Background: Adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. The mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models (i.e. 3T3-L1 and 3T3-F442A), revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors (C/EBPβ, C/EBPα and PPARγ). However, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells (hBMSCs), remain poorly understood. This study investigated cell cycle regulation and the roles of C/EBPβ, C/EBPα and PPARγ during adipocyte differentiation from hBMSCs. Results: Utilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPβ, C/EBPα and PPARγ were required for adipocyte differentiation from hBMSCs. C/EBPβ and C/EBPα individually induced adipocyte differentiation in the presence of inducers; PPARγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse. Conclusions: The characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.

Although obesity is associated with low-grade inflammation and metabolic disorders, clinical studies suggested some obese people were metabolically healthy with smaller adipocyte size compared with metabolically abnormal obese (MAO). This indicated adipocyte size may be an important predictor underlay the distinction between MAO and metabolically healthy obese. As recent study has shown that adipocytes expressed class II major histocompatibility complex (MHCII), which functioned as APCs during obesity. However, the relationship between adipocyte hypertrophy and MHCII expression was not involved. Here we hypothesize that hypertrophic adipocytes could be associated with upregulating MHCII to influence adipose tissue metabolism. Adipocytes were sorted by fluorescence-activated cell sorting (FACS) according to the cell size from MAO mice. The activation of MHCII, T cells and related signaling molecules were examined by FACS, ELISA and western blotting. 3T3-L1 cell line and primary adipocytes were used to examine the effect of free fatty acids (FFA) on adipocytes enlargement and MHCII expression. MAO mice had a significant increase in adipocytes size and FFA concentration. The large adipocytes from both obese and non-obese mice expressed higher levels of MHCII than small adipocytes. Importantly, large adipocytes from obese mice stimulated CD4⁺ T cells to secrete more interferon (IFN)-γ. Furthermore, the activation of the JNK-STAT1 pathway was involved in upregulation of MHCII in large adipocytes. In vitro FFA treatment promoted adipocyte hypertrophy and expression of MHCII-associated genes. This study demonstrates that large adipocytes highly express MHCII and function as APC to stimulate IFN-γ-expressing CD4⁺ T cells, in which FFA may have important roles before IFN-γ elevated. These findings suggest that adipocyte hypertrophy, rather than overall obesity, is the major contributor to adipose tissue inflammation and insulin resistance.

Background: Exposure to zearalenone (ZEN, a widespread Fusarium mycotoxin) causes reproductive toxicity and immunotoxicity in farm animals, and it then poses potential threats to human health through the food chain. A systematic understanding of underlying mechanisms on mycotoxin-induced toxicity is necessary for overcoming potential threats to farm animals and humans. The gastrointestinal tract is a first-line defense against harmful mycotoxins; however, it remains unknown whether mycotoxin (e.g., ZEN)-induced toxicity on the reproductive-immune axis is linked to altered gut microbial metabolites. In this study, using pigs (during the three phases) as an important large animal model, we investigated whether ZEN-induced toxicity on immune defense in the reproductive-immune axis was involved in altered gut microbial-derived metabolites. Moreover, we observed whether the regulation of gut microbial-derived metabolites through engineering ZEN-degrading enzymes counteracted ZEN-induced toxicity on the gut-reproductive-immune axis. Results: Here, we showed ZEN exposure impaired immune defense in the reproductive-immune axis of pigs during phase 1/2. This impairment was accompanied by altered gut microbial-derived metabolites [e.g., decreased butyrate production, and increased lipopolysaccharides (LPS) production]. Reduction of butyrate production impaired the intestinal barrier via a GPR109A-dependent manner, and together with increased LPS in plasma then aggravated the systemic inflammation, thus directly and/or indirectly disturbing immune defense in the reproductive-immune axis. To validate these findings, we further generated recombinant Bacillus subtilis 168-expressing ZEN-degrading enzyme ZLHY-6 (the Bs-Z6 strain) as a tool to test the feasibility of enzymatic removal of ZEN from mycotoxin-contaminated food. Notably, modified gut microbial metabolites (e.g., butyrate, LPS) through the recombinant Bs-Z6 strain counteracted ZEN-induced toxicity on the intestinal barrier, thus enhancing immune defense in the reproductive-immune axis of pigs during phase-3. Also, butyrate supplementation restored ZEN-induced abnormalities in the porcine small intestinal epithelial cell. Conclusions: Altogether, these results highlight the role of gut microbial-derived metabolites in ZEN-induced toxicity on the gut-reproductive-immune axis. Importantly, targeting these gut microbial-derived metabolites opens a new window for novel preventative strategies or therapeutic interventions for mycotoxicosis associated to ZEN.

Chemerin restricts cold-induced IL-33 production by beige adipocytes, impairing beige fat’s ability to dissipate excess energy stores.

CCAAT enhancer binding protein β (C/EBPβ) is required for both mitotic clonal expansion (MCE) and terminal differentiation during the 3T3-L1 adipocyte differentiation program. Whereas the mechanism of C/EBPβ during terminal differentiation is well understood, the mechanism of C/EBPβ in MCE is not. We provide evidence that histone H4, the most conserved cell cycle–related histone, the change of which is strictly correlated with DNA content change during the cell cycle, is transcriptionally activated by C/EBPβ during MCE. Expression of histone H4 is increased at 16 h after induction when 3T3-L1 preadipocytes synchronously reenter S phase, which is correlated with the sequential phosphorylation and activation of C/EBPβ, and expression was partially suppressed when A-C/EBP (dominant negative for C/EBP protein) was overexpressed. One C/EBP-binding site was identified in one of the histone H4 gene promoters (hist4h4), confirmed by both electrophoretic mobility shift assay and chromatin immunoprecipitation assay. C/EBP-binding sites were also found in 9 of 11 other histone H4 promoters, which can also be transactivated by C/EBPβ. Knockdown of C/EBPβ by stealth small interfering RNA partially decreased H4 gene expression and arrested cells in G1 phase as indicated by bromodeoxyuridine incorporation and fluorescence-activated cell sorting analysis of DNA content. This study provides new insights into why C/EBPβ is required for MCE during 3T3-L1 adipocyte differentiation and why C/EBPβ plays important roles in the proliferation of other cell types.