Adaptation of AMPK-mTOR-signal pathways and lipid metabolism in response to low- and high-rapeseed meal diet in Chinese perch ( Siniperca chuatsi )

Background: Chinese perch, a carnivorous fish, can accept artificial diet after domestication nowadays, and this farm way will gain high economic interest and sustainability. However, the high content and high quality requirement of dietary protein make it need the high cost in Chinese perch. Therefore, the aim of this study was to explore the effect of fish meal replacement by low- or high-rapeseed meal on growth performance, feeding, lipid and glucose metabolism. Methods: Three experimental diets were formulated with 0, 10% and 30% rapeseed meal, named as control, RSL and RSH, groups respectively. After the 8-week of feeding trial, growth performance, lipid metabolism and AMPK-mTOR-signal pathways were measured. Results: Chinese perch fed with RSH and RSL diets showed significantly decreased WG, SGR, BFR, VSI, MSI and the whole-body crude lipid compared to those fed with the control diet (P < 0.05). Fish in RSL group decreased feed intake, serum LDL-C, hepatic mRNA expression of LPL, PEPCK and phosphorylated Grb10 (P < 0.05). In visceral adipose tissue, mRNA expression of FAS, SREBP1, ACC1, HL, CPT1 and PEPCK were all significantly down-regulated (P < 0.05). Fish in RSH group showed phosphorylated AMPK, hepatic mRNA expression of SREBP1, ACC1, FAS, PPARα and CPT1 were down-regulated, while HSL, G6PD and PC were up-regulated (P < 0.05). In visceral adipose tissue, mRNA expressions of SREBP1, LPL, CPT1 and PEPCK were down-regulated, while mRNA expression of HSL was up-regulated (P < 0.05). Conclusions: Chinese perch fed with RSL and RSH diets showed decreased fat deposition in viscera. Fish fed with low level of rapeseed meal diet ate less diet, which caused inhibited lipid metabolism in the liver and visceral adipose tissues.Fish fed with high level of rapeseed meal diet inhibited hepatic FA synthesis, activated lipolysis, hence reducing


Background
Rapeseed is a crucial oilseed crop in the world, and its oil is one of the main edible oils source [1].
Rapeseed meal is the main co-products of the seed pressing and de-oiling processes [2], and it is an essential plant-protein source in mammals and fish [3]. Using the rapeseed meal replaced the fish meal partial or total for diets has been successfully attempted in some teleost fishes, such as rainbow trout (Oncorhynchus mykiss) [3], European seabass (Dicentrarchus labrax) [4],gilthead sea bream (Sparus aurata) [5]. In comparison with fish meal, the majority of plant-protein, such as rapeseed meal, is short of essential amino acids (EAA) and minerals besides it contains anti-nutritional factors (ANFs) and complex carbohydrates [5]. As an important kind of plant protein, the use of rapeseed is limited. However, despite these shortcomings, plant-protein has made great progress in its substitution in aquaculture. Nevertheless, recent studies have revealed that plant-protein affected lipid metabolism in the livers of salmonids [6][7][8][9].
The metabolism in mammals is regulated by nutrients and energy generally. AMPK is a serine/threonine protein kinase that regulates energy homeostasis, and is a chief kinase regulating mTOR signaling [10]. The mTOR protein kinase established a eukaryotic signaling network that coordinates the cell growth, nutrient metabolism and environmental conditions, Grb10 is key effector downstream of mTOR pathway [11]. As an essential regulator of lipid metabolism and energy homeostasis, Grb10 is relevant to lipolysis and thermogenesis [12].Moreover, activation of AMPK inhibited SREBP, which is a vital lipogenic transcription factor associated with biosynthesis of fatty acids. Activation of AMPK regulates PPARa positively, furthermore, activated fatty acid oxidation [13].
Activation of AMPK could also inhibit ACC activity directly, thereby reducing inhibition of CPT1, leading to an increase in fatty acid oxidation [14].
Chinese perch (Siniperca chuatsi) has great value in freshwater aquaculture of China for rapid growth and delicious flesh [15,16]. Chinese perch merely eat living bait from the initial feeding stage in wild [15]. However, after acceptance of artificial diet, Chinese perch gained high research and economic interest from aquaculture sector. As a carnivorous fish, Chinese perch has a high dietary protein requirement as other carnivorous fish, and the artificial diet of Chinese perch comprises a large portion of fish meal, which caused high dietary and aquaculture cost. As plant protein including rapeseed meal considered a good alternative cost-effective protein source, however the effect of plant protein on growth performance, lipid and glucose metabolism is still little explored, especially in carnivorous fishes. Therefore, the aim of the study was to explore the effect of fish meal replacement with low-and high-rapeseed meal diet on the adaptation of AMPK-mTOR-signal pathways and the lipid metabolism in Chinese perch (Siniperca chuatsi)..

