In the human body, adipose tissue is present as both visceral and subcutaneous fat. Apart from other functions, its main role is associated with the storage of energy. In periods of food overabundance, human adult adipose tissue maintains its ability to increase the number of adipocytes suggesting that differentiation of preadipocytes can occur at any time in response to nutritional or hormonal stimulants .
A large body of evidence indicates that certain plant extracts and their respective bioactive components might have direct effects on adipose tissue . Based on these data, the overall objective of our studies was to investigate the effects of a particular White Tea extract solution on fat accumulation and the expression of adipocyte-specific genes in primary human preadipocyte and adipocyte culture.
In previous studies polyphenols as well as various natural extracts were shown to induce apoptosis, decrease lipid accumulation and stimulate lipolysis in preadipocytes and adipocytes [6, 13, 17]. These results, however, were mainly obtained studying 3T3-L1 cells. Although this mouse cell line represents a well-established model system to study fat metabolism , 3T3-L1 cells differ in various aspects from primary cells isolated from fat tissue. Furthermore, it has been widely accepted that significant differences in adipose tissue biology exist between species. To account for these known differences, we exclusively utilized primary cultured human cells for our experiments. Compared to murine 3T3-L1 cells, human preadipocytes and adipocytes should better reflect the human lipid metabolism and, hence, the human in vivo situation.
Natural White Tea represents the least processed form of tea and comprises high levels of EGCG and several other polyphenols such as epigallocatechin and epicatechin as well as the methylxanthines theobromine and caffeine.
As our data show, incubation with White Tea extract solution decreases triglyceride incorporation in human preadipocytes during adipogenesis (Figure 1A, B). Importantly, this significant reduction in triglyceride accumulation is not due to toxic side effects (Figure 1C, D).
One possible explanation for the observed attenuation in triglyceride incorporation during adipogenesis could be the ability of White Tea extract solution to stimulate lipolytic activity as evidenced by an increased conversion of triglyceride to fatty acids and glycerol. Interestingly indeed, by measuring glycerol release we were able to demonstrate that White Tea extract solution augments lipolysis-activity in differentiated adipocytes (Figure 2). However, the degree of lipolysis-activity induced by White Tea extract solution is not sufficient enough to fully explain the observed reduction in triglyceride incorporation. In addition to this effect one could hypothesize that White Tea extract solution also inhibits adipogenesis on a molecular level. Accordingly, we investigated the effects of White Tea extract solution on the expression pattern of ADD1/SREBP-1c an essential adipogenesis-related transcription factor. ADD1/SREBP-1c is highly expressed in the liver and in adipose tissue and plays an important role in adipocyte differentiation . ADD1/SREBP-1c mRNA levels are increased in determined preadipocytes and to a greater extend augmented during the differentiation process . Interestingly, ADD1/SREBP-1c has been shown to promote PPARγ expression [1, 2] further stressing its physiological relevance.
To study ADD1/SREBP-1c regulation in more detail, we investigated its protein expression following treatment with White Tea extract solution. Compared to differentiated adipocytes, that display an augmented ADD1/SREBP-1c expression (control, Figure 3A), cells incubated in the presence of White Tea extract solution show only a weak ADD1/SREBP-1c signal (Figure 3B). This observation together with quantitative analysis (Figure 3C–E) clearly shows that White Tea extract solution influences adipocyte differentiation.
Beside ADD1/SREBP-1c, several transcription factors are involved in orchestrating adipocyte differentiation [2, 3]. Briefly, adipogenesis is initiated by the transient expression of C/EBPβ and C/EBPδ. In response to adipogenic signals, these transcription factors lead to the activation of PPARγ which in turn stimulates the expression of C/EBPα. C/EBPα again exerts positive feedback on PPARγ to maintain the differentiation process .
To gain more mechanistic insights into the signalling cascade that is stimulated by White Tea extract solution, we investigated the expression of the essential transcription factors PPARγ, ADD1/SREBP-1c, C/EBPα, C/EBPβ and C/EBPδ. Interestingly, our results illustrate that cells incubated with White Tea extract solution display a decrease in PPARγ, ADD1/SREBP-1c, C/EBPα and C/EBPδ mRNA levels during adipogenesis while the expression of C/EBPβ mRNA was not affected (Figure 4). Overall, the observed reduction of adipogenesis-related transcription factors supports the notion that White Tea extract solution acts on two different levels, by increasing lipolysis and by inhibiting adipogenesis.
