High Fatty Diet Effects on Rat Liver

Aim of this study was to investigate effects of high fat diet on rat liver and weight gain. By this purpose 30 Wistar Albino rats were divided into 4 groups. 1. High carbonhydrate diet group for 16 weeks (K16), 2. High fatty diet group for 16 weeks (D16), 3. High carbonhydrate diet group 20 weeks (K20), 4. High fatty diet group 20 weeks (D20). High fatty diet; %60 fat (1/3 canola, 1/3margarine, 1/3 sunflower oil), %20 protein, %20 carbohydrate and high carbohydrate control diet; %69 carbohydrate, %20 protein and %11 margarine was composed. There weren’t any significant differences between groups as compared to body weight, liver weight and epididymal fat weight. At the result of biochemical analysis, LDH in 16 weeks high carbohydrate diet and ALT in 20 weeks high carbonhydrate diet was significant higher than high fatty diet groups. In histological research, even though fibrosis, inflamation, steatosis findings observed at portal area of all groups, there wasn’t significant statistical differences. Similar to this, α-SMA and TGF-β accumulation in all groups were similar interms of immunohistological investigation. In conclusion; this study showed that comparing to relatively high fatty diet rich with omega-9, high carbonhydrate feeding caused liver injury and anointment and there wasn’t significant differences in terms of weight gain. By this way, we think that decreasing of carbonhydrate and increasing amount of olive oil, canola oil and hazelnut oil at which containing omega-9 in diet, liver can be protected.

Cervical dislocation was performed to the rats by ether anesthesia at the end of the sixteenth and twentieth weeks.Liver, epididymal adipose tissue, and blood samples were taken.After the tissues had been taken, weights were measured with precision scales and then placed in 10% formaldehyde solution.After these tissues had been fixed in formaldehyde solution for 48 hours, they were cut in appropriate size and then were embedded into paraffin block.Sections were stained with hematoxylin eosin and Gomori trichrome.Immunohistochemical staining was performed using by alpha smooth muscle actin (α-SMA)(Biocare Medical, Lot:062410) and transforming growth factor beta (TGF-β)(antibodies, Lot:360350) primary antibodies.Stained sections were examined and photographed with photomicroscope Olympus BX50.Serum levels of glucose, serum albumin, insulin, triglycerides, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), lactate dehydrogenase (LDH), alanin aminotransferase (ALT), and aspartate transaminase (AST) were measured.
Mann-Whitney U Test was used for statistical evaluation of biochemical data, histological data, rat weights, epydidimal fat and liver weights.Moreover, biochemical evaluation has been analyzed with independent 2-Sample T Test as well.

INTRODUCTION
Obesity is one of the most common and important diseases of today.Approximately, 1.2 billion people are overweight in the world and at least 300 million of them are obese.According to the World Health Organization, obesity is one of the most preventable ten diseases.Obesity is considered as associated with imbalance between energy intake and expenditure.However, studies show that genetic, physiological and behavioral factors play a role in the etiology of obesity (1).
In recent years, so many studies have been conducted about the relationship between diet and obesity.Previously, fats were supposed to play a leading role induce obesity because of including high-calorie, unlike this, some studies have been done about the carbohydrates induce obesity in recent years (1-3).According to Dr. Atkins trigger of obesity on foods are not fat and proteins, but carbohydrates.In Dr. Atkins diet method, daily carbohydrate intake is limited with maximum 20 grams protein and no restrictions are imposed on proteins and fats (1).
Obesity leads to steatosis and steatohepatitis on liver.While a normal liver has oxidative stress-resistant, fatty liver is vulnerable to oxidative stress.Researchers exemplify this with the two pulse hypothesis.In the lubricated liver as a result of obesity, inflammation, and fibrosis are developed and steatohepatitis is show up with effects of oxidative stres, cytokines such as TNF-α, mitochondrial dysfunction, hormones such as adiponectin and leptin (4).
In this study, we aimed to investigate effects of relatively high-fat diet rich in omega-9 on the liver.Furthermore, we aimed to reveal that high fat diet or isocaloric highcarbohydrate diet has more active role on weight gain and whether omega fatty acids in canola oil used in highfat diet content has any protective effect on the liver.

