Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone

The lipogenic capacity of rat liver is increased in animals fed a high carbohydrate, fat-free diet or by the administration of 2,2',5'-triiodo-L-thyronine. Underlying this change is a generalized induction of the enzymes involved in lipogenesis, including glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, which together serve to generate the additional NADPH required for increased fatty acid synthesis. This report presents evidence indicating that induction of the hexose-shunt dehydrogenases involves increased enzyme synthesis secondary to elevated enzyme specific mRNA levels, as has previously been shown for malic enzyme. Activities of specific mRNAs, estimated by cell-free translation of hepatic poly(A)-containing RNA in the mRNA dependent rabbit reticulocyte lysate, were compared with enzyme specific activities and relative rates of specific enzyme synthesis. The 2-fold increase in glucose-6-phosphate dehydrogenase specific activity in hyperthyroid rats and the 13-fold increase in rats fed a high carbohydrate, fat-free diet, relative to euthyroid, chow-fed controls were paralleled by comparable increases in the synthetic rates and mRNA levels of this enzyme. Similarly, consonant changes in the rate of enzyme synthesis and concentration of 6-phosphogluconate dehydrogenase mRNA accompanied the 2.5- and 3-fold increases in specific activity of this enzyme observed in response to hormonal and dietary induction, respectively. Thus, both thyroid hormone and carbohydrate feeding appear to induce glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase primarily by increasing the effective cellular concentrations of their respective mRNAs and, consequently, their rates of synthesis.     

Effects of dietary nutrients on lipogenic enzyme and mRNA activities in rat liver during induction

By feeding a carbohydrate diet (without protein) to fasted rats, malic enzyme mRNA activity in the liver was increased to the level in rats fed a carbohydrate and protein diet, whereas the enzyme activity itself was increased to 60% of that level. It appears that malic enzyme mRNA activity was increased by dietary carbohydrate, while dietary protein contributed to an increase in the translation of mRNA. In the animals fed carbohydrate without protein, glucose-6-phosphate dehydrogenase mRNA activity increased to 50% of the level in rats fed the carbohydrate and protein diet, whereas the enzyme activity increased to only 25%. By feeding a protein diet (without carbohydrate), glucose-6-phosphate dehydrogenase activity increased to 65% of the level in rats fed both carbohydrate and protein. This enzyme induction appears to be more dependent on protein than carbohydrate. With the carbohydrate diet, acetyl-CoA carboxylase was induced up to the level in the carbohydrate and protein diet group, whereas fatty acid synthetase was induced to only 33%. Acetyl-CoA carboxylase induction appears to be carbohydrate dependent. On the other hand, isotopic leucine incorporation studies showed that the magnitudes of the enzyme inductions caused by the dietary nutrients should be ascribed to the enzyme synthesis rates rather than the degradation. By fat feeding, the mRNA activities of malic enzyme and glucose-6-phosphate dehydrogenase were markedly decreased along with the enzyme induction. Fat appears to reduce these enzyme inductions before the translation of mRNA.     

Regulation of glucose-6-P dehydrogenase activity in primary rat hepatocyte cultures

The glucose-6-P dehydrogenase specific activity in rat hepatocytes increases approximately 10-fold when the cells are placed into culture for three days. The induction requires insulin with maximum enzyme levels occurring at 10^?7 M. Pulse-labeling experiments revealed a 10-fold increase in the enzyme's relative rate of synthesis after only 8 hours in culture.     

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A

Summary In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent K _m 's for NADP⁺ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.     

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.     

Immunological titration of rat liver glucose-6-phosphate dehydrogenase from animals fed high and low carbohydrate diets.

Recent work (Hizi and Yagil [1974] Eur. J. Biochem. $\text{45}$: 211–221, and Kelly $\text{et. al.}$ [1975] Fed. Proc. $\text{34}$: 881) suggests that the marked increase in rat liver glucose-6-phosphate dehydrogenase activity which is observed upon feeding an animal a high carbohydrate diet does not involve an increase in the total amount of enzyme present. In contrast, the data presented herein involving immunological titrations of rat liver glucose-6-phosphate dehydrogenase indicates that the increase in enzyme activity resulting from feeding a high carbohydrate diet does involve an increase in the total amount of enzyme present.     

Effect of manganese(ous) and sulfate on activity of human placental glucose 6-phosphate-dependent form of glycogen synthase.

