Altered glucose homeostasis in response to aluminium phosphide induced cellular oxygen deficit in rat
Raina Dua, Vijay Kumar, Aditya Sunkaria & K D Gill*
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India Received 29 October 2009; revised 26 April 2010
The present study was designed to analyze the effect of acute aluminium phosphide (ALP) (10 mg/kg body wt.) exposure on the glucose homeostasis in rat liver and brain. ALP has been implicated in the inhibition of cytochrome oxidase causing reduced oxygen uptake and decreased ATP synthesis eventually resulting in cellular energy crisis. A significant decrease in plasma glucose levels in the ALP treated rats has been observed. Therefore, decreased ATP levels coupled with hypoglycemia may further intensify the cellular energy deficits. In order to meet the sudden increase in the local energy demand, the brain tissue utilizes its stored energy in the form of glycogen breakdown as observed by a decrease in the glycogen levels in both liver and brain which was accompanied by a marked increase in the activity of glycogen phosphorylase in both the tissues. The glycolytic rate was found to be enhanced in brain tissue as evident by increased activities of hexokinase and phosphofructokinase enzymes, but decreased in liver of ALP treated rats. Lactate levels were increased in plasma and brain, but decreased in liver of ALP treated rats. Pyruvate levels increased in the plasma and liver, but no change was observed in the brain tissue. ALP did not cause any change in the gluconeogenic enzymes like glucose-6- phosphatase and fructose-1,6-bisphophatase in brain, but a significant increase was observed in the liver. Results of the study showed that ALP induced cellular energy deficit leads to compromised energy status of liver and brain coupled with substantial alterations in glucose homeostasis. However, the activity of glucose-6-phosphate dehydrogenase decreased significantly in both the tissues.
Keywords: Aluminium phosphide, Fructose-1,6-bisphosphate, Glucose homeostasis, Glucose-6-phosphate
Aluminium phosphide (ALP), a fumigant pesticide, is one of the most extensively used metal phosphides for the protection of stored products and crops because of its efficacy, lack of persistence and harmless decomposition products1. In view of its widespread and rather indiscriminate use, coupled with its ability to interact with biological systems other than its primary targets, this pesticide constitutes a very potent health hazard to both humans and animals2.
An appraisal of literature reveals a wide range of clinical features associated with ALP poisoning, an ill-defined pathophysiology and lack of consensus about treatment3. Clinically, cases of ALP poisoning present with nausea, vomiting, severe shock, acute respiratory distress, altered sensorium and coma.
Hypotension, tachycardia and marked bradycardia are the other symptoms associated with ALP poisoning4,5. Various neurobehavioral changes like ataxia, stupor, tremors and convulsions have also been observed6.
Acute hypoxic encephalopathy due to ALP exposure
has been reported7 which may lead to death as a result of complete depression of central nervous system and paralysis of the respiratory centers of the brain1.
Brain as a tissue is distinct from other body organs due to its high metabolic rate and near complete dependence on glucose for the maintenance of neural activity and synaptic transmission8. The brain’s requirement for glucose as the exclusive energy source9 makes it especially important to understand the events that govern glucose homeostasis under normal and pathological conditions. In brain, ALP exposure may enhance neuronal lipoperoxidation damage altering structural and functional status of brain10. Histological study of human brain reveals degenerated neurons after ALP exposure11.
ALP is rapidly absorbed throughout the gastrointestinal tract after ingestion in the form of phosphine and partly carried to the liver by the portal vein. Liver functional abnormalities have been reported following acute ALP exposure but detailed investigations regarding the potential role of liver in mediating toxicity of this metal phosphide are still warranted, although there are several reports documenting the toxic effects of metal phosphide
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*Correspondent author−Telephone: 0172-2747585 Ext. 516;
Fax: 091-0172-2744401 E mail: kdgill2002@yahoo.co.in
following accidental and intentional exposure in humans as well as in animal systems, but the involved metabolic pathways, remain to be envisaged.
Therefore, this aspect needs to be thoroughly investigated in view of the increasing risk of exposure of the general population to this widely used pesticide.