Experimental Diet
Diet composition of the three experimental diets are shown in Table 6. The replacement levels of rapeseed meal were 0, 10 and 30 %, respectively (named as Control, RSL and RSH). The ingredients were purchased from Coland (Wuhan, China). The soft granule diets were pelleted to cylinders form of 1cm in diameter and 1cm in length with the feed machine. Subsequently, the pellets were stored in a freezer at -20 ℃ until used.

Fish and Feeding Trial
Chinese perch were brought from the Innovation Base for Chinese Perch Breeding (Wuhan, China).
Before feeding trial, 250 fish were temporarily kept in 4 tanks (1000L) to weaned on artificial diet for 14 days. At the beginning of the experiment, fish were measured for initial weight and length after 24-hour starvation. Fish were randomly separated to three groups, and then every group has three randomly assigned tanks. Furthermore, every tank contained 15 fish (48.78 ± 4.37 g). Fish were fed to apparent satiation twice a day at 08:00 and 17:00 in the course of the 8-weeks feeding trial.
Uneaten feed was collected and dried after feeding for feed intake and FCR measurement. The filtered flow-through tap water was system was used at a water flow-rate of 3 L min-1. The dissolved oxygen (DO) value was 8.25 -8.76 mg L-1, the temperature ranged from 20 to 22 ℃, the ammonia content was 0.10 ± 0.02 mg L-1 and pH ranged from 7.20 to 7.60 in the course of the experimental period.

Sampling, Body composition and Serum Biochemistry Parameters
After 24-hour starvation, fish were anaesthetized with MS-222 (50 mg L-1 water). Afterwards, final body weight, fish length, visceral mass weight, liver weight and gut weight were measured. After that nine fish were randomly chosen from each tank for western-bloting and RT-PCR analysis. The livers, visceral adipose and brains of Chinese perch were dissected out, frozen in liquid nitrogen and stored at -80 ℃ for subsequent analysis. The blood was collected from caudle vein and placed at room temperature for 2h. Then serum was separated after centrifuged at 3,500 g for 10 min, and stored it at -80 ℃ until used. Three fish were chosen randomly from each tank to analyses whole body composition.
Proximate compositions (Crude protein, crude lipid, moisture and crude ash) of experimental diets, whole body, fish liver and fish muscle were measured according to AOAC, 1995 method [17]. Samples were dried at 105 ℃ for 6 h using the ovens, then the moisture was measured. Crude protein (N ×

Real-time PCR assay
Total RNA was extracted by RNAiso Plus reagent (Takara, Japan). The purity and quantity of RNA was examined by using the BioTek Synergy™ 2 Multi-detection Microplate Reader (BioTek Instruments, Winooski, VT) and by 1.5% agarose gel electrophoresis. Afterward we reverse transcribed 1 µg of RNA into cDNA using SuperScript™ II RT reverse transcriptase (Takara, Japan).
Gene-specific primers for Real-time PCR are shown in Table 7. The primers sequences are based on the published transcriptome sequences of Chinese perch [18]. We evaluated the stability of RPL13A, β-actin, HMBS, B2M, SDHA and YWHA2 following the previous studies [19]. That RPL13A gene was more stable and amplified as the internal control. Real-time PCR was evaluated using a quantitative thermal cycler (MyiQTM 2 Two-Color Real-Time PCR Detection System, BIO-RAD, USA). The reaction volume (20 µL) contained 10µL AceQ® qPCR SYBR® Green Master Mix (Vazyme, USA), 0.4µL forward and reverse primers (10 mM) respectively, 1 µL cDNA and 8.2 µL sterile double-distilled water. The PCR parameters were 95 ℃ for 1 min followed by 40 cycles at 95 ℃ for 10 sec, Tm for 30 sec and a melt curve step (from 95 ℃, gradually reducing 0.5 ℃ s-1 to 57 ℃, with acquisition data every 6 sec). The amplification efficiencies of reference gene and target genes ranged from 95.9 to 105.4 %.
Expressions of mRNA were quantified relative to the expression of RPL13A according to the optimized comparative Ct  value method [20]. Amplification was performed in triplicate for each cDNA sample.
All statistical analyses were carried out using the SPSS 19.0. Data are shown as means ± S. E.M of triplicates. Before one-way ANOVA, all data were subjected to One Sample T-test and normality and homogeneity test. Differences were considered significant at P < 0.05.

