Insulin Resistance And Metabolism Of Liver Fatty Acids In High-fat Diet Rat

Western-like-diet and lifestyle, especially excessive lipid intake, is accused for epidemic of obesity and insulin resistance, as well as the rapid increase of type 2 diabetes. Diabetes can be classified into two types, type 1 and 2. The latter accounts for more than 90% of diabetes. Insulin resistance, the decreased response to insulin, is a key feature of type 2 diabetes. Although how insulin resistance occurs remains unclear,the focus has been concentrated on the elevation of free fatty acid(FFA), such as palmitic acid (C16:0), stearic acid (C18:0), docosanoic acid (C22:0) and isoselachoceric acid (C24:0), and the accumulation of triglyceride in liver. Intralipid infused in vein, which can elevate the level of circulation FFA, results in insulin resistance, proving that FFA may be an independent risk factor in the developement of insulin resistance. Then, whether FFA is one of the key factors in the insulin resistance induced by high-fat diet.The liver is a vital organ in fatty acid metabolism and insulin resistance. The variations of fatty acids metabolism in liver have a profound effect on the levels of serum fatty acids and affect the fatty acid metabolism in other organizations. FFA is a natural ligand of peroxisome proliferator-activated receptorα(PPARα). In normal physiological conditions, the increased FFA in the blood activates PPARα, which enhances the expression of proteins involved in the intake and oxidation of fatty acids, and therefore accelerate the intake andβ- oxidation of fatty acids in mitochondria and peroxisome in liver. At the same time, the excessive FFA is esterified to fat in liver, which is catalyzed by diacylglycerol acyltransferases (DGAT), and then diverted to other organs by very low density lipoprotein (VLDL). In insulin resistance and diabetes, whether the elevated levels of total FFA and some fatty acids in liver are caused by the abnormal lipid metabolism and abatedβ-oxidation and whether the fat deposition in liver is related to enhanced expression of DGAT that is a limit enzyme in fat synthesis, have not been reported.Short, medial and long chain fatty acids can be catabolized in mitochondria byβ-oxidation, tricarboxylic cycle and oxidative phosphorylation to produce ATP and CO2. During this process, oxygen free radicals are produced in respiratory chain level. Very long chain fatty acids must be handled in peroxisomal byβ-oxidation. In this condition, Acyl-CoA oxidases act as a rate-limiting enzyme, which donates electrons directly to molecular oxygen and generates H2O2. Therefore, accumulation of very long chain fatty acids in cells can not only cause changes of fatty acids types contained in phospholipid of biomembrane, but also generate a lot of reactive oxygen species that damage the cell structure and function. Is the lipotoxicity to liver in insulin resistance and diabetes, which were induced by high-fat diet, caused by accumulation of fatty acids especially the very long chain fatty acids?In order to study these issues, we developed insulin resistance in Sprague-Dawley rat by high-fat diet, and further formation of diabetes, and primary culture hepatocytes in high level fatty acids environment. To explore the relation between peroximalβ-oxidation and the variance of quantity and kinds of FFA in liver of insulin resistance and type 2 diabetes states induced by high-fat diet, the changes in quantity and kinds of fatty acids of blood, liver and primary culture hepatocytes were detected, and the changes of fatty acids outlet(triglycerides synthesis and fatty acid oxidation) and the activities of differentβ-oxidation ways were also detected from integeral and cell level.PartⅠHigh fat diet and occurrence of insulin resistanceObjective: By observing the dynamic changes in blood biochemical parameters, the relationship between FFA and insulin resistance developement was explored in high-fat diet rat.Methods: Male SD rats were divided randomly into control group (Con group) and high fat group (HF group). The rats in Con group were fed with standard diet and HF group, high-fat diet. lipid in the high-fat diet consisted mainly of lard-based, accounting for 59.8 percent of calory. After feeding for 2, 