It has been previously reported that acute ALP exposure resulted in decreased oxygen uptake via inhibition of cytochrome oxidase, the terminal enzyme complex of the mitochondrial electron transport chain, hence hampered mitochondrial energy metabolism in both rat liver and brain12,13. Perturbed mitochondrial electron transport chain may have serious implications on the energy requirement of the liver as well as brain tissue which may be elicited in terms of altered glucose homeostasis. To examine the alterations in the liver and brain glucose homeostasis in response to perturbed mitochondrial energy metabolism due to ALP toxicity, the glucose and glycogen metabolic pathway associated enzyme activities from control and ALP treated animals have been observed.
Materials and Methods
ChemicalsAluminium phosphide was purchased from Sandhya Organic Chemicals Pvt Ltd., India.
Bovine serum albumin, tris-hydroxymethyl aminomethane, glucose-6-phosphate, fructose-1,6- bisphosphate, sodium pyruvate were purchased from Sigma Chemicals Co., St. Louis, MO, USA. Glycogen (Oyster muscle) was purchased from E. Merk, Darmstadt, Germany. All other chemicals used were highest grade commercial products.
Experimental design
All experiments were designed in accordance with the guidelines issued by the Institute’s Ethics Committee. Male albino rats (Wistar strain) in the weight range of 140-160 g were procured from the Institute Animal House. The animals were housed in polypropylene cages, kept in well ventilated rooms, and were provisioned standard rat pellet diet (Hindustan Lever Ltd., Bombay, India) and water ad libitum. The dose of ALP was based on our previous study13.
The animals were divided into two groups of five animals each:
Control groupAnimals received an equal volume of peanut oil (vehicle) as administered to the
animals in the aluminium phosphide treated group, intragastrically.
Aluminium phosphide treated groupIn this group, the animals were administered aluminium phosphide (10 mg/kg body wt.) intragastrically.
Animals were euthanized by decapitation, 24 h after dose administration. The brain and liver were removed, rinsed in ice cold physiological saline and homogenized using appropriate buffer. Before the animals were sacrificed, blood was drawn from the orbital sinus for the estimation of plasma glucose levels.
Analytical methods
GlucoseGlucose was estimated in plasma by glucose oxidase-peroxidase method using a commercially available kit (Oscar Biotech Pvt. Ltd.
New Delhi, India).
GlycogenLiver and brain tissue (500 mg) were homogenized in 3 ml of extraction buffer (NaF 50 mM/L; EDTA 5 mM/L; 60% glycerol in de-ionized water, pH 6.5). Samples (500 µl) of the homogenate were pipetted in 2 ml of 30% KOH and boiled for 60 min in a water-bath. After the tissue digestion, 200 µl of saturated Na2SO4 solution was added and glycogen was precipitated by the addition of ethanol and centrifuged (15 min at 2000 g). The precipitate was re-suspended in boiling water and this procedure was repeated once again. Finally, the glycogen pellet was resuspended in 2 ml of 1N HCl and hydrolyzed for 60 min in boiling water. This last solution was neutralized with an equal volume of 1N NaOH and the glucose concentration was determined14.
Glycogen phosphorylaseGlycogen phosphorylase was assayed as per the method of Niemeyer et al.15. The reaction mixture containing 67 mM citrate buffer (pH 6.0), 75 mM NaF, 25 mM glucose-1-phosphate, 2.5 mM AMP, 1% glycogen and the required amount of homogenate was incubated at 37°C for 60 min. The reaction was stopped by 10% trichloroacetic acid (TCA) and the contents spun at 3000 g for 10 min. The resultant supernatant was estimated for inorganic phosphate (Pi) according to the method of Fiske and SubbaRow16.
Glycolytic enzymes
HexokinaseHexokinase was assayed according to the method followed by Bergmeyer et al.17 with slight modifications. Reaction mixture containing Tris-EDTA buffer (0.1 M histidine, 0.1 M Tris HCl, 10 mM EDTA (pH 7.0), ATP-Mg2+ mixture (25 mM
ATP, 40 mM MgCl2), requisite amount of sample and glucose (1 mg/ml) was incubated at 37°C for 30 min.
The reaction was terminated by the addition of 2 ml of chilled 10% TCA followed by centrifugation at 3000 g for 10 min. The supernatant was assayed for phosphorus and the results expressed in terms of nmol glucose phosphorylated/min/mg protein.
