Abstract
Carbohydrates are the most economical source of energy in fish feeds, but most fish have limited ability to utilize carbohydrates. It has been reported that phosphoenolpyruvate carboxykinase 1 (pck1) is involved in carbohydrate metabolism, lipid metabolism, and other metabolic processes. However, direct evidence is lacking to fully understand the relationship between pck1 and glucose and lipid metabolism. Here, we generated a pck1 knockout zebrafish by CRISPR/cas9 system, and a high-carbohydrate diet was provided to 60 days post-fertilization (dpf) for 8 weeks. We found that pck1-deficient zebrafish displayed decreased plasma glucose, elevated mRNA levels of glycolysis-related genes (gck, pfk, pk), and reduced the transcriptional levels of gluconeogenic genes (pck1, fbp1a) in liver. We also found decreased triglyceride, total cholesterol, and lipid accumulation and in pck1−/− zebrafish, along with downregulation of genes for lipolysis (acaca) and lipogenesis (cpt1). In addition, the observation of HE staining revealed that the total muscle area of pck1−/− was substantially less than that of WT zebrafish and real-time PCR suggested that GH/IGF-1 signaling (ulk2, stat1b) may be suppressed in pck1-deficient fish. Taken together, these findings suggested that pck1 may play an important role in the high-carbohydrate diet utilization of fish and significantly affected lipid metabolism and protein synthesis in zebrafish. pck1 knockout mutant line could facilitate a further mechanism study of pck1-associated metabolic regulation and provide new information for improving carbohydrate utilization traits.
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Introduction
Carbohydrates are not only an important source of energy for the body, but also the most economical source of energy in fish feed. Carbohydrates have been widely added to fish feed to improve feed quality, reduce catabolism of other nutrients for energy, and supply metabolic intermediates for the production of numerous biological compounds (Wilson, 1994). However, the ability of different fishes to digest digestible carbohydrates varies greatly. In general, carnivorous fish have poorer carbohydrate digestibility than omnivorous and herbivorous fish (Krogdahl et al., 2005), manifested as persistent glucose intolerance such as hyperglycemia syndrome after intaking high-carbohydrate diet (HCD) (Moon, 2001). Long-term intake of HCD may also lead to vacuolation of liver parenchymal cells, decrease antioxidant capacity, inhibit immune function of fish, and induce inflammation, thereby limiting fish growth and ultimately affecting aquaculture profitability (Alexander et al., 2011; Goodwin et al., 2002). Hence, the study on the regulation of carbohydrate metabolism and the mechanism of glucose homeostasis in fish deserves special attention.
In the process of carbohydrate metabolism in fish, it is very important to maintain the balance between glucose anabolism and catabolism, among which one of the most important processes is gluconeogenesis pathway (Enes et al., 2009; Hemre et al., 2002). Phosphoenolpyruvate carboxykinase 1 (Pck1) is one of the rate-limiting steps of the gluconeogenesis pathway. It can catalyze the conversion of oxaloacetate to phosphoenolpyruvate, which can be converted to glucose through subsequent enzymatic reactions (Matte et al., 1997; Yang et al., 2009). In fish, pck includes cytoplasmic (C-type) and mitochondrial (M-type) subtypes, named pck1 and pck2, respectively. Based on previous reports, pck2 may not be regulated by dietary carbohydrate levels, whereas pck1 is sensitive to dietary carbohydrate responses (Yu et al., 2021). As a result, most review revealed that pck1 (but not pck2) is linked to gluconeogenesis, obesity, and diabetes (Arai et al., 2015; Beddow et al., 2019; Hanson and Hakimi, 2008). A study on the rainbow trout (Oncorhynchus mykiss) demonstrated that the increase of liver glycogen induced by glucagon stimulation was due to the transcriptional activation induction of pck1 and glycogen phosphorylase (Forbes et al., 2019). On the other hand, overexpression of pck1 in two hepatocellular carcinoma cells (hep3B and SMMC-7721) was associated with enhanced glucose production and limited production of pyruvate, lactate, citrate, and malate, and these effects were also observed in a rat model and was associated with inhibition of glycolysis and induction of gluconeogenic pathways (Tang et al., 2018). The above findings revealed that pck1 may be essential in protecting blood glucose homeostasis. However, it was observed that pck1 expression levels were maintained at high levels in studies of carnivorous fish such as rainbow trout, Perch (Perca fluviatilis), and Atlantic salmon (Salmo salar), which greatly inhibited the utilization of carbohydrates (Borrebaek and Christophersen, 2000; Enes et al., 2006; Moreira et al., 2008; Panserat et al., 2001). Additionally, in Chinese perch (Siniperca chuatsi), the expression level of pck1 was upregulated with the increase of dietary carbohydrate level, which promoted the further increase of plasma glucose, indicating that the lack of effective regulation of pck1 may lead to the poor utilization of carbohydrates in carnivorous fish (Li et al., 2019a; Marandel et al., 2015; Zhou et al., 2016). However, direct evidence is lacking to fully understand the relationship between pck1 and glucose and lipid metabolism.
Zebrafish has become an important model for fish nutrition and metabolism research (Seiliez et al., 2013; Fowler et al., 2020; Zhang et al., 2018; Wang et al., 2021; Idris et al., 2022; Williams 2021). Utilizing the zebrafish as the model, we generated zebrafish pck1 mutant using CRISPR/cas9 system. To explore the important role of pck1 in carbohydrate utilization, the pck1-deficient zebrafish were fed with high-carbohydrate diet condition (HCD) for 8 weeks, confirming that pck1 may play an important role in the utilization of high-carbohydrate diets in fish while negatively affect lipid metabolism and protein synthesis. These findings suggest that impaired pck1 signaling facilitates a further mechanism study of pck1-associated metabolic regulation and provides guidance for improving carbohydrate utilization in fish.
