Study On Metabolism And Regulation Of Cellular Triacylglycerol And Lipid-Lowering Mechanism Of Berberine

Obesity is a global health problem that puzzles the people. Obesity induced high cholesterol, hypertension, diabetes, fatty liver and other diseases, posed much dangers to human health. It has become the second largest threat to human health. Moreover, obesity has also caused many economical problems, leading to large economic loss every year. Therefore, it is imperative to find ways to solve obesity problem effectively. However, the pathogenesis of obesity and obesity-induced disease is not clear, how to effectively control the obesity and obesity-related diseases is still a difficult problem to solve right now.In the study of obesity, it was found that the disorder of triacylglycerols(TAG) synthesis and mobilization is the main factor leading obesity. Triacylglycerol is the most abundant lipids in cells, and the main stored form of fat in plant and animal. The disorder of triacylglycerol regulation produced excess fatty acids and excessive fatty acids, resulting accumulation of fatty acids in the subcutaneoust issue, liver and other tissues, induced lipotoxicity, and develop obesity. Lipotoxicity is an important cause of obesity-related diseases, such as fatty liver, which was caused by excessive fatty acids accumulated in the liver cells via the process of steatosis. The development of obesity and obesity-related diseases were always accompanied with abnormal elevation of triacylglycerols and fatty acids level. Disorder of triacylglycerol metabolism regulation is closely related to obesity, and inhibition of triacylglycerol synthesis has become an important strategy to control obesity and obesity-related diseases. Lots of related drugs were targeted at triacylglycerol synthesis pathway to control obesity, and phospholipid phosphatase mediated triacylglycerol synthesis pathway has been considered to be the only pathway of triacylglycerol synthesis. However, targeting at the triacylglycerol synthesis pathway to research and develop lipid-lowering drugs, limited the alternative targets, and slowed the development of related lipid-lowering drugs, and may lead to greater adverse effects. For examples, the lipid-lowering drugs, clenbuterol and propranolol targeted inhibitors, may cause headaches, heart palpitations, and other residues, which harmful to human health. Traditional Chinese antimicrobial medicine berberine(BBR) has a good lipid-lowering effect, lowering TAG activity with lower adverse effects. It is an excellent candidate compound for control obesity and obesity-related diseases. However, the lipid-lowering effect and antibacterial activity of berberine are overlaped, it is difficult to reveal the lipid-lowering mechanism of berberine. It can not be wide applied in treatment of obesity and obesity-related diseases untill its lipid-lowering mechanism is clear.In order to promote the progress of obesity and obesity-related diseases research and control above problems, We taked the advantages of the model organism Saccharomyces cerevisiae in studies of gene function and regulation of lipid metabolism, and studied the mechanism of triacylglycerol synthesis regulation and the lipid-lowering mechanism of berberine in S. cerevisiae in this paper. The main work of this paper includes the following three parts:Part one: The pathway of triacylglycerol synthesis plays an important role in lipotoxicity alleviating. Lipotoxicity is an important cause of obesity-related diseases, and it is often accompanied with the disorder of fatty acid metabolism. Triacylglycerol synthesis as an indispensable part of fatty acid metabolism, showed a lipotoxicity alleviating effect. However, its mechanism is not clear so far. Therefore, using of Oleic acid as lipotoxic agent, we studied the mechanism of lipotoxicity alleviating of triacylglycerol synthesis pathway. Lots of protein folding related genes were screened from S. cerevisiae overexpression library, including ZIM17?ALG2 and so on. Moreover, a significant of UPR activation was detected after treatment of 1 m M oleic acid by GRP78 expression and new UPR detect system. Under the stress of oleic acid, the synthesis of triacylglycerol was significantly promoted. Those founding indicated that, the mechanism of oleic acid-induced ERs may involve the protein folding; triacylglycerol synthesis plays an important role in lipotoxicity alleviating; the synthesis of triacylglycerol will be promoted to alleviate lipotoxicity after cell was exposure to oleic acid. In this part, we also found that, when PAH1 was deleted, the yeast is still able to convert oleic acid into triacylglycerol, indicating there may be other unknown pathway of triacylglycerol synthesis to maintain triacylglycerol synthesis.Part two: A novel bypass for PAH1-mediated triacylglycerol synthesis in yeast ——CDS1-involved triacylglycerol synthesis. The synthesis of triacylglycerol is regulated by phospholipase phosphatase(PAP) encoded by PAH1(LPIN1 in human). The dephosphorylation of phospholipid acid and production of diacylglycerol catalyzed by phospholipase phosphatase is the key step in the triacylglycerol synthesis in yeast and mammal. When PAP mediated triacylglycerol synthesis was blocked, such as PAH1 defects, it would lead 95% reduction in the number of lipid droplets and 63% decrease in triacylglycerol levels. PAPs are encoded by the PAH1, APP1, DPP1 and LPP1 genes. However, diacylglycerol(DAG) can still be detected in pah1?app1?dpp1?lpp1? qua-mutant, which suggested that there is another bypass pathway remedying the PAP-deficiency induced TAG synthesis defect to maintain triacylglycerol homeostasis. To find the new triacylglycerol synthesis pathway, we employed the mutant berberine-sensitive phenotype of to screen PAH1 functional remedy gene. As a result, we found 21 berberine tolerant strains and a total of 13 resistance gene, such as CDS1, NSA2, LSM3, DED8, CNS1 and SFH1 etc. Among those genes, CDS1 was the highest BBR-resistant and most lipid relevant gene. The level of triacylglycerol s recovered significantly after overexpression of CDS1 in pah1?