Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Cell Signal. 2021 Apr 17;84:110010. doi: 10.1016/j.cellsig.2021.110010

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

Siddharth Sunilkumar a, Scot R Kimball a, Michael D Dennis a
PMCID: PMC8169602  NIHMSID: NIHMS1696861  PMID: 33872697

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.

Keywords: glucagon, cyclic AMP, protein synthesis, liver, insulin, diabetes

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 [2–4].

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 [10–13]. 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.

Material and Methods

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) with EMEM (Invitrogen). Cells were cultured in medium 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 N6-Benzoyl-cAMP (100 μM; Axxora), or EPAC agonist 8-pCPT-2’-O-Me-cAMP AM (10 μM; Axxora) for the indicated times. Glucagon receptor was inhibited by pretreating cells with LY2409021 (30 μM; MedChemExpress). For specific studies cells were treated with PKA inhibitor H-89 (50 μM; N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulfonamide), EPAC inhibitor ESI-09 (10 μM; α-[2-(3-Chlorophenyl) hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile), PI3 kinase inhibitor LY294002 (50 μM) or mTOR complex inhibitor Torin 2 (10 nM; 9-(6-Amino-3-pyridinyl)-1-[3-(trifluoromethyl)phenyl]-benzo[h]-1,6-naphthyridin-2(1H)-one) for indicated time periods followed by glucagon addition to culture medium. The Flag-Rap1B plasmid (Addgene_118325) was gifted by Philip Stork. Cells were transfected using JetPRIME (Polyplus-transfection) following manufacturer’s instructions. A phospho-mimetic Rap1 variant (Rap1_Q63E) was generated by site-directed mutagenesis using and a QuickChange Lightning Kit (Agilent). Plasmids were validated by sequencing analysis.

cAMP measurements.

H4IIE cells were exposed to 25 nM GCG for 0–2 hours and intracellular cAMP concentrations were measured using the direct cAMP ELISA kit (Enzo Life Sciences) following manufacturer’s instructions. Cells were lysed in 0.1 M HCl. After centrifugation, supernatants of cell extracts were used for ELISA. cAMP concentrations were normalized to total protein content.

Rap1 activation assay

Rap1-GTP loading was assessed using the Active Rap1 Pull-Down and Detection Kit (Thermo Scientific; Cat# 16120) following the manufacturer’s instructions. In brief, 1 mg of protein from 16,000 × g supernatant fractions of cell lysates was incubated along with glutathione resin and GST-RalGDS-RBD for 1 h at 4°C. The beads were pelleted at 6000 × g and washed thrice in 1X lysis buffer. The beads were then resuspended in 2X reducing SDS sample buffer and eluted using spin columns at 600 × g for 30 sec. The eluate was boiled and Rap1-GTP loading was assayed by Western blot analysis.

SUnSET assay

Effect of glucagon stimulation on protein synthesis was assessed as previously described [28]. Rat H4IIE cells were deprived of serum for 16?h and exposed to vehicle or 25 nM GCG for 30 min followed by exposure to 1 μg/ml puromycin for an additional 30 min. In specific studies, cells were incubated with 10 μM ESI-09 for 30 min prior to exposure to GCG. Following puromycin incorporation, cells were washed with ice-cold PBS and lysed directly in 1X reducing SDS sample buffer. Samples were analyzed by western blotting and puromycin incorporation was assessed with a mouse monoclonal anti-puromycin [3RH11] antibody (Kerafast).

Western blot analysis

Cell lysates were separated on Criterion Precast 4–20% gels (Bio-Rad Laboratories). Proteins were transferred to a polyvinylidene fluoride membrane, blocked in 5% milk in 1X TBS-T buffer, and evaluated with the appropriate antibodies (Table S1).

PCR analysis

RNA from rat liver, rat H4IIE, rat R28 retinal neuronal precursor cells, and rat H9c2 cardiomyocytes was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) as previously described [29]. RNA (1 μg) was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and subjected to quantitative real-time PCR (Q12K Flex Real-Time PCR System; Applied Biosystems) using a QuantiTect SYBR Green Master Mix (Qiagen). GCG-R gene expression was determined using the appropriate primer sequences (Table S2) and values for mRNA transcripts were normalized to the levels of actin mRNA.

Statistical analysis

Data are expressed as mean ± SD. Data were analyzed overall with either one-way or two-way ANOVA, and pairwise comparisons were made using the Dunnett’s or Tukey’s test for multiple comparisons. Significance was defined as p < 0.05 for all analyses.

