The air-breathing singhi catfish (Heteropneustes fossilis) is frequently being challenged by different environmental insults such as hyper-ammonia, dehydration and osmotic stresses in their natural habitats throughout the year. The present study investigated the effect of hyperosmotic stress, due to exposure to hypertonic environment (300 mM mannitol) for 14 days, on gluconeogenesis in this catfish. In situ exposure to hypertonic environment led to significant stimulation of gluconeogenic fluxes from the perfused liver after 7 days of exposure, followed by further increase after 14 days in presence of three different potential gluconeogenic substrates (lactate, pyruvate and glutamate). Environmental hypertonicity also caused a significant increase of activities of key gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase, fructose 1, 6-bisphosphatase and glucose 6-phosphatase by about 2-6 fold in liver, and 3-6 fold in kidney tissues. This was accompanied by more abundance of enzyme proteins by about 1.8–3.7 fold and mRNAs by about 2.2–5.2 fold in both the tissues with a maximum increase after 14 days of exposure. Hence, the increase in activities of key gluconeogenic enzymes under hypertonic stress appeared to be as a result of transcriptional regulation of genes. Immunocytochemical analysis further confirmed the tissue specific localized expression of these enzymes in both the tissues with the possibility of expressing more in the same localized places. The induction of gluconeogenesis during exposure to environmental hypertonicity possibly occurs as a consequence of changes in hydration status/cell volume of different cell types. Thus, these adaptational strategies related to gluconeogenesis that are observed in this catfish under hypertonic stress probably help in maintaining glucose homeostasis and also for a proper energy supply to support metabolic demands mainly for ion transport and other altered metabolic processes under various environmental hypertonic stress-related insults.
Citation: Das M, Banerjee B, Choudhury MG, Saha N (2013) Environmental Hypertonicity Causes Induction of Gluconeogenesis in the Air-Breathing Singhi Catfish, Heteropneustes fossilis. PLoS ONE 8(12): e85535. https://doi.org/10.1371/journal.pone.0085535
Editor: Zhiyuan Gong, National University of Singapore, Singapore
Received: October 3, 2013; Accepted: November 27, 2013; Published: December 20, 2013
Copyright: © 2013 Das et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the DSA programme to the Department of Zoology and the UPE-Biosciences project to the North-Eastern Hill University, Shillong by the University Grants Commission, New Delhi. The financial support as meritorious research fellowship to M.D. from the University Grant Commission, New Delhi, is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Gluconeogenesis from lactate, pyruvate and amino acids is important for the maintenance of circulating glucose level during strenuous  and fasting conditions in vertebrates . Gluconeogenesis has been extensively studied in liver and kidney tissues of various fish species, since these two organs are the major sites of this metabolic pathway [3-5]. In some teleostean fish, gluconeogenesis occurs at relatively higher rates [6-10], and is thought to be a key process in maintaining glucose homeostasis , especially in carnivorous fish that have high protein and low carbohydrate diets . Further, carbohydrate may also be used for short term responses in acute stress situations as a last resort in fish . Even though most of the enzymes involved in glucose metabolism have been detected in fish, the regulation of carbohydrate metabolism differs in some aspects from that of mammals . The regulation of hepatic glucose metabolism in teleost fish is reported to be influenced by different stressful conditions, such as low dietary carbohydrates and changes in hepatocellular hydration status [15-17].
Cells respond to changes in osmotic pressure with compensatory molecular adaptations that allow them to reestablish homeostasis of osmotically disturbed aspects of cell structure and function . A remarkable property of living cells is their ability to maintain a comparatively constant cell volume under different physiological conditions (for reviews, see 19,20). Thus, cells restore their conserved ionic milieu, chiefly by adjusting the levels of compatible osmolytes . Cell volume may be challenged by a variety of factors such as the intestinal absorption of water, and of various amino acids and metabolites, or by exposure to different osmotic environments especially in the case of aquatic animals. Most cells possess various volume-regulatory mechanisms such as regulatory volume decrease (RVD) and regulatory volume increase (RVI) to maintain the constancy of cell volume and also the hydration status of the cell largely by changing the permeability of various ions such as K+, Na+, H+, Cl– and HCO3-, and certain organic osmolytes [19,22-24]. However, it has been noticed in many cell types that they remain either in a slightly swollen or shrunken state for the duration of the anisotonic exposure (for review, see 19). Irrespective of the route of RVD or RVI, increase in hepatic cell volume generally results in increased anabolism and curtailment of catabolic pathways, while the reverse is true during the decrease in hepatic cell volume [16,25-28].
More recently, it has been demonstrated that the liver cells of the air-breathing walking catfish (Clarias batrachus) possess efficient volume regulatory mechanisms, but remain in partly swollen or shrunken state as long as they are exposed to anisotonicity . These changes of cell volume due to anisotonicity have been reported to cause changes in glucose, pyruvate and lactate fluxes, glycogen metabolism , hexose monophosphate pathway , and also on gluconeogenesis  in the perfused liver of walking catfish. Hallgren et al.  also reported similar effects of cell volume changes at least on glycogen metabolism in the hepatocytes of three fish species. However, it has been noticed that teleost fish face more problems of osmotic stress in comparison with mammals primarily owing to osmolarity changes in their external environment. The air-breathing singhi catfish (Heteropneustes fossilis), found predominantly in tropical Southeast Asia, is reported to be more resistant to various environmental challenges such as high environmental ammonia, hypoxic and desiccation stresses (for reviews, see 31,32). Further, they are reported to be euryhaline, inhabiting fresh and brackish waters as well as muddy marshes, thus facing wide variations of external osmolarity changes ranging from 100-350 mOsmol.l-1 . They frequently encounter the problem of osmolarity changes in the same habitat during different seasons of the year, especially in summer when the ponds and lakes dry up, thus compelling them to migrate inside the mud peat to avoid total dehydration, and during the monsoon season when the water in the same habitat gets diluted. Thus, looking at its enormous capacity in challenging the external osmolarity changes, the present study was aimed at in elucidating the possible effect of environmental hypertonicity on gluconeogenesis in this singhi catfish.
Materials and Methods
The air-breathing singhi catfish (Heteropneustes fossilis) weighing 60 ± 10 g body mass were purchased from a single source that are bred and cultured in selected commercial ponds. Fishes were acclimatized in the laboratory approximately for 1 month at 28 ± 2 °C with 12 h:12 h light and dark photoperiods before experiments. No sex differentiation of the fish was done while performing these studies. Minced dry fish and rice bran (5% of body wt) were given as food every day, and the water, collected from a natural stream, was changed on alternate days. Experiments were conducted after one month of acclimatization when the food consumption became normal and mortality rate became zero. Food was withdrawn 24 h prior to experiments.
Fishes were purchased from single source that are bred and cultured in selected commercial ponds in Lumding situated in the state of Assam, India. Fishes were anaesthetized in neutralized 3-aminobenzoic acid ethyl ester (MS-222, 0.2 g.l-1) before sacrificing by decapitation. The study was approved by the Institutional Animal Ethics Committee (IAEC) of North-Eastern Hill University, Shillong, India.