4, 6 and 8 weeks in high-fat diet, the rats were detected for insulin resistance by oral glucose tolerance (OGTT), insulin sensitivity index (ISI) and euglycaemic-hyperinsulinaemic clamp. Blood sugar was test with rapid blood glucose detector. Blood triglycerides and cholesterol were measured by oxidase method and blood insulin concentration was detected by RIA, and serum total FFA concentration was detected with detection kit. After 8 weeks, some HF rats developed to dibetes by streptozotocin(DM group). All rats were fed continuely for 6 weeks, and sacrificed, and the blood fatty acids profiles were evaluated by gas chromatography.Results:1 Biochemical parameters in the bloodAt 4th week, the body weights of HF group exceeded that of Con group(HF. 286.0±16.0g,Con.250.2±15.1g,P<0.01), and so did the blood triglycerides levels (HF. 1.14±0.14 mmol/L,Con. 0.92±0.17 mmol/L,P<0.01) and the FFA levels (HF. 323.52±35.83μmol/L,Con. 272.65±50.21μmol/L,P<0.05). However, fasting blood sugar, cholesterol and blood insulin concentration showed no significant difference (P>0.05) between the two groups at this time point. At 6th week, blood cholesterol (2.05±0.29 mmol/L) and insulin (28.43±14.5μIU/ml) concentrations of HF group were are higher than those of Con group (cholesterol, 1.69±0.14 mmol/L and insulin, 15.28±3.97μIU/ml), there were statistical differences between two groups(P<0.01 and P<0.01). But there was no statistical difference in blood sugar levels between two groups. At 8th week, fasting blood sugar(5.85±0.10 mmol/L) of HF group was obviously higher than that of Ctrl group(5.32±0.39 mmol/L), and the difference was statistically significan(tP<0.05). Total blood FFA concentration in HF group (406.69±70.47μmol/L) was higher than that of Con group(299.52±59.17μmol/L) (P<0.01). Along with the duration of high-fat feeding increases, the differences of blood glucose, triglycerides, cholesterol, FFA and insulin concentration between the two groups were increasing. 2 insulin sensitivity index (ISI)2 weeks after high fat diet, ISIs in HF group and Con group were -4.53±0.19 and -4.41±0.28, respectively. there was no statistical difference(P>0.05)between the two groups. At the time points of 4,6,8 weeks, ISIs of HF and Ctrl group were -4.71±0.21 and -4.47±0.25, -4.95±0.52 and -4.45±0.30,-5.31±0.26 and -4.60±0.14, respectively. There were statistical differences(P<0.05, P<0.01 and P<0.01)between the two groups. 4 weeks after the rats were fed high fat diet, the insulin sensitivity in HF group rats began to decease, which progressed time dependently.3 Oral glucose tolerance test (OGTT) After fasting for 12 h, rats were gavaged with 50% glucose (2 g / kg), and then the blood glucose levels were tested. At the time point of 2 weeks, there was no difference at each time point in OGTT. At 4 weeks, 30 min and 60 min blood sugars were higher in HF group than that of Ctrl. group, there were statistical differences. In the rats fed in high-fat for 6 weeks, the blood sugar at 30 min, 60 min and 120 min time points were higer than that of Ctrl group, but at 0 min, the difference has no statistical significance.4 Result of euglycaemic hyperinsulinaemic clampAt the time point of 6 weeks, glucose infusion rate(GIR) of HF group was 20.44±1.99mg/kg.min ,which was lower than that of Ctrl. group(27.97±1.72 mg/kg.min) (P <0.01). The results showed that HF group rats developed obvious insulin resistance.5 Correlation between IR and blood lipid.At the time point of 4 weeks, correlation coefficient of triglycerides and ISI was -0.368 (P>0.05), that of cholesterol and ISI was -0.288 (P >0.05) and that of FFA and ISI was -0.511(P<0.05). At the time point of 8 weeks, correlation coefficient of triglycerides and ISI was -0.771 (P<0.01), that of cholesterol and ISI was -0.82 (P<0.01) and that of FFA and ISI was -0.856(P<0.01). Blood triglycerides, cholesterol and FFA were correlated to ISI.6 Changes of blood FFAs In our research conditions, C18:1,C18:2 and C18:3 could not be separated completely, while other fatty acids were separated well. When compared with Con, the concentrations of C20:0(18.91±2.49 mg/L), C24:0(4.18±1.05 mg/L)of DM group did not change(P>0.05), C20:5(19.33±4.47 mg/L)decreased, and the other fatty acids, such as C16:0, C18:0, C20:4, C22:0, C22:6 