| Obesity is a pathological body condition characterized by over accumulation of calories because of long-term more in-taking than consumption of energy. The excess calories were stored in the body and were distributed abnormal, leading to a body weight over-gain, which was named as a multi-factorial chronic metabolic disease. As we all know, obesity brings great harm to human health. obesity is a risk factor for diabetes, and fatty individual is more commonly to be complicated by diabetes mellitus. Seventy to eighty percent of diabetic patients over40years old have obesity history before diabetes mellitus. Obese people were susceptible to hypertension and30-50%of obese people suffered from hypertension. The incidence of hypertension in20-30year-old obese population is two times the normal. Obese people were susceptible to hyperlipidemia and coronary heart disease. The incidence of coronary heart disease in middle-aged male obese is two times the normal. Obesity is more easily to be complicated with cholelithiasis, gout, cancer and others. The synthesis of cholesterol and the excretion of cholesterol and bile were both increased in obesity, over the solubility of phospholipids and bile acids, leading to the supersaturation and crystallization of cholesterol. Snoring is more common in obese patients. Large number of fat was accumulated in the nasal mediastinal of obese people; gas exchange was interfered with normal breathing, resulting in snoring. Therefore, the prevention and control of obesity occurrence and development is of great significance to our health. Commonly, obesity is determined by body mass index (BMI), which means the squared value of weight (kg) divided by height (m). World Health Organization (WHO) prescribed BMI≥25as overweight, and BMI≥30as obese. The studies for obesity etiology found that the main factors leading to obesity included genetic factor, environmental factor, and personal behavioral factor. But their common core health hazard of above factors is the accumulation of excess fat cells. The function of excessive differentiation and increased in volume of fat cells were disordered. So, obesity is also defined as a pathological state reflected as an increase in number or volume fat cells, over accumulation of fat (mainly in triglycerides), and an overweight by20%than standard weight. Therefore, the molecular mechanisms of fat cell growth, differentiation and function is an important research focus in studies of pathogenesis of obesity.Transcription factor Twist1, a novel gene found in1987in Drosophila, can encode bHLH (basic helix-loop-helix). Numerous studies confirmed that Twist1participated in the formation of a variety of organizations, such as bone, muscle, nerve, heart, hematopoietic tissue and adipose tissue. The function of Twist1in adipose tissue is a research hot spot in recent years. The overexpression of Twist1in human white adipose tissue (WAT) and brown adipose tissue (BAT) was found by many researchers. The negative correlation between Twist1and obesity was also confirmed recently. Previous studies suggest that Twist1are closely related with the occurrence and development of obesity, but largely keeps unknown about the molecular mechanisms of Twist1in obesity occurrence and development. So, studies on the role of Twist1in adipocyte differentiation, and exploring on the middle links and molecular regulation mechanism between Twist1and obesity is clinically significant to the prevention and control of obesity. Experimental work is divided into two parts:Part I The expression of Twist1in adipose tissues of obese mice, rats and humanAim:To investigate the expression of Twist1in subcutaneous and visceral adipose tissues of obese mice and rats induced by high-fat diet, and in individuals suffering from clinical obesity, to clear the association between Twist1and obesity.Methods:1. The establishment of high-fat diet-induced obese C57/BL6mice:Twenty-four adult male C57/BL6mice at6weeks old were studied here. The mice were divided into two groups randomly after one week adaptation, with12/group and6/cage. The rearing environment was alternating12hours of day and night, room temperature at22-26℃, and45-60%relative humidity. Two groups were fed different feed, with the control group fed with the basal diet (fat content of5%), and obesity model group fed with high fat diet (20%fat content). Water and fodder was without limiting during experiment. The animal body weight was gained once a week. The model of obesity was considered successful when weight gain in obese group was increased by20%than the control group.2. The establishment of high-fat diet-induced obese Wistar rats:Twenty-four adult male