Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes

Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes

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Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes Carmen Pheiffer a,∗ , Zulfaqar Dudhia a , Johan Louw a , Christo Muller a , Elizabeth Joubert b,c a b c

Diabetes Discovery Platform, Medical Research Council, P.O. Box 19070, Tygerberg 7505, South Africa Post-Harvest and Wine Technology Division, Agricultural Research Council, Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa Department of Food Science, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

a r t i c l e Keywords: Cyclopia maculata Cyclopia subternata 3T3-L1 adipocytes Glycerol release Lipolysis

i n f o

a b s t r a c t We have previously, for the first time, demonstrated that hot water extracts of Cyclopia maculata and Cyclopia subternata, endemic South African plants that are consumed as herbal teas, inhibit adipogenesis in 3T3-L1 adipocytes. The aim of this study was to extend the anti-obesity investigations of these plants by quantifying lipolysis in mature 3T3-L1 adipocytes. Glycerol concentration in culture supernatants was used as a marker of adipocyte lipolysis. Isoproterenol, a ␤-adrenergic agonist and a known lipolytic agent, was used as a positive control in our assays. Lipolysis was stimulated by all extracts, although statistical significance was noted for fermented (oxidised) C. maculata only. A concentration of 80 ␮g/ml of C. maculata extract induced maximal lipolysis (1.8-fold, p < 0.001). The increased lipolysis was accompanied by an increase in the expression of hormone sensitive lipase (1.6-fold, p < 0.05) and perilipin (1.6-fold, p < 0.05). The plant extracts, at the concentration range assayed (0–100 ␮g/ml), were not cytotoxic in terms of mitochondrial dehydrogenase and adenosine-5 -triphosphate activity. These results showed that C. maculata stimulates lipolysis in mature 3T3-L1 adipocytes, providing further support for the anti-obesity effects of Cyclopia spp. © 2013 Elsevier GmbH. All rights reserved.

Introduction Obesity is a major source of morbidity and mortality worldwide, increasing the risk for chronic diseases such as type 2 diabetes, cardiovascular disease and certain types of cancer (Haslam and James, 2005). It is estimated that currently more than 1.4 billion adults are overweight and more than 500 million individuals are obese (Finucane et al., 2011). In 2008, the prevalence of overweight individuals already surpassed projections for 2030 (Kelly et al., 2008), with the current prevalence of obesity being close to 2030 predictions. These findings highlight the magnitude of the obesity pandemic and emphasise the urgent need for its prevention and treatment. Despite widespread acceptance that reduced energy consumption and increased physical activity are effective anti-obesity treatment options (Hunter et al., 2008; Lagerros and Rossner, 2013), people are reluctant to lifestyle modifications, resulting in the increased reliance on therapeutics and other supplements to treat and manage obesity. Currently only three drugs, Orlistat, Lorcaserin and Qsymia are approved by the Food and Drug Adminsitration

(FDA) for the long term treatment of obesity (Adan, 2013). However, these drugs are plagued by numerous side-effects which limit their usefulness. Plant bioactive compounds are receiving renewed interest as potential anti-obesity agents due to the perception that natural products are safer than their synthetic counterparts (Vermaak et al., 2011). We have previously reported that hot water extracts of two endemic South African plants, Cyclopia maculata and Cyclopia subternata inhibit adipocyte differentiaton in 3T3-L1 adipocytes, thus suggesting their anti-obesity potential (Dudhia et al., 2013). The aim of the present study was to extend the anti-obesity investigations of these plants by quantifying lipolysis in mature 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with varying concentrations of the extracts, whereafter the secretion of glycerol (a marker of lipolysis) into the culture medium, the protein expression of hormone-sensitive lipase (HSL) and perilipin, and possible cytotoxic effects of the extracts were determined. Materials and methods Cell culture and adipocyte differentiation

∗ Corresponding author at: Diabetes Discovery Platform, Medical Research Council, Cape Town, South Africa. Tel.: +27 219389202; fax: +27 219380456. E-mail address: [email protected] (C. Pheiffer).

The plant material, preparation of hot water extracts and their phenolic chracterization have been previously described (Dudhia et al., 2013). The reconstitution and dilution of the dried water

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Please cite this article in press as: Pheiffer, C., et al., Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.016

