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Cerebral palsy is a clinical syndrome characterized primarily, but not exclusively, by movement and posture disorders, which arise due to injury to the developing brain. This injury directly and indirectly leads to contractures, which limit range of joint motion. Contractures develop due to functional and structural alterations affecting the stretch reflex and skeletal muscle. In the early stages of spastic cerebral palsy joint motion is limited mainly due to increased muscle tone, which is caused by overactivity of the stretch reflex. Later on, structural alterations in skeletal muscle lead to fixed contractures, which are characterized by shorter muscle fibers and hypertrophy of the extracellular matrix. Mechanisms by which injury to the developing brain leads to structural alterations in skeletal muscle are obscure. Interestingly, recent findings suggest that the number of muscle satellite cells is reduced in cerebral palsy, which may directly contribute to development of contractures. This mechanism, if confirmed, might lead to novel therapeutic approaches for contractures in cerebral palsy.

Neurotrophins are a subgroup of growth factors acting within the nervous system. They are small proteins forming a family of several members. Among these, NT-3 is the most abundantly distributed within the developing and mature nervous system. NT-3 significantly contributes to the formation and maintenance of the nervous system and may be involved in various neurological diseases. In the future, it may also become a therapeutic agent for their treatment, therefore the mechanisms of its action are intensely investigated. The aim of our study was to investigate the effects of NT-3 on the ability of motor neurons to form functional neuromuscular junctions. An in vitro experimental model in which neurons extenaing from rat spinal cord explants form functional neuromuscular junctions onto human myotubes has been employed for this purpose. The effects of NT-3 were quantitatively determined by 1) counting neuronal outgrowths from the spinal cord explants 2) determining the percentage of contraction-positive explants 3) counting the number of contracting units per explant and 4) counting the number of nicotinic receptorclusters at neuromuscular junctions using rhodamine-labelled a-bungarotoxin. The statistical significance of differences between the treated and control groups of explants was tested using the independent samples Student t-test. NT-3 added to the culture medium at the concentration of 10 ng/ml, which proved the most efficient in our concentration testing, increased all the parameters of functional innervation. A higher capacity of the neurons to form functional neuromuscular junctions in NT-3-containing cocultures was thus demonstrated. Nevrotrofini, med katere sodi tudi nevrotrofin 3 (NT-3), so beljakovine iz skupine rastnih dejavnikov z učinki na živčevjc. Od vseh nevrotrofinov je NT-3najbolj razširjen v razvijajočem se in zrelem živčnem sistemu in ima pomembno vlogo pri razvoju in ohranjanju strukture in delovanja živčevja, pomemben pa bi lahko bil tudi pri nekaterih nevroloških boleznih in v prihodnosri morda tudi pri njihovem zdravljenju. Namen naše naloge je bil preučiti vpliv NT3 na zmožnost motonevronov, da tvorijo delujoče živčno-mišične stike. Pri tem smo se odločili uporabiti poskusni model, v katerem nevroni izraščajo iz eksplantov podganje embrionalne hrbtenjače in tvorijo delujoče živčno-mišične stike na mišičnih cevčicah človeške mišice. Tamodel omogoča opazovanje nastajanja živčno-mišičnega stika na vseh pomembnihstopnjah tega procesa in kvantitativno ocenjevanje uspešnosti funkcionalnega oživčenja. Na tak način učinki NT3 še niso bili kvantitativno ocenjeni. Vpliv NT-3 na funkcionalno oživčenje smo ocenjevali tako, da smo: 1.šteli živčne izrastke na robu eksplanta, 2. določali delež kontrakcijsko pozitivnih eksplamov, 3. šteli kontrahirajoče enote v polju oživčenja posameznega eksplanta in 4. šteli skupke acetilholinskih receptorjev v živčno-mišičnem stiku s pomočjo barvanja z a-bungarotoksinom, konjugiranim z rodaminom. Pod vplivom NT3, dodanega v koncentraciji 10 ng/ml, M se je v poizkusih testiranja različnih koncentracij izkazala kot najbolj učinkovim, sose povečali vsi merjeni parametri funkcionalnega oživčenja. S tem smo potrdili hipotezo, da NT-3 izboljša zmožnosti motonevronov za ustvarjanje delujočih živčno-mišičnih stikov.

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: Skeletna mišica je z izločanjem miokinov pomemben endokrini organ in mišična vlakna so eno izmed najbolj presnovno aktivnih tkiv. Interlevkin-6 (IL-6) je pleiotropen protein, ki ga izloča širok spekter celic in tkiv. Vnetje in presnovni učinki so odvisni od raznolikega časovno ali tkivno specifičnega delovanja IL-6. Vnetni učinki kronično povišanega IL-6 poslabšujejo prognozo bolezenskih stanj, medtem ko akutno izločen IL-6 pri fizioloških prilagoditvah v splošnem deluje pozitivno na zdravje in izboljšuje presnovo. Zaviralci delovanja IL-6 so pomembni pri zdravljenju vnetnih revmatičnih bolezni. Namen: Razumevanje uravnalnih mehanizmov IL-6 nam širi možnosti optimizacije terapevtskih pristopov in razvoja zdravil naslednjih generacij. Zaradi pomanjkanja natančnih definicij dejavnikov, ki privedejo do dvojne vloge IL-6, nas zanima uravnavanje njegove sinteze, izločanja in delovanja. Raziskovali smo mehanizme izražanja IL-6, receptorske signalizacije za IL-6 ter učinke terapevtske blokade delovanja IL-6 v skeletnih mišicah. Glavni namen je bil preveriti učinke zaviralcev IL-6 na njegovo izražanje in delovanje v kulturi skeletnomišičnih celic. Hipoteze: 1.) TLR4 in ERK1/2 sta pomembna za bazalno izražanje IL-6 v kulturi človeških skeletnomišičnih celic. 2.) Zaviralci IL-6 ne vplivajo na izražanje in izločanje IL-6 v kulturi človeških skeletnomišičnih celic. 3.) Zaviralci IL-6 in inzulin zmanjšajo bazalno fosforilacijo STAT3 v človeških skeletnomišičnih celicah. 4.) Učinek zaviralcev IL-6 na bazalno in z inzulinom spodbujeno aktivnost signalnih poti Akt in ERK1/2 je odvisen od koncentracije in trajanja izpostavitve IL-6. 5.) Zaviralci IL-6 zmanjšajo aktivnost z AMP aktivirane protein kinaze v človeških skeletnomišičnih celicah. Metode: Poskuse smo izvedli na primarni kulturi človeških mišičnih cevčic. Za določanje izražanja IL-6 mRNA v mišičnih celicah smo uporabili metodo kvantitativne verižne reakcije s polimerazo v realnem času. Izločen IL-6 smo določili z encimskoimunskim testom. Signalne odzive celic smo spremljali z metodo prenos western, ki smo jo uporabili za analizo fosforilacije ključnih signalnih proteinov STAT3. AKT, ERK1/2, AS160, GSK-3α/β, PAS in ACC. Rezultati: Izmerili smo bazalno izločanje IL-6. LPS aktivira TLR4, kar spodbudi izražanje IL-6 v skeletnomičičnih celicah. Dejavniki, ki zavirajo to pot, zavirajo izražanje IL-6 ob dodatku LPS. Noben uporabljen zaviralec ni spodbudil ali zavrl izražanja IL-6. Tocilizumab in zaviralec tofacitinib nista spodbudila ali zavrla fosforilacije s presnovo povezanih signalnih proteinov. Pri kronični izpostavitvi skeletnomišičnih celic z IL-6 smo opazili zmanjšanje inzulinskega odziva. Tofacitinib je učinkovito zaviral tudi bazalno fosforilacijo STAT3, zaviralni učinek tocilizumaba pa smo izmerili samo ob akutni izpostavitvi IL-6. Pri zaviralcih IL-6 nismo izmerili učinka na delovanje inzulina. Zaključki: Potrdili smo endogeno izločanje IL-6 v bazalnih razmerah. Izločili smo vpletenost mehanizmov spontane aktivnosti TLR4 in delovanja ERK1/2, kot pozitivne povratne zanke IL-6 za ta pojav. Sklepamo, da IL-6 sam ne vpliva na lastno izražanje/izločanje. Naši rezultati ne kažejo bistvenega vpliva zaviranja delovanja IL-6, ali kronične izpostavljenosti IL-6, na signalne poti STAT3, Akt, ERK1/2 in AMPK v kulturi človeških skeletnomišičnih celicah. Kronična izpostavljenost IL-6 v mišici bi lahko vplivala na delovanje inzulina. Background: Skeletal muscle is highly active metabolic tissue, and with its myokine secretion an important endocrine organ. Interleukin-6 (IL-6) is a pleiotropic cytokine that is secreted from a wide spectrum of cells and tissues. Inflammatory and metabolic responses depend mostly on different physiological environment, time, and tissue specific actions of IL-6. Chronically elevated IL-6 has proinflammatory effects and is linked to negative prognosis of disease but its acute elevation under physiological conditions exerts beneficial effects on metabolism and health in general. IL-6 inhibitors are important in treating chronic inflammatory diseases. Aim: Understanding the regulatory mechanisms of IL-6 broadens our possibilities in therapeutic optimisation and development of next generation drugs. Factors that determine the IL-6 double role are not yet well defined, therefore we are interested aim to gain the understanding of the regulation of IL-6 synthesis, secretion, and its actions. We study the mechanisms of IL-6 expression, receptor signalling and effects of therapeutic targeting of IL-6 in skeletal muscle. The main aim was to examine the effects of IL-6 inhibitors on IL-6 expression and action in skeletal muscle cell culture. Hypotheses: 1.) TLR4 and ERK1/2 are important for basal IL-6 expression in human skeletal muscle cell culture.2.) IL-6 inhibitors do not affect IL-6 expression and secretion in human skeletal cell culture. 3.) IL-6 inhibitors and insulin reduce basal STAT3 phosphorylation in human skeletal muscle cells. 4.) The effect of IL-6 inhibitors on basal and insulin-stimulated Akt and ERK1/2 signalling pathway activity is dependent on the concentration and duration of IL-6 exposure. 5.) IL-6 inhibitors reduce AMP-activated protein kinase activity in human skeletal muscle cells. Methods: Experiments were taken on primary human skeletal muscle cell culture. IL-6 mRNA expression in muscle cells was determined with quantitative real time PCR. IL-6 secretion was measured with the enzyme linked immunoabsorbent assay. Cellular signalling responses were assessed with western blotting by measuring the phosphorylation of key signalling proteins STAT3. AKT, ERK1/2, AS160, GSK-3α/β, PAS in ACC. Results: Constitutive IL-6 secretion from cultured skeletal muscle cells was measured. LPS treatment activated the receptor TLR4, which stimulated IL-6 expression in muscle cells. IL-6 expression was inhibited by TLR4 signalling inhibitors. Neither stimulation nor complete inhibition of IL-6 expression was noticed by any of the used inhibitors. Phosphorylation of metabolic signalling proteins after treatment with tocilizumab and tofacitinib was not significantly changed. Decreased insulin response was noticed after the chronic treatment of skeletal muscle cells with IL-6. Constitutive phosphorylation of STAT3 was effectively inhibited by tofacitinib. Inhibition of STAT3 phosphorylation by tocilizumab was measured only at the acute IL-6 treatment. Effect on insulin actions by IL-6 inhibitors was not detected. Conclusions: Endogenous secretion of IL-6 under basal conditions was confirmed. The mechanisms of spontaneous TLR4 activation and ERK1/2 activity as an IL-6 positive feedback loop were excluded for this state. We conclude that IL-6 itself does not influence its own expression/secretion. Our results show no significant effect of IL-6 inhibition, or chronic IL-6 exposure, on STAT3, Akt, ERK1/2 and AMPK signalling pathways in cultured human skeletal muscle cells. Chronic exposure to IL-6 in muscle could affect insulin action.

