Lotus leaf extract and L-carnitine influence different processes during the adipocyte life cycle

<p>Abstract</p> <p>Background</p> <p>The cellular and molecular mechanisms of adipose tissue biology have been studied extensively over the last two decades. Adipose tissue growth involves both an increase in fat cell size and the formation of mature adipocytes from precursor cells. To investigate how natural substances influence these two processes, we examined the effects of lotus leaf extract (<it>Nelumbo nucifera</it>-extract solution obtained from Silab, France) and L-carnitine on human preadipocytes and adipocytes.</p> <p>Methods</p> <p>For our <it>in vitro </it>studies, we used a lotus leaf extract solution alone or in combination with L-carnitine. Utilizing cultured human preadipocytes, we investigated lotus leaf extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. Studies on human adipocytes were performed aiming to elucidate the efficacy of lotus leaf extract solution to stimulate lipolytic activity. To further characterize lotus leaf extract solution-mediated effects, we determined the expression of the transcription factor adipocyte determination and differentiation factor 1 (ADD1/SREBP-1c) on the RNA- and protein level utilizing qRT-PCR and immunofluorescence analysis. Additionally, the effect of L-carnitine on beta-oxidation was analyzed using human preadipocytes and mature adipocytes. Finally, we investigated additive effects of a combination of lotus leaf extract solution and L-carnitine on triglyceride accumulation during preadipocyte/adipocyte differentiation.</p> <p>Results</p> <p>Our data showed that incubation of preadipocytes with lotus leaf extract solution significantly decreased triglyceride accumulation during adipogenesis without affecting cell viability. Compared to controls, adipocytes incubated with lotus leaf extract solution exhibited a significant increase in lipolysis-activity. Moreover, cell populations cultivated in the presence of lotus leaf extract solution showed a decrease in adipocyte differentiation capacity as indicated by a decrease in the ADD1/SREBP-1c signal. Importantly, our results demonstrated that a combination of lotus leaf extract solution and L-carnitine reduced triglyceride accumulation to a greater extent compared to incubation with either substance alone.</p> <p>Conclusions</p> <p>Overall, our data demonstrate that a combination of lotus leaf extract and L-carnitine reduced triglyceride accumulation in human (pre)adipocytes by affecting different processes during the adipocyte life cycle. For this reason, this combination might represent a treatment option for obesity-related diseases.</p

White Tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre)-adipocytes

<p>Abstract</p> <p>Background</p> <p>The dramatic increase in obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. To investigate how natural substances influence lipolysis and adipogenesis, we determined the effects of White Tea extract on cultured human subcutaneous preadipocytes and adipocytes.</p> <p>Methods</p> <p>For our in vitro studies we used a White Tea extract solution that contained polyphenols and methylxanthines. Utilizing cultured human preadipocytes we investigated White Tea extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. In vitro studies on human adipocytes were performed aiming to elucidate the efficacy of White Tea extract solution to stimulate lipolytic activity. To characterize White Tea extract solution-mediated effects on a molecular level, we analyzed gene expression of essential adipogenesis-related transcription factors by qRT-PCR and determined the expression of the transcription factor ADD1/SREBP-1c on the protein level utilizing immunofluorescence analysis.</p> <p>Results</p> <p>Our data show that incubation of preadipocytes with White Tea extract solution significantly decreased triglyceride incorporation during adipogenesis in a dose-dependent manner (n = 10) without affecting cell viability (n = 10). These effects were, at least in part, mediated by EGCG (n = 10, 50 μM). In addition, White Tea extract solution also stimulated lipolytic activity in adipocytes (n = 7). Differentiating preadipocytes cultivated in the presence of 0.5% White Tea extract solution showed a decrease in PPARγ, ADD1/SREBP-1c, C/EBPα and C/EBPδ mRNA levels. Moreover, the expression of the transcription factor ADD1/SREBP-1c was not only decreased on the mRNA but also on the protein level.</p> <p>Conclusion</p> <p>White Tea extract is a natural source that effectively inhibits adipogenesis and stimulates lipolysis-activity. Therefore, it can be utilized to modulate different levels of the adipocyte life cycle.</p

Identification of New Genes Involved in Human Adipogenesis and Fat Storage

Since the worldwide increase in obesity represents a growing challenge for health care systems, new approaches are needed to effectively treat obesity and its associated diseases. One prerequisite for advances in this field is the identification of genes involved in adipogenesis and/or lipid storage. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. The primary screen showed that 459 genes affected adipogenesis and/or lipid accumulation after knock-down. Out of these hits, 333 could be validated in a secondary screen using independent siRNAs and 110 genes were further regulated on the gene expression level during adipogenesis. Assuming that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we performed InCell-Western analysis for the most striking hits to distinguish between the two phenotypes. Beside well known regulators of adipogenesis and neutral lipid storage (i.e. PPARγ, RXR, Perilipin A) the screening revealed a large number of genes which have not been previously described in the context of fatty tissue biology such as axonemal dyneins. Five out of ten axonemal dyneins were identified in our screen and quantitative RT-PCR-analysis revealed that these genes are expressed in preadipocytes and/or maturing adipocytes. Finally, to show that the genes identified in our screen are per se druggable we performed a proof of principle experiment using an antagonist for HTR2B. The results showed a very similar phenotype compared to knock-down experiments proofing the “druggability”. Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point to identify anti-obesity compounds targeting the adipose tissue

