N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway*

Background: The hexosamine biosynthesis pathway to UDP-GlcNAc has been implicated in glucose homeostasis. Results: UDP-GlcNAc and Golgi N-acetylglucosaminyltransferases modify the N-glycans on glucagon receptor, which increases sensitivity to glucagon in vivo. Conclusion: The hexosamine biosynthesis pathway contributes to glucose homeostasis, in part through N-glycan branching on glucagon receptor. Significance: Hepatic Mgat5 and the N-glycan branching pathway may be a therapeutic target for control of glycemia.

Next Section Abstract Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5−/− mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5−/− cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5−/− hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor.

Previous Section Next Section Introduction The major biological function of the pancreatic hormone glucagon is to counteract the glucose-lowering action of insulin and maintain blood glucose concentration during fasting (1, 2). Indeed, the severe hyperglycemia and glucose intolerance that characterizes insulin-deficient mice is reversed by a deficiency in glucagon receptor (Gcgr), although the mice are more sensitive to prolonged fasting (3–6). These experiments appear to unmask more ancient levels of feedback regulation that underpin insulin and glucagon signaling. In Saccharomyces cerevisiae, glucose perception and import are separate modules that interact through post-translational modifications signaling to regulate metabolism and growth rates (7, 8). Kinases and phosphatases mediate rapid allosteric regulation of enzymes, whereas protein N-glycosylation adapts cell surface receptors and transporters to extracellular conditions (9). Biosynthesis of high energy post-translational modification substrates such as acetyl-CoA (10), and UDP-GlcNAc are key to nutrient sensing (11–15). Fructose-6P, glutamine, and acetyl-CoA are pivotal substrates in multiple pathways including the hexosamine biosynthesis pathway (HBP)3 to UDP-GlcNAc (9, 16). Both O-GlcNAcylation of transcription factors (17–19) and N-glycosylation of membrane proteins have been identified as effectors of UDP-GlcNAc in glucose homeostasis (12, 20). However, these effector pathways remain poorly understood (21). Membrane receptors and solute transporters are cotranslationally modified in rough endoplasmic reticulum (ER) by oligosaccharyltransferase, which transfers the N-glycan from Glc 3 Man 9 GlcNAc 2 -pp-dolichol to Asn at NX(S/T) (where X ≠ P) sites. N-Glycosylation is found in all domains of life (22, 23) and has an ancient function that promotes protein folding in the endoplasmic reticulum. Protein synthesis and chaperone-assisted homeostasis consumes a large portion of cellular resources, and under stress conditions, reduced biosynthesis of Glc 3 Man 9 GlcNAc 2 -pp-dolichol activates the unfolded protein response, thus a metabolic mechanism of stress tolerance (24). With metazoan evolution, the ER N-glycan modification became a platform for remodeling in the Golgi and additional functionality at the cell surface. The N-glycans are trimmed and rebuilt beginning with the branching N-acetyl-glucosaminyltransferases, encoded by the Mgat-1, Mgat-2, Mgat-4a/b/c, and Mgat-5 genes (25) (see Fig. 1A). The branching enzymes form a linear pathway and require the donor substrate UDP-GlcNAc. The GlcNAc branches are extended with galactose, poly-N-acetyllactosamine, fucose, and sialic acid, generating sequences recognized by galectins, C-type lectins, and siglecs at the cell surface. Galectin binding to Galβ1–4GlcNAcβ, a common sequence of N-glycan branches of membrane proteins, forms multivalent complexes, which results in a highly dynamic and heterogeneous lattice at the cell surface. Strong selective pressures on Golgi remodeling and its substrates during vertebrate evolution (26) have resulted in a layer of HBP-sensitive regulation for membrane receptors and solute transporters (12). Glucose transporters (Glut) are dependent on N-glycan