2025 Theses Doctoral

The TRIB1-C/EBPa axis in adipocytes

Hu, Krista Yumin

Cardiometabolic disease (CMD) remains the leading cause of death in the world. Central obesity, characterized by increased visceral adiposity, as well as elevated plasma cholesterol and triglycerides are highly heritable risk factors for CMD and subsequent mortality. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms in the 8q24 locus near the gene TRIB1 that associate with coronary artery disease, plasma lipid traits, and adiponectin, a metabolically beneficial adipocyte-specific protein, pointing to an adipocyte-specific role for TRIB1 in metabolism. Additionally, TRIB1’s best known function is facilitating turnover of C/EBPa, a key adipogenic transcription factor. In my thesis, I investigate the regulatory axis of the pseudokinase TRIB1 and the transcription factor C/EBPa in adipose tissue to understand how we may harness this mechanism to target CMD risk factors.

Our previously established adipocyte-specific Trib1 knockout mice (Trib1_ASKO mice) have decreased plasma cholesterol and increased plasma adiponectin translation, and the mechanisms driving these traits are still unclear. Using tandem affinity purification with mass spectrometry, we generated a list of potential binding partners through which TRIB1 may be mediating effects on adiponectin or lipids. Concerning the cholesterol phenotype, crossing Trib1_ASKO mice to the human ApoB transgenic mouse model revealed that the decrease in plasma cholesterol appears to be in the HDL fraction. RNA-seq results suggest that HDL uptake or efflux may be affected in Trib1_ASKO adipocytes, but we have yet to uncover the complete mechanism. Functional assays for HDL metabolism as well as knockdown experiments to query the importance of the TRIB1 binding candidates are the focus of future studies.

TRIB1’s best known mechanism is in facilitating the turnover of C/EBPa, reflected in increased C/EBPa in Trib1_ASKO adipose. We attempted to generate an adipocyte-specific Trib1 and Cebpa double knockout model to understand if the increased C/EBPa was contributing to the Trib1_ASKO phenotype. Instead, we found that Cebpa_ASKO mice have a severe and specific loss of gonadal white adipose tissue (WAT) mass, the largest visceral WAT depot in mice, while other WAT depots remain, pointing to a depot-specific function of C/EBPa.

We turned our focus to investigating depot-specific roles of adipocyte C/EBPa. Histology analysis showed that Cebpa_ASKO gonadal WAT (gWAT) development diverges from controls as early as between postnatal days 7 and 11, pointing to a requirement for expression of C/EBPa and its targets during this window. Meanwhile, inguinal WAT (iWAT) appears to develop normally, but there are fewer and larger adipocytes in the fat pad compared to controls. Bulk RNA-seq analysis suggested that the existing iWAT is dysfunctional with downregulation of lipolysis genes, and experimental validation in vivo and in vitro confirmed that Cebpa_ASKO adipocytes have reduced lipolysis function. RNA-seq data also points to decreased PPARg activity, but whether this is driving the dysfunction alongside Cebpa loss in Cebpa_ASKO iWAT remains to be seen. Lastly, Cebpa_ASKO mice cannot expand any of their WAT depots upon high fat diet (HFD) feeding, consistent with the idea that all Cebpa_ASKO WAT is dysfunctional. Regardless, these findings show that C/EBPa plays a specific and crucial role in development of visceral gWAT, while other WAT depots can develop but need C/EBPa for normal adipocyte function.

Cebpa_ASKO mice also have many other metabolic traits. Cebpa_ASKO BAT is lipid laden and enlarged, which may be from ectopic lipid deposition or could suggest that C/EBPa acts as a brake for whitening in BAT. Cebpa_ASKO mice also have larger livers with increased hepatic lipid deposition as evidenced by lipid assays, histology, and RNA-seq, which progresses upon HFD feeding. Further studies will investigate whether these phenotypes are a result of the loss of gWAT mass, but our current findings underscore the importance of having functional WAT.

In summary, we have investigated the functions of adipocyte TRIB1 and the depot-specific roles of adipocyte C/EBPa, shedding more light on the TRIB1-C/EBPa regulatory axis. We demonstrate that C/EBPa expression is required for visceral gWAT but not subcutaneous iWAT presence and now have a unique model to study the role of gWAT in whole body metabolism. Though we have many more questions to answer regarding TRIB1’s regulation of C/EBPa in various WAT depots, we have furthered our understanding of the differences between subcutaneous and visceral WAT. Our work points to the possibility of therapeutically targeting this regulatory axis to specifically alter visceral adipose tissue to avoid central obesity and reduce cardiometabolic disease risk.