Preliminary Study On The Protective Effect And Regulation Mechanism Of Caffeineon Hepatic Steatosis In Zebrafish Larvae

Background and objectiveNAFLD is becoming one of the most common causes of chronic liver diseases. It is estimated that approximately 20-30% of adults and 3-10% of children in western countries suffer from excessive fat accumulation in the liver. A recent meta-analysis showed that theoverall pooled prevalenceof NAFLD was 20.09% in china, male is 24.81%, female is 13.16%. Following the prevalence of obesity and its associated complications such as diabetes, insulin resistance and hyperlipidemia in the world, the incidence of NAFLD have been increasing in recent years. Indeed, NAFLD is considered to be a manifestation of metabolic syndrome in the liver, which range from non-progressive simple steatosis to non-alcoholic steatohepatitis (NASH) with ballooning degeneration, inflammation and fibrosis. NASH can progress from steatohepatitis to liver cirrhosis, and may eventually develop hepatocellular carcinoma after decade years. Due to lack of clear knowledges of molecular mechanism on NAFLD, the main treatment strategy of this disease at present consists of lifestyle modification, the promotion of physical activity and avoiding to sedentary lifestyle.There are many studies that have attempted to establish NAFLD models in rodents via the use of dietary and pharmacological induction, as well as genetic manipulation. Recently, zebrafish compare popularly to rodents as new experimental animals for researchers because zebrafish have a high reproductive rate, mature rapidly, and cost little regard to rearing space and daily maintenance due to their small size. More and more researchers use zebrafish as a model to study the pathogenesis and pharmacological therapy of NAFLD. In a review, Yoichi Asaoka et al summarized the various types of fish models in use for NAFLD, including those generated by mutation, transgenesis, or dietary or chemical treatment, and contrast them with rodent models. And recently Valerie Sapp et al have established a fructose-induced zebrafish model of NASH in 7 days post-fertilization (dpf) zebrafish larvae. However, these previous study for NAFLD using zebrafish model mostly focused on the period of 5dpf-7dpf zebrafish larvae and adult zebrafish, there is lack of knowledge about studying juvenile zebrafish larvae of NAFLD. Because of the advantage of transparent body in juvenile period suitable for identifying lipid accumulation in liver by whole-mount oil red 0 staining, in present study we attempted to determine whether juvenile zebrafish can be used as a model for diet-induced NAFLD.Coffee is probably the most frequently consumed beverage worldwide. Because of its consumption in most countries in the world, it is concerned to investigate its potential benefits or adverse aspects in relationship to human health in terms of a public and a scientific perspective. Caffeine is a main component of coffee, which also includes other ingredients such as diterphenoic alcohols, potassium, niacin, magnesium, and the anti-oxidants chlorogenic acid (CGA) and tocopherols. Although coffee and caffeine can increase the risk of cardiovascular disease, numerous evidences demonstrated that coffee and caffeine have a hepatoprotective effect on chronic liver diseases. Epidemiological and clinical investigations have suggested that its consumption could reduce the risk of alcoholic liver cirrhosis, type 2 diabetes, NAFLD, and HCC and the progression of NASH and the severity of fibrosis,as well as the activity of ALT in liver injury patients. There are many studies that show coffee and caffeine exhibit a beneficial role on liver injury in animal and in vitro studies. Sandra kal thoff et al have demonstrated that coffee mediate the protective and antioxidant properties through upregulation of expression of glucuronosyltransferases in liver and stomach. Experimental study has shown that caffeine can attenuate the development of liver cirrhosis and HCC through inhibition of transforming growth factor-beta (TGF-beta) and its downstream effectors. Recent study has demonstrated that caffeine reduces intrahepatic lipid content and stimulates β-oxidation in hepatic cells and liver by an autophagy-lysosomal pathway via using genetic, pharmacological, and metabolomic approaches. Similarly, researchers have shown that caffeine effectively depletes TG and cholesterol levels by inhibition of lipogenesis and stimulation of lipolysis through modulating AMPK-SREBP ignalling pathways in HepG2 cells. While coffee