Phosphatidic Acid Phosphatase Research Articles

Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana

Algal biomass is a viable source of chemicals and metabolites for various energy, nutritional, medicinal and agricultural uses. While stresses have commonly been used to induce metabolite accumulation in microalgae in attempts to enhance high-value product yields, this is often very detrimental to growth. Therefore, understanding how to modify metabolism without deleterious consequences is highly beneficial. We demonstrate that low-doses (1–5 Gy) of ionizing radiation in the X-ray range induces a non-toxic, hormetic response in microalgae to promote metabolic activation. We identify specific radiation exposure parameters that give reproducible metabolic responses in Chlorella sorokiniana caused by transcriptional changes. This includes up-regulation of >30 lipid metabolism genes, such as genes encoding an acetyl-CoA carboxylase subunit, phosphatidic acid phosphatase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase. The outcome is an increased lipid yield in stationary phase cultures by 25% in just 24 hours, without any negative effects on cell viability or biomass.

Protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

Lysosomal diacylglycerol pyrophosphate phosphatase is not essential in Trypanosoma brucei.

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of β-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.

Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope

Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.

Abstract 1437 Phosphatidate phosphatase Pah1 is required for sporulation in yeast

Abstract 1437 Phosphatidate phosphatase Pah1 is required for sporulation in yeast

Abstract 1485 Construction and analysis of the catalytic core of yeast Pah1 phosphatidate phosphatase

Abstract 1485 Construction and analysis of the catalytic core of yeast Pah1 phosphatidate phosphatase

Abstract 1381 Identification of three catalytic motifs in the HAD-like domain from Saccharomyces cerevisiae phosphatidate phosphatase Pah1

Abstract 1381 Identification of three catalytic motifs in the HAD-like domain from Saccharomyces cerevisiae phosphatidate phosphatase Pah1

Abstract 2048 Loss of ATP-dependent Citrate Lyase Abrogates Phosphatidate Phosphatase Activity in the oleaginous yeast Yarrowia lipolytica

Abstract 2048 Loss of ATP-dependent Citrate Lyase Abrogates Phosphatidate Phosphatase Activity in the oleaginous yeast Yarrowia lipolytica

Chloroplast phosphatases LPPγ and LPPε1 facilitate conversion of extraplastidic phospholipids to galactolipids.

Galactolipids comprise the majority of chloroplast membranes in plants, and their biosynthesis requires dephosphorylation of phosphatidic acid at the chloroplast envelope membranes. In Arabidopsis (Arabidopsis thaliana), the lipid phosphate phosphatases LPPγ, LPPε1, and LPPε2 have been previously implicated in chloroplast lipid assembly, with LPPγ being essential, as null mutants were reported to exhibit embryo lethality. Here, we show that lppγ mutants are in fact viable and that LPPγ, LPPε1, and LPPε2 do not appear to have central roles in the plastid pathway of membrane lipid biosynthesis. Redundant LPPγ and LPPε1 activity at the outer envelope membrane is important for plant development, and the respective lppγ lppε1 double mutant exhibits reduced flux through the ER pathway of galactolipid synthesis. While LPPε2 is imported and associated with interior chloroplast membranes, its role remains elusive and does not include basal nor phosphate limitation-induced biosynthesis of glycolipids. The specific physiological roles of LPPγ, LPPε1, and LPPε2 are yet to be uncovered, as does the identity of the phosphatidic acid phosphatase required for plastid galactolipid biosynthesis.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet β cells, which modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single-nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ∼8-fold and ∼3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Lipid- and phospho-regulation of CTP:Phosphocholine Cytidylyltransferase αassociation with nuclear lipid droplets.

Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTαtranslocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1β, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

The Saccharomyces cerevisiae Spo7 basic tail is required for Nem1–Spo7/Pah1 phosphatase cascade function in lipid synthesis

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

Catalytic core function of yeast Pah1 phosphatidate phosphatase reveals structural insight into its membrane localization and activity control

The PAH1-encoded phosphatidate (PA) phosphatase is a major source of diacylglycerol for the production of the storage lipid triacylglycerol and a key regulator for the de novo phospholipid synthesis in Saccharomyces cerevisiae. The catalytic function of Pah1 depends on its membrane localization which is mediated through its phosphorylation by multiple protein kinases and dephosphorylation by the Nem1-Spo7 protein phosphatase complex. The full-length Pah1 is composed of a catalytic core (N-LIP and HAD-like domains, amphipathic helix, and the WRDPLVDID domain) and non-catalytic regulatory sequences (intrinsically disordered regions, RP domain, and acidic tail) for phosphorylation and interaction with Nem1-Spo7. How the catalytic core regulates Pah1 localization and cellular function is not clear. In this work, we analyzed a variant of Pah1 (i.e., Pah1-CC (catalytic core)) that is composed only of the catalytic core. Pah1-CC expressed on a low-copy plasmid complemented the pah1Δ mutant phenotypes (e.g., nuclear/ER membrane expansion, reduced levels of triacylglycerol, and lipid droplet formation) without requiring Nem1-Spo7. The cellular function of Pah1-CC was supported by its PA phosphatase activity mostly associated with the membrane fraction. Although functional, Pah1-CC was distinct from Pah1 in the protein and enzymological properties, which include overexpression toxicity, association with heat shock proteins, and significant reduction of the Vmax value. These findings on the Pah1 catalytic core enhance the understanding of its structural requirements for membrane localization and activity control.

