The key finding from this work is that BME, a thiol donor and radical scavenger, induced the adipogenic program in murine F442A preadipocytes, evidenced by increased protein expression of transcription factors PPARgamma and C/EBPalpha as well as the mRNA expression of their downstream target genes and established markers for adipocyte differentiation, including aP2, SCD-1, LPL, and Glut4. In association with its pro-adipogenic effect, BME rapidly reduced expression of inflammatory cytokines known to be downstream of NFkappaB, including MCP-1, IL-6, and iNOS. This effect was found to occur early during induction of adipogenic gene expression. Furthermore, BME interacted with exogenously added TNFalpha, a strong inducer for NFkappaB activation, partially blunting the effect of each other on adipocyte differentiation. These findings are not entirely surprising as the master transcription factors for adipogenesis (PPARgamma) and for inflammation (NFkappaB) are both thio-regulated proteins and they are also mutually inhibiting 
. Mathematical modeling has predicted that whether a preadipocyte will differentiate or not is dependent on a dynamic interplay between PPARgamma and NFkappaB 
. As expected, our data demonstrated that BME suppressed NFkappaB and activated PPARgamma both within the cell context of differentiating preadipocytes and in a non-adipogenic cell type. This would explain the findings of BME-mediated reduction of cytokine expression and up-regulation of adipogenic genes in differentiating preadipocytes.
Although NFkappaB is well known for its sensitivity to redox changes, how it is regulated by BME remains not completely understood. It has been shown that an increase in intracellular GSH inhibits TNFalpha-induced IkappaBalpha phosphorylation 
, which is in good agreement with our current findings. However, there is also evidence that GSH can regulate NFkappaB activity through IkB-independent pathways 
. In agreement with these prior studies, we also noticed that exposure to TNFalpha for 15 min induced a similarly rapid loss of IkappB protein in both control and BME-treated cells, but phospho-p65 was markedly increased only in the control cells, implying additional mechanism by which BME impairs TNFalpha-induced phosphorylation (activation) of p65.
In addition to regulation of NFkappaB activity, changes in redox state can directly modulate other transcription factors and functional proteins, alter endoplasmic reticulum (ER) homeostasis, and even chromatin remodeling 
, all may have an effect on adipogenic differentiation. Many studies have documented that preadipocyte differentiation is inhibited by oxidant stress caused by either cytokines or free radicals 
, in a way similar to our findings with BME. However, others reported controversial findings 
. Of note, while BME is known to promote the reduction of cysteine to cystine, which is an important mechanism for intracellular GSH elevation, intracellular GSH levels and the GSH/GSSG ratio may increase or decrease
with the addition of extracellular BME 
. In this work, we have not measured intracellular GSH or GSH/GSSG ratio in part because of the technical difficulty to prevent BME contamination to the cell lysates. By co-addition of BME with buthionine sulfoximine (BSO, 0.2–2 mM), an inhibitor for GSH synthase 
, we found no suppression of the pro-adipogenic effect of either (data not shown). Indeed, we found that BSO alone enhanced adipocyte differentiation (data not shown) which is in agreement with others’ reports 
. Therefore, with the current data, we cannot draw a conclusion as to whether and how the changes in GSH or GSH/GSSG ratio per se
mediate the BME-induced adipocyte differentiation under our experimental conditions.
Another interesting observation from this work is that we found a dramatic increase of adiponectin expression in cells treated with BME, an effect that was largely blocked by co-treatment with TNFalpha. While this effect could be secondary to the changes in adipocyte differentiation, BME may also have specific effects on this adipokine. It has been shown that adiponectin oligomerization is redox-dependent 
. Whether this has any regulatory effect on its gene expression is not known. Besides, expression of leptin, another adipokine whose expression typically increases with differentiation, was found to respond to BME and TNFalpha in a very different manner from that of adiponectin. Hence, the changes in expression of these two adipokines may not be simply reflective of an overall stage of adipogenic differentiation. To date, adiponectin is one of the very few adipokines identified as positive regulators for systemic redox regulation, metabolism, and anti-inflammation 
. Studies in humans have shown that short-term supplementation with antioxidant vitamins increases systemic adiponectin levels in both lean and obese subjects 
. Our findings of BME-induced increase in expression of this anti-inflammatory adipokine coupled with its effect on expression of inflammatory cytokines and adipogenic genes can have useful clinical implications and is worth of further investigation.
In summary, this work provided novel evidence that BME, a thiol compound that may alter cellular redox state and scavenge reactive oxygen species, induced adipogenic differentiation in murine F442A preadipocytes, coupled with reduced expression of inflammatory cytokines and increased expression of anti-inflammatory adipokines. This effect was blunted by TNFalpha and nearly completely blocked by high concentration of TNFalpha. We suggest that BME may promote adipogenesis through its reciprocal effects on the master transcription factors NFkappaB and PPARgamma. Since “metabolically dysfunctional” obesity and aging are two well-established physiological conditions known to increase fat tissue inflammation and reduce preadipocyte differentiation 
, and in light of recent studies showing that supplementation of BME in drinking water improves metabolic health and extends longevity in mice 
, it will be interesting to test our in vitro findings in animal models with impaired fat tissue plasticity, such as those with metabolic syndrome or at late stage of aging.