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Journal of Cerebral Blood Flow and Metabolism

11:122-128 © 1991 Raven Press, Ltd., New York

Changes in Pyruvate Dehydrogenase Complex Activity During and Following Severe Insulin-Induced Hypoglycemia

Monika Cardell, Bo K. Siesjo, and Tadeusz Wieloch Laboratory for Experimental Brain Research, University of Lund, Lund Hospital, Lund, Sweden

Summary: The effect of severe insulin-induced hypogly­ cemia on the activity of the pyruvate dehydrogenase en­ zyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex during burst suppression EEG, after 10, 30, and 60 min of isoelectric EEG, and after 30 and 180 min and 24 h of recovery following 30 min of hypoglycemic coma. Changes in PDHC activity were correlated to levels of labile organic phosphates and gly­ colytic metabolites. In cortex from control animals, the rate of [1-14C]pyruvate decarboxylation was 7.1 ± 1.3 U/mg of protein, or 35% of the total PDHC activity. The activity was unchanged during burst suppression EEG whereas the active fraction increased to 81-87% during hypoglycemic coma. Thirty minutes after glucose­ induced recovery, the PDHC activity had decreased by 33% compared to control levels, and remained signifi­ cantly depressed after 3 h of recovery. This decrease in activity was not due to a decrease in the total PDHC

activity. At 24 h of recovery, PDHC activity had returned to control levels. We conclude that the activation of PDHC during hypoglycemic coma is probably the result of an increased PDH phosphatase activity following de­ polarization and calcium influx, and allosteric inhibition of PDH kinase due to increased ADP!ATP ratio. The de­ pression of PDHC activity following hypoglycemic coma is probably due to an increased phosphorylation of the enzyme, as a consequence of an imbalance between PDH phosphatase and kinase activities. Since some reduction of the ATPIADP ratio persisted and since the lactate! pyruvate ratio had normalized by 3 h of recovery, the depression of PDHC most likely reflects a decrease in PDH phosphatase activity, probably due to a decrease in intramitochondrial Ca2 +. Key Words: Pyruvate dehy­ drogenase complex-Hypoglycemia-Energy metabo­ lism-Calcium-Mitochondria.

Hypoglycemia, severe enough to cause cessation of EEG activity, is accompanied by membrane de­ polarization (Astrup and Norberg, 1976; Harris et aI. , 1984), elevated intracellular calcium concentra­ tions (Harris et aI. , 1984; Uematsu et aI. , 1989), and gross perturbation of cerebral metabolism (Tews et aI. , 1965; Hinzen and Muller, 1971; Lewis et aI. , 1974; Norberg and Siesjo, 1975 ; Agardh et aI. , 1978). During hypoglycemic coma, tissue concen­ trations of ATP and other nucleotide triphosphates are reduced to 20-40% of control within the first 5 min, and remain at these levels during sustained coma (Agardh et aI. , 1978; Chapman et ai, \981). In

the recovery period following glucose administra­ tion, brain metabolism is successively restored (Tews et aI. , 1965; Agardh et aI. , 1978; Chapman et aI. , 1981; Ghajar et aI. , 1982). During the recovery period, however, there is prolonged neurological deficit (Ghajar et aI. , 1982) and depressed cerebral metabolic rate (Abdul-Rahman and Siesjo, 1980; Ghajar et aI. , 1982) as well as reduced cerebral blood flow (Abdul-Rahman et aI. , 1980). In the case of transient cerebral ischemia, the postischemic neocortical metabolic rate is reduced to =50% for up to at least 48 h (Pulsinelli et aI. , 1982). We have recently shown that the activity of the pyruvate dehydrogenase complex (PDHC) is depressed to 5 0% of control levels up to 6 h post­ ischemia (Cardell et aI. , 1989). Subsequent studies have shown that the depression persists for 18 h (Lundgren et aI. , 1990), possibly even for up to 24 h (Cardell and Wieloch, unpublished observations). Pyruvate dehydrogenase is one of three intramito-

Received February 22, 1990; accepted April 27, 1990. Address correspondence and reprint requests to Dr. M. Cardell at Laboratory for Experimental Brain Research, Lund Hospital, S-221 85 Lund, Sweden. Abbreviations used: CoA, coenzyme A; DCA, dichloroace­ tate; EGTA, ethyleneglycol bis(aminoethylether) tetra acetate; PCr, phosphocreatine; PDHC, pyruvate dehydrogenase complex.