and caffeine show a hepatoprotective characteristic on NAFLD, the precise mechanism is completely unknown.In this study, we aimed to determine the effect and likely mechanism of caffeine on lipid accumulation in the liver of zebrafish larvae by establishment of diet-induced model of NAFLD in juvenile zebrafish, providing a good platform for studying the pathogenesis of NALFD and drug screening for this disease.Methods1. Identification of optimal quantity and duration of feeding on zebrafish larvaeBecause zebrafish have the feature of food preference, zebrafish larvae at 5 days post-fertilization(5dpf) were divided into 6 different feeding group(20mg/d,30mg/d, 60mg/d,80mg/d,120mg/d and 180mg/d) and fed for three different duration(10 days, 15 days and 20 days), each group have 100 zebrafish larvae. The growth of larva in different feeding quantity and duration group were evaluated through measuring weight and length of zebrafish. At the same time mortality of larva in different groups were analyzed. The rate of hepatic steatosis and hepatic pathological change of larvae in different feeding quantity and duration group were determined by whole-mount oil red 0 staining and frozen oil red O staining and HE staining. Next, the change of lipid content in zebrafish larvae with different feeding quantity were observed by examining the level of triglyceride(TG) and total cholesterol(TCH). According to the growth, liver pathology and lipid content in zebrafish larvae, the optimal feeding quantity and duration which could induce zebrafish to develop fatty liver were identified and provided model for evaluating the effect of caffeine on hepatic steatosis in zebrafish.2. Study on the impact of caffeine on fatty liver in diet-induced zebrafish larvaeTo investigate the effect of caffeine on hepatic accumulation in zebrafish, we added caffeine to zebrafish food. Larvae fed for 20days were divided into1%caffeine, 2.5%caffeine,5%caffeine,8%caffeine, control and overfed groups. Weight and the rate of hepatic steatosis of zebrafish larvae were measured in different groups. And the pathological change of liver in zebrafish were evaluated by frozen oil red O and HE staining. In addition, the level of TG and TCH were examined to evaluate the effect of caffeine on lipid content.3.Preliminary study on the mechanism of caffeine reducing hepatic accumulation in diet-induced zebrafish larvaThis study investigated transcriptionally the expression level of associated genes through real-time quantity PCR technology. Using this method,we determined the impact of caffeine on genes involved in lipid metabolism(PPAR-γ、SREBP1、 PPAR-α、ACC1、FASN, CD36, UCP2, CPT-1 and ACO), ER stress(ATF6, IRE1、PERK、BIP and CHOP), inflammation(IL-1β、TNF-α and NF-κB) and autophage(Beclin-1、ATG12 and ATG3).4. Nie Red fluorescent staining labeling and imaging hepatic lipid droplets and adipose tissues in overfed zebrafish larvae in vivoBecause of the property of transparent body in zsbrafish larvae, we used fluorescent dye Nile Red, which posses the function of labeling neutral lipid, to label and image hepatic lipid droplets and adipose tissues in overfed zebrafish larvae in vivo. Simultaneously, the change of hepatic lipid droplets and distribution of adipose tissues in zebrafish larvae in different feeding time were dynamically observed by using Nile Red staining. In addition, the dynamic change of hepatic lipid droplets and adipose tissues in zebrafish larvae were observed in vivo when zebrafish was under the condition of starvation andrefeed.Results1. Zebrafish larvae overfed diets remarkably developed hepatic steatosiswemeasured larval weight and length under different quantity and duration of feeding conditions.Analytic results shows that the body weight and length of larvae gradually increased with the increased amount of feeding, and the body weight of larvae fed for 20 days (2.063±1.624mg) was significantly increased than that fed for 15 days (1.108±0.736mg) (P<0.001). Similarly, the body length of larvae fed for 20 days (6.587±1.220mm) was markedly increased than that fed for 10 (4.885±0.5879mm) and 15 days (5.594±0.7336mm) (P<0.001). However, the mortality of larvae between the different feeding groups had no significant difference (P>0.05); The death rate of larvae fed for 15 (19.25%) and 20 (21.47%) days was significantly higher than that fed for 10 days (7.83%)(P<0.01), but it was not a significant difference between larvae fed for 15 and 20 days(P>0.05). The data suggested that the mortality of larvae was disassociated with the amount of feeding and