Wistar rats at3months old were studied here. The rats were divided into two groups randomly after one week adaptation, with12/group and4/cage. The rearing environment was alternating12hours of day and night, room temperature at22-26℃, and45-60%relative humidity. Two groups were fed with different feed, with the control group fed with the basal diet (fat content of5%), and obesity model group fed with high fat diet (20%fat content). Water and fodder was without limiting during experiment. The animal body weight was gained once a week. The model of obesity was considered successful when weight gain in obese group was increased by20%than the control group.3. The serology after successful establishment of obese animal models:The blood of anesthetized C57/BL6J mice and Wistar rats was all collected after the model was successful. In C57/BL6J mice, blood was taken by eyeball, and blood from femoral artery was collected in Wistar rats. The serum was obtained after centrifuge. Serum cholesterol (CHOL), triglycerides (TG), and serum glucose (GLU) levels were measured.4. Twist1expression was detected in the adipose tissues:Subcutaneous and visceral adipose tissue (mainly perirenal adipose tissue) were collected in anesthetized C57/BL6J mice and Wistar rats. RNA and protein were extracted from adipose tissue, respectively. RT-PCR and Western Blot testing were performed to analyze Twist1expression in subcutaneous and visceral adipose tissue of different signs of obesity in animals.5. Collection of different clinical signs of adipose tissue in obese patients:A total of90cases adipose tissue samples were collected from patients with benign cervical lesions who underwent surgeries in the department of Plastic Surgery of Qianfoshan Hospital Affiliated to Shandong University from September2012to December2013. All included patients had no obesity history. Preoperative patient’s informed consents were obtained in this experiment, and the conduct of this study was approved by Shandong University Medical Ethics Committee. These patients were divided in4groups based on their BMI (Body Mass Index, calculated as body weight (BW, kg) over squared height in meter):BMI≤20group (slim group,15cases);20<BMI<25group (normal group,26cases);25≤BMI<30group (overweight group,21cases); BMI≥30group (obesity group,28cases) group. Adipose tissues were collected during surgery, and were stored in-80℃refrigerator.6. Detection of Twist1transcription and protein expression levels in adipose tissue in obese individuals:RNA and protein were extracted after extraction of adipose tissue lipids. RT-PCR and Western Blot testing were performed, and Twist1transcription and protein expression levels in adipose tissue in obese individuals were analyzed.Results:1. The body weight of high-fat diet-induced obese C57/BL6mice increased significantly:Before experiment, the body weights of C57/BL6mice in control group was (19.71±1.08) g, and which in obesity model group was (19.90±1.02) g, with no significant difference (P>0.05). During breeding, the body weights of C57/BL6mice increased gradually. At the end of the experiments, the body weight of obese C57/BL6mice was (34.86±2.98) g, which was significantly higher than that of the control mice (26.15±2.95g; P<0.05). Weight difference between the two groups was33.31%.2. The serological changes of high-fat diet-induced obese C57/BL6mice:At the end of the experiments, serum CHOL and GLU levels were both obviously increased in obese mice compared with the control (P<0.05). However, no significant difference in TG levels was observed.3. The body weight of high-fat diet-induced obese Wistar rats increased significantly: During breeding, the body weights of Wistar rats increased gradually. The body weight of obese Wistar rats was (438.61±37.65) g, while that of the control rats was (342.66±31.46) g (P＜0.05). Weight difference between the two groups was28%.4. The serological changes of high-fat diet-induced obese Wistar rats:At the end of the experiments, serum CHOL and GLU levels were both obviously increased in obese rats compared with that in control (P＜0.05). However, no significant difference in TG levels was observed.5. The transcription level of Twist1in adipose tissues of obese C57/BL6mice and Wistar rats declined obviously:Taken the relative transcription level of Twist1in control subcutaneous and visceral fat as100%, that of Twist1in obese C57/BL6mice and Wistar rats were all declined, and semi-quantitative results showed a statistically significance (all P<0.05).6. The protein expression level of Twist1in adipose tissues of obese C57/BL6mice and Wistar rats declined significantly:Taken the relative expression level of Twist1in