Effects Of GLP-1 On Diet Induced Non-alcoholic Fatty Liver Disease And PKCε、TLR4/NF-κB Signaling In Rats

Background:Nonalcoholic fatty liver disease (NAFLD) begins with the accumulation of triacylglycerol (TAG) in the liver and is defined as the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes or TAG levels exceeding the 95th percentile for lean, healthy individuals without significant alcohol consumption and negative viral and autoimmune liver disease.With the the improvement of people’s living standars, an increased incidence NAFLD was reported, therefore, dramatic increase in the prevalence of NAFLD is becoming a major global health problem. The pathogenesis of NAFLD is not well understood yet, the classical two-hit hypothesis (insulin resistance, oxidative stress) has been accepted by most of the researchers. NAFLD is strongly associated with metabolic syndrome (MetS), type 2 diabetes and obesity as these diseases share a common pathophysiology that includes insulin resistance and systemic inflammation. Expansion and inflammation of adipose tissue results in adipose insulin resistance and increased lipolysis and thereby in an elevated flux of free fatty acids into the liver, an impaired hepatic fatty acid oxidation and a decrease in proteins inducing lipid oxidation, e.g., adiponectin, results in further accumulation of fat with in the liver. This stage is often referred to as benign steatosis. The overwhelming of lipid oxidation capacity eventually occurs resulting in the generation of reactive oxidative species (ROS), gut-derived signals (e.g.),bacterial endotoxins, short-chain fatty acids, inflammatory cytokines, and an imbalanced release of adipokines that then may result in the advance of this condition toward more severe stages such as steatohepatitis, fibrosis, and cirrhosis.Protein kinase C epsilon (PKCs) is a kind of insulin resistance accosiated protein, which has a great effect on the development of NAFLD and hepatic insulin resistance. PKCs are a family of protein that express lowly in the liver, howerer, PKCs that is a subtype of PKC s is express high in the liver. Some studies have suggested that PKCs can be activated in NAFLD. With a performance of PKCs decreases bcause it has a transposition from cytoplasm to the cytomembrane. Some studies suggest that the accumulation of lipids and diacylglycerol (DAG) in the liver leads to activation of PKCs, resulting in hepatic insulin resistance.Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors (PRR) that play a key role in innate and adaptive immunity by recognizing structural components unique to bacteria, fungi and viruses. TLR4 is one of the family and is the most studied of the TLRs, and its primary exogenous ligand is lipopolysaccharide, a component of Gramnegative bacterial walls. In the absence of exogenous microbes, endogenous ligands including damage-associated molecular pattern molecules from damaged matrix and injured cells can also activate TLR4 signaling. NF-κB is a protein complex that is activated from a sequestered, cytoplasmic form normally retained in the cytoplasm by binding to iκB (the NF-κB inhibitor protein) via pro-inflammatory extracellular signals and cellular stress. However, after iκB degradation, initiated by a complex signaling cascade initiated at the cell surface (for example, TLR4 signaling), the active form of NF-κB translocates into the nucleus, where it activates transcription. NF-κB activity can be perturbed in a variety of nonparenchymal and parenchymal liver cells during hepatic inflammation, fibrosis, protection of hepatocytes from TNF-a-induced cell death, and compensatory proliferation of hepatocytes in response to loss of hepatic mass after liver injury.Intact TLR4 signaling was identified in the liver especially the TLR4/NF-κB signaling mediates key responses including an inflammation, fibrogenesis and cell apoptosis. Therefore it pays an important role in the development of NAFLD.According to the pathogenesis, the main therapeutic strategies of NAFLD are improve insulin insistence and oxidative stress. Howere, they are so complicated that there are no