Methods
Fish maintenance
Wild-type zebrafish (AB line) were purchased from the National Zebrafish Resource Center, Institute of Hydrobiology, Chinese Academy of Sciences (http://www.zfish.cn/), and fish were raised in the environment of 28 °C and 14-h light/10-h dark per day. All experiments were approved by the Institutional Animal Care and Use Ethics Committee of Huazhong Agricultural University.
CRISPR/Cas9-induced mutagenesis
CRISPR/Cas9 target sites in pck1 were designed using CRISPR/Cas9 target online predictor (CHOPCHOP, http://chopchop.cbu.uib.no/) (Labun et al., 2016), and selected target sequences of pck1 was designed in the fourth exon and sequences were 5′-AACTGGCCCTGTAACCCGG-3′. The target guide RNA (gRNA) was synthesized using TranscriptAid T7 High Yield Transcription kit (Thermo, Scientific) and then recovered by lithium chloride precipitation. The Cas9 protein (NEB, USA) and gRNA (50 ng/μL) were microinjected into one/two-cell stage zebrafish embryos. After 48 h of injection, 10–20 embryos were collected and extracted genomic DNA to judge target efficiency. A pair of primers (forward: 5′-GGCGTGTGATGTACGTGAT-3′, reverse: 5′-CCCTCCTCTTTAGCGATGCG-3′) was amplified by PCR and sequenced. The F0 founders were crossed with wide-type, and F1 adult fish identified each mutant allele by PCR and sequencing analysis. The F1 zebrafish with the same mutation sequences were self-crossed to get F2 mutants, and homozygotes were screened. F3 generation was produced from F2 by sibling mating. All experiments were performed using the pck1−/− F3 mutants.
Experimental diets
The main nutrient components in the trial diet were bought from Gao Long Dietary Company (Wuhan, China) and passed a 180-μm mesh. The main nutrient components of the trial diet included 41% carbohydrates according to previous research (Yang et al., 2018). All larvae fed paramecia from 6 to 10 days post-fertilization (dpf) and thereafter fed newly hatched Artemia twice a day until 60 dpf. To avoid the effects of sex differences in metabolism (Fowler et al., 2019; Kamstra, 2021), wild-type (WT) zebrafish males and pck1−/− zebrafish males were randomly selected and cultured in triplicate under tank (13 × 9 × 9 cm) (10 zebrafish in per tank)-based recirculating systems. After training to fed diet for 2 weeks, all experimental fish were fed with the high-carbohydrate diet twice a day (09:00 and 18:00) for 8 weeks.
Sampling and chemical analyses
After the 8-week feeding, fish were starved for 12 h and anesthetized with 80 mg/L trivacaine methane sulfonate (MS-222, Sigma, USA) prior to sampling. Blood samples were collected at the caudal vein. For total RNA extraction and chemical analyses, the livers and muscles were kept at − 80 °C after being frozen in liquid nitrogen. Besides, parts of livers and muscles were preserved in 4% paraformaldehyde solution (PFA) for histological testing. The levels of glucose, total cholesterol, and total triglyceride in plasma and total triglyceride in livers were estimated by commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [glucose assay kit (catalog no. F006-1-1), total cholesterol assay kit (catalog no. A111-1-1), total triglyceride assay kit (catalog no. A110-1-1), and total protein assay kit (with standard: BCA method) (catalog no. A045-4-1)] following the producer’s guidelines.
Histology analysis
Liver (n = 3) and muscle (n = 3) tissues were dehydrated and frozen microtomy with thickness of 4 μm (Leica, Germany) and stained with hematoxylin-eosin (H&E) and Oil red O staining according to standard protocols. The slides were imaged by slice digital scanning (Pannoramic250, Pannoramic250 MIDI, 3D HISTECH). The liver sections were viewed at 40 × magnification. Three slides from each group were included in quantification, and 3 fields were randomly selected on each slide. The cross-section at the base of the cloaca was selected to quantify the area of muscle as previously described, and the muscle sections were viewed at 20 × magnification (Gao et al., 2016). Using ImageJ software, the number of cell nuclei of liver tissue and the area of muscle tissue in the H&E and the relative area of lipid droplet in the Red Oil O were all measured for each field.
Real-time quantitative PCR
Total RNA was extracted from each sample according to the RNAiso instruction and reversed transcribed to synthesize the first-strand cDNA using the HiScript® III 1st Strand cDNA Synthesis Kit (Vazyme, China). Each reaction mixture (20 μL) contained 1 μL cDNA template, 10 μL SYBR (Vazyme, China), 0.4 μL of each primer, and 8.2 μL ddH2O. The gene (actb2) was used as the endogenous reference and inflammation-related genes (il-1β and tgf-β), glycometabolism genes (pck1, fbp1a, fbp1b, g6pca.1, gck, pfk, and pk), lipid metabolism genes (acaca, fasn, pparγ, cpt1, pparα, and lpl), and protein synthesis genes (igf1, igf1rb, ulk2, mat2aa, stat1b, and 4ebp1) used in the present study are listed in Table 1. The reaction conditions were set as follows: 95 °C for 30 s, 40 cycles at 95 °C for 10 s, 58 °C for 30 s, and melting curve from 65 to 95 °C (gradually increasing 0.5 °C s−1) with data acquired every 6 s. The results were normalized to actb2, and relative transcript abundances of genes were performed using the 2−ΔΔCt value method (Livak and Schmittgen, 2001).