Z AMPK aktivirana protein kinaza (AMPK) je pomemben senzor energijskega stanja celice. V skeletni mišici se aktivira med telesno dejavnostjo in prispeva k uravnavanju presnovnih procesov. Ker aktivacija AMPK izboljša homeostazo glukoze in delovanja inzulina, je AMPK pomembna tarča za razvoj novih oblik zdravljenja metabolnih bolezni, med drugim tudi sladkorne bolezni tipa 2. Pri razvoju novih farmakoloških učinkovin z delovanjem v skeletni mišici so pomembni zanesljivi eksperimentalni modeli in vitro. Namen diplomskega dela je bil preveriti, kakšna je zanesljivost spremljanja farmakološke aktivacije AMPK v skeletnomišičnih celicah, v katerih območjih je signal linearen, in kako redčitve vzorca vplivajo na zanesljivost ocenjevanja aktivacije AMPK. Predvidevali smo, da je območje linearnosti signala za pAMPK in AMPK oziroma pACC in ACC pri različnih redčitvah različno ter, da bi z nanosom manjše mase proteinov lahko izboljšali zaznavanje z AICAR spodbujene aktivacije AMPK s prenosom western. Poskuse smo razdelili na dva dela, in sicer smo najprej testirali učinek AICAR in MTX na aktivacijo AMPK v podganjih skeletnomišičnih celicah celične linije L6, nato pa še zanesljivost spremljanja farmakološke aktivacije AICAR in MTX na aktivacijo AMPK pri različnih redčitvah vzorcev celičnih lizatov L6. Potrdili smo, da kombinacija AICAR in MTX statistično značilno (glede na kontrolo in tretma samo z AICAR ali z MTX) poveča fosforilacijo AMPK. Ugotovili smo tudi, da je bil signal linearen za vzorce pAMPK in pACC z 20-% do 80-% koncentracijo. Od 80-% koncentracije naprej se je strmina grafa zmanjšala in ni bila več v linearnem območju. Ugotovili smo tudi, da spremljanje farmakološke aktivacije AMPK s prenosom western lahko optimiziramo z nanosom manjših koncentracij proteina na gel (80 % običajne koncentracije proteina), saj tako dobimo boljše rezultate. Signal je tako bolj v linearnem območju, obenem pa porabimo manj vzorca. AMP-activated protein kinase (AMPK) is an important sensor of the energy state of the cell. It is activated in skeletal muscle during physical activity and contributes to the regulation of metabolic processes. Since the activation of AMPK improves glucose homeostasis of insulin action, AMPK is an important therapeutic target for the development of new treatments for metabolic diseases, including type 2 diabetes. Development of new skeletal muscle pharmacological agents requires reliable in vitro experimental models. The aim of the thesis was to check the reliability of monitoring the pharmacological activation of AMPK in skeletal muscle cells, in which range the signal is linear and how the dilution of samples affects the reliability of the assessment of AMPK activation. We assumed that the range of linearity of the signal for pAMPK and AMPK, or pACC and ACC is different at different dilutions and that by applying a lower protein mass, we could improve the knowledge of AICAR-stimulated activation of AMPK by western blotting. We divided the experiments into two parts, so we first tested the action of AICAR and MTX on AMPK activation in rat skeletal muscle cells of the L6 cell line, and then the reliability of monitoring the pharmacological activation of AICAR and MTX on AMPK activation. We confirmed that the combination of AICAR and MTX significantly (compared to control and treatment with AICAR or MTX alone) increases AMPK phosphorylation. We also found that the signal was linear for pAMPK and pACC samples with concentration from 20 % to 80 %. From 80 % concentration onwards, the slope of the graph decreased and was no longer in the linear range. We have also established that the monitoring of pharmacological activation of AMPK by western blotting can be optimized by applying lower protein concentrations in the gel (80 % of the normal protein concentration), as this way we get better results. The signal is thus better in the linear range, while at the same time we use less sample.

Skeletna mišica je v fizioloških razmerah največje endokrino oziroma sekretorno tkivo v telesu. Poleg ostalih funkcij ima pomembno vlogo kot sekretorni organ, saj med telesno dejavnostjo izloča vrsto miokinov in drugi peptidov. Eden pomembnejših miokinov je interlevkin-6 (IL-6), ki je prvi miokin, za katerega so dokazali, da se sprosti v obtok med mišičnim krčenjem. IL-6 je vpleten v procese miogeneze in mišične hipertrofije. Med miogenezo pride do fuzije enojedrnih mioblastov, prekurzorjev miocitov, v večjerdne mišične cevčice, ki kasneje formirajo mišična vlakna. IL-6 deluje protivnetno tako, da inhibira izražanje TNF in IL-1 in spodbudi tvorbo klasičnih protivnetnih citokinov, kot sta IL-10 in IL-1ra. Je eden od pomembnih fosforiliranih proteinov, ki se izločajo. Človeški genom vsebuje zapise za kinaze iz družine Fam20, pod katere spadajo Fam20A, Fam20B in Fam20C. Vsaka od njih ima različno funkcijo. Kinaza Fam20C fosforilira več kot sto izločenih proteinov in se alosterično aktivira z interakcijo s psevdokinazo Fam20A. Substrati Fam20C sodelujejo pri veliko pomembnih bioloških procesih, kot so biomineralizacija, metabolizem fosfatov, adhezija in migracija celic ter delovanje srca. Fam20B fosforilira ksilozne ostanke in s tem nadzoruje sintezo proteoglikanov. Vse tri kinaze iz družine Fam20 imajo signalni peptid, ki jih usmeri v sekrecijo. Osnovni namen diplomskega dela je bil ugotoviti, kako se kinaza Fam20C in psevdokinaza Fam20A izražata v kulturi primarnih človeških skeletnomišičnih celic in podganjih skeletnomišičnih celic L6. Za analizo izražanja kinaze Fam20C in psevdokinaze Fam20A v kulturi podganjih in primarnih človeških skeletnomišičnih celic smo uporabili metodi verižno reakcijo s polimerazo v realnem času in prenos western. Pokazali smo, da se vse tri kinaze Fam20 izražajo v človeških in podganjih skeletnomišičnih celicah in da stopnja diferenciacije teh celic vpliva na izražanje Fam20C in Fam20A. Prav tako smo ugotovili, da je vzorec izražanja kinaze Fam20B drugačen od vzorcev izražanja za Fam20C in Fam20A. Skeletal muscle is the biggest of the human body’s tissues in physiological conditions. It has an important function as a secretory organ, as it releases more than a hundred myokines and other peptides during physical activity. One of the most important myokines, which is released from skeletal muscle during muscle contraction, is interleukin-6 (IL-6). It is involved in processes such as myogenesis and muscle hypertrophy. During myogenesis, myoblasts fuse into multinucleated myotubes, which later form mucle fibers. IL-6 has anti-inflammatory properties, as it inhibits expression of cytokines TNF and IL-1. IL-6 also stimulates secretion of anti-inflammatory cytokines, such as IL-10 and IL-1ra. It is also one of the phosphorylated proteins of the secretory pathway. The human genome encodes genes for kinases from family Fam20, which include Fam20A, Fam20B and Fam20C. Each of these kinases has a different function. Kinase Fam20C phosphorylates more than a hundred secreted proteins and is allosterically activated by interaction with pseudokinase Fam20A. Fam20C substrates are involved in many biologically important processes, such as biomineralization, metabolism of phosphates, cell adhesion and migration and heart function. Fam20B phosphorylates xylose residues in proteoglicans. All three kinases have a signal peptide, that directs them into the secretory pathway. The purpose of this research was to determine the expression of kinase Fam20C and preusokinase Fam20A in primary human skeletal muscle cells and rat skeletal muscle cell line L6. Using a real-time polymerase chain reaction and Western blot, we showed that all three kinases are expressed in human and rat skeletal muscle cells. We demonstrated that differentiation of these cells in fact does have an effect on Fam20C and Fam20A expression. We also concluded that the pattern of Fam20B expression is different than the expression Fam20C and Fam20A.