Experimental set up
Two groups of fish of similar sizes having five fish in each group were placed in two plastic containers having 5 L each of 300 mM mannitol (equivalent to water osmolarity of 300 mOsmo.l-1l) solution prepared in bacteria-free filtered stream water (pH 7.15 ± 0.07). Another two groups of fish were kept in two plastic containers having 5 L each of bacteria-free filtered stream water (pH 7.05 ± 0.04) and served as controls. Solutions from each bucket were replaced with fresh media every day at a fixed time. After 7 and 14 days, five fish each from control and treated containers were anaesthetized in neutralized 3-aminobenzoic acid ethyl ester (MS-222, 0.2 g.l-1) for 5 min. Blood samples were collected from the caudal vasculature with a heparinized syringe, and liver and kidney tissues were dissected out, plunged into liquid nitrogen and stored at −80 °C. All analyses in different tissues were completed within 2-3 weeks of collecting tissues.
Another set of treated and control fish were used for perfusion experiments after 7 and 14 days of experimental set-up.
Blood sampling and osmolarity measurement
The blood was collected with a heparinized syringe from the caudal vein and centrifuged at 10,000×g for 10 min at 0 ± 2 °C for separating out the plasma from blood leucocytes. The plasma osmolarity was measured with a Camlab osmometer (Model 200) using the freezing point depression method.
Measurement of water content
The water content in cells of different tissues of both control and hypertonically-treated fish was determined by oven drying method following Goswami and Saha .
Liver perfusion technique
Fishes were anaesthetized in neutralized 3-aminobenzoic acid ethyl ester (MS-222, 0.2 g/l) for 5 min before operation to perform the liver perfusion. The livers, while remaining attached to the body, were perfused via the portal vein in a non-circulating manner with haemoglobin-free medium following the method described by Saha et al. . The isotonic medium (265 mOsmol.l-1, determined by freezing point depression method) contained 119 mM NaCl, 5 mM NaHCO3, 5.4 mM KCl, 0.35 mM Na2HPO4, 0.81 mM MgSO4, 0.44 mM KH2PO4 and 1.25 mM CaCl2 as a basic solution for perfusion. The perfusate was gassed with O2/CO2 (99:1, v/v) and its pH adjusted to 7.5. Livers were perfused at a flow rate of 4-5 ml/g liver/min and at a temperature of 30 °C. For determining the rates of gluconeogenic efflux from the perfused liver of both treated and control fish, livers were initially perfused for 30 min with isotonic medium, followed by infusion of gluconeogenic substrates (lactate, pyruvate or glutamate) separately in three sets of perfusion experiments each at a concentration of 5 mM (a concentration suitable for studying gluconeogenic efflux, Goswami et al. ) for 30 min. Effluents were collected at 2 min intervals for the determination of glucose efflux from the perfused liver and the steady-state efflux of glucose, obtained between 22 to 30 min of infusion of substrates, was used to calculate the rates of gluconeogenic fluxes. A steady state continuous efflux of glucose normally occurs from the perfused liver while perfusing with isotonic medium at least for 100-120 min (results not shown). Therefore, the rates of gluconeogenic fluxes were calculated by subtracting the value of steady-state efflux of glucose, obtained just before infusion, from the value of steady state efflux obtained after 20 min of infusion of gluconeogenic substrates .
For estimation of glucose in the perfusate, 10 µl of 2 M perchloric acid (PCA) was added to 1 ml of effluent collected at 2 min intervals, and the precipitated protein was removed by centrifugation. The supernatant was neutralized by adding 10 µl of 2M NaOH before estimation of glucose. Concentrations of glucose in effluents were measured enzymatically following the method of Bergmeyer et al. .
A 10% homogenate (w/v) of each frozen tissue was prepared in a homogenizing buffer containing 50 mM Tris-HCl buffer (pH 7.4), 0.25 M sucrose, 1 mM ethylene diamine tetra-acetic acid (EDTA), 2 mM MgCl2, 1 mM dithiothreitol (DTT), 3 mM 2-mercaptoethanol and a cocktail of protease inhibitor (Roche, Germany) using a motor driven Potter-Elvehjem type glass homogenizer with a Teflon pestle. The homogenate was treated with 0.5% Triton X-100 in 1:1 ratio for 30 min, followed by mild sonication for 30×2 s. The homogenate was then centrifuged at 10,000 × g for 10 min and the supernatant was used for assaying the enzymes. All steps were carried out at 4°C.
The phosphoenolpyruvate carboxykinase (PEPCK) was assayed following the method of Mommsen et al.  with two-step enzymatic reactions. Fructose 1, 6-biphosphatase (FBPase) was assayed following the method of Mommsen et al.  with three step enzymatic reactions. Glucose-6-phosphatase (G6Pase) was assayed following the method of Nordlie and Arion . In case of G6Pase, the reaction was stopped by the addition of 0.5 ml 10% perchloric acid after a specific period of time and the inorganic phosphate formed was estimated in the supernatant spectrophotometrically at 700 nm following Fiske and Subbarow  against a tissue blank, and expressed as enzyme activity. The decrease in absorbance (due to oxidation of NADH to NAD+) in case of PEPCK, the increase in absorbance (due to reduction of NADP+ to NADPH) in case of FBPase were recorded at 30 s interval at 340 nm in a UV-visible spectrophotometer (Varian, Model Cary 50) fitted with a peltier temperature-controlled device. One unit of enzyme activity was expressed as that amount of enzyme which catalyzed the oxidation of 1 µmol of NADH h-1 for PEPCK, or the reduction of 1 µmol of NADP+h-1at 30°C. For G6Pase, one unit of enzyme activity was expressed as that amount which catalyzed the formation of 1 µmol of inorganic phosphate h-1 at 30°C.
Western blot analyses of different gluconeogenic enzymes such as PEPCK, FBPase and G6Pase in different tissues of singhi catfish were performed following standard methods, the details of which were described in Saha et al. .
RNA extraction and cDNA synthesis
The total RNA was isolated from liver and kidney tissues using TRI® Reagent (Sigma Chemicals, St. Louis, USA), following Rio et al. . The RNA solution was then further purified using the RNAase miniprotocol for RNA cleanup (Qiagen, Germany). Purified RNA was quantified spectrophotometrically, diluted to 5 µg/µl and electrophoresed on 1% agarose gel stained with ethidium bromide to verify integrity. First strand cDNA was synthesized from 1 µg total RNA (DNase I-treated, Invitrogen) in a total volume of 20 µl with High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, USA) as per the standard protocol.
Quantitative Real-Time PCR (qPCR)
The qPCR was performed in the 7500 FAST RT-PCR (Applied Biosystems, USA) with Power SYBR® Green PCR Master Mix (Applied Biosytems, USA). The reaction mixture of 25 µl each contained 12.5 µl of 2x SYBR Green/ROX PCR Master Mix (Applied Biosystems, USA), 2.5 µl of cDNA, 8 pmoles of each primer and 6 µl of MilliQ H2O. The PCR conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 54 °C 1 min for PECK, 57 °C 1 min for FBPase and 55 °C 1 min for G6Pase. Data were collected at 54 °C, 57 °C and 55 °C for PEPCK, FBPase and G6Pase, respectively. The qPCR was performed in triplicate and negative controls using no cDNA were run for each gene. Melting curve analysis was used to re-confirm amplification of only a single PCR product. The level of β-actin was invariant between the control and treated fish validating its choice as an endogenous control. Fold changes of PEPCK, FBPase and G6Pase genes in treated fish compared to untreated controls were calculated using the modified delta-delta CT method [41,42].
