EFFECT OF PIOGLITAZONE ON HEPATIC ULTRA- STRUCTURE AND METABOLIC CHANGES INDUCED BY A HIGH SUCROSE DIET IN RATS

Pioglitazone (Pio) is a PPARγ agonist insulin sensitizer has anti-inflammatory a ctivity. Our novel aspect was to investigate its hepatic anti-inflammatory ac tivity at the level of ultra-structure and enzymati c changes in a high sucrose diet animal model. Forty male Sprague Dawley rats were divided into four equ al groups: Control; control Pio; high sucrose diet; hi gh sucrose diet Pio treated groups. Fourteen weeks later, serum glucose, insulin, lipogram, gamma glutamyle t ransferase, alanine transaminase, aspartate transaminase, alkaline phosphatase, fetuin-A and ad iponectin levels were measured. Hepatic tissue homogenate levels of tumor necrosis factorα, interleukin-6 and myeloperoxidase activity were d etermined. Microscopic and ultra-structure hepatic changes wer e conducted in all animal groups. Administration of Pio in HS+Pio group reduced significantly the hepatic i nflammatory markers and the hepatic enzymes compared with HS group. Both light and electron mic ros opic examination revealed a great improvement o f the hepatic tissue with Pio treatment. This study s uggested that Pio treatment in obesity; in addition insulin sensitizing activity; could protect the liv er from the development of hepatic inflammation ind uced by a high sucrose diet not only at the enzymatic bu t also at ultra-structure levels.


INTRODUCTION
High caloric intake has been increasing steadily in different societies carrying the risk for the development of obesity, diabetes, hepatic and cardiovascular complications (Nielsen and Popkin, 2004). Sugarsweetened beverages contain up to 15% sucrose promotes obesity and Insulin Resistance (IR) development (Collino et al., 2010). Although the mechanism of IR development not yet exactly known, it has been established that chronic inflammation in key metabolic tissues, including the liver due to fat accumulation is an important pathogenic factor in IR development (Bashir et al., 2014). Hepatic and systemic inflammatory cytokines produced in obesity activate intracellular kinases which capable to inhibit key elements of insulin signaling pathway with subsequent development of insulin resistance (Al-Musa and AL-Hashem, 2014). Tumor Necrosis Factor-α (TNF-α) and Interleukin-6 (IL-6) among several hepatic cytokines are playing an essential role in hepatic inflammatory conditions. Also, Fetuin-A is a recent discovered hepatokines produced exclusively by the liver in chronic inflammatory conditions. It has been reported that fetuin-A binds with the insulin receptor tyrosine kinase causing its inhibition in the hepatocytes with subsequent suppression of the insulin signal transduction resulting in insulin resistance (Rauth et al., 1992). A reduction in the serum Science Publications AJAS adiponectin level in obesity decreases the insulin sensitivity which leads to aggravation of the adverse effects of inflammatory mediators (Ismail et al., 2014). Adiponectin receptors type 2 are primarily expressed in hepatocytes (Lee et al., 2008) and their down expression in obesity induce liver enzymes over activity involved in the gluconeogenesis and the inflammatory hepatic processes result in inhibition of insulin sensitivity and decreases glucose uptake leads to more severe hepatic inflammatory reaction (Kadowaki et al., 2008).
Several trials have been preformed to alleviate the insulin resistance and the inflammatory hepatic reaction developed in obesity. Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-dependent transcription factors regulate target gene expression by binding to a specific peroxisome proliferator response elements in enhancer sites of regulated genes (Michalik et al., 2006). Moller and Berger (2003) reported that, PPAR-γ agonists beyond their insulin-sensitizing efficacy; has antiinflammatory properties. Pioglitazone is a PPAR-γ agonist belongs to the Thiazolidinedione (TZD) class of antidiabetic insulin sensitizers. It is able to manage obesity induced insulin resistance (Gerstein et al., 2006). However, to date, few studies carried out on the anti-inflammatory hepatic activity of pioglitazone in addition to its insulin sensitizing effect in obesity and metabolic syndrome.
High sucrose diet induces hepatic fat accumulation, with subsequent insulin resistance and chronic hepatic inflammation leading to overproduction of hepatic inflammatory markers. In addition, the reduction of adiponectin level in obesity aggravates hepatic metabolic disturbances and inflammation. A vicious circle could develop between the IR and the chronic hepatic inflammatory process leading to more and more hepatic inflammatory reaction and insulin resistance. Pioglitazone as PPAR-γ agonist has anti-inflammatory activity in addition to its insulin sensitizing effect reduces the hepatic inflammatory markers and increases adiponectin level. From the previous data, we could expect the potential protective effect of PPAR-γ agonist pioglitazone on the developing hepatic inflammatory process associated with high caloric intake by high sucrose diet.
The present study was undertaken to determine the effect of the long term administration of the selective PPAR-γ agonist pioglitazone on hepatic inflammatory reaction induced by a high sucrose diet in rats. We also try to uncover the different anti-inflammatory mechanisms of Pio and its relation to adiponectin and fetuin-A levels in the hepatic inflammatory cascade in such animal model. The novel point in our research is to investigate the hepatic ultra-structure changes occur in the experimental and in the Pio treated animals.