PhosphofructokinasePhosphofructokinase was assayed by the method of McClard et al.18. The reaction mixture contained 50 mM Tris-HCl (pH 7.0), 25 mM dithiothreitol, 60 mM MgCl2, aldolase (4 U/ml), auxillary enzyme mixture: α-glycerophosphate dehydrogenase-triose phosphate isomerase (44 U/ml), 40 mM fructose-6-phosphate, 10 mM ATP and the requisite amount of sample. The reaction was started by the addition of an appropriate amount of 10 mM NADH. The decrease in absorbance due to the oxidation of NADH was followed at 340 nm for 5 min. The enzyme activity was calculated on the basis of the molar extinction coefficient of NADH (6.22 × 10-3 M-1 cm-1) and expressed as nmol NADH oxidized/min/mg protein.
Lactate dehydrogenaseLactate dehydrogenase was assayed by the method followed by Lee et al.19. The reaction mixture contained 0.1 M Tris-HCl (pH 8.0), 1 mM sodium pyruvate, 0.15 mM NADH. The reaction was started by the addition of requisite amount of sample. The change in absorbance due to oxidation of NADH was followed at 340 nm for 2 min. The enzyme activity was calculated on the basis of molar extinction of NADH (6.22 × 10-3 M-1 cm-1) and expressed as nmol NADH oxidized/min/mg protein.
LactateLactate levels were estimated by the method followed by Bergmeyer and Bernt20 with slight modifications. The sample was deproteinized with 5% (w/v) TCA and centrifuged at 3000 g for 5 min. 20% CuSO4 and 50 mg Ca(OH)2 were added to the supernatant followed by 30 min incubation at room temperature with intermittent shaking and were finally centrifuged at 3000 g for 5 min. Two drops of 4% CuSO4 was added to the supernatant followed by addition of concentrated sulphuric acid. The contents were incubated in boiling water bath for 5 min and cooled before adding ρ-hydroxydiphenyl reagent. The reagent precipitated out on entering the concentrated acid, which was dispersed thoroughly by lateral shaking and were allowed to stand for 30 min. Finally the tubes were placed in boiling water bath for 90 s and absorbance read at 560 nm.
PyruvatePyruvate levels were measured according to the method followed by Pilkis et al.21 with slight modifications. Sample was deproteinized by the addition of 10% TCA. After centrifugation supernatant was added to dinitrophenyl hydrazine solution and allowed to react for 5 min. Toulene was added to the contents and stream of air was passed for 2 min. The lower layer was discarded and 10%
Na2CO3 was added to the upper phase. Tubes were shaken and to lower layer, 1.5 N NaOH was added.
Absorbance was read at 520 nm.
Gluconeogenic enzymes
Glucose-6-phosphataseGlucose-6-phosphatase was assayed according to the method followed by Koide and Oda22 with slight modifications. In brief, the reaction mixture containing 0.1 M citrate buffer, pH 6.2, 0.1 M glucose-6-phosphate and the appropriate amount of homogenate was incubated at 37°C for 60 min. The reaction was terminated by the addition of chilled 10% TCA and the contents spun at 3000 g for 10 min. The resultant supernatant was estimated for Pi and the results expressed as nmol Pi liberated/min/mg protein.
Fructose-1,6-bisphosphataseFructose-1,6-bis- phosphatase was assayed as per the method followed by Gancedo and Gancedo23 with slight modifications.
In brief, the reaction mixture containing 0.05 M borate buffer (pH 9.5), 0.05 M fructose-1,6- bisphosphate, 0.05 M MgCl2, 5 mM MnCl2 and required amount of sample was incubated at 37°C for 30 min. The reaction was stopped by addition of cold 10% TCA and the contents centrifuged at 3000 g for 10 min. The supernatant was estimated for Pi and the results expressed as nmol Pi liberated/min/mg protein.
ProteinProtein was determined by the method of Lowry et al.24 using bovine serum albumin as standard.
Statistical analysisAll values are expressed as mean ± SD of 5 animals in each group. Student’s t- test was used for analysis of the data and values with P<0.05 were considered statistically significant. All the calculations were carried out by Sigma Stat computer software program.
Results
Previous studies have revealed that PH3 gas, liberated from aluminium phosphide (ALP) under moist conditions, inhibits cytochrome oxidase that results in decreased oxygen uptake and hence disturbed energy metabolism12,13. The perturbed
mitochondrial electron transport chain may further compound the energy requirements of the neuronal tissue which in turn may be reflected through alterations in glucose homeostasis. Glucose is the sole energy source in brain and it utilizes an equivalent of 30% of the total body requirement of glucose. This made it pertinent to investigate the different aspects of glucose homeostasis in the wake of acute ALP exposure (10 mg/kg body wt.)
