The Administration of Chitosan-Tripolyphosphate-DNA Nanoparticles to Express Exogenous SREBP1a Enhances Conversion of Dietary Carbohydrates into Lipids in the Liver of Sparus aurata.

In addition to being essential for the transcription of genes involved in cellular lipogenesis, increasing evidence associates sterol regulatory element binding proteins (SREBPs) with the transcriptional control of carbohydrate metabolism. The aim of this study was to assess the effect of overexpression SREBP1a, a potent activator of all SREBP-responsive genes, on the intermediary metabolism of Sparus aurata, a glucose-intolerant carnivorous fish. Administration of chitosan-tripolyphosphate nanoparticles complexed with a plasmid driving expression of the N-terminal transactivation domain of SREBP1a significantly increased SREBP1a mRNA and protein in the liver of S. aurata. Overexpression of SREBP1a enhanced the hepatic expression of key genes in glycolysis-gluconeogenesis (glucokinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), fatty acid synthesis (acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2), elongation (elongation of very long chain fatty acids protein 5) and desaturation (fatty acid desaturase 2) as well as reduced nicotinamide adenine dinucleotide phosphate production (glucose-6-phosphate 1-dehydrogenase) and cholesterol synthesis (3-hydroxy-3-methylglutaryl-coenzyme A reductase), leading to increased blood triglycerides and cholesterol levels. Beyond reporting the first study addressing in vivo effects of exogenous SREBP1a in a glucose-intolerant model, our findings support that SREBP1a overexpression caused multigenic effects that favoured hepatic glycolysis and lipogenesis and thus enabled protein sparing by improving dietary carbohydrate conversion into fatty acids and cholesterol.

Metformin counteracts glucose-dependent lipogenesis and impairs transdeamination in the liver of gilthead sea bream ( Sparus aurata).

Metformin is an antidiabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis, but also by affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability in using dietary carbohydrates. To increase the current knowledge about the molecular mechanisms by which metformin can improve glucose homeostasis in carnivorous fish, we addressed the effect of intraperitoneal administration of metformin, in the presence or absence of a glucose load, on metabolic rate-limiting enzymes and lipogenic factors in the liver of gilthead sea bream ( Sparus aurata). Hyperglycemia markedly upregulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h posttreatment, it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1ß, Lpin1, and SREBP1). Metformin counteracted glucose-dependent effects, and downregulated glutamate dehydrogenase, alanine aminotransferase, and mammalian target of rapamycin 5 h posttreatment in the absence of a glucose load, leading to decreased long-term activity of PFK1 and IDH. The results of the present study suggest that hyperglycemia enhances lipogenesis in the liver of S. aurata and that metformin may exert specific metabolic effects in fish by decreasing hepatic transdeamination and suppressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model.

Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver.

Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53-55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture.

Author Correction: Disposition of a Glucose Load into Hepatic Glycogen by Direct and Indirect Pathways in Juvenile Seabass and Seabream.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Role of upstream stimulatory factor 2 in glutamate dehydrogenase gene transcription.

Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination of l-glutamate into α-ketoglutarate using NAD+ or NADP+ as cofactor. To gain insight into transcriptional regulation of glud, the gene that codes for Gdh, we isolated and characterised the 5' flanking region of glud from gilthead sea bream (Sparus aurata). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of S. aurata were also addressed. 5'-Deletion analysis of glud promoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation of glud Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver of S. aurata, while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding to glud promoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates S. aurata glud by binding to an E-box located in the proximal region of glud promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of glud transcription in the fish liver.

Disposition of a Glucose Load into Hepatic Glycogen by Direct and Indirect Pathways in Juvenile Seabass and Seabream.

