Polne Razlike u Regulaciji Ekspresije i Aktivnosti Inducibilne Azot-Monoksid-sintaze i Natrijum-Kalijumove Pumpe u Jetri Gojaznih Pacova

Abstract

Gojaznost i posledično poremećen metabolizam lipida i glukoze u jetri su glavni faktori rizika za razvoj bolesti jetre. Polne razlike u gojaznosti i razvoju rezistencije na insulin (IR) i nealkoholne bolesti masne jetre (NAFLD) pripisuju se estrogenima, ali mehanizmi kojima ovi hormoni ostvaruju pomenute efekte nisu dovoljno istraženi. Pored toga što ostvaruju genomske efekte, estrogeni aktiviraju i signalne molekule u citoplazmi: fosfatidilinozitol 3-kinazu (PI3K), protein kinazu B (Akt), Rho familiju malih GTP vezujućih proteina (Rho), kao i njihove nishodne efektore, Rho-vezane protein kinaze (ROCK), zatim AMP-aktivirajuće protein kinaze (AMPK) i kinaze regulisane ekstraćelijskim signalima (ERK1/2). Dosadašnja istraživanja su pokazala da, pored protektivne uloge koju inducibilna azot-monoksid-sintaza (iNOS) ima u jetri tokom različitih procesa (sepsa i ishemija), poremećaj u regulaciji njene ekspresije i aktivnosti mogu biti uključeni u razvoj IR u gojaznosti. Jedna od pratećih komplikacija IR je i poremećaj u regulaciji ekspresije i aktivnosti natrijum-kalijum adenozin trifosfataze (Na⁺ /K⁺ -ATP-aza), koja učestvuje u realizaciji brojnih procesa u jetri. Poznato je da estradiol reguliše ekspresiju iNOS i Na⁺ /K⁺-ATPaze, ali podaci koji ukazuju na polne razlike u regulaciji ekspresije i aktivnosti ovih enzima u jetri gojaznih životinja nedostaju u literaturi.

U eksperimentima ove doktorske disertacije korišćeni su adultne ženke i mužjaci pacova Wistar soja. Jedna grupa pacova je hranjena standardnom laboratorijskom hranom (kontrolni pacovi), a druga standardnom laboratorijskom hranom obogaćenom sa 42% masti (gojazni pacovi). Nakon 10 nedelja životinje su žrtvovane, izolovane su jetre, a iz pune krvi su izdvajani serum i plazma. U serumu pacova određivane su koncentracije: glukoze, insulina, ukupnog holesterola, dok je u plazmi određivana koncentracija slobodnih masnih kiselina (SMK) i koncentracija nitrita (NO₂ ^- ) i nitrata (NO₃ ^- ), kao krajnjih produkata NO. U lizatu jetre pacova određivana je koncentracija glukoze, holesterola i SMK, kao i aktivnost Rho proteina. Aktivnost Na⁺ /K⁺ -ATPaze je određivana u frakciji plazma membrana ćelija jetre. Nivo iRNK za iNOS i α1 subjedinicu Na⁺ /K⁺ -ATPaze određivan je metodom qPCR. Western blot metodom određivana je količina α1 subjedinice Na⁺ /K⁺-ATPaze, GLUT2 i FAT/CD36 u frakciji plazma membrana, dok je nivo ekspresije iNOS, NFκB IRS-1, PI3K-p85 i PI3K-p110, RhoA, ROCK1 i ROCK2, ERα i ERβ, kao i nivo fosforilacije mTOR, PDK1, Akt, ERK1/2 i AMPKα1 kinaza određivan u lizatima ćelija jetre. Asocijacija IRS-1 proteina sa PI3K-p85 u lizatu jetre određivana je metodom koimunoprecipitacije.

Rezultati ove doktorske disertacije pokazuju da, za razliku od mužjaka, kod ženki pacova nema promena u koncentraciji insulina u serumu i vrednosti HOMA indeksa, dok je koncentracija glukoze u serumu smanjena. U membranskoj frakciji proteina jetre nivo GLUT2 proteina je smanjen kod gojaznih pacova oba pola, dok je nivo FAT/CD36 proteina smanjen kod gojaznih ženki, a povećan kod gojaznih mužjaka pacova. Nivo iRNK za iNOS i koncentracija NO₂ ^- /NO₃ ^- je povećana, a nivoa iNOS proteina smanjen samo kod gojaznih mužjaka pacova. Nivo proteina α1-Na⁺ /K⁺ -ATPaze i aktivnost Na⁺ /K⁺ -ATPaze su smanjeni kod gojaznih pacova oba pola, dok je relativna ekspresija gena za α1 subjedinicu Na⁺ /K⁺ -ATPaze smanjenja samo kod gojaznih mužjaka pacova. Nivo fosforilacije PDK1 i Akt na Thr³⁰⁸ smanjen je u jetri pacova oba pola, dok je nivo fosforilacije mTOR i Akt na Ser⁴⁷³smanjen samo u jetri gojaznih mužjaka pacova. Nivo fosforilacije ERK1/2 i AMPKα1 je povećan u jetri gojaznih ženki, a smanjen u jetri gojaznih mužjaka. Takođe, povećanje aktivnosti Rho proteina uočeno je samo u jetri gojaznih mužjaka, dok je nivo RhoA proteina smanjen samo u jetri gojaznih ženki pacova.