Results

Glucagon elicits a biphasic response in 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 an additive effect, but rather the response was similar to that elicited by insulin alone. To further investigate the impact of GCG on mTORC1 signaling, H4IIE cells cultured in complete medium were exposed to either GCG or a vehicle control over a time course of 24 h. Exposure to vehicle for up to 24 h did not alter S6K1 phosphorylation (Fig. 1B). However, phosphorylation of S6K1 was attenuated in cells exposed to GCG for 24 h as compared to vehicle (Fig 1C–D). Consistent with the prior observation in serum deprived cells, GCG transiently stimulated mTORC1 activation, as assessed by phosphorylation of S6K1 and 4E-BP1 (Fig. 1D). GCG-induced mTORC1 activation was observed for up to 1 h, and followed by an inhibitory effect with more prolonged exposure. A similar biphasic mTORC1 response to GCG was observed in human HepG2 cell cultures (Fig. 1E). Together, the data support that GCG elicits acute activation of mTORC1 and the findings are consistent with activation of a convergent signaling axis by insulin and GCG.

Figure 1: Glucagon elicits biphasic mTORC1 signaling.

Figure 1:

Open in a new tab

A) Rat H4IIE cells that were serum deprived for 16 h prior were exposed to 5 nM insulin (open square), 25 nM glucagon (GCG, closed circle), or both (GCG + insulin, closed square) for 0–8 h. B) Rat H4IIE were exposed to B) vehicle (Veh) for 0–24 h. C) H4IIE cells were exposed to either Veh or 25 nM GCG for 24 h. D) H4IIE or (E) HepG2 cells were exposed to culture medium supplemented with Veh or 25 nM GCG for 0–24 h. Phosphorylation of S6K1 at Thr389 and 4E-BP1 at Ser65, as well as total S6K1, 4E-BP1, and actin protein expression, were evaluated in whole cell lysates by Western blotting. Representative blots are shown and protein molecular mass in kDa is shown to the left. Data are expressed as mean ± SD and significance was determined by one-way ANOVA with Dunnett’s testfor multiple comparisons. Significance between two groups was determined by Student’s T-test. *, p < 0.05 versus vehicle control.

Transient activation of mTORC1 signaling by GCG requires PI3K/Akt.

GCG action is facilitated by activation of the glucagon receptor (GCG-R). The mRNA encoding GCG-R was expressed at modest levels in H4IIE cells, but not H9c2 (heart) or R28 (retina) cell cultures (Fig S1D). GCG-R inhibition was sufficient to prevent the transient increase in S6K1 and 4E-BP1 phosphorylation in cells exposed to GCG (Fig. 2A). To confirm that the acute stimulatory effect of GCG was indeed mediated by mTORC1 activation, mTOR kinase activity was inhibited with Torin 2. Inhibition of mTOR kinase activity prevented GCG-induced S6K1 phosphorylation (Fig. 2B). To determine if the effects of GCG on mTORC1 correlated with an increase in mTORC2 activation, phosphorylation of Akt at Ser473 was evaluated (Fig. 2C). Akt phosphorylation was enhanced shortly after GCG addition to culture medium and remained so beyond 1 h of exposure (Fig. 2C). Importantly, GCG-induced phosphorylation of Akt preceded the change in S6K1 phosphorylation, suggesting the possibility that GCG mediates mTORC1 signaling changes via modulation of mTORC2. Indeed, inhibition of PI3K prevented the stimulatory effects of GCG on mTORC1 (Fig. 2D) and mTORC2 (Fig. 2E). Together, these data support that GCG transiently stimulates mTORC1 through a signaling axis that includes Akt/mTORC2.

Figure 2: Glucagon transiently stimulates mTORC1 activation through a convergent signaling axis with insulin.

Figure 2:

Open in a new tab

A) H4IIE cells were exposed to the glucagon receptor (GCG-R) antagonist LY2409021 for 20 min prior to GCG addition. B) H4IIE cells were exposed to the mTOR kinase inhibitor Torin 2 for 15 min prior to addition of either GCG or a vehicle (Veh) control. In A and B, whole cell lysates were collected for Western blot analysis 45 min after addition of GCG or Veh. C) Rat H4IIE cells were serum deprived for 16 h prior to manipulation. H4IIE cells were exposed to GCG for up to 2 h. D-E) H4IIE cells were exposed to the PI3K inhibitor LY294002 for 15 min followed by 45 min of exposure to GCG or vehicle (Veh) control. Phosphorylation of S6K1 at Thr389, CREB at Ser133 and Akt at Ser473, as well as total S6K1 and actin protein expression, were evaluated in whole cell lysates by Western blotting. Representative blots are shown and protein molecular mass in kDa is shown to the left. Data are expressed as mean ± SD. Data are expressed as mean ± SD and significance was determined by two-way ANOVA with Tukey’s test for multiple comparisons. *, p < 0.05versus time zero or Veh control; #, p< 0.05 versus GCG alone.

Activation of mTORC1 by GCG is mediated via EPAC.

