Pu-Erh Tea Down-Regulates Sterol Regulatory Element-Binding Protein and Stearyol-CoA Desaturase to Reduce Fat Storage in Caenorhaditis elegans

February Pu-Erh Tea Down-Regulates Sterol Regulatory Element-Binding Protein and Stearyol-CoA Desaturase to Reduce Fat Storage in Caenorhaditis elegans YiHong Ding 0 1 2 XiaoJu Zou 0 1 2 Xue Jiang 0 1 2 JieYu Wu 0 1 2 YuRu Zhang 0 1 2 Dan Chen 0 1 2 Bin Liang 0 1 2 0 1 Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan province, Kunming Institute of Zoology, Chinese Academy of Sciences , Kunming, 650223, China , 2 Department of Life Science and Technology, Key Laboratory of Special Biological Resource Development and Utilization of University in Yunnan Province, Kunming University , Kunming, 650214 , China 1 Funding: This work was supported by National Natural Science Foundation of China (NSFC) 31160216 to XJ Zou and 31171134 to B Liang; Yunnan Natural Science Foundation 2011FZ179 and Kunming University Science Project YJL11018 to XJ Zou; and Yunnan Provincial Science and Technology Department 2014HB022. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Hongyuan Yang, The University of New South Wales , AUSTRALIA Consumption of Pu-erh has been reported to result in numerous health benefits, but the mechanisms underlying purported weight-loss and lowering of lipid are poorly understood. Here, we used the nematode Caenorhaditis elegans to explore the water extract of Pu-erh tea (PTE) functions to reduce fat storage. We found that PTE down-regulates the expression of the master fat regulator SBP-1, a homologue of sterol regulatory element binding protein (SREBP) and its target stearoyl-CoA desaturase (SCD), a key enzyme in fat biosynthesis, leading to an increased ratio of stearic acid (C18:0) to oleic acid (C18:1n-9), and subsequently decreased fat storage. We also found that both the pharyngeal pumping rate and food uptake of C. elegans decreased with exposure to PTE. Collectively, these results provide an experimental basis for explaining the ability of Pu-erh tea in promoting inhibition of food uptake and the biosynthesis of fat via SBP-1 and SCD, thereby reducing fat storage. - Obesity is an outcome of energy intake exceeding energy expenditure, which leads to the extra energy becoming stored mainly as fat due to abnormal proliferation and over excessive volume of adipocytes. Globally, obesity and its comorbidities, type 2 diabetes, cardiovascular diseases, non-alcoholic fatty liver disease, among others, are increasingly prevalent, and present a rising public health concern, especially to the *1.46 billion overweight and the *502 million obese adults[1]. Given the alarming rise in these trends in both developed and developing countries, there is a stark need for more effective therapies that can both prevent and treat obesity and obesity-related diseases. One potential such therapy may lie in a long used traditional beverage produced in southwestern China known as Pu-erh tea. Pu-erh tea, a tea post-fermented by Competing Interests: The authors have declared that no competing interests exist. microorganisms[2] has long been consumed for its supposed benefits to health that are only recently being scientific explored, marked effects against oxidation [35], cancer[6], atherosclerosis [7], obesity[8,9], hypercholesterolemia, [1012], among others. Despite great progress in both in vivo and in vitro researches into the effects of Pu-erh tea on human, rodents, and cells to ability to reduce body weight, fat mass, and the level of serum triglycerides, cholesterol, and low-density lipoproteins (LDL) while also inducing the level of high-density lipoproteins, and the progression of steatosis[79,1317]. Unfortunately, the mechanisms by which Pu-erh tea consumption lowers body fat and lipid profiles is poorly understood. Over the past decade, however, the nematode C. elegans has become an increasingly popular model for investigating the regulation of fat metabolism and obesity genetics[1823], since C. elegans is quite similar to other organisms in its storage fat in lipid droplets[18,2426]. In the present study, we used this model to explore the underlying mechanisms of Pu-erh teas weight-cutting effects. First, we investigated the effect of Pu-erh tea on fat storage reduction using Nile Red staining [26], and quantitation via thin layer chromatography and gas chromatography (TLC/GC), followed by food uptake and absorption We also explored the effects of Pu-erh tea on stearoyl CoA desaturase (SCD), a key enzyme in the biosynthesis of fat, and its upstream regulator SREBP[27,28]. Pu-erh tea (100 g, LongRun Ripen Pu-erh; LongRun Group, Yunnan, China) was soaked in 500 ml boiling water for 20 min, then the water extract of Pu-erh tea (PTE) was centrifuged, filtered and sterilized with a filter membrane (0.45 m pore size) at room temperature. The composition of PTE is surprisingly complex, with polyphenols, polysaccharides, caffeine, protein, and amino acids at low concentrations[29] Consequently, quantifying the concentration of PTE based on one specific compound can present some difficulties. Here, we used a microplate reader (Biotek, Synergy H1) and found that the full wavelength absorbance of PTE displayed a peak at 590 nm (S1A Fig.) wavelength with absorbance, and also the absorbance OD of PTE presented a linear relationship (R2 = 0.9155) (S1B Fig.) with dilution. Accordingly, we set the absorbance of PTE at 590 nm as standard to make nematode growth medium (NGM) plates with the corresponding concentrations of PTE. Precisely which compound in PTE results in the peak at 590 nm wavelength is not clear, as PTE contains a large body of tea pigments that may contribute. Nevertheless, this simple method using a microplate reader helped us to control the concentrations of PTE in this study. The quantified PTE (0.2 g/ml) was added and mixed with nematode growth medium (NGM) before pouring the plates to the final concentrations of 0 g/ml, 0.0125 g/ml, 0.025 g/ml, and 0.05 g/ml (S1C Fig.). All prepared plates with PTE were used in 2 days. Worm strains and maintenance C. elegans were grown on nematode growth media (NGM) with OP50 strain of E. coli as a food source. The wild-type strain was N2. The following mutant strains were analyzed: sbp-1(ep79) [30], fat-6(tm331);fat-7(wa37)[31], fat-6::GFP{BX115, lin-15(n765)X;waEx16[fat-6WG::GFP]; lin-15(+)}[32], fat-7::GFP{BX113, lin-15(n765)X;waEx15[fat-7WG::GFP;lin-15(+)])}[32], KQ377(N2; ftISf [epEx307[unc-119(+); pSbp-1:SBP-1:GFP]), daf-1(e1370), age-1(hx546), akt-1 (mg144), and aak-2(gt33). Generally, synchronized L1 worms were placed on NGM plates with different concentrations of PTE, and were cultured under standard conditions. Lipids extraction and analysis Young adult worms with 13 eggs were washed off growing plates and immediately frozen in liquid nitrogen and stored at -80C freezer until further use. To determine the levels of triacylglycerol (TAG) and phospholipids, lipid extraction (...truncated)