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HYPOGLYCEMIA AND BRAIN PDHC ACTIVITY chondrial enzymes whose activity can be enhanced by increasing concentrations of calcium, and is con­ sidered generally important in the regulation of oxidative metabolism. A concept of hormonal influ­ ence on cell metabolism via cellular "stimulus­ response-metabolism" coupling has been pre­ sented, where Ca2 + acts as a second messenger (McCormack and Denton, 1986). Pyruvate dehy­ drogenase participates in the decarboxylation of py­ ruvate and formation of acetyl-CoA (Reed, 1981), and is a central enzyme in the metabolism of glu­ cose and acetylcholine synthesis (Tucek and Cheng, 1974; Gibson et ai., 1975). Phosphorylation­ dephosphorylation of the a-subunit protein of PDHC regulates the enzyme activity via reactions catalyzed by a PDH phosphatase that activates and a PDH kinase that inactivates the enzyme (for re­ views, see Reed, 1981; Randle, 1981). These en­ zymes, in turn, are modulated by metabolites of intermediary metabolism and by divalent ions (Wieland, 1983; Hansford, 1985). Brain PDH kinase and phosphatase have similar properties as those found in peripheral tissues (Booth and Clark, 1978; Sheu et ai., 1983, 1984), but the regulation of brain PDHC is still not fully understood (Lai and Sheu, 1985; Kauppinen and Nicholls, 1986). In view of the posthypoglycemic lowering of ce­ rebral metabolic rate, and the possible role of post­ hypoglycemic mitochondrial loading of Ca2 + in me­ diating neuronal damage, we found an investigation of the activity of PDHC merited. To that end, hy­ poglycemia with a burst suppression EEG pattern ("precoma" ) as well as with cessation of spontane­ ous EEG activity (' "coma" ) was induced by insulin injection, and the PDHC activity was measured. The activity of PDHC was also assessed in the re­ covery period up to 24 h.

MATERIALS AND METHODS Animals and operative techniques

All experiments were performed on male Wistar rats (SPF strain, Mollegaard's Breeding Center, Copenhagen, Denmark) weighing between 290 and 345 g. The rats were fasted overnight with free access only to tap water. About 60 min before operation, the rats were given 2 IU/kg of insulin (Actrapid Human, NOVO Industri, Copenhagen, Denmark) in Krebs-Henseleit solution, intraperitoneally. Anesthesia was induced with 3% Fluothane (halothane, ISC Chemicals, Ltd., Avonmouth, U.K.) in a mixture of nitrous oxide and oxygen (70:30). The animals were intu­ bated (lntramedic PE 240 polyethylene tubing, Clay Ad­ ams, Parsippany, NJ, U.S.A.) and placed on a respirator, whereafter the Fluothane concentration was reduced to 1% for the remainder of the operation. The tail artery and vein were cannulated for blood sampling, continuous blood pressure recording, and administration of drugs. A

123

central venous catheter was inserted via the external jug­ ular vein to allow for rapid exsanguination and reinfusion of blood for blood pressure control (Auer et aI., 1984). The skin was incised over the skull bone to accommodate a plastic funnel for later freezing of the brain in situ (Ponten et aI., 1973), and bipolar leads were placed in the temporal muscles for continuous EEG monitoring. Ani­ mals were immobilized with succinylcholine (Celocurin, Vitrum AB, Stockholm, Sweden), 1 mg/kg, by intrave­ nous injection, and were given heparin, 100 IU. Upon completion of the surgical procedures, the Fluothane was withdrawn and ventilation was adjusted to give arterial oxygen (Pa02) and carbon dioxide (PaC02) tensions of about 100 and 35-40 mm Hg, respectively. We monitored blood pressure and EEG continuously, await­ ing the period of isoelectricity, and checked the blood glucose level intermittently. Just before EEG isoelectricity, 0.1 mg of atropine (ACO Liikemedel AB, Solna, Sweden) was given intra­ venously. During the isoelectric period, blood was frozen directly in liquid nitrogen for later determination of glu­ cose. An excessive elevation of blood pressure was coun­ teracted by drawing blood via the central venous line. This blood was later reinfused. After the desired period of isoelectricity, rats desig­ nated to recover were given an injection of glucose 50% , followed by a continuous infusion of glucose in Krebs­ Henseleit solution (50:50) overnight (see Auer et aI., 1984). Control animals were treated similarly but were given glucose immediately so as to maintain a normal glucose level. In all experiments, body temperature was kept close to 3TC. At desired times, the rat brain was frozen in situ by pouring liquid nitrogen into the plastic funnel over the skull bone. The brain was chiseled out during irrigation with liquid nitrogen and subsequently stored at - 80°C until dissected for analysis. Determination of PDHC activity

