Abstract

   The peptide hormone glucagon is a fundamental metabolic regulator that
   is also being considered as a pharmacotherapeutic option for obesity
   and type 2 diabetes. Despite this, we know very little regarding how
   glucagon exerts its pleiotropic metabolic actions. Given that the liver
   is a chief site of action, we performed in situ time-resolved liver
   phosphoproteomics to reveal glucagon signaling nodes. Through pathway
   analysis of the thousands of phosphopeptides identified, we reveal
   “membrane trafficking” as a dominant signature with the vesicle
   trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being
   a top hit. Hepatocyte-specific loss- and gain-of-function experiments
   reveal that SEC22B was a key regulator of glycogen, lipid and amino
   acid metabolism, with SEC22B-S137 phosphorylation playing a major role
   in glucagon action. Mechanistically, we identify several protein
   binding partners of SEC22B affected by glucagon, some of which were
   differentially enriched with SEC22B-S137 phosphorylation. In summary,
   we demonstrate that phosphorylation of SEC22B is a hepatocellular
   signaling node mediating the metabolic actions of glucagon and provide
   a rich resource for future investigations on the biology of glucagon
   action.

   Subject terms: Hormones, Metabolism, Cell signalling, Membrane
   trafficking
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   Glucagon is hormone that signals via a dedicated g-protein coupled
   receptor, but downstream signaling is poorly understood. Here, Wu et
   al. uncover liver glucagon signaling using phosphoproteomics and define
   a role for the vesicle trafficking protein SEC22B in distinct metabolic
   actions.

Introduction

   Despite glucagon being discovered over 100 years ago^[70]1, and
   recognized as a key factor in the etiology of type 2
   diabetes^[71]2–[72]4, surprisingly, very little is known about how
   glucagon signaling works within its major target tissue, the liver, to
   induce its pleiotropic effects on metabolism and beyond^[73]4,[74]5.
   Glucagon is a peptide hormone mainly secreted from the alpha cells of
   pancreatic islets^[75]5, and was first identified as a hyperglycemic
   factor; it targets the liver to increase blood glucose by stimulating
   gluconeogenesis and glycogenolysis^[76]3,[77]5. Therefore, glucagon is
   known as a counter-regulatory hormone to insulin, and the balance
   between glucagon and insulin signaling is crucial for maintaining
   physiological euglycemia^[78]3,[79]5, although glucagon also has clear
   actions in the postprandial state^[80]6,[81]7. Glucagon levels are
   elevated in patients with type 2 diabetes, indicating that failure of
   glucose to suppress glucagon action plays a causal role in the
   pathology of type 2 diabetes^[82]3. However, glucagon is a pleiotropic
   hormone with multiple metabolic actions beyond glucose metabolism and
   these non-glycemic effects of glucagon include the modulation of food
   intake and satiety, amino acid and lipid homeostasis, insulin
   secretion, and energy expenditure^[83]2,[84]5. Aside from this, and
   spurred on by promising pre-clinical studies^[85]8–[86]11, peptides
   with glucagon receptor agonist activity are in development for treating
   obesity and type 2 diabetes in humans^[87]12,[88]13. Thus, it is
   paramount that we understand the mechanisms of action of glucagon, both
   from a basic- and medical-biology perspective.

   Glucagon action is mediated by the glucagon receptor (GCGR), a member
   of the family of class B G-protein coupled receptors that are highly
   conserved across mammalian species^[89]4. The binding of glucagon to
   the GCGR activates adenylyl cyclase through the Gs subtype G-protein,
   generating cellular adenosine-3’−5’-cyclic monophosphate (cAMP) and
   activating protein kinase A (PKA) as the major mode of intracellular
   signaling^[90]4. Aside from PKA and a few modes of action on glucose
   and amino acid metabolism, there is a dearth of knowledge as to which
   cellular mechanisms that post-receptor glucagon signaling engages to
   exert its multitude of effects^[91]4,[92]14. As protein phosphorylation
   is a rapid, potent, and dynamic means of manipulating intracellular
   protein action^[93]15, particularly affecting metabolism^[94]16, we
   sought to partially illuminate the ‘black box’ of glucagon signaling
   using phospho-proteomics.

   Since the liver is the chief site of glucagon action^[95]17, by taking
   advantage of a combination of key technologies including the perfused
   rat liver model, proteomics, and molecular manipulation using
   adeno-associated viruses, we have uncovered multiple glucagon-regulated
   proteins and show that a glucagon-regulated phosphoprotein, the
   vesicle-trafficking protein SEC22B, is a key intracellular signaling
   node that governs distinct metabolic actions of glucagon.