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extracts and the differentiation of 3T3-L1 (CL-173, American Type Culture Collection, Manassas, USA) adipocytes for bioassays were conducted as previously described (Dudhia et al., 2013). The effect of the plant extracts on lipolysis was quantified by adding various concentrations (0–100 ␮g/ml) of fermented (oxidised) and unfermented (unoxidised, “green”) C. maculata and unfermented C. subternata extract to differentiated adipocytes for 24 h. Isoproterenol (Merck, Darmstadt, Germany), a known stimulator of adipolysis (Gauthier et al., 2008), was used as a positive control in all assays. Glycerol release After treatment of differentiated adipocytes with the plant extracts, free glycerol content in cell supernatants was quantified using a glycerol quantification kit according to the manufacturer’s instructions (Biovision Inc., Milpitas, USA). Glycerol was quantified at (Ex/Em) 535/590 nm on a BioTek® ELX 800 plate reader (BioTek Instruments Inc., Winooski, USA). Western blot analysis The effect of the plant extracts on HSL and perilipin expression was determined using Western blot analysis as previously described (Dudhia et al., 2013). Briefly, differentiated 3T3-L1 adipocytes were treated with 80 ␮g/ml of the extracts for 24 h and proteins extracted and transferred to polyvinylidene fluoride (PVDF) membranes (Pierce, Rockford, USA). Membranes were probed with HSL, perilipin or ␤-tubulin antibodies (Cell Signalling, Danvers, USA), diluted 1:1000 at 4 ◦ C overnight, and proteins of interest detected using horseradish peroxidise (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotechnologies, Dallas, USA) and the LumiGLO Reserve kit (Santa Cruz Biotechnologies, Dallas, USA) according to the manufacturer’s instructions. Immunoreactive proteins were visualised and quantified by chemiluminescence using a ChemiDoc-XRS image analyser and Quantity One 1-D software (Biorad Laboratories Inc., Hercules, USA). ␤-tubulin was used as the normalisation control. Statistical analysis All experiments were done as three replicates in three independent experiments. Data are expressed as the mean ± standard deviation. Statistical significance was determined by one-way analysis of variance (ANOVA) and the Dunnett post hoc test (Graphpad Prism® version 6.01, GraphPad Software, La Jolla, USA). The student unpaired t-test was used to analyse Western blot data. A p value of less than 0.05 (p < 0.05) was considered to be statistically significant. Results and discussion Cyclopia spp. are consumed as herbal teas (honeybush tea) locally in South Africa and internationally (Joubert et al., 2011). Phenolic compounds in Cyclopia spp. have attracted considerable interest due to growing evidence of their medicinal properties, which include antioxidant, anti-mutagenic, anti-cancer and antidiabetic effects (Joubert et al., 2008; Muller et al., 2011). Recently, it was reported that some of the major phenolic compounds present in the Cyclopia spp. extracts investigated in the present study (Dudhia et al., 2013), i.e. the xanthone mangiferin, the benzophenone iriflophenone-3-C-␤-glucoside, and the flavanones hesperidin and eriocitrin, possess anti-obesity properties (Chiba et al., 2003; Guo et al., 2011; Miyake et al., 2006; Yoshikawa et al., 2002; Zhang et al., 2011). We have previously demonstrated that

Fig. 1. Effect of plant extracts on glycerol release. Differentiated 3T3-L1 adipocytes were untreated (clear bar) or treated with hot water extracts of fermented (fCM) or unfermented (uCM) C. maculata, unfermented C. subternata (CS) or 10 ␮g/ml of isoproterenol (solid shaded bar) for 24 h. Extracellular free glycerol was quantified with a commercial glycerol quantification kit and expressed as a percentage of glycerol release in untreated cells. Data are expressed as the mean ± standard deviation of three independent experiments, each performed in triplicate. ***p < 0.001 vs. untreated control.

aqueous extracts of C. maculata and C. subternata inhibit adipogenesis in 3T3-L1 adipocytes, suggesting their potential in the prevention and treatment of obesity (Dudhia et al., 2013). In this study we extended our anti-obesity investigations of these extracts by quantifying lipolysis in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the extracts increased the amount of glycerol secreted (Fig. 1), although statistical significance was noted for fermented C. maculata only. C. maculata extract concentrations between 60 ␮g/ml and 100 ␮g/ml induced lipolysis, with maximal stimulation observed at 80 ␮g/ml (1.8-fold, p < 0.001). C. maculata also increased the expression of HSL (1.6fold, p < 0.05) and perilipin (1.6-fold and p < 0.05) (Fig. 2). None of the plant extracts at the concentration range tested was cytotoxic as demonstrated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and adenosine-5 -triphosphate (ATP) assays (data not shown). The main function of adipose tissue is to store excess energy as triacylglycerols and to mobilise them using the process of lipolysis during starvation conditions (Gibson and Harris, 2002). The control of lipolysis represents a complex process involving multiple regulatory events, with HSL being the rate-limiting enzyme (Londos et al., 1999). Activation by cAMP-dependent protein kinase (PKA) leads to the translocation HSL from the cytosol to the lipid droplet, where it interacts with activated perilipin thereby stimulating lipolysis (Miyoshi et al., 2006; Shen et al., 2009). During the basal state, perilipin restricts lipolysis by coating the lipid droplet and protecting it from lipase activity, however, activation of perilipin by PKA modifies the lipid droplet, thus allowing access to translocated HSL and stimulation of lipolysis. This study has demonstrated that a hot water extract of fermented C. maculata, and to a lesser extent those of unfermented C. maculata and C. subternata, stimulates lipolysis, thus confirming the anti-obesity properties of these plants. Fermented C. maculata contains lower concentrations of polyphenols, in particular, mangiferin, iriflophenone-3-C-glucoside, eriocitrin and hesperidin, than unfermented C. maculata and C. subternata suggesting that other compounds may contribute to the anti-obesity properties of this extract. Alternatively, these compounds may improve bioactivity of the previously reported anti-obesity compounds (Chiba et al., 2003; Guo et al., 2011; Miyake et al., 2006; Yoshikawa et al., 2002; Zhang et al., 2011) using synergistic mechanisms (UlrichMerzenich et al., 2009; Wagner and Ulrich-Merzenich, 2009). No