control subcutaneous and visceral fat as100%, that of Twist1in obese C57/BL6mice and Wistar rats were all declined, and semi-quantitative results also showed a statistically significance (all P<0.05).7. Twist1transcription and protein expression levels were both declined in adipose tissues in obese individuals in clinic:Taken the relative transcription and expression level of Twist1in control group (20<BMI<25) as100%, the transcription level of Twist1in slim group (BMI≤20group), overweight group (25<BMI<30), and obesity group (BMI>30group) were (111.43±15.43)%,(89.97±12.54)%and (42.66±6.28)%; the protein expression level of Twist1in slim group (BMI<20), overweight group (25<BMI<30), and obesity group (BMI>30) were (130.31±15.82)%,(80.39±10.34)%and (25.43±2.48)%. Significant difference could be found in both transcription and expression level of Twist1in obese group (BMI>30), compared with the control group (20<BMI<25).Conclusions:Based on the animal and clinical specimen experiments, we proved the down-regulation of Twist1in the subcutaneous and visceral adipose tissues of obesity, which means that expression of Twist1is negatively correlated with obesity. Twist1is expected to become a new molecular indicator of obesity occurrence. Part Ⅱ Role of Twist1in the differentiation of cultured3T3-L1preadipocytes and its mechanism analysis in obesity occurrenceAim:To investigate the possible role of Twist1expression in adipogenesis of3T3-L1preadipocytes in vitro, and further analyze the probable molecular mechanism involved in Twist1expression and obesity occurrence.Methods:1. Differentiation induction and identification of3T3-L1preadipocytes in vitro:3T3-L1preadipocytes were cultured in vitro, and were induced to differentiate under insulin, dexamethasone and IBMX combination. Lipids in mature adipocytes were stained by oil-red O staining, and absorbance under510nm was detected by UV spectrophotometer after extraction by isopropanol. Adipocytes differentiated at day0th,2th,4th,8th and12th were collected during differentiation induction. RNA and protein were extracted, transcription and protein expression levels of peroxisome proliferator-activated receptor gamma (PPAR y), and adipocyte lipid binding protein (ALBP), two adipocyte differentiation marker molecules, were assayed.2. Dynamic expression changes of Twist1in3T3-L1preadipocytes differentiation: The transcription and protein expression levels of Twist1during3T3-L1preadipocytes differentiation (at differentiated0th,2th,4th,8th and12th) were analyzed based on the collected RNA and protein samples.3. The construction of lentiviral vector targetd to the interference expression of Twist1and viral packaging:Lentiviral vector carrying Twist1shRNA was constructed. Virals were packaged by cultured293FT cells, which were further used to infect3T3-L1preadipocytes. Single cell clones stably interfered with Twist1expression were selected and identified.4. Influence of Twist1interference to the differentiation of3T3-L1preadipocytes in vitro:Taken3T3-L1preadipocytes infected with the blank viral particles with vector as control (3T3-L1/NC), we analyzed the influence of Twist1interference to the differentiation of3T3-L1preadipocytes in vitro based on oil-red O staining and PPAR y expression changes.5. Influence of Twist1interference to the expression of PPARy:Taken3T3-LIpreadipocytes infected with the blank viral particles with vector as control (3T3-L1/NC), expression of PPARy at the same time point during differentiation was studied to analyze the role of Twist1in PPARy expression regulation.6. Detection of Twist1interference to the secretion of adipokines in the adipogenesis of3T3-L1adipocytes:The ability of3T3-L1adipocytes to secrete various adipokines into conditioned media after Twist1interference was evaluated and compared with that of control cells using a RayBio(?) Biotin Label-based Mouse Antibody Array I obtained from RayBiotech, Inc.(Norcross, GA).Results:1. The differentiation-induction system of3T3-L1preadipocytes in vitro was established successfully:We could see the fibroblast-like preadipocytes become round gradually under differentiation-induction, and small lipid droplets could be seen in cells distributed as ring shapes in cells. The lipid droplets become increasingly apparent with the extension of differentiation-induction. Big lipids could be seen clearly after fusion of small lipids. A large number of lipid droplets were filled with cytoplasm at the12th day of differentiation. 