Protective effects of d-3-hydroxybutyrate and propionate during hypoglycemic coma: clinical and biochemical insights from infant rats.

BACKGROUND: d-3-hydroxybutyrate (3OHB) is an alternative energy substrate for the brain during hypoglycemia, especially during infancy. Supplementation of 3OHB during sustained hypoglycemia in rat pups delays onset of burst suppression coma, but is associated with white matter injury and increased mortality. The biochemical basis for this ambivalent effect is not known. It may be related to an anaplerotic or gluconeogenetic deficit of 3OHB. METHODS AND RESULTS: We studied clinical alertness, EEG and brain metabolites (acyl-carnitines, amino acids, glycolytic and pentose phosphate intermediates) in 13 day-old rat pups during insulin induced hypoglycemic coma and after treatment with 3OHB alone or in combination with the anaplerotic substrate propionate. Clinically, treatment with 3OHB and propionate resulted in an alert state and EEG improvement, while treatment with 3OHB alone resulted in an improved EEG but animals remained clinically comatose. Biochemically, both treatments resulted in correction of cerebral glutamate and ammonia levels but not of gluconeogenetic substrates and pentose phosphate metabolites. CONCLUSION: 3OHB treatment restores glutamate metabolism but cannot restore a glycolytic or pentose phosphate pathway deficit. Additional treatment with propionate significantly improved the clinical protective effect of 3OHB in hypoglycemic coma.

Hypoglycemic brain damage.

Hypoglycemia was long considered to kill neurons by depriving them of glucose. We now know that hypoglycemia kills neurons actively rather than by starvation from within. Hypoglycemia only causes neuronal death when the EEG becomes flat. This usually occurs after glucose levels have fallen below 1 mM (18 mg/dL) for some period. At that time abrupt energy failure occurs, the excitatory amino acid aspartate is massively released into the limited brain extracellular space and floods the excitatory amino acid receptors located on neuronal dendrites. Calcium fluxes occur and membrane breaks in the cell lead rapidly to neuronal necrosis. Significant neuronal necrosis occurs after 30 min of electrocerebral silence. Other neurochemical changes include energy depletion to roughly 25% of control, phospholipase and other enzyme activation, tissue alkalosis, and a tendency for all cellular redox systems to shift towards oxidation. Hypoglycemia often differs from ischemia in its neuropathologic distribution, in that necrosis of the dentate gyrus of the hippocampus can occur and a predilection for the superficial layers of the cortex is sometimes seen. Cerebellum and brainstem are universally spared in hypoglycaemic brain damage. Hypoglycemia constitutes a unique metabolic brain insult.

Brain excitatory amino acid concentrations are lower in the neonatal pig: a buffer against excitotoxicity?

Hypoglycemic brain damage has been associated with high levels of the excitatory amino acids (EAA) aspartate and glutamate in the newborn and adult. We hypothesized that newborn piglet EAA would be different from those of older pigs when stressed with severe insulin-induced hypoglycemia (<30 mg/dl). Brain EAA were measured in piglets and adolescent pigs via microdialysis. Eleven of 12 newborn normoglycemic piglets had no detectable baseline levels (<0.5 microM) of EAA, while pigs had aspartate and glutamate concentrations of 1.78 +/- 0.44 and 3.43 +/- 1.14 microM (mean +/- SEM), respectively. Piglet aspartate and glutamate concentrations reached but did not significantly exceed normoglycemic pig levels after 2 h with plasma glucose values < or =20 mg/ml. Elevations in EAA were only detected in piglets whose EEG activity ceased. Aspartate and glutamate concentrations did not increase in insulin-treated pigs nor in control animals. We speculate that newborns with blood glucose less than clinically acceptable values (35 mg/dl) may be protected from EAA-associated neuronal damage during acute hypoglycemia. Lower normoglycemic and hypoglycemic levels of EAA in newborns when compared to older pigs provide this protection.

Extracellular pH in the rat brain during hypoglycemic coma and recovery.