MATERIALS AND METHODS
This experimental study was carried out with the approval of Law No. 2009-20 ethics committee of experimental animals.All rats were provided from Duzce University Experimental Animal Research Center.30 male Wistar albino rats were divided into 4 groups when they were 28 days old.1-16-weeks old high-carbohydrate diet group (K-16 group) (n:7)

Weights of rats
Weights of total 4 groups (16-weeks old high-carbohydrate diet group, 16-weeks old high-fat diet group, 20-weeks old high-carbohydrate diet group, 20-weeks old high-fat diet group) which are consisting of 30 rats were measured weekly regularly.The obtained data were statistically analyzed by Mann-Whitney-U test.During this assessment 16-weeks high-carbohydrate diet and 16-weeks high-fat diet groups were compared with each other, 20-weeks high-carbohydrate diet and 20-weeks high-fat diet groups were compared with each other (Figure 1).According to these statistical results, no significant difference was found in overall body weight change between16-weeks high-carbohydrate diet group and 16-weeks high-fat diet group (p>0.05).Moreover, according to these statistical results, no significant difference was found in overall body weight change between 20-weeks high-carbohydrate diet group and 20-weeks high-fat diet group (p> 0.05).

Epididymal fat and liver weights
The obtained data were statistically evaluated by Mann-Whitney-U test.During this assessment 16-weeks highcarbohydrate diet and 16-weeks high-fat diet groups were compared with each other, 20-weeks high-carbohydrate diet and 20-weeks high-fat diet groups were compared with each other.According to these statistical results there was no statistically significant difference between 16-weeks high-carbohydrate diet group and 16-weeks high-fat diet group in terms of both liver weight and epididymal fat weight (p> 0.05).Also, no significant difference was found between 20-weeks highcarbohydrate diet group and 20-weeks high-fat diet group (p >0.05) (Table 1).

Biochemical Evaluation
Serum insulin, glucose, albumin, triglycerides, total cholesterol, LDL, HDL, LDH, ALT, and AST levels were measured in all groups (Table 2).The data were statistically evaluated using by the Mann-Whitney-U test.Insulin were excluded from statistical analysis for all groups, because of lower than 2μu/ml.According to these statistical results no significant difference was found between 16-weeks high-carbohydrate diet group ).Emergence of these enzymes with elevated levels in high-carbohydrate diet shows that carbohydrates lead to liver damage (Table 3).

Histological Evaluation
Liver sections of rats in all groups were stained with  hematoxylin-eosin, Gomori trichrome.When the sections examined, fibrosis in portal area (Figure 2c, 2d), steatosis in hepatocytes (Figure 4c, 4d), and lobular inflammation in the portal area (Figure 2a, 2b, 4a, 4b) were observed in all groups.Hepatocyte nuclei and sinusoids observed in the normal structure, perisinusoidal fibrosis wasn't observed.Sections were stained with α-SMA primary antibodies to evaluate the hepatic fibrosis as immunohistochemically and were stained with TGF-β primary antibodies to determine hepatocyte damage.α-SMA was observed on the walls of blood vessels (Figure 3a, 3c, 5a, 5c).Especially, TGF-β was intensively observed around of vena centralis in hepatocytes (Figure 3b, 3d, 5b, 5d).Fibrosis, steatosis, and inflammation were scored for the statistical analysis.Rating: The rating has been done as unavailable /not exist:0, minimal: ±, focal: +, slightly:++, moderately +++, intensive (heavy) degree ++++ and 0:0, ±:5 and each + have been scored as 10 (7).Mann-Whitney-U test was statistically used for the assessment of significant differences between the groups.16-weeks high-carbohydrate diet group was compared with 16-weeks high-fat diet group;

