White adipose tissue (WAT) plays a crucial role in systemic energy homeostasis. Adipocyte dysfunction, particularly in visceral fat, is linked to metabolic disorders. Short-term caloric excess leads to WAT expansion via hypertrophy and hyperplasia (neoadipogenesis). Chronic inflammation, however, impairs neoadipogenesis, contributing to metabolic dysfunction. The molecular mechanisms regulating VA-MSC plasticity under chronic inflammation remain largely unexplored. This study aimed to identify conserved molecular pathways controlling the adipogenic potential of visceral MSCs.

Previous research on adipogenic differentiation has largely focused on mouse embryonic fibroblasts and committed preadipocytes, which do not fully represent the multipotent nature of primary adipose MSCs. While the modulatory effects of pro-inflammatory cytokines on terminal adipogenic differentiation have been studied in various committed preadipocytes (e.g., 3T3-L1), their impact on the adipogenic potential of VA-MSCs remains largely unknown. The effects of long-term IFNγ exposure on JAK-STAT pathway activation, excluding STAT1, also needed further investigation. Similarly, the regulation and natural function of TGFβ signaling during VA-MSC differentiation remained unexplored, despite evidence of its role in inhibiting adipogenesis in mouse models. The potential for cross-regulation between IFNγ and TGFβ pathways during adipogenic inhibition under chronic inflammatory conditions had not been fully elucidated.

Human and mouse VA-MSCs were isolated from visceral adipose tissue samples. An in vitro adipogenic differentiation screen was performed using 14 cytokines and growth factors to identify inhibitors of differentiation. The roles of IFNγ and TGFβ were investigated using various techniques including Oil Red staining, triglyceride assays, Western blotting, qRT-PCR, immunofluorescence, flow cytometry, co-immunoprecipitation, and siRNA knockdown. A chronic high-fat diet-fed murine model was used to study the in vivo effects of IFNγ signaling on visceral adipogenesis. Oral glucose tolerance tests (oGTT) and insulin secretion measurements were performed to assess systemic metabolic effects. Specific inhibitors for STAT proteins (STAT5i, fludarabine, stattic), JAK2 (ruxolitinib), and TGFβR1 kinase (galunisertib) were employed.

IFNγ and TGFβ were identified as potent inhibitors of VA-MSC adipogenic differentiation. IFNγ primarily impacted early adipogenic commitment by suppressing C/EBPβ and PPARγ expression. Chronic IFNγ exposure activated JAK2, STAT1, STAT3, and STAT5. STAT5, but not STAT1 or STAT3, was essential for IFNγ's anti-adipogenic effects. TGFβ also inhibited adipogenesis by suppressing C/EBPβ and PPARγ. IFNγ enhanced pSmad3 levels, and inhibiting Smad3 rescued adipogenesis. STAT5 and Smad3 physically interacted via Smad4, forming a complex that prevented their degradation and sustained the suppression of adipogenesis. This interaction was confirmed through co-immunoprecipitation assays. In a murine model, IFNγ receptor 1 knockout mice exhibited improved glucose tolerance and increased neoadipogenesis compared to wild-type mice on a high-fat diet.

This study demonstrates that IFNγ and TGFβ synergistically inhibit VA-MSC adipogenic differentiation via a novel STAT5-Smad3-Smad4 complex. This complex protects itself from proteasomal degradation, thus sustaining the inhibition of adipogenesis. The results highlight the importance of considering both acute and chronic inflammatory responses and the intricate interplay between multiple signaling pathways in regulating MSC plasticity. The identification of STAT5 and Smad3 as key regulatory factors opens up potential therapeutic avenues by targeting these pathways to restore adipogenesis in metabolic disorders and wasting syndromes.

The study revealed a critical mechanism by which chronic inflammation impairs adipogenesis through a STAT5-Smad3 dyad interacting through Smad4. Targeting this pathway may offer therapeutic strategies for metabolic dysfunction and wasting syndromes. Future research should investigate the precise mechanisms of STAT5-Smad3 interaction and the potential of specific inhibitors for therapeutic applications.

The study primarily used in vitro models and one in vivo model. Further in vivo studies are needed to confirm the findings and assess the long-term effects of targeting STAT5 and Smad3. The study focused on visceral adipose tissue, and the findings might not be directly generalizable to other adipose depots. The high concentrations of IFNγ used in some in vitro experiments might not fully reflect physiological conditions.