Influence of Severe Hypoglycemia on Mitochondrial and Plasma Membrane Function in Rat Brain

Abstract: Previous experiments have shown that severe hypoglycemia disrupts cerebral energy state in spite of a maintained cerebral oxygen consumption, suggesting uncoupling of oxidative phosphorylation. Other studies have demonstrated that hypoglycemia leads to loss of cerebral cortical phospholipids and phospholipid-bound fatty acids. The objective of the present study was, therefore, to study respiratory characteristics of brain mitochondria during severe hypoglycemia and to correlate respiratory activity to mitochondrial phospholipid composition. Mitochondria were isolated after 30 or 60 min of hypoglycemia with ceased EEG activity, and after a 90-min recovery period, and their resting (state 4) and ADP-stimulated (state 3) oxygen consumption rates and phospholipids and phospholipid-bound fatty acid content were measured. After 30 min of hypoglycemia, state 3 respiration decreased without any increase in state 4 respiration or change in ADP/O ratio. This decrease, which occurred with glutamate plus malate—but not with succinate—as substrates, was partly reversed by addition of bovine serum albumin and KCI. Chemical analyses of isolated mitochondria did not reveal changes in their phospholipid or fatty acid content. The results thus failed to account for the dissociation of cerebral energy state and oxygen consumption. It is emphasized, though, that uncoupling may well occur in vivo due to accumulation of free fatty acids and “futile cycling” of K⁺ and Ca²⁺. After 60 min of hypoglycemia, a moderate decrease in state 3 respiration was observed also with succinate as substrate, and there was some decrease in ADP/O ratios in KCI-containing media. However, the changes in ADP/O ratios were more conspicuous during recovery; in addition, state 4 respiration increased significantly. It is concluded that changes in mitochondrial function after 30 min of hypoglycemia are potentially reversible but that true mitochondrial failure develops in the recovery period following 60 min of hypoglycemia. This conclusion was corroborated by results demonstrating incomplete recovery of cerebral energy state. Since EEG and sensory evoked potentials return after 30 min but not after 60 min of hypoglycemia it seemed difficult to explain failure of return of electrophysiological function after 60 min of hypoglycemia solely by mitochondrial dysfunction; plasma membrane function was therefore assessed by measurements of extracellular potassium activity ([K⁺]_e). The results showed that whereas [K⁺]_e remained close to control in the recovery period following 30 min of hypoglycemia it rose progressively during recovery following 60 min of hypoglycemia. Possibly, inhibition of Na⁺ K⁺–activated ATPase could contribute to the permanent loss of spontaneous or evoked electrical activity.     

Cerebral Oxidative Metabolism and Blood Flow During Acute Hypoglycemia and Recovery in Unanesthetized Rats

Abstract: Progressive neurological depression leading to coma was produced in unanesthetized rats at a constant level of hypoglycemia induced by insulin. High-energy phosphate concentrations in brain remained normal during hypoglycemic lethargy, but ATP declined by 6% during stupor and by 40% during coma that was characterized by an isoelectric EEG. Cerebral blood flow (CBF) remained normal during hypoglycemia whereas the cerebral metabolic rates for oxygen (CMRo₂) and glucose (CMR_glucose) decreased by 45 and 73%, respectively, indicating oxidation of nonglucose fuels. A plot of CMRo₂ and CMR_glucose versus plasma glucose indicated increasing oxidation of alternate substrates (elevated CMRo²/CMR_glucose) at plasma glucose concentrations below 2.5 mm . The cerebral uptake of β-hydroxybutyrate increased during hypoglycemic stupor and its complete oxidation could account for the CMRo₂ in excess of glucose utilization. Brain ammonia, a byproduct of amino acid metabolism, reached a level during hypoglycemic coma sufficient to produce coma in normoglycemic animals. The rate and degree of recovery after glucose administration depended on the duration of hypoglycemia and the pretreatment neurological state of the animal. Following 10 min of glucose infusion, ATP levels that were modestly depressed in stuporous rats recovered fully, paralleling the animals' apparently full neurological recovery. Rats that had been in hypoglycemic coma for 1 min or less fully recovered high-energy phosphate concentrations in brain. However, when normalization of plasma glucose was delayed for more than 1 min of coma, the CMRo₂ remained depressed, CBF decreased to 40% of control, and high-energy substrates failed to normalize. In keeping with the depression of oxidative metabolism and blood flow, neurological function and the EEG remained abnormal even after 1 h of glucose infusion. The findings suggest that irreversible brain injury may develop within the first minutes of hypoglycemic coma.     

