| Summary: | 博士 === 國立中興大學 === 食品暨應用生物科技學系 === 95 === Obesity is an important topic in the world of public health and preventive medicine. Obesity has become a global epidemic in both developed and developing countries. It serves as a significant risk factor for various diseases such as diabetes, cancer, heart disease and hypertension. Adipose tissue is vital for maintaining whole body energy homeostasis, and it consists of adipocytes, which store triacylglycerol as a fuel for the body. Dietary fat is one of the most important environmental factors associated with the incidence of cardiovascular diseases. Excessive adipose tissue deposition is attributed to an imbalance between energy intake and energy expenditure. The literature regarding the in vitro and in vivo models’ evaluation of the inhibitory effects of naturally occurring polyphenolic antioxidants (phenolic acids and flavonoids) on cell growth and adipogenesis in adipocytes and their molecular mechanism remained unclear. Therefore, the objective of this study was to investigate the inhibitory effect of natural polyphenolic antioxidants on adipocytes and their molecular mechanism. There are six topics included in this study: (1) Effect of natural polyphenolic antioxidants in 3T3-L1 pre-adipocytes; (2) Effect of natural polyphenolic antioxidants in 3T3-L1 adipocytes; (3) Effect of natural polyphenolic antioxidants on the rats in preventive model of obesity; (4) Effect of natural polyphenolic antioxidants on the rats in a model of weight loss; (5) Study of the molecular mechanism of natural polyphenolic antioxidants in 3T3-L1 pre-adipocytes; (6) Study of the molecular mechanism of natural polyphenolic antioxidants in 3T3-L1 adipocytes.
(1) Obesity is an important topic in the world of public health and preventive medicine. Inhibition of pre-adipocyte proliferation plays an important role in the mechanisms of proposed antiobesity. In this in vitro study, the inhibitory effect of natural polyphenolic antioxidants on 3T3-L1 pre-adipocytes was evaluated and a relationship analysis was then conducted. The results showed that the addition of phenolic acids to the growth medium decreased the cell population growth of 3T3-L1 pre-adipocytes. The IC50 values of chlorogenic acid, gallic acid, o-coumaric acid and m-coumaric acid on 3T3-L1 pre-adipocytes were 72.3, 43.3, 48.2, and 49.2 μM, respectively. A relationship analysis indicated that there is a significant linear correlation between the influence of phenolic acids on cell population growth and their antioxidant activity (r=0.77, p‹0.01). The cell cycle assay indicated that the treatment of 3T3-L1 pre-adipocytes with chlorogenic acid, o-coumaric acid and m-coumaric acid caused cell cycle arrest in the G1 phase. Gallic acid did not affect the cell cycle profile; however, it increased the number of apoptotic cells (sub-G1 phase) in a time- and dose-dependent manner. Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis flow cytometry showed that gallic acid increased the number of early apoptotic (annexin V-FITC+/PI-) and late apoptotic cells (annexin V-FITC+/PI+) but not necrotic cells (annexin V-FITC-/PI+). The treatment of cells with gallic acid caused the loss of mitochondrial membrane potential (ΔΨm). The results showed that the inhibition of flavonoids (naringenin, rutin, hesperidin, resveratrol, naringin and quercetin) on 3T3-L1 pre-adipocytes was 28.3, 8.1, 11.1, 33.2, 5.6 and 71.5%, respectively. In an oxygen radical absorbance capacity (ORAC) assay, quercetin had the highest ORACROO˙value among the six flavonoids tested. Apoptosis assays showed that quercetin increased apoptotic cells in a time- and dose-dependent manner. Treatment of cells with quercetin decreased the mitochondrial membrane potential in the courses of time and dose. The cell apoptosis/necrosis assay showed that quercetin increased the number of apoptotic cells, but not necrotic cells. These results indicate that the inhibition of pre-adipocyte population growth by phenolic acids and flavonoids might have implication in in vivo antiobesity effects.
