Involvement of protein phosphatase 5 (pp5) in diet-induced obesity and insulin resistance

Jalševac, Florijan (author), Bratkovič, Tomaž (mentor), Caelles Franc, Maria del Carmen (co-mentor)

With 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.

• Language: English
• Keywords: diabetes, diet-induced obesity, insulin resistance, protein phosphatase 5
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-119765
• Publication date in RUL: 11.09.2020

Secondary language

• Language: Slovenian
• Title: Vloga protein fosfataze 5 (pp5) v razvoju s prehrano povzročene debelosti in inzulinske rezistence

Abstract

V današnjem svetu je vse več ljudi, katerim se diagnosticira diabetes, zaradi česar je ta bolezen ena izmed največjih nevarnosti, ki nam pretijo v prihodnosti. Zaradi našega sedečega načina življenja, uživanja večjih količin hrane in pomanjkanja gibanja je več kot verjetno, da se bo število novih pacientov le povišalo. Večina bolnikov po svetu ima diagnosticiran diabetes tipa 2, bolezen, ki sicer deli hiperglikemijo z diabetesom tipa 1, a je ta popolnoma drugega izvora. Medtem ko je diabetes tip 1 avtoimunska bolezen, kjer naš lastni imunski sistem napade in uniči β-celice ledvične žleze, ki proizvajajo inzulin, je tip 2 posledica odpornosti tarčnih tkiv, kot je jetrno, mišično in maščobno tkivo, na inzulinsko signalizacijo, kar pomeni, da inzulin ne opravi svoje naloge, tj. prevzema glukoze. Ta povišan sistemski nivo glukoze povzroči povišano sintezo in izločanje inzulina iz β-celic trebušne slinavke kot mehanizem proti hiperglikemiji, a tega mehanizma β-celice ne morejo vzdrževati v nedogled. Povečan pritisk na trebušno slinavko skupaj z ostalimi dejavniki, med katerimi so tudi okoliški in genetski, povzroči opešanje in propad β-celic, in zaradi tega tudi zmanjšanje izločanja inzulina, kar še bolj poslabša problem. Če to neravnovesje pustimo brez nadzora, se bo sčasoma le poslabšalo, kar na koncu lahko pripelje do mnogih zdravstvenih težav za osebe, ki trpijo za to boleznijo. Eden izmed pomembnih posredovalcev, ki so aktivni v razvoju inzulinske odpornosti, je c-Jun N terminalna kinaza (JNK), protein, ki ga aktivirata vnetje in stres, dve stanji, ki sta značilni za s prehrano povzročeno debelost. Ko se JNK aktivira, ta forforilizacija povzroči inaktivacijo substrata inulinskega receptorja (IRS). S to inaktivacijo JNK posledično prepreči, da se signal inzulina širi naprej, in preko te kinaze vnetje in stres spodbudita razvoj inzulinske odpornosti. Hkrati JNK poviša izražanje vnetnih citokinov, kot je dejavnik tumorske nekroze α, ki dodatno aktivirajo JNK in tako se ustvari začaran krog, ki pripelje do slabšanja odzivnosti tkiv na insulin in hiperglikemije. Aktivacija JNK poteka skozi kaskado mitogensko aktiviranih protein kinaz, med katerimi je tudi signal apoptoze regulirajoče kinaze 1 (ASK-1), le-ta pa je pod vplivom protein fosfataze 5 (Pp5). Ta fosfataza je vključena v mnoge celične funkcije, med katerimi so celična delitev, ravnotežje elektrolitov, apoptoza, popravilo poškodb DNA in druge. Natančna vloga tega proteina v razvoju inzulinske odpornosti ni še popolnoma jasna, saj nekatere raziskave kažejo, da Pp inhibira ASK-1 in posledično inhibira aktivacijo JNK, kar vodi v izboljšanje inzulinske signalizacije v tarčnih tkivih. Vendar, nasprotna dejstva so prisotna, saj so nekateri raziskovalci pokazali, da so miši, ki so imele onesposobljen gen za Pp5, imele povišane nivoje glukokortikoidov. Te spojine delujejo preko posebnih glukokortikoidnih receptorjev, spodbujajo izražanje protivnetnih mediatorjev, kot je interlevkin 10, in hkrati zavirajo izražanje vnetnih mediatorjev, kot so interlevkin 1, 2, 6 in ostali. Zmanjšanje vnetnih dejavnikov povzroči manjšo aktivacijo JNK, kar se izrazi kot izboljšano inzulinsko signaliziranje. Na koncu je vredno tudi omeniti vlogo jetrnih receptorjev X (angleško liver X receptor ali LXR) na inzulinsko občutljivost. Ti receptorji nadzorujejo izražanje genov, ki so vpleteni, med drugim, v biosintezo lipidov in