Experimental Animals
All animals received human care in compliance with the Public Health Service Policy on Human Care and Use of Laboratory Animals, published by the National Institutes of Health and were approved by the Ethical Committee of College of Medicine, King Khalid University, Saudi Arabia. This study followed a randomized-controlled animal experiment design. Forty male Sprague-Dawley rats were obtained from animal house of the physiology department, College of Medicine, King Khalid University, Saudi Arabia, weighed between 150-200 gm. Animals were fed with a standard chow diet, water, ad libitum and they were housed in the animal house of College of Medicine with a 12:12-h light/dark cycle. The rats were randomly divided into four groups (n = 10) as follows: Control rats (C) which were given 1 ml distilled water daily through oral gavage; Control Pio treated rats (C+Pio); which were the animals treated with Pio (Sigma-Aldrich) in a daily dose of 10 mg kg −1 dissolved in one ml volume of 0.5% sodium carboxy-methylcellulose by oral gavage (Koufany et al., 2008); A high sucrose diet group (HS) which were given daily 3mL of a thirty-percent sucrose solution by oral gavage for 14 weeks (Lombardo et al., 1996;Muñoz Cano et al., 2013). A high sucrose diet pioglitazone treated group (HS+Pio) which were given a high sucrose solution as HS group and were treated with Pio as C+Pio group. The body weight for each rat was determined at the start and at the end of the experimental protocol (14 weeks). The percent changes of the body weight were determined.

Blood Sampling and Biochemical Measurements
At the end of the experimental protocol and after overnight fasting, retro-orbital blood samples were obtained through non-heparinized capillary tubes. The samples were allowed to clot for 20 min in a 37°C water bath centrifuged at 14,000 rpm for 10 min for serum separation and were used for different determination.

Determination of Serum Glucose, Total
Cholesterol, Triglyceride, HDL-C and LDL-C The serum glucose was determined using the glucose oxidase method. The lipid profile estimation was performed Science Publications AJAS using commercially available kits for the total cholesterol, triglycerides and HDL-C (BioMerieux, France) whereas the LDL-C was mathematically calculated. The serum albumin, GGT, ALT, AST and ALP were determined by an enzymatic colorimetric method using Randox reagent kits (Sigma-Aldrich).

Determination of Fasting Serum Insulin Level
Determination of serum insulin was performed using an ELISA kit at wavelength 450 NM. (BioSource Europe, Nivelles, Belgium) according to the manufacturer's instruction (Matthews et al., 1985).

Calculation of HOMA Index
The euglycemic hyperinsulinemic clamp is the standard method for measuring insulin resistance. We used a common method using the homeostasis model for HOMA of IR follows the equation: HOMA = fasting glucose (mmol L −1 )×fasting insulin (mU L −1 )/22.5. Typically, a HOMA value>2 is used to identify significant IR (Matthews et al., 1985).

Determination of Serum Adiponectin Level
Serum adiponectin determination was performed using the rat adiponectin ELISA kit (B-Bridge international, Inc.), according to the manufacturer's instructions (Kubota et al., 2002).

