Overweight is one of the most common problems related to modern lifestyle. Severe overweight or obesity is one of the major risk factor for the development of many chronic diseases such as heart and respiratory diseases, diabetes mellitus, non-insulin dependent (or Type 2) diabetes, hypertension and some forms of cancer, as well as the risk of early death. The onset of obesity is a complex and multifactorial phenomenon in which different biological factors (hormones, genes), stress, drugs and aging are involved in addition to the type of dietary regimen. Among the genetic factors, the ob gene, first identified in the mouse and codifying for the hormone leptin, plays a primary role. This hormone is mainly produced by adipose tissue and is considered one of the most important regulator of energy balance also in humans. Though the complete leptin deficiency in humans is an extremely rare condition, it is estimated that about 15% of the population may show reduced leptin levels as the result of a mutation or a polymorphism in the ob gene. Previous studies carried out in our laboratory have characterized the ob/+ heterozygous mouse as an interesting model for the understanding of the interactions between genetic component and eating habits, showing that partial leptin deficiency is an aggravating factor for the development of obesity, insulin resistance and impaired cardiac function in the context of a high-calorie diet. The purpose of the thesis was to investigate the phenomenon of insulin resistance through the study of the molecular mechanisms that regulate insulin signal transduction, by comparing two different models of obesity: othe ob/ob mouse, which spontaneously develops obesity and insulin resistance as the result of the lone genetic defect ; othe ob/+ mouse, where the onset of obesity and metabolic alterations results from the interaction between the partial genetic defect and a high fat and high-calorie dietary regimen. Wild type (wt, control strain) and ob/+ male mice were fed with standard or High Fat diet for 18 weeks, starting from the age of 8 weeks. A group of ob/ob mice, comparable for age and sex to the other groups, was fed with standard diet for the same period of time. Insulin tolerance test and echocardiographic investigations were performed at baseline, at 10 and 18 weeks. At the end of the experimental period, plasma samples were collected for the analysis of blood glucose, insulin, leptin and adiponectin levels. Finally, the animals were sacrificed and cardiac and skeletal muscle samples were collected and frozen at -80°C for subsequent analysis of protein expression by Western Blot. In particular, some key points of glucose metabolism and insulin signaling, such as the insulin receptor (IR), the AKT and AMPK kinases and the insulin-dependent glucose transporter Glut-4, have been investigated. The results confirmed that partial leptin deficiency represents a negative variable in the model of diet-induced obesity, confirming previous data. The group of ob/ob animals, although under a standard diet, showed the same type of metabolic and functional alterations as ob/+ group fed with High Fat diet. The molecular analyses performed on cardiac tissues supported the hypothesis of the existence of two different mechanisms responsible for insulin resistance observed in both models, ob/+ and ob/ob. In particular, in ob/+ animals fed with High Fat diet, alterations in the pathway may depend on a reduced translocation of the glucose transporter to the plasma membrane. In contrast, in ob/ob animals, Glut-4 translocation appeared efficient, however the protein did not seem active; in this particular context, the protein kinase p38mapk is supposed to play an important role. These results are in line with what suggested by other authors, about the need of two steps in series for the proper functioning of the glucose transporter: translocation and activation. The comparison between cardiac and skeletal muscle seemed to reveal some tissue-specific regulatory mechanisms as regards to insulin transduction signaling; this topic, however, is presently under analysis. The final part of the thesis work also compared, in the two different models of obesity, the effect of treatment with apolipoprotein A1 mimetic peptide, L- 4F. According to previous studies, this peptide was able to reduce obesity and metabolic and heart disorders in ob/+ animals under High Fat diet. Treatment with L-4F provided mixed results among the different experimental groups: while in the heterozygous strain it proved to be effective in restoring plasma parameters, echocardiographic parameters and protein expression to the control levels, in the ob/ob strain the effects were negligible and not able to return the altered parameters within the normal levels. The difference in efficacy observed in response to treatment, as well as the differences at the molecular level in the regulation of insulin signaling pathways, are indicative of the multifactorial nature of obesity and its complications, and therefore of the need to develop personalized pharmacological approaches.
