Consumption of a high fat diet promotes protein O-GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

Abstract

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise in developed countries due to overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-diabetes, we evaluated the impact of a diet high in saturated fat (HFD) on O-GlcNAcylation of retinal proteins, as dysregulated O-GlcNAcylation contributes to diabetic complications and evidence supports a role in DR. Protein O-GlcNAcylation was increased in the retina of mice fed a HFD as compared to littermates receiving control chow. Similarly, O-GlcNAcylation was elevated in retinal Müller cells in culture exposed to the saturated fatty acid palmitate or the ceramide analog Cer6. One potential mechanism responsible for elevated O-GlcNAcylation is increased flux through the hexosamine biosynthetic pathway (HBP). Indeed, inhibition of the pathway's rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) prevented Cer6-induced O-GlcNAcylation. Importantly, expression of the mRNA encoding GFAT2, but not GFAT1 was elevated in both the retina of mice fed a HFD and in retinal cells in culture exposed to palmitate or Cer6. Notably, expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) was increased in the retina of mice fed a HFD and NR4A1 expression was sufficient to promote GFAT2 mRNA expression and O-GlcNAcylation in retinal cells in culture. Whereas palmitate or Cer6 addition to culture medium enhanced NR4A1 and GFAT2 expression, chemical inhibition of NR4A1 transactivation repressed Cer6-induced GFAT2 mRNA expression. Overall, the results support a model wherein HFD increases retinal protein O-GlcNAcylation by promoting NR4A1-dependent GFAT2 expression.

Keywords: Diabetic retinopathy; Hexosamine biosynthetic pathway; NR4A1; Type 2 diabetes.

Figure 1.. Relative GFAT isoform expression is tissue and cell type specific.

A. GFAT1 and GFAT2 mRNA expression was assessed by RT-PCR in rat liver, kidney, retina, brain, and gastrocnemius muscle and is expressed relative to GAPDH (n=5). B. Relative ratios of GFAT1 to GFAT2 mRNA in various rat tissues from A. C. GFAT1 and GFAT2 mRNA expression was assessed by RT-PCR in human retinal MIO-M1, endothelial HUVEC, and liver HepG2 cells in culture (n=3). D. Relative ratios of GFAT1 to GFAT2 mRNA in human cell lines from C. Results are expressed as means ± SEM. Statistical significance is denoted in B. and D. by “*” (p<0.05) versus either liver or HepG2 cells, respectively.

Figure 2.. Protein O-GlcNAcylation is increased in the retina of mice fed a high-fat diet (HFD) concomitant with increased GFAT2 mRNA expression.

A. Protein O-GlcNAcylation was assessed by Western blotting of retinal lysates from mice fed either a control chow (Chow) or HFD for 4 weeks. Protein molecular mass (kDa) is indicated at the right of blots. B. Retinal protein O-GlcNAcylation in A. was quantified and is expressed relative to GAPDH. Representative blots are shown. Protein loading was assessed by protein staining (Protein S.). C. Change in body weight is expressed as a percentage body weight prior to initiating the 4-week diet. D. Nonfasting blood glucose concentrations were assessed the day before retinal extraction. GFAT1 (E.) and GFAT2 (F.) mRNA expression were assessed in the retina of mice fed a HFD for 4 weeks by RT-PCR. Results are expressed as means ± SEM (n=5). Statistical significance is denoted by “*” (p<0.05) or “**” (p<0.01), versus control chow diet.

Figure 3.. Palmitate (PAL) and ceramide (Cer6) promote protein O-GlcNAcylation and increase GFAT2 expression in retinal cells in culture.