DISCUSSION
In this study, we aimed to investigate the role of a highfat diet on weight gain and effects on liver.Therefore, 16-weeks old and 20-weeks old rat groups were arranged for comparison with high-fat diet including %60 fat (1/3canola, 1/3 margarine and 1/3 sunflower oil) and standard rat diet including high-carbohydrate.At the end of the experiment body weight, liver weight and epididymal fat weight were measured.Glucose, albumin, insulin, triglycerides, total cholesterol, HDL, LDL, VLDL, LDH, ALT, and AST levels were examined in cardiac blood.Liver tissues were examined histologically.
Although there was no significant difference between the groups compared with body weight, liver weight and epididymal fat weight, LDH in 16-weeks high carbohydrate group and ALT in 20-weeks high-carbohydrate group was found significantly higher.
In spite of detecting fibrosis, inflammation, steatosis findings in portal area histological examination of all groups, no significant differences were detected statistically.α-SMA and TGF-β were similar in immunohistochemical examination.
However, Zeng-Jie Xu et al.(8), noticed that the high fat diet (HFD) fed rats were observed as significantly increased in point of body weight, epididymal fat weight and liver index (Liver indeks(LI): liver weight / body weight X 100%) comparing to the control group rats.Although, serum ALT, serum free fatty acid (FFA) and serum TNF-α concentrations were higher in HFD-fed rats, there is no significant difference between the two groups in serum triglyceride (TG) levels.
In the livers of rats fed a normal diet, both macroscopic and microscopic abnormality weren't observed, while in rats fed with HFD diet, especially from 24-weeks to 48-weeks severe steatosis, from 16-weeks hepatic fibrosis findings were observed.Immunohistochemical examination of liver sections of rats fed with HFD from 12-weeks, the α-SMA and TGF-β increased expression were observed(8).
Omagari et al. ( 15) compared high fat diet (HFD) with low fat standard diet(LFD).HFD included 45% fat (6%of soybean oil and 39% of lard, kcal) and LFD included 10% fat (6%of soybean oil and 4% of lard, kcal).No significant differences were found between the groups in body weight, epididymal fat weight, serum insulin, glucose, total cholesterol, triglyceride, leptin, adiponectin, ALT and AST levels.According to NASH Clinical Research Network scoring system, no significant differeneces were found histopatological examination among the three groups in frequency of steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis.
The lack of a statistically significant difference between high-fat diet and the control diet in biochemical and histopathological examination, suggest that oleic acid which has been in high fat diet content of soybean oil (13) and lard (14) would have protective effect (15).In another study, it was found that soybean oil decrease lipogenesis and liver fat density (16).
In some studies about high -fat diet, corn oil was used as the content of the high-fat diet and the negative effects such as liver steatosis, inflammation were observed (17,18).Corn oil includes 24.1% oleic acid, 61.9% linoleic acid, 11% palmitic acid, 2% stearic acid and linolenic acid 0.07% (19) and linoleic acid (omega-6) has inflammatory, thrombotic, mitogenic and hyperalgesic effects (20).These negative effects may depend on amount of omega -6 fatty acids.
Omega-3 rich fish oil was found more protective in a study of lard and fish oil rich diet (21).