Phosphatidate phosphatase Lipin1 involves in diabetic encephalopathy pathogenesis via regulating synaptic mitochondrial dynamics

Diabetic encephalopathy (DE) is a common central nervous system complication of diabetes mellitus without effective therapy currently. Recent studies have highlighted synaptic mitochondrial damages as a possible pathological basis for DE, but the underlying mechanisms remain unclear. Our previous work has revealed that phosphatidate phosphatase Lipin1, a critical enzyme involved with phospholipid synthesis, is closely related to the pathogenesis of DE. Here, we demonstrate that Lipin1 is significantly down-regulated in rat hippocampus of DE. Knock-down of Lipin1 within hippocampus of normal rats induces dysregulation of homeostasis in synaptic mitochondrial dynamics with an increase of mitochondrial fission and a decrease of fusion, then causes synaptic mitochondrial dysfunction, synaptic plasticity deficits as well as cognitive impairments, similar to that observed in response to chronic hyperglycemia exposure. In contrast, an up-regulation of Lipin1 within hippocampus in the DE model ameliorates this cascade of dysfunction. We also find that the effect of Lipin1 that regulating mitochondrial dynamics results from maintaining appropriate phospholipid components in the mitochondrial membrane. In conclusion, alterations in hippocampal Lipin1 contribute to hippocampal synaptic mitochondrial dysfunction and cognitive deficits observed in DE. Targeting Lipin1 might be a potential therapeutic strategy for the clinical treatment of DE.

Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Lipin-2 regulates the antiviral and anti-inflammatory responses to interferon.

Interferons (IFN) are crucial antiviral and immunomodulatory cytokines that exert their function through the regulation of a myriad of genes, many of which are not yet characterized. Here, we reveal that lipin-2, a phosphatidic acid phosphatase whose mutations produce an autoinflammatory syndrome known as Majeed syndrome in humans, is regulated by IFN in a STAT-1-dependent manner. Lipin-2 inhibits viral replication both in vitro and in vivo. Moreover, lipin-2 also acts as a regulator of inflammation in a viral context by reducing the signaling through TLR3 and the generation of ROS and release of mtDNA that ultimately activate the NLRP3 inflammasome. Inhibitors of mtDNA release from mitochondria restrict IL-1β production in lipin-2-deficient animals in a model of viral infection. Finally, analyses of databases from COVID-19 patients show that LPIN2 expression levels negatively correlate with the severity of the disease. Overall, these results uncover novel regulatory mechanisms of the IFN response driven by lipin-2 and open new perspectives for the future management of patients with LPIN2 mutations.

Effects of Mulberry Leaf Extract on the Liver Function of Juvenile Spotted Sea Bass (Lateolabrax maculatus).

In order to explore the effect of mulberry leaf extract (ELM) on the liver function of spotted sea bass, 360 fish with healthy constitution (average body weight 9.00 ± 0.02 g) were selected and randomly divided into six groups with three repetitions, and six groups of fish were randomly placed into 18 test tanks (200 L) with 20 fish per tank for the 52-day feeding test. Every day, the fish were fed the experimental feed with different concentrations (0, 3, 6, 9, 12, 15 g/kg) to the level of apparent satiation, with a crude protein content of 48.0% and a crude fat content of 8.6%. And the water temperature was maintained at 25-28°C with a salinity of 0.5%-1‰. After feeding, five fish were randomly selected to collect their livers and serum for detection of indicators. The results showed that, compared with the control group, ELM significantly increased the activities of lipase (LPS) and trypsin (TRS) in the liver, and reached the highest level when the amount of ELM added was 6 g/kg (P < 0.05). ELM significantly increased the activities of lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT) involved in the metabolic process in liver tissue, and GOT activity reached the highest when ELM was added at 9 g/kg, and LDH activity reached the highest when ELM was added at 15 g/kg (P < 0.05). ELM had no significant effect on liver antioxidant enzymes (P > 0.05), but the content of malondialdehyde was significantly reduced (P < 0.05). Compared with the control group, ELM significantly increased the activities of AKP and ACP in the liver, and the AKP activity reached the highest when the ELM addition amount was 3 g/kg, and the ACP activity reached the highest when the ELM addition amount was 9 g/kg (P < 0.05). Through comparative transcriptomic analysis, it was indicated that ELM enhanced the hepatic lipids and carbohydrates metabolism ability, as manifested in the upregulation of expression of phosphatidate phosphatase, glucuronosyltransferase, inositol oxygenase, carbonic anhydrase, and cytochrome c oxidase subunit 2. ELM can also increase the expression of signal transducer and activator of transcription 1, ATP-dependent RNA helicase and C-X-C motif chemokine 9 involved in the immune process. The above results show that the ELM can enhance the digestion, metabolism, and immunity of the liver by increasing the activity of digestive enzymes, metabolic enzymes, and the expression of metabolism and immune regulation genes. This study provides a theoretical basis for the application of ELM in the cultivation of spotted sea bass by exploring the effect of ELM on the liver function of spotted sea bass.