Uvod: Z AMP aktivirana protein kinaza (AMPK) ima pomembno vlogo pri uravnavanju presnovnih procesov in ohranjanju energijske homeostaze v skeletnih mišicah. Aktivacija skeletnomišične AMPK spodbudi privzem glukoze neodvisno od inzulina, s čimer zaobide okvare v inzulinski signalizaciji pri sladkorni bolezni tipa 2. Inzulinska rezistenca v skeletnih mišicah znatno prispeva k razvoju sladkorne bolezni tipa 2, zato predstavljajo farmakološki aktivatorji skeletnomišične AMPK obetavni pristop zdravljenja presnovnih obolenj. Metotreksat in druge zdravilne učinkovine s prijemališčem delovanja v presnovnih poteh purinskih nukleotidov so se izkazale učinkovite pri aktivaciji AMPK. Namen: AICAR, farmakološki aktivator AMPK, se presnavlja v sečno kislino, ki je neodvisni dejavnik tveganja nastanka inzulinske rezistence, sladkorne bolezni tipa 2 in številnih drugih obolenj. Sočasna aplikacija alopurinola, zaviralca ksantin oksidoreduktaze, bi zmanjšala obremenitev pacientov s sečno kislino. Namen magistrske naloge je bil raziskati vpliv alopurinola na delovanje AMPK in inzulina v skeletnomišičnih celicah L6. Hipoteze: 1) Alopurinol ne aktivira AMPK, ampak ojača z AICAR spodbujeno aktivacijo AMPK v skeletnomišičnih celicah L6 2) Alopurinol v prisotnosti metotreksata ne vpliva na z AICAR spodbujeno aktivacijo AMPK v skeletnomišičnih celicah L6 3) Alopurinol ne vpliva na z inzulinom spodbujeno aktivacijo Akt in ERK1/2 v skeletnomišičnih celicah L6 4) Alopurinol zveča z AICAR spodbujen privzem glukoze, a ne vpliva na z inzulinom spodbujen privzem glukoze v skeletnomišičnih celicah L6. Metode: Poskuse smo opravili na kulturi skeletnomišičnih celic L6. Aktivacijo AMPK smo spremljali z metodo prenos western, tako da smo merili fosforilacijo AMPK (Thr172) in acetil-CoA karboksilaze (ACC, Ser79). Določili smo tudi fosforilacijo kinaz Akt (Ser473) in ERK1/2 (Thr202/Tyr204). Presnovne učinke smo ovrednotili z merjenjem privzema 3H-2-deoksiglukoze. Izražanje ksantin oksidoreduktaze v vzorcih skeletnomišičnih celic in tkiv smo ocenili s kvantitativno verižno reakcijo s polimerazo (PCR) v realnem času. Rezultati: Alopurinol (100 µM) samostojno ali v kombinaciji z AICAR ni aktiviral skeletnomišične AMPK. Prav tako alopurinol ni vplival na z AICAR spodbujeno fosforilacijo AMPK ob prisotnosti metotreksata. Alopurinol je zmanjšal z inzulinom spodbujeno fosforilacijo Akt, na z inzulinom spodbujeno fosforilacijo ERK1/2 pa ni imel vpliva. Alopurinol ni vplival na z AICAR ali z inzulinom spodbujen privzem glukoze v skeletnomišične celice. Zaključek: 1) Alopurinol ni aktiviral AMPK ali ojačal z AICAR spodbujene aktivacije AMPK. 2) Alopurinol ob prisotnosti metotreksata ni vplival na z AICAR spodbujeno aktivacijo AMPK. 3) Alopurinol je zavrl z inzulinom spodbujeno aktivacijo Akt, na z inzulinom spodbujeno aktivacijo ERK1/2 pa ni vplival. 4) Alopurinol ni vplival na z AICAR ali z inzulinom spodbujen privzem glukoze v skeletnomišične celice. Naši rezultati torej podpirajo drugo hipotezo. Prva, tretja in četrta hipoteza pa so le delno skladne s pridobljenimi rezultati. Background: AMP-activated protein kinase (AMPK) has a significant role in regulating metabolic processes and maintaining energy homeostasis in skeletal muscle. AMPK activation in skeletal muscle stimulates glucose uptake independently of insulin action thereby bypassing defects in insulin signaling. Insulin resistance in skeletal muscle considerably contributes to development of type 2 diabetes, therefore pharmacological activators of AMPK in skeletal muscle present a promising strategy for its treatment. Methotrexate and various other compounds that target purine metabolic pathways were shown to promote AMPK activation. Aim: AICAR, a pharmacological activator of AMPK, is metabolized to uric acid, which is an independent risk factor of development insulin resistance, type 2 diabetes and numerous other diseases. AICAR application combined with alopurinol, inhibitor of xanthine oxidoreductase, reduces the plasma levels of uric acid. The aim of the master thesis was to investigate the effect of alopurinol on the action of AMPK and insulin in L6 skeletal muscle cells. Hypotheses: 1) Alopurinol does not activate AMPK, but enhances the AICAR-induced AMPK activation in L6 skeletal muscle cells. 2) In the presence of methotrexate alopurinol does not affect the AICAR-induced AMPK activation in L6 skeletal muscle cells. 3) Alopurinol does not affect the insulin-induced Akt and ERK1/2 phosphorylation in L6 skeletal muscle cells. 4) Alopurinol enhances the AICAR-induced glucose uptake, but does not affect the insulin-induced glucose uptake in L6 skeletal muscle cells. Methods: To investigate our hypotheses we used cultured L6 skeletal muscle cells. AMPK activation was monitored with western blot by measuring the phosphorylation of AMPK (Thr172) and acetyl-CoA carboxylase (ACC, Ser79). We also determined the phosphorylation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204). Metabolic effects were evaluated by estimating the upake of 3H-2-deoxyglucose. The mRNA expression of xanthine oxidoreductase was estimated with quantitative real-time PCR. Results: Alopurinol itself (100 µM) or in combination with AICAR did not promote the activation of AMPK in skeletal muscle cells. Alopurinol also did not affect the AICAR-induced AMPK phosphorylation in the presence of methotrexate. In addition, alopurinol reduced the insulin-induced phosphorylation of Akt, but did not affect the phosphorylation of ERK1/2. Finally, alopurinol did not affect the AICAR or insulin-induced glucose uptake in skeletal muscle cells. Conclusion: 1) Alopurinol did not activate AMPK or enhanced AICAR-induced AMPK activation. 2) In the presence of methotrexate alopurinol did not affect the AICAR-induced AMPK activation. 3) Alopurinol reduced the insulin-induced Akt activation, but did not affect the ERK1/2 activation. 4) Alopurinol did not affect the AICAR or insulin-induced glucose uptake in skeletal muscle cells. Our results therefore, support the second hypothesis. The first, third and fourth hypothesis are only partially consistent with the obtained results.