Effect of aluminium phosphide on the plasma glucose levelsPlasma glucose levels were monitored 24 h after ALP treatment. As evident from the data (Table 1), there was a significant decline in the plasma glucose level (49%) in the ALP administered rats as compared to the control group.
This may suggest that ALP poisoning may compound the cellular energy demand and lead to an excessive utilization of glucose by brain.
Effect of aluminium phosphide on the glycogen metabolism in rat liver and brain Glycogen levels in brain are low but are the main energy reserve in brain.
The results as depicted in Table 2, showed a significant depletion (16%) in the glycogen levels in brain following ALP administration when compared to the control group. As expected, the decrease in the levels of glycogen was accompanied by an increase in the activity of glycogen phosphorylase by 24% (Table 2), the principle glycogen degrading enzyme, in the ALP treated animals. Liver too demonstrated a similar increase in the activity of glycogen phosphorylase following toxic insult by ALP (Table 2).
Effect of aluminium phosphide on the glycolytic enzymes in rat liver and brainThe central nervous system predominantly utilizes the glycolytic pathway for the upkeep of neuronal homeostasis. Even mild impairments of cerebral glucose utilization can have profound effects on brain metabolism. ALP has been shown to have a strong bearing on the energy metabolism, therefore this made it imperative to study the effect of acute ALP (10 mg/kg body wt.) administration on the various enzymes of the
glycolytic pathway. The activity of hexokinase, the first and a major regulatory enzyme of the glycolytic pathway, responsible for the catalytic phosphorylation of glucose to glucose-6-phosphate, was assayed in rat brain following acute ALP exposure. The study revealed a significant increase (27%) in the activity of hexokinase in brain (Table 3). In contrast, liver showed a significant decline (21%) in the activity of hexokinase (Table 3) after ALP exposure, suggesting a compensatory mechanism following toxic insult.
Phosphofructokinase (PFK) catalyzes the reaction which constitutes the most important control point in the glycolytic cascade in transfer of a phosphate group from ATP to phosphorylate fructose-6- phosphate and yields fructose-1,6-diphosphate. ALP exposure resulted in two fold increase in the activity of PFK in brain (Table 3) depicting enhanced rate of glycolysis to meet the sudden increase in the local energy demand. On the contrary, in liver there was a marked decline (36%) in the specific activity of PFK following acute ALP exposure when compared to control group. Lactate dehydrogenase, the terminal
Table 2Effect of acute aluminium phosphide exposure on glycogen levels and the activity of glycogen phosphorylase in rat
brain and liver
[Values are mean ± SD of 5 animals in each group]
Control Aluminium phosphide treated group Glycogen levels Brain 485.19 ± 45.42 405.73 ± 19.29**
(mg/mg protein) Liver 1287.49 ± 29.30 979.14 ± 103.6***
Glycogen phosphorylase
Brain 4.35 ± 0.11 5.40 ± 0.24***
(nmol/min/mg protein)
Liver 5.35 ± 0.56 6.63 ± 0.41**
**P<0.01,***P<0.001, significantly different from control group Table 3Effect of acute aluminium phosphide exposure on the
activities of hexokinase, phosphofructokinase and lactate dehydrogenase in rat brain and liver
[Values are mean ± SD of 5 animals in each group]
Control Aluminium phosphide treated
group Hexokinase Brain 17.21 ± 1.51 21.93 ± 2.12**
(nmol/min/mg protein) Liver 14.63 ± 1.81 11.49 ± 1.18**
Phosphofructokinase Brain 0.38 ± 0.04 0.72 ± 0.07***
(nmol NADH oxidized/
min/ mg protein)
Liver 0.41 ± 0.07 0.26 ± 0.03***
Lactate dehydrogenase Brain 168.39 ± 9.48 207.28 ± 12.68***
(nmol NADH oxidized/
min/ mg protein)
Liver 156.00 ± 9.21 180.94 ± 9.05***
**P<0.01,***P<0.001, significantly different from control group Table 1Effect of acute aluminium phosphide exposure on
plasma glucose levels
[Values are mean ± SD of 5 animals in each group]
Control Aluminium phosphide treated group Plasma glucose levels
(mg/dl)
87.71 ± 5.62 48.68 ± 12.6***
***P<0.001, significantly different from control group
enzyme of glycolytic sequence, catalyzes the oxidation of NADH to NAD+ with subsequent reversible conversion of pyruvate to lactate. The results presented in Table 3, showed that acute ALP exposure elicited a significant increase of 23% and 15% in the specific activity of lactate dehydrogenase in brain and liver respectively resulting in excessive generation of lactate. From this observation, it was therefore inferred that there might be a shift from aerobic to anaerobic metabolism in brain after ALP exposure. To further substantiate our observations we assessed the effect of ALP exposure on the levels of pyruvate and lactate in rat blood, brain and liver. A significant (37%) increase was noted in blood pyruvate levels in the ALP treated animals suggesting an alternate pathway of energy generation. There was a marked increase in the pyruvate content in liver (3 fold) after ALP exposure suggesting the reversal of the glycolytic pathway via conversion of lactate to pyruvate and generating glucose. The decrease in pyruvate content as observed in present study however remained insignificant in brain tissue on ALP treatment. Enhanced lactic acid content is an index of anaerobic glycolysis. An increase of 31% in the brain lactate levels, followed by blood with an increase of 22% on ALP treatment has been observed. In contrast liver showed 19% decline in the lactate levels. These results clearly depicted the enhanced anaerobic metabolic status in brain following ALP exposure (Table 4).