It has previously been shown that hypoglycemic coma is accompanied by marked energy failure and by loss of cellular ionic homeostasis. The general proposal is that shortage of carbohydrate substrate prevents lactic acid formation and thereby acidosis during hypoglycemic coma. The objective of the present study was to explore whether rapid downhill ion fluxes, known to occur during coma, are accompanied by changes in extra- and/or intracellular pH (pHe and/or pHi), and how these relate to the de- and repolarization of cellular membranes. Cortical pHe was recorded by microelectrodes in insulin-injected rats subjected to 30 min of hypoglycemic coma, with cellular membrane depolarization. Some rats were allowed up to 180 min of recovery after glucose infusion and membrane repolarization. Arterial blood gases and physiological parameters were monitored to maintain normotension, normoxia, normocapnia, and normal plasma pH. Following depolarization during hypoglycemia, a prompt, rapidly reversible alkaline pHe shift of about 0.1 units was observed in 37/43 rats. Immediately thereafter, all rats showed an acid pH shift of about 0.2 units. This shift developed during the first minute, and pHe remained at that level until repolarization was induced. Following repolarization, there was an additional, rapid, further lowering of pHe by about 0.05 units, followed by a more prolonged decrease in pHe that was maximal at 90 min of recovery (delta pHe of approximately -0.4 units). The pHe then slowly normalized but was still decreased (-0.18 pH units) after 180 min when the experiment was terminated. The calculated pHi showed no major alterations during hypoglycemic coma or after membrane repolarization following glucose administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin-induced hypoglycemia on somatostatin level and binding in rat cerebral cortex and hippocampus.

The effects of severe insulin-induced hypoglycemia on somatostatin level and specific binding in the cerebral cortex and hippocampus were examined using 125I-Tyr11-somatostatin as a ligand. Severe insulin-induced hypoglycemia did not affect the level of somatostatin-like immunoreactivity in the brain areas studied. However, the number (but not the affinity) of specific somatostatin receptors was significantly decreased in membrane preparation from the hippocampus but not in the cerebral cortex at the onset of hypoglycemic coma (5-10 min). Administration of glucose at the onset of hypoglycemic coma brought about extensive recovery of hippocampal somatostatin receptor number. These results suggest that glucose modulates the somatostatin receptor in the rat hippocampus. The physiological significance of these findings remains to be clarified.

Improved brain metabolism with fructose 1-6 diphosphate during insulin-induced hypoglycemic coma.

The effect of fructose 1-6 diphosphate (FDP) on brain metabolism and brain function was investigated in hypoglycemic rabbits. The electroencephalogram and differences in oxygen content of arterial and cerebral venous blood were used as indicators for brain metabolic activity. Hypoglycemic coma was induced and maintained for 1 hour by insulin administration. At the onset of isoelectric EEG, six rabbits were treated with FDP and five rabbits received 0.9% saline. The animals were killed by an overdose of barbiturate 60 minutes after hypoglycemic recovery with glucose. FDP-treated rabbits had lower arterial glucose concentration after 40 minutes of treatment (p less than .05) and a significantly greater difference between the oxygen content of arterial and venous blood after 40 minutes (p less than .01), and after 60 minutes (p less than .025) of FDP infusion than saline-treated rabbits. FDP-treated rabbits also had a lower cerebral glucose-oxygen index than did saline-treated rabbits (p less than .005, after 20 and 40 minutes of FDP infusion). FDP administration was followed by a return of EEG activity during hypoglycemia, whereas saline produced no such effect. After glucose infusion, EEG activity was improved in FDP-treated rabbits; in saline-treated rabbits, minimal or no EEG activity was observed. The data suggest the possibility that, at the doses given in this study, FDP is taken up and used as a metabolic substrate by the brain.

[Enzyme activity and substrate levels of the Krebs cycle in the brain tissue of rats with insulin-induced hypoglycemia and during the recovery period]. / Aktivnost' fermentov i soderzhanie substratov tsikla Krebsa v tkani golovnogo mozga krys pri insulinovoi gipoglikemii i v vosstanovitel'nom periode.

Alterations in enzymatic activity and in content of substrates involved in the Krebs cycle and in content of glutamate were studied in brain of rats with insulin-dependent coma and in various periods of the coma restoration with glucose. After administration of glucose content of the Krebs cycle substrates and succinate dehydrogenase activity, which were lowered during the coma, were not normalized, whereas the content of glutamate and activity of cytoplasmic NAD-dependent malate dehydrogenase were increased in brain. Functional impairments of the nervous system in hypoglycemia and the restoration effect of glucose appear to involve some alterations in glutamate metabolism in brain.

Electrolyte shifts between brain and plasma in hypoglycemic coma.

Hypoglycemia of sufficient severity to cause cessation of EEG activity (coma) is accompanied by energy failure and by loss of ion homeostasis, the latter encompassing a marked rise in extracellular fluid (ECF) K+ concentration and a fall in ECF Ca2+ concentration. Presumably, ECF Na+ concentration decreases as well. In the present study, the extent that the altered ECF-plasma gradients give rise to net ion fluxes between plasma and tissue is explored. Accordingly, whole tissue contents of Ca2+, Mg2+, K+, and Na+ were measured. The experiments were carried out in anaesthetized and artificially ventilated rats given insulin i.p.; cerebral cortical tissue was sampled at the stage of slow-wave EEG activity, after 10, 30, and 60 min of coma (defined as isoelectric EEG), as well as after 1.5, 6, and 24 h of recovery. In the precomatose animals (with a slow-wave EEG pattern), no changes in electrolyte contents were observed. During coma, tissue Na+ content increased progressively and the K+ content fell (each by 20 mumol g-1 during 60 min). During recovery, these alterations were reversed within the first 6 h. The Mg2+ content remained unchanged. In spite of the appreciable plasma to ECF Ca2+ gradient, no significant calcium accumulation was observed. It is concluded significant calcium accumulation was observed. It is concluded that hypoglycemia leads to irreversible neuronal necrosis in the absence of gross accumulation of calcium in the tissue.