branching as reported for Glut2 in β-cells (20), Glut1 in tumor cells (27), and Glut4 (12, 28). The Mgat4a−/− mice display suppressed secretion of insulin in response to glucose caused by a failure to retain Glut2 at the surface of β-cells (20). The Mgat4a-branched N-glycan on Glut2 binds to galectin-9, which slows mobility at the cell surface and loss to endocytosis, thus increasing the transport of glucose, and thereby insulin secretion. Although Mgat4a−/− mice display hypoinsulinemia and excess weight gain on a high fat diet, Mgat5−/− mice are resistant to weight gain and display hyperglucagonemia (29), suggesting either a gain of function in glucagon secretion and/or a loss of function in hepatic Gcgr. Mice lacking Gcgr display resistance to obesity on a high fat diet with elevated circulating glucagon (30, 31). Receptor deficiencies often result inelevated levels of the cognate ligand caused by feedback up-regulation of ligand production or secretion and/or failure to internalize and clear the ligand in the absence of receptor. Therefore, we hypothesized that the Gcgr may be regulated by Mgat5-dependent N-glycan branching in an HBP-dependent manner. The Gcgr is a member of the class B G protein-coupled receptor superfamily (32), and activation results in the replacement of GDP with GTP bound to the G s α subunit of the α/β/γ heterotrimeric complex. The GTP-bound G s α activates adenylyl cyclase, and cAMP stimulates PKA, glycogen phosphorylase kinase, and glycogen phosphorylase leading to hepatic glycogenolysis and gluconeogenesis (33). Inhibition of glucagon action attenuates hyperglycemia in experimental models and in subjects with type 2 diabetes (34). Here we report that Mgat5−/− mice and primary hepatocytes are hyposensitive to glucagon. The Mgat5-branched N-glycans on Gcgr increases receptor binding to galectin-9, which slows mobility and increases responsiveness to glucagon. GlcNAc supplementation increases UDP-GlcNAc flux to N-glycan branching in primary hepatocytes, and GlcNAc supplementation to Mgat5−/− mice restored the glycemic response to glucagon. Our findings reveal a role for HBP and N-glycan branching on Gcgr as a positive regulator of glucagon responsiveness.

Previous Section Next Section EXPERIMENTAL PROCEDURES Mice Age- and sex-matched littermates on the C57BL/6 background were used in all the experiments. The Mgat5−/− mutation was described previously (35) and made isogenic on C57BL6 by 15 generations of backcrosses. The Mgat5−/− hypoglycemia phenotype has been validated on both 129/sv and C56BL6 backgrounds (29). Mice were maintained on a standard rodent chow (Teklad rodent diet, 18% protein, 6% fat, #2018) with a normal 12-h light/12-h dark cycle. In some experiments, mice were on low fat diet (Teklad rodent diet, 19% protein, 4% fat, #8604) or high fat diet (Teklad rodent diet, 19% protein, 9.8% fat, #2019) with or without GlcNAc (0.5 mg/ml) in the drinking water as indicated. Experiments were done according to protocols and guidelines approved by the Toronto Centre for Phenogenomics animal care committee. Glucose Tolerance, Glucagon Challenge, or Insulin Tolerance Test For intraperitoneal glucose tolerance tests (IPGTTs), mice were fasted for 16 h before intraperitoneal injection of 0.01 ml/g of body weight of a glucose solution containing 150 mg/ml. For oral glucose tolerance test (OGTT), a glucose solution was administered by oral gavage. Blood samples were drawn via the tail vain, and glucose was measured using a Glucometer Elite blood glucose meter (Bayer, Toronto, Canada). For the glucagon challenge (GC) test or insulin tolerance test (ITT), mice were fasted for 5 h and injected intraperitoneally with a glucagon solution of 1.6 μg/ml (0.01 ml/g body of weight) (16 μg/kg) or 0.75 units/kg of human insulin, respectively. Plasma glucagon and insulin were measured using a mouse endocrine LINCOplex kit (Linco Research) following the manufacturer's protocol. To measure the glycogen content in liver, 20–50 mg of tissue was acid-hydrolyzed in 2 m HCl at 95 °C for 2 h and neutralized using 2 m