specific drugs yet.The main therapeutic strategies of NAFLD are lifestyle changing and diet structrue adjusting and strengthening doing exercises. Seeking effective drugs for NAFLD treatment has become hot spot in the field of the NAFLD researches.Glucagon-like peptide 1 (GLP-1) is a kind of enterogenous insulin which can stimulate the P cell of pancreas to serete insulin with a dependency on the concentration of gucose. Liraglutide is an analogue of GLP-1, which has 97% homologous sequences with GLP-1. It can regulate the seretion of insulin according to the concentration of glucose. When the glucose elevated, the insulin secretion incrased, on the contrary, insulin secretion declined. Howere, when the glucose is low enough, insulin secretion is no longer changing. Our previous research has show that the GLP-1 effected on the inflammatory factors of TNF α and TGF β and hepatic insulin resistance. And there is a close relationship between hepatic insulin resistance and PKCε, the same as between oxidative stress and TLR4/NF-κB. Therefore, we proposed hypothesis that whether the GLP-1 effected the NAFLD through activating the PKCs and TLR4/ NF-κB. And we designed the research that used the Liraglutide to intervent the NAFLD rats which was induced by high fat diet for 12 weeks, so that to evaluate the influence of the GLP-1 on the lipid metabolism,the liver function and hepatic insulin resistance, PKCε and TLR4/ NF-κB.Aim:In order to find out new basis for potential drug treatment of NAFLD, we designed the research that used the Liraglutide to intervent the NAFLD rats for 4 weeks which was induced by high fat diet for 12 weeks. And to evaluate the influence of the GLP-1 on lipid metabolism,the liver function and resistance, PKCε and TLR4/NF-κB. The triglycerid (TG), total cholesterol (TC), aspartate aminotransferase(AST,) alanine aminotransferase(ALT),insulin, glucose (GLU) in the serum will be detected,the TG,TC of the liver homogenate will be tested and the mRNAs of PKCε and TLR4 in the liver tissues, the proteins of PKCε, TLR4 and NF-kB in the liver will be tested.Method:establishing the rat modle of NAFLD and grouping 32 SPF male Sprague Dawley rats were bought from Sun Yat-sen University. The weight of rats was about 130 g. They were randomly devided into tow groups, the first group including 21 rats were fed with a high fat diet which was composed by 88% normal diet,10% ladr and 2% cholesterol. And the second group which was called normal control (NC) including 11 rats were fed with a normal diet. After 12 weeks,1 rat was randomly selected from each group, fasting for 12 hours, chloral hydrate was used to cause anesthesia.10 ml venous blood would be drawn from the abdominal aorta of rats. And the TG, TC,AST, ALT of serum were tested by fully automatic biochemical analyser. Then taking out of the liver of the rats and making pathological HE stainming to observe the fatty change of the liver tissues. After making sure that the NAFLD rat modle was successfully established, the model rats were randomly divided into 2 equal groups:one group (HFD+GLP-1)was treated with glucagon-like peptide 1 analog (daily dose of 0.6 mg/kg) by intraperitoneal injection for 4 weeks, the other group (HFD) using saline as the control.The SD rats could have water and diet freely during intervention, they were randomly divided into 6 cages,5 to 6 rats each cage. The temperature of the animal room was 20℃ to 25℃, and the lighting was set as light in the first 12 h and shade in the next 12 h,light and shade alternated. Besides, the hair changing, appetite,urine and activities,body weight e.g of the rats should be observed. And adrusting the drug dose according to the body weight of rats.At the end of the 16th week, we weighed the body weight of all rats after fasting for 12 h. Then all of the rats were cause anesthesia by 10% chloral hydrate. Then we drew the 10 ml venous blood and took out the liver tissues, some of which were frozen by liquid nitrogen, and some of which were soaked in the 10% neutral formalin for observing histological change with optical micrope.Liver index First, we we weighed