Identification of dihydromyricetin as a natural DNA methylation inhibitor with rejuvenating activity in human skin

Changes in DNA methylation patterning have been reported to be a key hallmark of aged human skin. The altered DNA methylation patterns are correlated with deregulated gene expression and impaired tissue functionality, leading to the well-known skin aging phenotype. Searching for small molecules, which correct the aged methylation pattern therefore represents a novel and attractive strategy for the identification of anti-aging compounds. DNMT1 maintains epigenetic information by copying methylation patterns from the parental (methylated) strand to the newly synthesized strand after DNA replication. We hypothesized that a modest inhibition of this process promotes the restoration of the ground-state epigenetic pattern, thereby inducing rejuvenating effects. In this study, we screened a library of 1800 natural substances and 640 FDA-approved drugs and identified the well-known antioxidant and anti-inflammatory molecule dihydromyricetin (DHM) as an inhibitor of the DNA methyltransferase DNMT1. DHM is the active ingredient of several plants with medicinal use and showed robust inhibition of DNMT1 in biochemical assays. We also analyzed the effect of DHM in cultivated keratinocytes by array-based methylation profiling and observed a moderate, but significant global hypomethylation effect upon treatment. To further characterize DHM-induced methylation changes, we used published DNA methylation clocks and newly established age predictors to demonstrate that the DHM-induced methylation change is associated with a reduction in the biological age of the cells. Further studies also revealed re-activation of age-dependently hypermethylated and silenced genes in vivo and a reduction in age-dependent epidermal thinning in a 3-dimensional skin model. Our findings thus establish DHM as an epigenetic inhibitor with rejuvenating effects for aged human skin

Data correction and quality control of the primary screen.

<p>(A) Correction of neutral lipid values with respect to changes in cell number. Z-scores of lipid values are illustrated before and after the amount of DNA was factored in. (B) Q-Q plot of normally distributed quantiles against screening result (Z-score) quantiles (red circles = positive control/<i>PPARγ</i>-siRNA; blue circles = negative control/scrambled siRNA). A perfect fit to a normal distribution is represented by red dotted line. (C) Experiment-wide quality plot focusing on controls. Signal from positive (red dots; <i>PPARγ</i>-siRNA) and negative (blue dots; control (scrambled) siRNA) controls plotted against plate number. The distance between the two distributions was quantified by the Z′-factor (0.42). For data normalization, the method ‘normalized percent inhibition’ (NPI) was applied.</p

Effects of the <i>HTR2B</i>-antagonist RS127445 on neutral lipid accumulation during human primary (pre)adipocyte differentiation.

<p>(A) Increase in lipid concentration in maturing adipocytes after treatment with 50 µM RS127445. Neutral lipid accumulation is shown relative to untreated control cells set as 100%. Results are depicted as mean ± SD (n = 10). Significant differences are marked with an asterisk (* for p<0.0001). (B) Cell viability of differentiating adipocytes after treatment with 50 µM RS127445 is shown relative to untreated control cells set as 100%. Results are depicted as mean ± SD. (C) Fluorescence microscopy after incubation with or without 50 µM RS127445 and lipid staining (yellow: neutral lipids; scale bar: 200 µm).</p

Network of axonemal dyneins.

<p>(A) Dynein network: The green coloring indicates 4 out of 5 hits regarding axonemal dyneins. <i>DNAH7</i> (also identified in our screen) was not part of the network. Blue symbols represent dyneins which were part of the library but were not determined as hits. Dyneins colored in white could not be investigated using the druggable siRNA library. (B) IPA showed an accumulation of motor proteins including dyneins and kinesins. (C) Messenger RNA levels of <i>DNAH7</i>, <i>DNAH8</i> and <i>DNAH17</i> in the course of adipocyte differentiation. Expression data at day 0 (preadipocytes) were set as 100%. All CT-values analyzed were between 28 and 34.</p

Genes identified and validated in our siRNA-screening showing the most significant changes in gene expression during adipogenesis were again transfected with specific siRNAs to classify the corresponding phenotype.

<p>Knock-down of genes caused a decreased lipid accumulation (upper box) or increased lipid accumulation (Lower box). InCell-Western Analyses were performed using aP2- and Perilipin-specific antibodies to classify target phenotypes into the following categories: (1) target-phenotype that reduce differentiation, [aP2 and Perilipin A signals decreased ≥20% compared to controls, green arrow]; (2) target-knock-down that stimulate differentiation [aP2 and Perilipin A signals increased ≥20% compared to controls, red arrow] and (3) target-phenotype that caused no changes in differentiation [none alterations in aP2 and Perilipin signals]. For targets highlighted in grey, signals of both markers decreased or increased. For those targets under laid in yellow, none marker changed.</p