Nealkoholna bolest jetre (NAFLD) je najčešća bolest jetre. Sastav masnih kiselina može doprinijeti akumulaciji masnoća, a neke masne kiseline mogu imati blagotvoran utjecaj na NAFLD. Oksidacijski stres i otpornost na inzulin u jetri glavni su čimbenici u razvoju jetrene steatoze i progresije na NASH (bezalkoholni steatohepatitis). MUFA (mononezasićena masna kiselina) iz prehrane stvara čestice lipoproteina otporne na oksidativnu modifikaciju, a PUFA (polinezasićena masna kiselina) značajno povećava peroksidaciju lipida. Za procjenu utjecaja različitih nezasićenih masnih kiselina u razvoju NAFLD-a u našem eksperimentu korišteno je maslinovo ulje bogato MUFA-om, suncokretovo ulje s visokim udjelom PUFA i bučino ulje s umjerenom količinom obje vrste ulja. Nedavne studije o sjemenkama i ulju buče pokazale su hepatoprotektivni učinak. Štakori Wistar, muški, dva mjeseca stari, podijeljeni su u čeitri skupine. Štakori su bili 21 dan na standardnoj prehrani (SD) ili visokomasnoj dijeti (HFD). HFD pripremljene su miješanjem 30 % suncokretova ulja (MDS skupina) ili 30 % maslinova ulja (MDM) ili 30 % bundevina ulja u standardnu prehranu (MDB). Nakon eksperimentalnog razdoblja životinje su anestezirane i žrtvovane. Određivana je masa, masa jetre, količina konzumirane hrane i lipidi u plazmi te je rađena histološka analiza jetre. Dodatno, životinje SD i MDB korištene su za pokuse in vitro. Nije bilo statistički značajnih razlika u tjelesnoj masi, masi jetre i lipidnom statusu između skupina. Unos hrane u gramima značajno se razlikovao između životinja SD i HFD, ali u kJ razlike nije bilo. Histološki, u MDS-u steatoza je bila teška, u MDB-u umjerena, a u MDM-u blaga. U ranijim istraživanjima, naš laboratorij je prikazao neosjetljivost hepatocita na inzulin kod štakora hranjenih HFD-om sa suncokretovim uljem. U hepatocitima od štakora na HFD-u s maslinovim uljem, inzulin je značajno smanjio proizvodnju glukoze u usporedbi s kontrolnom skupinom. Bučino ulje nije promijenilo inzulinsku osjetljivost hepatocita. Omjer PUFA/MUFA u prehrani utjecao je na razinu nakupljanja lipida u hepatocitima.
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The liver is the central metabolic organ for the processing and storage of nutrients and glycemic homeostasis and has a central role in the development of metabolic syndrome. Metabolic syndrome consists of a cluster of risk factors that greatly increase a probability of developing the atherosclerotic cardiovascular disease and type two diabetes mellitus (DM2). Non-alcoholic fatty liver disease (NAFLD) is not included in defining criteria for metabolic syndrome, but it is considered the hepatic manifestation of the syndrome. NAFLD consists of an array of conditions from simple benign steatosis to cirrhosis and hepatocellular carcinoma. It is the main cause of dyslipidemia and is closely related to insulin resistance. The prevalence of NAFLD in the western world is about 30 %, and in the population, with DM2 the prevalence of fatty liver is up to 70 %. Chemical modification of biological molecules can be directly poisonous to liver cells or can stimulate the response of the host that is translated into inflammation, collagen production and progression of NAFLD. A composition of fatty acids can contribute to fat accumulation, and some fatty acids may have a beneficial influence on NAFLD. Vegetable oils with high content of unsaturated fatty acids were considered as “healthier” lipids because of their beneficial effects on metabolic syndrome. Polyunsaturated fatty acids from ω-6 (n-6) and ω-3 (n-series are precursors to potent lipid mediator signaling molecules, termed "eicosanoids", which have important roles in the regulation of inflammation. The main fatty acid in sunflower oil is linoleic acid (C18:2, n-6). Linoleic acid is not synthesized by the body and is, therefore, an essential fatty acid. In the olive oil, the prevalent fatty acid is monounsaturated oleic acid (C18:1, n-9). MUFA fatty acids have a strong positive effect on cardiovascular health. However, only olive oil and its main component, oleic acid, seem to be