الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Diabetes mellitus is one of the most common chronic diseases in nearly all countries, and continues to increase in numbers and significance, as economic development and urbanization lead to changing lifestyles characterized by reduced physical activity, and increased obesity. The increasing prevalence of Type 2 diabetes mellitus has stimulated an active search for novel risk factors. A number of peptide hormones have been identified and have been suggested to play a role in the pathogenesis of Type2 DM and metabolic syndrome. One of these hormones is ghrelin which is a peptide hormone from the stomach with growth hormone releasing activity. It is also able to modify glucose and insulin metabolism, blood pressure levels, adipogenesis, and inflammatory processes.
The majority of the studies addressing the relationship between ghrelin and diabetic states suggest that a correlation between ghrelin and diabetes mellitus might exist. There is a growing body of evidence indicating a suppressive role of ghrelin in the release of insulin from the pancreatic islets. Recent literature showed that fasting plasma concentrations of total ghrelin were lower among subjects with Type 2 diabetes compared to those without Type 2 diabetes.
The characterization of the gene encoding ghrelin and its overall genomic structure has made possible genomic screening of the ghrelin gene. Mutations of the ghrelin gene could potentially cause a defective or inactive ghrelin. One of the recently discovered mutations was identified at amino acid position 51 of the preproghrelin gene resulting in the substitution of arginine by glutamine (Arg51Gln). This mutation seems to be associated with low plasma ghrelin concentrations and might possibly increase the risk for Type 2 diabetes.
The present study was conducted to assess plasma ghrelin level in patients with type 2 DM as well as healthy controls, and to determine its relation to the ghrelin Arg51Gln mutation. The study also aimed to determine the relationship between plasma ghrelin level and age, sex, anthropometric indices, blood pressure, biochemical parameters, as well as nutritional factors and lifestyle habits.
A case control approach was used to conduct the study. Type 2 diabetic patients (cases) aged 35 years or more and from both sexes were selected, attending the Diabetes Clinic of the Alexandria Main University Hospital. As for controls, apparently healthy persons matched for sex and within a 5 year age group, from other clinics or departments of the Alexandria Main University Hospital were selected. The sample size was 124 subjects for each group.
All subjects were interviewed to answer a pre-designed structured questionnaire which included demographic and socioeconomic data, medical and family history as well as data on lifestyle habits (smoking and physical activity). Anthropometric measurements were recorded including weight, height, waist circumference and hip circumference then body mass index and Waist/Hip ratio were calculated. Blood pressure was also measured.
Blood samples (10 ml) were taken from both cases and controls after an overnight fast, then it was divided into two equal portions. The first 5 ml were delivered into plain tubes, left to clot then centrifuged and the obtained serum was assayed on the same day to determine serum glucose level, lipid profile, kidney function tests and liver function tests. The rest of the blood (5 ml) was delivered into two sterile EDTA tubes; one tube was used for DNA extraction from leucocytes and the extracted DNA was stored at -20°C until used for the detection of the Arg51Gln mutation in the ghrelin gene by polymerase chain reaction (PCR). The other tube was centrifuged and the obtained plasma was stored at -20°C until assayed for ghrelin and insulin levels using enzyme - linked immunosorbant assay (ELISA).
All subjects were also interviewed to answer a pre-designed quantitative food frequency questionnaire. It included data about the different food groups, as well as additional information about the preferred methods of cooking.
After completion of data collection, data were fed to the computer using the Foxpro for windows data base program. Then, after checking and correction of the data, the data base file was transferred to the SPSS program version 20.0 software for tabulation and statistical analysis. A nutrient database and analysis program was designed using Foxpro for windows software to calculate nutrient intakes. Univariate and multivariate analyses were performed to demonstrate the different sample characteristics and to establish the relationships of the different variables with plasma ghrelin level.
As regards the level of plasma ghrelin, the present study revealed that mean plasma ghrelin level was significantly lower in diabetics compared to controls in both males and females (P=0.000**) (Table VIII). Furthermore, the stepwise multiple linear regression revealed that the type of case (diabetic/control) was the first significant predictor for ghrelin level (β=0.402) (Table XLII).
The present study has investigated many factors that may affect plasma ghrelin level including age and sex, lifestyle habits (smoking and physical activity), dietary characteristics, anthropometric measurements and obesity indices, blood pressure, insulin level, blood glucose level and lipid profile. Concerning age, the present study has demonstrated a negative but non-significant correlation between ghrelin level and age in both cases and controls (Table XIV).