(73.95% of typical levels). Phosphatidate cytidylyltransferase encoded by CDS1 involves major phospholipids biosynthesis, and plays an important role in maintaining the balance of phospholipids. Overexpression CDS1 significantly increased inositol phospholipid(PI) level in pah1?, and promoted the synthesis of triacylglycerol via PI resynthesis pathway. More important, there is a PAP1-response feedback mechanism to regulate CDS1 expression to ensure TAG level in PAP1 dysfunction. In PAH1 defect, when triacylglycerol synthesis was blocked and phosphatidic acid was accumulated, the suppressor Opi1 p would fall off from the CDS1 upstream and binded to phosphatidic acid in nuclear membrane, and lost the inhibitory ability on CDS1. Once CDS1 was not control by Opi1 p, the expression level of CDS1 would increase significantly, which ensured the triacylglycerol homeostasis in pah1?. These findings revealed the triacylglycerol synthesis and regulation mechanisms, provided the basic datas for the study of obesity and obesity-related diseases.Part three: the study of triacylglycerol-lowing mechanism of Berberine. Pharmacological inhibition of triacylglycerol synthesis is an effective strategy to control obesity. Commonly used lipid-lowering drugs, such as propranolol and clenbuterol, can significantly reduce intracellular triacylglycerol levels, inhibit fat deposition, control obesity and obesity-related diseases. Berberine as a traditional Chinese antimicrobial drug showed strong lipid-lowering activity and could cure lipid metabolism and diabetes. Newresearch showed that the lipid-lowering activity, especially the triacylglycerol-lowing activity of berberine was closely related to its inhibition on PAP activity. In this part of the study, we used the yeast model S. cerevisiae to study the lipid-lowering mechanisms of berberine. As results, we found 0.5g/L berberine significantly reduced the yeast lipid droplets(47.12%) and triacylglycerol levels(47.34%). Lipid-lowering activity of berberine is closely related to triacylglycerol synthesis and its key regulatory gene PAH1. Berberine showed strong inhibition effect on PAH1 expression. There are several transcription factors on PAH1 promoter, including Fkh1 p, Rfx1 p, Tfc7 p, Yap1 p, Yap6 p and Zap1 p. Among these transcription factors, Yap1 p plays an important role in the inhibition of berberine on PAH1. When YAP1 defected, the inhibiting effect of berberine on PAH1 disappeared. There is a putative Yap1p(YAP1) recognition site(ARE: 5'-TTA[AG]TAA-3') in PAH1 promoter region(-960~-728bp). Yap1 p, as a transcriptional regulator response to oxidative stress, induced PAH1 when cell was exposure to oxygen free radical. However, berberine could remove these oxygen free radicals produced from cellular metabolism, inhibitite the induction of Yap1 p on PAH1, then decrease PAH1 expression levels, reduce intracellular triacylglycerol levels. These findings will enable us to have a deeper understanding of the relationship between obesity and oxidative stress, and lay the foundation for the application of berberine in the field of treatment of obesity and obesity-related diseases.Part four: The lipid-lowering activity of berberine plays an important role in antifungal therapy. Fungal infection is becoming prevalent internationally, especially these patients who uesed catheters, intubation and organ transplantation. Fungal infection increased the incidence and mortality of those patients. Medication therapy is the most effective means in treatment of fungal infections. Currently, the triazole antifungal azole drugs widely used in monotherapy of deep and systemic fungal infections is fluconazole. However, with a large number and long-term abuse of fluconazole, more and more fungals showed resistance to fluconazole. Nowadays, fluconazole resistance has become the main reason for treatment failure, and which has become a hot topic in the field of antifungal research.Berberine showed strong lipid-lowering activity, andantifungal activity. In previous studies we found that synergistic antifungal effect of berberine has a closely raltionship with its lipid-lowering activity. Berberine showed synergistic effects with fluconazole, econazole, amphotericin B and nystatin against fungal. However, this synergistic effect could be inhibited by oleic acid, triacylglycerol, phosphatidylcholine and ergosterol. In testing of the fluconazole sensitivity of triacylglycerol, phosphatidylcholine and ergosterol biosynthesis-related deficient strains, we found triacylglycerol synthesis raleted deficient strain pah1? and phosphatidylcholine biosynthesis related deficient strain psd1? showed slight fluconazole sensitivity, and phosphatidylethanolamine transition related deficient strain cho2? exhibited a strong resistance to fluconazole. As previous studies showed phosphatidylethanolamine affected cell drug efflux pump activity, and the fluconazole sensitivity of yeast. However, Berberine reduced phosphatidylethanolamine synthesis via inhibiting expression of PSD1, and inhibited drug efflux pump activity and increased fluconazole sensitivity of yeast. These findings will give us insight into the synergism antifungal and lipid-lowering mechenism of berberine, and provide the strategy for antifungal treatment.By studying on metabolism and regulation of cellular triacylglycerol and lipid-lowering mechanism of berberine, on the one hand, we clarified the lipotoxicity alleviating mechanism of triacylglycerol synthesis pathway, and found a novel CDS1 mediated triacylglycerol synthesis pathway independent of phosphatide phosphatase for related drug development. On the other hand, we found lipid-lowering mechanism of berberine is working through inhibiting the expression of phosphatide phosphatase and reducing the synthesis of triacylglycerol via its antioxidant activity, and the synergism mechanism of berberine with fluconazole in antifungal is working through inhibiting the synthesis of phosphatidylethanolamine and reducing drug efflux pump activity. Those founding will promote the development of obesity and obesity-related diseases research and control, improve the value of clinical application of berberine.