Obesity is a significant health concern, characterized by excessive lipid storage in adipose tissue, leading to metabolic disorders like type 2 diabetes, cardiovascular disease, and certain cancers. Adipose tissue expansion involves adipocyte hypertrophy (enlargement) and hyperplasia (increased number) through adipogenesis. Understanding the molecular mechanisms of adipogenesis is crucial for developing obesity treatments. While transcriptional factors regulating adipogenesis are well-studied, post-transcriptional regulation remains less understood. RNA-binding proteins (RBPs) play a vital role in mRNA processing, impacting stability, translation, and decay. Several RBPs, such as SFRS10, Sam68, KSRP, IMP2, and PSPC1, have been implicated in adipocyte regulation by affecting various aspects of RNA processing. However, the functions of most RBPs in adipocytes remain largely unexplored. HuR (ELAVL1) is a ubiquitously expressed RBP that binds to AU-rich elements (AREs) in the 3' untranslated regions (UTRs) of target mRNAs, often enhancing mRNA stability and translation. HuR's roles in various cell types have been established, including its essentiality for hematopoietic progenitor cell survival, T cell function, B cell responses, and regulation of inflammation. Despite extensive research, HuR's functions in major metabolic tissues remain largely unknown. This study investigates the role of HuR in adipogenesis, specifically addressing the gap in understanding its physiological function in metabolic tissues.

Previous research has identified numerous transcriptional factors that control various aspects of adipogenesis by regulating downstream genes at the transcriptional level. However, the post-transcriptional regulation of adipogenesis remained less understood. Studies have shown that several RNA-binding proteins (RBPs) modulate adipocyte development and lipid metabolism by influencing alternative splicing (SFRS10 and Sam68), reducing microRNA expression (KSRP), or inhibiting translation efficiency (IMP2). PSPC1 has been shown to facilitate the export of adipocyte RNAs from the nucleus, contributing to adipogenesis. Ybx2 has also been identified as a regulator of brown adipose tissue activation through mRNA stability control. However, most RBPs' roles in adipogenesis were still under-investigated. This research aims to establish a more complete picture of post-transcriptional regulation in adipogenesis by investigating the function of HuR, a key RBP involved in several cell types' development and function.

The study utilized both *in vitro* and *in vivo* approaches to investigate HuR's role in adipogenesis. *In vitro*, primary brown and white preadipocytes were isolated from mice and humans. HuR was knocked down using retroviruses expressing shRNAs, and conversely, HuR was overexpressed using retroviral vectors. Adipogenesis was assessed by measuring the expression of adipogenic marker genes via real-time PCR and by evaluating lipid accumulation using Oil Red O staining. For *in vivo* studies, mice with a fat-specific deletion of the HuR gene (HuR-FKO) were generated by crossing *Hur^flox/flox* and *Adipoq-Cre* mice. Control littermates (*Hur^flox/flox*; Cre-) were also used. Body composition was analyzed using EchoMRI, and glucose and insulin tolerance tests were performed. RNA sequencing (RNA-seq) was conducted on epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) from HuR-FKO and control mice to identify differentially expressed genes. Gene Set Enrichment Analysis (GSEA) was used to identify enriched pathways. To determine HuR's mechanism of action, RNA immunoprecipitation followed by sequencing (RIP-seq) was performed on eWAT to identify HuR's target mRNAs. Ribosome profiling was used to assess the impact of HuR on translational efficiency. The interaction between HuR and Insig1 mRNA was confirmed using RIP-PCR and RNA pull-down assays. The effect of HuR on Insig1 mRNA stability was investigated by measuring the decay rate of Insig1 mRNA in the presence and absence of HuR using actinomycin D to inhibit transcription. Furthermore, functional interactions between HuR and Insig1 were examined through simultaneous overexpression of HuR and knockdown of Insig1 during adipogenesis.

The study revealed that HuR acts as a repressor of adipogenesis. *In vitro*, HuR knockdown in primary brown and white preadipocytes significantly enhanced adipogenesis, whereas HuR overexpression strongly inhibited it. These results were consistent in both mouse and human adipocytes. *In vivo*, fat-specific HuR knockout (HuR-FKO) mice exhibited significantly increased fat mass, primarily in the epididymal white adipose depot, accompanied by glucose intolerance and insulin resistance. RNA-seq and GSEA analyses of eWAT from HuR-FKO mice showed an enhanced adipogenesis pathway and upregulated inflammatory response pathways. In iWAT, HuR knockout promoted both adipogenesis and browning, indicated by increased expression of brown fat markers such as Ucp1. In BAT, HuR knockout repressed the myogenesis program, leading to enhanced brown adipogenesis. Interestingly, the effects of HuR knockout were depot-specific; eWAT displayed enhanced inflammation, iWAT showed increased browning, and BAT showed repressed myogenesis. Mechanistically, RIP-seq identified hundreds of HuR target mRNAs, including Insig1, a known negative regulator of adipogenesis. HuR was shown to bind to and stabilize Insig1 mRNA. Knockdown of Insig1 attenuated the pro-adipogenic effects of HuR overexpression, further supporting Insig1 as a critical downstream target of HuR. Analysis of mRNA decay rates demonstrated that HuR significantly enhances Insig1 mRNA stability. The study suggests that HuR’s repressive effect on adipogenesis is at least partly mediated through its stabilization of Insig1 mRNA.

This study provides strong evidence for HuR's role as a negative regulator of adipogenesis. The consistent findings from *in vitro* and *in vivo* experiments demonstrate that HuR's downregulation is associated with enhanced adipogenesis and metabolic dysfunction. The identification of Insig1 as a key target of HuR's post-transcriptional regulatory action provides a mechanistic explanation for these observations. The depot-specific effects highlight the complexity of adipose tissue regulation and suggest that HuR's influence may vary depending on the specific adipose depot. The findings have implications for understanding the intricate interplay between RNA processing and metabolic regulation. Further research into HuR's other targets and how it affects RNA processing beyond mRNA stability could provide further insights into the mechanisms of adipogenesis and obesity.

This research establishes HuR as a critical post-transcriptional regulator of adipogenesis, acting as a repressor by stabilizing Insig1 mRNA. The depot-specific effects of HuR knockout suggest a complex role in adipose tissue homeostasis. Future studies should focus on identifying additional HuR targets and investigating the detailed mechanisms of its function in various adipose depots. Understanding these mechanisms could potentially lead to novel therapeutic strategies for obesity and related metabolic disorders.

The study primarily focused on male mice, limiting the generalizability of the findings to female mice. The use of AdipoQ-Cre resulted in HuR deletion in all adipose tissues, making it challenging to isolate the specific contribution of HuR in each depot. While Insig1 was identified as a key target, it is likely that HuR affects many other targets contributing to the overall phenotype. Further research is needed to completely elucidate the mechanisms of HuR-mediated regulation of adipogenesis and its depot-specific effects.