and C26:0, increased(P<0.05) In HF group, the concentrations of C16:0 (73.37±13.03 mg/L), C18:0(51.40±7.87 mg/L), C18:1+C18:2+C18:3 (176.07±22.27 mg/L), C22:6(5.33±1.48 mg/L) increased, C20:5 (17.71±2.95 mg/L) decreased ( P<0.05 ), and the concentrations of other fatty acid, such as C20:0, C20:4, C22:0, C24:0 and C26:0, were unchanged (P>0.05), when compared with control group. Percentages of different fatty acids in the total fatty acid were also changed. C18:1+C18:2+C18:3 (32.14±3.50) %, C20:4(1.85±0.26) %, C22:6 (9.49±1.35) % and C26:0(1.18±0.18) % in the serum of DM group increased, and the other fatty acids (C16:0, C18:0, C20:0, C20:5, C22:0 and C24:0) showed no change. C18:1+C18:2+C18:3 (4.96±3.21)% in the serum of HF group increased, C20:5(0.64±0.09)% of HF group decreased(P<0.05), and the other fatty acids (C16:0, C18:0, C20:0, C20:4, C22:0, C22:6, C24:0 and C26:0) showed no change (P<0.05).Conclusion:1 Occurrence and degree of insulin resistance were related to the inducing time, blood sugar concentration began to increase obviously when insulin resistance reached to certain degree.2 Increase of FFA had a close relation with occurrence of insulin resistance induce by high-fat diet.PartⅡChanges of hepatic lipids in rats with insulin resistance induced by high-fat dietObjective: To observe changes of hepatic lipids(fat and FFA) and fatty acidβ-oxidation, and investigate the relation between insulin resistance inducing by high fat diet and hepatic fatty acid metabolism.Methods: In order to establish quickly the animal model with insulin resistance and diabetes, HF group rats that had developed insulin resistance, were administered with 2% STZ (27 mg/kg) by peritoneal injection. The blood sugar levels of tail vains were evaluated 72 hours later. The rats whose fasting blood sugar was higher than 7.8 mmol/L were fed continuely with high-fat diet for 6 weeks, and grouped as insulin resistance complicated with diabetes group (DM). Con and HF groups were also fed for 6 weeks with standard diet and high-fat diet respectively. Eight rats were selected randomly from each group for next test. The rats were intraperitoneally anesthetized with pentobarbital sodium (60 mg/kg) in the fasting state. Blood was collected by carotid artery bloodletting, and the serum was used for the detection of blood total FFA and FFA profiles, and the liver was used for the detection of liver fatty deposition(oil red o staining), amount of fat (oxidase way), fatty acidβ-oxidation activities (ultraviolet absorption) and liver FFA profiles.Results:1 Contents of liver fatCompared with Con group(11.30±1.89 mg/g), content of liver fat of HF group(34.94±4.93 mg/g) and DM group(51.47±7.77mg/g) increased remarkly (P<0.01).2 Detected fatty deposition in liver by oil red o stainingLittle fat drops could be found in Ctrl. group, but more fat drops were found in DM and HF groups. Results of oil red o staining showed that rats with insulin resistance reduced by high-fat diet had severe fat deposition.3 Fatty acidβ-oxidation of liver tissue3.1 Total activities of fatty acidβ-oxidationCompared with Con group (6.46±1.07 mU/mg pro), total activities ofβ-oxidation of DM group (10.76±0.81mU/mg pro) and HF group (9.10±1.31mU/mg pro) increased remarkablely (P<0.01).3.2 Activities ofβ-oxidation of peroxisome Compared with Con group (3.21±0.55 mU/mg pro), the activities of peroximalβ-oxidation of DM group (4.41±1.10 mU/mg pro) and HF group (4.13±0.82mU/mg pro) increased (P<0.05). 3.3 Activities ofβ-oxidation of mitochondriaCompared with Con group (3.25±1.20 mU/mg pro), the activities of mitochondriaβ-oxidation of DM group (6.35±1.85mU/mg pro) and HF group (4.97±1.86mU/mg pro) increased (P<0.01, P<0.05).4 Total FFA contents in liversCompared with Con group (66.03±15.00μmol/g), the total FFA contents of DM group (112.79±22.24μmol/g) and HF group (97.01±16.02μmol/g) increased (P<0.01, P<0.05).5 Changes of liver FFA profiles measured by gas chromatographyC20:5 could not be detected and C18:1, 18:2, 18:3 could not be separated completely by the gas chromatography equipment we used. In comparison with Con, C18:1+C18:2+C18:3 (24.31±3.50 mg/g), C20:4 (5.06±0.82 mg/g), C22:6 (1.41±0.23 mg/g) and C26:0 (2.49±0.46 mg/g) contained in liver increased (P<0.05), C18:0(5.56±1.09 mg/g), C20:0 (0.50±0.08 mg/g), C22:0 (0.13±0.02 mg/g) and C24:0 (0.12±0.02 mg/g) decreased (P<0.05), and C16:0 was unchanged(P>0.05)in DM group。