The signaling events mediated by GCG-R activation are largely driven by an increase in intracellular cAMP levels [30]. Measurement of intracellular cAMP levels in H4IIE cells indicated a transient spike in cAMP concentrations upon GCG addition to culture medium (Fig. 3A). Acting as a second messenger, cAMP can activate various pathways by signaling through either PKA or EPAC [31,32]. To investigate signaling downstream of cAMP, H4IIE cells were exposed to selective agonists of either PKA or EPAC. The PKA selective agonist 6-Benz cAMP enhanced phosphorylation of the PKA substrate CREB in H4IIE cells, but did not alter S6K1 phosphorylation as compared to a vehicle control (Fig. 3B). However, addition of the PKA inhibitor H89 to culture medium enhanced S6K1 phosphorylation. Notably, the stimulatory effect of H89 on S6K1 phosphorylation was additive with that of GCG (Fig. 3C). The EPAC specific agonist 8-CPT cAMP enhanced S6K1 phosphorylation and EPAC inhibition with ESI-09 prevented the effect (Fig. 3B). Similarly, in H4IIE cells exposed to ESI-09, GCG addition to culture medium failed to enhance S6K1 phosphorylation (Fig. 3C-D). Whereas GCG enhanced phosphorylation of Akt (Fig. 3E) and TSC2 (Fig. 3F) in H4IIE cells, ESI-09 was sufficient to prevent the stimulatory effect. A similar repressive effect of EPAC inhibition on GCG-stimulated mTORC1/2 activation was also observed in HepG2 cells (Fig. S2). These results support that GCG acts via cAMP-EPAC signaling to acutely stimulate mTORC1/2 activation.

Figure 3: Glucagon transiently stimulates mTORC1/2 by activation of EPAC.

Figure 3:

Open in a new tab

A) Intracellular cAMP levels were measured in H4IIE cells at various times till 2 hours after glucagon (GCG) addition to culture medium. B-C) H4IIE cells were exposed to culture medium containing either the PKA inhibitor H89, the EPAC inhibitor ESI-09, or a vehicle (Veh) control for 30 minutes. The PKA agonist 6-benz cAMP or the EPAC agonist 8 CPT cAMP was added to culture medium as indicated. Whole cell lysates were collected for analysis 45 min after addition of cAMP analogs. C) H4IIE cells were exposed to H89 or ESI-09 as indicated for 30 min followed by addition of either GCG or a vehicle (Veh) control for 45 min. D-F) Cells were exposed to ESI-09 or Veh for 30 min prior to GCG addition for 45 min. Phosphorylation of S6K1 at Thr389, CREB at Ser113, Akt at Ser473, and TSC2 at Thr1462 was evaluated in whole cell lysates by Western blotting. Total S6K1, TSC2 and actin protein expression were also determined. Representative blots are shown and protein molecular mass in kDa is shown to the left. Data are represented as mean ± SD and significance was determined by two-way ANOVA with Tukey’s test formultiple comparisons. *, p < 0.05 compared to Veh alone; #, p< 0.05 versus GCG alone.

Glucagon signals to mTORC1 via an EPAC/Rap1 signaling axis.

The best-known mediator of EPAC signaling is the small GTPase Rap1 [32]. Expression of wild-type Rap1 (Rap1 WT) or a constitutively active variant (Rap1 Q63E) [34] in HEK293 cells enhanced phosphorylation of S6K1 (Fig. 4A). In H4IIE cells, expression of Rap1 WT enhanced S6K1 phosphorylation (Fig. 4B). The stimulatory effect of GCG on S6K1 phosphorylation was not additive with Rap1 expression, suggesting a similar mechanism of action. Notably, Rap1 overexpression also increased Akt S473 phosphorylation in both HEK293 (Fig. 4A) and H4IIE cells (Fig. 4C). To evaluate activation of Rap1 by GCG, Rap1-GTP pull down was performed on H4IIE cell lysates. In H4IIE cells exposed to GCG, the proportion of Rap1 bound to GTP was increased concomitant with enhanced S6K1 phosphorylation (Fig. 4D). Overall, the findings support a working model wherein GCG acts to stimulate activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP, which is followed by latent repression of mTORC1 (Fig. 4E).

Figure 4: Glucagon promotes Rap1-GTP loading.

Figure 4:

Open in a new tab

A) HEK293 cells were transfected to express either an empty vector (EV) control plasmid, FLAG-tagged Rap1 wild-type (WT), or the FLAG-tagged Rap1 Q63E variant. B-C) H4IIE cells were transfected to express either EV or Rap1 WT and exposed to culture medium containing either glucagon (GCG) or vehicle (Veh) control for 45 min. D) Rap1-GTP loading was examined in H4IIE cell lysates following 45 min glucagon stimulation by immunoprecipitation (IP) of GST-tagged Ral1GDS Rap-binding domain (RBD). Samples incubated with GTPγS or GDP were used as positive and negative controls, respectively. Phosphorylation of S6K1 at Thr389 and Akt at Ser473 were evaluated in whole cell lysates by western blotting. Total S6K1, FLAG-tag, actin, and Rap1 were also evaluated. Representative blots are shown and protein molecular mass in kDa is shown to the left. Data are represented as mean ± SD and significance was determined by two-way ANOVA with Tukey’s test for multiple comparisons. *, p < 0.05 versus EV; #, p< 0.05 compared to Veh. E) Working model for the mechanism whereby glucagon (GCG) stimulates biphasic activation of mTORC1/2 via an EPAC/Rap1 signaling axis.