Effect of acute aluminium phosphide exposure on the gluconeogenic enzymes in rat liver and brainGluconeogenesis is responsible for
converting non-carbohydrate precursors to glucose or glycogen under inadequate availability of glucose which is necessary for the metabolism of brain, erythrocytes and for the anaerobic metabolism of skeletal muscle. The conversion of fructose-1,6- bisphosphate to fructose-6-phosphate, necessary to achieve a reversal of glycolysis, is catalyzed by a specific enzyme, fructose-1,6-bisphosphatase. The results in Table 5 show no change in the activity of fructose-1,6-bisphosphatase in brain of ALP treated animals. The conversion of glucose-6-phosphate to glucose is catalyzed by glucose-6-phosphatase which allows a tissue to add glucose to the blood. Similar to fructose-1,6-bisphosphate no significant change in the activity of glucose-6-phosphatase in brain was observed (Table 5). However, in liver 29% increase was observed in ALP treated animals.
Discussion
Glucose is the major fuel for the metabolic upkeep of brain tissue, which is almost entirely dependent on a continuous supply of glucose to meet its high- energy requirements9. It is regarded that the vital processes associated with the supply of cellular energy are extremely sensitive to insult by xenobiotics. Liver plays a pivotal role in regulating the concentration of glucose in blood. It exhibits net uptake of sugar when the concentration of glucose in portal blood is high and provides a release of glucose when the blood sugar is low. Brain in contrast to other tissues, does not function autonomously, but is incorporated into a complex network where its functional activity is integrated with other parts of the central nervous system. Thus, it is obvious, that any process, which disrupts the integrity of the network,
Table 4Effect of acute aluminium phosphide exposure on lactate and pyruvate levels in blood, brain and liver of rat
[Values are mean ± SD of 5 animals in each group]
Control Aluminium phosphide treated
group Blood (mg/dl) 15.87 ± 0.76 19.44 ± 1.24***
Lactate levels Brain (µg/mg protein)
12.02 ± 1.34 15.78 ± 1.82**
Liver (µg/mg protein)
13.81 ± 1.43 11.19 ± 0.72**
Blood (mg/dl) 2.03 ± 0.39 2.79 ± 0.48*
Pyruvate levels Brain (µg/mg protein)
0.53 ± 0.07 0.52 ± 0.05NS Liver (µg/mg
protein)
0.11 ± 0.03 0.37 ± 0.07***
*P<0.05, **P<0.01, ***P<0.001, significantly different from control group. NS: not significant
Table 5 Effect of acute aluminium phosphide exposure on the activity of fructose 1,6 bisphosphatase and glucose-6-phosphatase
in rat brain and liver
[Values are mean ± SD of 5 animals in each group]
Control Aluminium phosphide treated
group Fructose-1,6-bisphosphate Brain 1.10 ± 0.23 1.12 ± 0.09NS (nmol Pi liberated/ min/
mg protein)
Liver 8.32 ± 0.39 9.35 ± 0.84*
Glucose-6-phosphatase Brain 1.49 ± 0.04 1.49 ± 0.14NS (nmol Pi liberated/ min/
mg protein)
Liver 20.63 ± 1.70 26.64 ± 2.10**
NS: not significant, *P<0.05,**P<0.01, significantly different from control group
would inevitably cause alterations in the normal metabolic pattern, which may get reflected through changes in glucose metabolism.
