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. 2022 Mar 19;23(6):3326.
doi: 10.3390/ijms23063326.

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders

Izabela Baryła  1 , Ewa Styczeń-Binkowska  1 , Elżbieta Płuciennik  1 , Katarzyna Kośla  1 , Andrzej K Bednarek  1
Affiliations

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders

Izabela Baryła et al. Int J Mol Sci. .

Abstract

Recent reports indicate that the hypoxia-induced factor (HIF1α) and the Warburg effect play an initiating role in glucotoxicity, which underlies disorders in metabolic diseases. WWOX has been identified as a HIF1α regulator. WWOX downregulation leads to an increased expression of HIF1α target genes encoding glucose transporters and glycolysis' enzymes. It has been proven in the normoglycemic mice cells and in gestational diabetes patients. The aim of the study was to determine WWOX's role in glucose metabolism regulation in hyperglycemia and hypoxia to confirm its importance in the development of metabolic disorders. For this purpose, the WWOX gene was silenced in human normal fibroblasts, and then cells were cultured under different sugar and oxygen levels. Thereafter, it was investigated how WWOX silencing alters the genes and proteins expression profile of glucose transporters and glycolysis pathway enzymes, and their activity. In normoxia normoglycemia, higher glycolysis genes expression, their activity, and the lactate concentration were observed in WWOX KO fibroblasts in comparison to control cells. In normoxia hyperglycemia, it was observed a decrease of insulin-dependent glucose uptake and a further increase of lactate. It likely intensifies hyperglycemia condition, which deepen the glucose toxic effect. Then, in hypoxia hyperglycemia, WWOX KO caused weaker glucose uptake and elevated lactate production. In conclusion, the WWOX/HIF1A axis downregulation alters glucose metabolism and probably predispose to metabolic disorders.

Keywords: WW domain-containing oxidoreductase (WWOX); glycolysis; hypoxia-inducible factor 1α (HIF1α); metabolic disorders.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Confirmation of 1BR.3.N cell line transfection on the protein level (a,b), calculated relative protein expression (OD WWOX/OD GAPDH) (c,d) and relative WWOX mRNA expression (e,f). The values are mean ± SEM. ***, p < 0.001.
Figure 2
Figure 2
Obtained variants of 1BR.3.N cell line (1BR.3.N CONTR and 1BR.3.N WWOX KO) was transfected with a luciferase reporter gene under the regulation of Hypoxia-Responsive Elements (HRE) and subjected to all tested conditions for 6 h. Extracts were analyzed for luciferase activity. The values are mean ± SEM. *, p < 0.05; **, p < 0.01.
Figure 3
Figure 3
Glucose uptake in WWOX KO and CONTR variants of 1BR.3.N cell line incubated all tested conditions (Normoxia Normoglycemia, Normoxia Hyperglycemia, Hypoxia Normoglycemia, Hypoxia Hyperglycemia) with or without insulin for 48 h. The values are mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4
Relative expression fold change of glycolysis-associated genes detected by RT-qPCR in WWOX-silencing 1BR.3.N cells after 48 h incubation in four tested condition. Bar graphs showing the relative mRNA expression (the ratio of the target gene relative to the reference genes RPS17, RPLP0, H3F3A) of WWOX (a), HIF1A (b), the WWOX/HIF1A ratio (c) and all WWOX/HIF-related genes including those involved in glucose transport (SLC2A1 (d), SLC2A4 (e)), glycolytic pathway (HK2 (f), ENO1 (g), PFK (h), PKM2 (i), LDHA (j)), PDK (k), PDHA (l), CS (m) and ACLY (n). Results of ANOVA with a post-hoc Tukey-Test are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5
Figure 5
Colorimetric method study of aerobic glycolysis metabolic enzymes and metabolites in 1BR.3.N CONTR and WWOX KO cells after 48 h incubation in four tested condition. Bar graphs showing the enzyme activity of Hexokinase (a), Pyruvate Dehydrogenase (b), Lactate Dehydrogenase (c) Citrate Synthase (e) and Lactate concentration (d). Results of ANOVA with a post-hoc Tukey-Test are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 6
Figure 6
WWOX and HIF1α western blot analysis of cytoplasmic (a) and nuclear (b) fractions of CONTR and WWOX KO 1BR.3.N cells after 48 h incubation in four tested condition. Results of ANOVA with a post-hoc Tukey-Test are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NN—normoxia normoglycemia, NH—normoxia hyperglycemia, HN—hypoxia normoglycemia, HH—hypoxia hyperglycemia.
Figure 7
Figure 7
The western blot analysis of selected proteins (HK2, PKM2, LDHA, GLUT1) of CONTR and WWOX KO 1BR.3.N cells after 48h incubation in four tested condition (a). Bar graphs showing the relative level OD density of HK2 (b), PKM2 (c), LDHA (d), GLUT1 (e) to ACTIN. Results of ANOVA with a post-hoc Tukey-Test are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. NN—normoxia normoglycemia, NH—normoxia hyperglycemia, HN—hypoxia normoglycemia, HH—hypoxia hyperglycemia.
Figure 8
Figure 8
Immunocytochemistry analysis of WWOX and HIF1α in 1BR.3.N CONTR and WWOX KO cells after 6h incubation in normoxia (a) and hypoxia (b). Calculation of Total Corrected Cell Fluorescence (TCCF) (c). Results of ANOVA with a post-hoc Tukey-Test are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 9
Figure 9
Effect of WWOX KO on mRNA and protein expression, and enzyme activity of genes involved in glucose transport and glycolysis pathway of 1BR.3.N fibroblast cell line in normoxia normoglycemia condition. Based on free images from Servier Medical Art; CC BY 3.0.
Figure 10
Figure 10
(top) Effect of hyperglycemia on mRNA and protein expression, and enzyme activity of genes involved in glucose transport and glycolysis pathway of 1BR.3.N fibroblast cell line in normoxia. (bottom) Effect of WWOX KO on mRNA and protein expression, and enzyme activity of genes involved in glucose transport and glycolysis pathway of 1BR.3.N fibroblast cell line in normoxia hyperglycemia condition. Based on free images from Servier Medical Art; CC BY 3.0.
Figure 11
Figure 11
(top) Effect of hyperglycemia on mRNA and protein expression, and enzyme activity of genes involved in glucose transport and glycolysis pathway of 1BR.3.N fibroblast cell line in hypoxia. (bottom) Effect of WWOX KO on mRNA and protein expression, and enzyme activity of genes involved in glucose transport and glycolysis pathway of 1BR.3.N fibroblast cell line in hypoxia hyperglycemia condition. Based on free images from Servier Medical Art; CC BY 3.0.
Figure 12
Figure 12
The scheme of experiment.
See this image and copyright information in PMC

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