As for sex, the present results show that in cases; the mean plasma ghrelin level was higher in females (457.8±64.21 pg/ml) than in males (447.7±50.94 pg/ml). While in controls; the level was lower in females (566.3±79.50 pg/ml) compared to males (602.5±79.10 pg/ml) (Table VIII). Concerning the association of ghrelin level with sex, the present study demonstrated that in controls; the frequency distribution according to quartiles was significantly different between males and females (P=0.011*), where the highest percentage of male controls (61.9%) was present in the highest ghrelin quartile, while the highest percentage of female controls (45.2%) was present in the third ghrelin quartile (Table XXI). This observation of the lower ghrelin level in female controls might be explained by the higher BMI in female subjects.
The present study has shown the influence of lifestyle factors (smoking and physical activity) on ghrelin level. Regarding smoking, it was found that ghrelin was negatively correlated with Ferris index of present smoking in both cases and controls, with a significant correlation in cases (P=0.050*) (Table XIV). However, there is an observation worth mentioning, in cases; the highest percentage of non-smokers (47.1%) came in the lowest ghrelin quartile while that in present smokers (52.2%) came in the second ghrelin quartile, which might unravel a raising effect of smoking on the ghrelin level (Table XXVII). As for controls, it was observed that more than half of present smokers (62.5%) compared to only 44.0% of non-smokers came in the highest ghrelin quartile, again implying a raising effect of smoking on ghrelin (Table XXVIII).
As for the association between physical activity and plasma ghrelin level, the present study showed, in controls; a significant difference in the frequency distribution according to ghrelin quartiles between sedentary, light and moderate occupational activity (P=0.010*) (Table XXXII). It was also noticed that more than half of diabetics enrolled in moderate occupational activity (55.6%) compared to 38.5% of those doing sedentary activity came in the lowest ghrelin quartile (Table XXXI), thus suggesting a lowering effect on ghrelin level with higher levels of physical activity.
Regarding the relationship between ghrelin level and energy balance, the present study has clearly demonstrated that ghrelin was inversely and significantly correlated with mean daily energy intake (P=0.000**), as well as mean daily intakes of all macronutrients (proteins, fats, carbohydrates) in both cases and controls (Table XVIII). Furthermore, the stepwise multiple linear regression showed that the mean daily protein intake was the fourth significant predictor for ghrelin level (β=0.106) (Table XLII), showing that ghrelin is inversely and independently associated with the protein content of the meal.
In the present study, it is obvious that ghrelin was significantly and negatively correlated with most of the obesity indices in both cases and controls (Table XIV). An inverse significant correlation was observed for BMI in both cases and controls (P=0.000**), as well as for waist circumference in cases (P=0.000**). And finally, ghrelin was inversely and significantly correlated with Waist/Hip ratio in cases (P=0.048*). In addition, the stepwise multiple linear regression showed that BMI was the third significant predictor for ghrelin level (β=0.188), showing that ghrelin is inversely and independently associated with BMI (Table XLII).
Concerning the effect of blood pressure on plasma ghrelin, the present study demonstrated that ghrelin level was inversely correlated with both systolic and diastolic blood pressures in both cases and controls (Table XIV); the correlation being significant (P=0.000**) in diabetics, suggesting that the inverse relationship between fasting ghrelin level and both systolic and diastolic BP was stronger in the diabetic state.
As for the relationship between ghrelin and insulin, the present study has clearly demonstrated that ghrelin level was negatively and significantly correlated with insulin level in both cases and controls (P=0.000**) (Table XVI). In addition, the stepwise multiple linear regression revealed that plasma insulin was the second significant predictor (β=0.379) for ghrelin level (Table XLII), thus supporting the fact that ghrelin and insulin are independently associated with one another.
Laying shadow on the interplay between ghrelin level and blood glucose, the current study has demonstrated that ghrelin was inversely correlated with fasting blood glucose in both cases and controls, the correlation being highly significant in cases (r=-0.449, P=0.000**) (Table XVI).
The present study has also investigated the relationship between ghrelin and lipid profile. It has been demonstrated that in diabetics, ghrelin had an inverse relationship with total cholesterol, LDL cholesterol and triglycerides; where the correlation was significant with TG (P=0.004**) (Table XVI). On the other hand, ghrelin showed a direct but non-significant correlation with HDL cholesterol in both cases and controls. These data further support the fact that low ghrelin level is related to the less favorable levels of each of these variables.