The primer pairs were chosen from the published cDNA sequences of Heteropneustes fossilis PEPCK (FJ594279), FBPase (GQ860954), G6Pase (GU131155) and β-actin (FJ409641). The primers for PEPCK were: forward (5′-CGG GAA CCT CAC TGA AGA CAA-3′) and reverse (5′-GTG AAT ATC GTG TTC TTT GAA-3′), for FBPase forward (5′-GCA GCG CCA CCA TGA TAG T-3′) and reverse (5′-TCC AGC ATG AAG CAG TTG ACA-3′), for G6Pase forward (5′-TGA AGG CTG TGG GTG TGGAT-3′) and reverse (5′-ACG CAC CAT GTC TGA GCT TTT-3′), and for β-actin the primers were: forward (5'-CG TGA CAT CAA GGA GAA GCT-3') and reverse (5'-TGC CCA TCT CCT GCT CAA AG-3'), which were designed with the help of Primer Express Software 3.0 (Applied Biosystems, USA).
Liver and kidney of both control and treated fish were excised and processed for immunostaining following Choudhury and Saha . The PEPCK and G6Pase antibody rose in goat and FBPase antibody rose in rabbit (1:20) were applied for 2 h in a wet chamber at room temperature. After washing with PBS, the slides were incubated for 2 h in Cy3-conjugated rabbit anti-goat IgG for PEPCK and G6Pase and Cy3-conjugated goat anti-rabbit IgG for FBPase (1:500) in a dark wet chamber. After final washing, the sections were covered with Vectashield mounting medium with DAPI (Vector Laboratories, USA). Another set of slides were processed in the same way except incubation with primary antibodies, which served as negative controls. Immunostained sections were analyzed in a confocal laser microscope (Leica, TCS SP5, Germany). Cross-talk of fluorochromes was excluded by the use of the acousto optical tunable filter. The entire depth of a section was scanned in 1 µm steps. The resulting stacks of pictures were mounted as single projections.
Enzymes, co-enzymes, substrates and oligonucleotide primers were purchased from Sigma Chemicals (St. Louis, USA). The PEPCK, G6Pase goat and FBPase rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (USA). Other chemicals were of analytical grades and were obtained from local sources. MilliQ water was used in all preparations.
The data collected from different replicates, were statistically analyzed and presented as mean ± S.E.M (n = number of animals in each set of experiment). Student’s t-test followed by multiple comparisons of means by Student-Newman-Keuls multiple range test were performed to evaluate differences between means where applicable. Differences with P<0.05 were regarded as statistically significant.
Effect of environmental hypertonicity on blood osmolarity and tissue water content
In situ exposure of singhi catfish in hypertonic environment (300 mOsmol.l-1) led to a significant (P<0.05) increase of blood osmolarity from 265 ± 4 to 320 ± 5 mOsmol.l-1 (21%) after 7 days and to 332 ± 6 mOsmol.l-1 (25%) after 14 days (Table 1). This also led to decreases of water content in liver, and kidney tissues by 11.2 and 9.5%, respectively, after 7 days with no further changes at later stages of exposure (Table 2).
|Blood osmolarity (mOsmol.l-1)|
|Control||7 days treated||14 days treated|
Effect of environmental hypertonicity on gluconeogenic fluxes from the perfused liver
Effect of environmental hypertonicity on gluconeogenic fluxes from the liver organ of singhi catfish, as a measure of gluconeogenic activity, was studied by the perfusion technique in presence of three different potential gluconeogenic substrates separately such as lactate, pyruvate and glutamate (Figure 1). In control fish, the maximum gluconeogenic efflux from the perfused liver was recorded in presence of glutamate (22.2 ± 0.08 µmoles.g-1 liver.h-1), followed by the presence of lactate (20.4 ± 0.12 µmoles.g-1 liver.h-1) and pyruvate (15.6 ± 0.12 µmoles.g-1 liver.h-1). Interestingly, the gluconeogenic fluxes from the perfused liver of fish exposed to hypertonic environment increased significantly by 1.61, 2.38 and 1.51 fold, respectively, in presence of lactate, pyruvate and glutamate after 7 days, which further rose to 3.30, 5.13 and 3.44 fold after 14 days.
The changes of gluconeogenic fluxes (µmoles.g-1 liver.h-1) from the perfused liver of singhi catfish were measured both in control and in fish exposed to hypertonic environment for different time intervals. Values are plotted as mean ± S.E.M (n = 5).
Livers of both control and hypertonically-treated fish were perfused with isotonic medium for 30 min, followed by infusion of gluconeogenic substrates (5 mM) for 30 min, and then again without the substrate for 20 min. The steady state fluxes of glucose between 22-30 min of perfusion and between 52-60 min of perfusion were used to calculate the rate of gluconeogenic fluxes in presence of different gluconeogenic substrates (mentioned in details in materials and methods section).
Effect of environmental hypertonicity on activities of gluconeogenic enzymes
In control fish, significant levels of activities of three key gluconeogenic enzymes namely PEPCK, FBPase and G6Pase were detected both in liver and kidney tissues (two key gluconeogenic tissues) of singhi catfish, which further enhanced significantly in fish exposed to hypertonic environment (Figure 2). In liver, the activities of PEPCK, FBPase and G6Pase increased significantly by 2.00, 2.89 and 3.84 fold, respectively, after 7 days, followed by further increase by 4.88, 3.57 and 6.16 fold after 14 days of exposure. In kidney, the activities of PEPCK, FBPase and G6Pase increased significantly by 2.92, 6.05 and 4.47 fold, respectively, after 7 days, which increased further by 4.66, 6.09 and 5.25 fold after 14 days of exposure.
Changes in activities (units.g-1 wet wt) of different gluconeogenic enzymes in singhi catfish were analysed both in control and in fish exposed to hypertonic environment for different time intervals. Values are plotted as mean ± S.E.M (n = 5).
One unit of enzyme activity was expressed as that amount of enzyme that catalyzed the oxidation of 1 µmol of NADH h-1 at 30 °C in case of PEPCK, reduction of 1 µmol of NADP+ h-1 at 30 °C in case of FBPase and 1 µmol of inorganic phosphate formed h-1 at 30 °C in case of G6Pase.
c :P value significant at <0.001 level compared to respective controls (Student’s t-test).
