Involvement of protein phosphatase 5 (pp5) in diet-induced obesity and insulin resistanceJalševac, Florijan (Avtor) Bratkovič, Tomaž (Mentor) Caelles Franc, Maria del Carmen (Komentor) diabetesdiet-induced obesityinsulin resistanceprotein phosphatase 5With an ever-increasing number of people who are getting diagnosed with diabetes, this disease presents one of the modern world’s biggest dangers. With our sedentary lifestyle, together with increased food consumption, it is more than likely that this increase is not going to slow down any time soon. Most patients around the world are diagnosed with type 2 diabetes, a disease that shares the hyperglycaemia with diabetes type 1, but which origins are completely different. While the type 1 diabetes is an autoimmune disease, where our own immune system attacks and destroys the insulin-producing pancreatic β-cells thus causing hyperglycaemia, type 2 diabetes is caused because target tissues, such as liver, muscle and adipose tissues, develop insulin resistance, meaning that insulin fails in its role of stimulating glucose intake. This elevated systemic level of glucose causes the increase in the insulin production and its secretion from the pancreatic β-cells, as a way to battle hyperglycaemia, but this mechanism cannot be sustained by the β-cells indefinitely. This increased burden on the β-cells, combined with other factors, which include environmental and genetic components, results in pancreatic failure with the concomitant decrease in insulin secretion, exacerbating the problem and worsening the blood glucose levels. If this unbalance is left unchecked, it will progressively deteriorate, and it can lead to a multitude of health complications for the affected individual. One of the important mediators that are active in this development of insulin resistance is c-Jun N-terminal kinase (JNK), which is a protein that is stimulated by inflammation and stress, two conditions that are regularly present in diet-induced obesity. These two conditions activate the JNK, phosphorylating the protein and activating it, thus concomitantly inactivating insulin receptor substrate (IRS). By inactivating the IRS, JNK consequently prevents the insulin signalling cascade propagating its signal and through this kinase, the inflammation and stress cause the inhibition of insulin effect. Moreover, JNK also increases the expression of inflammatory cytokines, such as tumour necrosis factor α (TNF-α), which then in turn again activate JNK, resulting in the spiralling effect, all culminating in the worsening of the tissue insulin signalling and increasing the systemic glucose levels. Activation of JNK occurs through a mitogen-activated protein kinase cascade, and one of these kinases, known as apoptosis signal-regulating kinase 1 (ASK-1), is under the control of protein phosphatase 5 (Pp5). This phosphatase is involved in a multitude of different cellular functions, including cell proliferation, electrolyte balance, apoptosis and DNA repair, among others. The exact role of Pp5 in the development of insulin resistance is still unclear, as some research suggests that Pp5 inhibits ASK-1, and consequently, inhibits the activation of JNK, resulting in improved insulin signalling in the insulin targeted tissues. However, conflicting evidence is presented, as some researchers showed that mice with inactivated Pp5 gene had increased levels of glucocorticoids. These compounds, through their specialised glucocorticoid receptors, promote the expression of anti-inflammatory proteins, like interleukin 10, and inhibit the expression of inflammatory ones, such as interleukins 1, 2, 6 and others. Lowering of inflammation also causes less inflammatory cytokines, leading to less JNK activation, and consequently, better insulin signalling in the targeted tissues. Finally, it is important to also consider the role liver X receptors (LXR) have in the insulin sensitivity. These receptors regulate the expression of genes that partake in lipid biosynthesis and carbohydrate metabolism, among others. With its variety of effects on multiple genes involved in lipid metabolism and anti-inflammatory effect, and with studies showing that LXR ligands activate the expression of Pp5, these receptors and their ligands could present a novel new way for the treatment of diabetes. GW3965 is one of these LXR agonists, and this non-steroidal compound could represent a novel new group of antidiabetic drugs. In our study, we were interested how different physiological mediators, such as JNK, are involved in the development of diet-induced obesity and insulin resistance, and how they influence the onset of type 2 diabetes. Another aspect we were interested in was how these mediators interact with Pp5 and how they change its role, if it all has any, in the progress of the insulin resistance. For this purpose, we analysed how inflammation induced by the obesity is expressed and how it influences the insulin signalling cascade in three different mice tissues: skeletal muscle, liver and epididymal white adipose tissue. We used wild-type and genetically altered mice with inactivated Pp5 gene. These animals were then further divided into 2 groups, where one was kept on a standard diet, and the other on the high-fat diet. After 10 weeks, we divided the subjects into 2 groups and treated one with the LXR ligand GW3965. Glucose tolerance test and insulin tolerance test were performed, half of the animals in the experiment received insulin just before they were sacrificed, and their tissues were collected. With the use of immunoblotting, we evaluated the activation levels of protein kinase B (AKT), which is a protein activated in the process of insulin signalling, and JNK. In addition, we analysed the effect of LXR agonist GW3965 on the development of insulin resistance. Finally, using quantitative polymerase chain reaction, we analysed the expression of Slc2a4, the gene that encodes glucose transporter type 4 (GLUT4), one of the main transporters for glucose intake, and the pro-inflammatory cytokine TNF-α in the skeletal and epididymal adipose tissues. From the AKT data, we were able to conclude that animals, which were fed with the high-fat diet, developed resistance to insulin signalling. Analysis of the JNK activation showed that in epididymal white adipose tissue GW3965 lowered the phosphorylation of the protein, making it less active and lowering the inflammation signalling. With this decrease of inflammation, insulin signalling is improved, and these data indicate that GW3965 is a potential new antidiabetic medication. However, it is difficult to draw more concrete conclusions, as the liver tissue results did not corroborate results from adipose tissue, indicating that the influence of LXR agonists could be limited to certain tissues. RNA experiments in the muscle and epididymal white adipose tissue displayed lowered GLUT4 expression levels in the mice which had developed insulin resistance. But interestingly, epididymal white adipose tissue taken from mice that were treated with GW3965 showed a significant increase in the Slc2a4 gene expression, adding additional evidence that this tissue is where this compound could be used with the most benefits. The role of Pp5 in diet-induced obesity and insulin resistance remains elusive though, as we could not draw any concrete conclusions from the experiments. From the data we gathered we can state that Pp5 deletion, with concurrent use of GW3965, inhibits the activation of JNK, thus improving insulin signalling pathway, with the consequence being a higher expression of the GLUT4 gene. But some contradictory data also seem to be present, as liver samples especially displayed unusual behaviour, with Pp5 deletion causing an increase of JNK activation in the presence of GW3965.20202020-09-11 08:45:02Magistrsko delo/naloga119765sl