Regional differences in the effects of insulin-induced hypoglycemia on phospholipid metabolism of rat brain

Insulin-induced hypoglycemia in rats may lead to stimulated brain activity and if severe enough, they may develop a stupor-coma condition. In this study, the effects of insulin-induced hypoglycemia on brain phospholipid metabolism were examined in rats which were prior injected with ³²Pi. Three hours after insulin injection (1 or 5 units/100 g body wt, i.p.), there was an increase (25%) in radioactivity of the lipid phase of cerebral cortex, but radioactivity in the cerebellum tended to decrease instead. Radioactivity in the aqueous phase of cortex was not altered after insulin injection, but that in the cerebellum was decreased by 30%. Differences were observed in labeling of individual phospholipids in response to the hypoglycemic treatment. A marked decrease in labelled phosphatidate was observed in the cerebellum from the hypoglycemic samples, but not in the cerebral cortex. In the cortex, hypoglycemic condition resulted in an increase in ³²Pi uptake into the phospholipids. However, the differences in the amount of label among individual phospholipids suggest that phosphatidylinositol and phosphatidylcholine are turning over more rapidly than other phospholipids. The hypoglycemic rats also showed a 3-fold increase in the brain free fatty acid level, but the level of diacylglycerol was not changed. Results thus suggested a correlation between the free fatty acid release and the increased turnover of phosphatidylinositol and phosphatidylcholine during brain stimulation due to insulin-induced hypoglycemia.     

Cerebral Phosphoinositide and Energy Metabolism During and After Insulin-Induced Hypoglycemia

During and after insulin-induced hypoglycemia, changes in levels of cerebral phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA), triacylglycerol (TAG), diacylglycerol (DAG), and free fatty acids (FFAs) as well as the cerebral energy state were studied in relation to the EEG. In hypoglycemic rats with an EEG pattern of quasiperiodic sharp or slow sharp waves, which preceded the development of an isoelectric EEG, PIP2 levels increased significantly, together with a slight decrease in PI content. Levels of the other lipids did not change during this period. The cerebral energy state was affected only slightly in spite of profound decreases in plasma and tissue glucose levels. With 30 min of an isoelectric EEG, levels of all phosphoinositides and PA decreased significantly; total FFA and DAG contents increased seven- and twofold, respectively; the TAG-palmitate level decreased, and that of TAG-arachidonate increased. Plasma and tissue glucose were nearly depleted, and the cerebral energy state deteriorated severely. The increment in fatty acids in the DAG and FFA pools was less than their loss from phosphoinositides and PA, an observation suggesting vascular washout or oxidation of a portion of the FFAs produced. Following 90 min of glucose infusion, PIP and PA levels recovered to control values; however, the PIP2 content exceeded control levels, and that of PI remained below control levels. DAG and FFA contents returned to normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA

—Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattern of slow waves and polyspikes. In animals with slow waves and polyspikes, there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate, and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5–15 min, there were further decreases in citrate, isocitrate, α-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5 min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids.     

Effect of Insulin-Induced Hypoglycemia on the Concentrations of Glutamate and Related Amino Acids and Energy Metabolites in the Intact and Decorticated Rat Neostriatum

Abstract The glutamate (Glu) terminals in rat neostriatum were removed by a unilateral frontal decortication. One to two weeks later the effects of insulin-induced hypoglycemia on the steady-state levels of amino acids [Glu, glutamine (Gin), aspartate (Asp), γ-aminobutyric acid (GABA), tau-rine] and energy metabolites (glucose, glycogen, α-ketoglu-tarate, pyruvate, lactate, ATP, ADP, AMP, phosphocre-atine) were examined in the intact and decorticated neostriatum from brains frozen in situ. The changes in the metabolite levels were examined during normoglycemia, hypoglycemia with burst-suppression (BS) EEG, after 5 and 30 min of hypoglycemic coma with isoelectric EEG, and 1 h of recovery following 30 min of isoelectric EEG. In normoglycemia Glu decreased and Gin and glycogen increased significantly on the decorticated side. During the BS period no significant differences in the measured compounds were noted between the two sides. After 5 min of isoelectric EEG Glu, Gin, GABA, and ATP levels were significantly lower and Asp higher on the intact than on the decorticated side. No differences between the two sides were found after 30 min of isoelectric EEG. After 1 h of recovery from 30 min of isoelectric EEG Glu, Gin, and glycogen had not reached their control levels. Glu was significantly lower, and Gin and glycogen higher on the decorticated side. The Asp and GABA levels were not significantly different from control levels. The results indicate that the turnover of Glu is higher in the intact than in decorticated neostriatum during profound hypoglycemia.     