The human placental glucose-6-P-dependent form of glycogen synthase, in the absence of glucose-6-P, can be activated by MnSO4. Separately, Mn2+ and SO4(2-) have no significant effect. In the presence of glucose-6-P, Mn2+ activates the enzyme, but SO4(2-) inhibits; MnSO4 synergetically increases the enzyme activity. Mn2+ reduces the Ka for glucose-6-P to one-tenth of the control value; SO4(2-) increases the Ka 5-fold; however, MnSO4 has no effect on Ka. MnSO4, like glucose-6-P, increases the Vmax of the enzyme in the presence of its substrate, UDP-glucose; it slightly increases the Km for UDP-glucose. In the presence of glucose-6-P, Mn2+ increases and SO4(2-) decreases the Vmax of the enzyme, but neither has an effect on the Km for UDP-glucose. At physiological concentrations of UDP-glucose and glucose-6-P, either Mn2+ or MnSO4 at concentrations less than 1 mM increases the enzyme activity as much as 8 mM glucose-6-P does. At physiological concentrations of UDP-glucose and glucose-6-P, Mn2+ or MnSO4 reverses the inhibition of the enzyme by ATP.     

Regulation of pentose phosphate pathway dehydrogenases by NADP+/NADPH ratios.

Equilibrium dialysis indicates that rat liver glucose-6-P dehydrogenase binds two molecules of NADP⁺ per subunit with a dissociation constant of 0.6 × 10^?6 M. The NADP⁺ free enzyme will not bind glucose-6-P indicating a compulsory order of substrate binding. Development of an isotopic assay allowed a direct measurement of the effect of physiological alterations in the NADP⁺/NADPH ratio on the activity of glucose-6-P and 6-phosphogluconate dehydrogenases. A combination of enzyme induction and altered NADP⁺/NADPH ratios could produce 30–50 fold changes in the capacity of these enzymes to produce NADPH during alterations in the nutritional state of the animal.     

Cellulose synthesis by extracts of Acanthamoeba castellanii during encystment. Stimulation of the incorporation of radioactivity from UDP-(14C)glucose into alkali-soluble and insoluble beta-glucans by glucose 6-phosphate and related compounds.

1. The activity of a particulate enzyme prepared from encysting cells of Acanthamoeba castellanii (Neff), previously shown to catalyze the incorporation of glucose from UDP-[14C]glucose into both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans, was stimulated several fold by glucose-6-phosphate and several related compounds. 2. Incorporation was observed when [14C]glucose-6-P was incubated with the particles in the presence of UDP-glucose. The results of product analysis by partial acid hydrolysis indicated that glucose-6-P stimulates the formation of both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans from UDP-[14C]glucose and was itself incorporated into an alkali-insoluble beta-(1 leads to 4)glucan. 3. When particles incubated with UDP-[14C]glucose and glucose-6-P were reisolated and then reincubated with unlabeled UDP-glucose and glucose-6-P, a loss of counts from the alkali-soluble fraction was detected along with a corresponding rise in the radioactivity of the alkali-insoluble fraction. This suggests that the alkali-soluble beta-glucan was converted to an alkali-insoluble product and possibly may be an intermediate stage in cellulose synthesis.     

Dietary induction of hepatic glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme in lean and obese female Zucker rats

Responses of the hepatic lipogenic enzymes, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME) to starvation refeeding and diet shifting were determined in lean and obese female Zucker rats. Rats were either fed nonpurified diet, starved 48 hr, and then refed nonpurified diet or one of the refined carbohydrate diets containing either glucose, fructose, cornstarch, or sucrose for 72 hr, or shifted from nonpurified diet directly to one of the refined carbohydrate diets for 72 hr. Initial activities were greater in obese than lean rats for all three enzymes studied. Similar to other strains of female rats, lean Zucker rats failed to demonstrate a starve-refeed response when refed nonpurified diet. Obese female littermates showed a statistically significant increase in enzymes when refed a nonpurified diet. Both lean and obese female Zucker rats demonstrated increases in enzyme activities above controls when starved and refed any of the refined carbohydrate diets. The greatest responses were observed when female rats were starved and refed sucrose; activities increased 2.6- to 3.5-fold in lean and 3.0- to 4.3-fold in obese Zuckers. In lean females 50-70% of the starve-refeed response observed with G6PDH and ME can be accounted for by simply shifting from a nonpurified diet to the respective refined carbohydrate diet, whereas in obese females only 33-55% of the increase could be attributed to diet shifting. Plasma testosterone/estrogen ratios were consistently 1.5 times higher in obese than in lean female rats. This phenotypic difference may potentiate the heightened starve-refeed overshoot response observed in obese rats.     

