GDF15 expression decreases in the early stages of adipocyte differentiation
We conducted animal experiments to investigate GDF15 expression during adipocyte proliferation and differentiation in response to obesity. One group of mice was fed a normal diet (ND), whereas in the other group, obesity was induced using a high-fat diet (HFD) fed for 8 weeks (Fig. 1A; Supplementary Fig. S1). The findings revealed no significant differences in feed intake between the two groups. Upon examining the expression level of FABP4, an adipocyte differentiation marker, in epididymal white adipose tissue (eWAT), it was found to be higher in the HFD group compared to the ND group (Supplementary Fig. S1) (Supplementary Fig. S1). GDF15 expression in eWAT monitored every 2 weeks showed significantly decreased expression in the HFD group than that in the ND group on week 4, which increased afterward from week 6 (Fig. 1B, C). On the contrary, GDF15 expression in inguinal white adipose tissue remained unchanged by week 4 but increased on week 8 (Supplementary Fig. S1). Furthermore, on week 4, GDF15 level decreased significantly in the blood samples of the HFD group compared to that in those of the ND group; however, on week 8, no significant difference was observed between the two groups (Supplementary Fig. S1). The findings showed that GDF15 expression decreased during visceral fat proliferation and differentiation in the early stages of obesity.
We confirmed these results using human adipose-derived stem cells (ADSCs). Estimation of the mature GDF15 levels in the differentiation media (DM) revealed a decrease in GDF15 levels until day 8, followed by an increase (Fig. 1D). Furthermore, the expression levels of GDF15 in cells decreased 6 h after culture in DM, whereas the GDF15 mRNA levels remained unchanged (Fig. 1E), which could be attributed to post-translational modifications.
GDF15 levels decrease through autophagy–lysosomal degradation
Autophagy significantly affects adipocyte differentiation14,22; therefore, we investigated whether autophagy-mediated degradation is associated with the observed decreased level of GDF15 during ADSC differentiation. We treated the cells with an autophagy inhibitor, including CQ, NH4Cl, Leupeptin, Pepstatin, and E-64, which restored the decrease in GDF15 expression during the treatment of human ADSCs with DM (Fig. 2A). However, the autophagy inhibitor induced no changes in the expression of GDF15 mRNA (Supplementary Fig. S2). Next, we investigated whether the degradation of GDF15 was mediated by lysosomes using LysoSensor staining and fluorescence-activated cell sorting analysis. The results showed an approximately 1.3-fold increase in lysosomal activity with the progression of differentiation (Supplementary Fig. S2).
The ubiquitin-proteasome system (UPS) is another major cellular pathway involved in protein degradation. To understand whether the UPS contributed to GDF15 degradation, we treated human ADSCs with the proteasome inhibitor bortezomib. The results showed that bortezomib restored the reduced GDF15 levels (Supplementary Fig. S2) and increased the GDF15 mRNA levels (Supplementary Fig. S2). These findings suggest that the increase in expression rather than the degradation was suppressed by the proteasome inhibitor. To confirm this, we treated cells with cycloheximide to stop protein biosynthesis, followed by the application of the proteasome inhibitor, which revealed no changes in the expression of GDF15 (Supplementary Fig. S2). Together, these findings demonstrated that proteasomal degradation is not the primary mechanism responsible for the decrease in GDF15 levels.
Therefore, we investigated whether the observed protein degradation involves lysosomal degradation. We administered chloroquine, an autophagy inhibitor, to prevent degradation of both Sequestosome1(SQSTM1), an autophagy marker and GDF15. Subsequently, we utilized immunocytochemistry to determine whether GDF15 and SQSTM1,co-localized (Fig. 2B and 2C). Through the same experimental method, staining of LAMP1, a lysosomal marker and GDF15 revealed enhanced correlation after DM treatment (Fig. 2D, E), suggesting that GDF15 is being targeted for degradation in the lysosomes. In addition, the knockdown of autophagy-related genes ATG5, ATG16L, and SQSTM1 increased the expression of GDF15 (Fig. 2F–H), indicating that autophagy plays the primary role in GDF15 degradation. Together, these findings demonstrated that GDF15 is degraded in the lysosomes through autophagy rather than being targeted for degradation by UPS.
GDF15 inhibits C/EBPa expression and adipocyte differentiation
Next, we examined the direct role of GDF15 in adipocyte differentiation by overexpressing GDF15 during the differentiation process and performing Oil Red O staining to visualize lipid accumulation. The results indicated a decrease in the number of stained cells (Fig. 3A). Quantification of absorbance from lysed stained cells further supported this observation, revealing a reduction in staining intensity by approximately 50% upon GDF15 overexpression (Fig. 3B).
Furthermore, we measured the mRNA levels of the genes associated with early adipocyte differentiation in cells overexpressing GDF15. The findings revealed a significant decrease in C/EBPa expression, whereas no changes were observed in the expression levels of C/EBPb, C/EBPd, and PPARg mRNAs (Fig. 3C–F). We induced adipocyte differentiation for 7 days, and when we checked the protein levels, the expression of key genes for adipocyte differentiation such as C/EBPa and PPARg decreased. Also, the expression of Perilipin, a gene important for lipid droplet formation, decreased as well (Fig. 3G).
The active form of GDF15 is known as mature GDF15 23. To verify whether the same results are obtained when the expression of GDF15 is increased or when treated with mature GDF15, we conducted experiments by treating recombinant GDF15 (Supplementary Fig. S3). When proGDF15 expression increases, mature GDF15 also increases simultaneously. Same as the previous experiment, when treated with recombinant GDF15, the mRNA level of C/EBPa decreases. Also protein level analysis further demonstrates inhibition of adipocyte differentiation. As a result, we have discovered that GDF15 can directly or indirectly suppress the expression of C/EBPa, thereby regulating adipocyte differentiation.
GDF15 inhibits C/EBPa expression by increasing HOP2 expression
Next, we investigated the direct inhibitory effect of GDF15 on C/EBPa expression. A recent study showed that the expression of the homologous chromosome pairing protein 2 (HOP2) represses the C/EBP-dependent transcriptional activation of reporter genes for all three C/EBP isoforms and suppresses adipocyte differentiation 24. In this study, we showed that GDF15 overexpression increased HOP2 expression (Fig. 4A). Analysis of the changes in HOP2 mRNA levels revealed increased HOP2 mRNA levels with increased GDF15 (Supplementary Fig. S4). Furthermore, the expression of C/EBPa was reduced by approximately 50% in HOP2-ovedrexpressing cells compared to that in the mock(Fig. 4B–E). Overexpression of HOP2 significantly reduced C/EBPa, PPARg on day 7 of differentiation (Fig. 4F).
The inhibition of adipocyte differentiation upon HOP2 overexpression was confirmed using Oil Red O staining (Fig. 4G). A decrease in the number of stained cells was observed in the overexpression group, and the staining intensity of lysed cells also decreased (Supplementary Fig. S4).
Finally, to determine whether the suppression of C/EBPa expression during adipogenesis was mediated by HOP2, we overexpressed GDF15 and simultaneously knocked down HOP2 using siHOP2 RNA. The results showed that the DM-induced increase in C/EBPa levels was suppressed with increased GDF15 expression, whereas HOP2 knockdown reversed this inhibition (Fig. 4H). Oil Red O staining also revealed similar results, suggesting that the inhibitory effect of GDF15 on adipocyte differentiation resulted from the enhancement of HOP2 expression (Fig. 4I; Supplementary S4). These findings demonstrated that GDF15 increases HOP2 expression, which consequently suppresses C/EBPa expression.