Effect of environmental hypertonicity on the abundance of gluconeogenic enzyme proteins
As evidenced by Western blot analysis, the increases of activities of PEPCK and FBPase and G6Pase in liver and kidney tissues of singhi catfish during exposure to environmental hypertonicity was accompanied by a significant increase in the abundance of these enzyme proteins in both the tissues (Figures 3-5). In case of PEPCK, the enzyme protein concentration increased by 1.8 and 1.9 fold in liver and kidney, respectively, after 7 days, with a further increase by 3.4 and 3.2 fold after 14 days of exposure (Figure 3). In case of FBPase, it increased by 2.2 and 2.1 fold in liver and kidney tissues, respectively, after 7 days of exposure, which further rose to 3.4 and 3.2 fold after 14 days (Figure 4). Similarly, the abundance of G6Pase enzyme protein also increased by 2.4 and 2.8 fold after 7 days of exposure, followed by further increase by 3.7 and 3.6 fold after 14 days of exposure in liver and kidney tissues, respectively (Figure 5).
Western blot analysis showing changes in the levels of expression of PEPCK enzyme protein in liver (L) and kidney (K) of singhi catfish following exposure to environmental hypertonicity at different time intervals. (A) A representative plot of 5 individual experiments. GAPDH was taken as a protein loading control. (B) Densitometric analysis showing the fold increase of PEPCK protein concentration in treated fish compared to respective controls. Values are plotted as mean ± S.E.M. (n = 5).
c :P value significant at <0.001 level compared to respective controls (Student’s t-test).
Western blot analysis showing changes in the levels of expression of FBPase enzyme protein in liver (L) and kidney (K) of singhi catfish following exposure to environmental hypertonicity at different time intervals. (A) A representative plot of 5 individual experiments. GAPDH was taken as a protein loading control. (B) Densitometric analysis showing the fold increase of FBPase protein concentration in treated fish compared to respective controls. Values are plotted as mean ± S.E.M. (n = 5).
c :P value significant at <0.001 level compared to respective controls (Student’s t-test).
Western blot analysis showing changes in the levels of expression of G6Pase enzyme protein in liver (L) and kidney (K) of singhi catfish following exposure to environmental hypertonicity at different time intervals. (A) A representative plot of 5 individual experiments. GAPDH was taken as a protein loading control. (B) Densitometric analysis showing the fold increase of G6Pase protein concentration in treated fish compared to respective controls. Values are plotted as mean ± S.E.M. (n = 5).
c :P value significant at <0.001 level compared to respective controls (Student’s t-test).
Effect of environmental hypertonicity on the expression of mRNAs for gluconeogenic enzymes
Real-time qPCR analysis on the expression of different mRNAs of three gluconeogenic enzymes indicated that the abundance mRNAs for all the enzymes got significantly elevated both in liver and kidney tissues following exposure to hypertonic environment (Figure 6). In case of PEPCK, the mRNA level increased significantly by 2.5 and 3.6 fold in liver and kidney, respectively, after 7 days, which further rose to 4.7 and 5.2 fold after 14 days of exposure. Similarly, in case of FBPase, the mRNA level increased by 2.7 and 2.2 fold in liver and kidney tissues, respectively, after 7 days, followed by further increase by 3.5 and 4.7 fold after 14 days of exposure. The level of mRNA for G6Pase also increased significantly by 2.2 and 3.1 fold, respectively, in liver and kidney tissues after 7 days, which further rose to 3.4 and 4.6 fold after 14 days of exposure to environmental hypertonicity.
Immunocytochemical analysis showing the localized expression of PEPCK in liver and kidney tissues of singhi catfish following exposure to hypertonic environment at different time intervals. Representative pictures of three independent experiments are shown. Nucleus – blue (DAPI); PEPCK – red (cy3). Scale bar: 55 µm.
Immunolocalization of gluconeogenic enzymes under environmental hypertonicity
The expression pattern and zonal localization of PEPCK, FBPase and G6Pase enzymes were observed by immunocytochemical analysis under confocal laser scanning microscope in two main gluconeogenic tissues (liver and kidney) of control and also in fish after exposure to hypertonic environment by using a monoclonal antibodies specific to PEPCK, FBPase and G6Pase (Figures 7-9). Labeling specificity was confirmed by the absence of signal in parallel control sections treated without the primary antibody (data not shown). In the liver of control fish, the signals for these gluconeogenic enzymes were mainly localized in the cluster of hepatic sinusoidal endothelial cells. After exposing the fish in hypertonic environment, the signals became more intense, but in the same localized places. In the kidney of control fish, the signals for these gluconeogenic enzymes were mainly localized in the proximal and distal tubules in the cortex region with further enhancement of signals after exposing the fish in hypertonic environment.
Immunocytochemical analysis showing the localized expression of FBPase in liver and kidney tissues of singhi catfish following exposure to hypertonic environment at different time intervals. Representative pictures of three independent experiments are shown. Nucleus – blue (DAPI); FBPase – red (cy3). Scale bar: 55 µm.
Immunocytochemical analysis showing the localized expression of G6Pase in liver and kidney tissues of singhi catfish following exposure to hypertonic environment for different time intervals. Representative pictures of three independent experiments are shown. Nucleus – blue (DAPI); G6Pase – red (cy3). Scale bar: 55 µm.
qPCR analysis showing the levels of relative expression of mRNAs for different gluconeogenic enzymes in liver and kidney tissues of singhi catfish following exposure to environmental hypertonicity at different time intervals. Values are plotted as mean ± S.E.M. (n = 5)
c : P value significant at <0.001 level, compared to respective controls (Student’s t-test)
Reports on the influences of various environmental factors such as temperature, hypoxia, starvation, and certain hormones on carbohydrate metabolism including gluconeogenesis in different fish species are well documented by several workers (for review, see 14). There are also reports on the influence of dietary carbohydrates on gluconeogenesis in trout, carp and sea bream [15,44,45]. However, reports on the influence of environmental hypertonicity on gluconeogenic activity in teleosts are scanty. More recently, it has been demonstrated that the alterations of hepatic cell volume due to anisotonicity lead to changes in carbohydrate and oxidative metabolisms in the perfused liver of air-breathing walking catfish [16,17,29], and also the autophagic proteolysis  and the rates of protein synthesis in isolated hepatocytes of the walking catfish .
The present work clearly demonstrated that the gluconeogenic activity and expression of different gluconeogenic enzyme genes viz. PEPCK, FBPase and G6Pase could be stimulated by environmental hypertonicity in singhi catfish by exposing the fish in situ in 300 mM mannitol (equivalent to 300 mOsmol.l-1osmolarity). As a consequence, the gluconeogenic fluxes from the perfused liver of fish exposed to hypertonic environment with all the three substrates (lactate, pyruvate and glutamate), which are considered to be most potential gluconeogenic substrates at least in another closely related species of air-breathing catfish (C. batrachus) , got significantly elevated. The maximum elevation was seen with lactate and pyruvate, indicating that an active Cori and alanine cycle is prevailing in this singhi catfish. Thus, lactate and pyruvate gluconeogenesis could be one of the major sources of energy in this catfish under various environmental constraints including that of hypertonicity. Further, this catfish is predominantly carnivorous in its feeding habit, and primary depends on high protein and low carbohydrate diets . Fishes are known to use lactate as an energy substrate during acclimation to hypertonic stress as evidenced from the previous studies of changes in plasma lactate levels, as well as lactate content and lactate dehydrogenase expression/activity in osmoregulatory organs [48-52]. Amino acid gluconeogenesis, which has great physiological significance, was reported in walking catfish and also in trout [17,53]. A sufficient and timely energy supply is a prerequisite for the operation of iono- and osmoregulatory mechanisms in fish. Carbohydrate metabolism appears to play a major role in the energy supply for iono- and osmoregulation, and liver is known to be the major source supplying carbohydrate metabolites to osmoregulatory organsduring acclimation to hypertonic stress. Many genes associated with many metabolic processes such as electron transport chain, TCA cycle, glycolysis, polysaccharide metabolism, fatty acid catabolism, peptide cleavage and proteolysis are reported to be up-regulated in different fish species under hypertonic stress . Hypertonicity is also reported to stimulate the autophagic proteolysis in walking catfish liver . Therefore, stimulation of proteolysis in response to hypertonicity should favour gluconeogenesis from proteolysis-derived amino acids as a coordination of a functionally linked physiological process in response to changes of cell volume under hypertonic stress.
