Search

NAMEN: Splošni namen doktorske disertacije je bil preučiti molekularne mehanizme, prek katerih skeletne mišice uravnavajo delovanje z AMP-aktivirane protein kinaze (AMPK) v fizioloških in patofizioloških stanjih, katerih posledica je indukcija imunskega odziva. Poleg tega je bil cilj pojasniti tudi molekularne mehanizme, prek katerih farmakološki modulatorji metabolizma vplivajo na AMPK v skeletnih mišicah. Specifični cilji so bili: (1) opredeliti molekularne mehanizme, prek katerih dikloroacetat (DCA) vpliva na piruvat dehidrogenaza kinazo 1 (PDK1) in AMPK v presnovno različnih tipih celic (2) raziskati, kako aktivacija signalne poti STING-TBK1 vpliva na presnovni status celic in delovanje AMPK in (3) preučiti vlogo AMPK pri vplivu signalne poti STING-TBK1 na delovanje inzulina v skeletnomišičnih celicah. HIPOTEZE: Preverili smo tri hipoteze: (1) Dikloroacetat zniža raven kinaze piruvat dehidrogenaze in spodbudi aktivacijo AMPK v skeletnomišičnih celicah. (2) Signalna pot STING-TBK1 zavira delovanje AMPK v skeletnomišičnih celicah. (3) Farmakološka aktivacija AMPK zmanjša s TBK1 spodbujeno inzulinsko rezistenco v skeletnomišičnih celicah. METODE: Za raziskovanje molekularnih procesov povezanih s presnovnimi odzivi smo uporabili in vitro modele skeletne mišice (primarne (hSkMC) in iz induciranih pluripotentnih matičnih celic (iPSC)-razvite človeške skeletnomišične celice (id-SkMC) ter podganje (L6) skeletnomišične celice). Diferenciacijo skeletnomišičnih celic, razvitih iz iPSC, smo spremljali z RT-qPCR, imunofluorescenco in odtisom western. Za preučevanje odziva tarčnih proteinov in analizo celične signalizacije smo uporabili molekularnobiološke metode (odtis western, RT-qPCR, gensko utišanje z metodo siRNA, ELISA in Magnetic Luminex test za analizo citokinov). Vpliv farmakoloških učinkovin na presnovne procese smo določali s pomočjo analize tvorbe laktata in privzema glukoze. REZULTATI: Ad H1: Napačno delovanje PDK je povezano s presnovnimi motnjami, sladkorno boleznijo tipa 2, debelostjo in rakom. Neposredno z inhibicijo delovanja PDK in posredno z zaviranjem transkripcije gena za PDK1, DCA spodbuja delovanje kompleksa piruvat dehidrogenaze (PDC) in izboljša presnovno homeostazo. Tretiranje z DCA je povzročilo selektiven upad proteina PDK1 v presnovno različnih tipih celic, kar ni bilo povsem odvisno le od regulacije transkripcije. Pri dodatnih posttranskripcijskih mehanizmih, prek katerih je DCA deloval, AMPK ni imela bistvene vloge. Ad H2: Debelost in sladkorna bolezen tipa 2 sta povezana s kroničnim vnetjem. V adipocitih lahko pride do indukcije vnetja tudi zato, ker debelost spodbuja sprostitev mitohondrijske DNA v citosol in posledično aktivacijo signalne poti cGAS-STING-TBK1. V človeških skeletnomišičnih celicah in cevčicah L6 smo signalno pot STING-TBK1 aktivirali z eksogenim 2'3'-cGAMP oziroma z aktivatorjem proteina STING, DMXAA. Aktivacija signalne poti STING-TBK1 je povzročila povečanje aktivnosti AMPK. Ad H3: Z debelostjo povezano kronično vnetno stanje prispeva k razvoju inzulinske rezistence. cGAMP in TBK1 spodbudita aktivacijo protein kinaze B (Akt), ki je glavna kinaza v inzulinski signalni poti. 2'3'-cGAMP je v človeških skeletnomišičnih celicah razvitih iz iPSC stimuliral aktivacijo Akt, DMXAA pa je v cevčicah L6 zavirala z inzulinom spodbujeno aktivacijo Akt in privzem glukoze. Aktivacija AMPK z AICAR je dodatno povečala, medtem ko je posredna inhibicija AMPK s STO-609 preprečila s TBK1-posredovane učinke. ZAKLJUČKI: (1) DCA selektivno zmanjša raven proteina PDK1 in PDK2 ter inducira aktivacijo AMPK v cevčicah L6, kar potrjuje našo prvo hipotezo. (2) Skeletnomišične celice imajo funkcionalno signalno pot cGAS-STING-TBK1, katere aktivacija spodbudi delovanje AMPK, kar ne podpira druge hipoteze. (3) Čeprav 2'3'-cGAMP in DMXAA različno vplivata na inzulinsko signalno pot, je funkcionalna AMPK bistvena za učinke navzdol od TBK1 v skeletnomišičnih celicah. Farmakološka aktivacija AMPK okrepi učinke, posredovane s TBK1, kar ne podpira tretje hipoteze. AIMS: General purpose