Decreased vascular endothelial growth factor response to acute hypoglycemia in type 2 diabetic patients with hypoglycemic coma

IntroductionPlasma vascular endothelial growth factor (VEGF) was shown to increase during acute hypoglycemia and could mediate rapid adaptation of the brain. In this study we examined the neuroendocrine response in patients with type 2 diabetes mellitus (T2DM) in hypoglycemic coma or with acute neuroglycopenic symptoms.MethodsWe prospectively studied 135 consecutive T2DM patients admitted for severe hypoglycemia during a 2-year period. We collected clinical variables and measured plasma concentrations of VEGF, epinephrine, norepinephrine, cortisol and growth hormone at admission and 30 min afterwards.ResultsThirty two patients developed hypoglycemic coma and 103 did not lose consciousness. Median plasma VEGF level of coma patients was 3.1-fold lower at baseline than that of non-coma patients, and even 5.3-fold lower 30 min afterwards. Plasma epinephrine concentration was significantly lower just at baseline in coma patients. On the contrary, there were no differences in concentrations of the other hormones. Multivariate logistic regression analysis showed that VEGF concentration (OR 0.68; CI 0.51–0.95) was a protective factor against the development of coma.ConclusionsVEGF and epinephrine responses to acute hypoglycemia are reduced in T2DM patients who develop hypoglycemic coma. An increased plasma VEGF concentration appeared to be a protective factor against the development of hypoglycemic coma.     

Brain Cortical Fatty Acids and Phospholipids During and Following Complete and Severe Incomplete Ischemia

Abstract: To explore the possibility that peroxtdative degradation of brain tissue lipid constituents is an important mechanism of irreversible ischemic damage, we measured cortical fatty acids and phospholipids during reversible brain ischemia in the rat. Neither complete nor severe incomplete ischemia (5 and 30 min) caused any measurable breakdown of total or individual fatty acids or phospholipids. Except for a small (and reversible) decrease of inositol plus serine phosphoglycerides in the early postischemic period following 30 min of incomplete ischemia, there were no significant losses of fatty acids or phospholipids during recirculation. Since peroxidation, induced in brain cortical tissue in vitro , characteristically involves degradation of polyenoic fatty acids (arachidonic and docosahexaenoic acids) and of ethanolamine phosphoglycerides, the present in vivo results fail to support the hypothesis that peroxidation of membrane lipids is of primary importance for ischemic brain cell damage. Both complete and severe incomplete ischemia caused a similar increase in the tissue content of free fatty acids (FFA). Thus the FFA pool increased by about 10 times during a 30-min ischemic period, to constitute 1 - 2% of the total fatty acid pool. Since there was a relatively larger increase in polyenoic FFA (especially in arachidonic acid) than in saturated FFA, the release of FFA may be the result of activation of a phospholipase A₂ unbalanced by reesterification. Increased levels of FFA persisted during the initial recirculation period, but a gradual normalization occurred and the ischemic changes were essentially reversed at 30 min after restoration of circulation. The pathophysiological implications of the changes in FFA are discussed with respect to mitochondrial dysfunction, formation of cellular edema and prostaglandin-mediated deterioration of postischemic circulation.     

Fructose 2, 6-Bisphosphate in Hypoglycemic Rat Brain

Abstract: Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-l-kinase and glycolysis.     

The role of short chain fatty acid substrates in aerobic and glycolytic metabolism in primary cultures of renal proximal tubule cells

Summary This study examined the role of odd and even short-chain fatty acid substrates on aerobic and glycolytic metabolism in well-aerated primary cultures of rabbit renal proximal tubule cells (RPTC). Increasing oxygen delivery to primary cultures of RPTC by shaking the dishes (SHAKE) reduced total lactate levels and lactate dehydrogenase (LDH) activity and reduced net glucose consumption compared to RPTC cultured under standard conditions (STILL). The addition of butyrate, valerate, heptanoate, or octanoate to SHAKE RPTC produced variable effects on glycolytic metabolism. Although butyrate and heptanoate further reduced total lactate levels and net glucose consumption during short-term culture (<24 h), no fatty acid tested further reduced total lactate levels, net glucose consumption, or LDH activity during long-term culture (7 days). During the first 12 h of culture, maintenance of aerobic metabolism in SHAKE RPTC was dependent on medium supplementation with fatty acid substrates (2 mM). However, by 24 h, SHAKE RPTC did not require fatty acid substrates to maintain levels of aerobic metabolism equivalent to freshly isolated proximal tubules and greater than STILL RPTC. This suggests that SHAKE RPTC undergo adaptive changes between 12 and 24 h of culture, which give RPTC the ability to utilize other substrates for mitochondrial oxidation, therefore allowing greater expression of mitochondrial oxidative potential in SHAKE RPTC than in STILL RPTC.