Glucagon/EPAC signaling increases mRNA translation.

A major consequence of mTORC1 activation is increased rates of global protein synthesis [33]. The SUnSET method was used to evaluate protein synthesis by incorporation of puromycin into nascent peptide chains. Protein synthesis was increased in H4IIE cells after exposure to either GCG or insulin (Fig. 5A). By contrast, H4IIE cells exposed to ESI-09 failed to exhibit an increase in puromycin incorporation following GCG exposure (Fig. 5B). This supports that the transient stimulatory effect of GCG on mTORC1 is sufficient to promote an acute increase in protein synthesis.

Figure 5: Glucagon acutely stimulates global rates of protein synthesis via EPAC.

Figure 5:

Open in a new tab

A) Rat H4IIE cells were serum deprived for 16 h and then exposed to 25 nM GCG or 5 nM insulin for 30 min followed by an additional 30 min of exposure in the presence of 1 μM puromycin. B) Serum deprived H4IIE cells were exposed to culture medium containing 10 μM ESI-09 for 30 min, followed by addition of 25 nM GCG for 30 min, and addition of 1 μM puromycin for 30 min. Puromycin incorporation, phosphorylation of S6K1 at Thr389, actin and total S6K were evaluated in whole cell lysates by western blotting. Representative blots are shown and protein molecular mass in kDa is shown to the left. Data are represented as mean ± SD and significance was determined by two-way ANOVA with Tukey’s test for multiple comparisons. *, p < 0.05 compared to Veh alone; #, p< 0.05 versus GCG alone.

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 reported, we found that in H4IIE and HepG2 cell cultures the hormone elicited transient stimulation of mTORC1 signaling and promoted an acute increase in protein synthesis. This observation is consistent with the prior report wherein a similar acute increase in protein synthesis was observed in cultured hepatocytes exposed to GCG [27]. In the present study, the transient activation of mTORC1 upon GCG addition to culture medium preceded a repressive effect that was anticipated based on prior investigations.

It is well established that insulin acts via Akt/PI3K-dependent signaling to promote activation of mTORC1/2. In the present study, GCG acutely stimulated phosphorylation of Akt at Ser473 and S6K1 at T389. Notably, the GCG-induced change in Akt phosphorylation was observed prior to the effect on S6K1. Moreover, GCG-induced mTORC1 activation was not additive with insulin, suggesting that these effects were mediated by a convergent signaling axis. GCG-induced signaling in H4IIE cells is consistent with site-specific potentiation of Akt S473 phosphorylation in the liver of mice 1 h after administration of the GCG-R agonist IUB288 [23]. In the prior study [23], GCG enhanced insulin-stimulated Akt phosphorylation at S473 and Akt kinase activity in primary hepatocytes. However, GCG alone was not sufficient to do so. An important consideration when comparing the prior observation to those herein is the difference in dose and time course of exposure, as the absence of an effect in primary hepatocytes was observed after 2.5 min of exposure to 10 nM glucagon [23]. In the present study, a modest increase (1-fold) in Akt S473 phosphorylation was observed in H4IIE cells 5 min after exposure to 25 nM GCG, with a maximal response (6-fold) observed after 30 min. Thus, we suspect that with more prolonged exposure a similar response may also be observed in primary hepatocytes. Regardless of whether or not GCG is sufficient to elicit the effect, both studies support that activation of GCG-R by GCG acts to promote mTORC2-dependent Akt phosphorylation.

GCG action on the GCG-R leads to a conformational change in Gas-coupled proteins, activation of adenylate cyclase, and increased synthesis of the intracellular secondary messenger cAMP from ATP. Levels of cAMP influence a multitude of signaling pathways via the effector proteins PKA and EPAC. In the present study, GCG enhanced cAMP levels in H4IIE cells and the stimulatory effect of GCG on mTORC1/2 signaling required EPAC. Moreover, an EPAC-specific cAMP analog was sufficient to enhance phosphorylation of S6K1. Our findings support the prior report that EPAC activation in 1-LN prostate cancer cells increases cellular proliferation by stimulation of mTORC1 signaling [35]. Furthermore, our observations that GCG-induced protein synthesis is dependent on EPAC signaling expands on previous findings that demonstrate enhanced cAMP levels stimulate protein synthesis in rat heart [36] and C6–2B rat glioma cells [37]. Although evidence suggests a role for glucagon like peptide 1 receptor (GLP-1R) stimulation in the activation of mTORC1 [38] and increased EPAC-mediated signaling [39], lack of the GLP-1R in the liver of rodents [40], non-human primates [41], as well as rat liver and H4IIE cells (Fig. S1D), advocates for the specificity of GCG-R in the effects of GCG on mTORC1.