of doctoral dissertation was to study molecular mechanisms that regulate AMP-activated protein kinase (AMPK) activity in skeletal muscles in physiological and pathophysiological conditions related to induction of immune response. Also, the aim was to elucidate molecular mechanisms by which pharmacological modulators of metabolism affect AMPK in skeletal muscles. Specific aims of doctoral thesis were: (1) to determine molecular mechanisms through which dichloroacetate (DCA) affects pyruvate dehydrogenase kinase 1 (PDK1) and AMPK in metabolically different cell types (2) to investigate how the activation of STING-TBK1 signaling pathway affects cell’s metabolic status and AMPK activity, and (3) to examine the role of AMPK in the effect of STING-TBK1 signaling pathway on insulin action in skeletal muscle cells. HYPOTHESES: We tested three hypotheses: (1) Dichloroacetate decreases pyruvate dehydrogenase kinase level and induces the activation of AMPK in skeletal muscle cells. (2) STING-TBK1 signaling pathway suppresses AMPK activity in skeletal muscle cells. (3) Pharmacologic activation of AMPK decreases TBK1-induced insulin resistance in skeletal muscle cells. METHODS: In vitro models of the skeletal muscle (primary human skeletal muscle cells (hSkMCs), induced pluripotent stem cells (iPSCs)-derived human skeletal muscle cells (id-SkMCs), and rat (L6) skeletal muscle cells) were used to investigate molecular processes associated with metabolic responses. The differentiation of skeletal muscle cells developed from iPSCs was monitored with RT-qPCR, immunofluorescence and western blot. In order to study target protein response and cell signalization, we used molecular methods (western blot, RT-qPCR, gene silencing with siRNA, ELISA and Magnetic Luminex Assay). Effects of pharmacological agents on metabolic processes were determined by analysis of lactate production and glucose uptake. RESULTS: Ad H1: Dysfunction of PDK is associated with metabolic disorders, type 2 diabetes, obesity and cancer. By directly inhibiting PDK and indirectly suppressing PDK gene transcription, dichloroacetate (DCA) promotes the activity of pyruvate dehydrogenase complex (PDC) and improves metabolic homeostasis. Treatment with DCA selectively decreased PDK1 protein abundance in metabolically different cell types, which was partially independent of the regulation at the transcription level. Activated AMPK was not required for additional post-transcriptional mechanisms underlying DCA action. Ad H2: Obesity and type 2 diabetes are associated with chronic inflammation. In adipocytes, the inflammation is likely to develop, also, because obesity promotes release of mitochondrial DNA into the cytosol and, consequently, the activation of the cGAS-STING-TBK1 signaling pathway. In human SkMCs and L6 myotubes the activation of STING-TBK1 was induced with exogenous 2'3'-cGAMP or with STING activator DMXAA, respectively. STING-TBK1 activation increased AMPK activity. Ad H3: Obesity-related chronic inflammation contributes to the development of insulin resistance. cGAMP and TBK1 promote the activity of protein kinase B (Akt), the major kinase within the insulin signaling pathway. Treatment of id-SkMCs with 2'3'-cGAMP increased Akt activity, while DMXAA suppressed insulin-induced Akt activity and glucose uptake in L6 myotubes. AMPK activator AICAR further enhanced, whereas indirect inhibition of AMPK activity with STO-609 prevented TBK1-mediated effects. CONCLUSIONS: (1) DCA selectively decreases PDK1 and PDK2 protein levels and induces the activation of AMPK in L6 myotubes, which supports our first hypothesis. (2) Skeletal muscle cells have functional cGAS-STING-TBK1 signaling pathway which promotes AMPK activity, which does not support the second hypothesis. (3) 2'3'-cGAMP and DMXAA exhibit divergent effects on insulin signaling, but functional AMPK is required for TBK1 downstream effects in skeletal muscle cells. The pharmacologic activation of AMPK further enhances TBK1-mediated effects, which does not support the third hypothesis.