In this study, parallel to induction of gluconeogenesis, increases in the activity of key gluconeogenic enzymes by 2-6 fold, accompanied by increases in the abundance of enzyme proteinsby about 2-4 fold and mRNAs by about 2-5 for in liver and kidney tissues of fish exposed to hypertonic environment were observed. Thus, the induction of PEPCK, FBPase and G6Pase activities appeared to be mainly associated with transcriptional regulation of genes of these enzymes under hypertonic stress.
The enzyme PEPCK is known to occur in two isoforms (the mitochondrial and the cytosolic forms) with different distribution and regulatory patterns in various groups of vertebrates . A full length PEPCK cDNA coding for mitochondrial isoform has been cloned in rainbow trout liver . It has been demonstrated that in animals in which both the mitochondrial and the cytosolic forms occur such as in chicken , only the cytosolic form is acutely regulated by diet and hormones, whereas the gene for mitochondrial PEPCK is largely constitutive in its pattern of expression . Similarly, in rainbow trout, the PEPCK gene, which is exclusively codes for the mitochondrial type of PEPCK, could not be regulated by dietary carbohydrates . But, with our present data and with partial sequence data of PEPCK (FJ594279), also for FBPase (GQ86094) and G6Pase (GU131155) genes from this singhi catfish, which could not discriminate between cytosolic and mitochondrial isoforms, it may be difficult to conclude about which isoforms were regulated at the transcriptional level resulting to an increase of activity of these enzymes in this singhi catfish during hypertonicity. However, compartmentalization of gluconeogenic enzymes could be of regulatory significance in this catfish as suggested in other fish species such as plaice (Pluronectis platessa)  and in chicken . Upregulation of PEPCK and FBPase genes at transcriptional level has been demonstrated in perfused rat liver and in H4IIE rat hepatoma cells within 3-6 h of hypertonic exposure and correlated with the hydration status of hepatic cells [58,59]. In situ exposure of singhi catfish in hypertonic environment led to a significant increase of blood osmolarity, which was accompanied by a decrease of water content both in liver and kidney tissues. In walking catfish, the hepatocytes were reported to remain partly swollen/shrunken state in hypo-/hypertonic conditional though it possesses a very efficient volume regulatory mechanisms, shown both in intact liver organ [23,25] and also in isolated cells . The same is also probably true in case of singhi catfish, since the water content both in liver and kidney tissues decreased significantly during exposure to hypertonic environment. Therefore, the induction of activities of PEPCK, FBPase and G6Pase along with more abundance of enzyme proteins and mRNAs, following in situ exposure to hypertonic environment, could also be as a result of decreasing water content or cell volume in both the gluconeogenic tissues of this catfish. Further, it was demonstrated that the increase of cell volume due to hypotonicity and decrease of cell volume due to hypertonicity cause decrease and increase of gluconeogenic activity, respectively, from different substrates using an intact liver organ of walking catfish under perfusion condition . More recently, stimulation of gluconeogenesis during exposure to high saline environment (150 mM NaCl) has also been reported in the walking catfish . Thus, the reports on the regulation of gluconeogenesis by changing the hydration status or alterations of cell volume in different gluconeogenic tissues add a new event to the complex regulation of PEPCK, FBPase and G6Pase genes of gluconeogenic enzymes in air-breathing catfish.
A major question arises now from this study concerning the mechanisms by which environmental hypertonicity, thereby decreasing the cellular hydration status of different tissues, exerts an effect on PEPCK, FBPase and G6Pase genes transcription and also enhances the gluconeogenic activity. Modulation of all the three mRNA levels due to hypertonicity appears to be due to up-regulation of gene transcription rather than mRNA stability, since increases in mRNA levels were also accompanied by more abundance of all the three enzyme proteins. In mammals, the PEPCK activity is generally altered by transcriptional regulation of expression of its gene . Further, the PEPCK gene in mammals encoding the cytosolic isoform is known to be under nutritional and hormonal control, which is not the case for mitochondrial isoform and is known to be constitutively expressed independently of nutritional status of the animal, unfed versus fed with or without carbohydrate or fed with increased dietary proportion of protein levels [44,61-64]. As noticed in mammalian system during varied physiological stimuli, including dietary carbohydrate content, nutritional status, and various hormones [54,65], the transcription of PEPCK in singhi catfish may also be tightly controlled by various pre-existing transcription factors that bind to PEPCK promoter due to altered phosphorylation status in response to hypertonicity. In rainbow trout, insulin was found to inhibit the expression of PEPCK at the transcriptional level  through the activation of the protein kinase AKT . In addition to transcriptional regulation of PEPCK, TIP60-dependent acylation of PEPCK, as a posttranslational modification, could be another means of induction of activity during exposure to environmental hypertonicity and other environmentally-related insults, as shown recently as a cause for increasing its activity in mammals during fasting . In mammals, FBPase gene expression is regulated both by transcriptional and post transcriptional mechanisms . In rainbow trout, expression of FBPase was suggested to be poorly regulated by feeding and re-feeding [56,63,70], whereas starvation was found to significantly increase the expression of FBPase gene in zebrafish . Again in mammals, the hepatic expression of G6Pase is subjected to hormonal and nutritional regulation. Increasing of cAMP, due to starvation and hormones, was reported to stimulate G6Pase gene expression, whereas re-feeding and insulin both developed opposite effect [72,73]. Similarly, food deprivation was reported to increase hepatic expression of G6Pase in gilthead sea bream [61,74,75]. In case of singhi catfish, in addition to transcriptional regulation of gluconeogenic enzymes, there could be allosteric modulation of certain gluconeogenic enzymes under hypertonic stress to ensure a prompt adaptation to gluconeogenic fluxes leading to glucose homeostasis, and energy supply during ono- and osmoregulatory processes. However, to understand better about the possible mechanism(s) of regulation of gluconeogenesis during osmotic stress in this air-breathing catfish one requires to study further.