(2) Obesity has become a global epidemic in both developed and developing countries. It serves as a significant risk factor for various diseases such as diabetes, cancer, heart disease and hypertension. In the present study, the effect of naturally occurring polyphenolic antioxidants on the inhibition of adipogenesis in 3T3-L1 adipocytes was investigated. The results reveal that o-coumaric acid and rutin had the highest inhibition of intracellular triacylglycerol (61.3 and 83.0%, respectively) among 15 phenolic acids and 6 flavonoids being tested. But, the oil red o stained material (OROSM) showed that cell number in 3T3-L1 adipocytes was not influenced by the naturally occurring polyphenolic antioxidants. In GPDH (glycerol-3-phosphate dehydrogenase) activity, our data indicated that o-coumaric acid and rutin had the highest inhibition of GPDH activity (54.2 and 66.8%, respectively) among the 15 phenolic acids and 6 flavonoids. Western blot analysis indicated that o-coumaric acid and rutin also inhibited the expression of PPARγ, C/EBPα and leptin and then up-regulated expression of adiponectin at the protein level. These results suggest that naturally occurring polyphenolic antioxidants efficiently suppress adipogenesis in 3T3-L1 adipocytes.
(3) Antioxidants are a naturally abundant plant phenolic compound in the human diet and are known to reduce the risk of disease. Previous studies showed that there is a significant linear correlation between the influence of phenolic acids and flavonoids on 3T3-L1 pre-adipocytes population growth and their antioxidant activity. However, the inhibitory effect of these on high-fat diet (HFD)-induced oxidative stress in obese rats has not yet been investigated. In this study, the anti-obesity effect of gallic acid (GA) and quercetin (Q) in an animal model of diet-induced obesity was investigated. Obesity was induced in male Wistar rats by feeding them a HFD. GA or Q was supplemented at the levels of 0.1 and 0.2% for a period of 10 weeks. Growth parameters, weights of organ and adipose tissues, serum biochemical parameters, histology and antioxidant defense systems were measured in rats fed various diets. The results showed that the body weight, liver organ and adipose tissue weights of the peritoneal and epididymal in the HFD+GA or HFD+Q groups were significantly decreased as compared to the HFD group (p‹0.05). Serum triacylglycerol, phospholipid, total cholesterol, LDL-cholesterol, insulin and leptin levels in the HFD+GA or HFD+Q groups were significantly decreased as compared to the HFD group (p‹0.05). Histological study showed that the lipid droplets in the HFD+GA or HFD+Q groups were less as compared to those in the HFD group. Liver triacylglycerol and cholesterol levels in the HFD+GA or HFD+Q groups were significantly decreased as compared to the HFD group (p‹0.05). Moreover, the consumption of GA or Q reduced oxidative stress and glutathione disulfide (GSSG), and enhanced glutathione (GSH), GSH peroxidase (GPx), GSH reductase (GRd) and GSH S-transferase (GST) in the hepatic tissue of rats with HFD-induced obesity (p‹0.05). These results demonstrated that intake of gallic acid or quercetin could be beneficial for the suppression of high fat diet-induced dyslipidemia, hepatosteatosis and oxidative stress in rats.