Preparation of Hepatic Tissue Homogenate and Histopathological Hepatic Preparation
After obtaining blood samples, all rats were sacrificed using the lethal dose of thiopental sodium by IP injection. The rat's abdomens were opened and the rat's whole livers were dissected, excided and weighed individually. Then, part of the rat's right liver lobes for each animal was cut and fixed in 10% formalin then was embedded in paraffin blocks and stained with hematoxylin and eosin for light microscopic examination. The other parts of the right liver lobes were cut into 2-3 mm 3 and immediately fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.2 at 4o C for 2-3 h. Samples were post-fixed in 1% osmium tetroxide, dehydrated in an ascending series of ethyl alcohol and embedded in Spurr's resin. Semi-thin sections (0.5 µm) were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Jeol JEM-1011 Transmission Electron Microscope (TEM) at 80 KV to observe the hepatic ultra-structure changes by electron microscope. Electron microscope was used to assess the degree of hepatic damage. While the rat's left liver lobes for each rat was homogenized using an Omni tissue homogenizer (Omni international, Gainesville, VA, USA) in ice-cold buffer [0.1 M phosphate, pH 7.4, 1 mM EDTA, 10 µM indomethacin (Cayma Chemical, Ann Arbor, MI, USA)] using a tube pestle. Acetone was added (2×sample volume) and samples were centrifuged at 1500×g for 10 min at 4°C. The supernatants were then stored at -80°C for determination of hepatic tissue homogenate levels of TNF-α and IL-6 and Myeloperoxidase activity (MPO). At the end of the experiment, the visceral, epididymal and retroperitoneal fats were collected and weighed for each rat individually to ensure the occurrence of general obesity.

Liver Homogenate TNF-α and IL-6 assay
Hepatic TNF-α and IL-6 levels were determined with a double antibody sandwich ABC-ELISA using a rat TNF-α and IL-6 kits according to the manufacturer's instructions (Shanghai Senxiong Technology industry Co. Ltd, China). The samples were compared with the standard curve and expressed as pg/mL (Xu et al., 2008).

Liver Homogenate MPO Activity Assay
The myeloperoxidase activity in hepatic tissue was determined by the spectrophotometric method. This method uses 3, 3', 5, 5'-Tetramethyl Benzidine (TMB) as an oxidizable dye and the reaction was started by adding Hydrogen Peroxide (H 2 O 2 ) in the medium (Xu et al., 2008). The assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Statistical Analysis
The data were expressed as mean ± Standard Deviation (SD). The data were processed and analyzed using the SPSS version 10.0 (SPSS, Inc., Chicago, Ill., USA). One-way ANOVA was done followed by Tukey's post hoc test. Pearson correlation statistical analysis was done for detection of a Science Publications AJAS probable significance between two parameters. The results were considered significant if p≤0.05.

RESULTS
Pioglitazone treatment in normal rats in the C+Pio group did not produce any significant changes in all measured parameters and the light and the electron microscopic histopathological examination compared with the C group except the body weight which increased significantly by Pio treatment compared with the control group (Table 1and 2).

Body Weight, Liver Weight, Liver/Body Weight Ratio, Visceral, Epididymal And Retroperitoneal Fats
High sucrose diet in rats resulted in significant increased in the total body weight, liver weight, the liver/total body weight ratio, visceral, epididymal and retroperitoneal fat (

Serum Fetuin-A and Adiponectin levels
The serum fetuin-A level increased significantly whereas the serum adiponectin level decreased significantly in the HS group (251.2±23.43, 14.51±1.33 respectively) compared with the control group (132.9±12.08, 23.92±2.18 respectively). Pio treatment in the HS+Pio group produced a significant reduction in the serum fetuin-A (172.8±16.94) with a significant elevation of serum adiponectin (22.16±2.15) levels compared with the HS group ( Fig. 2a and b).

Serum GGT, ALT, AST, ALP and Albumin
High sucrose diet in rats resulted in a significant elevation of hepatic serum enzymes GGT, ALT, AST and ALP levels (  Fig. 1 and 3c).

Correlation of The Serum Fetuin-A With: Serum Adiponectin And Serum GGT
There is a significant negative correlation between serum fetuin-A and adiponectin levels (r = -0.8836) (p<0.0001) for the all groups (Fig. 2a). There is a significant positive correlation between the serum fetuin-A and the serum GGT levels (r = 0.7062) (p<0.0001) for the all groups (Fig. 4).

Hepatic Tissue Homogenate Levels of TNFα, IL-6 and MPO Activity
High sucrose diet in rats resulted in a significant elevation of hepatic tissue inflammatory markers TNF-α and IL-6 levels and MPO activity (4.38±0.41, 25.24±2.57, 0.43±0.06 respectively) compared with the control group (1.94±0.18, 12.72±1.26, 0.23±0.04 respectively). Treatment with PPAR-γ agonist Pio produced a significant reduction in hepatic homogenate levels of TNF-α and IL-6 levels and MPO activity (3.15±0.31, 19.21±1.53, 0.32±0.05 respectively) compared with the HS group (Fig. 2b, c and d). Results are expressed as means ± SD (n = 10). Significance was considered when P value was <0.05. † Significantly different from control (C) group, ‡ Significantly different from control groups (C and C+Pio), § Significantly different from HS group