In carnivorous fish, conversion of a glucose load to hepatic glycogen is widely used to assess their metabolic flexibility towards carbohydrate utilization, but the activities of direct and indirect pathways in this setting are unclear. We assessed the conversion of an intraperitoneal glucose load (2 g.kg-1) enriched with [U-13C6]glucose to hepatic glycogen in juvenile seabass and seabream. 13C-NMR analysis of glycogen was used to determine the contribution of the load to glycogen synthesis via direct and indirect pathways at 48-hr post-injection. For seabass, [U-13C6]glucose was accompanied by deuterated water and 2H-NMR analysis of glycogen 2H-enrichment, allowing endogenous substrate contributions to be assessed as well. For fasted seabass and seabream, 47 ± 5% and 64 ± 10% of glycogen was synthesized from the load, respectively. Direct and indirect pathways contributed equally (25 ± 3% direct, 21 ± 1% indirect for seabass; 35 ± 7% direct, 29 ± 4% indirect for seabream). In fasted seabass, integration of 2H- and 13C-NMR analysis indicated that endogenous glycerol and anaplerotic substrates contributed an additional 7 ± 2% and 7 ± 1%, respectively. In fed seabass, glucose load contributions were residual and endogenous contributions were negligible. Concluding, direct and indirect pathways contributed equally and substantially to fasting hepatic glycogen repletion from a glucose load in juvenile seabream and seabass.

A transcriptomic approach to study the effect of long-term starvation and diet composition on the expression of mitochondrial oxidative phosphorylation genes in gilthead sea bream (Sparus aurata).

BACKGROUND: The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. RESULTS: After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. CONCLUSIONS: Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming.

Viability-reducing activity of Coryllus avellana L. extracts against human cancer cell lines.

The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studiedagainst three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chormatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-ß-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer.

Effect of dietary macronutrients on the expression of cholecystokinin, leptin, ghrelin and neuropeptide Y in gilthead sea bream (Sparus aurata).

Endocrine factors released from the central nervous system, gastrointestinal tract, adipose tissue and other peripheral organs mediate the regulation of food intake. Although many studies have evaluated the effect of fed-to-starved transition on the expression of appetite-related genes, little is known about how the expression of appetite-regulating peptides is regulated by the macronutrient composition of the diet. The aim of the present study was to examine the effect of diet composition and nutritional status on the expression of four peptides involved in food intake control in gilthead sea bream (Sparus aurata): neuropeptide Y (NPY), ghrelin, cholecystokinin (CCK) and leptin. Quantitative real-time RT-PCR showed that high protein/low carbohydrate diets stimulated the expression of CCK and ghrelin in the intestine and leptin in the adipose tissue, while downregulation of ghrelin and NPY mRNA levels was observed in the brain. Opposite effects were found for the expression of the four genes in fish fed low protein/high carbohydrate diets or after long-term starvation. Our findings indicate that the expression pattern of appetite-regulating peptides, particularly CCK and ghrelin, is modulated by the nutritional status and diet composition in S. aurata.

Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.).

Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium ((2)H) enrichment was determined following 6 days in (2)H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day(-1)) were calculated for hepatic TG-glyceryl and FA moieties through (2)H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL.

Chitosan-Mediated shRNA Knockdown of Cytosolic Alanine Aminotransferase Improves Hepatic Carbohydrate Metabolism.

Alanine aminotransferase (ALT) catalyses a transamination reaction that links carbohydrate and amino acid metabolism. In this study, we examined the effect of silencing cytosolic ALT (cALT) expression on the hepatic metabolism in Sparus aurata. A number of siRNA and shRNA designed to down-regulate cALT expression were validated in HEK-293 cells transfected with plasmids expressing S. aurata cALT or mitochondrial ALT (mALT) isoforms: ALT silencing significantly decreased the expression levels of S. aurata mRNA cALT1 to 62% (siRNA) and 48% (shRNA) of the values observed in control cells. The effect of cALT silencing was analysed in the liver of S. aurata 72 h after intraperitoneal injection of chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a shRNA to down-regulate cALT expression (pCpG-si1sh1). In fish fed diets with different ratio of protein to carbohydrate and treated with chitosan-TPP-pCpG-si1sh1, cALT1 and cALT2 mRNA levels significantly decreased irrespective of the diet. Consistently, ALT activity decreased in liver of treated animals. In the liver of S. aurata treated with chitosan-TPP-pCpG-si1sh1 nanoparticles, down-regulation of cALT expression increased the activity of key enzymes in glycolysis (6-phosphofructo-1-kinase and pyruvate kinase) and protein metabolism (glutamate dehydrogenase). Besides showing for the first time that administration of chitosan-TPP-pCpG-si1sh1 nanoparticles silences hepatic cALT expression in vivo, our data support that down-regulation of cALT could improve the use of dietary carbohydrates to obtain energy and spare protein catabolism.

