Concentration Of Glycerol 3-phosphate Research Articles

Studies on immunotoxicity induced by emamectin benzoate in zebrafish embryos based on metabolomics.

Emamectin benzoate (EMB) is an insecticide for the control of agricultural lepidoptera pests, and also an anti-parasiticide for the control of exoparasites in aquaculture industry. Increased studies suggest that EMB could cause toxicity to non-targeted organisms, but its immunotoxicity to human remains unclear. In this study, zebrafish were used to investigate the immunotoxic effects induced by environmentally relevant doses of EMB. We observed that EMB exposure led to embryo mortality and delayed hatching, as well as increased malformations. Meanwhile, zebrafish exposed to EMB exhibited a significant decrease in the number of neutrophils and macrophages. In addition, untargeted metabolomics approach was developed to elucidate the mechanism of EMB-induced immunotoxicity. We found that a total of 10 shared biomarkers were identified in response to EMB exposure. Furthermore, pathway analysis identified glycerophospholipid metabolism was the most relevant pathway. Within this pathway, it was observed abnormal increases in glycerol 3-phosphate content, which could be attributed to the increased expression of GK5 and decreased expression of GPAT3. Our study provided novel and robust perspectives, which showed that EMB exposure to zebrafish embryos could cause metabolic disturbances that adversely affected development and immune system.

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

Effects of adrenaline on triacylglycerol synthesis and turnover in ventricular myocytes from adult rats.

Ca2+-tolerant myocytes were isolated with endogenous triacylglycerol (TAG) stores prelabelled with [3H]palmitate and subsequently incubated for a 1h chase period with [14C]palmitate, 2% albumin and 5mM glucose. Measurements were then made of [14C]palmitate conversion into TAG and phospholipids, of loss of [3H]TAG, of glycerol release and of change in the total TAG content. Rates of de novo synthesis of TAG were calculated by a balance method. With 0. 5mM palmitate present, 5 microM adrenaline increased de novo synthesis of TAG by 81% and incorporation of [14C]palmitate into phospholipids by 59%. Significant increases in these processes with adrenaline were also seen with 0.08, 0.14 and 0.26 mM palmitate. The beta-agonist isoprenaline had little effect on de novo synthesis of TAG and had no effect on [14C]palmitate conversion into phospholipids. The alpha1-agonist phenylephrine mimicked adrenaline in increasing [14C]palmitate conversion into phospholipids but had no effect on de novo synthesis of TAG. Adrenaline did not significantly alter the myocyte glycerol 3-phosphate content but caused a persistent 40% increase in the activity of the form of glycerolphosphate acyltransferase found predominantly in the sarcoplasmic reticulum. With 0.5 mM palmitate present, the value [14C]TAG formed -decrease in [3H]TAG consistently exceeded the enzymically measured change in cell TAG content. From this it was suggested that the specific radioactivity of [3H]TAG pool(s) mobilized during the chase period was lower than that of the overall cell TAG. In the basal state, complete mobilization of TAG measured as glycerol release was low, but cycling of TAG to diacylglycerol or monoacylglycerol and back to TAG appeared to be high. With adrenaline present, glycerol release was increased 5-6-fold but recycling of lower acylglycerols to TAG was abolished. Glycerol release was inhibited by increasing extracellular palmitate from 0.08 to 0.5 mM. Adrenaline partially over-rode this effect.

Relationship between the stimulation of citric acid cycle oxidation and the stimulation of fatty acid esterification and inhibition of ketogenesis by lactate in isolated rat hepatocytes

Isolated hepatocytes from fasted rats were used to study the effects of lactate on palmitate metabolism. Lactate was found to stimulate fatty acid esterification and citric acid cycle oxidation and to inhibit ketone body synthesis. These effects of lactate were largely maintained when gluconeogenesis was inhibited with either quinolinate or perfluorosuccinate, but were overcome by α-cyano-4-hydroxycinnamate. However, the responses of hepatocytes to lactate could be restored in the presence of α-cyano-4-hydroxycinnamate by the further addition of propionate. The stimulation of triacylglycerol synthesis by lactate was not associated with an increase in the concentration of glycerol 3-phosphate. Rather, there was a correlation between flux through the citric acid cycle and the rate of triacylglycerol synthesis. In all instances reduction of ketone body formation in the presence of lactate was accompanied by a stimulation of citric acid cycle oxidation.