Histopathological Studying of Liver
The light microscopic examination revealed that a high sucrose diet in HS group resulted in inflammatory cellular infiltration of liver tissue with dilatation of the central vein and discontinuity of the endothelial lining. The blood sinusoids dilated and filled with excessive bleeding and fat deposition. There was an infiltration of dark inflammatory cells with shrunken and pyknotic nuclei (Fig. 5c). Treatment with pioglitazone in HS+Pio resulted in a partial preservation of the hepatic cellular structures with less fat infiltration, while the other hepatocytes appeared with normal morphology and intact nuclei. The hepatic sinusoids showed less bleeding with discontinuity of some its epithelial lining compared with HS group (Fig. 5d). The electron microscopic examination showed that a high sucrose diet in rats resulted in swollen-degenerated hepatocytes with cell membrane discontinuity and damage blood sinusoid and excessive bleeding, disrupted endoplasmic reticulum, abnormal shaped mitochondria, excessive lipid droplets and collagen fibers with swollen nuclei (Fig. 6c).

AJAS
Whereas Pio treatment in HS+Pio rats improved the hepatocytes and the cell membrane structure with less fat vacuolation and collagen fibers and normal histological appearance of both endoplasmic reticulum and mitochondria with a healthy nucleus (Fig. 6d).

DISCUSSION
Several studies documented that diet composition is indicted in the development of obesity and insulin resistance (Bessesen, 2001). Fourteen weeks of high sucrose diet in rats resulted in a significant increase in body weight accompanied with hyperglycemia, dyslipidemia and hyper-insulinemia with insulin resistance development. This induced hepatic inflammatory reaction revealed by a significant elevation of the hepatic inflammation markers TNF-α, IL-6, fetuin-A levels and MPO activity reflecting the number of polymorphonuclear neutrophils in the liver. Hepatic inflammation deteriorated the liver function proved by an elevation of serum GGT, ALT, AST and ALP levels together with a reduction of serum albumin significantly compare with control groups. The light microscopic study showed hepatocellular infiltration with inflammatory cells and excessive fat vacuolation with pyknotic nuclei. The central veins dilated and the endothelial lining of the hepatic sinusoids showed discontinuity with excessive bleeding. All these finding confirmed the biochemical and metabolic disturbances induced by a high sucrose diet in rats. The electron microscopic examination of the liver tissue of the HS rats revealed degeneration of the hepatocytes with an abnormal cell membrane, excessive vacuolar degeneration and collagen fibers with swollen nucleus and damage blood sinusoid. The cellular organelles showed disrupted endoplasmic reticulum and swollen mitochondria with cristae destruction. Our results are in agreement with several studies (Yoshimoto et al., 1997;Yang et al., 2005). Few researches concerned with the hepatic anti-inflammatory effect of PPAR-γ agonist pioglitazone at the level of ultra-structure changes and its relation to improvement of insulin sensitivity, fetuin-A and adiponectin levels in high sucrose diet. The aim of this study was to investigate the hepatic antiinflammatory effect of the insulin sensitizer pioglitazone on the hepatic, ultra-structure and metabolic changes induced by a high sucrose diet in rats. In addition, we addressed the mechanisms by which pioglitazone mediates its anti-inflammatory action.
Pioglitazone is a specific potent PPAR-γ ligand having substantial anti-inflammatory accompanied its insulin sensitizing activities. Moller and Berger (2003) documented the presence of a higher level of PPAR-γ expression in the liver of obese rats and its activation in addition to the insulin sensitizing effect has selected antiinflammatory activity. The mechanisms of the antiinflammatory and insulin-sensitizing activities of PPARγ ligands are complex. It has been reported that PPAR-γ activation by pioglitazone induces expression of fatty acid transporters involved in fatty acid catabolism such as lipoprotein lipase, fatty acid acetyl-CoA synthase, oxidase and carnitine palmitoyl-transferase I (Berger and Moller, 2002). Activation of PPAR-γ in adipocytes induces the expression of many PPRE-containing genes involved in lipid metabolism. This could lead to a gradual increase in liver insulin sensitivity (Gurnell, 2006). Improvement of lipid metabolism by PPAR-γ reduced the circulating level of FFAs and a more gradual reduction in liver steatosis (Guan et al., 2002). This proved in the present study by the significant improvement of the dyslipidemia, the elevated liver enzymes and the serum albumin in the Pio treated group.
An important contributor to the insulin-sensitizing efficacy of PPAR-γ agonists is the suppression of local and systemic cytokine production (Moller and Berger 2003). It has been found that treatment of the obese patients with PPAR-γ agonists reduces circulating levels of inflammatory cytokines and other proinflammatory markers leading to improve insulin sensitivity. Samaha et al. (2006) reported that binding of PPAR-γ to co-activators reduces the levels of coactivators available for binding to pro-inflammatory transcription factors such as NF-κB. This causes a decrease in transcription of a number of pro inflammatory genes including various interleukins and tumor necrosis factors. This was confirmed in the present study by the significant reduction in hepatic inflammatory markers TNF-α, IL-6, fetuin-A levels and MPO activity in the Pio treated group (HS+Pio group). Importantly, it has been reported that, PPAR-γ agonists can potently suppress TNF-α production and its action in adipocytes. This could result in ablation of TNFalpha's ability to inhibit insulin signaling and to down regulate the glucose transporter expression leads to inhibition of TNF-α induced insulin resistance and other hepatic adverse effects (Moller, 2000). The light microscopic histopathological examination revealed an improvement of hepatocytes, structure and their cell membrane with less dilation of the central vein. The hepatic sinusoids showed less bleeding with regular endothelial lining. While electron microscopic transmission study in the Pio treated group showed continuity of the hepatic cell membrane and preseved Science Publications AJAS cellular structure with less fat vacuolation and collagen fibers. The hepatocellular organelles revealed a normal histological appearance of the endoplasmic reticulum and the mitochondria with regular cristae and a normal nuclear histological appearance.
Recent research has shown that PPAR-γ agonists increase plasma level of adiponectin in obese animal models and type II diabetic patients (Kadowaki et al., 2006). Our study showed a significant increase in the adiponectin level in high sucrose diet rats treated with Pio. Induction of adiponectin secretion by PPAR-γ agonists might have a key role in the mechanism by which Pio treatment ameliorates the hepatic inflammatory processes induced by high sucrose diet. Recent evidences suggested that the normal level of adiponectin inhibits the progression of hepatic steatosis to fibrosis through an AMPK-dependent pathway (Ix and Sharma, 2010).
Different other mechanisms of the anti-inflammatory activity of Pio treatment have been reported. Pioglitazone has the ability to attenuate the diet induced the expression of the inflammatory enzymes iNOS and COX-2 whose role in the development of insulin resistance has been recently documented in obese rats and in cultured hepatocytes (Fujimoto et al., 2005;Hsieh et al., 2009). Other studies suggested that PPARγ activation suppresses the monocyte-macrophage migration, the expression of the adhesion molecule ICAM-1 (Moore et al., 2001), the inflammatory action of eicosanoid leukotriene B4 (Devchand et al., 1996), the induction of apoptosis in cytokine-activated macrophages and NF-kB signaling (Delerive et al., 2001). It has been reported that PPAR-γ agonists reduce adipose 11b-HSD-1 level, "the enzyme regulates the conversion of cortisone to cortisol". Since hypercorticosteroidism is a well-known cause of insulin resistance with subsequent inflammatory cascades in obese patients (Masuzaki et al., 2001).The limitation of our study was we didn't investigate the hepatic enzymes homogenate levels, pancreatic tissue ultra-structure changes and insulin gene expression that could highlighting on the hepato-protective mechanism of Pio.

