In this study, we found that FXR negatively regulates β-cell apoptosis by repressing Cox6a2 transcription. Specifically, FXR achieves this by diminishing p300-mediated acetylation of H3K27 at the Cox6a2 promoter. Importantly, we further revealed that COX6A2 facilitates the mitochondrial translocation of Bax by interacting with VDAC1, which in turn leads to islet β-cell apoptosis. These findings suggest that the diabetogenic situations-reduced FXR leads to the increase of COX6A2, thereby inducing β-cell apoptosis through a mitochondria-dependent pathway, ultimately contributing to the exacerbation of type 2 diabetes.
COX6A2 is one of the 13 subunits of cytochrome c oxidase. Deficiency of Cox6a2 in mice has shown protection against insulin resistance and obesity induced by high-fat diets [20]. COX-2, another subunit of cytochrome c oxidase, has been implicated in the apoptosis of human colon cancer, non-small-cell lung cancer cells, and endothelial cells [25–27]. However, there is no existing evidence that COX6A2 plays a role in regulating β-cell apoptosis. Based on our findings, we present the first evidence that COX6A2 promotes β-cell apoptosis in diabetes. This claim is supported by three key pieces of evidence. First, elevated expression of COX6A2 was observed in islets from diabetic animals (Fig. 1A-C). Second, overexpression of COX6A2 led to increased expression of cleaved-caspase3 in INS-1 832/13 cells, which was reduced in sh-Cox6a2 INS-1 832/13 cells under lipotoxic stress, as well as in islets from GK rats injected with AAV9-Cox6a2-KO virus, and in islets from Cox6a2−/− mice fed with high-fat diet when compared to their respective controls (Fig. 1D, F, H, I; Fig. 2A, B). Third, COX6A2 overexpressing INS-1 832/13 cells exhibited a higher number of apoptotic cells, whereas Cox6a2 knockdown cells displayed a reduced number of apoptotic cells under lipotoxic stress (Fig. 1E, G) compared to their controls.
To investigate how COX6A2 triggers β-cell apoptosis, we utilized mass spectrometry to identify proteins interacting with COX6A2 in COX6A2 overexpressing INS-1 832/13 cells. Among the proteins, VDAC1 exhibited the highest fold enrichment (Fig. 4A). VDAC1 plays a critical role in regulating mitochondrial function and can induce apoptosis by facilitating the release of cytochrome c through its oligomerization and formation of a channel within the VDAC1 homo-oligomer [28]. Our findings reveal that COX6A2 promotes β-cell apoptosis via modulation of VDAC1, supported by several key observations. First, the Co-IP assay confirmed the interaction between COX6A2 and VDAC1 (Fig. 4B). Second, the protein level of VDAC1 was notably elevated in COX6A2 overexpressing INS-1 832/13 cells but decreased in COX6A2 knockdown cells under lipotoxic stress (Fig. 4D). Third, either silence or inhibition of VDAC1 diminished the elevated level of cleaved-caspase3 protein induced by COX6A2 overexpression (Fig. 4F; Supplementary Fig. S4).
It is well established that VDAC1 can interact with Bax, which forms oligomers in the mitochondrial outer membrane in response to apoptotic signals. This interaction facilitates the release of cytochrome c into the cytoplasm, thereby promoting apoptosis. VDAC1 works synergistically with Bax to increase the permeability of the mitochondrial outer membrane, jointly fostering the apoptotic process. Reduction of VDAC1 expression via RNA interference significantly diminishes Bax activation, and inhibits mitochondrial outer membrane permeability, thereby precluding cytochrome c release and caspase 3 activation [29–31]. This ultimately leads to suppressed cell apoptosis. Notably, our data reveal that overexpression of COX6A2 increases the interaction between VDAC1 and Bax (Fig. 4C), enhancing mitochondrial translocation of Bax, an effect reduced by VDAC1 inhibition (Fig. 4E). Furthermore, the knockdown of Bax counteracted the elevated levels of cleaved-caspase3 induced by COX6A2 overexpression (Fig. 4G). This underscores the critical role of Bax in COX6A2-mediated β-cell apoptosis. Nonetheless, the precise mechanisms by which COX6A2 affects VDAC1 and Bax expression require further investigation.
A study [17] has reported a decrease in the mRNA levels of Cox6a2 in islets from db/db mice. Contrarily, our findings show an increase in the COX6A2 protein levels in islets from db/db mice compared to the control group (Fig. 6B). The discrepancy may stem from differences in experimental conditions and the levels at which the Cox6a2 gene was assessed.
FXR is a transcription factor that regulates the transcriptional expression of target genes through epigenetic mechanisms [32]. We demonstrate that Cox6a2 is a target gene negatively regulated by FXR in β-cells. The following evidence supports this concept: First, FXR binding sites (FXRE) are present at the Cox6a2 promoter (Fig. 5A). Second, activation of FXR by CDCA or GW4064 reduces the expression of Cox6a2 mRNA and protein (Fig. 5H, I; Supplementary Fig. S5A, B). Third, a deficiency in FXR leads to increased Cox6a2 expression (Fig. 5C–F). A key finding of this study is that FXR knockdown promotes the recruitment of p300 to the Cox6a2 promoter (Fig. 5K), which results in increased ACH3K27 at the promoter (Fig. 5J). This acetylation is associated with enhanced transcription of the Cox6a2 gene, as ACH3K27 is known to be a marker of gene activation [24, 33]. Furthermore, the inability of FXR knockdown to increase Cox6a2 expression in the presence of p300 inhibitor (Fig. 5L) further underscores the critical role of p300 in mediating FXR-dependent regulation of Cox6a2 expression.
Previous studies have indicated that FXR can suppress hepatocyte apoptosis [4]. In this research, we expand on these findings by demonstrating that FXR inhibits the apoptosis of β-cells by repressing the transcription of Cox6a2 in diabetic situations. This hypothesis is supported by several lines of evidence. First, FXR expression was found to be decreased in islets from diabetic animals (Fig. 6A-C). Correspondingly, FXR deficiency resulted in increased β-cell apoptosis both in vivo and in vitro (Fig. 6E, F; Supplementary Fig. S8A, B). Conversely, the overexpression of FXR led to reduced β-cell apoptosis under lipotoxic conditions (Supplementary Fig. S8D, E). Additionally, while the inhibition of FXR induced apoptosis in scramble INS-1 832/13 cells, it did not have the same effect in Cox6a2 knockdown cells (Fig. 6M). This suggests that COX6A2 may mediate FXR-regulated β-cell apoptosis.
Collectively, our findings indicate that increased COX6A2 levels may enhance β-cell apoptosis through the modulation of VDAC1-mediated cytochrome c release from the mitochondria. This rise in COX6A2 expression might result from the reduced expression of FXR in diabetes. Diabetic conditions likely drive β-cell apoptosis by modulating the FXR/p300/COX6A2 pathway. These insights reveal a novel regulatory mechanism for β-cell apoptosis and highlight COX6A2 as a possible therapeutic target for type 2 diabetes.