Growth performance
After the 8-week of feeding trial, the growth performance of Chinese perch fed with high and low levels of rapeseed meal diets are shown in Table 1, and Table 2 respectively. Fish fed with RSH and RSL diets showed significantly inhibited growth performance including the weight gain and SGR, with compared to control diet (P < 0.05) ( Table. 1). Fish fed with RSH diet showed significantly ascended in FCR, as compared with the control diet (P < 0.05). Moreover, CF in RSH group was significantly reduced as compared to control group diet (P < 0.05) ( Table 2). The BFR and MSI in RSH and RSL groups were significantly reduced as compared to control group (P < 0.05), while there was no significant difference observed in HSI ratio among all groups (P > 0.05) ( Table 2).

Feed intake and appetite genes expression
The feed intake in RSL group was significantly inhibited in comparison to control group (P < 0.05) ( Figure 1). The genes expressions involved in appetite regulation of Chinese perch are shown in Figure 2. The gene expression of NPY was significantly decreased in RSH group, while the gene expression of AgRP was significantly increased in RSL group, whereas gene expression of leptin A was significantly increased in RSH group (P < 0.05).

Whole body, tissue composition
The composition of whole body in Chinese perch fed with high and low levels of rapeseed meal diets was shown in Table 3. The crude lipid and moisture in whole body was significantly decreased in RSH and RSL groups as compared with control group (P < 0.05), the crude ash in whole body were significantly increased in RSL group as compared with control group (P < 0.05). The tissue composition of Chinese perch fed with high and low levels of rapeseed meal diets was presented in Table 4. The moisture in liver was significantly increased in RSH and RSL groups by comparison to control group (P < 0.05). No significant difference was found in crude protein and crude lipid of liver.
Moreover, no significant difference was found in muscle composition including moisture, crude protein and crude lipid (P > 0.05).

Serum biochemistry parameters
The serum biochemistry parameters of Chinese perch fed with high and low levels of rapeseed meal diets are shown in Table 5. Serum LDL-C were significantly decreased in RSH and RSL groups as compared to control group (P < 0.05). The serum glucose and cholesterol were significantly decreased in RSH group as compared to control group (P < 0.05). No significant difference was observed in TG and HDL-C (P > 0.05).

Gene expressions of glucose and lipid metabolism in liver of Chinese perch
Expressions of genes involved in glucose and lipid metabolism of Chinese perch liver fed with high and low levels of rapeseed meal diets are shown in Figure 3. The metabolic regulatory factors involved in lipid metabolism, such as SREBP1 and PPARα, were all decreased in RSH group significantly (P < 0.05) ( Figure 3A). Moreover, genes involved in fatty acid synthesis, such as FAS and ACC1, were all decreased significantly in RSH group, while expression of ACC1 in RSL group was increased significantly (P < 0.05) ( Figure 3B). Genes involved in FA transport, such as HL and LPL, were checked. The expression of LPL in RSL group was decreased significantly (P < 0.05), while no significant difference was found in the expression of HL (P > 0.05) ( Figure 3C). the expression of HSL, an important gene involved in lipolysis, was increased in RSH group (P < 0.05) ( Figure 3D). Expression of CPT1, an important gene involved in fatty acid β-oxidation, was decreased in RSH group (P < 0.05) ( Figure 3E). the expression of PEPCK, involved in glycerol synthesis, was increased significantly in RSH group as compared to RSL group (P < 0.05) ( Figure 3F). In addition, genes involved in glycolysis, such as G6PD and PC, were significantly increased in RSH group (P < 0.05) ( Figure 3G).

Gene expressions of lipid metabolism in visceral adipose tissue of Chinese perch
The expressions of genes involved in glucose and lipid metabolism of Chinese perch visceral adipose tissue are shown in Figure 4. The expression of SREBP1 was significantly decreased in RSL and RSH groups (P < 0.05) ( Figure 4A), while the expression of ACC1 was significantly decreased in RSL group (P < 0.05) ( Figure 4B). the expression of HL was significantly decreased in RSL group, while expression of LPL was significantly decreased in RSH group (P < 0.05) ( Figure 4C). the gene expression of HSL was significantly increased in RSH group (P < 0.05) ( Figure 4D). The expressions of CPT1 and PEPCK were significantly decreased in RSL and RSH groups (P < 0.05) ( Figure 4E, F).

