Identification of a novel association for the WWOX/HIF1A axis with gestational diabetes mellitus (GDM)

Abstract

Background: Although the WW-domain-containing oxidoreductase (WWOX)/Hypoxia-inducible factor 1 (HIF1) pathway is a well-known regulator of cellular glucose and energy metabolism in pathophysiological processes, its role in gestational diabetes mellitus (GDM), remains elusive. We undertook this study to determine the effect of WWOX/HIF1A signaling on the expression of glucose metabolism genes in GDM patients.

Methods: Leukocytes were obtained from 135 pregnant women with (n = 98) or without (n = 37) GDM and, in turn, 3 months (n = 8) and 1 year (n = 12) postpartum. Quantitative RT-PCR was performed to determine gene expression profiles of the WWOX/HIF1A-related genes, including those involved in glucose transport (SLC2A1, SLC2A4), glycolytic pathway (HK2, PKM2, PFK, LDHA), Wnt pathway (DVL2, CTNNB1), and inflammatory response (NFKB1).

Results: GDM patients displayed a significant downregulation of WWOX with simultaneous upregulation of HIF1A which resulted in approximately six times reduction in WWOX/HIF1A ratio. As a consequence, HIF1A induced genes (SLC2A1, HK2, PFK, PKM) were found to be overexpressed in GDM compared to normal pregnancy and negative correlate with WWOX/HIF1A ratio. The postpartum WWOX expression was higher than during GDM, but its level was comparable to that observed in normal pregnancy.

Conclusions: The obtained results suggest a significant contribution of the WWOX gene to glucose metabolism in patients with gestational diabetes. Decreased WWOX expression in GDM compared to normal pregnancy, and in particular reduction of WWOX/HIF1A ratio, indicate that WWOX modulates HIF1α activity in normal tissues as described in the tumor. The effect of HIF1α excessive activation is to increase the expression of genes encoding proteins directly involved in the glycolysis which may lead to pathological changes in glucose metabolism observed in gestational diabetes.

Keywords: Gestational diabetes mellitus (GDM); Glycolysis; Hypoxia-inducible factor 1α (HIF1α); WW domain-containing oxidoreductase (WWOX).

Figure 1. The classification of subjects to the study groups during diabetic pregnancy and the postpartum period based on the OGTT results.

Figure 2. The gene expression profiles of WWOX, HIF1A and WWOX/HIF1A-related genes.

Boxplots showing the relative leukocyte mRNA expression (the ratio of the target gene relative to the reference genes RPS17, RPLP0, H3F3A) of WWOX (A), HIF1A (B) and all WWOX/HIF-related genes including those involved in glucose transport (SLC2A1 (C), SLC2A4 (D)), glycolytic pathway (HK2 (E), PKM2 (F), PFK (G), LDHA (H)), Wnt pathway (DVL2 (I), CTNNB1 (J)), and inflammatory response (NFKB1 (K)), along with the WWOX/HIF1A ratio (L) in the patients with GDM (n = 98) vs the subjects with NGT (n = 37). Data are expressed as median (indicated by horizontal bars) ± interquartile range (25–75%), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 as assessed by the Mann–Whitney U-test. NS, not significant.

Figure 3. Postpartum leukocyte WWOX mRNA expression in the study groups.

The relative leukocyte WWOX mRNA expression in the group B (at the second visit) and group C (at the third visit) vs corresponding diabetic groups A1 and A2 (pregnant women diagnosed as having GDM at the first study visit; n = 8 and n = 12, respectively) vs pregnant subjects who were classified as NGT (n = 37) and GDM (n = 98) at the first visit. Data are expressed as median (indicated by horizontal bars) ± interquartile range (25–75%), ^∗p < 0.05, ^∗∗p < 0.01, as assessed by the Wilcoxon Matched-Pairs Signed Rank Test for paired results and the Mann–Whitney U test for unpaired.

Figure 4. The Spearman’s correlation analysis of genes expression in the GDM group.

The Spearman’s multiply correlation plot with R representing correlation coefficient and p the statistical significance (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001) for the transcripts of interest in the GDM group. R was presented in different colors; the legend is the color range of different R values.

Figure 5. The Spearman’s correlation analysis of genes expression in the NGT group.

The Spearman’s multiply correlation plot with R representing correlation coefficient and p the statistical significance (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001) for the transcripts of interest in the GDM group. R was presented in different colors; the legend is the color range of different R values.

Figure 6. Correlations of HbA1C with HIF1A (A), SLC2A1 (B), HK2 (C), PFK (D) and LDHA (E) in the GDM group. Spearman correlation coefficients at p < 0.05 are shown.

Figure 7. Proposed model of molecular connections between the components of the WWOX/HIF axis and the upregulation of glycolytic energy metabolism in leukocytes of diabetic patients.

Based on free images (“https://smart.servier.com/smart_image/cell-membrane-14/”; “https://smart.servier.com/smart_image/channel-119/”) from Servier Medical Art (2020a) and Servier Medical Art (2020b); CC BY 3.0.