EPAC1/2 act as guanine nucleotide exchange factors (GEF) for the Ras-like GTPase Rap1 and have been previously implicated in a range of diverse cellular signaling responses (reviewed in [42]). Evidence supports that Rap1 directly interacts with mTORC2 to promote kinase activity in a manner that is potentially analogous to stimulation of mTORC1 by Rheb-GTP [43]. Indeed, the catecholamine norepinephrine acts through an EPAC-dependent signaling cascade to activate mTORC2 in brown adipocytes [44]. In prostate cancer cells, the EPAC-selective agonist 8CPT cAMP increases Akt phosphorylation at S473 to promote cellular proliferation [45]. Evidence also supports that EPAC-dependent activation of Rap1 enhances Akt phosphorylation in primary cortical neurons [46]. Herein, GCG addition to culture medium promoted Rap1-GTP loading in H4IIE cells. Moreover, the stimulatory effect of GCG on mTORC1 and mTORC2, as assessed by phosphorylation of S6K1 at T389 and Akt at S473, respectively, in H4IIE cell cultures was EPAC-dependent. Rap1 expression was also sufficient to enhance phosphorylation at both sites.

Kim et.al. [47] have suggested a model wherein cAMP promotes mTORC1 activation by decreasing Rheb sequestration by phosphodiesterase (PDE) 4D5 to allow for enhanced Rheb binding to mTORC1. However, the activation of mTORC1 by Rheb is dependent on the binding of GTP, as neither Rheb-GDP nor the nucleotide free protein are sufficient to do so [8]. T he findings here support that cAMP acts to promote mTORC1 signaling via an EPAC-dependent effect that is associated with enhanced phosphorylation of Akt and TSC2. Importantly, the findings here are not necessarily in conflict with the prior report, but rather provide evidence that cAMP also acts through signaling inputs upstream of the TSC complex to activate mTORC1.

Cyclic AMP mediated regulation of Akt/mTOR signaling has to date provided contrary evidences [35,47–49]. This is likely due to opposing effects of EPAC and PKA on the pathway [50]. We previously demonstrated a suppressive effect of GCG on mTORC1 in perfused liver that was associated with the activation of PKA [2,3]. Following the transient stimulatory effects of GCG on mTORC1 in H4IIE and HepG2 cultures, we observed latent suppression. Further, the stimulatory effect of an EPAC-specific cAMP analog on mTORC1 was not recapitulated with a PKA-specific cAMP analog. In fact, cells exposed to the well-known PKA inhibitor H89 exhibited enhanced phosphorylation of S6K1. This observation suggests the possibility that PKA may be moderately active in this cell culture model in absence of GCG stimulation. However, H89 has also been shown to act directly on a number of other kinases, that could also potentially act to repress mTORC1 [51,52]. Regardless, the latent repression seen here are largely consistent with prior reports that demonstrate a suppressive effect of PKA on mTORC1/2 through either complex disassociation [48] or the direct phosphorylation of the raptor [49]. Recent reports on rapid deubiquitination and endocytosis of agonist-activated GCG-R [53] provide an alternate hypothesis for the delayed mTORC1/2 repression observed in this study.

In summary, the studies herein provide evidence that GCG elicits biphasic regulation of mTORC1. Acute activation of mTORC1/2 signaling by GCG was associated with an increase in cAMP, dependent on EPAC signaling, and occurred concomitant with an increase in Rap1-GTP loading and enhanced protein synthesis. These observations are consistent with a growing body of evidence that supports an expanded role for GCG action beyond its traditional role during fasting and hypoglycemia as an opposing signal to insulin. While the physiological outcomes associated with the transient GCG-induced signaling events identified herein remain to be resolved, it is tempting to speculate that our findings may offer a potential foundation to understanding some of the surprising benefits of glucagon pharmacotherapy in diabetes. Indeed, the translation of specific mRNAs encoding hepatokines linked to the development of metabolic syndrome have recently been shown to be particularly sensitive to mTORC1 [17,54].

Supplementary Material

1

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.