Induction of lipogenic enzymes in primary cultures of rat hepatocytes. Relationship between lipogenesis and carbohydrate metabolism

Using primary cultures of adult rat hepatocytes, the regulation of the following lipogenic enzymes was studied: glucose-6-phosphate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase, and stearoyl-CoA desaturase. The addition to the culture medium of either insulin or triiodothyronine produced a 2-3-fold increase in each of the individual enzyme activities whereas glucagon slightly decreased enzyme activities. The addition to the medium of 8-bromoguanosine 3,'5'-monophosphate had no effect on any of the enzyme activities unless glucose was also added to the culture medium. Glucose addition alone to the culture medium was without any effect; however, glucose enhanced the stimulation of enzyme activity due to insulin. The addition of fructose or glycerol, even in the absence of insulin, increased the activities of each of the enzymes studied 2-3-fold. The increases in enzyme activity brought about by insulin or fructose were apparently the result of de novo enzyme synthesis, as indicated by the observation that the increases were not noted in the presence of cordycepin or cycloheximide. Immunoprecipitation of ATP-citrate lyase from hepatocytes pulse-labeled with [3H]leucine indicated that the induction of this enzyme in response to the addition of fructose or glycerol to the culture medium was the result of an increase in the rate of synthesis of the enzyme. These results indicate that the activity and synthesis of individual enzymes involved in lipogenesis are increased in response to the metabolism of carbohydrate independently in part from hormonal effects.     

Immunological Difference between Glucose-6-P Dehydrogenase and Hexose-6-P Dehydrogenase from Human Liver

SHAW and Barto¹ have demonstrated the presence of an autosomally inherited glucose-6-P dehydrogenase (G6PD) in the deer mouse. Subsequently, Ohno et al.² found a similar enzyme in trout and showed that this enzyme and the autosomally inherited mouse enzyme differed from the sex-linked G6PD in possessing marked catalytic activity with galactose-6-P. This autosomally inherited G6PD was therefore named hexose-6-P dehydrogenase (H6PD)^2,3. It was shown to oxidize glucose-6-P, galactose-6-P, mannose-6-P and 2-deoxy glucose-6-P with a K_m of the order of 10^?5 M. It also oxidizes glucose with a K_m of 0.7 M³. It appears to be identical to the so-called “glucose dehydrogenase”. The enzyme utilizes both NAD and NADP and is microsome-bound. G6PD is localized in the soluble fraction of the cells of various tissues. Although it has been shown that two dehydrogenases from liver have different substrate specificity, molecular weight and elec-trophoretic mobility^3,4, it has been suggested that the two enzymes are merely isozymes and they might be interconvertible^5–7. We have now partially purified the two enzymes from human liver and show that they have different immunological properties.     

Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase. Nonenzymic formation of glucose 6-vanadate

Inorganic vanadate (Vi) activates catalysis by glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+. As the concentration of Glu-6-P dehydrogenase is increased, the rate of the vanadate-activated glucose oxidation becomes less sensitive to increases in enzyme concentration. The rate of glucose oxidation in the absence of Vi increases linearly with Glu-6-P dehydrogenase concentration. These results are interpreted in terms of nonenzymic formation of glucose 6-vanadate. At high enzyme concentration, vanadate ester formation becomes partially rate-limiting, and extrapolation to infinite Glu-6-P dehydrogenase concentration allows determination of the second order rate constant for formation of the ester. In separate experiments designed to test the proposed mechanism, it was found that Vi, at concentrations at which it strongly activates catalysis by Glu-6-P dehydrogenase of glucose oxidation, has no effect on the rates of oxidation of glucose 6-phosphate or 6-deoxyglucose catalyzed by Glu-6-P dehydrogenase. Sulfate, which is known to activate glucose oxidation and to inhibit glucose 6-phosphate oxidation, strongly activates 6-deoxyglucose oxidation. These experiments show that the 6-hydroxyl group of glucose is essential for the observed activation by Vi and are also consistent with the formation of glucose 6-vanadate. Also, the rate of the sulfate-activated glucose oxidation increases linearly with Glu-6-P dehydrogenase concentration. These results are consistent with the proposed mechanism for sulfate activation which involves sulfate binding to the enzyme (Anderson, W. B., Horne, R. N., and Nordlie, R. C. (1968) Biochemistry 7, 3997-4004). The second order rate constant calculated for formation of glucose 6-vanadate at pH 7.0 is 2.4 M-1 s-1. The corresponding values for glucose 6-phosphate and glucose 6-arsenate formation are approximately 9 X 10(-11) M-1 s-1 and 6.3 X 10(-6) M-1 s-1 (Lagunas, R. (1980) Arch. Biochem. Biophys. 205, 67-75).     