(4) Dietary fat is one of the most important environmental factors associated with the incidence of cardiovascular diseases. Previous studies showed that o-coumaric acid and rutin had the highest inhibition on intracellular triacylglycerol and GPDH activity among 15 phenolic acids and 6 flavonoids being tested. Nevertheless, the literature data regarding the effect of o-coumaric acid and rutin on high fat-diet induced dyslipidemia, hepatosteatosis and oxidative stress in rats is unclear. The objective of this study was to investigate the anti-obesity effect of o-coumaric acid (oCA) and rutin (R) in Wistar rats fed a high fat-diet. The rats (n=6/group) were divided into normal and obese groups and then obese rats were pre-fed a high fat-diet containing 40% beef tallow for 4 weeks. After, the oCA or R was given as a supplement at the levels of 0.1 and 0.2% for a period of 8 weeks. Growth parameters, weights of organs and adipose tissues, serum biochemical parameters, histology and the antioxidant defense system were measured in rats fed various diets. The results showed that the body weight, liver organ and adipose tissue weights of peritoneal and epididymal in the HFD+oCA or HFD+R groups were significantly decreased as compared to those in the HFD group (p‹0.05). Serum levels of phospholipid, low density lipoprotein (LDL)-cholesterol, high density lipoprotein (HDL)-cholesterol, insulin and leptin in the HFD+oCA and HFD+R groups were significantly decreased as compared to those in the HFD group (p‹0.05). Hepatic triacylglycerol and cholesterol levels in the HFD+oCA and HFD+R groups were significantly decreased as compared to those in the HFD group (p‹0.05). Moreover, the consumption of o-coumaric acid or rutin reduced oxidative stress (decreased lipid peroxidation and glutathione disulfide, and enhanced the levels of glutathione and antioxidant enzymes activity) in the hepatic tissue of rats with HFD-induced obesity (p‹0.05). In the hepatic and adipose tissue of the peritoneal and epididymal, GPDH activity in the HFD+oCA and HFD+R groups were significantly decreased as compared to those in the HFD group (p‹0.05). These results demonstrate that intake of o-coumaric acid and rutin can be beneficial for the suppression of high fat diet-induced dyslipidemia, hepatosteatosis and oxidative stress in rats.
(5) Gallic acid is a naturally abundant plant phenolic compound. Previous studies showed that phenolic acids (such as gallic acid) caused better inhibition of cell growth and induction of apoptosis in 3T3-L1 pre-adipocytes. However, the molecular mechanism of gallic acid in the induction of cell apoptosis is still unclear. In the present study, we investigated the effect of gallic acid on the apoptotic pathway in 3T3-L1 pre-adipocytes. PI and DAPI staining showed that apoptotic bodies appeared when cells were treated with 100 μM of gallic acid for 48 h. Western blot data revealed that gallic acid stimulated an increase in the protein expression of Fas, FasL and p53. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was changed by gallic acid treatment. Gallic acid could induce release of mitochondria cytochrome c into the cytosol, and subsequently induce the activation of caspase-9 and caspase-3, which were followed by the cleavage of PARP. Pre-treatment with a general caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) prevented gallic acid inhibition of cell viability in the 3T3-L1 pre-adipocytes. Our data indicate that treatment of gallic acid also inhibited HDAC activity in 3T3-L1 pre-adipocytes. These results demonstrate that gallic acid induces apoptosis in 3T3-L1 pre-adipocytes through the Fas- and mitochondria-pathways. The induction of cell apoptosis by gallic acid may prove to be a pivotal mechanism for decreased pre-adipocyte proliferation.
(6) Obesity is one of the main public health problems in developed countries. Phenolic acids and flavonoids have been reported to have important biological and pharmacological properties and may have benefits to human health. Previous studies showed that o-coumaric acid and rutin had the highest inhibition of intracellular triacylglycerol and GPDH activity among 15 phenolic acids and 6 flavonoids being tested. However, the molecular mechanism of o-coumaric acid and rutin in the inhibition of adipogenesis is still unclear. In the present study, we investigated the effect of o-coumaric acid and rutin on mRNA expression of transcription factors and adipocytic specific genes in 3T3-L1 adipocytes. In 3T3-L1 adipocytes, transcription factors (PPARγ, C/EBPα and C/EBPβ) and adipocytic specific genes (aP2, FAS, LPL, MCP-1 and resistin) were reduced by o-coumaric acid and rutin treatment. Our data shows that o-coumaric acid and rutin has anti-adipogenic effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic transcription factors.
These results evidence that naturally occurring polyphenolic antioxidants efficiently induces apoptosis and inhibits adipogenesis in 3T3-L1 pre-adipocytes and adipocytes. Intake of naturally occurring polyphenolic antioxidants could be beneficial for the suppression of high fat diet-induced dyslipidemia, hepatosteatosis and oxidative stress in rats. Antioxidant induces apoptosis in 3T3-L1 pre-adipocytes through Fas- and mitochondria-pathway. Antioxidants have anti-adipogenic effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic transcription factors. These results evidence that naturally occurring polyphenolic antioxidants may be useful for the treatment of obesity.
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