Statistical analysis
Dates were analyzed by SPSS 25 software (SPSS, USA). All results are presented as mean ± standard error of the mean (S.E.M), and the normality of the data was first tested by the Shapiro-Wilk test. The differences between WT and pck1−/− were compared by independent-sample t test, and the significance level was set at P < 0.05.
Results
Generation of the zebrafish pck1 −/− mutant lines
We used the CRISPR/Cas9 system to construct pck1 mutant zebrafish to explore the role of pck1 in glucose and lipid metabolism. The target was designed in the fourth exon and a mutant line with 7 base-pair deletion (pck1−/−∆7) was obtained (Fig. 1A). And the deletion of DNA resulted in premature stop of the encoded PCK1 protein, disrupting the PEPCK_N and PEPCK_GTP domains of pck1 (Fig. 1B). The development of pck1−/− zebrafish was morphologically normal and fertilizable (data not shown). They generally became mature at 60–90 dpf in our aquarium system.
Establishment of pck1 zebrafish knockout line. A Schematic diagram of pck1 knockout. Yellow rectangles indicated the exons of pck1. Red rectangles indicated the locations of guide RNA. The sequencing wave diagram is displayed below the genomic structure. B Comparison of pck1−/−∆7 and with wild-type pck1 protein structure. Zebrafish PCK1 protein contained conserved phosphoenolpyruvate carboxykinase N-terminal domain (PEPCK_N) and phosphoenolpyruvate carboxykinase C-terminal P-loop domain (PEPCK_GTP)
Plasma glucose and glucose metabolism in pck1-deficient zebrafish
The levels of glucose in the plasma in pck1-deficient zebrafish were significantly decreased compared to WT fish (P < 0.05) (Fig. 2A). To investigate if pck1 depletion increased the expression of glycolysis genes, the relative mRNA level of glucokinase (gck), phosphofructokinase (pfk), and pyruvate kinase L/R (pk) was analyzed by quantitative PCR. The transcriptional levels of gck, pfk and pk were markedly upregulated in the liver of pck1−/− zebrafish, compared with those in the liver of WT zebrafish (P < 0.05) (Fig. 2B). Furthermore, the expression of gluconeogenic genes pck1 and fbp1a was significantly decreased in pck1-deficient zebrafish (P < 0.05) (Fig. 2C). However, no differences were detected in the transcription of fbp1b and g6pca.1 (P > 0.05) (Fig. 2C).
The effect of the loss of pck1 on glucose metabolism. A The levels of glucose in the plasma in WT and pck1−/− zebrafish (n = 9). Relative mRNA expression levels of glycolysis (B) and gluconeogenic (C) genes in liver of WT and pck1−/− zebrafish fed with the high-carbohydrate diet (n = 6). Data are represented as mean ± SEM and *P < 0.05 by Student’s t test
HCD reduces lipid accumulation in pck1 −/− zebrafish
To investigate the role of pck1 in the accumulation, we performed histological analyses of the liver using sectioning and hematoxylin and eosin (H&E) staining and Oil Red O staining. The amounts of hepatic cell nuclei were significantly increased, while the relative area of lipid droplets in the liver was significantly decreased in pck1-deficient zebrafish (P < 0.05) (Fig. 3A–D). Simultaneously, the total triglyceride level in the liver of pck1−/− zebrafish showed a significant decrease compared to WT zebrafish (P < 0.05) (Fig. 3E). Additionally, the expression of interleukin 1, beta (il-1β) was strongly enhanced in the liver tissue of pck1-deficient zebrafish (P < 0.05) (Fig. 3F).
Effects of pck1 deletion zebrafish on lipid accumulation and transcript levels of inflammation-related genes in the liver. H&E staining (A) and Oil Red O staining (B) (40 × magnification) of the hepatic tissue (n = 3) for WT and pck1−/−. C Measurement of hepatic cell nuclei. D Oil red O relative area in the liver. Ld, lipid droplet; Nu, nuclei. E Hepatic triglyceride (TG) content of WT and pck1−/− (n = 5). Relative expression levels of il-1β (F) and tgf-β (G) (n = 6). Values are represented as mean ± SEM (n = 5), *P < 0.05
Transcriptional level of lipid metabolism-related genes
To further illustrate the function of pck1 in lipid metabolism, we first detected the level of triglycerides and cholesterol in plasma. The concentrations of triglyceride (TG) (A) and total cholesterol (T-CHO) in pck1−/− mutants were significantly lower than in WT zebrafish (P < 0.05) (Fig. 4A, B). We also compared the mRNAs levels of key genes involved in lipolysis and lipolytic between pck1−/− and WT zebrafish. The relative mRNA levels of acetyl-CoA carboxylase alpha (acaca), a fatty acid synthesis gene, displayed a decrease in pck1−/− livers compared to WT zebrafish (P < 0.05) (Fig. 4C). The transcriptional levels of fatty acid synthase (fasn) and peroxisome proliferator-activated receptor gamma (pparγ) were not significantly different between pck1−/− and WT fish (P > 0.05) (Fig. 4C). pck1−/− zebrafish had significantly reduced levels of carnitine palmitoyltransferase 1 (cpt1) compared to wild-type (P < 0.05) (Fig. 4D), although there were no differences in peroxisome proliferator-activated receptor alpha a (pparα) and lipoprotein lipase (lpl), and both are related to lipolysis (P > 0.05) (Fig. 4D).