Regulation of carbohydrate metabolism by indole-3-carbinol and its metabolite 3,3′-diindolylmethane in high-fat diet-induced C57BL/6J mice

Indole glucosinolates, present in cruciferous vegetables have been investigated for their putative pharmacological properties. The current study was designed to analyse whether the treatment of the indole glucosinolates—indole-3-carbinol (I3C) and its metabolite 3,3′-diindolylmethane (DIM) could alter the carbohydrate metabolism in high-fat diet (HFD)-induced C57BL/6J mice. The plasma glucose, insulin, haemoglobin (Hb), glycosylated haemoglobin (HbA1c), glycogen and the activities of glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were analysed in liver and kidney of the treated and HFD mice. Histopathological examination of liver and pancreases were also carried out. The HFD mice show increased glucose, insulin and HbA1c and decreased Hb and glycogen levels. The elevated activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase and subsequent decline in the activity of glucokinase and glucose-6-phosphate dehydrogenase were seen in HFD mice. Among treatment groups, the mice administered with I3C and DIM, DIM shows decreased glucose, insulin and HbA1c and increased Hb and glycogen content in liver when compared to I3C, which was comparable with the standard drug metformin. The similar result was also obtained in case of carbohydrate metabolism enzymes; treatment with DIM positively regulates carbohydrate metabolic enzymes by inducing the activity of glucokinase and glucose-6-phosphate dehydrogenase and suppressing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase when compared to I3C, which were also supported by our histopathological observations.     

Dietary protein and the control of fatty acid synthesis in rat adipose tissue

Fatty acid synthesis in adipose tissue normally proceeds at a high rate when fasted animals are refed a diet containing carbohydrate, protein, and low levels of fat. This study investigated the effect of omitting protein from the refeeding diet. Rats were fasted for 48 hr and refed either a protein-free diet or a balanced diet, and the rate of fatty acid synthesis from glucose, pyruvate, lactate, and aspartate was measured. Refeeding the animals a diet devoid of protein resulted in a low rate of fatty acid synthesis from each of these substrates as well as a reduction in carbon flow over the citrate cleavage pathway. The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-malate dehydrogenase, and ATP-citrate lyase were also reduced in epididymal fat pads from these rats. On the other hand, adipose tissue phosphoenolpyruvate carboxykinase activity was five times as great as that in tissue from animals refed a balanced diet. This difference could be eliminated if actinomycin D was injected coincident with refeeding. Refeeding rats diets high in carbohydrate is not, therefore, capable of inducing high rates of fatty acid synthesis in adipose tissue in the absence of dietary proteins. Thus, liver and adipose tissue respond differently to dietary protein.     

Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Carbohydrate metabolism of goldfish (Carassius auratus L.)

Summary The effect of hypoxia was studied in cold (15°C) and warm (30°C) acclimated goldfish. The hypoxic thresholds, defined as the lowest sustainablePO₂ were found to be 1.6 and 4.0 kPa O₂ at, respectively, 15°C and 30°C. At these levels the fish did not loose either weight or appetite over a 2-months period. While during starvation under normonic conditions a significant weight loss and breakdown of lactate dehydrogenase (90%) was observed, no such changes were found in fed hypoxic animals. In red lateral muscle, white epaxial muscle and liver of goldfish from 4 differently acclimated groups the maximal activities were measured of: glycogen phosphorylase, hexokinase, malate dehydrogenase, glycerol-3-P dehydrogenase, glucose-6-P dehydrogenase, malic enzyme, succinate oxidase, pyruvate carboxylase, phosphoenol-pyruvate carboxykinase, fructose-bisphosphatase and glucose-6-phosphatase. Thermal compensation, according to Precht's typology, was predominantly observed in red muscle and to a lesser extent in white muscle. The liver glucose-6-P dehydrogenase showed a strong inverse response, which points to enhanced synthetic activity at the higher temperature. Hypoxia acclimation exerted weaker responses at 15°C than at 30°C. Changes in liver enzyme activities suggest depressed protein synthesis and enhanced gluconeogenesis in hypoxic animals. In muscle of 30°C-acclimated goldfish hypoxia induces a significant increase of succinate oxidase activity, indicating adaptation of the aerobic energy metabolism. The occurrence of pyruvate carboxylase, never before observed in vertebrate muscle, probably plays an important role in pyruvate catabolism. Because its action produces oxalo-acetate, the enzyme may stimulate pyruvate oxidation and thus prevent early lactate accumulation. Since all gluconeogenic enzymes were shown to be active in goldfish muscle, the possible occurrence of gluconeogenesis in muscle (albeit at low rate) must be accepted. Enzyme activities in goldfish muscle were compared with literature data for a number of other fish species. This comparison indicates that maximal glycolytic flux in goldfish muscle tissue is rather low, although muscular glycogen levels are very high. It is suggested that this is part of the gold-fish's strategy to cope with hypoxia.