Sublethal effects of pentachlorophenol in the abalone (haliotis rufescens) as measured by in vivo 31P NMRSpectroscopy

The sublethal biochemical effects of pentachlorophenol (PCP) were investigated in live, intact red abalones (Haliotis rufescens), using a flow-through exposure system, by in vivo 31P NMR spectroscopy. Based on rangefinding tests (6-hr LC50 = 1.6 mg/L; 6-hr no-observable-effect-level (NOEL) = 0.8 mg/L), three abalones were separately exposed to a sublethal concentration (1.2 mg/L) for 5 hr, followed by a 13 hr recovery period. Effects in foot muscle included both a decrease in phosphoarginine and an increase in inorganic monophosphate concentrations ([PA] and [Pi], respectively); both foot muscle concentrations of adenosine triphosphate [ATP] and intracellular pH (pHi) also declined. Parallel in vitro experiments revealed that concentrations of glycerol 3-phosphate, lactate, citrate, succinate, malate, and alanine (Ala) all increased, while those of glyceraldehyde 3-phosphate and glutamine (Gln) remained stable. Also, these effects were not evident until 2 hr into exposure, possibly the time required for PCP to attain an effective concentration in foot muscle. During recovery, while Pi declined to pre-exposure levels, [PA] completely recovered in only one individual. Also, realkalinization of pHi was similar to recovery of [Pi], and ATP returned to near-initial levels, as did glycerol 3-phosphate, lactate, succinate, malate, and Ala; glyceraldehyde 3-phosphate, citrate, and Gln levels declined. Recovery responses corresponded to the time for PCP clearance from foot muscle. The effects of PCP were similar to those of hypoxia, fatigue, hypersalinity, and arginine kinase inhibitors, and so sublethal PCP concentrations may also inhibit electron transport and arginine kinase as well as uncouple mitochondrial oxidative phosphorylation in intact molluscs. Thus, the effects of pollutants on key biochemical processes may now be measured in intact aquatic organisms as they occur, improving our ability to accurately assess the environmental effects of pollutants in the laboratory.

Role of plastidial acyl-acyl carrier protein: Glycerol 3-phosphate acyltransferase and acyl-acyl carrier protein hydrolase in channelling the acyl flux through the prokaryotic and eukaryotic pathway

In order to investigate whether the relative activities of the plastidial acyl-acyl carrier protein (ACP):glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and acyl-ACP hydrolase play a role in controlling the acyl flux through the prokaryotic and eukaryotic pathway, we determined these enzymic activities in stroma fractions from 16:3- and 18:3-plants using glycerol 3-phosphate and labelled acyl-ACP as substrates. Several factors were examined which might influence the activities within plastids, such as leaf development, salts at physiological concentrations, stroma pH and substrates available to the enzymes. An appreciable alteration of the two enzymic activities was only observed with changes in the pH and substrate concentration, especially the concentration of glycerol 3-phosphate. An increase in pH from 7 to 8 resulted in a decreased ratio of acyltransferase versus hydrolase activity in stroma fractions from both pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), whereas exogenously added glycerol 3-phosphate, which only influenced the acyltransferase, raised this ratio. On the other hand, the relative activities of the two enzymes stayed rather constant at oleoyl-ACP concentrations between 1 and 2 μM not only when it was offered alone but also in a mixture with palmitoyl-ACP. At pH 8, the stroma pH of illuminated chloroplasts, and at physiologically relevant substrate concentrations we observed clear differences between the 16:3-plants spinach and mustard (Sinapis alba ssp. alba L.) and the 18:3-plants pea and maize (Zea mays L.). In accordance with the different proportions of prokaryotic glycerolipids in the two groups of plants, pea and maize showed distinctly lower ratios of acyltransferase versus hydrolase activity than spinach and mustard. Consequently the relative activities of the plastidial glycerol 3-phosphate acyltransferase and acyl-ACP hydrolase can play a decisive role in controlling the acyl flux through the different pathways at least in these plants.

Short and long term effects of ethanol on glycerolipid synthesis from palmitate in fasted rat hepatocytes

1. Prolonged ethanol administration to rats increased the rates of glycerolipid synthesis from added [U- 14C]palmitate in fasted hepatocytes; this increase was more than 2-fold in triglyceride synthesis. 2. Prolonged ethanol administration to rats completely eliminated the acute ethanol-induced increase in triglyceride synthesis from palmitate in hepatocytes from fasted rats. This adaptive change occurred in a short initial period of about 10 days of ethanol feeding. 3. In hepatocytes from fasted control rats, addition of ethanol produced a rapid and strong increase in the concentration of glycerol 3-phosphate. 4. By contrast, this acute effect of ethanol disappeared in hepatocytes from fasted alcoholic rats after a prolonged—5 weeks—administration of ethanol in a liquid diet.

Metabolic effects of D-glyceraldehyde in isolated hepatocytes.

