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. 2016 Mar 14:6:23043.
doi: 10.1038/srep23043.

Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways

Michael C Ryczko  1   2 , Judy Pawling  1 , Rui Chen  3   4 , Anas M Abdel Rahman  1   5 , Kevin Yau  1 , Julia K Copeland  6 , Cunjie Zhang  1 , Anu Surendra  6 , David S Guttman  6 , Daniel Figeys  3   7 , James W Dennis  1   2   8
Affiliations

Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways

Michael C Ryczko et al. Sci Rep. .

Abstract

De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhibition of Golgi N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase enzymes of the N-glycan branching pathway (Mgat1,2,4,5) display multistep ultrasensitivity to UDP-GlcNAc, as well as branching-dependent compensation. Indeed, oral GlcNAc rescued fat accumulation in lean Mgat5(-/-) mice and in cultured Mgat5(-/-) hepatocytes, consistent with N-glycan branching compensation. Our results suggest GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the UDP-GlcNAc supply to N-glycan branching pathway.

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Figures

Figure 1
Figure 1. Oral GlcNAc increases tissue UDP-GlcNAc and promotes weight-gain in young mice.
(A) Change in body-weight for wild-type C57BL/6 male mice on diets containing different percentages of fat. Data shown are mean ± SEM, n = 8–11. (B) Respiratory exchange ratio (RER) for night and day in mice fed different percentage fat diets for 50 weeks. Data shown are mean ± SEM, n = 8–11, analyzed by one-way ANOVA followed by Tukey’s multiple comparison test, with significant differences indicated as *p < 0.05, **p < 0.01 and ***p < 0.001. (C) Change in body-weight of C57BL/6 male mice on 4% fat diet beginning at 9 weeks of age with GlcNAc supplemented drinking water at 0.5, 5.0 and 15 mg/ml, n = 10, p < 0.001 ANOVA. (D) Relative abundance of ~150 liver metabolites measured by targeted LC-MS/MS and represented as principle component analysis at 90 days of GlcNAc treatment. Specific data for (E) amino acids and (F) glycolytic and gluconeogenic hepatic metabolites expressed as fold change for GlcNAc-treated mice compared to untreated controls. Data shown are mean ± SEM, n = 10, analysed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with vehicle control, with significant differences represented vertically as *p < 0.05, **p < 0.01 and ***p < 0.001. (G) Time-course and relative abundance of serum 13C6-GlcNAc and Glc-D7 in mice gavaged with bolus administration of 13C6-GlcNAc alone or together with Glc-D7, n = 1. (H) At 180 min following gavage with 13C6-GlcNAc, UDP-13C6-GlcNAc was detected as a strong peak in different mouse tissues, with arbitrary units normalized to tissue weight.
Figure 2
Figure 2. Oral GlcNAc promotes weight-gain and lipid accumulation in adult mice.
(A) Change in body-weight for wild-type C57BL/6 male mice on 4% or 9% fat diet with GlcNAc or GlcN supplemented drinking water at 0.5 mg/ml. Data shown are mean ± SEM, n = 10, analyzed by 2-tailed unpaired Student’s t-test, with significant differences indicated as *p < 0.05 for 9% fat control versus 9% fat on GlcNAc. (B) Body-weight and (C) calorie-intake per mouse per day following 21 weeks of GlcNAc treatment. (D) Lean and fat tissue composition measured by dual-energy X-ray absorptiometry (DEXA). Data shown are mean ± SEM, n = 10, *p < 0.05 and **p < 0.01 GlcNAc-treated versus control on either 4% or 9% fat diet. (E) Respiratory Exchange Ratio (RER = VCO2/VO2) over 20 h period, (F) RER quantification by night and day, (G) energy expenditure and (H) total activity in mice supplemented with oral GlcNAc on 9% fat diet. Data shown are mean ± SEM, n = 5, *p < 0.05 and **p <  < 0.01. (I) Serum free fatty-acids (FFA) and triglycerides (TG), and (J) serum metabolite changes in mice on 9% fat diet and supplemented with 0.5 mg/ml oral GlcNAc for 90 days. Data shown are mean and error bars represent ± SEM, n = 5, *p < 0.05 and **p < 0.01 GlcNAc-treated versus control with 2-tailed, unpaired Student’s t-test.
Figure 3
Figure 3. Oral GlcNAc alters liver metabolism.
(A) Liver free fatty-acids (FFA) and (B) triglycerides (TG) in wild-type C57BL/6 male mice on 4% or 9% fat diet over 30 weeks on oral GlcNAc at 0.5 mg/ml. Data shown are mean ± SEM, n = 5, *p < 0.05, **p < 0.01 and ***p < 0.001 GlcNAc-treated versus control in fasted or fed state with 2-tailed, unpaired Student’s t-test. Immunoblot analysis of metabolic signaling pathways, with fatty-acid synthase (FASN) and phosphorylated forms of Akt kinase, ribosomal protein S6 (S6), AMP-Activated Protein Kinase (AMPK-α) and Acetyl-CoA Carboxylates Kinase (ACC) in liver lysates from mice maintained on 9% fat diet and supplemented with GlcNAc for 30 weeks in ad libitum fed (C) or 18 h fasted (D) states. (E) Intraperitoneal glucagon tolerance test and (F) intraperitoneal glucose tolerance test, with area under the curve (AUC) quantification in mice supplemented with 0.5 mg/ml oral GlcNAc for 23 weeks. Data shown are mean ± SEM, n = 5, *p < 0.05.