NaOH. The liberated glucose was assayed spectrophotometrically using the glucose reagent (hexokinase method) (Amresco, Solon, OH) following the manufacturer's protocol. Primary Hepatocytes and Glucose Secretion Murine hepatocytes were isolated as previously described (36), seeded in 6-well plates at 4 × 105 cells/well in Williams E medium containing 5% FBS and 0.7 mm insulin for 3 h to allow attachment. The cells were washed with PBS and DMEM without glucose and phenol red. Cells were stimulated with 20 nm of glucagon, and medium was collected after 30 min, 1 h, 3 h, and 6 h and stored at −20 °C. To determine the residual glucose concentration in the cells, 2 n ice-cold HCl was added to cells, and lysates were incubated for 2 h at 95 °C followed by neutralization using 2 n NaOH. Glucose concentration in media and lysates were measured using the glucose reagent (hexokinase method) (Amresco, Solo, OH). LC-MS/MS Analysis of Metabolites Frozen liver tissue (80–100 mg) was crushed using the CellCrusherTM cryogenic tissue pulverizer under liquid nitrogen, and 1 ml of ice-cold solution of (40% acetonitrile, 40% methanol, and 20% water) was added for metabolite extraction. For cells grown in cell culture plates, metabolites were extracted by adding 1 ml of ice-cold extraction solution (40% acetonitrile, 40% methanol, and 20% water) to the plate, scraping the cells and collecting in 1.5-ml vials (16). Samples were separated twice on a reversed phase HPLC column Inertsil ODS-3 of 4.6-mm internal diameter, 150-mm length, and 3 μm particle size (Dionex Corporation, Sunnyvale, CA) for MS analysis in positive and negative modes. The eluted metabolites were analyzed at the optimum polarity in MRM mode on electrospray ionization triple-quadrupole mass spectrometer (4000 QTRAP; ABSciex, Toronto, Canada) as previously described (16). Glucagon Signaling Primary hepatocytes seeded on 24-well plates at a density of 1 × 105cells/ml were cultured in Williams E medium without FBS for 16 h, then supplemented with 100 μmol/liter isomethyl butyl xanthine containing 0, 0.01, 0.1, 1.0, 10, 100, or 1000 nm glucagon, and incubated for 10 min at 37 °C. The reaction was stopped with ice-cold ethanol, and cAMP was measured by radioimmunoassay kit or targeted mass spectrometry using electrospray ionization triple-quadrupole ABSciex 4000 QTRAP (LC-MS/MS). HEK293 Flp-In-TREx cells were purchased from Invitrogen and maintained in DMEM (Sigma) supplemented with 10% FBS, 2 mm Gln, penicillin/streptomycin, 3 μg/ml blastocidin, and 100 μg/ml Zeocin. Human Mgat5 cDNA was FLAG-tagged at the N terminus and cloned into the pcDNA5/FRT/TO expression vector. The plasmid was integrated into the genome at a preintegrated FRT recombination site, by cotransfection with Flp recombinase encoding POG44 plasmid, using Lipofectamine (Invitrogen) and OptiMEM medium lacking FBS or antibiotics. Following selection in 200 μg/ml of hygromycin, clones displayed 5–10-fold increase in Mgat5 enzyme activity when induced by 1 μg/ml tetracycline for 24 h. For Gcgr signaling in HEK293 Flp-In-TREx cells were transfected with 1 μg of Gcgr plasmid DNA using Lipofectamine 2000 reagent (Invitrogen). The next day cells were incubated with medium containing 1 μg/ml tetracycline and GlcNAc as indicated for 24 h. Cells were stimulated with glucagon as described, and cAMP was measured by targeted LC/MS-MS. Membrane Preparation and Competitive Glucagon Binding Assay Plasma membranes were prepared from primary hepatocytes using a cell surface isolation kit (Pierce) following the manufacturer's protocol. Plasma membrane preparation were analyzed via Western blot, and the ImageJ software was used to quantify signal intensity (37). For liver membrane preparations, ∼2 g of liver tissue were homogenized in 15 ml of 0.32 m sucrose solution at 4 °C using a Teflon tissue grind tube SZ23 (Kontess Class Co., Vinland, NJ). Homogenates were centrifuged at 4 °C for 10 min at 600 × g. The supernatant was transferred to an ultra-clear ultracentrifuge tube (25 × 89 mm; Beckmann Coulter, Inc., Brea, CA) and underlayed with 15 ml of a 41% sucrose solution, followed by centrifugation at 100,000 × g for 17 h at 4 °C. Membranes were collected at the interface and washed twice with 15 ml of 50 mm Tris/HCl buffer, pH 7.5, and protein concentration was measured using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). 