

Discussion

The historical dogma regarding GCG action positions the hormone as a guard against starvation/hypoglycemia that acts counter-regulatory to insulin. More recently, the role of GCG action has extended to prandial metabolism. The specific signaling events that mediate the non-canonical effects of GCG action remain to be fully established. In the present study, we investigated the regulatory effect of GCG on mTORC1. While a suppressive effect of GCG action on mTORC1 signaling has been previously

Grants

This research was supported by the American Diabetes Association Pathway to Stop Diabetes Grant 1-14-INI-04, National Institutes of Health grants R01 EY029702 (to M.D.D.), and R01 DK13499 (to S.R.K.).

Declaration of Competing Interest

No conflicts of interest, financial or otherwise, are declared by the authors.

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