Interaction Between PYY Gene And Dietary Fat In The Susceptibility To Obesity

Obesity is defined medically as a state of increased body weight, more specifically adipose tissue, of sufficient magnitude to produce adverse health consequences. It is not only a disease but also an independent risk factor for many chronic diseases including type 2 diabetes, coronary heart diseases, stroke, hypertension, fatty livers , gallstones, some cancers , gout and osteoporosis. Affected individuals usually experience more negative interpersonal treatment and are discriminated in many aspects of social life, and have low self-esteem and higher rate of depression and suicide than normal weight subjects. The prevalence of overweight and obesity has increased with alarming speed over the past twenty years. It has been described by the World Health Organization as a 'global epidemic'. So it has been listed in the top 10 public health problems worldwide by WHO and there is a pressing need to understand the mechanism and to determine the potential to which they can be exploited in the prevention and treatment of obesity.Only a small fraction of human obesity can be explained by single gene defects. In most human obesity, it is the interaction of many susceptibility gene-gene and gene-environment contribute to the complex phenotype. Nutrition is a key environmental factor involved in the pathogenesis and progression of obesity. When placed on a high-fat diet, a subset of subjects becomes very obese (diet induced obesity, DIO), whereas others remain as lean as subjects fed a low fat diet (diet induced obesity resistant, DIO-R), suggesting a role for interaction between genes and diet on obesity risk. Further research on gene-dietary interactions on the susceptibility to obesity will advance our understanding of the underlying mechanisms of obesity and facilitate progress towards obesity prevention and treatment.Obesity results from an energy imbalance ,where energy intake exceeds total body energy expenditure. Many obese people do have a problem of hyperphagia which contribute to the positive energy balance. Adequate food intake under normal appetite regulation plays a key role in maintaining energy balance and normal body weight. Energy homeostasis is a complex mechanism involving both the gastrointestinal tract and the brain. The hypothalamus is a region of the brain critical for regulation of homeostatic processes by sensing neural, endocrine, and metabolic signals, integrating these inputs, and engaging distinct effector pathways, resulting in behavioral, autonomic, and endocrine responses. Peptide tyrosine tyrosine (Peptide-YY, PYY) is a 36-amino acid secreted from the L-cells primarily located in the distal part of the intestine following food intake in propotion to the calorie content of a meal and then cleaved by the enzyme dipeptidyl peptidase-IV(DPP-IV) to yield the major circulating form, PYY3-36. More recently, intraperitoneal administration of PYY3-36 was reported to inhibit food intake in rodents and, after intravenous injection, to decrease appetite in humans. PYY3-36 is a specific agonist for the Y2 receptor, which is highly expressed on neuropeptide Y(NPY) neuron in the arcuate nucleus of the hypothalamus, and is a putatively presynaptic autoreceptor with inhibitory effects on NPY release. In addition, it is shown that endogenous fasting and postprandial levels of PYY3-36 are significant lower in obese compared with lean individuals, and thus it has been suggested that deficiency of PYY3-36 may contribute to the pathogenesis of obesity and PYY3-36 infusion may become apromising method for the prevention and treatment of obesity.The main aim of this study is to explore the interaction between PYY gene and dietary fat in the development of obesity. The primary goal of this research is to establish diet induced obesity (DIO) and diet induced obesity resistant (DIO-R) rat models for further study. The second goal is to study the PYY gene expression and circulating levels of PYY, blood lipid, and their relationship with food intake and obesity. A third goal is to investigate the influence of dietary fat on the expression and release of PYY in DIO rats. The main results are summarized as follows:Part one Diet-induced Obesity and Obesity Resistantin the Sprague-Dawley RatObjective To investigate the different susceptibility to obesity of free feedingSprague-Dawley rats when chronically fed a high-fat and high energy diet, andto establish DIO and DIO-R models for further study.Methods Seventy-two Sprague-Dawley rats (weighing 150g~160g on arrival, one-half males) were randomly divided into high-fat diet group (n=54) and control group (n=18) on the