Characterization of the lncRNA-mediated ceRNA regulatory networks in preeclampsia by integrated bioinformatics

Preeclampsia (PE) is a significant threat to all pregnancies that is highly associated with maternal mortality and developmental disorders in infants. However, the etiopathogenesis of this condition remains unclear. This study aims to explore the regulatory roles of long noncoding RNAs (lncRNAs) and the mediated competing endogenous RNAs (ceRNA) in the etiopathogenesis of PE through analysis of lncRNA expression patterns in PE and healthy pregnant women (HPW), as well as the construction of lncRNA-mediated ceRNA regulatory networks using bioinformatics. A total of 896 significant differentially expressed lncRNAs, including 586 upregulated lncRNAs and 310 downregulated lncRNAs, were identified in comparison between PE and HPW. Analysis of these differential expressed lncRNAs revealed their predominant enrichment in molecular functions such as sphingosine-1-phosphate phosphatase activity, lipid phosphatase activity, phosphatidate phosphatase activity, thymidylate kinase activity, and UMP kinase activity. Moreover, these differential expressed lncRNAs were predominantly enriched in KEGG analyses such as fat digestion and absorption, lysine degradation, ether lipid metabolism, glycerolipid metabolism, and sphingolipid metabolism. Two ceRNA regulatory networks were constructed based on ceRNA score, including one that had 31 upregulated lncRNAs, 11 downregulated miRNAs, and 34 upregulated mRNAs, while the other contained 128 downregulated lncRNAs, 40 upregulated miRNAs, and 113 downregulated mRNAs. These results may provide a clue to explore the roles of lncRNAs in the etiopathogenesis of PE.

Molecular cloning and tissue distribution of glucokinase and glucose-6-phosphatase catalytic subunit paralogs in largemouth bass Micropterus salmoides: Regulation by dietary starch levels and a glucose load

The dysregulation of glucose-G6P (glucose-6-phosphate) interconversion is thought to be one of the main reasons for the low glucose disposal of carnivorous fish, but is not yet well understood in largemouth bass Micropterus salmoides (LMB). In this study, the full length cDNA sequences of genes encoding glucokinase (Gck, catalyzing glucose phosphorylation) and glucose-6-phosphatase catalytic subunit (G6pc, catalyzing glucose dephosphorylation) were cloned by the RACE method from the liver of LMB. Subsequently, the distribution of g6pc and gck as well as their transcriptional regulation by dietary starch levels and a glucose load were investigated. Only one gck gene was identified, while the tandem duplication of g6pca.1 gene was named as g6pca.2 in LMB. The full cDNA sequences of g6pca.1, g6pca.2 and gck in LMB were 1585, 1813 and 2115 bp in length, encoding 478, 352 and 359 amino acids, respectively. Gck was predicted to contain two hexokinase domains, an ATP-binding domain and multiple functional sites, while G6pca.1 and G6pca.2 contained nine transmembrane helices, a PAP2 (type-2 phosphatidic acid phosphatase) domain and multiple functional amino acid sites. Both g6pca.1 and g6pca.2 were predominantly distributed in the liver and to some extent in the intraperitoneal fat, intestine and pyloric caeca, while gck was mainly transcribed in the liver and to some extent in the heart, intestine and brain. Both feeding a high starch diet and a glucose load stimulated the mRNA expression of gck in the liver of LMB. An increase of dietary starch from 9% to 14% down-regulated the transcription of g6pca.1 in the liver of LMB. However, both the mRNA levels of hepatic g6pca.1 and g6pca.2 were sharply up-regulated in LMB during 1–3 h after a glucose load. Overall, the results of this study suggested that the functions of G6pc (G6pca.1 and G6pca.2) and Gck in LMB were highly conserved in evolution. Though hepatic glucose-G6P interconversion was well regulated at the transcript level in LMB fed high starch diets, a futile cycle between glucose and G6P was induced in the liver after a glucose load.