Uvod: Sladkorna bolezen tipa 2 je najpogostejša oblika sladkorne bolezni. Pri sladkorni bolezni tipa 2 se razvije izrazita inzulinska rezistenca v skeletnih mišicah, ki so v normalnih razmerah glavno mesto privzema glukoze po obroku. Farmakološka aktivacija z AMP-aktivirane protein kinaze (AMPK) predstavlja obetaven pristop za zmanjšanje inzulinske rezistence v skeletnih mišicah. Učinkovine, ki delujejo kot aktivatorji AMPK, bi zato lahko bile uporabne pri zdravljenju sladkorne bolezni tipa 2. Namen: Sulfasalazin, podobno kot antirevmatik metotreksat, zavira 5-aminoimidazol-4-karboksamid ribonukleotid formiltransferazo/inozin monofosfat ciklohidrolazo (ATIC) in tako zmanjša očistek ZMP. ZMP (AICAR-monofosfat) je farmakološko aktivna oblika 5-aminoimidazol-4-karboksamid-1-&#946 -D-ribonukleozida (AICAR), ki je najbolj pogosto uporabljen eksperimentalni aktivator AMPK, kar nakazuje da bi lahko tudi sulfasalazin deloval kot aktivator AMPK. Salicilna kislina pa se veže na AMPK in jo aktivira. Namen naše naloge je bil preveriti, ali sulfasalazin in/ali njegova presnovka sulfapiridin in 5-aminosalicilna kislina spodbudijo aktivacijo AMPK v skeletni mišici. Hipoteze: (1) Sulfasalazin spodbudi aktivacijo AMPK v skeletnomišičnih celicah neodvisno od svojih presnovkov 5-aminosalicilne kisline in sulfapiridina (2) Sulfasalazin ojača z AICAR spodbujeno aktivacijo AMPK v skeletnomišičnih celicah neodvisno od svojih presnovkov 5-aminosalicilne kisline in sulfapiridina (3) Derivati salicilne kisline balsalazid, olsalazin in diflunisal aktivirajo AMPK v skeletnomišičnih celicah. Metode: Kot poskusni model smo uporabili primarno kulturo človeških skeletnomišičnih celic in linijo podganjih skeletnomišičnih celic L6. Aktivacijo AMPK smo ocenili s prenosom western in sicer z merjenjem fosforilacije AMPK (Thr172) in njenega substrata acetil-CoA karboksilaze (ACC, Ser79). Spremljali smo tudi fosforilacijo kinaze uravnavane z zunajceličnim signalom 1/2 (ERK1/2, Thr202/Tyr204). Rezultati: Naši rezultati osemurnega tretiranja podganjih skeletnomišičnih cevčic L6 s sulfasalazinom so nakazali, da sulfasalazin spodbuja aktivacijo AMPK, medtem ko v primarnih človeških skeletnomišičnih celicah ne deluje kot aktivator AMPK. Sulfapiridin in 5-aminosalicilna kislina nista spodbudila fosforilacije AMPK in ACC in torej ne delujeta kot aktivatorja AMPK v podganjih ali človeških skeletnomišičnih celicah. Prvo hipotezo smo torej z našimi rezultati delno potrdili. Rezultati so hkrati tudi nakazali, da sulfasalazin in njegova presnovka ne ojačajo z AICAR spodbujene aktivacije AMPK v podganjih ali človeških skeletnomišičnih celicah, s čimer smo ovrgli drugo hipotezo. Tretjo hipotezo smo lahko delno potrdili, saj je diflunisal po 1-urnem in po 8-urnem tretiranju podganjih skeletnomišičnih cevčic L6 statistično značilno povečal fosforilacijo ACC, medtem ko balsalazid in olsalazin nista dala jasnih rezultatov glede aktivacije AMPK. Zaključki: Sulfasalazin spodbuja aktivacijo AMPK v liniji podganjih skeletnomišičnih celic L6, in ne v primarni kulturi človeških skeletnomišičnih celic, medtem ko njegova presnovka ne delujeta kot aktivatorja AMPK. Sulfasalazin in njegova presnovka ne delujejo kot ojačevalci z AICAR spodbujene aktivacije