basis of body weight after acclimated to the environment and chow diet for 1 week. Then either high-fat or chow diet were given for thirteen weeks. The chow diet contained 3.29kcal/g with 21.88% as protein, 13.68% as fat, and 64.44% as carbohydrate. The high-fat diet (HF) was composed of 15% pork fat, 10% egg yolk powder, 8% skim milk powder, 5% casein, 2% sugar, and 60% chow diet (w/w%), and contained 4.62kcal/g with 20.00% as protein, 49.85% as fat, and 30.15% as carbohydrate. Rats were housed individually in stainless steel cages. Food and water were supplied ad libitum. Tail blood was collected before the rats were treated with different diet after 10h fasting. Plasma samples were prepared and stored at -20°C until the analysis for concentrations of glucose, total cholesterol(TC), high-density lipoprotein cholesterol(HDL-C) and triglycerides(TG). General condition and dietary intake were observed and recorded daily, and body weight were measured weekly and body length monthly. At the end of thirteenth week, the HF group was subdivided into diet induced obesity (DIO) and diet induced obesity resistant (DIO-R) groups by final body weight (weights more than x|-+ 1.96s of control group are classified as DIO and less than x|-+1s are classified as DIO-R, weights between were discarded). Then all female rats were killed by decapitation after 10h fasting, plasma and serum sample were prepared and stored at -80°C for further analysis. Liver, kidneys, spleen, heart, Ovaries and the visceral fat masses of periovarian, perirenal and retroperitoneal were rapidly separated and weighed. Liver tissue were immediately excised, frozen in liquid nitrogen for frozen tissue section and adipose tissue from perirenal fat pad were immediately excised for paraffin tissue section.Results HF rats had varied body weight gain than chow diet controls and were subdivided into DIO and DIO-R groups . The levels of body weight, body length, Lee's index, all 3 dissected white adipose tissue depots , body fat percent and organs relative weight were greater in DIO than in DIO-R or control rats. The adipose cells morphology were normal in control group, and adipocytes were rounded and enlarged in DIO rats, while adipocytes were rounded but not enlarged in DIO-R rats by hematoxylin-eosin stain. The liver tissues submitted to sudan IV staining showed small lipid droplets in control group, while the size and quantity of lipid droplets were markedly increased in DIO group and in DIO-R group and there were vacuoles existed in the lipid droplets of DIO group. Fasting blood glucose, TC, LDL-C and TG were all higher significantly in DIO group than in control group(P<0.05), while no significant difference was found between DIO-R and control groups(P>0.05), except that TG were increased in DIO-R group.Conclusion We established SD rat models of DIO and DIO-R after 3 months of high-fat feeding. DIO rats were different from DIO-R rats not only in body weight, but also in body fat percent and physiological aspects such as blood lipids . The successful establishment of DIO and DIO-R models provided an attractive tool for further study.Part two Plasma and Gene Expression Levels of PYY in RatsSusceptible or Resistant to Diet Induced Obesity Objective To characterize the food intake of DIO and DIO-R rats after chronic high-fat feeding, and identify the role of PYY and NPY in the development of DIO andDIO-R.Methods Animal treatment was the same as the part one. SD rats were fed with chow diet (control group) or high-fat diet. After 13 weeks, two groups of rats with different response respect to the high-fat diet were identified, and were assigned as diet induced obesity (DIO) and diet induced obesity resistant (DIO-R) rats. Dietary intake were weighed and recorded daily and blood sample were collected, plasma were prepared and stored at -80°C for further assay. All female rats were killed by decapitation after 10h fasting, hypothalamus tissues were homogenized and the supernatant were stored at -80°C for further assay of NPY concentration. Samples from hypothalamus, white adipose tissue , ileum and colon were immediately excised and frozen in liquid nitrogen and then store at -80°C until analysis. Total RNA were extracted using Trizol Reagent and the mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) for PYY of gut, NPY, Y1, Y2, Y5 receptors of hypothalamus and UCPs and PPARy2 of white adipose tissues.Results When transferred from chow diet to HF diet , calorie intake were increased in DIO rats in comparison to DIO-R and control group, but no difference was found between DIO-R and control group. In addition, feeding efficiency (the ratio of weight gained to calories consumed) was