125I-Labeled glucagon (50,000 cpm; PerkinElmer Life Sciences) was diluted in 100 μl of buffer. 40 μg of membrane preparation was dissolved in 200 μl buffer (25 mm HEPES, 2.5 mm CaCl 2 , 1.0 mm MgSO 4 , 0.05% bacitracin, 2% BSA, 0.003% Tween 20). Tracer, membrane preparation, and 200 μl of glucagon solution at a final concentration from 10−12 to 10−6 m were incubated for 1 h at room temperature. To measure nonspecific binding, 1 mm nonlabeled glucagon was added to control samples. Binding reaction was stopped with 3 ml of buffer and immediately filtered through glass microfiber filters (Whatman GF/B). Filters were washed twice, and radioactivity was measured in a γ-scintillation counter. Experiments were carried out in duplicate with four independent liver membrane preparations. Insulin binding was measured with 125I-labeled insulin (50,000 cpm; PerkinElmer Life Sciences) in 100 μl of buffer incubated with 40 μg of membrane preparation (200 μl). Expression Vectors Mouse Gcgr (NCBI clone NM_008101.2) was subcloned via EcoRI and HindIII restriction sites into a pcDNA3 (−) vector (Invitrogen). An internal FLAG tag (DYKDDDK) followed by a linker sequence containing four glycine residues were inserted at the N terminus of the Gcgr after the putative cleavage signal site at amino acid 27 using a double-joint PCR strategy (38). To generate a construct with an internal GFP tag or FLAG tag, the same double-joint PCR strategy was applied. GFP-Gcgr was subcloned into pEGFP-C1 vector with restriction enzymes AgeI and EcoRI. The glycosylation site mutants of Gcgr (i.e. N47Q, N60Q, N75Q, N79Q, and N118Q) were generated using site-directed mutagenesis. Mouse galectin-9 coding sequence (NCBI clone NM_001159301.1) was subcloned via Xho and EcoRI into pERFP-C1 vector (Clontech). Western Blotting and Immunoprecipitation Endogenous Gcgr was detected with ST-18 antibody (39). For chemical cross-linking to detect cell surface galectin and Gcgr, hepatocytes transfected with RFP-galectin-9 overnight were treated with 0.1 mg/ml 3,3′-dithiobis (sulfosuccinimidylpropionate) for 15 min at room temperature. Complexes were pulled down with rabbit anti-RFP antibody and protein G-Sepharose beads (GE Healthcare). For detection of FLAG-Gcgr, the anti-FLAG antibody M2 (Sigma-Aldrich) was used. For leucoagglutinin (L-PHA) binding, cells in 96-well plates were fixed for 15 min with 4% paraformaldehyde, washed with PBS, and incubated for 1 h at 20 °C in 50 μl of PBS containing 1/5,000 of Hoechst 33342 and 2 μg/ml Alexa Fluor 488-conjugated L-PHA (Invitrogen). After washing with PBS, cell staining was quantified by IN Cell Analyzer 1000 automated fluorescence imaging. Fluorescence Recovery after Photobleaching (FRAP) Analyses Primary hepatocytes were seeded on 35-mm glass-bottomed culture dishes and transfected with 1 μg of GFP-Gcgr and/or RFP-galectin-9 using Lipofectamine 2000 reagent. The next morning FRAP analyses was performed at room temperature on a confocal FV1000 Olympus microscope, with a 405-nm laser at full power in a circular region of interest with 50-pixel diameter. Cells were incubated with 30 mm lactose for 4 h prior to the FRAP experiment or overnight with 100 μm castanospermine or 20 mm GlcNAc supplementation in Williams E medium. Fluorescence during recovery was normalized to the pre-bleach intensity, and the data were averaged for a minimum of four to five animals in independent experiments in which 6–10 cells were bleached. Statistical Analyses Statistical significance was assessed by one-way or two-way analysis of variance using Bonferroni's multiple comparison post-test and, where appropriate, by Student's t test using GraphPad Prism 5 (GraphPad Software, San Diego, CA). A p value of <0.05 was considered to be statistically significant.

