Investigation Of Mechanisms Of Fatty Liver Induced By High-fructose-and High-fat-diet

Non-alchholic fatty liver disease (NAFLD) is a syndrome of clinical pathological disorders with a fat deposit of more than 5% of liver weight in liver in the absence of significant alcohol consumption (daily intake:<10g alcohol/day). It is characterized by fatty degeneration and steatosis in hepatic parenchymal cells. NAFLD represents a spectrum of liver impairment including simple fatty liver, non-alchholic steatohepatitis, progressive liver fibrosis and liver cirrhosis. Most of NAFLD patients stay in the stage of simple fatty liver, which is also called"fatty liver"for short.In recent years, with the change of diet strcture and lifestyles, the incidence of NAFLD has been significantly increased. The average incidence of NAFLD is approximately 20% in general population; meanwhile, the incidence in obese populations and diabetes patients are 60% and 70% respectively. NAFLD has become one of the most common liver diseases threatening the health of human beings. NAFLD has already been the first chronic liver disease and is the most important reason for an abnormal peptase. In USA, the incidence of NAFLD is 24%. In Asian countries, the incidence of NAFLD has also been increasing gradually. In the past 7-10 years, the incidence of fatty liver in developed regions in China has nearly doubled, and the incidence in adults is as high as 15%, most of which are NAFLD.NAFLD is closely related with metabolic syndrome (MS) and the incidence of cardiovascular disease (CVD). Researchers have gradually realized that NAFLD is one of important components of MS and is the hepatic manifestations of MS. Because of the high incidence of NAFLD and its close association with metabolism and CVD, NAFLD has become the hotspot in medical research worldwidely. Most of the patients with NAFLD stayed in the stage of simple fatty liver for a long time. Some patients stayed in this stage lifelong, and some patients develop into the stage of NASH after more than 10 years. Therefore, simple fatty liver is more associated with MS. The mechanism study of NAFLD is surely meaningful for the prevention and treatment of MS and CVD and can provide the possible treatment targets.Diet factors have been thought to be an important environmental factors leading to the development of fatty liver. A variety of diet factors can either prevent or promote the development of fatty liver. Fructose has especially attracted the attention of researchers as a factor contributing to the development of fatty liver. Fructose exists abundantly in food such as soft drinks. With the introduction of high fructose corn syrup (HFCS) into the food industry, the intake of fructose in the populations has increased year by year, particularly, some adolescence take the soft drinks instead of water. Epidemiology studies indicated the over-intake of fructose is related to overweight, dyslipidemia and MS. Animal studies showed that high-fructose-diet can lead to the dyslipidemia, higher blood glucose, abdominal obesity and higher blood pressure, which are typical manifestations of metabolic syndrome. Therefore, the harmfulness of high-fructose-diet has been increasingly recognized. In spite of the inconsistence of reports, both human and animal studies proved that the over-consumption of fructose can lead to the development of fatty liver. Long-term feeding of high-fructose- and high-fat-diet can induce the fat steatosis in liver. Massive animal studies indicated that high-fructose diet cause the fat deposition in liver; rodents studies showed that either short-term or long-term high fructose feeding can induce fatty liver. The underlying mechanisms have not been clarified completely. According to previous studies, with the development of liver steatosis and liver insulin resistance (IR) induced by chronic high-fructose- and high-fat-feeding, diverse cellular incidents occurs concomitantly, including dysfunction of fatty acid oxidation in mitochondrial, increased de novo lipogenesis, endoplasm reticulum stress (ERS) and dysfunction of mitochondrial. However, the initiating mechanisms of the occurrence of fatty liver, the sequence of various intra-cellular events and their association with fatty liver remain to be clarified. On the other hand, the influence of short-term over-intake of fructose on liver steatosis and liver insulin sensitivity remain to be investigated.In the present study, we first observed the change of hepatic lipid content after a short-term (1 week) high-fructose-feeding. After the establishment of fatty liver, the whole-body and liver insulin sensitivity were evaluated. From the respects of both function and molecular biology, the fatty acid oxidation in mitochondrial and de novo lipogenesis were accessed during the early happening of fatty liver. In addition, by detecting the protein and gene expression of ERS and inflammation markers, the association of development of fatty liver with ERS and inflammation were observed. At the same time, to investigate the effect of different