The effect of sulphasalazine on regulation of energy metabolism in skeletal muscle

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