Uvod: Sladkorna bolezen zajema več presnovnih motenj z različno etiopatogenezo. Vsem je skupna hiperglikemija, ki je posledica motenega izločanja inzulina in/ali inzulinske rezistence v na inzulin odzivnih tkivih kot so jetra, maščevje in skeletne mišice. Presežek endogenih ali eksogenih glukokortikoidov, kot sta na primer kortizol in deksametazon, lahko vodi v inzulinsko rezistenco, moteno toleranco za glukozo in sladkorno bolezen (t.i. steroidni diabetes). Skeletne mišice so pomembno mesto z inzulinom spodbujenega privzema glukoze in eden izmed najpomembnejših organov, ki jih prizadene inzulinska rezistenca. Farmakološka aktivacija z AMP-aktivirane protein kinaze (AMPK), celičnega energijskega senzorja, v skeletnih mišicah poviša občutljivost na inzulin in s tem pripomore k izboljšanju homeostaze glukoze na ravni celega telesa. Namen: Natančen mehanizem z glukokortikoidi povzročene inzulinske rezistence v skeletnih mišicah ni poznan. Preveriti smo želeli, ali bi k inzulinski rezistenci, ki jo povzročijo glukokortikoidi, lahko prispevala zavora delovanja AMPK. Hipoteze: 1) Kortizol in deksametazon zmanjšata aktivnost inzulinske signalne poti v skeletnomišičnih celicah L6. 2) Kortizol in deksametazon zmanjšata z inzulinom spodbujeni privzem glukoze v skeletnomišičnih celicah L6 kljub povišanju izražanja prenašalcev GLUT1 in GLUT4. 3) Kortizol in deksametazon zmanjšata z A769662 spodbujeno fosforilacijo AMPK in acetil-CoA karboksilaze (ACC) v skeletnomišičnih celicah L6. 4) Kortizol in deksametazon zmanjšata z A769662 spodbujeni privzem glukoze v skeletnomišičnih celicah L6 kljub povišanju izražanja prenašalcev GLUT1 in GLUT4. Metode: Za poskusni model smo uporabili podganje skeletnomišične celice L6. Aktivnost inzulinske signalne poti in AMPK smo ovrednotili s prenosom western, s katerim smo merili fosforilacijo AKT (Thr308 in Ser473), AS160 (Thr642), ERK1/2 (Thr202/Tyr204), ribosomskega proteina S6 (Ser235/236, S6RP), AMPK (Thr172) in ACC (Ser79). Privzem glukoze v skeletnomišične celice L6 smo ocenili z merjenjem privzema 3H-2-deoksiglukoze. Izražanje prenašalcev GLUT1 in GLUT4 smo določili s kvantitativno reakcijo verižne polimeraze v realnem času. Za farmakološko aktivacijo AMPK smo uporabili učinkovino A769662. Rezultati: Kortizol in deksametazonom (0,1 µM in 1 µM) sta v prisotnosti inzulina (12 in 120 nM) povzročila porast fosforilacije AKT (Thr308 in Ser473). V inzulinsko spodbujenih vzorcih kortizol in deksametazon na fosforilacijo AS160 (Thr642) nista vplivala (24-urno tretiranje) oziroma sta jo zmanjšala (72-urno tretiranje). V vzorcih z inzulinom (12 in 120 nM) sta kljub temu povzročila porast privzema glukoze. Fosforilacija ERK1/2 (Thr202/Tyr204) in S6RP (Ser235//236) se je pod vplivom kortizola in deksametazona zmanjšala v prisotnosti inzulina. Izražanje mRNA za prenašalce GLUT1 in GLUT4 se je pod vplivom kortizola in deksametazona povišalo. Kortizol in deksametazon nista spremenila fosforilacije AMPK (Thr172). Z A769662 spodbujena fosforilacija ACC (Ser79) se je ob dodatku kortizola in deksametazona zmanjšala. Kortizol in deksametazon sta v prisotnosti A769662 zvečala privzem glukoze. Zaključek: 1) Kortizol in deksametazon nista zmanjšala aktivnosti inzulinske signalne poti v skeletnomišičnih celicah L6. 2) Kortizol in deksametazon sta zvečala tako izražanje prenašalcev GLUT1 in GLUT4 kot z inzulinom spodbujeni privzem glukoze. 3) Kortizol in deksametazon sta zmanjšala z A769662 spodbujeno fosforilacijo ACC, nista pa vplivala na fosforilacijo AMPK. 4) Kortizol in deksametazon sta zvečala z A769662 spodbujeni privzem glukoze. Introduction: Diabetes mellitus is comprised of several metabolic disorders with different aetiopathogenesis. They are characterized by hyperglycaemia, which results from impaired insulin secretion and/or insulin resistance in insulin-responsive tissues, such as liver, fat and skeletal muscle. Excess endogenous or exogenous glucocorticoids, such as cortisol and dexamethasone, can lead to insulin resistance, impaired glucose tolerance and diabetes mellitus (so-called steroid diabetes). Skeletal muscle is an important site of insulin-stimulated glucose uptake and one of the most important organs affected by insulin resistance. Pharmacological activation of AMP-activated protein kinase (AMPK), a cellular energy sensor, in skeletal muscle increases insulin sensitivity and thus helps to improve glucose homeostasis of the whole body. Aim: The exact mechanism of glucocorticoid induced insulin resistance in skeletal muscle is unknown. The aim of our study was to test whether inhibition of AMPK action could contribute to glucocorticoid-induced insulin resistance. Hypotheses: 1) Cortisol and dexamethasone suppress insulin signalling in L6 skeletal muscle cells. 2) Cortisol and dexamethasone suppress insulin-stimulated glucose uptake in L6 skeletal muscle cells despite an increase in the expression of GLUT1 and GLUT4 transporters. 3) Cortisol and dexamethasone decrease A769662-induced AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in L6 skeletal muscle cells. 4) Cortisol and dexamethasone decrease A769662-stimulated glucose uptake in L6 skeletal muscle cells despite an increase in the expression of GLUT1 and GLUT4 transporters. Methods: The experiments were performed on rat L6 skeletal muscle cells. Insulin signalling pathway and AMPK activity were evaluated by western blotting, which assessed phosphorylation of AKT (Thr308 and Ser473), AS160 (Thr642), ERK1/2 (Thr202 and Tyr204), S6 ribosomal protein (Ser235/236, S6RP), AMPK (Thr172) and ACC (Ser79). The glucose uptake in cultured cells was estimated by 3H-2-deoxyglucose uptake assay. The expression of GLUT1 and GLUT4 transporters was evaluated with real time quantitative polymerase chain reaction. A769662 was used for pharmacological activation of AMPK. Results: In the presence of insulin (12 and 120 nM), cortisol and dexamethasone (0.1 µM and 1 µM) increased the AKT (Thr308 and Ser473) phosphorylation. In insulin stimulated samples, cortisol and dexamethasone did not affect (24-hour treatment) or reduced (72-hour treatment) phosphorylation of AS160 (Thr642), while they increased GLUT1 and GLUT4 mRNA expression and enhanced the insulin-stimulated (12 and 120 nM) glucose uptake. In the presence of insulin, phosphorylation of ERK1/2 (Thr202 and Tyr204) and S6RP (Ser235/236) was reduced by the treatment with glucocorticoids. While cortisol and dexamethasone did not alter the phosphorylation of AMPK (Thr172), they suppressed the A769662-induced phosphorylation of ACC (Ser79) and enhanced the A769662-stimulated glucose uptake. Conclusions: 1) Cortisol and dexamethasone did not decrease the activity of insulin signaling in L6 skeletal muscle cells. 2) Cortisol and dexamethasone increased both the expression of GLUT1 and GLUT4 transporters and insulin-stimulated glucose uptake. 3) Cortisol and dexamethasone diminished A769662-stimulated phosphorylation of ACC but not AMPK. 4) Cortisol and dexamethasone elevated A769662-stimulated glucose uptake.

Z AMP aktivirana protein kinaza (AMPK) je osrednji energijski senzor na ravni celice in organizma ter obetavna tarča v terapiji presnovnih motenj. Raziskave kažejo, da metotreksat, zaviralec dihidrofolat reduktaze, in sulfanilamid sulfasalazin lahko vplivata na aktivnost AMPK v skeletni mišici. Mehanizem delovanja je inhibicija encima ATIC (5-amino-4-imidazolkarboksamid ribonukleotid formiltransferaza/inozin monofosfat ciklohidrolaza). S tem se upočasni presnova ZMP, ki je fosforilirana oblika AICAR, farmakološkega aktivatorja AMPK. Tako se zmanjša prag za z AICAR spodbujeno AMPK aktivacijo. Posledično se v celici povečata privzem glukoze in lipidna oksidacija, kar zmanjša hiperglikemijo in inzulinsko rezistenco pri osebah s sladkorno boleznijo tipa 2. Namen naloge je bil proučiti, ali na delovanje AMPK v skeletni mišici poleg metotreksata in sulfasalazina vplivajo tudi nekateri drugi klinično uporabni zaviralci dihidrofolat reduktaze (trimetreksat in trimetoprim) oziroma sulfanilamidi, kot je sulfametoksazol, ki se uporablja skupaj s trimetoprimom. AMP-activated protein kinase (AMPK) is a central energy sensor at the cellular and organismal level and a promising target in the therapy of metabolic disorders. Studies show that methotrexate, a dihydrofolate reductase inhibitor, and the sulphanilamide sulfasalazine can affect AMPK activity in skeletal muscle. The mechanism of action is inhibition of the ATIC (5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase/IMP cyclohydrolase) enzyme, which suppresses the metabolism of ZMP, a phosphorylated (active) form of pharmacological AMPK activator AICAR. In this way, the threshold for AICAR-stimulated AMPK activation is decreased. Consequently, cellular glucose uptake and lipid oxidation are increased, thus reducing hyperglycaemia and insulin resistance in persons with type 2 diabetes. Our aim was to investigate whether, in addition to methotrexate and sulfasalazine, AMPK function in skeletal muscle is modulated by some other clinically useful inhibitors of dihydrofolate reductase (trimetrexate and trimethoprim) or by sulphanilamides, such as sulfamethoxazole, which is used in combination with trimethoprim.