dietary factors on mice livers and the possible mechanisms underlying early development of fatty liver, the present study used high-fat-fed mice as another fatty liver model in comparison to high-fructose-fed mice. By using two different diet models, we expect for a better understanding of the harmfulness of high-fructose-diet and the pathogenesis of fatty liver.Part One The effect of short-term high-fructose-and high-fat feeding on liver triglyceride content and hepatic insulin sensitivity in miceObjective: To investigate the effect of short-term high-fructose- and high-fat feeding on liver triglyceride content and to observe the change in hepatic insulin sensitivity in miceMethod: Seventy-three C57BL/J6 mice aged from 12 to 15 weeks were used in the study (Provided by Perth Animal Center in Australia). The animals were kept in a temperature-controlled room (22±1°C) in animal feeding center on a 12-h light/dark cycle. After a week of acclimation feeding, the mice were divided into 3 groups: Control group(Con) (n=25), high-fructose group(HFru)(n=24) and high-fat group(HF) (n=24). Con group were fed with standard lab chow diet (Gordon's Specialty Stock Feeds, Yanderra, Australia)with the energy contents as follows (see table 1): 71% calories from carbohydrate ,8% calories from fat, and 21% calories from protein with total energy of 260kcal/100g; HFru group were fed with a high-fructose-diet with the energy contents as follows: 70% calories from carbonhydrates with 35% of fructose, 9% calories from fat and 21% calories from protein with total energy of 260kcal/100g;HF group were fed with a high-fat-diet with the energy contents as follows: 20% calories from carbohydrate, 59% calories from fat and 21% calories from protein with total energy of 540kcal/100g. Eight mice were randomly chosen from each group after 1 week of feeding. Intraperitoneal glucose test (ipGTT) was performed in the 8 mice. After 1 week feeding, blood samples were taken from 8 mice in every group. Blood samples were taken from mice tails and the plasma was collected after centrifuged in refrigerated centrifuge for detection of plasma insulin. For At the end of the test, mice were sacrificed by cervical dislocation and liver tissues were collected immediately and stored in-70℃liquid nitrogen. The frozen liver tissues were homogenized and dried by nitrogen. TG contents were measured by GPO-PAP method. For evaluation of hepatic insulin sensitivity, after 1 week feeding, 12 mice were randomly chosen from each group. Six of the mice were injected intraperitoneally with physiological saline; six of the mice were injected with a mixed solution containing glucose (3g/kg BW) and insulin (2U/kg.BW). Mice were sacrificed 40 minutes after injection and liver tissues were collected and stored in-70℃. The total and phosphorylated protein kinase B (PKB/Akt) and glycogen synthase kinase 3 (GSK-3α/β) protein expression were detected by western blot in liver tissues in mice injected with above mixed solution. Hepatic insulin sensitivity was evaluated by comparing the change in protein expression of p-Akt/t-Akt and p- GSK- 3α/β/t- GSK-3α/βin mice livers.Results: 1 Basic data in 3 groups: After 1 week feeding by different diets, there are no significant difference in body weight among 3 groups(p>0.05); the epididymal weight corrected by body weight were significantly high in HFru and HF group compared with Con group(P<0.01). Fasting blood glucose, insulin and plasma TG were no difference among the 3 groups(p>0.05). 2 The change in liver TG contents: Compared with Con group, liver TG were significantly increased in HFru and HF group. Moreover, the liver TG were higher in HFru group than that in HF group (P<0.01).3 Intraperitoneal glucose tolerance test(ipGTT): Compared with Con group, blood glucose at 30′, 60′and 90′was significantly higher in HFru and HF group. The difference was statistically significant (P<0.05 or P<0.01). Compared with Con group, the area under curves in HFru and HF group were significantly increased compared with Con group (58% and 64% increased respectively). The difference was statistically meaningful (P<0.01).4 Evaluation of liver insulin sensitivity: Compared with Con group, the ratios of p-Akt/t-Akt and p- GSK-3α/β/t- GSK-3α/βwere no difference in HFru and HF group under basic state(p>0.05). Forty minutes after injection of insulin, compared with Con group, p-Akt/t-Akt decreased by 57% and 42% respectively in HFru and HF group. The difference was statistically meaningful (Both P<0.01). Compared with Con group, p-GSK-3α/β/t- GSK-3α/βdecreased by 53% and 52% respectively in HFru and HF group after insulin injection. The difference was statistically meaningful (Both P<0.01). The above results indicate the insulin sensitivity was attenuated and insulin signalling transduction was weakened in mice livers in HFru and HF group. After insulin injection, the ration of p-Akt/t- Akt and p- GSK-3α/β/t- GSK-3α/βwere no significant difference between HFru and HF group(p>0.05).Conclusions:1 Both short-term high-frucotse-diet and high-fat-diet can