gamma-Aminobutyric acid and taurine release in the striatum of the rat during hypoglycemic coma, studied by microdialysis.

Extracellular levels of striatal gamma-aminobutyric acid (GABA) and taurine were monitored during insulin-induced hypoglycemia using microdialysis. At the onset of isoelectricity in the electroencephalogram (EEG), a transient 5-fold increase in the levels of GABA occurred. Taurine levels increased 5 min following the onset of isoelectricity and continued to increase during the entire isoelectric period. The results demonstrate that events associated with the onset of isoelectricity during hypoglycemia trigger an increase in extracellular concentrations of GABA and taurine. The discrepancy in time-course of these changes may reflect differences in compartmentation, function and metabolism of the two amino acids.

Effects of hypoglycemia on rat brain polyribosome sedimentation pattern.

Insulin-induced hypoglycemia provokes polyribosome disaggregation and accumulation of monomeric ribosomes in the brain of rats with hypoglycemic paresis and coma. The extent of brain polyribosome disaggregation depends on the decrease of blood glucose concentration, and in comatose animals on the duration of hypoglycemia. Cycloheximide prevents the disaggregation of brain polyribosomes induced by hypoglycemia, indicating that hypoglycemia affects brain protein synthesis, decreasing the rate of initiation relative to the rate of elongation of polypeptide chain synthesis.

Impaired growth hormone response to growth hormone releasing factor and insulin-hypoglycaemia in obesity.

We have previously reported an impaired growth hormone (GH) response and abnormal prolactin release to insulin-hypoglycaemia in obesity. We suggested that obese women with an absent prolactin response to hypoglycaemia ('non-responders') have a disorder of hypothalamic function. We have now investigated the GH response to i.v. growth hormone releasing factor, GHRF (1-29)NH2, in 14 obese women and nine age-matched normal-weight women. We found a significantly reduced GH response to GHRF in the obese women as compared with controls (mean peak +/- SEM: obese 8.9 +/- 2 mu/l, controls 28 +/- 2 mu/l; P less than 0.01). When the obese women were divided on the basis of their prolactin response to insulin-hypoglycaemia (seven 'non-responders', mean weight 102 +/- 5 kg; seven responders, mean weight 108 +/- 8 kg) a similar GH response to GHRF was found between the two groups but the GH response to hypoglycaemia was significantly less in the 'non-responder' women (mean peak 'non-responders' 10.5 +/- 3 mu/l, responders 27 +/- 4 mu/l; P less than 0.05). We conclude that obesity may be characterized by an impaired GH response to both i.v. GHRF and insulin-hypoglycaemia, which suggests altered hypothalamic-pituitary function. The finding that the GH response to hypoglycaemia is significantly less in the obese prolactin 'non-responder' women supports the hypothesis for a hypothalamic disorder.

Lactate reverses insulin-induced hypoglycemic stupor in suckling-weanling mice: biochemical correlates in blood, liver, and brain.

The recovery of weanling mice from insulin-induced hypoglycemic stupor-coma after injection of sodium -L(+)-lactate (18 mmol/kg) was as rapid (10 min) as in litter-mates treated with glucose (9 mmol/kg). Stimulated by this dramatic action, we studied the effects of lactate injection on brain carbohydrate and energy metabolism in normal and hypoglycemic mice; blood and liver tissue were also studied. Ten minutes after lactate injection in normal mice, plasma lactate levels increased by 15 mmol/L; plasma glucose levels were unchanged, but the beta-hydroxybutyrate concentration fell 59%. In the brains of these animals, glucose levels increased 2.3-fold, and there were significant increases in brain glycogen (10%), glucose-6-phosphate (27%), lactate (68%), pyruvate (37%), citrate (12%), and malate (19%); the increase in alpha-ketoglutarate (32%) was not significant. Lactate injection reduced the cerebral glucose-use rate 40%. These changes were not due to lactate-induced increases in blood [HCO-3] and pH (examined by injection of 15 mmol/kg sodium bicarbonate). Although lactate injection of hypoglycemic mice doubled levels of glucose in plasma and brain (not significant) and most of the cerebral glycolytic intermediates, values were far below normal (still in the range seen in hypoglycemic animals). By contrast, citrate and alpha-ketoglutarate levels returned to normal; the large increase in malate was not significant. Reduced glutamate levels increased to normal, and elevated aspartate levels fell below normal. Thus, recovery from hypoglycemic stupor does not necessarily depend on normal levels of plasma and/or brain glucose (or glycolytic intermediates).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-induced hypoglycemic coma and regional cerebral energy metabolism.