the body weight (g) and the liver weight (g) of all rats. And the liver index=liver weight/body weight×100%.Blood processing and related indicators testing The blood stood at a room temperature for 30 mins, then they were centrifugaled for 15 mins with a rotate speed of 3500 r/min. And the upper layer was the serum. We moved them to the new EP tubes and made tags. At last, put all of them in the -80℃ refrigerator that were under tested. The TG, TC, AST, ALT of serum were tested by fully automatic biochemical analyser. And the glucose of the serum was detected with a glucose detection kit. The fating insulin (FIN) of serum was tested by an enzyme-linked immunosorbent (ELISA) kit. The insulin resistance index (HOMA-IR)= (FPG×FIN) 122.5 (FPG’s unit:mmol/L; FIN’s unit:uIU/mL)10% liver homogenate preparing and chemistric indicators testing Firstly, we weighed 100 mg liver tissues which were from the same area. Then we rinsed them some times by frozen saline,until the blood was washed up and put them into the 2ml EP tubes. And we added 1.9ml homogenate medium (anhydrous ethanol) into the tubes. At last, the tubes were centrifugaled for 5 mins with a rotate speed of 2000 r/min. All of the upper layers were moved to the new tubes and the tags should be made. The TG,TC of the 10% liver homogenate were tested with a GPO-PAP kit.Liver tissue Paraffin embedding and HE staining The liver tissues should be cut into the sizes of 0.5 cm×0.5 cm×0.5 cm. We put them into the 10% neutral formalin for 24 h, then used the paraffin to embed them. After HE staining, we observed histological change with optical micrope to evaluate the fatty degeneration. The detail assessment methods will be descried in the text.RT-PCR Total RNA isolating Firstly we grinded 50-100 mg liver tissues in the motar with liquid nitrogen, unti they turned into powder form. Then we transferred them to RNase-free EP tubes and added 1 ml trizol into the tubes, incubate in ice for 10 mins. Then added 0.2 ml chloroform and shade tubes vigorously by hand for 15 seconds. Incubated for 3 mins at room temperature. Then centrifuged the samples at 12,000×g for 15 mins at 4 ℃. Added 0.5 ml 100% isopropanol to the aqueous phase and incubated at room temperature for 5 mins. Centrifuged at at 12,000×g for 15 mins at 4 ℃. Removed the supernatant from the tubes, leaving only the RNA pellet. Then washed the RNA pellet with 1 ml 75% ethanol. Vortexed the sample brefly, then centrifuge the tubes at 7500xg for 8 mins at 4 ℃. Air dried the RNA pellet for 5-10 mins. Added 30-70 ml RNase-free water to dissolved the RNA pellet. After cDNA synthesis, the mRNAs of PKCε and TLR4 in the liver tissues would be detected by RT-PCR. GAPDH would be the internal control. Repeated 3 times each sample, including negative control without cDNA template and blank control with RNase-free water. We took the average value of CT which was a cycle number of the amplification products achieving the threshold value. We made a relative quantitative analysis the testing results with 2-△△Ct to evaluate the relative mRNAs of PKCs and TLR4 expression. △△Ct=(CtPKCε/ TLR4 - CtGAPDH) GLP-lgroup -(CtPKCε/TLR4 - CtGAPDH)NC group.Western blot Briefly,50μg of crude cytosol protein extracts were resolved by SDSPAGE using 8% gel and electroblotted onto polyvinylidene difluoride membrane using a wet-transfer cell (Bio-Rad). The membrane was then blocked for 2 h at room temperature in TBS-T containing 5% (w/v) nonfat dried milk, washed twice, and then incubated overnight with rabbit anti-peptide antibody against PKCe(Abcam) and TLR4(Santa Cruz) diluted 1:500 and 1:200 in rinsing solution. After further washings, membranes were incubated with horseradish peroxidase-conjugated IgG fraction of goat anti-rabbit IgG diluted 1:3000 in TBS-T for 1h. Exposured the membrane with ECL Detection Kit (Key GEN Bio TECH). Then analyzed the grey values with FlurChem 8900 software.Immunohistochemical detection The liver tissues were fixed in 10%neutral formaldenhyde for 24 h and embedded by paraffin. Pathological sections were waxed paraffin and washed with water. Then antigen should be repaired