Contribution of dietary starch to hepatic and systemic carbohydrate fluxes in European seabass (Dicentrarchus labrax L.).

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30% digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5% (2)H2O over the last six feeding days, and quantifying the incorporation of (2)H into blood glucose and hepatic glycogen by (2)H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P < 0.001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P < 0.001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (sem 1) µmol/kg per h; P < 0.001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding.

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction) while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.

Activating transcription factor 4 mediates up-regulation of alanine aminotransferase 2 gene expression under metabolic stress.

Alanine aminotransferase (ALT) provides a molecular link between carbohydrate and amino acid metabolism. In humans, two ALT isoforms have been characterized: ALT1, cytosolic, and ALT2, mitochondrial. To gain insight into the transcriptional regulation of the ALT2 gene, we cloned and characterized the human ALT2 promoter. 5'-deletion analysis of ALT2 promoter in transiently transfected HepG2 cells and site-directed mutagenesis allowed us to identify ATF4 as a new factor involved in the transcriptional regulation of ALT2 expression. Quantitative RT-PCR assays showed that the metabolic stressors histidinol and tunicamycin increased ATF4 levels and up-regulated ALT2 in HepG2 and Huh7 cells. Consistently, knock-down of ATF4 decreased ALT2 mRNA levels in HepG2 and Huh-7 cells. Moreover, ATF4 silencing prevented the activating effect of histidinol and tunicamycin on ATF4 and ALT2 expression. Our findings point to ALT2 as an enzyme involved in the metabolic adaptation of the cell to stress.

Effects of food-deprivation and refeeding on the regulation and sources of blood glucose appearance in European seabass (Dicentrarchus labrax L.).

Sources of blood glucose in European seabass (initial weight 218.0±43.0g; mean±S.D., n=18) were quantified by supplementing seawater with deuterated water (5%-(2)H2O) for 72h and analyzing blood glucose (2)H-enrichments by (2)H NMR. Three different nutritional states were studied: continuously fed, 21-day of fast and 21-day fast followed by 3days of refeeding. Plasma glucose levels (mM) were 10.7±6.3 (fed), 4.8±1.2 (fasted), and 9.3±1.4 (refed) (means±S.D., n=6), showing poor glycemic control. For all conditions, (2)H-enrichment of glucose position 5 was equivalent to that of position 2 indicating that blood glucose appearance from endogenous glucose 6-phosphate (G6P) was derived by gluconeogenesis. G6P-derived glucose accounted for 65±7% and 44±10% of blood glucose appearance in fed and refed fish, respectively, with the unlabeled fraction assumed to be derived from dietary carbohydrate (35±7% and 56±10%, respectively). For 21-day fasted fish, blood glucose appearance also had significant contributions from unlabeled glucose (52±16%) despite the unavailability of dietary carbohydrates. To assess the role of hepatic enzymes in glycemic control, activity and mRNA levels of hepatic glucokinase (GK) and glucose 6-phosphatase (G6Pase) were assessed. Both G6Pase activity and expression declined with fasting indicating the absence of a classical counter-regulatory stimulation of hepatic glucose production in response to declining glucose levels. GK activities were basal during fed and fasted conditions, but were strongly stimulated by refeeding. Overall, hepatic G6Pase and GK showed limited capacity in regulating glucose levels between feeding and fasting states.

Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration.