Glycogen is the single largest energy reserve in brain, which may serve a protective function during ischemia and hypoglycemia or can be subjected to an active stimulus responsive metabolism under normal conditions. It also acts as an immediate accessible energy reserve for meeting the sudden increase in local energy demand25. The present study involved an assessment of the changes in the levels of plasma glucose, brain glycogen and glycogen phosphorylase, the principle glycogenolytic enzyme following exposure to ALP. Acute ALP exposure elicited a significant decrease in plasma glucose levels (Table 1).
Our results are in line with those of Singh et al.26, who reported decreased blood sugar levels in a case study of a patient exposed to ALP. ALP exposure has been reported to cause bradycardia and hypotension4,5 which might also interfere with the cerebral blood supply thereby enhancing the energy requirements of the brain tissue. Hypoglycemia coupled with cellular oxygen deficits compound the energy requirements of brain, which may have serious bearing on the structural and functional integrity of the central nervous system.
Acute ALP exposure elicited a marked decrease in the glycogen content in brain along with a concomitant increase in the activity of glycogen phosphorylase (Table 2). Glycogen breakdown is known to be rapidly activated as a result of changes in both extracellular and intracellular environments.
Massive glycogenolysis has been reported to occur due to a shortfall of oxygen in tissues27. Mammalian astrocytes possess extensive glycogen stores and it has been suggested that the glucose stored in astrocytic glycogen might be released for neuronal use, depending upon the requirements signaled to these cells. This indicates that the cerebral glycogen deposits may be strongly influenced by the metabolic status of the cell28,29. Acetylcholine has been reported to cause an activation of glycogen phosphorylase with consequent depletion of glycogen30. Rastogi et al.31 reported low cholinesterase activity in serum following ALP exposure, which eventually leads to accumulation of acetylcholine. Mitra et al.32 also reported plasma cholinesterase inhibition by ALP exposure.
Liver also exhibited decreased glycogen content accompanied by enhanced activity of glycogen
phosphorylase. The decrease in glycogen content following ALP treatment could be a possible effect of phosphine interference with oxygen transport to tissues. Massive glycogenolysis has been reported to occur in liver in the presence of potassium cyanide, due to short fall of oxygen in tissues33,27 and also in an attempt to maintain cytosolic ATP levels. Hypoxic conditions in tissue have also been reported to increase the catalytic efficiency of glycogen phosphorylase34. Therefore, the observed depletion of glycogen content in brain and liver with concurrent increased efficiency of glycogen phosphorylase following ALP exposure may be a compensatory phenomenon to meet the increased energy demand.
Acute ALP exposure caused a significant increase in the activity of hexokinase in rat brain (Table 3).
The increased activity of hexokinase favors the rapid formation of glucose-6-phosphate from glucose, which is the main source of energy in the brain35. Glucose uptake is determined by the maximum phosphorylation rate and is relatively independent of glucose transport across the blood brain barrier, even if plasma glucose levels are reduced considerably.
Brain hexokinase is > 95% saturated if plasma glucose is half of its normal value36. Thus, we may speculate that even though the concentration of glucose in blood was observed to be reduced, hexokinase, rapidly converts it into glucose-6- phosphate in the cells, thus maintaining a concentration gradient. Hexokinase is one of the rate limiting enzymes of glycolysis in brain and therefore of cerebral glucose utilization37. Even mild impairment of cerebral glucose utilization can have profound effects on brain metabolism, including particularly the metabolism of acetylcholine and other neurotransmitters38. Liver hexokinase demonstrated a decline in its activity after ALP treatment indicating a reduction in the glycolytic flux so that less of glucose gets phosphorylated to glucose-6-phosphate and is available to maintain the constant concentration of blood glucose.