associated with this effect. Oxidative stress and insulin resistance in the liver are interdependent and are major factors in the development of liver steatosis and its progression to NASH (Non-alcoholic steatohepatitis) and cirrhosis. MUFA from diets generates lipoprotein particles that are resistant to oxidative modification. Diet rich in PUFA increases lipid peroxidation significantly and may raise the susceptibility of tissues to injury. The Mediterranean diet, although having a very high energy intake from fat (30-40 %), is considered to have a beneficial effect on health, by protecting the vascular system and reducing the factors that affect the occurrence of metabolic syndrome. To evaluate the influence of different unsaturated fatty acids in the development of NAFLD in our experiment used three types of unsaturated fatty acids sources, olive oil rich in MUFA, sunflower oil with high content of polyunsaturated fatty acids (PUFA) and pumpkin seed oil with the moderate amount of both oils. In recent years pumpkin seed oil gained attention as "healthy " oil and even sold as a supplement. In our region of central Europe, pumpkin seed oil is traditional oil for consummation along with olive and sunflower oil. Recent studies on pumpkin seed and pumpkin seed oil have shown promising results on glycaemia and dyslipidemia and even showed hepatoprotective effect. To our knowledge, there was no study on the impact of high-fat diet (HFD) with pumpkin seed oil on liver or plasma lipid profile on healthy laboratory animals. We used an animal model which is the closest to the pathogenesis of NAFLD in humans.
Animals and methods
The experiments were carried out according to the guidelines on the use of animals for biomedical research (Croatian Animal Welfare Regulation Acts NN135/06; NN 37/13; NN55/13) and were approved by Ethical committees of Zagreb University School of Dental Medicine and School of Medicine. We used animals from the University of Zagreb, School of Medicine.
24 male Wistar rats, two months old, weighing 265 ± 27 g, were randomly divided into four groups. Rats were housed individually, to monitor individual feed consumption, at constant room temperature of 21 ± 2°C, with 12-h light-dark cycle, and ad libitum access to diet and water. Diet consumption and body mass were monitored daily by the same trained handler at the end of the light cycle. The rats were 21 days on the standard diet (diet #4RF21-GLP certificate, Mucedola, Milan, Italy) as Control Diet group (SD). According to certificate standard food energy density was 11.17 kJ per gram and composition was carbohydrate 53.5 %, fat 3 %, and protein 18.5 %. The high-fat diets (HFD) were prepared by mixing 30 % sunflower oil to the standard diet (MDS group) or 30 % olive oil (MDM) or 30 % of pumpkin seed oil to the standard diet (MDB). Edible oils were from the same manufacturer (Zvijezda, Zagreb, Croatia). After three weeks of the experimental period, animals were anesthetized by an intraperitoneal administration of sodium thiopental and sacrificed by bleeding from v. cava inferior. Three mL of venous blood from v. cava inferior was collected in EDTA test tubes to determine lipid profile. The concentrations of triacylglycerol (TG), total cholesterol (Total-CH), and HDL- cholesterol (HDL) were measured enzymatically using commercial kits. LDL-cholesterol (LDL) was calculated using formula by Friedewald and Non-HDL-cholesterol was used as suggested by Sánchez-Muniz and Bastida. The liver was washed with saline, dried with filter paper and weighed, and then cut into small sections for histological procedures. Immediately after cutting the liver, samples were put in the Carnoy fixative and after an appropriate time the samples were set in paraffin blocks, cut and stained with hematoxylin and eosin stain (HE) and Masson's trichrome stain. We used Kleiner’s grading system of NAFLD modified for rodents as proposed by Liang. Steatosis was expressed as the percentage of the hepatocytes with fatty infiltration in random slides, in twelve fields at 200 x magnification per group, against 25 x 33 grid.