And finally we come to the effect of the Arg51Gln mutation, where it was found that the percentage of the Arg51Gln mutation was quite similar between the two study groups, where the Arg/Gln allele was present in 4.8% of diabetics compared to 4.0% of controls, with no statistically significant difference between them (Table X), thus implying that the mutation was not associated with an increased risk of Type 2 diabetes. As regards the association of plasma ghrelin level with Arg51Gln mutation, our study has demonstrated that there was a highly significant difference in the frequency distribution according to ghrelin quartiles among subjects having the Arg/Arg genotype and those having the Arg/Gln genotype (P=0.000**) in both diabetics and controls (Table XXXIX). These results suggest that low ghrelin level is strongly associated with the Arg51Gln mutation in both groups of the study.
from the present study we came to the following conclusions:
• Fasting plasma ghrelin level was significantly lower in diabetics than in controls. In addition, the type of case was the most significant predictor for ghrelin level.
• Plasma ghrelin was inversely and significantly correlated with insulin level in both diabetics and controls. Moreover, insulin level was found to be the second predictor for ghrelin level.
• Plasma ghrelin was inversely correlated with fasting blood sugar and the correlation was highly significant in diabetics.
• In diabetics, plasma ghrelin was inversely correlated with TC, LDLC and TG, and directly correlated with HDLC in both diabetics and controls.
• Plasma ghrelin was found to be inversely correlated with age in both cases and controls. As for sex, ghrelin level was lower in female controls compared to males which might be explained by the higher BMI in females.
• An interesting observation was unraveled from the association studies; a raising effect of smoking on the ghrelin level in both cases and controls, which goes in agreement with available research. Concerning the association between physical activity and plasma ghrelin level, a lowering effect was observed on ghrelin level with higher levels of occupational physical activity in cases.
• Fasting ghrelin level was found to be inversely and significantly correlated with total energy intake as well as mean daily intakes of all macronutrients (proteins, fats, carbohydrates) in both cases and controls. Moreover, mean daily protein intake was found to be the fourth significant predictor for ghrelin level, showing that ghrelin is inversely and independently associated with the protein content of the meal.
• Plasma ghrelin was inversely and significantly correlated with most of the obesity indices (BMI, waist circumference, and waist/hip ratio) in both cases and controls. Moreover, BMI was found to be the third significant predictor for ghrelin level, showing that ghrelin is inversely and independently associated with BMI.
• Plasma ghrelin was found to be inversely correlated with both systolic and diastolic blood pressure in both cases and controls, where the correlation was significant in diabetics.
• The percentage of the Arg51Gln mutation was quite similar between the two study groups, where the Arg/Gln allele was present in 4.8% of diabetics compared to 4.0% of controls, with no statistically significant difference between them, thus implying that the mutation was not associated with an increased risk of Type 2 diabetes.
• As regards the association of plasma ghrelin level with Arg51Gln mutation, the study has demonstrated that there was a highly significant difference in the frequency distribution according to ghrelin quartiles among subjects having the Arg/Arg genotype and those having the Arg/Gln genotype in both diabetics and controls. These results support the hypothesis that low ghrelin level is strongly associated with the Arg51Gln mutation.
The most important recommendations that have been suggested by the present study include aspects for future research and studies:
• Prospective studies clarifying whether ghrelin plays a role in the development of insulin resistance, diabetes, and cardiovascular disease.
• Studies with large study populations and long follow-up times are needed to clarify the predictive value of ghrelin for the development of metabolic abnormalities and Type 2 diabetes.
• More studies are needed to show whether and to what extent, the genetics of ghrelin and its receptors are involved in the pathogenesis of Type 2 diabetes and metabolic syndrome.
• More research is needed to better understand the role of different forms of ghrelin in Type 2 diabetes and metabolic disorders, because measuring acylated and des-acyl ghrelin separately in a similar study design might provide further information on the role of ghrelin in the regulation of glucose homeostasis.
• Studies focusing on identification of the stimuli and pathways regulating ghrelin synthesis and release are fundamental to discovering clinical uses for ghrelin, which can open a vast field in the management of Type 2 diabetes, metabolic syndrome and obesity.