depend on inherent quality of zebrafish larvae per se. The incidence f hepatic steatosis of larvae was gradually increased with the increased amount of feeding when estimated by whole-mount oil red O (whole-mount ORO) staining, and it was more than 80% in larva fed on 120mg and 180mg per day, but there was no difference between different feeding times (P>0.05). The results of whole-mount ORO and Frozen ORO and HE staining showed that the liver in zebrafish larvae fed for 10 days had lipid droplets accumulation and the droplets became more intensive and bigger when larva fed for 15 and 20 days. However, the liver in zebrafish larvae fed 20 or 30mg/d basically haven’t lipid accumulation, indicating this amount of feeding can maintain the normal need of energy for zebrafish larva. And the larvae fed 120 or 180mg/d shown considerable lipid accumulation in the liver, which suggested that in this condition zebrafish larvae have surpassed the need of energy. In addition, we examined the level of triglyceride (TG) and total cholesterol (TCH) in zebrafish larvae. The results revealed that the content of TG was significantly higher in larvae fed 180mg/d (0.0142±0.0035mmol/gprotein) compared to larvae fed 30mg/d (0.0073±0.0034mmol/gprotein) for 20 days (P<0.01), and there was no difference in the level of TCH between the two groups (P>0.05). Thus, these data demonstrated that zebrafish larvae were quite readily to have hepatosteatosis under the overfeed dietary conditions and we successfully established an diet-induced model for fatty liver in zebrafish larvae,and defined zebrafish larvae fed on 30mg/d for 20days as control group and zebrafish larvae fed 180mg/d for 20 days as overfed group.2.Caffeine attenuated hepatic lipid accumulation in overfed zebrafish larvaeMany epidemiological and clinical evidences have demonstrated that coffee and caffeine have protective effects on the chronic liver diseases. So we investigated whether caffeine can reduce hepatic steatosis in overfeeding zebrafish larvae or not. First, we evaluated the effect of caffeine on the body weight of zebrafish. We found that body weight of zebrafish was remarkably reduced in 2.5%,5% and 8% caffeine groups compared with the overfed group (180mg/d), but there was no significant difference in 1% caffeine group compared to the overfed group. As like the body weight, the hepatic steatosis rate and the TG content of zebrafish significantly decreased in 2.5%,5% and 8% caffeine group and there was no difference between 1% caffeine and overfed group, and the content of TCH have no significance between all groups. Then frozenHE and oil red O (ORO) staining of consecutive serialsections shown that the amount and size of lipid droplets in the liver of zebrafish was markedly reduced in 2.5%,5% and 8% caffeine group compared with 1% caffeine and model group. These data showed that caffeine can alleviate hepatic steatosis in overfed zebrafish larvae.3.Caffeine got involved in modulating the expression of genes associated with lipid metabolism pathways, endoplasmic reticulum (ER) stress and hepatic inflammatory cytokinesTo investigate the mechanism by which caffeine reduced hepatic lipid accumulation, we examined the expression levels of genes involved in lipid metabolism. QRT-PCR showed that the level of gene expression of fatty acid transport protein exhibited notably downregulation in 5% caffeine group compared with overfed group, including fatty acid translocase (FAT)/CD36. The mRNA levels of uncoupling protein -2(UCP-2) and gene involved in hepatic lipogenesis transcription factor as sterol regulatory element binding transcription factor 1(SREBP1) was decreased. The level of gene expression of key lipogenic enzymes involved in fatty acid synthesis was significantly downregulated, including acetyl-CoA carboxylase 1(ACC1) and fatty acid synthase (FASN). But the mRNA level of gene acyl-CoA oxidase (ACO) involved in fatty acid β-oxidation was significantly increased in 5% caffeine group.Thenwe explored whether caffeine can impact on the function of ER to perform protective effects in hepatocytes in overfed zebrafish larvae. We found that the mRNA level of genes involved in ER stress were significantly upregulated in the liver of model group compared to control group (30mg/d), including IRE1, BIP, CHOP. After caffeine treatment, the gene expression level of IRE1 and BIP were decreased, and the mRNA level of genes ATF6 and PERK were also remarkably reduced in the liver of caffeine treatment larvae. These date suggested that caffeine can reduce the expression of genes