Protein activation levels Analysis of AMPK and mTOR pathway of Chinese perch
Phosphorylation levels of AMPK, Grb10 in liver of Chinese perch fed with high and low levels of rapeseed meal diets are shown in Figure 5-6. In comparison with control group, the phosphorylation level of AMPK was improved significantly in RSH group (P < 0.05) ( Figure 5), In addition, the phosphorylation level of Grb10 had significant difference between RSL and RSH groups (P < 0.05), while no significant difference was observed control group and RSH group (P > 0.05) ( Figure 6).

Discussion
As an important source of plant-protein, rapeseed meal was used to replace dietary fish meal in many teleost fishes successfully [3][4][5]. Nevertheless, due to poor palatability, high anti-nutrititional factors (ANFs) and amino acid imbalance of rapeseed meal, fish will contribute to poor appetite and slow growth [21]. In our study, Chinese perch fed with low or high levels of rapeseed meal both lead to impoverished weight gain and SGR. Similar results were found in juvenile cobia [21], rainbow trout [22], hybrid tilapia [23] and Japanese seabass [24]. This result suggests that the nutrient content of rapeseed meal could not satisfy the growth of Chinese perch compared to fish meal. However, FCR in RSH group was significantly higher than the other two groups in the present study, indicating that low level replacement of rapeseed meal diet caused no significant negative effect on FCR of Chinese perch, while high level replacement of rapeseed meal diet enhanced FCR significantly. It is suggested that excessive rapeseed meal in diet could affect the utilization of Chinese perch.
Some studies showed that less feed intake was one of the reasons for reduced growth performance in fish [22]. In our study, feed intake in RSL group was lower than other two groups. Studies have confirmed that plant-protein rich in ANFs, which makes diet less palatability [25]. This might be the reason why Chinese perch accepted less low-level rapeseed meal diet in the present study. The consequence also illustrated the reason why Chinese perch grew slowly in RSL group. The conclusion was consistent with Chinook salmon [26], rainbow trout [27] and cobia [21]. However, feed intake in RSH group was not affected by high level replacement of rapeseed meal diet, which might be for the reason that the feed utilization ability of Chinese perch in RSH group was poor and they had to take in the diets to meet their body needs. The conclusion was consistent with Japanese seabass [24]. Fish appetite is regulated by appetite-regulating hormones [28]. In the mammalian, NPY and AgRP located at appetite stimulating neurons jointly, while the POMC located at appetite suppressing neurons [29][30][31]. Leptin is recognized as peripheral appetite genes to suppress appetite [32]. Expression of NPY was significantly down-regulated, while expression of Leptin A was significantly up-regulated in RSH group, suggesting that high level replacement of rapeseed meal diet inhibited appetite of Chinese perch by down-regulating mRNA level of NPY and Leptin A. Grass carp fed with plant-protein diets showed low expression of NPY after 24 hours of starvation [33], the result was consistent with our study, this suggests that plant-protein affected the expression of appetite-related genes. In combination with feed intake in RSH group, although fish accepted high level replacement of rapeseed meal diet a lot, but the appetite was inhibited. Expression of AgRP was significantly upregulated in RSL group, that shows that fish in RSL group was in the active state of appetite. Similar results were found in Japanese seabass [24]. In combination with feed intake in RSL group, Chinese perch had an active appetite due to the low feed intake. The expression of appetite-related genes showed a compensatory response.
The CF of a fish is an indicator of nutrition and adaptation to the environment. It is also relevant to the meat content of fish. The low and high CF, means that the fish is thin or fat [34]. Fish fed with high level of rapeseed meal diet had a lower CF compared to other two groups. Similar results were found in gilthead sea bream fed with plant protein sources diets containing rapeseed meal [35], this shows that high level rapeseed meal affected fat deposition. However, the VSI, BFR and MSI in fish fed with low and high levels of rapeseed meal diets decreased as compared to control group, while no significant difference was found in HSI. Similar results were found in gilthead sea bream [35], and in Salminus brasiliensis [36]. The results further proved that the replacement of rapeseed meal could decrease fat deposition in viscera rather than the liver. The composition of whole body, liver and muscle tissue were measured to further prove this hypothesis. Crude lipid in whole body of fish fed with low and high levels of rapeseed meal diets decreased as compared to control group, while no significant difference was found in liver and muscle. Combined with the above results, the replacement of rapeseed meal with fish meal could decrease fat deposition in viscera rather than in liver.