Rezultati u okviru ove doktorske disertacije pokazuju da HF ishrana dovodi do razvoja sistemske IR i poremećaja metabolizma glukoze i lipida samo u jetri mužjaka pacova. Takođe, pokazane su polne razlike u regulaciji ekspresije i aktivnosti iNOS i Na⁺ /K⁺-ATP-aze, koja je posredovana učešćem signalnih molekula mTOR, Akt, ERK1/2, AMPKα i RhoA. Rezultati ove doktorske disertacije doprinose razumevanju polnih razlika u razvoju IR i mogu imati primenu u razvoju novih terapijskih pristupa u lečenju komplikacija u gojaznosti.

Alternate abstract:

Obesity and altered hepatic lipid and glucose metabolism are main risk factors for the development of liver diseases. Sex differences in obesity and development of insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) are attributed to estrogens, but the exact mechanisms of their effects have not been fully understood. In addition to genomic effects, estrogens also activate signaling molecules in the cytoplasm: phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), Rho family of small GTP binding proteins (Rho), as well as their effectors, Rho-associated protein kinases (ROCK), AMP-activating protein kinase (AMPK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Besides the protective role of inducible nitric oxide synthase (iNOS) in the liver during various processes (sepsis and ischemia), the altered expression and activity of this enzyme may be involved in the development of IR in obesity. One of the common comorbidities of obesity-induced IR is altered expression and activity of sodium/potassium-adenosine triphosphatase (Na⁺/K⁺-ATPase), which participates in numerous processes in the liver. It is known that estradiol regulates the expression of iNOS and Na⁺/K⁺-ATPase, but there is no evidence on sex-specific differences in hepatic iNOS and Na⁺/K⁺-ATPase expression/activity in obese animals.

For the experiments of this doctoral dissertation adult female and male Wistar rats were used. Control groups of rats were fed standard laboratory diets (control rats) and the other two groups were fed standard laboratory food enriched with 42% fat (obese rats). After 10 weeks, the animals were sacrificed, and livers, serum and plasma were isolated. Serum glucose, insulin and total cholesterol concentrations were determined, as well as plasma free fatty acids (FFA) and nitrite (NO₂^-) and nitrate (NO₃^-) concentrations. The concentrations of glucose, cholesterol and FFA, as well as the activity of Rho proteins, were determined in liver lysates. The hepatic Na⁺/K⁺-ATPase activity was determined in the plasma membrane fraction. The iNOS and α1 subunit of Na⁺/K⁺-ATPase mRNA levels were determined by qRT-PCR. Western blot method was used to determine protein levels of α1-Na⁺/K⁺-ATPase, GLUT2 and FAT/CD36 in the plasma membrane fraction, while protein levels of iNOS, NFκB IRS-1, PI3K-p85 and PI3K-p110, RhoA, ROCK1 and ROCK2, ERα and ERβ, as well as phosphorylation levels of mTOR, PDK1, Akt, ERK1/2 and AMPKα1 were determined in liver lysates.The association of IRS-1/PI3K-p85 in liver lysates was determined by coimmunoprecipitation.

The results of this doctoral dissertation show that, unlike males, there are no changes in serum insulin levels and HOMA indices in female rats, while serum glucose levels are reduced. GLUT2 protein level in the plasma membrane fraction in liver was decraesed in obese rats of both sexes, while FAT/CD36 protein level was decreased in obese females, and increased in obese male rats. The iNOS mRNA level in liver and plasma NO₂ ^- /NO₃ ^- concentration were increased, and iNOS protein level was decreased only in obese male rats. The hepatic α1-Na⁺ /K⁺ -ATPase protein level and Na⁺ /K⁺ - ATPase activity were decreased in obese rats of both sexes, while α1-Na⁺ /K⁺ -ATPase mRNA level was decreased only in livers of obese male rats. The phosphorylation levels of PDK1 and Akt (Thr³⁰⁸) were reduced in livers both female and male obese rats, while the phosphorylation levels of mTOR and Akt (Ser⁴⁷³) were reduced only in livers of obese male rats. The phosphorylation level of ERK1/2 and AMPKα1 was increased in livers of obese females and decreased in livers of obese males. Also, the increase of hepatic Rho activity was observed only in obese male rats, while the hepatic RhoA protein level was decreased only in obese female rats.

The results of this doctoral dissertation show that HF diet leads to the development of systemic IR and disturbance of hepatic glucose and lipid metabolism only in male rats. Also, obtained results show sex specific differences in the regulation of iNOS and Na⁺ /K⁺-ATPase expression/activity which are mediated by the participation of mTOR, Akt, ERK1 / 2, AMPKα and RhoA signaling molecules. These results contribute to better understanding of pathogenesis and sex differences in obesityinduced IR in the liver and may lead to discovering new therapeutic approaches in the treatment of obesity complications.