Previous Section Next Section DISCUSSION In this report, we tested the hypothesis that Gcgr sensitivity is dependent on modification by Mgat5 and more broadly, the N-glycan branching pathway. We demonstrate that Mgat5−/− mice are hypoglycemic, display improved glucose tolerance, and display decreased sensitivity to glucagon. HBP and glycolytic metabolites were decreased, whereas glycogen storage and free amino acids were increased in Mgat5−/−livers, a regulatory imbalance similar to that reported for Gcgr−/− mice (42). The Mgat5−/− mice displayed a remarkably similar glucoregulatory phenotype to mice deficient in either glucagon processing or Gcgr signaling (3, 30, 31). Furthermore, experiments with primary hepatocytes confirmed that glucagon-dependent cAMP signaling is impaired, and gluconeogenesis is reduced by the Mgat5 deficiency. Reduced hepatic catabolism of amino acids and glycogen may also contribute to lower glucose production and systemic hypoglycemia. The EC 50 for glucagon-dependent activation of adenylyl cyclase was increased ∼4-fold in Mgat5−/− primary cells. Plasma membrane levels of Gcgr were similar, but glucagon binding sites were reduced by 18%, and affinity was reduced ∼10-fold in Mgat5−/− membrane preparations. GlcNAc supplementation and Mgat5 and N-glycan branching were shown to promote Gcgr association with galectin-9, which slows receptor mobility and enhances sensitivity to glucagon in primary hepatocytes. Finally, GlcNAc supplementation in the drinking water rescued glucagon sensitivity in Mgat5−/− mice, concordant with the GlcNAc rescue of Gcgr signaling and dynamics in cultured primary hepatocytes. Importantly, GlcNAc supplementation did not alter glucose tolerance in either wild type or mutant mice, suggesting that insulin action was not impaired by GlcNAc. The results suggest a model for metabolic feedback through HBP and N-glycan branching in the regulation of Gcgr (Fig. 11). View larger version: Download as PowerPoint Slide FIGURE 11. A model of metabolic feedback via HBP and N-glycan branching. Steps 1 and 2, in the fed state, hepatic glucose transport into the cell (step 1) and flux to HBP (step 2) increase intracellular UDP-GlcNAc supply to the medial Golgi. Step 3, UDP-GlcNAc is a rate-limiting substrate for the Mgat enzymes. Step 4, N-glycan branching on Gcgr and other glycoproteins increases (left to right). Step 5, equilibrium shifts in a cyclical manner with feeding and fasting. Mgat5 N-glycan branching enhances galectin-9 binding and sensitivity to glucagon. The galectin lattice may promote Gcgr receptor dimerization and/or recruit coreceptors. Increasing Gcgr sensitivity during feeding may be an adaptation that precedes and anticipates fasting in the normal feeding/fasting cycles. Step 6, increasing Gcgr sensitivity may contribute to glucagon signaling in the hyperglycemic state of diabetes. Step 7, high protein diet or salvage of amino acids during starvation may support UDP-GlcNAc concentrations and Gcgr sensitivity. Experimental deletion of the N-glycosylation sites in Gcgr, GIP, and GLP-1 receptors blocks protein expression at the cell surface (50, 51), which is likely due to receptor instability or misfolding. The removal of NX(S/T) (where X ≠ P) sites is a blunt instrument that precludes the analysis of Golgi N-glycan remodeling pathways. The present study is the first to reveal that N-glycan remodeling is an effector downstream of HBP that regulates responsiveness of a critical receptor in glucose homeostasis. Individual sites in Gcgr were not critical, because mutation of each site did not disrupt receptor activities measured in HEK293 cells. This suggests that functionality of N-glycan branching may be cumulative and distributed over the five sites in Gcgr. We show that galectin-9 binds to Gcgr and slows receptor mobility, with a dependence on Mgat5 activity and UDP-GlcNAc supply to the N-glycan branching pathway. Galectin-9 binding promotes cross-linking of glycoproteins and may facilitate Gcgr dimerization (52) or association with other glycoproteins. Dimerization of the GLP-1R promotes coupling with G protein-coupled receptors and sensitivity to ligand (53). All nine mammalian adenylyl cyclases share conserved N-glycosylation sites in extracellular loops 5 and 6 (54). N-Glycosylation of adenylyl cyclase 8 is required to target the enzyme into lipid raft domains (55), and Golgi modifications