Immunocytochemical analysis clearly demonstrated the localized expression of gluconeogenic enzyme proteins in liver and kidney tissues and more expression of all the three gluconeogenic enzymes under hypertonic stress. In liver, the expression PEPCK, FBPase and G6Pase enzyme proteins were noticed in clusters of endothelial cells of sinusoids. This zonation of gluconeogenic enzymes and to remain in same localized place could as a consequence of predominance of gluconeogenesis over glycolysis as suggested by many workers in mammals [76-79]. In kidney of singhi catfish, all the three gluconeogenic enzymes were found to express mainly in proximal and distal tubular cells localized in the kidney cortex, indicating that the glucose synthesis is compartmentalized to the proximal tubule with more expression of all the three enzymes in the same localization after exposure to hypertonic environment.
In conclusion, environmental hypertonicity leads to a stimulation of gluconeogenesis in the air-breathing singhi catfish by up-regulating the activities of key gluconeogenic enzymes as a consequence of transcriptional regulation of genes for gluconeogenic enzymes, since the induction of activities of gluconeogenic enzymes was accompanied by more abundance of key gluconeogenic enzyme proteins and mRNAs in liver and kidney tissues during exposure to hypertonic environment. Further, the gluconeogenic enzymes show localized expression in liver and kidney tissues with the possibility of more expression of these enzymes in same localized places. Furthermore, in addition to lactate and pyruvate gluconeogenesis, amino acid gluconeogenesis is also very much prevalent in this fish probably as a consequence of more abundance of amino acids due to induction of autophagic proteolysis during by hypertonic cell shrinkage shown in one related catfish (C. batrachus) . These biochemical adaptational strategies, possibly as a consequence of changes of hydration status/cell volume of different cell types during environmental hypertonicity, would assist in maintaining glucose homeostasis and proper energy supply mainly to support metabolic demands for ion transport and other altered metabolic processes in this air-breathing singhi catfish.
Conceived and designed the experiments: NS. Performed the experiments: MD BB MGC. Analyzed the data: MD BB MGC. Contributed reagents/materials/analysis tools: NS. Wrote the manuscript: NS MD.
- 1. Petersen KF, Price TB, Bergeron R (2004) Regulation of net hepatic glycogenolysis and gluconeogenesis during exercise: impact of type 1 diabetes. J Clin Endocrinol Metab 89: 4656-4664. doi:10.1210/jc.2004-0408. PubMed: 15356077.
- 2. Chandramouli V, Ekberg K, Schumann WC, Kalhan SC, Wahren J et al. (1997) Quantifying gluconeogenesis during fasting. Am J Physiol 273: E1209-E1215. PubMed: 9435538.
- 3. Mommsen TP (1986) Comparative gluconeogenesis in hepatocytes from salmonid fishes. Can J Zool 64: 1110-1115. doi:10.1139/z86-166.
- 4. Moon TW (1988) Adaptation, constraint, and the function of the gluconeogenic pathway. Can J Zool 66: 1059-1068. doi:10.1139/z88-156.
- 5. Suarez RK, Mommsen TP (1987) Gluconeogenesis in teleost fishes. Can J Zool 65: 1869-1882. doi:10.1139/z87-287.
- 6. Bever K, Chenoweth M, Dunn A (1981) Amino acid gluconeogenesis and glucose turnover in kelp bass (Paralabrax sp.). Am J Physiol 240: R246-R252. PubMed: 7212097.
- 7. Hayashi S, Ooshiro Z (1979) Gluconeogenesis in isolated liver cells of the eel, Anguilla japonica. J Comp Physiol B 132: 343-350.
- 8. Moon TW, Walsh PJ, Mommsen TP (1985) Fish hepatocytes: A model metabolic system. Can J Fish Aquat Sci 42: 1772-1782. doi:10.1139/f85-222.
- 9. Renaud JM, Moon TW (1980) Characterization of gluconeogenesis in hepatocytes isolated from the American eel, Anguilla rostrata LeSueur. J Comp Physiol B 135: 115-125.
- 10. Renaud JM, Moon TW (1980) Starvation and the metabolism of hepatocytes isolated from the American eel, Anguilla rostrata LeSeur. J Comp Physiol B 135: 127-137.
- 11. Carneiro NM, Amaral AD (1983) Effects of insulin and glucagon on plasma glucose levels and glycogen content in organs of the freshwater teleost Pimelodus maculatus. Gen Comp Endocrinol 49: 115-121. doi:10.1016/0016-6480(83)90014-X. PubMed: 6337926.
- 12. de la Higuera M, Cardenas P (1986) Hormonal effects on gluconeogenesis from (U-14C) glutamate in rainbow trout (Salmo gairdneri). Comp Biochem Physiol B 85: 517-521. PubMed: 3539501.
- 13. Christiansen DC, Klungsøyr L (1987) Metabolic utilization of nutrients and the effects of insulin in fish. Comp Biochem Physiol B 88: 701-711. doi:10.1016/0300-9629(87)90686-4. PubMed: 3322660.
- 14. Moon TW, Foster GD (1995) Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences. In: PW HochachkaTP Mommsen. Metabolic Biochemistry. Elsevier. pp. 65-100.
- 15. Enes P, Panserat S, Kaushik S, Oliva-Teles A (2009) Nutritional regulation of hepatic glucose metabolism in fish. Fish Physiol Biochem 35: 519-539. doi:10.1007/s10695-008-9259-5. PubMed: 18791853.
- 16. Goswami C, Saha N (1998) Glucose, pyruvate and lactate efflux by the perfused liver of a teleost, Clarias batrachus during aniso-osmotic exposure. Comp Biochem Physiol A 119: 999-1007. doi:10.1016/S1095-6433(98)00017-8.
- 17. Goswami C, Datta S, Biswas K, Saha N (2004) Cell volume changes affect gluconeogenesis in the perfused liver of the catfish Clarias batrachus. J Biosci 29: 337-347. doi:10.1007/BF02702616. PubMed: 15381855.
- 18. Kültz D, Fiol D, Valkova N, Gomez-Jimenez S, Chan SY et al. (2007) Functional genomics and proteomics of the cellular osmotic stress response in `non-model' organisms. J Exp Biol 210: 1593-1601. doi:10.1242/jeb.000141. PubMed: 17449824.
- 19. Häussinger D (1996) The role of cellular hydration in the regulation of cell function. Biochem J 313: 697-710. PubMed: 8611144.
- 20. Perlman DF, Goldstein L (1999) Organic osmolyte channels in cell volume regulation in vertebrates. J Exp Zool 283: 725-733. doi:10.1002/(SICI)1097-010X(19990601)283:7. PubMed: 10222593.
- 21. Somero GN, Yancey PH (1997) Osmolytes and cell volume regulation: physiological and evolutionary principles. In: JF HoffmannJD Jamieson. Handbook of Physiology, Cell Physiology. New York: Oxford University Press. pp. 441-484.
- 22. Fugelli K, Kanli H, Terreros DA (1995) Taurine efflux is a cell volume regulatory process in proximal renal tubules from the teleost Carassius auratus. Acta Physiol Scand 155: 223-232. doi:10.1111/j.1748-1716.1995.tb09967.x. PubMed: 8669295.
- 23. Goswami C, Saha N (2006) Cell volume regulation in the perfused liver of a freshwater air-breathing catfish Clarias batrachus under aniso-osmotic conditions: roles of inorganic ions and taurine. J Biosci 31: 589-598. doi:10.1007/BF02708411. PubMed: 17301497.