CONCLUSION
The present study suggests that pioglitazone as a member of PPAR-γ agonist insulin sensitizer has a hepatic anti-inflammatory activities through different mechanisms. It has the ability to reduce the serum lipids, hepatic fat accumulation, cytokines overproduction and insulin resistance resulting in amelioration of hepatic inflammatory reaction induced by high sucrose diet. Adiponectin may play a major hepatic anti-inflammatory role in such condition treated with Pio. The improvement by the pioglitazone treatment was not only at the level of biochemical changes, but also at the histopathological and ultrastructures changes. Further studies should be carried out on hepato-protective role of Pio treatment in order to uncover the exact mechanism of action. Also, investigation of Pio with different doses and therapy regimens in other animal tissues for ultra-structure changes is required to ensure its efficacy and safety.

ACKNOWLEGEMENT
We kindly appreciate all staff members and technicians of the Physiology and Biochemistry Labs, College of Medicine, King Khalid University, Saudi Arabia and College of Medicine, Menoufiya University, Egypt; for their great help in every step in this study. Special thanks to Dr. Mohamed Soliman for his kind help in the biochemical investigation and to Mrs. Asmaa E Hassan for her great effort during the preparation of the statistics for this manuscript.

CONFILICT INTEREST
The researchers declare that they have no competing interests.