metabolizem ogljikovih hidratov. Z mnogimi različnimi efekti na gene, vključene v lipidni metabolizem, in proti vnetnim učinkom, dodatno podprto s podatki, ki kažejo, da LXR ligandi aktivirajo izražanje Pp5, prestavljajo ti receptorji in njihovi ligandi nov način zdravljenja diabetesa. GW3965 je eden izmed teh ligandov, ki aktivirajo LXR in bi mogoče lahko omogočil razvoj nove skupine antidiabetičnih zdravil. V tej raziskavi nas je zanimalo, kako so različni fiziološki mediatorji, kot je JNK, vpleteni v razvoj s prehrano povzročene debelosti in inzulinske odpornosti in kako vplivajo na razvoj diabetesa tipa 2. Dodatno nas je zanimalo tudi, kako ti mediatorji vplivajo na Pp5 in kako spremenijo vlogo te fosfataze, če jo sploh ima, v razvoju inzulinske odpornosti. Zato smo analizirali, kako se zaradi debelosti povzročeno vnetje izrazi in kakšen je vpliv le-tega na inzulinsko signaliziranje v treh različnih mišjih tkivih: skeletnem mišičnem, jetrnem in belem maščobnem tkivu. Uporabili smo miši divjega tipa in gensko spremenjene miši z inaktiviranim genom Pp5. Živali smo naprej razdelili v dve skupini, kjer smo eno skupino hranili s standardno dieto, drugo pa z dieto z veliko količino maščob. Po desetih tednih smo miši naprej razdelili v dve skupini, kjer je ena skupina prejemala ligand LXR (GW3965). Izvedli smo glukozni tolerančni test in inzulinski tolerančni test, polovica živali pa je tik pred žrtvovanjem prejela inzulin, nakar smo odvzeli izbrana tkiva. Z metodo prenosa western smo preverili stopnjo aktivacije proteinske kinaze B (AKT), ki je protein, aktiviran v procesu inzulinskega signaliziranja, in stopnjo aktivacije JNK. Dodatno smo preverili tudi, kakšen vpliv ima agonist LXR GW3965 na razvoj inzulinske odpornosti. Na koncu smo z uporabo verižne reakcije s polimerazo v mišičnem in v maščobnem tkivu analizirali izražanje gena Slc2a4, ki kodira glukozni transporter (GLUT4), enega izmed glavnih mediatorjev za prevzem glukoze v prej omenjenih tkivih, in gena za vnetni citokin TNF-α.. Iz podatkov, ki smo jih dobili iz analiz AKT, lahko sklenemo, da so živali, ki smo jih hranili s hrano bogato z maščobami, razvile rezistenco na inzulinsko signaliziranje. Analiza podatkov o JNK v maščobnem tkivu je pokazala, da je GW3965 znižal fosforilizacijo te kinaze (tj. znižal je stopnjo njene aktivacije), kar je pomenilo zmanjšanje vnetnega odziva. S tem znižanjem se inzulinsko signaliziranje izboljša in ti podatki nakazujejo, da bi v prihodnosti ta mehanizem lahko izkoriščala nova antidiabetična zdravila. Vendar bolj konkretni zaključki le niso možni, saj nam rezultati analiz iz jetrnega tkiva niso potrdili prej razložene rezultate, kar možno nakazuje, da je vpliv agonistov LXR omejen na le nekatera tkiva. Kvantifikacija izražanja genov na nivoju RNA v mišičnem in belem maščobnem tkivu je pokazala, da je izražanje prenašalca GLUT4 veliko nižje pri miših, ki so razvile inzulinsko odpornost kot posledico prehrane, a smo v maščobnem tkivu zaznali povišano izražanje gena za ta transporter, in sicer pri živalih, ki so prejele GW3965. To je dodaten dokaz, da je prav to tkivo tisto, kjer bi spojine, kot je GW3965, lahko imele najbolj koristen vpliv na zdravljenje sladkorne bolezni tipa 2. Vloge Pp5 v s prehrano povzročeni debelosti in razvoju inzulinske odpornosti nismo uspeli razjasniti bolj podrobno, saj nam niti analiza aktivacije encimov niti analiza izražanja genov na nivoju RNA nista podali statistično značilnih rezultatov. Iz dobljenih podatkov lahko rečemo, da inaktivacija gena Pp5 skupaj z uporabo GW3965 zmanjša fosforilizacijo in s tem aktivacijo JNK ter da tako izboljša inzulinsko signaliziranje, saj se zviša izražanje gena za prenašalec GLUT4. A še vedno obstajajo nasprotujoči si podatki, saj je še posebej jetrno tkivo pokazalo nasprotno vedenje, kjer je bila aktivacija JNK pri enakih pogojih višja kot pa v primeru, ko nismo aplicirali GW3965 in ko je bil gen Pp5 funkcionalen.

• Keywords: diabetes, s hrano povzročena debelost, inzulinska odpornost, protein fosfataza 5