Swiss-Albino female mice weighing 20 g were rendered hypoglycemic by injecting insulin (2 units/kg). Animals were sacrificed at 40 min (pre-coma), 2 h (coma) and 4.5 h (recovery) after insulin injection by rapid submersion in liquid N2. Following sectioning at 20 micrometer, samples from the ascending reticular activating system and the inferior colliculus were freeze-dried and assayed for glucose, lactate, ATP and phosphocreatine (PCr). There was a preferential effect of hypoglycemia on ATP and PCr in cells of the ascending reticular activating system. ATP was depleted 30%, and PCr 55% in the pre-coma stage. ATP and PCr in cells from the inferior colliculus were not decreased. This selective effect on cells of the ascending reticular activating system followed by coma suggests that the coma per se may not represent total failure of the organism, but rather a compensatory mechanism designed to permit the animal to correct its compromised energy status.

The metabolism of purine and pyrimidine nucleotides in rat cortex during insulin-induced hypoglycemia and recovery.

Brains of paralysed rats with insulin-induced hypoglycemia were frozen in situ after spontaneous EEG activity had been absent for 5 or 15 min ("coma"). Recovery (30 min) was achieved in a different group of rats by administering glucose after a 30-min coma period. Purine and pyrimidine nucleotides, nucleosides and free bases were determined in the cortical extracts by high pressure liquid chromatography (HPLC). The ATP values obtained with the HPLC method were in excellent agreement with those obtained using standard enzymatic/fluorometric techniques, while values for ADP and AMP obtained with the HPLC method were significantly lower. Comatose animals showed a severe (40-80%) reduction in the concentrations of all nucleoside triphosphates (ATP, GTP, UTP and CTP) and a simultaneous increase in the concentrations of all nucleoside di- and monophosphates, including that of IMP. The adenine nucleotide pool size decreased to 50% of control level. The concentrations of the nucleosides adenosine, inosine, and uridine increased 50- to 250-fold, while the concentrations of the purine bases, xanthine and hypoxanthine, rose 2- and 30-fold, respectively. There were no increases in the concentrations of adenine, guanine, or xanthosine. Following glucose administration there was a partial (ATP, UTP and CTP) or almost complete (GTP) recovery of the nucleoside triphosphate levels. During recovery, the levels of nucleoside di- and monophosphates and of adenosine decreased to values close to control; the rise in the inosine level was only partially reversed, and the concentrations of hypoxanthine and xanthine rose further. The adenine nucleotide pool size was only partially restored (to 67% of control value). The adenine nucleotide pool size was not increased by i.p. injection of adenosine or adenine under control condition, or during the posthypoglycemic recovery period.

Regional CNS levels of acetylcholine and choline during hypoglycemic stupor and recovery.

During insulin stupor in mice, acetylcholine levels in cerebral cortex, cerebellum, brainstem, striatum, and hippocampus were unchanged from control values despite brain glucose concentrations 3-10% of normal, whereas choline levels rose 2.4-3.6-fold in all five CNS regions. Brain acetylcholine and choline levels did not change during recovery following glucose injection. The data suggest that, in hypoglycemic stupor, (1) overall rates of acetylcholine synthesis and degradation remain balanced within each of the CNS regions studied; (2) the biochemical mechanism that elevates brain choline levels is unlikely to be related only to cholinergic synaptic processes; and (3) brain choline levels need not rise for stupor to occur.

[Cerebral GABA, glutamate, aspartate and transaminases in the insulin nervous syndrome]. / GAMK--glutamat--aspartat i transaminazy mozga pri insulinovom nervnom sindrome.

Alterations in content of glutamate, aspartate and gamma-aminobutyric acid /GABA/ as well as in activity of some enzymes involved in amino acid transamination were studied in rat brain tissue under conditions of insulin coma and in dynamics of its treatment using glucose. The comatose state caused in the brain tissue a decrease in content of glutamate and an increase in aspartic acid content but the concentration of GABA and the activity of transaminases were unaltered. Treatment of the coma using glucose was accompanied by an increase in concentration of the amino acids studied, especially of glutamic acid as well as by a decrease in the activity of transaminases.