The effect of the loss of pck1 on lipid metabolism. The levels of triglyceride (TG) (A) and total cholesterol (T-CHO) in the plasma of pck1−/− mutants (B) (n = 8). The qRT-PCR results of lipolysis (C) and lipolytic (D) genes in the liver between WT and pck1−/− zebrafish (n = 6). Results are represented as mean ± SEM and *P < 0.05
Disruption of pck1 inhibits protein synthesis in muscle tissue
The observation of HE staining revealed that the total muscle area of pck1−/− was substantially less than WT zebrafish (P < 0.05) (Fig. 5A, B). Additionally, in pck1−/− zebrafish muscle tissue, the mRNA expression levels of genes involved in protein synthesis were confirmed. In pck1−/− group, the transcription levels of u nc-51-like autophagy activating kinase 2 (ulk2) and signal transducer and activator of transcription 1b (stat1b) in muscle were significantly decreased than WT zebrafish (P < 0.05) (Fig. 5C). By contrast, no significant difference in the expression of insulin-like growth factor 1 (igf1), insulin-like growth factor 1b receptor (igf1rb), methionine adenosyl-transferase II alpha a (mat2aa), and eukaryotic translation initiation factor 4E-binding protein 1 (4ebp1) in muscle was observed between pck1−/− and WT zebrafish (P > 0.05) (Fig. 5C).
General features of the muscle tissues of the pck1−/− mutant. A H&E straining of (20 × magnification) of the muscle tissue (n = 3) for WT and pck1−/−. B Total muscle area of WT and pck1−/− (n = 3). C Transcript levels of protein synthesis genes in the muscle of WT and pck1−/− zebrafish after feeding a high-carbohydrate diet (n = 6). All data are represented as mean ± SEM, and statistical significance between WT and statistical significance (*) was evaluated as P < 0.05
Discussion
As a key gluconeogenic enzyme, the role of pck1 is primarily defined by the high level of transcriptional control it catalyzes in response to hormonal signals (Shi et al., 2020a). In zebrafish, PEPCK have two isoforms, cytoplasmic (PCK1 or PCK-C) and mitochondrial (PCK2 or PCK-M). Previous research has demonstrated that PCK1 activity is modulated by hormonal conditions such as glucagon (mediated by cAMP), glucocorticoids, thyroid hormones, and nutritional conditions such as diet, whereas PCK2 activity is unaffected (Hanson and Reshef, 1997; Li et al., 2019b; Pilkis and Granner, 1992). In recent years, pck1 has been identified as a key pathway for hepatic gluconeogenesis and widely involved in many other biosynthetic and oxidative processes (Semakova et al., 2017; Yang et al., 2009). Therefore, pck1 is considered essential for glucose homeostasis. In this study, we utilized CRISPR/Cas9 approach to generate a pck1 mutant line with 7 base-pair deletions that caused premature termination of the phosphoenolpyruvate carboxykinase N-terminal (PEPCK_N) domain and phosphoenolpyruvate carboxykinase C-terminal P-loop (PEPCK_GTP) domain in PCK1 (Fig. 1). We also detected a significant decrease in the mRNA expression level of pck1 in the liver (Fig. 2C). Zebrafish as a model for the investigation of energy homeostasis. Here, we examined biochemical, histological, and differentiation expressed genes in glucose metabolism, lipid metabolism, and protein synthesis under the condition of high-carbohydrate diet condition (HCD) between pck1−/− and WT zebrafish (males were chosen in this study), confirming that pck1 may play an important role in the utilization of high-carbohydrate diet in fish.
In general, the activation of the gluconeogenic pathway in response to a high-carbohydrate diet will further increase plasma glucose levels, resulting in a hyperglycemic syndrome (Wassana et al., 2017; Zhang et al., 2021). Various physiological processes of gluconeogenesis are impaired when pck1 is knocked out, especially the physiological processes of glucose utilization. Pck1−/− zebrafish fed with high-carbohydrate diet exhibited reduced plasma glucose compared to WT (Fig. 2A), in agreement with decreased plasma glucose observed in hepatic pck1 downregulation in mice (Yuan et al., 2019), demonstrating that decreased gluconeogenesis leads to decreased plasma glucose levels. She et al. (2000) used Cre/loxP strategy to obtain a systemic cytoplasmic phosphoenolpyruvate carboxykinase knockout mice (PCK1-KO); these mice developed severe hypoglycemia and did not survive past 3 days of birth. Moreover, overexpression of pck1 in hepatocellular carcinoma (HCC) manifested as increased glucose production (Tang et al., 2018). These data were consistent with plasma glucose levels in the pck1−/− zebrafish. We further examined the expression levels of genes related to glycolysis and gluconeogenesis in liver to explore the role of pck1 in glucose metabolism. Deletion of pck1 greatly increased the expression levels of gck, pfk, and pk involved in glycolysis, while downregulated the expression levels of gluconeogenic related genes (pck1, fbp1a) in liver tissues (Fig. 2B, C). The glycolysis pathway is an essential pathway of glucose oxidation for energy supply, and the upregulation of the mRNA levels of related genes means that the utilization of glucose is more active, which will facilitate more glucose to enter the tricarboxylic acid cycle for oxidative energy supply, thus leading to the reduction of plasma glucose (Polakof et al., 2012). Interestingly in mammals, plasma glucose levels can be rapidly reduced to maintain glucose homeostasis by inhibiting the gluconeogenesis pathway (Kuo et al., 2015). When fed with high-carbohydrate diets, the decrease of plasma glucose will help fish to relieve the metabolic pressure of high-glucose diet metabolism, implying that pck1 is associated with the utilization of carbohydrates by a high-carbohydrate diet.