AMPK. Diflunisal je od preučevanih derivatov salicilne kisline najučinkovitejši aktivator AMPK v podganjih skeletnomišičnih celicah L6. Background: Type 2 diabetes is the most common form of diabetes. Type 2 diabetes is characterized by insulin resistance in skeletal muscles, which represent a key site of postprandial glucose uptake. Pharmacological activation of AMP-activated protein kinase (AMPK) is a promising strategy to reduce insulin resistance in skeletal muscle. Drugs that act as AMPK activators, may be useful in treatment of type 2 diabetes. Aim: Sulfasalazine, like antirheumatic drug methotrexate, inhibits 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) and reduces clearance of ZMP. ZMP (AICAR-monophosphate) is a pharmacologically active form of the most widely used experimental AMPK activator 5-aminoimidazole-4-carboxamide-1-&#946 -D-ribonucleoside (AICAR), indicating sulfasalazine might also act as an AMPK activator. The aim of our study was to determine whether sulfasalazine and/or its metabolites sulfapyridine and 5-aminosalicylic acid promote AMPK activation in skeletal muscle. Hypotheses: (1) Sulfasalazine promotes AMPK activation in skeletal muscle cells independently of 5-aminosalicylic acid and sulfapyridine (2) Sulfasalazine enhances AICAR-induced AMPK activation in skeletal muscle cells independently of 5-aminosalicylic acid and sulfapyridine (3) Derivatives of salicylic acid, balsalazide, olsalazine and diflunisal, activate AMPK in skeletal muscle cells. Methods: As an experimental model we used primary human skeletal muscle cells and rat L6 skeletal muscle cells. AMPK activation was estimated with western blot, namely with measuring AMPK phosphorylation (Thr172) and phosphorylation of his substrate acetyl-CoA carboxylase (ACC, Ser79). We also measured phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2, Thr202/Tyr204). Results: Our results of 8-hour treatment of cultured myotubes with sulfasalazine indicate, that sulfasalazine promotes AMPK activation in rat L6 myotubes. While it does not act as an AMPK activator in primary human myotubes. Sulfapyridine and 5-aminosalicylic acid did not stimulate AMPK activation in rat or human skeletal muscle cells. So our results partially confirm our first hypothesis. Our results also show that sulfasalazine or its metabolites do not increase AICAR-induced AMPK activation in rat L6 or human myotubes, which does not support our second hypothesis. We partially confirmed our third hypothesis, because after an 1 hour and an 8 hour treatment of rat L6 myotubes, diflunisal increased ACC phosphorylation with statistical significance, while balsalazide and olsalazine did not give us clear results regarding AMPK activation. Conclusions: Sulfasalazine promotes AMPK activation in rat L6 skeletal muscle cells and does not act as an AMPK activator in primary human skeletal muscle cells, while its two metabolites, sulfapyridine and 5-aminosalicylic acid, do not act as AMPK activators in these experimental models. Sulfasalazine and its metabolites do not act as enhancers of AICAR-induced AMPK activation. We also found that diflunisal is the most effective AMPK activator from the studied derivatives of salicylic acid in rat L6 skeletal muscle cells.