Uvod: Ouabain je kardiotonični glikozid, ki svoje učinke sproži preko natrij-kalijeve črpalke (NKA), ki je doslej njegov edini znan receptor. Skeletne mišice so največji organ v človeškem telesu in zato vsebujejo največjo količino NKA ter tako predstavljajo pomembno tarčno tkivo za ouabain. Ouabain s svojo vezavo na NKA ne vpliva samo na njeno transportno funkcijo, ampak tudi na znotrajcelično signalizacijo, kar bi lahko vplivalo tudi na endokrino funkcijo skeletnih mišic (npr. izločanje interlevkinov). Skeletnomišične celice izločajo interlevkin-6 (IL-6) med telesno aktivnostjo, med katero se najverjetneje iz nadledvičnih žlez izloča tudi endogeni ouabain. Namen: Ugotoviti, ali bo ouabain v skeletnomišičnih celicah vplival na izražanje NKA in ali bo aktiviral signalne poti, ki so lahko prožene preko NKA in so za odziv mišice na telesno aktivnost še posebej pomembne. Hipoteze: 1) Ouabain zniža izražanje proteina NKAα1 v človeških skeletnomišičnih celicah 2) Ouabain aktivira signalni poti Src/ERK1/2 in Akt/mTOR v človeških skeletnomišičnih celicah 3) Ouabain zavira signalno pot IL-6/JAK/STAT3 v človeških skeletnomišičnih celicah 4) Ouabain ne vpliva na izražanje NKAα1 in ne proži signalnih poti v podganjih skeletnomišičnih celicah. Metode: Kot model smo uporabili človeške skeletnomišične celice in podganje skeletnomišične celice L6 ter določili izražanje proteinov z metodo prenosa western, izražanje genov pa z metodo RT-qPCR. Rezultati: V človeških skeletnomišičnih celicah je ouabain znižal izražanje proteina NKAα1, aktiviral signalni poti Src-ERK1/2 in Akt/mTOR in zavrl je signalno pot IL-6/JAK/STAT3. V podganjih skeletnomišičnih celicah ouabain ni vplival na izražanje NKAα1 in ni imel pomembnejših učinkov na aktivacijo znotrajceličnih signalnih poti. Zaključki: Ugotovili smo, da je mehanizem znižanja izražanja proteina NKAα1 v človeških skeletnomišičnih celicah posttranskripcijski. Odzivnost signalne poti Src-ERK1/2 je relativno majhna, aktivacija signalne poti Akt/mTOR pa je najverjetneje neodvisna od Src. Pri z ouabainom povzročenim zavrtjem signalne poti IL-6/JAK/STAT3 je udeleženih več mehanizmov, za razjasnitev katerih bi bili potrebni dodatni poskusi. Na podlagi rezultatov na podganjih skeletnomišičnih celicah smo ugotovili, da so signalni učinki ouabaina odvisni od prisotnosti na ouabain občutljive NKAα1. Background: Ouabain, cardiotonic steroid, trigggers its effects via the sodium-potassium pump (NKA), which is the only known receptor for ouabain so far. Skeletal muscles, which are the largest organ in human body, contain the largest pool of NKA and consequently represent the major target tissue for ouabain. In addition to being an inhibitor of ion transport, ouabain is also capable of inducing signals, which may influence the endocrine function of skeletal muscle. Skeletal muscle cells secrete Interleukin-6 during exercise, when endogenous ouabain is also secreted, probably from the adrenal gland. Aim: To determine whether ouabain has an effect on the expression of NKA and whether it activates any of the signalling pathways known to be activated through NKA and which are especially important for muscle response during exercise. Hypotheses: 1) Ouabain lowers the expression of protein NKAα1 in human skeletal muscle cells, 2) Ouabain activates the Src-ERK1/2 and Akt/mTOR signaling pathway in human skeletal muscle cells, 3) Ouabain inhibits the IL-6/JAK/STAT3 signalling pathway in human skeletal muscle cells, 4) Ouabain has no effect on the expression of NKAα1 and on signalling pathways in rat skeletal muscle cells. Methods: As an experimental model, we used human skeletal muscle cells and L6 rat skeletal muscle cells. We determined the expression of proteins with Western Blot and gene expression with RT-qPCR. Results: Ouabain reduced the expression of NKAα1 protein and activated the Src-ERK1/2 and PI3K/Akt/mTOR signalling pahways in human skeletal muscle cells. The IL-6/JAK/STAT3 signalling pathway in human skeletal muscle cells was inhibited by ouabain. In contrast, in rat skeletal muscle cells ouabain had no effect on the expression of protein NKAα1 and did not display major effects on intracellular signalling. Conclusions: We concluded ouabain reduces the expression of NKAα1 protein in human skeletal muscle cells in post-trancriptional manner. Src-ERK1/2 signalling pathway is barely responsive on treatment with ouabain, activation of Akt/mTOR signalling pathway is probably Src independent. Ouabain inhibits IL-6/JAK/STAT3 signalling pathway in human skeletal muscle cells through more than one mechanism, which has to be addressed by future studies. Based on results in rat skeletal muscle cells, we concluded that ouabain's effect on intracellular signalling is dependent on the presence of ouabain-sensitive NKAα1.

Uvod: Rak je med vodilnimi vzroki smrti. Pri ženskah je najpogostejši rak dojke, ki predstavlja 21 % primerov raka. Presnovne motnje, kot sta debelost in sladkorna bolezen tipa II, so pomemben dejavnik tveganja za razvoj raka dojke. Po drugi strani pa so presnovne spremembe ena temeljnih značilnosti rakavih celic. Učinkovine, ki vplivajo na presnovo, bi torej lahko bile uporabne pri zdravljenju raka dojke. Med takšnimi učinkovinami je tudi metformin, ki se sicer uporablja za zdravljenje sladkorne bolezni. Namen: Nedavno je bilo ugotovljeno, da metformin, ki inhibira kompleks I dihalne verige, v kombinaciji z 2-deoksiglukozo (2-DG), ki inhibira glikolizo, v celični kulturi povzroči odlepljanje rakavih celic. V nasprotju s splošno sprejetim prepričanjem, da so odlepljene celice mrtve, te celice v velikem deležu ostanejo žive in sposobne za nadaljnjo proliferacijo. Namen našega dela je bil preveriti, ali na celice raka dojke podobno kot metformin delujejo tudi drugi inhibitorji dihalne verige. Kot poskusni model smo uporabili celice MDA-MB-231, celično linijo trojno negativnega raka dojke. Hipoteze: (1) Za preživetje pritrjenih in odlepljenih celic MDA-MB-231 je glikoliza pomembnejša od oksidativne fosforilacije (2) Sočasno tretiranje z metforminom in inhibitorjem glikolize 2-DG ima podoben učinek na celice MDA-MB-231 kot sočasno tretiranje z drugimi inhibitorji dihalne verige in inhibitorjem glikolize 2-DG (3) Hipoksija inducira izražanje HIF-1α v pritrjenih in odlepljenih celicah MDA-MB-231 (4) Inhibicija glikolize z 2-DG poslabša preživetje pritrjenih in odlepljenih celic MDA-MB-231 v pravi hipoksiji ne pa v kemični hipoksiji. Metode: Število in preživetje celic MDA-MB-231 smo ocenjevali z barvilom propidijev jodid in metodo pretočne citometrije, nastajanje reaktivnih kisikovih zvrsti smo ocenjevali z barvilom CM-H2DCFDA in metodo pretočne citometrije, izražanje proteinov in signalne poti pa smo ocenjevali z odtisom western. Rezultati: Pri uporabi različnih inibitorjev energijske presnove je bilo od 20 do 95 % odlepljenih MDA-MB-231 celic živih. Preživetje celic je bilo manjše pri inhibiciji glikolize z 2-DG kot pri inhibiciji dihalne verige z metforminom ali inhibitorji dihalne verige, kot so antimicin A, natrijev azid in oligomicin. Inhibicija dihalne verige je pomembno zmanjšala preživetje celic MDA-MB-231 šele, ko smo celice gojili v odsotnosti glukoze. To nakazuje, da imajo celice MDA-MB-231 funkcionalno dihalno verigo, vendar je njihovo preživetje bolj odvisno od glikolize kot od oksidativne fosforilacije. S tem smo potrdili prvo hipotezo. Sočasno tretiranje z 2-DG in metforminom ali 2-DG in drugimi inhibitorji dihalne verige je imelo podoben učinek na odlepljenje živih celic MDA-MB-231. Ta rezultat podpira drugo hipotezo. Pri pritrjenih celicah MDA-MB-231 je hipoksija (1 % O2) inducirala izražanje transkripcijskega dejavnika HIF-1α. V odlepljenih celicah je bilo izražanje HIF-1α manjše tako v normoksičnih kot tudi hipoksičnih razmerah. Ti rezultati delno podpirajo tretjo hipotezo. Ugotovili smo tudi, da inhibicija glikolize z 2-DG poslabša preživetje celic MDA-MB-231 tako v pravi kot v kemični hipoksiji, inducirani s CoCl2, kar ne podpira naše četrte hipoteze. Zaključki: Inhibicija dihalne verige in/ali glikolize povzroči odlepljanje živih rakavih celic. Odlepljene rakave celice se po svojih funkcijskih lastnostih razlikujejo od prilepljenih celicah. Pri preučevanju modulatorjev energijske presnove in njihovih učinkov na rakave celice je pomembno, da se v analizo vključijo tako prilepljene kot odlepljene celice. Background: Cancer is one of the leading causes of mortality. Among women the most common cancer is breast cancer, which represents 21 % of all cancers. Metabolic disorders, such as obesity and type 2 diabetes mellitus, are important risk factors for development of breast cancer. On the other side, metabolic changes are one of the hallmarks of cancer cells, so the substances that affect metabolism, could be used in treatment of breast cancer. One of those substances is metformin, which is used for the treatment of diabetes. Aim: It was recently discovered that metformin, which inhibits complex I of respiratory chain in combination with 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, causes detachment