In HF group the fatty acids C18:1+C18:2+C18:3 (21.89±3.92 mg/g) contained in liver increased (P<0.05), C20:0 (0.82±0.19 mg/g), C22:0 (0.19±0.08 mg/g) and C24:0 (0.14±0.08 mg/g) decreased (P<0.05), and the other fatty acids (C16:0,C18:0,C20:4,C22:6和C26:0) were unchanged (P>0.05) when compared with control group. There were statistic differences of contents of C20:0,C22:6 and C26:0 between DM group and HF group.The percentage of each fatty acid in the composition of total fatty acid was also changed. Compared with Con, the percent of C18:1+C18:2+C18:3 (36.67±3.31)%, C22:6 (2.59±0.49)% and C26:0 (3.16±0.64) % increased (P<0.05), that of C20:0 (0.16±0.04) %, C22:0 (0.16±0.01) %, C24:0 (0.44±0.09) % decreased(P<0.05), and that of other fatty acids (C16:0,C18:0 and C20:4) were unchanged in DM group. Compared with Con group, percent fatty acid composition of C18:1+C18:2+C18:3 (37.24±3.06)% of HF group increased (P<0.05), that of C20:0 (0.12±0.03) %, C22:0 (0.19±0.04) % and C24:0 (0.51±0.11) % decreased (P<0.05), and that of other fatty acids (C16:0,C18:0,C20:4,C22:6 and C26:0) were unchanged (P>0.05). Percent fatty acid compositions of C22:6 and C26:0 of DM group were higher than HF group (P<0.05).Conclusion:1 Increase of blood FFA of the rats fed with high-fat diet for a long time promoted the activities of mitochondria, peroximal and total oxidation. Although having increased activities ofβ-oxidation, the liver failed to disposal the large amount of fatty acid and it resulted in increase of fat, total fatty acid and some very long chain fatty acid.2 Insulin resistance induced by high-fat diet correlated with increased hepatic triglycerides, total fatty acids and some very long chain fatty acids.PartⅢChanges in expression of genes involved in lipid metabolism in liver of high-fat diet induced insulin resistance ratObjective: To investigate the changes in lipid metabolism of liver by detecting the expression of the genes involved in fatty acid metabolism in liver of insulin resistance rats and diabetes rats.Methods: The expression of genes involved in fatty acid metabolism was detected with RT-PCR. The level of the D-bifunctional protein(DBP) was detected with western blotting.Results:1 The mRNA expression of diacylglycerol acyltransferase 1 (DGAT1) involved in triglyceride synthesisThe DGAT1 mRNA of DM and HF groups (0.38±0.06,0.26±0.06) were higher than Con group (0.12±0.02, P<0.01, P<0.05), which indicated that triglyceride synthesis in DM and HF group increased.2 The mRNA expression of carnitine palmitoyl transferaseⅠ(CPTⅠ), the rate-limiting enzyme in mitochondria fatty acidβ-oxidation.The CPT I mRNA of DM and HF groups (0.58±0.10,0.37±0.04) were higher than Con group (0.26±0.02, P<0.01,P<0.05) and that of DM group was higher than HF group (P<0.01), which indicated that mitochondria fatty acidβ-oxidation in DM and HF groups increased. 3 The mRNA expression of fatty acyl-CoA oxidase 1(ACOX1), fatty acyl-CoA oxidase 3 (ACOX3), D-bifunctional protein(DBP), L-bifunctional protein (LBP)The ACOX1 mRNA expression of DM and HF groups (1.51±0.10,1.74±0.25) was higher than Con group (1.12±0.22, P<0.01,P<0.05) and the LBP mRNA expression of DM and HF groups (0.66±0.12,0.59±0.04) was higher than Con group (0.47±0.09, P<0.01,P<0.05), which indicated that peroxisomal fatty acid oxidation involved of LBP of HF and DM groups was increased.DM and HF groups compared to Con group(0.88±0.15,1.08±0.19 vs 1.01±0.10, p>0.05), the changes of expression of ACOX3 mRNA have no statistical significance. The DBP mRNA of DM group(0.13±0.01)was lower than Con and HF groups (0.21±0.05,0.26±0.06, P<0.05), which indicated that the activity of fatty acid oxidation in peroxisome involved of DBP was decreased.4 The changes of peroxisome proliferator-activated receptorα(PPARα) mRNA expressionThe PPARαmRNA of DM and HF groups (0.96±0.07,0.90±0.08) were higher than Con group (0.78±0.07, P<0.01,P<0.05) and the transcription of downstream genes (CPTⅠ,ACOX1,LBP) increased , which indicated that mitochondria fatty acid oxidation in DM and HF group increased5 The expression of DBP protein in liverThe expression of DBP protein in DM group (0.43±0.05, n=4)was lower than Con group(0.75±0.12,P<0.01)and HF group(0.81±0.11,P<0.01). It was coincident with the expression of DBP mRNA, which indicated that the expression of DBP decreased.Conclusion:1 In high-fat diet induced insulin resistance rats and diabetes rats, triglyceride accumulated in liver. It associated with increased expression of DGAT1, the rate-limiting enzyme in triglyceride synthesis. 