- 24. Michel F, Fossat B, Porthé-Nibelle J, Lahlou B, Saint-Marc P (1994) Effects of hyposmotic shock on taurine transport in isolated trout hepatocytes. Exp Physiol 79: 983-995. PubMed: 7873166.
- 25. Biswas K, Khongsngi JL, Häussinger D, Saha N (2009) Influence of cell volume changes on autophagic proteolysis in the perfused liver of air-breathing walking catfish (Clarias batrachus). J Exp Zool A Ecol Genet Physiol 311: 115-124. PubMed: 18988235.
- 26. Lang F, Ritter M, Gamper N, Huber S, Fillon S et al. (2000) Cell volume in the regulation of cell proliferation and apoptotic cell death. Cell Physiol Biochem 10: 417-428. doi:10.1159/000016367. PubMed: 11125224.
- 27. Saha N, Stoll B, Lang F, Häussinger D (1992) Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver. Eur J Biochem 209: 437-444. doi:10.1111/j.1432-1033.1992.tb17307.x. PubMed: 1396717.
- 28. Weiergräber O, Häussinger D (2000) Hepatocellular hydration: signal transduction and functional implications. Cell Physiol Biochem 10: 409-416. doi:10.1159/000016373. PubMed: 11125223.
- 29. Saha N, Goswami C (2004) Effects of anisotonicity on pentose-phosphate pathway, oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish, Clarias batrachus. J Biosci 29: 179-187. doi:10.1007/BF02703416. PubMed: 15286415.
- 30. Hallgren NK, Busby ER, Mommsen TP (2003) Cell volume affects glycogen phosphorylase activity in fish hepatocytes. J Comp Physiol B 173: 591-599. doi:10.1007/s00360-003-0369-1. PubMed: 12898165.
- 31. Saha N, Ratha BK (1998) Ureogenesis in Indian air-breathing teleosts: adaptation to environmental constraints. Comp Biochem Physiol A 120: 195-208. doi:10.1016/S1095-6433(98)00026-9.
- 32. Saha N, Ratha BK (2007) Functional ureogenesis and adaptation to ammonia metabolism in Indian freshwater air-breathing catfishes. Fish Physiol Biochem 33: 283-295. doi:10.1007/s10695-007-9172-3.
- 33. Sen TK (1985) The fish fauna of Assam and the neighbouring North-Eastern states of India. Records of the Zoological Survey of India. Calcutta: Miscellaneous Publication. p. 217.
- 34. Saha N, Dkhar J, Ratha BK (1995) Induction of ureogenesis in perfused liver of a freshwater teleost, Heteropneustes fossilis, infused with different concentrations of ammonium chloride. Comp Biochem Physiol B 112: 733-741. doi:10.1016/0305-0491(95)00115-8.
- 35. Bergmeyer HU, Brent E, Schmidt FH, Stork H (1974) D-glucose determination with hexokinase and glucose-6-phosphate dehydrogenase. In: HU Bergmeyer. Methods of Enzymatic Analysis. New York: Academic Press. pp. 1196-1201.
- 36. Mommsen TP, Walsh PJ, Moon TW (1985) Gluconeogenesis in hepatocytes and kidney of atlantic salmon. Mol. Physiol (Bethesda, Md.) 8: 89-99.
- 37. Nordlie RC, Arion WJ (1966) Glucose-6-phosphatase. In: WA Wood. Methods of Enzymology. New York: Academic Press. pp. 619–624.
- 38. Fiske CH, Subbarow Y (1957) Method for estimation of phosphate. In: SP ColowickNO Kaplin. Methods in Enzymology. New York: Academic Press. pp. 843-844.
- 39. Saha N, Jyrwa LM, Das M, Biswas K (2011) Influence of increased environmental water salinity on gluconeogenesis in the air-breathing walking catfish, Clarias batrachus. Fish Physiol Biochem 37: 681-692. doi:10.1007/s10695-011-9468-1. PubMed: 21327497.
- 40. Rio DC, Ares M, Hannon GJ, Nilsen TW (2010) Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010doi:10.1101/pdb.prot5439.
- 41. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25: 402-408. doi:10.1006/meth.2001.1262. PubMed: 11846609.
- 42. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034. PubMed: 12184808.
- 43. Choudhury MG, Saha N (2012) Influence of environmental ammonia on the production of nitric oxide and expression of inducible nitric oxide synthase in the freshwater air-breathing catfish (Heteropneustes fossilis). Aquat Toxicol 116-117: 43-53. doi:10.1016/j.aquatox.2012.03.006. PubMed: 22466354.
- 44. Panserat S, Capilla E, Gutierrez J, Frappart PO, Vachot C et al. (2001) Glucokinase is highly induced and glucose-6-phosphatase poorly repressed in liver of rainbow trout (Oncorhynchus mykiss) by a single meal with glucose. Comp Biochem Physiol B Biochem Mol Biol 128: 275-283. doi:10.1016/S1096-4959(00)00322-5. PubMed: 11207441.
- 45. Panserat S, Plagnes-Juan E, Kaushik S (2002) Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata). Biochim Biophys Acta 1579: 35-42. doi:10.1016/S0167-4781(02)00501-8. PubMed: 12401217.
- 46. Biswas K, Jyrwa LM, Häussinger D, Saha N (2010) Influence of cell volume changes on protein synthesis in isolated hepatocytes of air-breathing walking catfish (Clarias batrachus). Fish Physiol Biochem 36: 17-27. doi:10.1007/s10695-008-9275-5. PubMed: 18989741.
- 47. Munshi JSD, Ghosh TK (1994) Metabolic wheel hypothesis as applied to air-breathing fishes of India. In: HR Singh. Advances in Fish Biology. Delhi: Hindustan. pp. 70–78.
- 48. Vijayan M, Mommsen T, Glémet HC, Moon T (1996) Metabolic effects of cortisol treatment in a marine teleost, the sea raven. J Exp Biol 199: 1509-1514. PubMed: 9319410.
- 49. Sangiao-Alvarellos S, Laiz-Carrión R, Guzmán JM, del Río MPM, Miguez JM et al. (2003) Acclimation of Sparus aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs. Am J Physiol 285: R897-R907.
- 50. Sangiao-Alvarellos S, Arjona FJ, del Río MPM, Míguez JM, Mancera JM et al. (2005) Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus. J Exp Biol 208: 4291-4304. doi:10.1242/jeb.01900. PubMed: 16272252.
- 51. Polakof S, Arjona FJ, Sangiao-Alvarellos S, Martín del Río MP, Mancera JM et al. (2006) Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus. J Comp Physiol B 176: 441-452. doi:10.1007/s00360-006-0065-z. PubMed: 16432730.
- 52. Tseng Y-C, Hwang P-P (2008) Some insights into energy metabolism for osmoregulation in fish. Comp Biochem Physiol C 148: 419-429. PubMed: 18539088.
- 53. Polakof S, Soengas JL (2008) Involvement of lactate in glucose metabolism and glucosensing function in selected tissues of rainbow trout. J Exp Biol 211: 1075-1086. doi:10.1242/jeb.014050. PubMed: 18344481.
