doi: 10.1038/s41467-019-13914-8.
O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity
Affiliations
- PMID: 31924761
- PMCID: PMC6954210
- DOI: 10.1038/s41467-019-13914-8
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O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity
Nat Commun.
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Abstract
Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.
Conflict of interest statement
The authors declare no competing interests.
Figures
a Breeding strategy used to generate wild-type (WT) control mice and adipose-specific Ogt-knockout (OGT AKO) mice. b Schematic diagram showing the localization of mouse adipose tissues including BAT, iWAT, and eWAT; the localization of corresponding human adipose tissues was shown as a reference. c Western blot analysis of OGT and β-actin in various adipose tissues from WT and OGT AKO mice. d, e Energy expenditure and respiratory exchange ratios (RERs) of WT and OGT AKO mice (n = 4 WT, 4 KO). f, g Area under curve (AUC) analyses of RER results shown in e. h, i Fat mass and lean mass of WT and OGT AKO mice fed on a normal chow (NC) (n = 19 WT, 11 KO for h; n = 11 WT, 6 KO for i). j Dynamic change of fat mass of WT and OGT AKO mice during fasting/refeeding (n = 23 WT, 18 KO for fasted 24 h; n = 8 WT, 5 KO for other groups). k Representative images of BAT, iWAT, and eWAT from WT and OGT AKO mice in fed and 24 h fasted states, scale bar is 1 cm. l, m Tissue weights, presented as % of body weight, in mice described in k (n = 8 WT, 6 KO for l; n = 6 WT, 8 KO for m). Data are presented as mean ± s.e.m. Statistical analysis: Student’s t test, *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.
a Tissue weight of eWAT from WT and OGT AKO mice in fed, 6 h fasted, 24 h fasted, and 6 h refed states (n = 3–5 WT, 3–4 KO). b Changes in eWAT tissue weight during fasting/refeeding (n = 3–5 WT, 3 KO). c Hematoxylin–eosin (H&E) staining of eWAT from 2-month-old WT and OGT AKO mice in fed and 24 h fasted states, scale bar is 20 μm. d, e Quantification of adipocyte size in eWAT shown in c (three experiments). f Dynamic changes in serum FFA level in WT and OGT AKO mice during fasting/refeeding (n = 4–8 WT, 4–6 KO). g Basal and stimulated (10 μM CL-316,243) lipolysis measured by glycerol released from explants of eWAT and iWAT and isolated brown adipocytes from WT and OGT AKO mice (n = 2–6/group, three experiments). Data are presented as mean ± s.e.m. Statistical analysis: Student’s t test, *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.
a 3D-reconstructed confocal images of differentiated C3H/10T1/2 cells treated with DMSO or ST045849 (ST04), an OGT inhibitor, cultured in 0.4 mM oleic acid-supplemented medium and serum-depleted medium, and stained with BODIPY 493/503 (lipid droplets, green), and DAPI (nucleus, blue), scale bar is 20 μm. b, c Cumulative frequency distribution and mean average of lipid droplet size shown in a (three experiments). d Time-lapse imaging of differentiated C3H/10T1/2 cells treated with DMSO and ST04, and labeled with BODIPY 558/568 C12 fatty acid (lipid droplets, red), scale bar is 20 μm.; e Quantification of lipid droplet size shown in d (n = 3/group, three experiments). Data are presented as mean ± s.e.m. Statistical analysis: ANOVA with Dunnett multiple comparisons for c and Student’s t test for e, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.
a Western blot analysis of PLIN1, p-HSL, HSL, OGT, and β-actin in eWAT of fed and 6 h fasted WT and OGT AKO mice (three experiments). b, c Phos-tag gel analysis and quantification of PLIN1 in eWAT of fed and 6 h fasted WT and OGT AKO mice; λ-PPase was used to remove phosphate groups from phosphorylated serine, threonine, and tyrosine residues (n = 3/group for c, three experiments). d–f Immunofluorescence staining and quantification of PLIN1 phosphorylation at serine 492 (p492) and serine 517 (p517) in primary adipocytes differentiated in vitro from the WT and OGT AKO eWAT SVFs (n = 4/group, three experiments); IBMX and forskolin (Fsk) were used to stimulate cAMP/PKA pathway; lipid droplets were stained with BODIPY 493/503 (green) and nuclei were stained with DAPI (blue), scale bar is 20 μm. g, h Representative Western blots and quantification of PLIN1 phosphorylation at serine 517 (p517) in eWAT and iWAT of fed and 6 h fasted WT and OGT AKO mice (n = 3/group for h, three experiments). i, j Western blot analysis and quantification of overall O-GlcNAcylation levels in eWAT and iWAT of fed and 6 h fasted WT and OGT AKO mice; RL2 recognizes O-GlcNAc modification on proteins (n = 3/group, three experiments). k Ratios of Ogt/Oga mRNA levels (full-length transcripts) in human subcutaneous fat (Sub.) and visceral fat (Vis.); original raw data was from the Genotype-Tissue Expression (GTEx) database (n > 100/group). Data are presented as mean ± s.e.m. Statistical analysis: ANOVA with Dunnett’s multiple comparisons for k and Student’s t test for the rest, *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.
