Physiology - Theses

GLUT4 is the major insulin-sensitive glucose transporter expressed predominantly in skeletal & cardiac muscles, and adipose tissue (AT) and mediates insulin-stimulated glucose transport into these tissues. Due to the major role of skeletal muscle in glucose disposal, considerable attention has focused on this tissue in order to understand how glucose homeostasis is affected by changes in muscle glucose metabolism. However, AT insulin-mediated glucose uptake and GLUT4 expression have been shown to be of importance for both glucose homeostasis and whole body insulin action. Interestingly, reduced GLUT4 expression in AT is a common defect found in insulin resistant states, including obesity, metabolic syndrome, and diabetes, whereas muscle GLUT4 expression is unaltered. Exercise increases AT-GLUT4 expression in rodents, and our lab has previously shown that 4 weeks of exercise training normalizes the expression of GLUT4 in the AT of patients with T2DM. However, the mechanisms involved in exercise-induced up-regulation of AT-GLUT4 are unknown. The broad aim of my research project was to examine the effect of exercise on AT-GLUT4 expression. With this purpose, we conducted three interventions using mice, human primary adipocytes and human subjects, in an attempt to gain a better understanding of: 1) how a high fat diet (HFD) affects the expression of AT-GLUT4, 2) how exercise may be beneficial to prevent this effect; and 3) how this effect may occur. Results showed that AT-GLUT4 protein is rapidly reduced by a HFD, suggesting this could be an early defect contributing to HFD-induced insulin resistance; exercise training increases GLUT4 protein in HFD-fed mice, short-term (10 d) exercise training did not affect GLUT4 levels in human, subcutaneous AT; and serum from exercised subjects increased GLUT4 protein and mRNA in human primary adipocytes suggesting that circulating factor(s) may mediate exercise effects on AT GLUT4 expression. Adipose tissue (AT) glucose transporter GLUT4 is reduced in insulin resistance (IR). Exercise training (EX) (4 weeks) normalized AT-GLUT4 expression in diabetic patients. However, mechanisms involved are unknown. Results showed: 1) AT-GLUT4 is reduced by high fat diet (HFD), which may contributes IR; 2) EX increased GLUT in HFD-fed mice, 3) short-term (10 d) EX didn’t affect GLUT4 in human AT; and 4) exercise serum increased GLUT4 in human primary adipocytes, suggesting circulating factor(s) may mediate EX-effects on AT GLUT4.

Current scientific investigations have shown that obesity is associated with several alterations affecting not only the metabolic state of adipose tissue, since it also interferes with functional outcomes in target organs including liver and skeletal muscle. Metabolic alterations linked with obesity in the adipose tissue include early development of insulin resistance, impaired glucose transport and increased adipogenesis. In this regards, glucose uptake is the rate limiting step for glucose metabolism in adipose tissue. Insulin-mediated glucose uptake provides the cells not only with glucose as an energy substrate towards glycolysis, but also participates in de novo synthesis of fatty acids, fatty acid re-esterification, and a small amount of the incoming glucose is stored as glycogen. Glycogen is the mechanism by which cells store and mobilize glucose to meet their energetic and synthetic demands. During feeding, high blood glucose and plasma insulin command glycogen synthesis. Conversely, glycogen degradation during fasting requires sequential inactivation of glycogen synthase and activation of glycogen phosphorylase – the glycogen-metabolising enzymes – both actions orchestrated by the upstream kinase PKA. Little is known about the regulatory mechanisms controlling glycogen metabolism in the adipose tissue and how it is affected by pathological states related to obesity. The aim of the project was to examine adipose glycogen metabolism and the effects of fasting on glycogen-metabolising enzymes in order to determine whether adipose glycogen metabolism is altered as consequence of the obesity-induced insulin resistance. It was hypothesised that obesity imposes a blunted response to fasting that is reflected by impaired glycogen mobilization. Data obtained in this study showed that lean Zucker rats activated the lipolytic pathway in response to fasting, and NEFA were released into circulation. In contrast, fed and fasted plasma NEFA and glycerol levels were elevated several fold in obese rats, although with a reduced response to fasting. Considering that lipolysis occurs in parallel with glycogen degradation, we then tested the hypothesis that the blunted fasting response was associated with altered glycogen metabolism. This study showed for the first time that glycogen is not mobilized by 24-h fasting in the adipose tissue of obese Zucker rats. It could be initially attributed to an impaired degradation due to the lack of GP activation, although the finding of an impaired GS inactivation could similarly contribute to higher glycogen content following fasting. Additionally, obesity induced a differential effect on the protein content of GS and GP; increasing GS whereas reducing GP proteins expression. Following fasting, protein contents of both enzymes were reduced which could be interpreted as an attempt to counterbalance the unaltered glycogen content during fasting. Accordingly, it can be argued that obesity in Zucker rats may be associated with a higher content of glycogen after 24-h of fasting mainly caused by obesity-induced dysregulation upon GS and GP protein expression, with a lack of GP activation and defective inactivation of GS. These latter findings led us questioning about the involvement of upstream steps into the activation and inactivation of glycogen-metabolising enzymes. Therefore we next examined the activity of the upstream kinase PKA. The PKA activity was increased by fasting in lean rats. However, PKA activity was reduced in obese fasted rats, which was associated with the observed high adipose tissue glycogen levels found among fasted animals. In order to confirm this finding, we tested the activation of CREB, the downstream effector of PKA signalling. CREB phosphorylation was increased 3-fold by fasting in the lean group in parallel with increase in PKA activity. Interestingly, a similar level of phosphorylation was found in obese fed rats when compared with lean fed rats, which implies basal activation of this transcription factor. This over-activated CREB could be explained considering the role of insulin in the activation of CREB. On the other hand, after 24-h fasting, the pCREB/totalCREB ratio was significantly reduced in the obese rats which means that CREB is less active during fasting in obese Zucker rats. Taking into consideration the repressive effect exerted by CREB on the expression of GLUT4, we analysed GLUT4 content in the adipose tissue. As anticipated, fasting reduced GLUT4 protein content in the adipose tissue of lean animals. In the obese rats, GLUT4 protein level was markedly reduced to one third of lean fed content, whereas fasting did not modify GLUT4 protein content in this group, which led us to consider that GLUT4 protein content in the obese was decreased secondary to obesity and that fasting did not induce any further repressive effect within the time course of 24-h of food deprivation. In sum, these novel findings have shed light on how obesity may alter adipose tissue glycogen metabolism impairing its degradation during fasting. Obesity impairs cAMP-PKA signalling leading to dysregulation of glycogen-metabolising enzymes. Additionally, the obesity-induced repressive effect on the protein contents of GLUT4 and GP in association with increased protein level of GS might be related to alterations in carbohydrate metabolism in the adipose tissue. Further analyses are required in order to investigate other potential mechanisms involved in the regulation of glycogen metabolism, impaired cAMP-PKA signalling and GLUT4 expression with obesity.

Physiology - Theses

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