Glucose (excess) conversion of fatty acids is also a major pathway in glucose homeostasis (Cai et al., 2018). It has been observed that some fish alleviate hyperglycemia by activating the lipid synthesis pathway, and these synthetic fats could be transferred to peripheral tissues via apolipoproteins in the plasma to improve hepatic lipid deposition (Wang et al., 2018; Yang et al., 2020). In the current study, pck1-deficient zebrafish significantly decreased the expression levels of the genes related to fatty acid synthesis (acaca) and fatty acid oxidation (cpt1) (Fig. 3C, D), thereby reducing fat deposition, and these results were confirmed by liver histology (Fig. 2A, B). Fatty acid β-oxidation is regulated by acaca and cpt1, which interact through the intermediate malonyl-CoA (Schreurs et al., 2010). In hybrid grouper, feeding a bile acid diet supplemented with antibiotic mixture (ASBD) reduced lipid deposition and downregulated the mRNA of acaca and cpt1 in the liver which is consistent with our results (Xu et al., 2022b). In juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), intraperitoneal injection of guggulsterone (an FXR antagonist, I-TGU) significantly enhanced hepatic lipid deposition and upregulated the mRNA of acaca and ctp1 (Xu et al., 2022a). However, no significance was detected in the transcriptions of fatty acid synthesis–related genes (fasn and pparγ) and fatty acid oxidation–related genes (pparα and lpl) between pck1−/− and WT zebrafish (Fig. 4). In addition, the reduced TG content in plasma and liver also implied the inactivity of lipid transport (Figs. 2E and 3A), indicating that hepatic endogenous triglycerides are rarely transported through the blood circulation to other peripheral tissues. In a previous study, it was shown that inhibition of the gluconeogenesis pathway also reduces the synthesis of fat, thereby reducing the content of triglycerides (Olswang et al., 2002).
What is more, pck1 was reported to be involved in regulating the adaptive response to inflammation, and the deletion of pck1 in macrophages led to an increase in lactic acid production, which promoted the proinflammatory phenotype (Ko et al., 2018). Il-1β is a pro-inflammatory cytokine that plays a key role in acute and chronic inflammation, and tgf-β is another significant pleiotropic cytokine with powerful immunomodulatory properties (Ren and Torres, 2009; Yoshimura et al., 2010). Our results displayed that il-1β expression was significantly increased in pck1-null zebrafish (Fig. 3F), illustrating that loss of pck1 increases the pro-inflammatory phenotype of the liver, which is in line with findings in a mouse model of disruption of specific pck1 to myeloid cells (Ko et al., 2018). These results suggested that there may be inflammation in the liver of pck1-deficient zebrafish.
Pck1 mutant zebrafish affected glucose and lipid metabolism, and we further demonstrate the function of pck1 in protein synthesis. In our study, pck1−/− fish showed reduced muscle area after feeding high-carbohydrate diet (Fig. 5A, B). As previously shown by Semakova et al. (2017), PEPCK-C might be involved in the robustness of at least some muscle fiber types, confirming the functional role of endogenous PEPCK-C in muscle. Overexpression of PCK1 in skeletal muscle of mice showed great levels of physical activity that persisted into old age, possibly due to increased mitochondrial numbers and high triglyceride concentrations in skeletal muscle (Koo et al., 2018). In addition, PEPCK-C is a key step of glycerol regeneration, and is most likely to participate in the triglyceride-fatty acid cycle in skeletal muscle (Reshef et al., 2003). Therefore, we hypothesized that the loss of pck1 disrupted the triglyceride-fatty acid cycle in skeletal muscle, leading to a reduction in muscle mass. In addition, previous research demonstrated that GH/IGF-1 signaling is crucial for regulating the skeletal muscle growth (Schiaffino and Mammucari, 2011). Consistently, we checked that pck1 deficiency zebrafish fed HCD showed significantly downregulated expression of ulk2 and stat1b, while the expression of igf1, igfbr, mat2aa, and 4ebp1 all showed a downward trend with no significant difference (Fig. 5C). These results suggested that, although the mechanism was unknown, GH/IGF-1 signaling may be suppressed in pck1-deficient fish. In mouse, ulk2 is highly expressed in muscle and acts as a potentially significant regulator of protein homeostasis in skeletal muscle, playing an essential role in retaining skeletal muscle strength, mass, and muscle fiber integrity (Fuqua et al., 2020; Fuqua et al., 2019). Regarding stat1, studies have shown that stat1 disruption led to increased autophagy in mouse skeletal muscle, which affected skeletal muscle protein metabolism (Goldberg et al., 2017; Mofarrahi et al., 2012). These results indicated that disruption of pck1 affects protein synthesis in muscle, and protein synthesis is involved in protein homeostasis and metabolism, which might be due to the damage of GH/IGF-1 signals on protein synthesis. Further studies are needed to fully understand the mechanism of reduced muscle protein synthesis in pck1−/− zebrafish.
In summary, taking pck1 knockout zebrafish with high carbohydrate diet as the model, our studies reveal that pck1 plays an important role in maintaining both gluconeogenesis and glycolysis. Lack of pck1 inhibits lipid catabolism by promoting glycolysis, thereby reducing the accumulation of liver fat, and additionally reducing muscle protein synthesis, which may be related to GH signaling (Fig. 6).
Data availability
All data are available from the corresponding author by request.