Figure 4
Figure 4. GlcNAc increases UDP-GlcNAc, β-1,6-GlcNAc branched N-glycans and lipid accumulation in AML12 cells.
(A) Fold changes of distal HBP metabolites upon GlcNAc treatment. (B) Analysis of Mgat5-dependent N-glycan branching on cell surface glycoproteins quantified with Alexa-488-conjugated lectin L-PHA. (C) Analysis of oligomannose-type and hybrid-type N-glycans on cell surface glycoproteins with Alexa-488-conjugated lectin ConA. (D) Lipid droplet content, quantified microscopically with lipophilic fluorescent probe BODIPY 493/503. (E) Western blot analysis of enzyme fatty-acid synthase (FASN) and loading control tubulin, used for relative quantification of immunoblot. (F) Fold change in specific metabolites involved in fat metabolism, normalized to cell number. (G) Glucose uptake in cells treated with GlcNAc for 20h, grown in the presence of fluorescent Glc-analog 2-NBD-Glc for 1h, and quantified using flow cytometry as mean fluorescent intensity (MFI). (H) Analysis of heavy-isotope dual-labelled Gln uptake in cells pretreated with GlcNAc, pulsed with 15N2-Gln for designated times, and quantified using mass spectrometry. (I) Tri- and tetra-antennary Mgat5-modified N-glycans and (J) lipid droplet content in the absence and presence of GlcNAc and/or Swainsonine (SW), quantified microscopically with Alexa-488-conjugated lectin L-PHA or BODIPY 493/503. Data shown are mean ± SEM, analyzed by 2-tailed unpaired Student’s t-test or one-way ANOVA followed by Dunnett’s multiple comparison test compared with vehicle control (A-F), with significant differences represented as *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 5
Figure 5. Oral GlcNAc partially rescues lipid storage in Mgat5−/− mice.
(A) Change in body-weight for Mgat5+/+ and Mgat5−/− male mice on 9% fat diet supplemented with 0.5 mg/ml oral GlcNAc in drinking water for 30 weeks. Data shown are mean ± SEM, n = 4–5, with significant difference indicated as *p < 0.05 for wt control versus wt on GlcNAc. (B) Fat and lean tissue mass content, measured by EchoMRI. (C) Diurnal and nocturnal respiratory exchange ratio (RER) and (D) total activity (locomotion, grooming and rearing on hind legs) during the same time period. (E) Relative abundance of liver GlcNAc-P and UDP-GlcNAc determined in the same cohort of mice and expressed as fold change. Data shown in panels above are mean ± SEM, n = 4–5, with statistical significance indicated as *p < 0.05 and **p < 0.01 for GlcNAc-treated versus untreated control groups of the same genotype with 2-tailed, unpaired Student’s t-test. (F) Lipid droplet content in primary hepatocytes derived from 3 month old Mgat5+/+ and Mgat5−/− mice and cultured overnight with exogenous GlcNAc supplementation. Lipid was quantified with the lipophilic fluorescent probe BODIPY 493/503. Data shown are mean ± SEM, n = 4, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared to 0 mM GlcNAc control of respective genotype, with significant differences indicated as *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 6
Figure 6. Proposed model of HBP and N-glycan branching dependent regulation of cellular metabolism.
GlcNAc salvaged by HBP increases UDP-GlcNAc, the substrate for branching N-acetylglucosaminyltransferases (Mgat1,2,4,5), which modify glycoproteins trafficking through the Golgi en route to the cell surface. Km values for UDP-GlcNAc decline from Mgat1, Mgat2, Mgat4 to Mgat5, thus biosynthesis of tri- and tetra-antennary N-glycans is sensitive to UDP-GlcNAc levels. Titration of N-glycan branching via UDP-GlcNAc increases the affinity of glycoproteins for galectins, which cross-link and oppose loss of receptors and transporters to endocytosis. This may stabilize cell surface residency and thereby activity of glucose (Glc), glutamine (Gln) and fatty-acid (FA) transporters (Glut, Slc and FA transporters, respectively). More nutrient uptake contributes to increase in FA synthesis and lipid accumulation via Fasn. A positive-feedback loop is formed by increasing uptake and flux of Glc, Gln and Ac-CoA through de novo HBP to UDP-GlcNAc and Golgi N-glycan branching on glycoprotein transporters and receptors.
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