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.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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

References:

  • [1].Kimball SR, Jefferson LS, Nguyen HV, Suryawan A, Bush JA, Davis TA, Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process, Am J Physiol Endocrinol Metab. 279 (2000) E1080–1087. 10.1152/ajpendo.2000.279.5.E1080. [DOI] [PubMed] [Google Scholar]
  • [2].Kimball SR, Siegfried BA, Jefferson LS, Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase, J Biol Chem. 279 (2004) 54103–54109. 10.1074/jbc.M410755200. [DOI] [PubMed] [Google Scholar]
  • [3].Baum JI, Kimball SR, Jefferson LS, Glucagon acts in a dominant manner to repress insulin-induced mammalian target of rapamycin complex 1 signaling in perfused rat liver, Am J Physiol Endocrinol Metab. 297 (2009) E410–E415. 10.1152/ajpendo.00042.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Dennis MD, Baum JI, Kimball SR, Jefferson LS, Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids, J Biol Chem. 286 (2011) 8287–8296. 10.1074/jbc.M110.209171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Inoki K, Li Y, Xu T, Guan K-L, Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling, Genes Dev. 17 (2003) 1829–1834. 10.1101/gad.1110003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Gan X, Wang J, Su B, Wu D, Evidence for direct activation of mTORC2 kinase activity by phosphatidylinositol 3,4,5-trisphosphate, J Biol Chem. 286 (2011) 10998–11002. 10.1074/jbc.M110.195016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Potter CJ, Pedraza LG, Xu T, Akt regulates growth by directly phosphorylating Tsc2, Nat Cell Biol. 4 (2002) 658–665. 10.1038/ncb840. [DOI] [PubMed] [Google Scholar]
  • [8].Sato T, Nakashima A, Guo L, Tamanoi F, Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein, J Biol Chem. 284 (2009) 12783–12791. 10.1074/jbc.M809207200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM, Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids, Cell. 141 (2010) 290–303. 10.1016/j.cell.2010.02.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Root MA, Ellis J, Staub A, Effect of glucagon on insulin hypoglycemia, Proc Natl Acad Sci. 85 (1954) 507–511. 10.3181/00379727-85-20934. [DOI] [PubMed] [Google Scholar]
  • [11].Jiang G, Zhang BB, Glucagon and regulation of glucose metabolism, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E671–E678. 10.1152/ajpendo.00492.2002. [DOI] [PubMed] [Google Scholar]
  • [12].Ramnanan CJ, Edgerton DS, Kraft G, Cherrington AD, Physiologic action of glucagon on liver glucose metabolism, Diabetes Obes Metab. 13 Suppl 1 (2011) 118–125. 10.1111/j.1463-1326.2011.01454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Ahrén B, Larsson H, Impaired glucose tolerance (IGT) is associated with reduced insulin-induced suppression of glucagon concentrations, Diabetologia. 44 (2001) 1998–2003. 10.1007/s001250100003. [DOI] [PubMed] [Google Scholar]
  • [14].Jennings AS, Cherrington AD, Liljenquist JE, Keller U, Lacy WW, Chiasson JL, The roles of insulin and glucagon in the regulation of gluconeogenesis in the postabsorptive dog, Diabetes. 26 (1977) 847. 10.2337/diab.26.9.847. [DOI] [PubMed] [Google Scholar]
  • [15].Perry RJ, Zhang D, Guerra MT, Brill AL, Goedeke L, Nasiri AR, Rabin-Court A, Wang Y, Peng L, Dufour S, Zhang Y, Zhang X-M, Butrico GM, Toussaint K, Nozaki Y, Cline GW, Petersen KF, Nathanson MH, Ehrlich BE, Shulman GI, Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis, Nature. 579 (2020) 279–283. 10.1038/s41586-020-2074-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Ikeda T, Hoshino T, Honda M, Takeuchi T, Mokuda O, Tominaga M, Mashiba H, Effect of glucagon on glucose output from bivascularly perfused rat liver, Exp Clin Endocrinol Diabetes. 94 (2009) 383–386. 10.1055/s-0029-1210925. [DOI] [PubMed] [Google Scholar]
  • [17].Welles JE, Dennis MD, Jefferson LS, Kimball SR, Glucagon-dependent suppression of mTORC1 is associated with upregulation of hepatic FGF21 mRNA translation, Am J Physiol Endocrinol Metab. 319 (2020) E26–33. 10.1152/ajpendo.00555.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Mothe-Satney I, Gautier N, Hinault C, Lawrence JC, Van Obberghen E, In rat hepatocytes glucagon increases mammalian target of rapamycin phosphorylation on serine 2448 but antagonizes the phosphorylation of its downstream targets induced by insulin and amino acids, J Biol Chem. 279 (2004) 42628–42637. 10.1074/jbc.M405173200. [DOI] [PubMed] [Google Scholar]