a Immunoprecipitation (IP) and Western blot analysis showing that OGT overexpression enhances PLIN1 O-GlcNAcylation and the catalytic dead mutation of OGT greatly impairs its ability to catalyze PLIN1 O-GlcNAcylation; RL2 is an antibody against O-GlcNAc moieties. b Overlapping of predicted O-GlcNAcylation sites determined by three independent analyses. c Final candidate sites, known PKA phosphorylation sites (red colored) were included, common sites from b that are adjacent to the PKA sites (green colored) were also included. d, e IP, Western blot analysis, and quantification results showing that OGT catalyzes PLIN1 O-GlcNAcylation primarily at serine 492 and serine 517 (n = 10 for WT positive control, 4–5 for other groups). f, g IP, Western blot analysis, and quantification results showing that serine 492 to alanine and serine 517 to alanine double mutation in PLIN1 (PLIN1-AA) largely eliminated its overall O-GlcNAcylation (n = 8 for WT positive control, 3–8 for other groups); data are presented as mean ± s.e.m. Statistical analysis: ANOVA with Dunnett’s multiple comparisons, *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.
a IP and Western blot analysis showing the effects of OGT on PLIN1 O-GlcNAcylation and phosphorylation; p-Ser antibody recognizes proteins phosphorylated on serine residues; cells were treated with IBMX/Fsk before analysis. b IP and Western blot analysis showing the effects of OGT knockout on endogenous PLIN1 O-GlcNAcylation and phosphorylation; hollow arrowheads indicate IgG heavy chains. c IP and Western blot analysis showing that OGT overexpression attenuates IBMX/Fsk-induced PLIN1 phosphorylation at serine 517. d IBMX/Fsk-induced lipolysis measured by glycerol release (n = 3/group). e IBMX/Fsk-induced lipolysis measured by glycerol released from cells transfected with HA-PLIN1 (WT), HA-PLIN1-AA (AA), and HA-PLIN1-EE (EE) (n = 6–11/group). f Basal and IBMX/Fsk-induced lipolysis measured by glycerol released from cells transfected with Myc or Myc-OGT together with HA, HA-PLIN1, HA-PLIN1-AA, or HA-PLIN1-EE (n = 4/group). g Immunofluorescence images of cells transfected with HA-PLIN1, HA-PLIN1-AA, or HA-PLIN1-EE, cultured in 0.4 mM oleic acid, treated with or without IBMX/Fsk, and stained with antibody against HA (Red), BODIPY 493/503 (green), and DAPI (blue); scale bar is 10 μm. h, i Mean average and cumulative frequency distribution of lipid droplet size shown in g (n > 150/group). j Immunofluorescence images of primary adipocytes differentiated from WT and OGT AKO eWAT SVFs; adipocytes were treated with IBMX/Fsk, fixed, and stained with antibody against CGI-58 (red), BODIPY 493/503 (green), and DAPI (blue), scale bar is 20 μm. k IP and Western blot analysis showing the interaction between endogenous CGI-58 and ATGL in eWAT of 6 h fasted WT and OGT AKO mice; hollow arrowheads indicate IgG heavy chains. l IBMX/Fsk-induced lipolysis measured by glycerol released from primary adipocytes differentiated from WT and OGT AKO eWAT SVFs (n = 4 for Atglistatin-treated WT, six for other groups); Atglistatin (10 μM) was used to inhibit ATGL activity. Data are presented as mean ± s.e.m. Statistical analysis: Student’s t test for i and ANOVA with Dunnett multiple comparisons for the rest, *p < 0.05, **p < 0.01, and ***p < 0.001; n.s., not significant. Source data are provided as a Source Data file.
a Breeding strategy used to generate WT control mice and OGT AKI mice. b Western blot analysis of OGT and β-actin in eWAT from WT and OGT AKI mice. c Body weight of WT and OGT AKI mice fed on HFD (n = 11 WT, 10 KI). d–f Fat mass and lean mass of WT and OGT AKI mice fed on HFD for 4 weeks (n = 8 WT, 7 KI). g, h Images and tissue weights of fat pads from HFD-fed WT and OGT AKI mice (n = 4 WT, 6 KO for h). i IP and Western blot analysis showing the effects of OGT knockin on endogenous PLIN1 O-GlcNAcylation and phosphorylation; hollow arrowheads indicate IgG heavy chains. j Basal and stimulated (10 μM CL-316,243) lipolysis measured by glycerol released from explants of eWAT and iWAT from HFD-fed WT and OGT AKI mice (n = 2/group, three experiments). k–n Blood glucose and area under curve (AUC) analyses of glucose tolerance test and insulin tolerance test of HFD-fed WT and OGT AKI mice (n = 7 WT, 4 KI). o Molecular model for OGT function in lipolysis. Data are presented as mean ± s.e.m. Statistical analysis: Student’s t test, *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.
a Correlation between the ratio of white fat Ogt/Oga transcript levels and serum free fatty acid (FFA) levels in 41 different mouse strains from the GeneNetwork database (the EPFL LISP3 Cohort); NC-fed and HFD-fed male mice at the fasted state were used for the analysis. b Correlation between the ratio of white fat Ogt/Oga transcript levels and glycemia during oral glucose tolerance test (glucose AUC); mice described in a were used. c, d Correlations between ratios of Ogt/Oga transcript levels (full-length transcripts) in human subcutaneous fat (Sub.) and visceral fat (Vis.) and body mass index (BMI); data from men and women were used for the analysis; original raw data was from the GTEx database (dbGaP study accession: phs000424.v7.p2) (n = 441 for c; n = 356 for d). e, f The average ratio of Ogt/Oga transcripts in subcutaneous fat (Sub.) and visceral fat (Vis.) of human subjects diagnosed with type 2 diabetes (T2D) and non-diabetic normal controls (n = 348 normal, 91 T2D for e; n = 286 normal, 68 T2D for f); data were obtained from the same study described in c, d. Data are presented as mean ± s.e.m. Statistical analysis: Student’s t test for e, f and linear regression for the other panels. Source data are provided as a Source Data file.
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