lead to liver steatosis in mice.2 Both short-term high-frucotse- and high-fat feeding can lead to an impaired glucose tolerance and liver IR.3 After 1 week of high-frucotse- and high-fat feeding, liver steatosis coexists with hepatic IR.Part Two The effect of short-term high-fructose and high-fat feeding on endogenous lipid synthesis in mice liver Objective: From both functional and molecular level, the changes in de novo lipogenesis in mice liver were studied after a short-term high-fructose and high-fat feeding.Method: Animal grouping and samples collection was the same as those in part one. From functional level, the rate of hepatic de novo lipogenesis was evaluated by radioactive substance tracing method. The mRNA expression of liver sterol regulatory element binding protein (SREBP) and carbohydrate response element binding protein (ChREBP) were measured by real time PCR. The protein expression of the key enzymes of liver de novo lipogenesis-including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD-1)-were analyzed by western-blot.Results:1 The comparison of hepatic de novo lipogenesis in 3 groups of mice: Compared with Con group, the rate of de novo lipogenesis in HFru group was significantly increased by 63% (p<0.01); the difference is statistically meaningful. Compared with Con group, the rate of de novo lipogenesis in HF group was significantly decreased by 32% (p<0.05); the difference is statistically meaningful. Above results indicate high fructose feeding promote de novo lipogenesis in liver while high fat feeding inhibit hepatic de novo lipogenesis.2 The comparison of gene expression of SREBP and ChREBP in liver in 3 groups: Compared with Con group, the mRNA expression of SREBP and ChREBP increased by 330% (p<0.01) and 44% (p<0.05) respectively in HFru group. Compared with Con group, the mRNA expression of SREBP increased by 50% (p<0.05) while the mRNA expression of ChREBP was unchanged in HF group.3 The protein expression of key enzymes of hepatic de novo lipogenesis in 3 groups: Compared with Con group, in HFru group, FAS protein expression increased by 2.7 times(p<0.01), ACC protein expression increased by 6.4 times(p<0.01) and SCD-1 protein expression increased by 8.0 times(p<0.01). Conversely, in HF group, FAS protein expression has no change, ACC protein expression decreased by 45%(p<0.01)and SCD-1 protein expression decreased by 78%(p<0.01).Conclusions:1 The effect on hepatic de novo lipogenesiss of high-fructose-diet and high-fat-diet are converse. High-fructose-diet increases the rate of de novo lipogenesis, while high-fat-diet inhibit the rate of de novo lipogenesis.2 Short-term high-fructose feeding increases the gene expression of SREBP and ChREBP (upstream transcriptional factors of lipid synthesis enzymes) in liver significantly. Short-term high-fat-feeding increases the gene expression of SREBP, while the upregulation of SREBP is significantly lower in HF group compared with that in HFru group. High-fat-feeding doesn't change the gene expression of ChREBP.3 High-fructose-diet promote the protein expression of liver de novo lipogenesis enzymes (ACC, SCD-1 and FAS) significantly. In contrast, the protein expression of ACC and SCD-1 were inhibited by high-fat feeding.Part Three Investigation of liver mitochondrial function in fatty liver induced by short-term high-fructose and high-fat feedingObjective: To investigate the change in mitochondrial function in liver cells in mice with fatty livers fed on high-fructose and high-fat diet for a short term.Method: Animal grouping and samples collection is the same as those in part one. From function level, the rate of mitochondrial substrate oxidation in liver was evaluated by radioactive substance tracing method. Palmitate acid labelled with [1-14C] was used to detect the palmitate acid oxidation rate in liver mitochondrial. Glutamate acid labelled with [1-14C] was used to detect the glutamate acid oxidation rate in liver mitochondrial. Western Blot was used to measure the protein expression of the following proteins: proliferator–activated receptor-coactivator -1α(PGC1α: an upstream factor of liver mitochondrial biogenesis), voltage dependent anion channel (VDCA: a protein indicating the quantities of mitochondrial), carnitine palmitoyltrans- ferase-1 (CPT-1: the key enzyme of fatty acid oxidation in mitochondrial), ComplexI, II, III, V(an antibody cocktail that recognizes several subunits of the mitochondrial respiratory chain) and COX-1(cytochrome oxidase (complex IV) subunit 1) .Results:1 Palmitate acid oxidation rate in liver mitochondrial in 3 groups: Compared with Con group, the palmitate acid oxidation rate is not significantly increased or decreased. The difference are not statistically significant(both P>0.05).2 Glutamate acid oxidation rate in liver mitochondrial in 3 groups: Compared with Con group, the glutamate acid oxidation rate is not significantly increased or decreased. The difference are not statistically significant(both P>0.05).3 Comparison of the protein expression of markers of mitochondrial metabolism in 3 group: There are no significant difference in protein expression in PGC1-α(indicating the biogenesis of mitochondrial) in 3 groups(P>0.05);There are no significant difference in protein expression in VDCA (indicating the quantities of mitochondrial) in 3 groups(P>0.05); There are no significant difference in protein expression in CPT-1 (indicating the metabolism of fatty acid oxidation in mitochondrial) in 3 groups(P>0.05).4 Comparison of protein expression of mitochondrial respiratory chain proteins: Compared with Con group, there are no significant difference in the protein expression in the mitochondrial respiratory chain proteins ( ComplexI,ComplexII,ComplexIII,ComplexV) (P>0.05); The protein expression of COX-1 which is another indicator of mitochondrial metabolsim is no difference in 3 groups(P>0.05).Conclusions:1 With the induction of fatty liver by high-fructose- and high-fat-feeding, mitochondrial substrate oxidation rates have no significant change in liver cells.2 With the induction of fatty liver by high-fructose- and high-fat-feeding, the markers of mitochondrial biogenesis and metabolism (including PGC1α, VDCA, CPT-1, mitochondrial respiratory chain complex(ComplexI,II,III,V) and COX-1)in liver cells have no change in protein expression.Part Four The relationship of endoplasm reticulum stress (ERS) in liver to fatty liver induced by high-fructose- and high-fat-dietObjective: To study the function of ER during the induction of fatty liver by high-fructose- and high-fat-feeding and to explore the mechanism by which different dietary factors induce liver steatosis and its association with ERS.Methods: Animal grouping and samples collection is the same as those in part one. Protein expression of markers of liver ERS including the phosphorylated proteins of pancreatic ER kinase (PERK), inositol-requiring kinase 1(IRE-1) and eukaryotic translation initiation factor 2α(eIF2α) were measured by Western Blot. The mRNA level of spliced XBP-1 was measured by PCR.Results:1 Compared with Con group, the phosphorylated proteins of upstream regulatory protein PERK increased by 2.2 times in HFru group (P<0.01). The phosphorylation of the downstream factor-eIF-2α(p-eIF-2α/-t-eIF-2α) increa- sed by 2.6 times(P<0.01). Meanwhile, the phosphorylation of the upstream regulatory protein of another pathway of ERS-IRE-1 (p-IRE-1/-t-IRE-1) increased by 2.1 times(P<0.01); correspondingly, the mRNA level of its downstream factor-spliced XBP1(XBP-1s) increased significantly by 1.15 (P<0.01), indicating the occurrence of ERS after 1 week of high fructose feeding.2 Compared with Con group, the protein expression of p-PERK, p-eIF- 2α/-t-eIF-2αand p-IRE-1/-t- IRE-1 were unchanged significantly (P all>0.05) in HF group. No significant change was observed in mRNA level of XBP-1s in HF group (P >0.05), indicating an absence of ERS in HF group.Conclusions:1 With the occurrence of liver steatosis and liver IR induced by a short-term high-fructose feeding in mice, ERS occurred in liver in mice. ERS might intermediate the development of fatty liver and liver IR in mice. 2 The converse effect of high-fructose-diet and high-fat-diet on de novo lipogenesis and ERS indicate that ERS is closely associated with lipid synthesis. The cause-effect relationship between ERS and de novo lipogenesis needs further investigation.3 Fatty liver and IR induced by short-term high fat feeding occur earlier than the occurrence of ER, indicating ERS is not the initiating mechanism of the development of liver steatosis and liver IR in mice.Part Five The change of inflammation markers in fatty liver induced by short-term high-fructose and high-fat feedingObjectives: To study the change of inflammation pathway during the early occurrence of fatty liver induced by high-fructose and high-fat-diet and to investigate the mechanisms by which different dietary factors induce liver steatosis and its relationship with IR.Methods: Animal grouping and samples collection is the same as those in part one. The protein phosphorylation of markers of two inflammation pathways-c-Jun NH2-terminal Jun kinase (JNK) pathway and inhibitor of kappa B kinase complexα/β- nuclear factorκB (IKKα/β-NF-B ) pathway- was measured by Western Blot.Results:1 Compared with Con group, the protein expression of p-JNK/tJNK, p -IKKα/β/t-IKKα/βand t-IKBαwere unchanged(all P>0.05).2 Compared with Con group, the phosphorylation of JNK(p-JNK/ tJNK)in liver increased by 2.0 time(p<0.01)s, indicating the JNK pathway was activated by high fat feeding. Compared with Con group, the protein expression of p-IKKα/β/t-IKKα/βand t-IKBαwere unchanged(both P>0.05).Conclusions:1 The occurrence of fatty liver and IR induced by short-term high fructose feeding does not accompany the activation of intra-liver inflammation pathways including JNK pathway and IKKα/β-NF-κB pathway.2 With the induction of fatty liver and IR induced by short-term high fat feeding, JNK pathway is activated in liver while IKKα/β-NF-κB pathway is unchanged, indicating JNK pathway might mediate the development of liver IR induced by high-fat-feeding.