2. The content of lipid droplets increased significantly under oil red O staining:The lipid droplets were identified by oil red O staining because of the property of oil red O which could combine with triglycerides filled in the cytoplasm. Quantification of oil red O staining was conducted under UV spectrophotometer. The control cells without differentiation-induction were not colored under oil red O staining, while the differentiated mature adipocytes were colored bright orange clearly. And oil red O staining was enhanced with the degree of differentiation. Result of quantification showed that significant difference could be found from the4th day of differentiation (P<0.05).3. The expressions of two important adipocytes differentiation markers, PPARy and ALBP, were up-regulated during differentiation:Little of PPARy and ALBP, two important adipocytes differentiation markers, were detected in3T3-L1preadipocytes. Expressions of both PPARy and ALBP were up-regulated with differentiation, and significant difference could be found from the4th day of differentiation, compared with the blank control cells (P<0.05).4. Transcription and protein expression levels of Twist1were both up-regulated with differentiation:The transcription and protein expression levels of Twist1were both up-regulated with differentiation, and significant difference could be found from the4th day of differentiation, compared with the blank control cells (P<0.05).5. The interference expression of Twist1did not influence the progress of3T3-L1differentiation:Western blot analysis demonstrated that the expression of Twist1was reduced to20-30%in Twist1shRNA-treated cells compared with its expression in vector-treated control cells, which proved the effectiveness of our recombinant vector. When induced to differentiate, the Twist1shRNA-treated cells showed no obvious differences in intracellular lipid accumulation (P>0.05). The expression levels of the PPARy and ALBP proteins were also increased in the Twist1shRNA-treated cells similar with the vector-treated control cells under hormone-induced differentiation (P>0.05). These results proved that retroviral interference of Twist1expression did not significantly impair the process of lipid formation.6. The interference expression of Twist1enhanced the expression of PPARy on day 4th of differentiation induction:We determined the expression of PPARy in preadipocyte clones, with or without Twist1interference, at the same time points of differentiation induction. Compared with the retroviral vector-transformed control cells, retroviral interference of Twist1expression in3T3-L1preadipocytes upregulated the mRNA and protein levels of PPARy on day4th of differentiation induction.7. Parts of adipokines were influenced by Twist1interference expression:The levels of adipokines in the conditioned medium of3T3-L1/Twist1-cells were determined based on adipokine antibody arrays. The results showed changes in the secretion of multiple adipokines, and the targeted adipokines were mainly divided into three categories, interleukins (including IL-3, IL-6, IL-7, IL-9, IL-10, IL-17, IL-21, and IL-27) and their receptors, growth factors and their receptors, especially FGF, GHR, HGF, VEGF, TNF, and their receptors, and chemokine family members and related proteins, including CXCR4, Cys-Cys receptor6(CCR6), and monocyte chemotactic protein-1(MCP-1).Conclusions:(1) Twist1expression was up-regulated in the adipogenesis of3T3-L1preadipocytes, but retroviral interference of Twist1expression did not significantly impair the process of lipid formation.(2) Retroviral interference of Twist1expression in3T3-L1preadipocytes upregulated the mRNA and protein levels of PPARy, which was believed to play important role in multiple physiological functions, on day4th of differentiation induction. This provide critical insight into the molecular mechanism linking Twist1expression and obesity.(3) Recent studies reported the active function of adipose tissue as an important endocrine organ. The secretions of multiple adipokines were changed under retroviral interference of Twist1expression in3T3-L1preadipocytes, mainly in interleukins (including IL-3, IL-6, IL-7, IL-9, IL-10, IL-17, IL-21, and IL-27) and their receptors, growth factors and their receptors, especially FGF, GHR, HGF, VEGF, TNF, and their receptors, and chemokine family members and related proteins, including CXCR4, Cys-Cys receptor6(CCR6), and monocyte chemotactic protein-1(MCP-1), which provided an important value to the molecular mechanism studies linking Twist1expression and obesity.Above all, transcription factor Twist1is closely related with the occurrence and development of obesity, but the target is not in adipocytes differentiation, and maybe related with another one or more physiological processes involved in PPARy or adipokines. |
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