of cancer cells in cell culture. In contrary with general accepted belief, that detached cells are dead, high percent of those cells are viable and able of continuing proliferation. Aim of our work was, to find out if other inhibitors of respiratory chain work on breast cancer cells, like metformin. We used MDA-MB-231 cell, the cell line of triple negative breast cancer as our experimental model. Hypotheses: (1) For survival of floating and attached MDA-MB-231 cells, glycolysis is more important than oxidative phosphorylation. (2) Combined treatment with metformin and glycolysis inhibitor 2-DG has a similar effect on MDA-MB-231 cells as combination of other inhibitors of the respiratory chain and 2-DG. (3) Hypoxia induces expression of HIF-1α in floating and attached MDA-MB-231 cells. (4) Glycolysis inhibition with 2-DG impair survival of floating and attached MDA-MB-231 cells in real hypoxia, and not in chemically-induced hypoxia. Methods: We evaluated the cell number and survival of MDA-MB-231 cells with propidium iodide staining and the method of flow cytometry. For detection of reactive oxygen species, we used CM-H2DCFDA staining and the method of flow cytometry. Expression of proteins and signal pathways were evaluated with western blot. Results: The analysis of the floating MDA-MB-231 cells has shown that 20 to 95 % of cells remain viable when different metabolism inhibitors are used. Survival of cells was lower with inhibition of glycolysis with 2-DG, than with inhibition of respiratory chain with metformin or others respiratory chain inhibitors. Inhibition of respiratory chain has significantly lowered the survival of MDA-MB-231 cells only when the cells were grown in the absence of glucose, indicating that MDA-MB-231 cells have a functional respiratory chain, but their survival is more dependent on glycolysis than oxidative phosphorylation. We thus confirmed our first hypothesis. Combined treatment with 2-DG and metformin or 2-DG and other mitochondrial respiratory chain inhibitors, like antimycin A, sodium azide and oligomycin, had a similar synergic effect on detached living MDA-MB-231 cells. This result support hypothesis two. In attached MDA-MB-231 cells hypoxia (1 % O2) induced expression of transcription factor HIF-1α, while in floating MDA-MB-231 cells HIF-1α expression was reduced in both normoxia and hypoxia. This result partially confirmed our third hypothesis. We also found that the inhibition of glycolysis with 2-DG reduced survival of attached MDA-MB-231 cell in real and chemically-induced hypoxia, induced with CoCl2, which does not support our fourth hypothesis. Conclusions: Inhibition of the respiratory chain and/or glycolysis cause the living cancer cell to detach. Functional characteristic in floating cancer cells are different than in attached cancer cells. While studying metabolism modulators and their effect on cancer cells, it is important that we analyze both, floating and attached cells.

Uvod: Pri uravnavanju normalne koncentracije glukoze v krvi sodelujejo skeletne mišice, presnovno eno najbolj aktivnih tkiv in pomembno mesto od inzulina odvisnega privzema glukoze po obroku. Za ohranjanje energijske homeostaze je pomembna z AMP aktivirana kinaza (AMPK). AMPK je tudi zanimiva tarča za razvoj novih oblik zdravljenja inzulinske rezistence in sladkorne bolezni tipa 2, saj njena aktivacija izboljša homeostazo glukoze. Namen: AMPK se aktivira alosterično in s fosforilacijo Thr172 na katalitični podenoti α, medtem ko fosforilacija Ser485 njeno aktivnost zmanjša. Namen naloge je bil preveriti, kako inzulin in adrenalin vplivata na delovanje AMPK oziroma farmakoloških aktivatorjev AMPK v skeletnomišičnih celicah. Hipoteze: 1) Inzulin zmanjša aktivnost AMPK v skeletnomišičnih celicah L6 tako, da zavre fosforilacijo AMPKα Thr172 in spodbudi fosforilacijo AMPKα Ser485. 2) Adrenalin poveča aktivnost AMPK v skeletnomišičnih celicah L6, saj spodbudi fosforilacijo AMPKα Thr172 in zavre fosforilacijo AMPKα Ser485. 3) Inzulin zmanjša, adrenalin pa poveča učinke aktivatorja AMPK A-769662 na fosforilacijo AMPKα Thr172 in Ser485. 4) Neposredni in posredni farmakološki aktivatorji AMPK v skeletnomišičnih celicah L6 povečajo fosforilacijo Thr172 in hkrati zmanjšajo fosforilacijo Ser485 na podenoti AMPKα. 5) Adrenalin kljub aktivaciji AMPK zmanjša bazalni in z inzulinom spodbujen privzem glukoze v skeletnomišičnih celicah L6. Metode: Uravnavanje AMPK smo raziskovali na podganji skeletnomišični celični liniji L6. Z metodo prenosa western smo analizirali fosforilacijo AMPKα Thr172 in Ser485. Aktivnost AMPK smo določili posredno, z analizo fosforilacije acetil-CoA karboksilaze (Ser79), ki je neposredna tarča AMPK. Privzem glukoze v celice smo izmerili s [3H]-2-deoksiglukozo. Rezultati: Potrdili smo prvo hipotezo, saj je inzulin spodbudil fosforilacijo AMPKα Ser485, zavrl fosforilacijo AMPKα Thr172 in zmanjšal aktivnost AMPK. V nasprotju z drugo hipotezo je imel adrenalin na fosforilacijo in aktivnost AMPK podobne učinke kot inzulin. Tretjo hipotezo smo podprli le delno, saj je predhodna stimulacija z inzulinom in adrenalinom zmanjšala učinke A-769662 na fosforilacijo AMPKα Thr172, vendar povečala učinke A-769662 na fosforilacijo AMPKα Ser485. Večina testiranih farmakoloških aktivatorjev je stimulirala fosforilacijo AMPKα Thr172, a na fosforilacijo AMPKα Ser485 niso imeli vpliva oziroma so jo celo povečali, kar je v nasprotju s četrto hipotezo. Peto hipotezo smo zavrgli, saj je adrenalin povečal bazalni in z inzulinom spodbujen privzem glukoze. Zaključek: Naši rezultati kažejo, da adrenalin in inzulin na uravnavanje AMPK in privzem glukoze v skeletnomišičnih celicah L6 delujeta podobno in da nekateri farmakološki aktivatorji hkrati povečajo fosforilacijo AMPKα Thr172 in AMPKα Ser485, kar kaže na obstoj številnih regulacijskih signalnih poti, ki lahko predstavljajo nove možnosti za razvoj novih oblik zdravljenja sladkorne bolezni tipa 2. Background: Skeletal muscles, metabolically one of the most active tissues, and an important site of insulin-dependent postprandial glucose uptake, are involved in maintaining normoglycaemia. For maintaining energy homeostasis is important AMP activated protein kinase (AMPK). AMPK is also a promising target for the development of new therapies for insulin resistance and type 2 diabetes, as activated enhances glucose homeostasis Aim: AMPK is activated allosterically and by phosphorylation of Thr172 on the catalytic α subunit, while phosphorylation of the Ser485 decreases its activity. Our aim was to determine how insulin and adrenaline affect the function of AMPK or AMPK pharmacological activators in skeletal muscle cells. Hypotheses: 1) Insulin impairs AMPK activity in skeletal muscle cells by suppressing AMPKα Thr172 phosphorylation and promoting AMPKα Ser485 phosphorylation. 2) Adrenaline increases AMPK activity in skeletal muscle cells, as it stimulates AMPKα Thr172 phosphorylation and inhibits AMPKα Ser485 phosphorylation. 3) Insulin decreases while adrenaline increases the effects of AMPK activator A-769662 on phosphorylation of AMPKα Thr172 and Ser485. 4) Direct and indirect AMPK pharmacological activators increase the phosphorylation of AMPKα Thr172 and decrease the phosphorylation of AMPKα Ser485 in skeletal muscle cells. 5) Adrenaline, despite AMPK activation, reduces basal and insulin-induced glucose uptake by L6 skeletal muscle cells. Methods: We investigated the regulation of AMPK in the rat skeletal muscle cell line L6. The phosphorylation sites of AMPKα Thr172 and Ser485 were analyzed with the Western blot method. Activation of AMPK was estimated by measuring phosphorylation of acetyl-CoA carboxylase (Ser79), a direct downstream target of AMPK. Glucose uptake in cells was assessed with the [3H]-2-deoxyglucose uptake assay. Results: We confirmed the first hypothesis, as insulin stimulated AMPKα Ser485 phosphorylation, inhibited AMPKα Thr172 phosphorylation and decreased AMPK activity. In contrast to the second hypothesis, adrenaline had similar effects on phosphorylation and AMPK activity as insulin. The third hypothesis was only partially supported, as prior stimulation with insulin and adrenaline reduced the effects of A-769662 on AMPKα Thr172 phosphorylation but increased the effects of A-769662 on AMPKα Ser485 phosphorylation. Most of the tested pharmacological activators stimulated the phosphorylation of AMPKα Thr172, but they did not affect or even incrased the AMPKα phosphorylation, which is contrary to the fourth hypothesis. We rejected the fifth hypothesis because adrenaline increased basal and insulin-induced glucose uptake in L6 skeletal muscle cells. Conclusion: Our results highlight that adrenaline and insulin act similarly on regulation of AMPK and glucose uptake in L6 skeletal muscle cells and that some pharmacological activators simultaneously increase phosphorylation of AMPKα Thr172 and AMPKα Ser485, indicating existence of multiple regulatory signalling pathways that may present new opportunities for the development of new strategies for treatment of type 2 diabetes.