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ALT levels, insulin resistance and IL-1β levels were found higher in another study of high trans-fat diet (22).Parthasaraty et al. (24), fed rabbits either a newly developed variant sunflower oil, containing more than 80% oleic acid and only 8% linoleic acid, or conventional sunfloweroil, containing only 20% oleic acid and 67% linoleic acid.LDL isolated from the plasma of animals fed with the variant sunflower oil was highly enriched in oleic acid and very low in linoleic acid.These oleate-rich LDL particles were significantly resistant to oxidative modification.These results show that diets sufficiently enriched in oleic acid, in addition to their LDL-lowering effect, may slow the progression of atherosclerosis by generating LDL that is highly resistant to oxidative modification (23).
Berry et al. (25), conducted a study which was randomly assigned to 24-weeks crossover study of monounsaturated fatty acid (MUFA) vs polyunsaturated fatty acid (PUFA) diets (50% carbohydrate, 32% fat, 18% protein) fed alternately during two 12-week periods.Total plasma cholesterol (TC) decreased significantly by 10% and 16% on the MUFA and PUFA diets, respectively.Lowdensity-lipoprotein cholesterol (LDL-C) decreased with an additional significant effect between periods in both groups.
Concentrations of high density lipoprotein cholesterol did not change significantly.There was significantly higher tendency toward lipid peroxidation on the PUFA diet, and lower to oxidative stress of LDL susceptibility on the MUFA diet (24).Riveros et al. (26), suggested that high-oleic acid provided higher protection against lipid oxidation (25).Nakbi et al. (27) concluded that protective effect of oleic acid against oxidative damage (26).These findings clarifies that how a high-fat diet protects the liver in our study.
Gomez-Lechona et al. (28), designed a study to define an experimental model of hepatocellular steatosis, and found that a high proportion of palmitic acid (oleate/ palmitate, 0:3 ratio) might represent a cellular model of steatosis (27).
In another study, Ricchi et al. (29) found that palmitic acid impaired insulin signalling.Despite the higher amount of fat resulting from incubation of the two fatty acids combined, the apoptosis rate and impaired insulin signalling were found lower than in cells treated with palmitic acid alone, indicating a protective effect of oleic acid (28).
Hussein et al. (30) aimed to evaluate the effects of different types of dietary fats on the hepatic lipid content and oxidative stress parameters in rat liver with experimental non-alcholic fatty liver disease.A total of 32 rats were divided into five groups.The rats in the control group were on chow diet, rats on methionine cholinedeficient diet (MCDD), rats on MCDD enriched with olive oil, rats on MCDD with fish oil, rats on MCDD with butter fat.According to this study, olive oil decreases the accumulation of triglyceride in the liver of rats (29).
As mentioned in literature, depending on reduce to oxidative stress on LDL, Omega-9 decrease tissue damage.Only Ricchi et al. (29) found that oleic acid is protective against apoptosis, but more steatogenic than palmitic acid.One of the reason we couldn't find any significant differences between control group and high fat diet group, would be protective properties of omega-9 fatty acid.
Romestaing et al. (31), fed 21 day-old rats for 14-weeks, with either coconut oil or butter, and they were compared with rats fed with a standard diet or a methionine choline-deficient (MCD) diet, a nonphysiological model of nonalcoholic steatohepatitis (NASH).Long term high saturated fat feeding led to increased "peripheral" fat storage and brown adipose tissue thermogenesis but did not induce hepatic steatosis and NASH (30).The reason of this, compared to the standard diet rich in coconut oil and butter diet, would be having higher rate of oleic acid.Some studies have stated that high-carbohydrate diet increases hepatic fatty acid synthesis (31), and on the contrary, some studies reported that a high-fat diet increases hepatic lipid level (8,9).Berke et al. (32) fed obese and non-obese rats either a high fat diet or high carbohydrate diet.The rats fed with high fat diet in first 6-weeks, afterwards some of them fed with high carbohydrate diet until 10-weeks.Carbohydrate feeding resulted in increased hepatic fatty acid synthesis regardless of the earlier feeding of the fat diet, but the obese rats were more responsive to the carbohydrate diet than the non-obese rats (31).This study support our findings in case of carbohydrate diet increases hepatic fatty acid synthesis.
We couldn't find any significant differences between high-carbohydrate diet and high-fat diet both in terms of weight gain and liver damage in this study.In the mentioned studies, while any histopatologic finding concerning steatohepatitis wasn't found in high-carbohydrate diet groups, we found steatosis, fibrosis, and inflamation findings involved in steatohepatis in both high-carbohydrate diet groups and high-fat diet group but couldn't find any statistically significant differences between these two groups.We found that only LDH levels in high-carbohydrate diet group at 16-weeks and ALT levels in high-carbohydrate diet group at 20-weeks was significantly higher in biochemical evaluation.The primary reason of this, a high-fat diet rich in omega-9 fatty acids and the relative protective effect on the liver also the second reason diet rich in carbohydrates have adverse effects on the liver.
High-carbohydrate diet leads to hyperglycemia, and hyperglycemia leads to fast insulin secretion, hyperinsulinemia, insulin and leptin resistance (2,3).As a result of this, fat deposition in the hepatocytes, the increase in lipid peroxidation, the emergence of free oxygen radicals, oxidative stress and mitochondrial dysfunction occur (2).Oboh et al. (33) found that high-carbohydrate, low-fat (HCLF) diet led to hypertriglyceridemia, as an indicator of hepatocellular injury, reduced serum protein and also increased AST, ALT, urea levels (32).
Lomba et al. (34) compared with high-sucrose (HS) diet and isocaloric control diet.HS diet increased adiposity, decreased plasma total cholesterol and HDL levels.Although there weren't any significant differences in terms of serum glucose, insulin, adiponectin, free fatty acids and liver malondialdehyde levels, slight increase was observed in serum and liver triglyceride levels.
This study has showed that LDH get higher in 16-weeks high-carbohydrate diet group, ALT get higher in 20-weeks high-carbohydrate diet group, carbohydrates cause to hepatocyte damage and relatively high-fat diet rich in oleic acid is protective.Also high-fat diet rich in omega-9 has protective effect on the liver and it does not cause weight gain compared with high-carbohydrate diet and high-carbohydrate diet leading more liver damage.As a result of this, in order to prevent weight gain and protect the liver, we suggest low carbohydrate diet rich in oleic acid.