2 In the liver of high-fat diet induced insulin resistance rats and diabetes rats, mitochondria and peroxisome fatty acid oxidation increased. It associated with increased transcription and activation of PPARαand up-regulated genes of CPTⅠ,ACOX1,LBP.3 In the liver of high-fat-induced insulin resistance and diabetes rats, some very long chain fatty acids ,such as C26:0, were increased. It associated with decreased expression of DBP and decreased activity of fatty acid oxidation involved of DBP.PartⅣEffects of free fatty acids on metabolism of fatty acids in primary cultured rat hepatocytesObjective: To observe the effects of FFA which outside of hepatocytes on IR and on expression of the genes which involve in the metabolism of fatty acids based on the primary cultured hepotocytes.Methods: SD rats weighing between 200 and 250 g were anesthetized with thiopental and hepatocytes were isolated after collagenaseⅣperfusion of the livers. Primary cultured hepatocytes were divided randomly into: control group (Con group), palmitic acid group(PA group). PA group was administered with 200μmol/L PA for 24 h. The expression of PEPCK1, CPTⅠ, DGAT1, AOX1, LBP and DBP mRNA were observed by RT-PCR and the expression of DBP protein were observed by Western blotting.Results:1 Expression of PEPCK1 mRNAThe PEPCK1 mRNA level in PA group (1.12±0.13) was significantly higher than that of Con group (0.73±0.15) ( P < 0. 01). The results showed that the effect of insulin's inhibition to glyconeogenesis was reduced, and IR was proved.2 Expression of mRNA of genes involved in the metabolism of fatty acidsThe DGAT1 mRNA level was higher in PA group (0.39±0.09) than that of Con group (0.25±0.07) (P<0.05). The CPTⅠmRNA level was higher in PA group (1.07±0.23) than that of Con group (0.72±0.16) (P<0.05), the ACOX1 mRNA level was higher in PA group (2.00±0.22) than that of Con group (1.61±0.15) (P<0.05) and the LBP mRNA level was higher in PA group (0.76±0.13) than that of Con group (0.50±0.09) (P<0.01). However, difference of the DBP mRNA level between PA(0.78±0.18) and Con (0.90±0.22) have no statistic significance (P>0.05). The results showed that the metabolism of fatty acids in primary cultured liver cells altered.3 The protein level of DBPThe DBP protein levels between PA group (0.87±0.11) and Con group (0.80±0.13) have no statistic significance (P>0.05). The result was similar to DBP expression of HF group, but was different from DM group in integral experiment.Conclusion:1 The expressions of some genes involved in fatty acids metabolism were altered in transcriptive level by FFA. These changes were similar to the changes of rat liver of insulin resistance induced by high-fat diet.2 Expression of DBP gene of liver cell cultured in palmitic acid (200μmol/L) was not altered, which is different from that of DM group in integrated research.Summary:1 Increase of free fatty acids in blood and accumulations of fat and free fatty acids resulted from the increase of free fatty acids in blood were vital factors that contributed to insulin resistance induced by a long term of high-fat diet.2 In high-fat-induced insulin resistance rats and diabetes rats, triglyceride accumulates in liver. It associated with increased expression of DGAT1, the rate-limiting enzyme in triglyceride synthesis.3 In the liver of high-fat-induced insulin resistance rats and diabetes rats, fatty acidβ-oxidation increased. It associated with increased transcription and activation of PPARαand up-regulated genes of CPTⅠ, ACOX1 and LBP. But it failed to consume the large mount of fatty acid uptaken by liver.4 In the liver of high-fat-induced insulin resistance and diabetes rats, some very long chain fatty acid (C26:0) was increased. It associated with decreased expression of DBP and decreased activity of fatty acid oxidation involved of DBP.