References
Alexander C, Sahu N, Pal A, Akhtar M (2011) Haemato-immunological and stress responses of Labeo rohita (Hamilton) fingerlings: effect of rearing temperature and dietary gelatinized carbohydrate. J Anim Physiol An N 95(5):653–663. https://doi.org/10.1111/j.1439-0396.2010.01096.x
Arai T, Kano F, Murata M (2015) Translocation of forkhead box O1 to the nuclear periphery induces histone modifications that regulate transcriptional repression of PCK1 in HepG2 cells. Genes Cells 20(4):340–357. https://doi.org/10.1111/gtc.12226
Beddow SA, Gattu AK, Gattu DF, Paolella L, Alqarzaee L, Tashkandi N, Popov VB, Church CD, Rodeheffer MS, Cline GW, Geisler JG, Bhanot S, Samuel VT (2019) PEPCK1 antisense oligonucleotide prevents adiposity and impairs hepatic glycogen synthesis in high-fat male fed rats. Endocrinology 160(1):205–219. https://doi.org/10.1210/en.2018-00630
Borrebaek B, Christophersen B (2000) Hepatic glucose phosphorylating activities in perch (Perca fluviatilis) after different dietary treatments. Comp Biochem Physiol B 125(3):387–393. https://doi.org/10.1016/S0305-0491(99)00185-6
Cai W, Liang X-F, Yuan X, Liu L, He S, Li J, Li B, Xue M (2018) Different strategies of grass carp (Ctenopharyngodon idella) responding to insufficient or excessive dietary carbohydrate. Aquaculture 497:292–298. https://doi.org/10.1016/j.aquaculture.2018.07.042
Enes P, Panserat S, Kaushik S, Oliva-Teles A (2009) Nutritional regulation of hepatic glucose metabolism in fish. Fish Physiol Biochem 35(3):519–539. https://doi.org/10.1007/s10695-008-9259-5
Enes P, Panserat S, Kaushik S, Oliva-Teles AA (2006) Effect of normal and waxy maize starch on growth, food utilization and hepatic glucose metabolism in European sea bass (Dicentrarchus labrax) juveniles. Comp Biochem Phys A 143(1):89–96. https://doi.org/10.1016/j.cbpa.2005.10.027
Forbes JL, Kostyniuk DJ, Mennigen JA, Weber J-M (2019) Glucagon regulation of carbohydrate metabolism in rainbow trout: in vivo glucose fluxes and gene expression. J Exp Biol 222(24):jeb211730. https://doi.org/10.1242/jeb.211730
Fowler LA, Williams MB, D'Abramo LR, Watts SA (2020) Zebrafish nutrition—moving forward. In: The zebrafish in biomedical research. Academic Press, pp 379–401. https://doi.org/10.1016/B978-0-12-812431-4.00033-6
Fowler LA, Williams MB, Dennis-Cornelius LN, Farmer S, Barry RJ, Powell ML, Watts SA (2019) Influence of commercial and laboratory diets on growth, body composition, and reproduction in the zebrafish Danio rerio. Zebrafish 16(6):508–521. https://doi.org/10.1089/zeb.2019.1742
Fuqua JD, Hughes J, Kuennen D, Harris M, Turner K, Kronemberger A, Ebert S, Dierdorff J, Zhu ZY, Zingman L, Adams C, Lira VA (2020) Skeletal muscle ULK1 and ULK2 jointly couple muscle mass with force and are required for survival under low nutrient availability. FASEB J 34(S1):1–1. https://doi.org/10.1096/fasebj.2020.34.s1.03974
Fuqua JD, Mere CP, Kronemberger A, Blomme J, Bae D, Turner KD, Harris MP, Scudese E, Edwards M, Ebert SM, Sousa L, Bodine SC, Yang L, Adams CM, Lira VA (2019) ULK2 is essential for degradation of ubiquitinated protein aggregates and homeostasis in skeletal muscle. FASEB J 33(11):11735–12745. https://doi.org/10.1096/fj.201900766R
Gao Y, Dai Z, Shi C, Zhai G, Jin X, He J, Lou Q, Yin Z (2016) Depletion of myostatin b promotes somatic growth and lipid metabolism in zebrafish. Front Endocrinol 7:88. https://doi.org/10.3389/fendo.2016.00088
Goldberg AA, Nkengfac B, Sanchez AMJ, Moroz N, Qureshi ST, Koromilas AE, Wang S, Burelle Y, Hussain SN, Kristof AS (2017) Regulation of ULK1 expression and autophagy by STAT1. J Biol Chem 292(5):1899–1909. https://doi.org/10.1074/jbc.M116.771584
Goodwin AE, Lochmann RT, Tieman DM, Mitchell AJ (2002) Massive hepatic necrosis and nodular regeneration in largemouth bass fed diets high in available carbohydrate. J World Aquacult Soc 33(4):466–477. https://doi.org/10.1111/j.1749-7345.2002.tb00026.x
Hanson RW, Hakimi P (2008) Born to run; the story of the PEPCK-Cmus mouse. Biochimie. 90(6):838–842. https://doi.org/10.1016/j.biochi.2008.03.009
Hanson RW, Reshef L (1997) Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu Rev Biochem 66(1):581–611. https://doi.org/10.1146/annurev.biochem.66.1.581
Hemre GI, Mommsen T, Krogdahl A (2002) Carbohydrates in fish nutrition: effects on growth, glucose metabolism and hepatic enzymes. Aquacult Nutr 8(3):175–194. https://doi.org/10.1046/j.1365-2095.2002.00200.x
Idris RIR, Taufek NM, Nordin NFH, Al-Saari N (2022) Zebrafish nutrition: promoting fish health and welfare of the animal model in Halal science research. Halalpshere 2(1):106–121
Kamstra K (2021) Leptin and glucose homeostasis in the Zebrafish. University of Otago http://hdl.handle.net/10523/10918
Ko C-W, Counihan D, Wu J, Hatzoglou M, Puchowicz MA, Croniger CM (2018) Macrophages with a deletion of the phosphoenolpyruvate carboxykinase 1 (Pck1) gene have a more proinflammatory phenotype. J Biol Chem 293(9):3399–3409. https://doi.org/10.1074/jbc.M117.819136