may play a role in efficient coupling of this adenylyl cyclase with Gcgr. RAMP-2 (receptor activity-modifying protein 2) is another transmembrane glycoprotein shown to be associated with Gcgr (56). It is likely that galectin-9 slows down not only the mobility of Gcgr but also other glycoproteins and regulates signaling efficiency. Thus galectin-9 binding to branched N-glycan on Gcgr may slow mobility and thereby enhance interactions with other regulatory glycoproteins. It is also possible that branched N-glycans interact within the receptor fold to enhance dimerization and/or affinity for ligand (57). In the absence of Mgat5, compensating amounts of N-acetyllactosamine (Galβ1–4GlcNAcβ) branches can be made by Mgat1, Mgat2, and Mgat4 when supplied with GlcNAc (12, 58), which is converted into UDP-GlcNAc. Indeed, glucagon sensitivity in Mgat5−/− mice was rescued by GlcNAc supplied in the drinking water. GlcNAc supplementation at 0.5 mg/ml increased liver HBP metabolites in Mgat5−/− mice and rescued Gcgr membrane dynamics, as well as cAMP signaling in cultured primary hepatocytes. The interaction between Mgat5, UDP-GlcNAc, Gcgr, and cAMP signaling was engineered into HEK293 cells and found to be very similar to primary hepatocytes. Gcgr sensitivity to glucagon was highest when Mgat5 was induced in the presence of GlcNAc supplementation. Mgat5 has a low affinity for UDP-GlcNAc (K m = ∼10 mm) relative to Mgat1, Mgat2, and Mgat4. Therefore, Mgat5 activity is highly dependent on UDP-GlcNAc concentration and enzyme levels. Conversely, expression of Mgat5 is relatively low in liver compared with intestine and brain (59), suggesting that regulation of branching may be highly dependent on HBP and central metabolites. Mgat5 gene expression is stimulated by hepatic stress and growth factor-dependent activation of Ets transcription factors (60, 61). In β-cells, Mgat4a gene expression is inhibited by the Foxa2 transcription factor under replete conditions (62). Hepatic Foxa2 is activated downstream of glucagon signaling (fasting) and inhibited by insulin-Pi3k-Akt signaling (63). Additional studies are needed to map interactions between the expression of the Mgat genes and metabolite sensing both β-cells and hepatocytes. Transgenic mice overexpressing the HBP enzyme, glutamine:fructose-6-phosphate amidotransferase in liver display obesity, glucose intolerance, and insulin resistance after 8 months of age (64). Here we have extended these observations by identifying the Golgi N-glycan branching pathway as an effector downstream of HBP that adapts hepatocyte responsiveness to glucagon. In addition to Gcgr, many other glycoproteins are known to be substrates for Mgat5 modification (65) and may contribute to glucose regulation. For example, TGF-β receptor II is up-regulated at the cell surface by HBP and Mgat5 in cultured cells (12, 44). TGF-β/Smad3 signaling regulates glucose homeostasis, and Smad3−/− mice have a phenotype similar to that of Mgat5−/− (66). As discussed earlier, GLUT-1, -2, and -4 glucose transporters are up-regulated at the cell surface by N-glycan branching (12, 20, 27, 28). Herein we have focused on Gcgr, because the action of glucagon is critically important for the development of hyperglycemia and insulin resistance (1, 2). Hence, selective reduction of Mgat5 activity or HBP in the liver may represent a novel approach for suppression of glucagon action and restoration of euglycemia in the setting of type 2 diabetes.

Previous Section Next Section Acknowledgments We thank Cecilia Unson for the ST-18 antibody and Cecile Boscher and Oliver Rocks for experiment advice.

Previous Section Next Section Footnotes ↵* This work was supported by Ontario Research Fund Grant GL-2, Canadian Institutes for Health Research Grants MOP-79405 and MOP-62975, Grant s from the Canada Research Chairs Program (to J. W. D. and D. J. D.), Canadian Institutes for Health Research Grants MOP-93749 and MOP-123391 (to D. J. D.), the Banting & Best Diabetes Centre (BBDC)-Novo Nordisk Chair in Incretin Biology (to D. J. D.), and Canadian Liver Foundation Graduate Studentship (to M. R.).

↵3 The abbreviations used are: HBP hexosamine biosynthesis pathway ER endoplasmic reticulum Glut glucose transporter Gcgr glucagon receptor L-PHA leucoagglutinin IPGTT intraperitoneal glucose tolerance test OGTT oral glucose tolerance test GC glucagon challenge ITT insulin tolerance test.