significantly increased in DIO as compared to DIO-R and control rats. Regarding PYY, a significant increase in both PYY plasma level and ileum and colon mRNA expression was observed in DIO-R rats in comparison with other groups (control and DIO). Significant increase in both hypothalamic NPY concentration and mRNA expressions of NPY, Y1,Y2 and Y5 receptor gene were also observed in DIO rats as compared with the other groups. But no significant difference was found in plasma NPY level among the three groups. Significant decrease in UCP2 and UCP3 mRNA expressions and increase in PPARγ2 mRNA expressions were also observed in DIO rats as compared with the other groups. Significant increase in UCP2, UCP3 and PPARγ2 were observed in DIO-R group in comparison to control group.Conclusion These data suggest that the susceptibility to obesity of DIO rats might be secondary, at least in part, to an increment of hypothalamic NPY release, which could lead to alterations in food intake (hyperphagia). The higher level of plasma PYY might contribute to the normal hypothalamic NPY release of DIO-R rats and confer resistant to over intaking of high-fat diet. Impaired PYY release of DIO rats after HF feeding might contribute to the higher hypothalamic NPY release and hyperphagia and result in the development of obesity. Higher energy efficiency of DIO rats may correlated with less energy expenditure because of decreased UCP2 ,UCP3, and increased PPARy2 expression.Part three PYY Response to Dietary Intervention inDiet Induced Obesity RatsObjective To explore the PYY response of dietary intervention in DIO rats and the effects on caloric intake.Methods After 15 wks of either a high-fat diet or chow fed diet feeding and establishing the DIO and DIO-R animal model as described in part one, the DIO rats were randomly divided into two subgroups for a 8-week secondary dietary intervention. One of the subgroup was switched to chow diet(DIO-HF/LF), whereas the other continued on high-fat diet (DIO-HF). DIO-R and control rats were continuously fed with HF and chow diet respectively. Weight gain, food intake were measured and caloric intake was calculated, the concentration of plasma NPY and PYY , hypothalamic NPY were assayed. Hypothalamic NPY, Y1, Y2 and Y5 receptor mRNA expression and PYY mRNA expression of ileum and colon were determined by RT-PCR.Results At the end of 15th wk, the levels of body weight and caloric intake were significantly higher in DIO group than in DIO-R or control group(P<0.01), while no significant difference was found between DIO-R and control group (P>0.05) . After switching the DIO rats to chow fed diet, The DIO-HF/LF group consumed significantly less calories when compared with DIO-HF group, and their body weight gains during the following 8 weeks were significantly lower than that of the any other groups(DIO-HF, DIO-R and control group). The calories intake and body weight were significantly higher in DIO-HF rats than in DIO-R or in control group. All 4 dissected white adipose tissue depots and body fat percent were significantly lower in DIO-HF/LF group than in DIO-HF group. Relative liver weight were significantly lower in DIO-HF/LF group than in DIO-HF group and DIO-R group. PYY mRNA and plasma PYY level increased significantly in DIO-HF/LF rats than in DIO-HF rats, and PYY mRNA were significantly higher in DIO-R group than those in DIO-HF or control group, whereas plasma PYY level were higher in DIO-R group than in DIO-HF group, but was not different from that of control group. Hypothalamic NPY mRNA were decreased in both DIO-HF/LF and DIO-R group than in DIO-HF or control group. Hypothalamic NPY concentration were lower significantly in DIO-HF/LF group than that of any other groups, and higher significantly in DIO-HF group than in DIO-R or control group.Conclusion Both genetic background and dietary composition affect the development of obesity. When chronically fed a high-fat and high energy diet, individuals predisposed to obesity down-regulated PYY gene expression and released less PYY, with higher hypothalamic NPY concentration and overeating resulted in obesity, whereas DIO-R individuals might avoid overload by regulate calories intake through spontaneously decrease food intake because of increased PYY release and decreased hypothalamic NPY concentration. High-fat diet contributed to the down-regulation of PYY gene and less PYY release of DIO individuals. DIO individuals had normal calories intake and PYY gene expression and release when placed on low fat chow diet either before or after exposed to high-fat diet. Taken together, the results showed that impaired PYY response and high-fat diet might predispose to obesity.