associated ER stress. Next, we examined the expression level of inflammatory cytokine genes to confirm whether caffeine can regulate inflammatory response. The results shown that the inflammatory cytokines, including IL-1beta and TNF-alpha, were markedly increased in the liver of overfed group compared with control group. The mRNA level of IL-1beta and TNF-alpha were significantly decreased in 5% caffeine group compared to overfed group. No significant difference was observed in the expression of NF-κB. In addition, the mRNA level of ATG12 and Beclin-1, which is invovled in autophagy, were significantly decreased in the liver of 5% caffeine treatment group. Taken together, these results indicated that caffeine may inhibit lipogenesis and fatty acidinput, and increase lipid oxidation, and probably improve ER stress and reduce hepatic inflammatory response to ameliorate hepatic lipid accumulation in zebrafish larvae.4. Nile Red staining labeling and imaging hepatic lipid droplets and adipose tissues in zebrafish larvae in vivoWe selected zebrafish larvae overfed for 20 days and used Nile Red solution to treat it, and then observed it using fluorescent microscopy. We found that intensive orange signal appear in liver and abdominal cavity, suggesting the presence of hepatic lipid droplets and visceral adipose tissues in larvae. Whole-mount oil red O staining, bright microscopy and HE staining further demonstrated presence of hepatic lipid droplets and visceral adipose tissues. Then we observed the development and distribution of adipose tissues in zebrafish larvae at different feeding time by Nile Red staining. The results showedincompletely absorbed yolk sac in 5dpf larva. Accumulation of neutral lipid appeared in pancreas in zebrafish larva overfed 2 days (9dpf), indicating that pancreas is the first position of adipose cells. Little lipid droplet turned up in viscera in zebrafish larva overfed 5days (12dpf), indicating of accumulation of adipose cell in abdominal cavity. Accumulation of visceral adipose cell increased over feeding time. Except for abdominal cavity, adipose cell appear in other location such as pectoral fin plate, periorbital region, tail fin region, pericardial region, jaw,, subcutaneous positions, and dorsal region. This suggested that the amount of adipose tissues and adipose location gradually increased over feeding time, demonstrating that the appearance of adipose tissues in different location is not synchronous and the development of adipose tissues in zebrafish is temporally and spatially controlled in a certain order. In addition, there was markedly hepatic lipid accumulation in zebrafish larva overfed 5 days and it consistently existed afterward, suggesting zebrafish is quite readily to develop hepatic lipid accumulation. Then we observed longitudinally the change of adipose tissues and hepatic lipid droplets in zebrafish larva after starvation. First, Nile Red staining shown that abdominal adipose tissues and hepatic lipid droplets accumulated in overfed 27 dpf zebrafish. After starved for 1,2 and 4 days, zebrafish shown markedly reduced abdominal adipose tissues and the size of liver andthe number of lipid droplets. The abdominal adipose tissues and hepatic lipid droplets basically disappeared after zebrafish starved for 5 days. This phenomenon suggested that lipid of adipose tissues and hepatic lipid droplets were mobilized to maintain energy supply in zebrafish under condition of starvation. To confirm the reappearance of adipose tissues in refed zebrafish, we refed on zebrafish starved for 5 days. Nile Red staining shown that little lipid droplets appear in abdominal cavity in zebrafish refed for 2 days and increased in zebrafish refed for 5 days, but hepatic lipid droplets didn’t changed remarkably. This demonstrated that adipose tissues can reassemble lipid droplets in refed zebrafish after starvation.Conclusions1.Successeful establishment of diet-induced zebrafish larva model for fatty liver, providing a new model for studying the pathogenesis of NAFLD and drug screening of treatment.2.Caffeine attenuate hepatic lipid accumulation in zebrafish larvae through reducing the expression level of genes associated fatty acid intake and lipogenesis and inflammation, upregulating lipid oxidation associated genes, and improving function of ER.3.Nile Red fluorescent staining can label and image in vivo hepatic lipid droplets and adipose tissues in zebrafish larvae, providing a perfect method for observing the development of adipose tissues and accumulation of hepatic lipid droplets in vivo.