In the present study, serum glucose and cholesterol were significantly decreased in RSH group, while no significant difference was observed in TG. The effect of plant protein on blood glucose in fish depends on fish species, the source and level of plant protein [37]. Japanese seabass fed with a full plant protein diet decreased blood glucose level [38]. However, there was no significant difference in blood glucose of Trachinotus carolinus fed with soybean meal diet was observed [37,39]. Our study revealed that high level of rapeseed meal could decrease blood glucose, which suggests that glucose uptake into tissues and glucose phosphorylation levels might be improved. Numerous studies had found that plant protein diets can reduce serum cholesterol levels in rainbow trout [40], European seabass [41] and Psetta maxima [42]. Our results were consistent with the conclusion, that the effect of high level of rapeseed meal on lowering plasma cholesterol in fish might be related to the low cholesterol content in feed [43]. LDL-C carries cholesterol from the liver to tissues, while HDL-C carries cholesterol back from tissues back to the liver for metabolism. In our study, serum LDL-C was significantly decreased in RSH and RSL groups as compared to control group, while no significant difference was observed in HDL-C. This result suggests that the replacement of rapeseed meal reduced the transport of cholesterol from the liver to tissues.
Chinese perch fed with low and high level of rapeseed meal diet all decreased fat deposition in viscera. Grb10 could promote lipolysis and thermogenesis by negative regulation of the mTOR signaling pathway [12]. In our study, fish fed with low level replacement of rapeseed meal diet phosphorylated Grb10, indicated that diet with low level of rapeseed meal could activate Grb10, and inhibit mTOR pathway, that enhanced lipolysis and thermogenesis of Chinese perch. Similar results were found in grass carp [44] and juvenile white shrimp [45], the results are in line with other studies.
Furthermore, lipid and glucose metabolism related genes were measured to demonstrate the specific mechanism of Chinese perch response to rapeseed meal. In liver tissue, mRNA expression of ACC1, which is involved in FA synthesis, was up-regulated, while mRNA expression of LPL, which is involved in lipid uptake, was down-regulated in Chinese perch fed with low level of rapeseed meal diet. It is suggested that Chinese perch fed with low level of rapeseed meal diet promoted FA synthesis, and inhibited lipid uptake in liver tissue. Similar results were found in Japanese seabass [38]. It is suggested that the reduction in fat accumulation caused by low level of rapeseed meal did not occur in the liver mainly. In visceral adipose tissue, mRNA expression of FA synthesis related genes (SREBP1, ACC1),, lipid uptake relative gene (HL),, fatty acid β-oxidation relative gene (CPT1) and glycerol synthesis relative gene (PEPCK) were all down-regulated. It is suggested that the reduction in fat accumulation caused by the low level of rapeseed meal occurred in the visceral adipose mainly, all fat metabolic activity was suppressed. Combined with food intake results, the results show that fish in RSL group ate less food and lipid, which caused inhibited lipid metabolism in the liver and visceral adipose tissues. AMPK has been considered as a sensitive energy sensor in cells [46]. In our study, high level of rapeseed meal diet significantly activated AMPK pathway in Chinese perch. This shows that the lack of energy intake of Chinese perch in RSH group activated the energy-sensing pathway. Similar results were observed in turbot [47]. In liver tissue, mRNA expression of FA synthesis relative genes (SREBP1, ACC1 and FAS) were down-regulated, mRNA expression of fatty acid β-oxidation relative genes (PPARα and CPT1) were all down-regulated, mRNA expression of lipolysis relative gene (HSL) was upregulated, while mRNA expression of glycolysis related genes (G6PD and PC) were up-regulated. It is suggested that FA synthesis was inhibited, while lipolysis was promoted, these metabolic processes caused Acetyl-CoA pool reduced. The fatty acid β-oxidation were inhibited, and glycolysis was promoted, lipid accumulation was decreased when Chinese perch fed with high level replacement of rapeseed meal diet. At the same time in visceral adipose tissue, mRNA expressions of SREBP1, LPL, CPT1 and PEPCK were down-regulated, while mRNA expression of HSL was up-regulated. It is suggested that lipid uptake was inhibited, that caused FA synthesis, and fatty acid β-oxidation, glycerol synthesis were inhibited, which caused improved lipolysis.