  • [19].Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ, AMPK phosphorylation of raptor mediates a metabolic checkpoint, Mol Cell. 30 (2008) 214–226. 10.1016/j.molcel.2008.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Inoki K, Zhu T, Guan K-L, TSC2 mediates cellular energy response to control cell growth and survival, Cell. 115 (2003) 577–590. 10.1016/s0092-8674(03)00929–2. [DOI] [PubMed] [Google Scholar]
  • [21].Heppner KM, Habegger KM, Day J, Pfluger PT, Perez-Tilve D, Ward B, Gelfanov V, Woods SC, DiMarchi R, Tschöp M, Glucagon regulation of energy metabolism, Physiol Behav. 100 (2010) 545–548. 10.1016/j.physbeh.2010.03.019. [DOI] [PubMed] [Google Scholar]
  • [22].Kazda CM, Ding Y, Kelly RP, Garhyan P, Shi C, Lim CN, Fu H, Watson DE, Lewin AJ, Landschulz WH, Deeg MA, Moller DE, Hardy TA, Evaluation of efficacy and safety of the glucagon receptor antagonist LY2409021 in patients with type 2 Diabetes: 12-and 24-Week phase 2 studies, Diabetes Care. 39 (2016) 1241–1249. 10.2337/dc15-1643. [DOI] [PubMed] [Google Scholar]
  • [23].Kim T, Holleman CL, Nason S, Arble DM, Ottaway N, Chabenne J, Loyd C, Kim J-A, Sandoval D, Drucker DJ, DiMarchi R, Perez-Tilve D, Habegger KM, Hepatic glucagon receptor signaling enhances insulin-stimulated glucose disposal in rodents, Diabetes. 67 (2018) 2157–2166. 10.2337/db18-0068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Ang T, Bruce CR, Kowalski GM, Postprandial aminogenic insulin and glucagon secretion can stimulate glucose flux in humans, Diabetes. 68 (2019) 939. 10.2337/db18-1138. [DOI] [PubMed] [Google Scholar]
  • [25].Finan B, Capozzi ME, Campbell JE, Repositioning glucagon action in the physiology and pharmacology of diabetes, Diabetes. 69 (2020) 532–541. 10.2337/dbi19-0004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Yoshizawa F, Endo M, Ide H, Yagasaki K, Funabiki R, Translational regulation of protein synthesis in the liver and skeletal muscle of mice in response to refeeding, J Nutritional Biochem. 6 (1995) 130–136. 10.1016/0955-2863(95)00018-U. [DOI] [Google Scholar]
  • [27].Hayashi S, Ooshiro Z, Effects of glucagon, insulin, and the eel serum in the eel liver cells in primary culture, Nippon Suisan Gakkaishi. 51 (1985) 1123–1127. 10.2331/suisan.51.1123. [DOI] [Google Scholar]
  • [28].Schmidt EK, Clavarino G, Ceppi M, Pierre P, SUnSET, a nonradioactive method to monitor protein synthesis., Nat Methods. 6 (2009) 275–277. 10.1038/nmeth.1314. [DOI] [PubMed] [Google Scholar]
  • [29].Miller WP, Sunilkumar S, Giordano JF, Toro AL, Barber AJ, Dennis MD, The stress response protein REDD1 promotes diabetes-induced oxidative stress in the retina by Keap1-independent Nrf2 degradation., J Biol Chem. 295 (2020) 7350–7361. 10.1074/jbc.RA120.013093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Sutherland EW, Studies on the mechanism of hormone action, Science. 177 (1972) 401. 10.1126/science.177.4047.401. [DOI] [PubMed] [Google Scholar]
  • [31].Walsh DA, Perkins JP, Krebs EG, An adenosine 3’,5’-monophosphate-dependant protein kinase from rabbit skeletal muscle, J Biol Chem. 243 (1968) 3763–3765. [PubMed] [Google Scholar]
  • [32].de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL, Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP, Nature. 396 (1998) 474–477. 10.1038/24884. [DOI] [PubMed] [Google Scholar]
  • [33].Wang X, Proud CG, The mTOR pathway in the control of protein synthesis, Physiology. 21 (2006) 362–369. 10.1152/physiol.00024.2006. [DOI] [PubMed] [Google Scholar]
  • [34].Dao VT, Dupuy AG, Gavet O, Caron E, de Gunzburg J, Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis, J. Cell Sci. 122 (2009) 2996. 10.1242/jcs.041301. [DOI] [PubMed] [Google Scholar]
  • [35].Misra UK, Pizzo SV, Epac1-induced cellular proliferation in prostate cancer cells is mediated by B-Raf/ERK and mTOR signaling cascades, J Cell Biochem. 108 (2009) 998–1011. 10.1002/jcb.22333. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • [36].Xenophontos XP, Watson PA, Chua BH, Haneda T, Morgan HE, Increased cyclic AMP content accelerates protein synthesis in rat heart., Circ Res. 65 (1989) 647–656. 10.1161/01.res.65.3.647. [DOI] [PubMed] [Google Scholar]
  • [37].Barovsky K, Pedone C, Brooker G, Forskolin-stimulated cyclic AMP accumulation mediates protein synthesis-dependent refractoriness in C6–2B rat glioma cells., J Cyclic Nucleotide Protein Phosphor Res. 9 (1983) 181–189. [PubMed] [Google Scholar]