- 54. Hanson RW, Reshef L (1997) Regulation of phosphoenolpyruvate carboxykinase (GTP). Gene Expression - Annu Rev Biochem 66: 581-611. doi:10.1146/annurev.biochem.66.1.581.
- 55. Weldon SL, Rando A, Matathias AS, Hod Y, Kalonick PA et al. (1990) Mitochondrial phosphoenolpyruvate carboxykinase from the chicken. Comparison of the cDNA and protein sequences with the cytosolic isozyme. J Biol Chem 265: 7308-7317. PubMed: 2110163.
- 56. 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: 2351-2360. PubMed: 11507117.
- 57. Moon T, Johnston I (1980) Starvation and the activities of glycolytic and gluconeogenic enzymes in skeletal muscles and liver of the plaice, Pleuronectes platessa. J Comp Physiol B 136: 31-38.
- 58. Kaiser S (1998) Cell volume regulates liver phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase genes. Am J Physiol 274: G509-G517. PubMed: 9530152.
- 59. Newsome WP, Warskulat U, Noe B, Wettstein M, Stoll B et al. (1994) Modulation of phosphoenolpyruvate carboxykinase mRNA levels by the hepatocellular hydration state. Biochem J 304: 555-560. PubMed: 7998992.
- 60. Saha N, Jyrwa LM, Das M, Biswas K (2011) Influence of increased environmental water salinity on gluconeogenesis in the air-breathing walking catfish, Clarias batrachus. Fish Physiol Biochem 37: 681-692. doi:10.1007/s10695-011-9468-1. PubMed: 21327497.
- 61. Kirchner S, Kaushik S, Panserat S (2003) Low protein intake is associated with reduced hepatic gluconeogenic enzyme expression in rainbow trout (Oncorhynchus mykiss). J Nutr 133: 2561-2564. PubMed: 12888637.
- 62. Plagnes-Juan E, Lansard M, Seiliez I, Médale F, Corraze G et al. (2008) Insulin regulates the expression of several metabolism-related genes in the liver and primary hepatocytes of rainbow trout (Oncorhynchus mykiss). J Exp Biol 211: 2510-2518. doi:10.1242/jeb.018374. PubMed: 18626086.
- 63. Lansard M, Panserat S, Seiliez I, Polakof S, Plagnes-Juan E et al. (2009) Hepatic protein kinase B (Akt)-target of rapamycin (TOR)-signalling pathways and intermediary metabolism in rainbow trout (Oncorhynchus mykiss) are not significantly affected by feeding plant-based diets. Br J Nutr 102: 1564-1573. doi:10.1017/S000711450999095X. PubMed: 19664314.
- 64. Skiba-Cassy S, Lansard M, Panserat S, Médale F (2009) Rainbow trout genetically selected for greater muscle fat content display increased activation of liver TOR signaling and lipogenic gene expression. Am J Physiol Regul Integr Comp Physiol 297: R1421-R1429. doi:10.1152/ajpregu.00312.2009. PubMed: 19710390.
- 65. Beale EG, Hammer RE, Antoine B, Forest C (2004) Disregulated glyceroneogenesis: PCK1 as a candidate diabetes and obesity gene. Trends Endocrinol Metab 15: 129-135. doi:10.1016/j.tem.2004.02.006. PubMed: 15046742.
- 66. Hall RK, Yamasaki T, Kucera T, Waltner-Law M, O'Brien R et al. (2000) Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin: The role of winged helix/forkhead proteins. J Biol Chem 275: 30169-30175. doi:10.1074/jbc.M004898200. PubMed: 10913147.
- 67. Barthel A, Schmoll D (2003) Novel concepts in insulin regulation of hepatic gluconeogenesis. Am J Physiol Endocrinol Metab 285: E685-E692. PubMed: 12959935.
- 68. Lin Y-y, Lu J-y, Zhang J, Walter W, Dang W et al. (2009) Protein acetylation microarray reveals that NuA4 controls key metabolic target regulating gluconeogenesis. Cell 136: 1073-1084. doi:10.1016/j.cell.2009.01.033. PubMed: 19303850.
- 69. Okar DA, Lange AJ (1999) Fructose-2,6-bisphosphate and control of carbohydrate metabolism in eukaryotes. Biofactors 10: 1-14. doi:10.1002/biof.5520100101. PubMed: 10475585.
- 70. Kirchner S, Panserat S, Lim PL, Kaushik S, Ferraris RP (2008) The role of hepatic, renal and intestinal gluconeogenic enzymes in glucose homeostasis of juvenile rainbow trout. J Comp Physiol B 178: 429-438. doi:10.1007/s00360-007-0235-7. PubMed: 18180932.
- 71. Drew RE, Rodnick KJ, Settles M, Wacyk J, Churchill E et al. (2008) Effect of starvation on transcriptomes of brain and liver in adult female zebrafish (Danio rerio). Physiol Genomics 35: 283-295. doi:10.1152/physiolgenomics.90213.2008. PubMed: 18728227.
- 72. Argaud D, Zhang Q, Pan W, Maitra S, Pilkis SJ et al. (1996) Regulation of rat liver glucose-6-phosphatase gene expression in different nutritional and hormonal states: Gene structure and 5′-flanking sequence. Diabetes 45: 1563-1571. doi:10.2337/diabetes.45.11.1563. PubMed: 8866562.
- 73. Minassian C, Montano S, Mithieux G (1999) Regulatory role of glucose-6 phosphatase in the repletion of liver glycogen during refeeding in fasted rats. Biochim Biophys Acta 1452: 172-178. doi:10.1016/S0167-4889(99)00130-5. PubMed: 10559470.
- 74. Caseras A, Metón I, Vives C, Egea M, Fernández F et al. (2002) Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream (Sparus aurata). Br J Nutr 88: 607-614. doi:10.1079/BJN2002701. PubMed: 12493082.
- 75. Metón I, Caseras A, Fernández F, Baanante IV (2004) Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from gilthead sea bream (Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition. Comp Biochem Physiol B Biochem Mol Biol 138: 145-153. doi:10.1016/j.cbpc.2004.03.004. PubMed: 15193269.
- 76. Botini FF, Suzuki-Kemmelmeier F, Nascimento ÉA, Ide LT, Bracht A (2005) Zonation of alanine metabolism in the bivascularly perfused rat liver. Liver Int 25: 861-871. doi:10.1111/j.1478-3231.2005.01093.x. PubMed: 15998438.
- 77. Jungermann K (1995) Zonation of metabolism and gene expression in liver. Histochem Cell Biol 103: 81-91. doi:10.1007/BF01454004. PubMed: 7634156.
- 78. Jungermann K, Kietzmann T (1996) Zonation of parenchymal and nonparenchymal metabolism in liver. Annu Rev Nutr 16: 179-203. doi:10.1146/annurev.nu.16.070196.001143. PubMed: 8839925.
- 79. Sáez DE, Slebe JC (2000) Subcellular localization of aldolase B. J Cell Biochem 78: 62-72. doi:10.1002/(SICI)1097-4644(20000701)78:1. PubMed: 10797566.