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

Abstract

Background: 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 (Nelumbo nucifera-extract solution obtained from Silab, France) and L-carnitine on human preadipocytes and adipocytes.

Methods: For our in vitro 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.

Results: 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.

Conclusions: 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.

Figure 1

Lotus leaf extract solution decreased triglyceride accumulation during preadipocyte/adipocyte differentiation. (A) Images after triglyceride staining (yellow) from cell populations incubated with and without 1% lotus leaf extract solution. Scale bar: 200 μm. (B) Triglyceride accumulation of maturing human adipocytes incubated with 0.5% or 1% (n = 12, each) lotus leaf extract solution relative to untreated control cells (n = 12) set as 100%. Results are depicted as mean ± SD. (C) Cell viability of maturing adipocytes cultivated in 'differentiation medium' with 0.5% or 1% lotus leaf extract solution (n = 12, each) compared to untreated control cells set as 100%. Results are depicted as mean ± SD. Significant differences are marked with an asterisk (* for p < 0.0001 against control).

Figure 2

Increase in glycerol release from differentiated adipocytes after incubation with lotus leaf extract solution. Glycerol release from differentiated human adipocytes after treatment with 0.5 or 1% lotus leaf extract solution (n = 18) compared to untreated control cells set as 100%. Results are depicted as mean ± SD. Significant differences are marked with an asterisk (* for p < 0.0001 against control).

Figure 3

Effect of lotus leaf extract solution on ADD1/SREBP-1c expression during adipogenesis. (A) ADD1/SREBP-1c gene expression in differentiating preadipocytes after incubation with 1% lotus leaf extract solution compared to control cells set as 100%. Expression of ADD1/SREBP-1c is normalized to GAPDH. Three independent experiments were prepared both for control and incubation with lotus leaf extract solution (n = 3). Results are depicted as mean ± SD. (B) Human preadipocyte populations were cultured in 'differentiation medium' without (a, c) or with (b, d) 1% lotus leaf extract solution for 9 days. Immunofluorescence staining of ADD1/SREBP-1c (red) and of DNA (Hoechst-33342 (blue)) was performed. a and b: Scale bar: 200 μm; c and d: Scale bar: 50 μm.

Figure 4

L-carnitine stimulated beta-oxidation in preadipocytes and differentiated adipocytes. (A) Preadipocytes were incubated with 0.1% L-carnitine, 0.5% and 1% lotus leaf extract solution or left untreated as control for 18 h (n = 7). (B) Differentiated adipocytes were cultivated with 0.01%, 0.025% or 0.1% L-carnitine and untreated cells served as control (n = 14). ¹⁴CO₂ counts emitted by control samples were set as 100%. The threshold for relevant differences was set at 150%. Results are depicted as mean ± SD. Significant differences are marked with an asterisk (* for p < 0.0001 against control).

Figure 5

Additive effect of lotus leaf extract solution and L-carnitine on triglyceride accumulation during preadipocyte/adipocyte differentiation. Treatment of preadipocytes with 0.01% L-carnitine (n = 10), 0.5% lotus leaf extract solution (n = 12) or a combination of 0.01% L-carnitine and 0.5% lotus leaf extract solution (n = 12) on triglyceride accumulation during preadipocyte/adipocyte differentiation. Triglyceride levels of the respective control samples were set as 100%. Results are depicted as mean ± SD. Significant differences are marked with an asterisk (* for p ≤ 0.0001 against control; ** for p ≤ 0.0001 against control and all other treatments).