An assessment of the activity of phosphofructokinase (PFK) in rat brain following ALP intoxication was also carried out. As evident from the data, acute ALP exposure brought about a nearly two-fold increase in the activity of PFK in brain (Table 3). PFK constitutes the most important control point in brain glucose homeostasis and is activated by increase in the positive allosteric effectors ADP, AMP, Pi, fructose-1,6-bisphosphate
and decrease in ATP, which is a potent inhibitor of PFK39. Ksiezak and Gibson40 also reported an activation of PFK following reduction in ATP/ADP ratio thereby enhancing lactate synthesis41. The activity of PFK was found to be decreased significantly in liver on ALP treatment. This further substantiates our previous findings which showed that inhibition of liver hexokinase and thus decrease in glycolytic flux. PFK exerts significant regulatory control on the oxidation of glucose via glycolysis, therefore an increase in the activity of this enzyme may have a marked effect on the ensuing sequence of glycolytic reactions in ALP treated animals.
An investigation regarding the effect of acute ALP administration on lactate dehydrogenase (LDH) in rat brain revealed an increase in the activity of this enzyme (Table 3). LDH brings about the reversible conversion of pyruvate to lactate. The levels of this enzyme are indicative of the oxidoreductive state of the tissue42. Under anaerobic conditions, the reoxidation of NADH to NAD+ is achieved by the conversion of pyruvate to lactate by LDH, without any concomitant ATP production. This would in turn stimulate LDH, resulting in an accumulation of lactate in the brain of treated animals, a situation that may elicit severe neurological deficits in animals exposed to an acute dose of ALP.
These results suggest that ALP exposure diverted the aerobic glycolytic pathway towards anaerobic glycolysis. This speculation was confirmed by estimation of the lactate and pyruvate levels in blood, brain and liver of the rats subjected to ALP treatment.
Under anoxic conditions, lactate accumulates in blood due to activation of glycolysis and is regarded as a good indicator for tissue anoxia43. Our results demonstrated 31% increase in the brain lactate levels followed by blood, which showed an increase of 22%
(Table 4). It has been reported that hypoxic rats increased brain glucose and lactate concentration and decreased brain glycogen44. Increase in lactate levels was also observed in insects (R. dominica) subjected to anoxic conditions45. As reported earlier and warranted by our results, the rats exposed to ALP suffer from cellular oxygen deficits and hence result in increased lactate content in both brain and blood.
The increased levels of lactic acid are suggestive of glycogenolysis under anaerobic conditions46 as documented by our results. This is also consistent with the increased activity of lactate dehydrogenase in ALP treated rats. Liver demonstrated a 19% decline in the
lactate levels on ALP administration (Table 4). Lactate generated in various tissues due to enhanced anaerobic glycolysis under anoxic conditions is conveyed to liver, where it reforms glucose which again becomes available via circulation for oxidation in the tissues.
This may justify the lowered lactate levels observed in liver. Pyruvate levels were also analyzed in the ALP treated animals to substantiate the above observations.
We noted a significant increase in the blood (37%) pyruvate (3 fold) levels (Table 4). Brain pyruvate content remained unaltered following ALP exposure.
The increased pyruvate levels in blood may be suggestive of the fact that the normal route of pyruvate oxidation has been disrupted due to altered oxygen metabolism, after ALP treatment. Cyanide exposure to insects (R. dominica) also resulted in increased levels of pyruvate, supporting our observations45. In liver, lactate first gets converted to pyruvate and then back to glucose which is an energy requiring process and under the conditions of energy deficits this conversion may be slowed down leading to accumulation of pyruvate in liver.
Glycolysis and gluconeogenesis share the same pathway but are reciprocally controlled. The activities of fructose-1,6-bisphosphatase and glucose-6- phosphatase were estimated after administration of ALP, but no significant change was observed in the activities of these two gluconeogenic enzymes.
However, in liver the rate of glycolysis is inhibited in the wake of anoxic insult, leading to lowered blood glucose levels. These observations suggested that capacity of glucose generation from other sources enhanced in liver, so as to balance the lowered glucose concentration in blood, by increasing the activities of fructose-1,6-bisphosphatase and glucose- 6-phosphatase (Table 5).
Results indicated that following acute ALP exposure, the brain cells probably adjusted themselves to the increased energy requirements, through an increased mobilization of glycogen and enhanced activities of glycolytic enzymes. However there was clear trade-off in upregulating glycolysis, the glycogen stores become a limiting factor and may results in self-pollution through build-up of anaerobic end products, eventually leading to neuronal dysfunctioning.
Acknowledgement
This work has been accomplished with funds from Indian Council of Medical Research, New Delhi, Postgraduate Institute of Medical Education and
Research, Chandigarh. Aditya Sunkaria was supported by fellowship grant from the Council of Scientific and Industrial Research (CSIR), India.
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