Figure 3
Figure 3.a.α-SMA involvement is observed on the walls of blood vessels of 16-weeks old high-carbohydrate diet group (α -SMA x200).b.TGF-β is observed in hepatocytes especially around of vena centralis of 16-weeks old high-carbohydrate diet group (TGF-β x200).c. α -SMA is observed on the walls of blood vessels of 16-weeks old high-fat diet group (α -SMA x200).d.TGF-β is observed around of vena centralis as intensive in 16-weeks old high-fat diet group (TGF-β x200).

Figure 4 .
Figure 4. a. Fibrosis and inflammation is observed in portal area of 20-weeks old high-fat diet group (HEx100).b.Foci of inflammation is observed in 20-weeks old high-carbohydrate diet group (HEx200).c.Steatosis is observed in hepatocytes of 20-weeks old high-carbohydrate diet group (HEx400).d.Steatosis in hepatocydes and dilatation in sinusoids are observed on 20-weeks old high-fat diet group (HE x400).
20-weeks high-carbohydrate diet group was compared with 20-weeks high-fat diet group (Figure6); 16-weeks high-fat diet group was compared with 20-weeks highfat diet group and 16-weeks high-carbohydrate diet group was compared with 20-weeks high-carbohydrate diet group.No significant difference was found statistically in these comparisons (p> 0.05).

Table 1 .
Liver and epydidimal fat weight analysis of all groups (Mean±SD) and 16-weeks high-fat diet group in terms of glucose, albumin, triglycerides, cholesterol, LDL, HDL, ALT, and AST parameters.Only LDH levels were found significantly higher in 16-weeks high-carbohydrate diet group than 16-weeks high-fat diet group (p<0.05).No significant difference was found statistically between 20-weeks high-carbohydrate diet group and 20-weeks high-fat diet groups in terms of glucose, albumin, triglycerides, cholesterol, LDL, HDL, LDH, and AST parameters.Only ALT levels were found significantly higher in high-carbohydrate diet group (p<0.05).No significant difference was found between 16-weeks high carbohydrate group and 20-weeks high carbohydrate group in terms of LDH and ALT levels.Significant difference was found statistically between16-weeks high-carbohydrate diet group and 20-weeks high-carbohydrate group only in terms of glucose level (p=0.003).According to this, the mean glucose level was higher in 16-weeks high carbohydrate group.Independent 2-sample T-test was used for this comparison.Statistically

Table 3 .
Statistical analysis of all groups (Mean and standard deviation)Figure 2. a. Intralobuler inflammation is observed around of V. Centralis of 16-weeks old high-carbohydrate diet group (HE x200).b.Inflammation is observed in 16-weeks old high-fat diet group (HE x200).c.Fibrosis is observed in 16-weeks old high-carbohydrate diet group (Gomori-tricrom x200).d.Increase of collagen fiber is observed in portal area of 16-weeks old high-fat diet group (Gomori-tricrom x200).
** This project is supported by Düzce University Research Fund.(Project Number: BAP 2010.04.01.047) , *** The following study cited below has been approved by the Local Ethical Committee of Düzce Medical School.Crescenzo R, Bianco F, Falcone I, at al. Dietary fructoseinduced obesity and insulin resistance: is there a role for altered hepatic mitochondrial energetics.18th European Congress on Obesity Proceedings Book, pp:149.18th European Congress on Obesity, Istanbul, Turkey, 25-28 May 2011.40.Fuente-Martin E, Garcia-Caceres C, Diaz F, at al. Interaction between neonatal over-nutrition and a subsequent sucrose-enriched diet on adipose tissue acquisition.18th European Congress on Obesity Proceedings Book, pp:47.18th European Congress on Obesity, Istanbul, Turkey, 25-28 May 2011.