Koo BC, Kwon MS, Nam Y, Kim T (2018) Expression of PCK1 gene in skeletal muscle cells using muscle-specific promoter. In: The 18th International Symposium on Developmental Biotechnology, p 78-78
Krogdahl A, Hemre GI, Mommsen T (2005) Carbohydrates in fish nutrition: digestion and absorption in postlarval stages. Aquacult Nutr 11(2):103–122. https://doi.org/10.1111/j.1365-2095.2004.00327.x
Kuo T, McQueen A, Chen T-C, Wang J-C (2015) Regulation of glucose homeostasis by glucocorticoids. In: Glucocorticoid signaling, pp 99–126
Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (2016) CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44(W1):W272–W276. https://doi.org/10.1093/nar/gkw398
Li S, Li Z, Zhang J, Sang C, Chen N (2019a) The impacts of dietary carbohydrate levels on growth performance, feed utilization, glycogen accumulation and hepatic glucose metabolism in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus♂). Aquaculture 512:734351. https://doi.org/10.1016/j.aquaculture.2019.734351
Li RX, Liu HY, Li SY, Tan BP, Dong XH, Chi SY, Yang QH, Zhang S, Chen LQ (2019b) Nutritional regulation of gene expression and enzyme activity of phosphoenolpyruvate carboxykinase in the hepatic gluconeogenesis pathway in golden pompano (Trachinotus ovatus). Aquacult Res 50(2):634–643. https://doi.org/10.1111/are.13941
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Marandel L, Seiliez I, Véron V, Skiba-Cassy S, Panserat S (2015) New insights into the nutritional regulation of gluconeogenesis in carnivorous rainbow trout (Oncorhynchus mykiss): a gene duplication trail. Physiol Genomics 47(7):253–263. https://doi.org/10.1152/physiolgenomics.00026.2015
Matte A, Tari LW, Goldie H, Delbaere LT (1997) Structure and mechanism of phosphoenolpyruvate carboxykinase. J Biol Chem 272(13):8105–8108. https://doi.org/10.1074/jbc.272.13.8105
Mofarrahi M, Sigala I, Guo Y, Godin R, Davis EC, Petrof B, Sandri M, Burelle Y (2012) Autophagy and skeletal muscles in sepsis. PloS One 7(10):e47265. https://doi.org/10.1371/journal.pone.0047265
Moon TW (2001) Glucose intolerance in teleost fish: fact or fiction? Comp Biochem Physiol B 129(2-3):243–249. https://doi.org/10.1016/S1096-4959(01)00316-5
Moreira I, Peres H, Couto A, Enes P, Oliva-Teles A (2008) Temperature and dietary carbohydrate level effects on performance and metabolic utilisation of diets in European sea bass (Dicentrarchus labrax) juveniles. Aquaculture 274(1):153–160. https://doi.org/10.1016/j.aquaculture.2007.11.016
Olswang Y, Cohen H, Papo O, Reshef L (2002) A mutation in the peroxisome proliferator-activated receptor γ-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice. P Natl Acad Sci USA 99(2):625–630. https://doi.org/10.1073/pnas.022616299
Panserat S, Plagnes-Juan E, Kaushik S (2001) Nutritional regulation and tissue specificity of gene expression for proteins involved in hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). J Exp Biol 204(13):2351–2360. https://doi.org/10.1242/jeb.204.13.2351
Pilkis SJ, Granner D (1992) Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Biochem 54(1):885–909. https://doi.org/10.1146/annurev.ph.54.030192.004321
Polakof S, Panserat S, Soengas JL, Moon TW (2012) Glucose metabolism in fish: a review. J Comp Physiol B 182(8):1015–1045. https://doi.org/10.1007/s00360-012-0658-7
Ren K, Torres R (2009) Role of interleukin-1β during pain and inflammation. Brain Res Rev 60(1):57–64. https://doi.org/10.1016/j.brainresrev.2008.12.020
Reshef L, Olswang Y, Cassuto H, Blum B, Croniger CM, Kalhan SC, Tilghman SM, Hanson RW (2003) Glyceroneogenesis and the triglyceride/fatty acid cycle. J Biol Chem 278(33):30413–30416. https://doi.org/10.1074/jbc.R300017200
Schiaffino S, Mammucari C (2011) Regulation of skeletal muscle growth by the IGF1-Akt/PKB pathway: insights from genetic models. Skelet Muscle 1(1):1–14 http://www.skeletalmusclejournal.com/content/1/1/4
Schreurs M, Kuipers F, Van Der Leij F (2010) Regulatory enzymes of mitochondrial β-oxidation as targets for treatment of the metabolic syndrome. Obes Rev 11(5):380–388. https://doi.org/10.1111/j.1467-789X.2009.00642.x
Seiliez I, Médale F, Aguirre P, Panserat S, Skiba-Cassy S (2013) Postprandial regulation of growth-and metabolism-related factors in zebrafish. Zebrafish 10(2):237–248. https://doi.org/10.1089/zeb.2012.0835
Semakova J, Hyroššová P, Méndez-Lucas A, Cutz E, Bermudez J, Burgess S, Alcántara S, Perales JC (2017) PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1del/del mice, unmasking role in non-gluconeogenic tissues. J Physiol Biochem 73(1):89–98. https://doi.org/10.1007/s13105-016-0528-y
She P, Shiota M, Shelton KD, Chalkley R, Postic C, Magnuson MA (2000) Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism. Mol Cell Biol 20(17):6508–6517. https://doi.org/10.1128/MCB.20.17.6508-6517.2000