Uvod: Pri cerebralni paralizi ali statični encefalopatiji kot posledici trajne in nenapredujoče okvare razvijajočih se možganov, je značilen čezmerno vzdražen refleks na razteg. S pomočjo standardizirane modificirane Ashworthove lestvice subjektivno ocenimo od hitrosti raztega mišice odvisno povečanje odpora na pasiven razteg (tj. spastičnost). Namen: Ovrednotiti zanesljivost posameznega preiskovalca in zanesljivost med preiskovalci za modificirano Ashworthovo lestvico ter sočasno veljavnost modificirane Ashworthove lestvice in Tardieujeve lestvice pri otrocih s cerebralno paralizo. Metode dela: Vključenih je bilo 24 otrok s cerebralno paralizo. Zanesljivost posameznega preiskovalca in zanesljivost med preiskovalci smo ugotavljali za modificirano Ashworthovo lestvico, pri izračunu sočasne veljavnosti smo dodali še rezultate Tardieujeve lestvice. Za vrednotenje zanesljivosti in veljavnosti je bil uporabljen koeficient intraklasne korelacije (ICC), pri veljavnosti smo izvedli tudi analizo s Spearmanovim koeficientom korelacije. V izračun smo vključili vse preiskovane mišice, ne glede na anatomsko razvrstitev gibalne oviranosti. Rezultati: Zanesljivost posameznega preiskovalca je bila v vzorcu visoka pri vseh preiskovanih mišicah (ICC = 0,91–0,99). Zanesljivost med preiskovalci je bila v vzorcu dobra, koeficient intraklasne korelacije je znašal od 0,80 do 0,89. V vzorcu smo pri vseh preiskovanih mišicah izračunali zmerno povezanost med Tardieujevo lestvico in modificirano Ashworthovo lestvico. Razprava in zaključek: Izsledki raziskave so v vzorcu pokazali visoko zanesljivost posameznega preiskovalca. Zanesljivost med preiskovalci se je v vzorcu izkazala za dobro. V vzorcu smo potrdili zmerno povezanost vključenih lestvic. Z namenom posplošitve na populacijo, bi bilo statistično analizo smotrno preveriti na večjem vzorcu. Smiselno bi bilo poenotenje protokola, ponovna standardizacija lestvice, vključujoč objektivnejšo, kvantitativno opredelitev spastičnosti. Introduction: Cerebral palsy or static encephalopathy as a permanent and unprogressing impairment of developing brain, results in overactivity of the stretch reflex. Standarised Modified Ashworth Scale can be used for subjective assessment of a velocity-dependent response of muscle to passive stretching, (i.e. spasticity). Purpose: The aim of this work was to evaluate intra- and interrater reliability of modified Ashworth scale and concurrent validity of modified Ashworth scale in comparison to Tardieu scale in children with cerebral palsy. Methods: 24 children with cerebral palsy were subjected. Intra- and interrater reliability was assessed with Modified Ashworth Scale. Outcomes of Tardieu Scale were added for assessment of concurrent validity. For the assessment of reliability and concurrent validity, intraclass correlation coefficient (ICC) was used. Furthermore, Spearman's correlation coefficient for validity was tested. All the assessed muscles were included in the calculation, regardless of the anatomical classification of physical impairment. Results: Intrarater reliability in our sample was excellent in all investigating muscle (ICC = 0,91– 0,99). Good interrater reliability in the sample was observed, with intraclass correlation coefficient from 0,80 to 0,89. Moderate positive correlation in all investigating muscles between Tardieu scale and modified Ashworth scale has been calculated. Discussion and conclusion: The outcomes of our research confirmed excellent intrarater reliability in the sample. Interrater reliability in the sample was assessed as good. In the sample moderate positive correlation was confirmed. For the generalization of the outcomes on population level, statistical analysis should be repeated in a larger sample. An unification, restandardization of the scale, including more objective, quantitative definition of spasticity should be considered in further research.

Uvod: Za razvoj sladkorne bolezni tipa 2 je značilna inzulinska rezistenca. Glavni periferni porabnik glukoze po obroku so skeletne mišice, zato je preprečevanje oziroma zdravljenje inzulinske rezistence v tem tkivu ključno za obvladovanje hiperglikemije. Z AMP-aktivirana protein kinaza (AMPK) kot osrednji regulator celične energije predstavlja novo farmakološko tarčo za zdravljenje sladkorne bolezni tipa 2. Aktivacija AMPK v skeletni mišici spodbudi privzem glukoze v tkivo, oksidacijo maščobnih kislin in mitohondrijsko biogenezo, kar pripomore k zmanjšanju hiperglikemije in inzulinske rezistence pri sladkorni bolezni tipa 2. Inzulinska rezistenca in sladkorna bolezen sta nemalokrat pridobljeni tudi po transplantaciji organov, k čemur v veliki meri prispevajo imunosupresivna zdravila. Mofetilmikofenolat se uporablja v trojnem režimu preprečevanja zavrnitve presajenega organa in deluje kot zaviralec inozin-monofosfat-dehidrogenaze (IMP-dehidrogenaza), ki katalizira pretvorbo IMP v ksantozin monofosfat. Namen: Mofetilmikofenolat z zaviranjem IMP-dehidrogenaze povzroči kopičenje IMP in ostalih intermediatov de novo sinteze gvanozin monofosfata. Podobno kot antirevmatik metotreksat bi s svojim delovanjem lahko zmanjšal očistek ZMP (5-aminoimidazol-4-karboksamid-1-?-D-ribofuranozil 5’-monofosfat), iz katerega v zadnjih dveh reakcijah de novo sinteze purinov nastane IMP. ZMP je analog AMP in aktivna oblika AICAR, najbolj pogosto uporabljenega farmakološkega aktivatorja AMPK. ZMP aktivira AMPK z vezavo na vezavno mesto za AMP. Namen naše raziskave je bil preveriti, ali mofetilmikofenolat in alanozin, ki zavira pretvorbo IMP v adenilosukcinat, vplivata na delovanje AMPK v skeletni mišici. Hipoteze: 1) Mofetilmikofenolat spodbudi aktivacijo AMPK v podganjih in človeških skeletnomišičnih celicah 2) Mofetilmikofenolat in alanozin sinergistično aktivirata AMPK v podganjih skeletnomišičnih celicah 3) Mofetilmikofenolat vpliva na aktivacijo signalne poti Akt v podganjih skeletnomišičnih celicah. Metode: Poskuse smo izvedli na celični liniji podganjih (L6) skeletnomišičnih celic in primarni kulturi človeških skeletnomišičnih celic. Učinke na znotrajcelično signalizacijo smo ovrednotili z metodo prenosa western, s katero smo spremljali aktivacijo AMPK z merjenjem fosforilacije AMPK (Thr172) in njenega substrata acetil-CoA karboksilaze (ACC, Ser79). Določali smo tudi aktivacijo Akt signalne poti z merjenjem fosforilacije Akt (Ser473), njenega substrata AS160 (Ser588) in glikogen sintaze kinaze-3?/? (GSK-3?/?, Ser21/9). Rezultati: Mofetilmikofenolat (5?M) je povečal z AICAR spodbujeno fosforilacijo AMPK in ACC v podganjih skeletnomišičnih celicah, v odsotnosti AICAR pa ni imel učinka na aktivacijo AMPK. V človeških skeletnomišičnih celicah ni dosegel z AICAR spodbujene aktivacije AMPK, zato smo našo prvo hipotezo le delno potrdili. Sočasno tretiranje podganjih skeletnomišičnih celic z mofetilmikofenolatom in alanozinom je zvišalo raven fosforilacije ACC. Ta rezultat nakazuje, da mofetilmikofenolat in alanozin sinergistično aktivirata AMPK, kar podpira našo drugo hipotezo. Preverili smo še vpliv mofetilmikofenolata na signalno pot Akt v podganjih skeletnomišičnih celicah, preko katere poteka od inzulina odvisen privzem glukoze v skeletno mišico. Inzulin je spodbudil povečanje fosforilacije Akt tudi v prisotnosti mofetilmikofenolata. V odsotnosti inzulina je mofetilmikofenolat povečal fosforilacijo Akt, kar podpira našo tretjo hipotezo. Zaključek: 1) Mofetilmikofenolat v podganjih skeletnomišičnih celicah ne aktivira AMPK, temveč okrepi z AICAR spodbujeno aktivacijo AMPK. V človeških skeletnomišičnih celicah mofetilmikofenolat ni spodbudil aktivacije AMPK. 2) Mofetilmikofenolat in alanozin sinergistično aktivirata AMPK v podganjih skeletnomišičnih celicah. 