The effects of D-glyceraldehyde on the hepatocyte contents of various metabolites were examined and compared with the effects of fructose, glycerol and dihydroxyacetone, which all enter the glycolytic/gluconeogenic pathways at the triose phosphate level. D-Glyceraldehyde (10 MM) caused a substantial depletion of hepatocyte ATP, as did equimolar concentrations of fructose and glycerol. D-Glyceraldehyde and fructose each caused a 2-fold increase in fructose 1,6-bisphosphate and the accumulation of millimolar quantities of fructose 1-phosphate in the cells. D-Glyceraldehyde caused an increase in the glycerol 3-phosphate content and a decrease in the dihydroxyacetone phosphate content, whereas dihydroxyacetone increased the content of both metabolites. The increase in the [glycerol 3-phosphate]/[dihydroxyacetone phosphate] ratio caused by D-glyceraldehyde was not accompanied by a change in the cytoplasmic [NAD+]/[NADH] ratio, as indicated by the unchanged [lactate]/[pyruvate] ratio. The accumulation of fructose 1-phosphate from D-glyceraldehyde and dihydroxyacetone phosphate in the hepatocyte can account for the depletion of the intracellular content of the latter. Presumably ATP is depleted as the result of the accumulation of millimolar amounts of a phosphorylated intermediate, as is the case with fructose and glycerol. It is suggested that the accumulation of fructose 1-phosphate during hepatic fructose metabolism is the result of a temporary increase in the D-glyceraldehyde concentration because of the high rate of fructose phosphorylation compared with triokinase activity. The equilibrium constant of aldolase favours the formation and thus the accumulation of fructose 1-phosphate.

Regulation of ketogenesis, gluconeogenesis, and glycogen synthesis by insulin and proinsulin in rat hepatocyte monolayer cultures.

The metabolic actions of porcine insulin and biosynthetic human proinsulin on fatty acid and glucose metabolism were studied in rat hepatocytes cultured in monolayer for 24 h. Our aim was to establish whether proinsulin action in the liver is similar to insulin action and whether the relative potencies of the two hormones are the same for different metabolic processes. Proinsulin and insulin exerted a similar maximal inhibitory effect on ketone body formation from palmitate and on gluconeogenesis from pyruvate. The half-maximal effective concentration of proinsulin was 11-13 times that of insulin. The antiketogenic effects of insulin and proinsulin were associated with an increased glycerol 3-phosphate content and a decreased affinity of carnitine palmitoyltransferase for its substrate palmitoyl-CoA. When the basal rate of ketogenesis was increased with isobutyl methylxanthine, the half-maximal effective concentrations of both proinsulin and insulin were decreased, but the relative potency of the two hormones was unchanged. Proinsulin and insulin exerted similar maximal stimulatory effects on glycogen synthesis and on the activities of pyruvate kinase, glucose 6-phosphate dehydrogenase, phosphogluconate dehydrogenase, and malic enzyme. The half-maximal effective concentration of proinsulin was 10-30 times that of insulin. These findings are consistent with receptor binding studies on liver membranes that suggest that proinsulin interacts with insulin-specific and not proinsulin-specific receptors. Our findings also suggest that proinsulin action does not differ from insulin action at a postreceptor site.

Accumulation of triacylglycerol in cultured rat hepatocytes is increased by ethanol and by insulin and dexamethasone.

Isolated hepatocytes from female rats were cultured in HI-WO/BA medium for 6 days. To the medium was added oleate, ethanol, dexamethasone and insulin. With oleate To alone, triacylglycerol accumulated; ethanol augmented the accumulation by 90%. To the best of our knowledge, this is the first demonstration that ethanol in vitro increases the content of triacylglycerol in liver cells. Further addition of dexamethasone or insulin did not alter the accumulation of triacylglycerol, indicating that these hormones did not play permissive roles for the effect of ethanol in the present system. Dexamethasone and insulin, in the absence of ethanol, increased the accumulation of triacylglycerol by 30% and 50% respectively. The concentration of glycerol 3-phosphate was increased in the presence of ethanol; however, with time the concentration of glycerol 3-phosphate declined almost to control values, while the accumulation of triacylglycerol continued linearly; this suggests that the effect of ethanol was not mediated via fluctuations in the concentration of glycerol 3-phosphate. These results are discussed in relation to earlier investigations in vivo and in vitro.

Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content

Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis.

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V(max.) of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30-40% in starvation. There was no change in apparent K(m) for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3-0.4 and 0.5-0.65mumol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3mumol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44mumol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10 6 cells. Incorporation of [1,3- 14C]glycerol into total cell lipids was 0.29 nmol/h per 10 6 cells. In the presence of saturating glycerol concentrations, production of 3H 2O from [2- 3H]glycerol was 13 times greater than incorporation of [ 14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.

Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.