Type 1 diabetes subjects are characterized by impaired direct pathway synthesis of hepatic glycogen that is unresponsive to insulin therapy. Since it is not known whether this is an irreversible defect of insulin-dependent diabetes, direct and indirect pathway glycogen fluxes were quantified in streptozotocin (STZ)-induced diabetic rats and compared with STZ rats that received subcutaneous or intraperitoneal insulin (I-SC or I-IP). Three groups of STZ rats were studied at 18 days post-STZ treatment. One group was administered I-SC and another I-IP as two daily injections of short-acting insulin at the start of each light and dark period for days 9-18. A third group did not receive any insulin, and a fourth group of nondiabetic rats was used as control. Glycogen synthesis via direct and indirect pathways, de novo lipogenesis, and gluconeogenesis were determined over the nocturnal feeding period using deuterated water. Direct pathway was residual in STZ rats, and glucokinase activity was also reduced significantly from control levels. Insulin administration restored both net glycogen synthesis via the direct pathway and glucokinase activity to nondiabetic control levels and improved the lipogenic pathway despite an inefficient normalization of the gluconeogenic pathway. We conclude that the reduced direct pathway flux is not an irreversible defect of insulin-dependent diabetes.

Hepatic glycogen synthesis in farmed European seabass (Dicentrarchus labrax L.) is dominated by indirect pathway fluxes.

Hepatic glycogen synthesis fluxes from direct and indirect pathways were quantified in seabass by postmortem (2)H NMR analysis of plasma water (PW) and glycogen glucosyl (2)H enrichments from (2)H-enriched seawater. Eighteen fish (28.0 ± 1.7 cm and 218.0 ± 43.0 g) were divided into three groups of 6 and studied over 24 days with transfer to 5% (2)H-seawater after day 21. Over this period, one group was fed daily with fishmeal, a second group was fasted, and a third group was fasted for 21 days followed by 3 days refeeding. Glycogen turnover and sources were determined from the ratio of glucosyl position 5 enrichment to that of plasma water (H5/PW). Glycogen levels of fed fish were significantly higher than fasted (665.4 ± 345.2 µmol.g(-1) liver versus 77.2 ± 59.5 µmol.g(-1) liver, P<0.05) while refed fish had comparable levels to fed (584.6 ± 140.4 µmol.g(-1) liver). Glycogen enrichment of fed fish was undetectable indicating negligible turnover over 3 days. For fasted fish, H5/PW was ~50% indicating that half of the glycogen had turned over via indirect pathway flux. For refed fish, H5/PW was ~100% indicating that the indirect pathway accounted for all net glycogen synthesis. Direct pathway conversion of dietary carbohydrate to glycogen was not detected in any of the groups.

Hepatocyte nuclear factor 4α transactivates the mitochondrial alanine aminotransferase gene in the kidney of Sparus aurata.

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5'-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L: -alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.

Effects of alanine aminotransferase inhibition on the intermediary metabolism in Sparus aurata through dietary amino-oxyacetate supplementation.

In liver, through the reaction catalysed by alanine aminotransferase (ALT), alanine becomes an effective precursor for gluconeogenesis. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients.

Transactivation of cytosolic alanine aminotransferase gene promoter by p300 and c-Myb.

Alanine aminotransferase (Alt) provides a molecular link between carbohydrate and amino acid metabolism. In the cell context, the predominant Alt isozyme is located in the cytosol. To gain insight into the transcriptional regulation of the cytosolic alt gene (calt), we cloned and characterized the calt promoter from gilthead sea bream (Sparus aurata). Transient transfection of sea bass larvae cells with deleted calt promoter constructs and electrophoretic mobility shift assays allowed us to identify p300 and c-Myb as new factors in the transcriptional regulation of calt expression. Transfection studies carried out with an acetylase-deficient mutant p300 (p300DY) revealed that the acetyltransferase activity of p300 is essential for the p300-mediated transcriptional activation of S. aurata calt. We had previously found up-regulation of liver cAlt2, an alternatively spliced isoform of calt, under gluconeogenic conditions and in streptozotocin (STZ)-treated S. aurata. Quantitative RT-PCR assays showed that increased p300 and c-Myb mRNA levels in the liver of starved S. aurata contribute to enhancing the transcription of cAlt2. Consistently, the administration of insulin decreased both p300 and c-Myb expression. The mRNA levels of p300 and c-Myb were also analyzed in the liver of STZ-induced diabetic S. aurata. Treatment with STZ increased the expression of p300, whereas it decreased c-Myb. Our findings suggest an involvement of p300 and c-Myb in up-regulation of cAlt2 in the liver of S. aurata under starvation. In addition, these results provide evidence for a role of p300 in diabetes.