Please cite this article in press as: Pheiffer, C., et al., Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.016

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Fig. 2. Effect of plant extracts on HSL and perilipin expression. Differentiated 3T3-L1 adipocytes were exposed to 80 ␮g/ml of hot water extracts of fermented (fCM) or unfermented (uCM) C. maculata, unfermented C. subternata (CS) or 10 ␮g/ml of isoproterenol for 24 h. Proteins were extracted from cell lysates and the expression of HSL and perilipin quantified by Western blotting. (a) Densitometry images of the protein bands detected. (b) Protein expression normalised to ␤-tubulin. Data are presented as the mean ± standard deviation of three independent experiments, each performed in triplicate. *p < 0.05, **p < 0.01 vs. untreated control.

cytotoxicity was observed using the MTT and ATP assays. Our results are in agreement with other studies that have also shown that plant polyphenols mediate their anti-obesity effects by inhibiting adipogenesis and stimulating lipolysis (Peng et al., 2011; Sohle et al., 2009). An interesting observation of this study was increased adiponectin secretion by 3T3-L1 adipocytes after C. maculata treatment (data not shown). Adiponectin has a protective effect against obesity, type 2 diabetes and cardiovascular disease (Shibata et al., 2009; Spranger et al., 2003). Thus, in addition to its sensory properties, C. maculata when consumed as a herbal tea, may have the added advantage of modulating adipocyte function and mediating anti-obesity effects. Consumption of honeybush tea (Cyclopia spp.) may present a practical approach to ameliorate the underlying causes of obesity and thereby prevent or delay the development of obesity-related complications such as type 2 diabetes. This idea is supported by studies reporting that phloridzin, the 2 -O-glucoside of phloretin, has anti-diabetic properties (Dimitrakoudis et al., 1992; Najafian et al., 2012; Rastogi et al., 1997; Rossetti et al., 1987). Previously, we hypothesised that phloretin-3 ,5 -di-C-glucoside, the most abundant phenolic compound present in C. subternata (Dudhia et al., 2013), could contribute to the plant’s anti-diabetic properties (Mose Larsen et al., 2008) by increasing the expression of PPAR␥1, a protein whose expression is decreased during insulin-deficient diabetes (Vidal-Puig et al., 1996). In our previous study, the concentration of phloretin-3 ,5 -di-C-glucoside in plant extracts correlated with PPAR␥1 expression, with fermented C. maculata having no detectable concentration of this phloretin derivative and also the lowest expression of PPAR␥1 (Dudhia et al., 2013). Recently, Muller

et al. reported that another Cyclopia species (C. intermedia), exhibited glucose-lowering properties in Wistar rats (Muller et al., 2011). Taken together, these studies suggest that Cyclopia spp., in addition to their anti-obesity effects (Dudhia et al., 2013), may have the added ability to regulate glucose levels. Future studies to determine whether phloretin-3 ,5 -di-C-glucoside and/or unidentified compounds contribute to the glucose lowering properties of Cyclopia spp., in addition to those with anti-diabetic effects such as mangiferin (Miura et al., 2001; Giron et al., 2009) and hesperidin (Jung et al., 2004; Shen et al., 2012), are warranted. Conflict of interest The authors have no conflict of interest to disclose. Acknowledgements This work is based on the research supported in part by the National Research Foundation of South Africa (Grant 70525 to EJ). The Grantholder acknowledges that opinions, findings and conclusions or recommendations expressed in any publication generated by the NRF supported research are that of the authors and that the NRF accepts no liability whatsoever in this regard. References Adan, R.A., 2013. Mechanisms underlying current and future anti-obesity drugs. Trends in Neurosciences 36, 133–140.

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Please cite this article in press as: Pheiffer, C., et al., Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.016