MODULAZIONE DEL SEGNALE DELL'INSULINA IN MODELLI MURINI DI OBESITA' GENETICA E INDOTTA DA DIETA
2014
Abstract
Overweight is one of the most common problems related to modern lifestyle. Severe overweight or obesity is one of the major risk factor for the development of many chronic diseases such as heart and respiratory diseases, diabetes mellitus, non-insulin dependent (or Type 2) diabetes, hypertension and some forms of cancer, as well as the risk of early death. The onset of obesity is a complex and multifactorial phenomenon in which different biological factors (hormones, genes), stress, drugs and aging are involved in addition to the type of dietary regimen. Among the genetic factors, the ob gene, first identified in the mouse and codifying for the hormone leptin, plays a primary role. This hormone is mainly produced by adipose tissue and is considered one of the most important regulator of energy balance also in humans. Though the complete leptin deficiency in humans is an extremely rare condition, it is estimated that about 15% of the population may show reduced leptin levels as the result of a mutation or a polymorphism in the ob gene. Previous studies carried out in our laboratory have characterized the ob/+ heterozygous mouse as an interesting model for the understanding of the interactions between genetic component and eating habits, showing that partial leptin deficiency is an aggravating factor for the development of obesity, insulin resistance and impaired cardiac function in the context of a high-calorie diet. The purpose of the thesis was to investigate the phenomenon of insulin resistance through the study of the molecular mechanisms that regulate insulin signal transduction, by comparing two different models of obesity: othe ob/ob mouse, which spontaneously develops obesity and insulin resistance as the result of the lone genetic defect ; othe ob/+ mouse, where the onset of obesity and metabolic alterations results from the interaction between the partial genetic defect and a high fat and high-calorie dietary regimen. Wild type (wt, control strain) and ob/+ male mice were fed with standard or High Fat diet for 18 weeks, starting from the age of 8 weeks. A group of ob/ob mice, comparable for age and sex to the other groups, was fed with standard diet for the same period of time. Insulin tolerance test and echocardiographic investigations were performed at baseline, at 10 and 18 weeks. At the end of the experimental period, plasma samples were collected for the analysis of blood glucose, insulin, leptin and adiponectin levels. Finally, the animals were sacrificed and cardiac and skeletal muscle samples were collected and frozen at -80°C for subsequent analysis of protein expression by Western Blot. In particular, some key points of glucose metabolism and insulin signaling, such as the insulin receptor (IR), the AKT and AMPK kinases and the insulin-dependent glucose transporter Glut-4, have been investigated. The results confirmed that partial leptin deficiency represents a negative variable in the model of diet-induced obesity, confirming previous data. The group of ob/ob animals, although under a standard diet, showed the same type of metabolic and functional alterations as ob/+ group fed with High Fat diet. The molecular analyses performed on cardiac tissues supported the hypothesis of the existence of two different mechanisms responsible for insulin resistance observed in both models, ob/+ and ob/ob. In particular, in ob/+ animals fed with High Fat diet, alterations in the pathway may depend on a reduced translocation of the glucose transporter to the plasma membrane. In contrast, in ob/ob animals, Glut-4 translocation appeared efficient, however the protein did not seem active; in this particular context, the protein kinase p38mapk is supposed to play an important role. These results are in line with what suggested by other authors, about the need of two steps in series for the proper functioning of the glucose transporter: translocation and activation. The comparison between cardiac and skeletal muscle seemed to reveal some tissue-specific regulatory mechanisms as regards to insulin transduction signaling; this topic, however, is presently under analysis. The final part of the thesis work also compared, in the two different models of obesity, the effect of treatment with apolipoprotein A1 mimetic peptide, L- 4F. According to previous studies, this peptide was able to reduce obesity and metabolic and heart disorders in ob/+ animals under High Fat diet. Treatment with L-4F provided mixed results among the different experimental groups: while in the heterozygous strain it proved to be effective in restoring plasma parameters, echocardiographic parameters and protein expression to the control levels, in the ob/ob strain the effects were negligible and not able to return the altered parameters within the normal levels. The difference in efficacy observed in response to treatment, as well as the differences at the molecular level in the regulation of insulin signaling pathways, are indicative of the multifactorial nature of obesity and its complications, and therefore of the need to develop personalized pharmacological approaches.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