Primary hepatocytes cultures were obtained from SD and MDB animals via modified collagenase perfusion technique via cannulation of vena Porte. After suspending cells in the medium, trypan blue viability test was performed, and cells were counted and suspended in the concentration of one million per liter. Three ml of suspension was put in a petri dish and incubated for four hours. After four hours, the medium was replaced, and culture put in incubation for further 20 hours. After 24 hours in incubation, incubation medium was removed, cultures were washed three times with Hanks Hepes medium and incubated with glucose free Hanks Hepes with addition of 10 mmol/l pyruvate and without added hormones or with insulin (80 nmol/l), or glucagon (200 nmol/l) or both hormones. Samples were taken every hour over three-hour period. Glucose production and protein content of dish were determined enzymatically. Results were expressed as nmol of glucose per mg of protein
Results are shown as mean ± SD unless stated otherwise and values of p ≤ 0.05 were considered statistically significant. Statistical analyses were performed by Student's t-test or ANOVA one-way analysis of variance followed by the Tukey HSD test.
Body mass gain was stable in all groups through 21 days of the experimental period. There were no statistically significant differences in the final body mass and body mass gain between the HFD groups at in comparison to SD group. Liver mass and liver/body mass ratio didn’t differ significantly between groups. Daily diet intake differed significantly between animals on standard diets and those on HFD diets but no differences between HFD groups were found. However, daily and total energy intake was similar for control and high-fat diet groups. Macroscopically, there was an accumulation of visceral fat which was prominent in MDS and MDB groups while in MDM group fat accumulation was mild. Livers from HFD groups were slightly yellowish in comparison to a rich dark color of SD liver. There was no difference in plasma lipid status between control and high-fat diet groups.
Histological examination of livers slides from SD groups and HFD groups of animals revealed that NAFLD developed in livers from HFD rats without evidence for nonalcoholic steatohepatitis (NASH). Livers from SD group showed normal histological acinar organization without fat infiltration in hepatocytes. Livers from MDM group developed mild microvesicular hepatic steatosis, with infiltration predominantly in the periportal zone (grade 1). In MDS group the microvesicular steatosis was severe and observed predominantly in zones one and two and reaching zone three (grade 3) with some mild hepatocyte hypertrophy. In MDB group microvesicular steatosis was moderate and in zones one and two (grade 2). No evidence of inflammation or progression to NASH was observed in SD or HFD groups. Trichrome staining showed no periportal or perisinusoidal fibrosis in SD or HFD groups. There was no differences in basal and glucagone stimulated glucose production and insulin sensitivity in hepacotites obtained from animals on high fat diet with pumpkin seed oil and from animals on standard diet.
In the previous studies, our laboratory demonstrated hepatic insulin resistance in rats fed HFD with sunflower oil and raw sunflower seeds. In hepatocytes from rats fed the HFD with dietary intake of olive oil, insulin significantly and strongly decreased glucose production compared to control group. In a murine model olive oil and pumpkin seed oil had a positive effect on serum lipid concentrations. That effect was greater in the group receiving olive oil. A daily dose of 0.2 mL of olive or pumpkin seed oil per animal lowered LDL cholesterol and TG levels. HDL levels in groups receiving oils were higher in comparison to control indicating a protective role of olive oil and pumpkin seed oil on cardiovascular health. We didn't find that effect of olive oil and pumpkin seed oil in our study, but that may be due to a very high intake of fats in our high-fat diets groups. In a study on rats, MUFA from olive oil decrease lipid content in NASH caused by methionine choline-deficient diet, while PUFA from fish oil showed no protective effect on fat accumulation in the liver. Olive oil, in the high-fat diet, manages to prevent excessive fat accumulation in the liver as seen in sunflower oil group. Hepatocytes from MDB group showed preserved glucagon and insulin sensitivity, and in contrast to olive oil, which showed increased insulin sensitivity, had a mild effect on preventing lipid accumulation.
In conclusion, high-fat diet, without increased caloric intake, weight gain, or lipid abnormality, plays a role in the pathogenesis of the nonalcoholic fatty liver disease. The study findings suggested the association between the amount of lipid accumulation in hepatocytes and the composition and ratio of fatty acids in the diet. The ratio and composition of fatty acids found in sunflower oil showed the highest risk factor that led to severe lipid accumulation in the liver cells. There is no expected evidence of beneficial effects of the pumpkin seed oil. The free fatty acid composition and ratio in the pumpkin seed oil led to moderate liver steatosis. By contrast, olive oil was the most favorable and led to a mild accumulation of lipids in hepatocytes, indicating that the ratio and composition of fatty acids in olive oil may delay or prevent the development of NAFLD.