A-C. TR-MUL cells were exposed to culture medium containing either BSA or palmitate bound to BSA (PAL, 0.5 mM) for 8 h (n=3). Protein O-GlcNAcylation, GFAT1, GFAT2, and GAPDH protein expression were assessed by Western blotting in A. and D. Representative blots are shown. Protein molecular mass (kDa) is indicated at the right of blots. Protein Staining (Protein S.) is shown as a loading control. GFAT1 (B. & E.) and GFAT2 (C. & .F). mRNA expression were assessed by RT-PCR. D. TR-MUL cells were exposed to culture medium containing the cell permeable ceramide Cer6 or DMSO vehicle (Veh) for up to 16 h (n=3). E-H. TR-MUL cells were treated with either Cer6 or Veh for 16 h. TR-MUL cells were exposed to culture medium containing GFAT inhibitors 6-diazo-5-oxonorleucine (DON) or Azaserine (Aza) for 1 h prior to Cer6 addition in G. and H., respectively. Results are expressed as means ± SEM (n=3). Statistical significance is denoted by “*” (p<0.05), “**” (p<0.01), or “***” (p<0.001), versus control treatment. Protein O-GlcNAcylation and GAPDH protein expression were assessed by Western blotting in G-H.

Figure 4.. ER stress promotes O-GlcNAcylation and GFAT1 but not GFAT2 mRNA expression.

TR-MUL cells were exposed to culture medium containing the ER stress inducer thapsigargin (TG) for up to 16 h (n=3). A. Protein O-GlcNAcylation and phosphorylation of PERK and eIF2α were evaluated by Western blotting. Representative blots are shown. Protein molecular mass (kDa) is indicated at the right of blots. Protein Staining (Protein S.) is shown as a loading control. XBP1 processing (B.) as well as GFAT1 (C.) and GFAT2 (D.) mRNA expression were assessed by RT-PCR. Results are expressed as means ± SEM (n=3). Statistical significance is denoted by the presence of different letters above bars on the graphs. Bars with different letters are statistically different; p<0.05. Results are representative of two experiments; within each experiment, three independent samples were analyzed.

Figure 5.. NR4A1 mRNA expression is increased in the retina of mice fed a HFD and in retinal cells exposed to PAL or Cer6.

A. NR4A1 mRNA expression was assessed in the retina of mice fed either a control chow (Chow) or HFD for 4 weeks (n=6). B. TR-MUL retinal cells were exposed to culture medium containing either BSA or palmitate bound to BSA (PAL) for 8 h (n=3). C. TR-MUL cells were exposed to culture medium containing the cell permeable ceramide Cer6 for up to 16 h (n=3). NR4A1 mRNA expression was assessed by RT-PCR. Results are expressed as means ± SEM. Statistical significance is denoted by the presence of different letters above bars on the graphs. Bars with different letters are statistically different; p<0.05. Results in B & C are representative of two experiments; within each experiment, three independent samples were analyzed.

Figure 6.. NR4A1 promotes GFAT2 expression in retinal cells in culture.

A-E. TR-MUL cells were transfected with either an empty vector (EV) or NR4A1 expression plasmid for 48 h. A. FLAG-tagged NR4A1, GFAT1, GFAT2, and GAPDH protein expression were assessed by Western blotting. Protein molecular mass (kDa) is indicated at the right of blots. Protein Staining (Protein S.) is shown as a loading control. NR4A1 (B.), GFAT1 (C.), and GFAT2 (D.) mRNA expression were assessed using RT-PCR. E. TR-MUL cells were transfected with either EV or NR4A1 plasmid for 48 h prior to treatment with the GFAT inhibitor DON for 16 h. Protein O-GlcNAcylation and GAPDH expression were measured by Western blotting. F-H. TR-MUL cells were exposed to culture medium containing either DMSO (Veh) or Cer6 for 16 h in the presence or absence of the NR4A1 inhibitor DIM-C-pPhOH (DIM). NR4A1 (F.), GFAT1 (G.), and GFAT2 (H.) mRNA expression were assessed by RT-PCR. Results are expressed as means ± SEM (n=3). Statistical significance is denoted by the presence of different letters above bars on the graphs. Bars with different letters are statistically different; p<0.05. Results are representative of two experiments; within each experiment, three independent samples were analyzed. I. Working model for mechanism whereby high fat diet (HFD) promotes retinal O-GlcNAcylation via enhanced NR4A1 and GFAT2 expression.