Conclusion
Chinese perch fed with RSL and RSH diets showed decreased fat deposition in viscera. Fish fed with low level of rapeseed meal diet showed decreased in feed intake, serum LDL-C, hepatic mRNA expression of LPL and PEPCK and phosphorylated Grb10. In visceral adipose tissue, mRNA expression of FA synthesis related genes (SREBP1, ACC1),, lipid uptake relative gene (HL),, β-oxidation relative gene (CPT1) and glycerol synthesis relative gene (PEPCK) were all down-regulated, this show that fish in RSL group ate less diet, which caused inhibited lipid metabolism in the liver and visceral adipose tissues.
Fish fed with high level of rapeseed meal diet showed phosphorylated AMPK, hepatic mRNA expression of FA synthesis relative genes (SREBP1, ACC1 and FAS) was down-regulated, while lipolysis relative gene (HSL) was up-regulated, and these metabolic processes reduced Acetyl-CoA pool. In turn, β-oxidation relative genes (PPARα and CPT1) were all down-regulated, while glycolysis relative genes (G6PD and PC) was up-regulated, thus lipid accumulation were decreased. In visceral adipose tissue, mRNA expression of SREBP1, LPL, CPT1 and PEPCK was down-regulated, while mRNA expression of HSL was up-regulated, which shows that lipid uptake were inhibited, caused inhibited FA synthesis, β-oxidation, glycerol synthesis, and improved lipolysis.

Consent for publication
Not applicable Availability of data and materials All data used to arrival at conclusions of this paper are present in this manuscript. The raw data is available from the authors on request.

Competing interests
The authors declare that they have no competing interests.  Tables   Table 1 Compositions of diets replacement with low and high level of rapeseed meal

Table 2
Primers used in this experiment Table 3 Growth performance and feed utilization of Chinese perch fed with different levels of rapeseed meal diet. Data are presented as mean ±S.E.M of three replicates (n = 6). The same row with different letters indicates significant differences between groups based on one-way analysis of variance (ANOVA) followed by the post hoc test (P < 0.05).

Table 4
Fatness of Chinese perch fed with different levels of rapeseed meal diet. Data are presented as mean ±S.E.M of three replicates (n = 6). The same row with different letters indicates significant differences between groups based on one-way analysis of variance (ANOVA) followed by the post hoc test (P < 0.05). Data are presented as mean ±S.E.M of three replicates (n = 6). The same row with different letters indicates significant differences between groups based on one-way analysis of variance (ANOVA) followed by the post hoc test (P < 0.05). Data are presented as mean ±S.E.M of three replicates (n = 6). The same row with different letters indicates significant differences between groups based on one-way analysis of variance (ANOVA) followed by the post hoc test (P < 0.05).

Table 7
Serum biochemistry parameters of Chinese perch fed with different levels of rapeseed meal diet.      Phosphorylation of AMPK of Chinese perch fed with different levels of rapeseed meal diet.
Data are presented as mean ±S.E.M of three replicates (n = 6). The same row with different letters indicates significant differences between groups based on one-way analysis of variance (ANOVA) followed by the post hoc test (P < 0.05).

Figure 6
Phosphorylation of Grb10 of Chinese perch fed with different levels of rapeseed meal diet.
Data are presented as mean ±S.E.M of three replicates (n = 6). The same row with different letters indicates significant differences between groups based on one-way analysis of variance (ANOVA) followed by the post hoc test (P < 0.05).

Figure 7
Metabolic process of Chinese perch fed with RSL diet. Fish fed with low level replacement of rapeseed meal diet decreased in feed intake and phosphorylated Grb10, which was improved lipolysis, inhibited glycerol synthesis and lipid uptake. These metabolic activities decreased transportation of lipid from liver to visceral adipose tissue, then FA synthesis, βoxidation and glycerol synthesis were inhibited. Finally, lipid accumulation of Chinese perch were decreased.

Figure 8
Metabolic process of Chinese perch fed with RSH diet. Fish fed with RSH diets phosphorylated AMPK, inhibited FA synthesis, while improved lipolysis, and these metabolic processes reduced Acetyl-CoA pool. In turn, -oxidation was inhibited, while glycolysis was improved, and then lipid accumulation was decreased. In visceral adipose tissue, lipid uptake were inhibited, inhibiting FA synthesis, β-oxidation, glycerol synthesis, improving lipolysis.