  • [38].Park SW, Mansur RB, Lee Y, Lee J-H, Seo MK, Choi AJ, McIntyre RS, Lee JG, Liraglutide activates mTORC1 signaling and AMPA receptors in rat hippocampal neurons under toxic conditions, Front Neurosci. 12 (2018) 756. 10.3389/fnins.2018.00756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Mangmool S, Hemplueksa P, Parichatikanond W, Chattipakorn N, Epac is required for GLP-1R-mediated inhibition of oxidative stress and apoptosis in cardiomyocytes, Mol Endocrinol. 29 (2015) 583–596. 10.1210/me.2014-1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Dunphy JL, Taylor RG, Fuller PJ, Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression, Mol and Cell Endocrinol. 141 (1998) 179–186. 10.1016/S0303-7207(98)00096–3. [DOI] [PubMed] [Google Scholar]
  • [41].Pyke C, Heller RS, Kirk RK, Ørskov C, Reedtz-Runge S, Kaastrup P, Hvelplund A, Bardram L, Calatayud D, Knudsen LB, GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody, Endocrinology. 155 (2014) 1280–1290. 10.1210/en.2013-1934. [DOI] [PubMed] [Google Scholar]
  • [42].Roscioni SS, Elzinga CRS, Schmidt M, Epac: effectors and biological functions, Naunyn-Schmiedeberg’s Arch. Pharmacol. 377 (2008) 345–357. 10.1007/s00210-007-0246-7. [DOI] [PubMed] [Google Scholar]
  • [43].Khanna A, Lotfi P, Chavan AJ, Montaño NM, Bolourani P, Weeks G, Shen Z, Briggs SP, Pots H, Van Haastert PJM, Kortholt A, Charest PG, The small GTPases Ras and Rap1 bind to and control TORC2 activity, Sci Rep. 6 (2016) 25823. 10.1038/srep25823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Albert V, Svensson K, Shimobayashi M, Colombi M, Muñoz S, Jimenez V, Handschin C, Bosch F, Hall MN, mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue, EMBO Mol Med. 8 (2016) 232–246. 10.15252/emmm.201505610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Misra UK, Pizzo SV, Upregulation of mTORC2 activation by the selective agonist of EPAC, 8-CPT-2Me-cAMP, in prostate cancer cells: Assembly of a multiprotein signaling complex, J Biol Chem. 113 (2012) 1488–1500. 10.1002/jcb.24018. [DOI] [PubMed] [Google Scholar]
  • [46].Nijholt IM, Dolga AM, Ostroveanu A, Luiten PGM, Schmidt M, Eisel ULM, Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation, Cellular Signalling. 20 (2008) 1715–1724. 10.1016/j.cellsig.2008.05.001. [DOI] [PubMed] [Google Scholar]
  • [47].Kim HW, Ha SH, Lee MN, Huston E, Kim D-H, Jang SK, Suh P-G, Houslay MD, Ryu SH, Cyclic AMP Controls mTOR through regulation of the dynamic interaction between Rheb and phosphodiesterase 4D, Mol. Cell. Biol. 30 (2010) 5406. 10.1128/MCB.00217-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [48].Xie J, Ponuwei GA, Moore CE, Willars GB, Tee AR, Herbert TP, cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity, Cellular Signalling. 23 (2011) 1927–1935. 10.1016/j.cellsig.2011.06.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Jewell JL, Fu V, Hong AW, Yu F-X, Meng D, Melick CH, Wang H, Lam W-LM, Yuan H-X, Taylor SS, Guan K-L, GPCR signaling inhibits mTORC1 via PKA phosphorylation of Raptor, Elife. 8 (2019) e43038. 10.7554/eLife.43038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Mei FC, Qiao J, Tsygankova OM, Meinkoth JL, Quilliam LA, Cheng X, Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation, J Biol Chem. 277 (2002) 11497–11504. 10.1074/jbc.M110856200. [DOI] [PubMed] [Google Scholar]
  • [51].Davies SP, Reddy H, Caivano M, Cohen P, Specificity and mechanism of action of some commonly used protein kinase inhibitors, Biochem J. 351 (2000) 95–105. 10.1042/0264-6021:3510095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Lochner A, Moolman JA, The many faces of H89: a review, Cardiovasc Drug Rev. 24 (2006) 261–274. 10.1111/j.1527-3466.2006.00261.x. [DOI] [PubMed] [Google Scholar]
  • [53].Kaur S, Chen Y, Shenoy SK, Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling, J Biol Chem. 295 (2020) 16630–16642. 10.1074/jbc.RA120.014532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Welles JE, Toro AL, Sunilkumar S, Stevens SA, Purnell CJ, Kimball SR, Dennis MD, Retinol binding protein 4 (Rbp4) mRNA translation in hepatocytes is enhanced by activation of mTORC1, Am J Physiol Endocrinol Metab. (2020). 10.1152/ajpendo.00494.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1696861-supplement-1.pdf (395KB, pdf)

RESOURCES