and endogenous peroxidase shoul be blocked. Then we added the NF-kB rabbit anti-peptide antibody (1:100) (Santa Cruz) to the sections, then added IgG fraction of goat anti-rabbit IgG (1:100), chromogenic reaction with DAB, redyed nucleus and dehydrated. At last, we observed the slices with optical micrope which were mounted. We analyzed the results as the way below: first, selected five high magnification views ramdonly each slice , which there were more than 500 cells each view. And calculated the positive expression cells, the positive rate = positive cells /total cellsx 100%.statistics The SPSS 13.0 software were used to analyze the datas. All values were represented as the mean±S.D (standard deviation), For multiple comparisons between groups, if the variances were neat, ANOVA was performed followed by LSD’s t test. On the contrary, Welch correction method was chosen followed by Dunnett’s T3. For ranked datas, Kruskal Wallis - H nonparametric test would be used. And significance was accepted at P < 0.05.Results:liver index the liver index of HFD group increased compared with NC group (30.96±3.46 versus23.02±1.52,p<0.05). In contrast, the liver index of HFD+GLP-1 group decreased compared with HFD group (27.53±3.23 versus 30.96±3.46,p<0.05).The indicators of serum the ALT,AST,TG,TC of HFD group increased compared with NC group((87.14±19.04 versus 67.00±17.64,176.14±30.04 versus 98.00±44.37, 1.94±0.76 versus 0.62±0.23, 2.44±0.25verms 1.30±0.12, 2.44±0.25verms 1.30±0.12,P<0.05 ) In contrast, the ALT,AST,TG,TC of HFD+GLP-1 group decreased compared with HFD group ( 59.00±15.82 versus 87.14±19.04, 124.25±24.52 versus 176.14±30.04, 1.35±0.54 versus 1.94±0.76, 2.05±0.23ver.ms 2.44±0.25^P<0.05) .The indicators of 10% liver homogenate the TG,TC of HFD group increased compared with NC group (2.89±0.19 versus 1.74±0.22 ,2.20±0.22 versusl.30±0A2,p<0.05). In contrast, the TG ,TC of HFD+GLP-1 group decreased compared with HFD group (1.35±0.54 versus 2.89±0.19, 1.17±0.16 versus 2.20±0.22,,p<0.05).HE staining Compared with NC group, the degree of liver steatosis in HFD group was significantly increased (P < 0.05), after GLP-1 interventions for 4 weeks, the liver steatosis turned back to normal (P < 0.05).Insulin and insulin resistance index (HOMA-IR) the insulin and the HOMA-IR of HFD group increased compared with NC group (24.02±2.11 versus 10.90±0.75 , 3.50±0.38 versus 1.59±0.10,p<0.05). In contrast, the insulin and the HOMA-IR of HFD+GLP-1 group decreased compared with HFD group (13.59±2.56 versus 24.02±2.11,2.12±0.18 versus3.50±0.38,p<0.05).The expression of PKCε The mRNA and cytoplasm protein of PKCεin HFD grou] decreased compared with NC group (p<0.05). In contrast, the mRNA and cytoplasn protein of PKCe in HFD+GLP-1 group increased compared with HFD grou] (p<0.05).The expression of TLR4 The mRNA and protein of TLR4 in HFD group increase compared with NC group (p<0.05). In contrast, the mRNA and protein TLR4 i(?) HFD+GLP-1 group decreased compared with HFD group (p<0.05).The expression of NF-kB The protein of NF-kB in HFD group increased compare(?) with NC group (p<0.05). In contrast, the protein NF-kB in HFD+GLP-1 grou] decreased compared with HFD group (p<0.05).Conclusion:this study suggests that a 12-week high fat diet can induce to establis(?) NAFLD rat modle, which has a metabolic disorder (hyperlipidemia), and abnorma liver function, ballooning pathological histology change in the liver. And it shared th(?) same pathological process with the NAFLD of humanbeing.In conclusion, the data presented in this manuscript support a causal role fo intracellular hepatic fat accumulation in the pathogenesis of hepatic insulin resistance GLP-1 shows good therapeutic effect on a rat model of nonalcoholic fatty live disease possibly by controlling lipid metabolism, lowering insulin resistance, whicl may be related to PKCe. Fat induced hepatic insulin resistance may result fron activation of PKCe. And GLP-1 can inhibit the activation of PKCε. At the same way Fat induced inflammation and the GLP-1 can inhibit the expression of TLR4 an(?) NF-κB which closely related to the inflammation.