Chronic Uridine Administration Induces Fatty Liver and Pre-Diabetic Conditions in Mice

Abstract

Uridine is a pyrimidine nucleoside that exerts restorative functions in tissues under stress. Short-term co-administration of uridine with multiple unrelated drugs prevents drug-induced liver lipid accumulation. Uridine has the ability to modulate liver metabolism; however, the precise mechanism has not been delineated. In this study, long-term effects of uridine on liver metabolism were examined in both HepG2 cell cultures and C57BL/6J mice. We report that uridine administration was associated with O-GlcNAc modification of FOXO1, increased gluconeogenesis, reduced insulin signaling activity, and reduced expression of a liver-specific fatty acid binding protein FABP1. Long-term uridine feeding induced systemic glucose intolerance and severe liver lipid accumulation in mice. Our findings suggest that the therapeutic potentials of uridine should be designed for short-term acute administration.

Fig 1. Uridine induces O-GlcNAc glycosylation of FOXO1 in HepG2 cells.

(A) Antagonistic effects of uridine and deglycosylases on the protein O-linked glycosylation profiles of HepG2 cells. Total cell extracts were evaluated with 1D Western blots using an antibody that recognizes O-GlcNAc. (B) Synergistic effects of uridine and deglycosylases inhibitor PUGNAc on protein O-linked glycosylation of HepG2 cells. Total cell extracts were evaluated with 1D Western blots using an antibody that recognizes O-GlcNAc. (C) Evidence of uridine-induced O-GlcNAc modification of purified recombinant FOXO1. Immunoprecipitated recombinant FOXO1 protein was evaluated with 1D Western blots using antibodies that recognize FLAG tag (upper panel) and O-GlcNAc (lower panel). (D) Antagonistic effects of uridine and deglycosylases on the pI shifts of FOXO1 following O-GlcNAc modification. Total cell extracts were evaluated with 2D Western blots using an antibody that recognizes FLAG-tagged FOXO1.

Fig 2. Chronic uridine administration induces liver lipid accumulation and elevated blood glucose level.

(A) Assessment of liver lipid content with CARS microscopy. Images presented are 3D stacked of 30 frames at 1-micron increment along the vertical axis. (B) Quantitative analysis of average liver lipid content using CARS signal intensity. (C) Average liver weight and (D) body weight. (E) Blood glucose level as a function of time after glucose injection. Error bars are standard deviation values across 9 mice measured per animal group. (F) 1D Western blot analysis of liver Akt and FOXO1 expression and phosphorylation level. (G) Quantitative analysis of liver Akt and FOXO1 expression and phosphorylation level. Error bars are standard deviation across triplicate measurements. Asterisks indicate p-value <0.01 versus C57BL/6J+LD.

Fig 3. Glycosylation of FOXO1 detected with 2D Western blot and capillary isoelectric focusing (cIEF) immunoassay.

(A) Total liver protein was evaluated with 2D WB using an antibody that recognizes FOXO1. (B) Chemluminescence intensity as a function of pI of the 2D WB shown in (A). Note an additional FOXO1 protein spot at high pI value was detected in the liver sample of C57BL/6J+LDU mice but not in C57BL/6J+LD mice. (C) Liver FOXO1 distribution as a function of pI detected with cIEF immunoassay. Peak chemiluminescence was normalized to 1. Peaks from pI 7.5 to 8.5 are due to FOXO1 glycosylation in the liver tissue of C57BL/6J+LDU mice. (D) Quantitative area under the curve analysis (pI 7.5–8.5) of glycosylated FOXO1 isoform as a function of liver tissues. Error bars are standard deviation values across 9 measurements. Asterisk indicates p-value < 0.01 versus C57BL/6J+LD.

Fig 4. Uridine stimulates hepatic gluconeogenesis.

(A) Increased expression of liver PEPCK and MnSOD following chronic uridine feeding. (B) Quantitative analysis of Western blot data on protein expression level. (C) Glucose production a function of time after uridine addition in HepG2 cells. Error bars are standard deviation values across three mice per animal group or triplicated HepG2 cell cultures. Asterisks indicate p-value <0.01 versus control.

Fig 5. Differential short- and long-term effects of uridine on protein expression level.

(A) Reduced expression of FABP1 following chronic uridine feeding. (B) Quantitative analysis of Western blot data on FABP1 expression level. (C) Expression level of PEPCK, MnSOD, and FABP1 are unchanged following 5 days of feeding with uridine supplemented diet. (D) Quantitative analysis of Western blot data on protein expression level in (C). Error bars are standard deviation values across three mice per animal group. Asterisks indicate p-value <0.01 versus control. (E) Liver FOXO1 distribution as a function of pI detected with cIEF immunoassay. Peak chemiluminescence was normalized to 1. (F) Quantitative area under the curve analysis (pI 7.5–8.5) of glycosylated FOXO1 isoform as a function of liver tissues. Error bars are standard deviation values across 9 measurements.