Results

Phosphoproteomics reveals glucagon signaling nodes

   To uncover aspects of glucagon signaling, we employed a perfused rat
   liver model to resolve the time-course of glucagon signaling. This in
   situ model has the advantages of a continuous supply of a fixed
   glucagon concentration via the natural anatomical route in fully
   differentiated liver, and allows a distinct assessment of glucagon
   action. To qualify the model, we assessed liver glycogen concentration,
   which as expected^[96]18, decreased in a time-dependent manner (Fig.
   [97]S1a). We also assessed protein kinase A activation, a canonical
   GPCR-Gs signaling node^[98]4. Indeed, we could see rapid and sustained
   PKA activation with glucagon as judged by higher levels of
   phosphorylation of liver proteins at a classic PKA motif (Fig. [99]1a;
   Fig. [100]S1b). We therefore proceeded and assessed the phosphoproteome
   from these samples at each time point (2, 8 and 32 min; Supplementary
   Data [101]1). A total of 11,234 unique phosphopeptides were identified
   across all time points and using stringent filter criteria, we managed
   to quantify 8,996 phosphopeptides. Interestingly, we did not detect
   CREB-S133, which is considered a classic glucagon-regulated
   phosphoprotein^[102]14, and we validated a lack of change in this
   phosphoprotein by western blot (Fig. S1c-d). This further strengthened
   the case for a more global approach to uncovering glucagon signaling
   nodes.

Fig. 1. Glucagon induces rapid and dynamic changes in the liver
phosphoproteome.

   [103]Fig. 1
   [104]Open in a new tab

   a Male Sprague Dawley rats were treated with vehicle (V) or glucagon
   (G) (1.15 nM) for 2, 8 and 32 min (n = 4 rats per group) in situ.
   Western blot images of liver phospho-protein kinase A (pPKA) motif
   protein substrates and control vinculin (VCL) were performed. b
   Principal component analysis (PCA) analysis via the Phospho-Analyst
   platform from liver samples (using pooled VEH and GCG data sets, n = 11
   for VEH and n = 12 for GCG). c VENN diagram of liver phosphoproteomics
   data from samples as in b (n = 4 rats per group except VEH, 32 min:
   n = 3). d Heatmap plot of up-regulated (log2 fold change > 4) and
   down-regulated (log2 fold change < −3) phosphopeptides across all time
   points from samples as in a (n = 4 rats per group except VEH, 32 min:
   n = 3). The red arrow indicates SEC22B-S137. Source data are provided
   as a Source Data file. e Pathway enrichment analysis using EnrichR
   (Reactome Pathway Database) via the Phospho-Analyst platform (using
   pooled VEH and GCG data sets, n = 11 for VEH and n = 12 for GCG).
   EnrichR using Fisher’s exact test and corrected with Benjamini–Hochberg
   multiple testing. The red arrow indicates the pathway in which SEC22B
   is involved. f Bubble plot of predicated kinase activation from
   phosphoproteomics data. Statistical significance was assessed using a
   one-sided Fisher’s exact test, with p-values adjusted by the
   Benjamini–Hochberg method. Detailed statistical analysis is provided in
   the “Phosphoproteomics-Based Kinase Prediction” section of the Methods.

   The principal component analysis (PCA) showed a clear divergent
   distribution in phosphoproteomes between glucagon-treated and control
   samples (Fig. [105]1b). Comparing only glucagon-treated to control
   samples within each time point, 1,029 phosphosites were observed to be
   regulated by glucagon with an overlap of 108 phosphopeptides across all
   time points (Fig. [106]1c; considering an adjusted p-value cutoff of
   0.05 and a log2 fold change cutoff of 1). Comparing glucagon (GCG) vs.
   vehicle (VEH) sample data, the heatmap plot (Fig. [107]1d) showed some
   representative overlapped phosphopeptides (using more stringent
   criteria, log2 fold change >4 or <−3), which indicates the
   glucagon-induced phosphorylation is rapid and sustained. The pathway
   enrichment of pooled GCG vs VEH sample data revealed that “membrane
   trafficking” and “vesicle-mediated transport” pathways are the dominant
   signatures, both in which SEC22B was found (Fig. [108]1e). Using the
   entire data set, we could predict kinase activation based on motif
   preferences^[109]19, and certain kinase classes such as PKA, AKT, PKG,