However, it should be noted that the results presented here were achieved using subcutaneous human (pre)-adipocytes. When using visceral human (pre)-adipocytes, neither White Tea extract solution nor EGCG significantly reduced triglyceride accumulation (data not shown). One explanation for this phenomenon could be that PPARγ activity is considerably lower in primary human visceral adipocytes  compared to subcutaneous adipocytes. Since our results show that White Tea extract solution acts by decreasing PPARγ mRNA expression in subcutaneous (pre)-adipocytes these data might explain the different actions observed in subcutaneous and visceral cells.
Another interesting facet that is a matter of intense research is the role of sirtuins in fat metabolism. Sirtuins belong to a family of enzymes implicated in apoptosis and fatty acid metabolism, to name two physiological functions. Mammalian sirtuins comprises of the seven members, Sirt1 – Sirt7 . Sirt1 regulates adipogenesis by inhibiting the expression of genes that control adipocyte differentiation and also triglyceride accumulation in 3T3-L1 cells. Sirt1 over-expression results in a reduction of PPARγ, C/EBPα and C/EBPδ but not C/EBPβ mRNA indicating that Sirt1 functions as a repressor of genes that control adipocyte differentiation .
Along these lines it is interesting to note that the established Sirt1 activator resveratrol, a polyphenol, inhibits adipogenesis in maturing 3T3-L1 preadipocytes . Accordingly, it appears plausible that polyphenols present in White Tea extract solution could reduce adipogenesis by means of an increased Sirt1 enzyme expression.
To tackle this question we determined Sirt1 mRNA expression after stimulation with White Tea extract solution. Interestingly, our data showed a slight reduction in Sirt1 gene expression suggesting that the polyphenols present in White Tea extract solution do not reduce adipogenesis via modulation of Sirt1 levels (Figure 5). The observed decrease in Sirt1 mRNA levels could be explained by the fact that Sirt1 expression is per se increased during fat cell differentiation . Therefore, a decrease in adipogenesis induced by White Tea extract solution could indirectly affect Sirt1 mRNA expression.
Moreover, no increase in Sirt1-activity was detected in the presence of 0.5% White Tea extract solution using a cell free Sirt1 activity assay (unpublished data). However, further studies need to be conducted in the future to elucidate the exact mechanism in more detail and to corroborate this finding.
Our results discussed above show that treatment of primary human (pre)-adipocytes with White Tea extract solution modulates both adipogenesis and lipolysis. These White Tea extract solution-induced effects are associated with a decrease in the expression of adipogenesis-associated transcription factors as well as a diminished gene expression of Sirt1. One possible explanation to account for these rather complex effects could be the fact that White Tea extract solution is composed of several active ingredients, among them polyphenols and methylxanthines.
To elucidate the possible effects of polyphenols in more detail, we investigated the influence of EGCG on triglyceride accumulation during differentiation in human pre-adipoyctes. EGCG represents the most abundant catechin present in White Tea extract solution. In that context it is important to remark that conflicting data have been published with respect to the effects of EGCG on (pre-)adipocytes. While EGCG inhibits adipogenesis in 3T3-L1 cells  it did not affect the conversion of preadipocytes to adipocytes in human AML-1 cells . Our results using human primary preadipocytes show that EGCG reduces triglyceride incorporation during adipogenesis (Figure 6). Considering our hypothesis the EGCG-mediated decrease in triglyceride accumulation might be due to an EGCG-induced inhibition of adipogenesis.
Previous studies also showed that EGCG did not promote lipolysis in both 3T3-L1  and C3H10T1/2 cells . These observations indicate that the stimulated lipolytic activity induced by White Tea extract solution might not be mediated by EGCG.
On the other hand, methylxanthines have been reported to stimulate triglyceride conversion by inhibiting phosphodiesterase 3B activity and subsequently increasing cAMP levels [18, 19]. Unpublished data from our lab show that caffeine (5 mM) increases lipolysis-activity in human adipocytes. Therefore, the observed White Tea-mediated increase in lipolysis-activity might, at least in part, be caused by methylxanthines.
Adding more to the complexity, EGCG not only influences triglyceride accumulation during adipogenesis but also the process of apoptosis. In both 3T3-L1 and AML-1 cells stimulation with EGCG induced apoptotic cell death [15, 16]. Our observation that the viability of primary human preadipocytes is reduced in the presence of EGCG appears to be in line with these studies. However, treatment with White Tea extract solution did not impair cell viability. Although we did not specifically study apoptosis, it can be speculated that other natural substances present in White Tea extract solution might counteract the EGCG-mediated reduction of cell viability.