Shi L, An S, Liu Y, Liu J, Wang F (2020) PCK1 regulates glycolysis and tumor progression in clear cell renal cell carcinoma through LDHA. Oncotargets Ther 13:2613. https://doi.org/10.2147/OTT.S241717
Tang Y, Zhang Y, Wang C, Sun Z, Li L, Cheng S, Zhou W (2018) Overexpression of PCK1 gene antagonizes hepatocellular carcinoma through the activation of gluconeogenesis and suppression of glycolysis pathways. Cell Physiol Biochem 47(1):344–355. https://doi.org/10.1159/000489811
Wang A, Zhang Z, Ding Q, Yang Y, Bindelle J, Ran C (2021) Intestinal Cetobacterium and acetate modify glucose homeostasis via parasympathetic activation in zebrafish. Gut Microbes 13(1):1–15. https://doi.org/10.1080/19490976.2021.1900996
Wang J, Liang X-F, He S, Li J, Huang K, Zhang Y-P, Huang D (2018) Lipid deposition pattern and adaptive strategy in response to dietary fat in Chinese perch (Siniperca chuatsi). Nutr Metab 15(1):1–11. https://doi.org/10.1186/s12986-018-0315-6
Wassana P, Prathomya P, Jakovlić I, Liu H, Zhao Y-H, Wang W-M (2017) Transcriptomics, metabolomics and histology indicate that high-carbohydrate diet negatively affects the liver health of blunt snout bream (Megalobrama amblycephala). BMC Genomics 18(1):856. https://doi.org/10.1186/s12864-017-4246-9
Williams MB (2021) Importance of the zebrafish model for understanding nutrition. The University of Alabama at Birmingham
Wilson RP (1994) Utilization of dietary carbohydrate by fish. Aquaculture 124(1-4):67–80. https://doi.org/10.1016/0044-8486(94)90363-8
Xu J, He GL, Chen LT, Xie SW, Chi SY, Zhang S, Cao JM, Tan BP (2022a) Farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5) signaling pathways improved the hepatic lipid metabolism in hybrid grouper. Aquacult Rep 22:100997. https://doi.org/10.1016/j.aqrep.2021.100997
Xu J, Shi ML, Chi SY, Zhang S, Cao JM, Tan BP, Xie SW (2022b) The effects of a mixture of antibiotics on the intestinal microbiota and hepatic lipid deposition in hybrid grouper fed bile acids diet. Aquacult Rep 24:101169. https://doi.org/10.1016/j.aqrep.2022.101169
Yang B-Y, Zhai G, Gong Y-L, Peng X-Y, Shang G-H, Han D, Jin J-Y, Liu H-K, Du Z-Y, Yin Z, Xie S-Q (2018) Different physiological roles of insulin receptors in mediating nutrient metabolism in zebrafish. Am J Physiol-Endoc M 315(1):E38–E51. https://doi.org/10.1152/ajpendo.00227.2017
Yang J, Kalhan SC, Hanson RW (2009) What is the metabolic role of phosphoenolpyruvate carboxykinase? J Biol Chem 284(40):27025–27029. https://doi.org/10.1074/jbc.R109.040543
Yang M, Deng K, Pan M, Zhang Y, Sampath W, Zhang W, Mai K (2020) Molecular adaptations of glucose and lipid metabolism to different levels of dietary carbohydrates in juvenile Japanese flounder Paralichthys olivaceus. Aquacult Nutr 26(2):516–527. https://doi.org/10.1111/anu.13013
Yoshimura A, Wakabayashi Y, Mori T (2010) Cellular and molecular basis for the regulation of inflammation by TGF-β. J Biochem 147(6):781–792. https://doi.org/10.1093/jb/mvq043
Yu S, Meng S, Xiang M, Ma H (2021) Phosphoenolpyruvate carboxykinase in cell metabolism: Roles and mechanisms beyond gluconeogenesis. Mol Metab 53:101257. https://doi.org/10.1016/j.molmet.2021.101257
Yuan X, Dong D, Li Z, Wu B (2019) Rev-erbα activation down-regulates hepatic Pck1 enzyme to lower plasma glucose in mice. Pharmacol Res 141:310–318. https://doi.org/10.1016/j.phrs.2019.01.010
Zhang Y, Liang X-F, He S, Wang J, Li L, Zhang Z, Li J, Chen X, Li L, Alam MS (2021) Metabolic responses of Chinese perch (Siniperca chuatsi) to different levels of dietary carbohydrate. Fish Physiol Biochem 47(5):1449–1465. https://doi.org/10.1007/s10695-021-00965-2
Zhang Y, Qin C, Yang L, Lu R, Zhao X, Nie G (2018) A comparative genomics study of carbohydrate/glucose metabolic genes: from fish to mammals. BMC Genomics 19:1–14. https://doi.org/10.1186/s12864-018-4647-4
Zhou S-S, Xu J, Zhu H, Wu J, Xu J-D, Yan R, Li X-Y, Liu H-H, Duan S-M, Wang Z, Chen H-B, Shen H, Li S-L (2016) Gut microbiota-involved mechanisms in enhancing systemic exposure of ginsenosides by coexisting polysaccharides in ginseng decoction. Sci Rep 6(1):1–13. https://doi.org/10.1038/srep22474
Acknowledgements
Xu-Fang Liang and Ke Lu designed the experiments and wrote the paper; Ke Lu and Jiaqi Wu performed the experiments and analyzed the data; Ke Lu and Yanpeng Zhang fed fish and sampled; Ke Lu and Wuyuan Zhuang prepared the experimental diets.
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This work was funded by National Key R&D Program of China (2018YFD0900400), the National Natural Science Foundation of China (31972809), and Key Research and Development Project of Hubei Province (2020BBA035).
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Lu, K., Wu, J., Zhang, Y. et al. Role of phosphoenolpyruvate carboxykinase 1 (pck1) in mediating nutrient metabolism in zebrafish. Funct Integr Genomics 23, 67 (2023). https://doi.org/10.1007/s10142-023-00993-6
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DOI: https://doi.org/10.1007/s10142-023-00993-6