3) Mofetilmikofenolat aktivira Akt v podganjih skeletnomišičnih celicah. Background: Type 2 diabetes is characterized by insulin resistance. Skeletal muscle is the main site for glucose uptake postprandially, thus representing an important target tissue to prevent or treat insulin resistance and hyperglicemia. AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis and represents a new pharmacological target in treatment of type 2 diabetes. In skeletal muscle AMPK activation promotes glucose uptake into the tissue, fatty acid oxidation and mitochondrial biogenesis, which helps to ameliorate metabolic dysregulation such as hyperglicemia and insulin resistance in type 2 diabetes. Metabolic dysregulation, such as type 2 diabetes is a common condition, also developed after an organ transplantation and is at least partially caused by immunosupressive therapy. Mycophenolate mofetil is a part of commonly used triple transplantation immunosupressive regimen. It inhibits inosine monophosphate dehydrogenase (IMP-dehydrogenase), preventing the coversion of IMP to xanthosine monophosphate. Aim: Inhibition of IMP dehydrogenase results in accumulation of IMP and its precursors in de novo synthesis of guanosine monophosphate. Therefore, mycophenolate mofetil, like antirheumatic drug methotrexate, could reduce clearance of ZMP (5-aminoimidazole-4-carboxamide-1-?-D-ribofuranosyl 5’-monophosphate), a precursor of IMP in the last two steps of the de novo purine synthesis. ZMP is an AMP analogue and an active form of AICAR, the most commonly used experimental AMPK activator. Aim of our research was to investigate, if mycophenolate mofetil and alanosine, an inhibitor of conversion of IMP to adenylosuccinate, could activate AMPK in skeletal muscle. Hypotheses: 1) Mycophenolate mofetil promotes AMPK activation in cultured rat and human skeletal muscle cells 2) Mycophenolate mofetil and alanosine synergistically activate AMPK in rat skeletal muscle cells 3) Mycophenolate mofetil modulates the Akt signalling pathway in rat skeletal muscle cells. Methods: To investigate our hypotheses we used cultured rat skeletal muscle cells (L6) and primary human skeletal muscle cells. We evaluated the effect of mycophenolate mofetil and other agents on AMPK activation with western blot, by measuring the phosphorylation of AMPK (Thr172) and its substrate acetyl-CoA carboxylase (ACC, Ser79). We assessed activation of Akt signalling pathway by measuring the phosphorylation of Akt (Ser473), its substrate AS160 (Ser588) and glycogen synthase kinase-3?/? (GSK-3?/?, Ser21/9). Results: Mycophenolate mofetil (5?M) increased AICAR-stimulated phosphorylation of AMPK and ACC in rat skeletal muscle cells, but did not activate AMPK when used alone. In human skeletal muscle cells, mycophenolate mofetil did not increase AICAR stimulated AMPK activation. These results only partially support our first hypothesis. In rat skeletal muscle cells mycophenolate mofetil and alanosine increased phosphorylation of ACC, indicating that they activate AMPK sinergistically, which supports our second hypothesis. Finally, we investigated the effect of mycophenolate mofetil on the Akt signalling pathway. Insulin increased Akt phosphorylation in the presence of mycophenolate mofetil. Further, mycophenolate mofetil alone increased Akt phosphorylation, which supports our third hypothesis. Conclusions: We can conclude that: 1) Mycophenolate mofetil alone does not activate AMPK, but enhances AICAR-stimulated AMPK activation in rat skeletal muscle cells, demonstrating it promotes AMPK activation. In human skeletal muscle cells mycophenolate mofetil did not promote AMPK activation. 2) Mycophenolate mofetil and alanosine synergistically activate AMPK in rat skeletal muscle cells. 3) Mycophenolate mofetil promotes activation of the Akt signalling pathway in rat skeletal muscle cells.

Uvod: Skeletne mišice so pomembno tarčno tkivo pri zdravljenju sladkorne bolezni tipa 2, saj predstavljajo glavno mesto z inzulinom stimuliranega privzema glukoze v postprandialnem stanju. Pri preučevanju delovanja signalnih poti, ki vplivajo na inzulinsko rezistenco, lahko izhajamo iz salicilne kisline. Njeni derivati imajo poleg protivnetnih učinkov, ugodne učinke na delovanje inzulina in presnovo glukoze, tudi zaradi aktivacije z AMP-aktivirane protein kinaze. Namen: Sulfasalazin je predzdravilo iz 5-aminosalicilne kisline in sulfapiridina. Klinični primeri in živalski modeli so nakazali, da bi lahko vplival na koncentracijo glukoze ter zaviral nastanek zapletov sladkorne bolezni. Ker njegov mehanizem na molekularni ravni pri diabetesu ni raziskan, smo želeli ugotoviti, ali učinkuje na inzulinsko signalno pot prek kinaze Akt. Hipoteze: 1) Sulfasalazin poveča bazalno in z inzulinom ali rastnim dejavnikom iz trombocitov (PDGF) spodbujeno fosforilacijo kinaze Akt. 2) Sulfasalazin poveča bazalno in z inzulinom ali s PDGF spodbujeno fosforilacijo AS160. 3) Sulfasalazin ne vpliva na z inzulinom ali s PDGF spodbujeno fosforilacijo kinaze ERK1/2. Metode: Učinke sulfasalazina na znotrajcelično signalizacijo smo preučili na in vitro modelu linije podganjih skeletnomišičnih celic L6. Aktivnost signalnih poti smo ovrednotili z merjenjem fosforilacije proteinov: Akt (Ser473), AS160 (Ser588), ERK1/2 (Thr202/Tyr204), 4E-BP1 (Thr37/Thr46) in acetil-CoA karboksilaze (Ser79). Uporabili smo metodo gelske elektroforeze, metodo prenosa western in zaznali proteine z metodo ojačane kemiluminiscence. Rezultati: Sulfasalazin je zmanjšal z inzulinom spodbujeno fosforilacijo Akt, ni pa vplival na bazalno niti s PDGF spodbujeno fosforilacijo Akt. Sulfasalazin je inhibiral fosforilacijo AS160, tudi ob prisotnosti inzulina ali PDGF. S tem smo zavrgli prvo in drugo hipotezo. Sulfasalazin je zmanjšal s PDGF spodbujeno fosforilacijo ERK1/2, s čimer smo delno podprli tretjo hipotezo. Zaključki: Ugotovili smo, da sulfasalazin zavira z inzulinom in s PDGF spodbujeno fosforilacijo AS160. Kljub temu da naj bi sulfasalazin deloval kot farmakološki aktivator z AMP-aktivirane protein kinaze, predvidevamo, da do translokacije GLUT4 na površino mišičnih celic ne bi prišlo, saj je fosforilacija AS160 pri tem ključen dogodek. Potrebne bi bile dodatne raziskave, s katerimi bi ugotovili, zakaj sulfasalazin zmanjšuje fosforilacijo AS160. Background: Skeletal muscles are important target tissues for treatment of type 2 diabetes, as they represent a main site of insulin stimulated glucose uptake in the postprandial state. When examining the signalling pathways related to insulin resistance, we can turn our focus on salicylic acid. Its derivatives not only have anti-inflammatory properties, but also beneficial effects on insulin and metabolism of glucose also because of activation of AMP-activated protein kinase. Aim: Sulfasalazine is a prodrug from 5-aminosalicylic acid and sulfapyridine. Some clinical reports and animal studies suggested that sulfasalazine could have beneficial effect on concentration of glucose in plasma and prevent the complications of diabetes. Our aim was to determine whether sulfasalazine modulates insulin signalling via Akt kinase. Hypotheses: 1) Sulfasalazine promotes basal and insulin- or PDGF-stimulated phosphorylation of Akt. 2) Sulfasalazine promotes basal and insulin- or PDGF-stimulated phosphorylation of AS160. 3) Sulfasalazine does not alter insulin- or PDGF-stimulated phosphorylation of ERK1/2. Methods: We examined the effects of sulfasalazine by using in vitro model of L6 rat skeletal muscle cells. Activation of signalling pathways was assessed by measuring phosphorylation of kinase Akt (Ser473), AS160 (Ser588), ERK1/2 (Thr202/Tyr204), 4E-BP1 (Thr37/Thr46), acetyl-CoA carboxylase (Ser79), using polyacrylamide gel electrophoresis, western blotting and detection of specific proteins with chemiluminescent imaging method. Results: Sulfasalazine inhibited the insulin-stimulated phosphorylation of Akt, but it did not alter basal or PDGF-stimulated phosphorylation. Sulfasalazine inhibited the phosphorylation of AS160, also in the presence of insulin or PDGF. These results do not support our first and second hypothesis. Sulfasalazine inhibited PDGF-stimulated phosphorylation of ERK1/2, what partially support the third hypothesis. Conclusions: We found out that sulfasalazine inhibits insulin- and PDGF-stimulated phosphorylation of AS160. Even though sulfasalazine should act as an activator of AMP-activated protein kinase, our results suggest that there is no translocation of GLUT4 on the surface of muscle cells, because phosphorylation of AS160 is the key event in the insulin signalling pathway leading to increased glucose uptake. Additional research should be done to uncover molecular mechanisms by which sulfasalazine suppresses phosphorylation of AS160.

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.