The frozen brains were brought to - 20°C and the neo­ cortex (200-250 mg) was dissected, homogenized (Thom­ as homogenizer) in 9 ml of 0.29 M sucrose, 1 mM EGTA, 2.5 mM DCA, 0.01 mM mercaptoethanol in 10 mM Tri­ cine buffer, pH 7.4, on ice at 800 rpm with 15 strokes, and subsequently diluted three times (see Cardell et at., 1989). All of the following steps were done on ice, unless stated otherwise. Following centrifugation at 1,300 g for 3 min (4°C), a crude supernatant was obtained that was divided into two halves. Each supernatant was centrifuged twice at 20,000 g for 10 min, and washed in between with 0.29 M sucrose in Tricine buffer, pH 7.4. The determination of PDHC was made essentially according to Baudry et at. (1982). One pellet was dissolved in 0.5 ml of 2 mM EGTA, 2.5 mM DCA. 0.2% Triton X-100, and 8 mM sodium flu­ oride in 10 mM Tricine buffer, pH 7.9, to prevent further PDH kinase and PDH phosphatase activities, sonicated for 1 min, and preincubated for 5 min. This portion was used to determine the active fraction of PDHC activity (PDH act.). The other pellet was dissolved in 0.5 ml of 1.5 mM CaCI2, 10 mM MgCI2, 2.5 mM DCA, 0.2% Triton X-IOO, and 0.1 mM dinitrophenol in 10 mM Tricine buffer, pH 7.9, sonicated for 1 min, and preincubated at 30°C for 15 min. This portion was used to determine the total PDHC activity (PDH tot.). Protein content was de-

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M. CARDELL ET AL.

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termined by the procedure of Lowry et al. (I9SI), with human serum albumin as standard. After lysis in incubation buffers, IS ILl of the homoge­ nate were added to 200 ILl of an assay mixture containing 2 mM EGTA, 0.1 mM thiamine pyrophosphate, 2.S mM NAD, I mM dithiothreitol, 1.5 mM CoA, 2 ILg of lactate dehydrogenase, I ILg of phosphotransacetylase, and 10 mM MgCl2 in 20 mM Tricine buffer, pH 7.9, and prein­ cubated at 2SoC for 2 min. All reagents were analytical grade and obtained from Sigma (St. Louis, MO, U.S.A.). The reaction was started by adding [I_14C]pyruvate (100 cpm/pmol; Amersham International, U.K.) to yield a final pyruvate concentration of O.S mM. The vessel was im­ mediately sealed with a rubber cap holding a plastic well containing a filter paper soaked in ISO ILl of tissue solu­ bilizer (TS-l, Zinsser Analytic, Frankfurt, F.R.G.), and shaken in a water bath at 2SoC for 10 min. The liberated 14C02 was captured on the filter paper. The reaction was terminated by injecting 0.2 ml of 10% trichloroacetic acid through the rubber cap. The mixture was then allowed to stand an additional 4S min at 2SOC, after which the filter paper was transferred to a scintillation vial containing 10 ml of scintillation fluid (Quickscint SOI, Zinsser Analytic) and counted in a Beckman �-scintillation counter (Beck­ man Instruments Inc., Fullerton, CA, U.S.A.). In both cases, PDHC activity was measured as the initial rate of conversion of [1-14C]pyruvate into 14C02, expressed as units/mg of protein (I unit I nmol of 14CO)minl. or as the ratio PDH act./PDH tot. The enzyme activities were within the linear range of the assay, both with respect to protein concentration and incubation time. =

Analysis of energy and glycolytic metabolites

The frozen tissue was brought to - 20°C and 2S-S0 mg of cortical tissue was dissected. These samples were taken from the same brains as were used for determina­ tion of PDHC activity. The analysis of phosphocreatine (PCr), creatine (Cr), ATP, ADP, AMP, glucose, glyco­ gen, lactate, and pyruvate was performed according to Folbergrova et al. (1972). Analysis of blood gases, pH, and blood glucose

Arterial blood (lS0-200 ILl) was collected anaerobically and Paco2, Pao2, and pH were determined with micro­ electrodes (Eschweiler, Kiel, F.R.G.; Radiometer, Copenhagen, Denmark), operated at 37"C. Blood glucose was determined either directly, with Glucostix reagent strips and a reflectometer system (Ames Co., Elkhart, IN, U.S.A.), or the hexokinase method (Folbergrova et al.,1972).

mM/kg, while animals with an isoelectric EEG had a blood glucose of 1.01 ± 0.30, 0.57 ± 0.26, and 0.85 ± 0.37 mM/kg at 10, 30, and 60 min, respectively. Other variables measured during burst suppression and isoelectric EEG did not differ significantly from control. Blood glucose measured after 15 min of the recovery was 12.4 ± 5.4, 10.1 ± 7.3, and 8.9 ± 1.6 mM/kg in the 30 min, 180 min, and 24 h groups, respectively. Apart from a lower pH in the 30 min recovery group 00.25 ± 0.06 (p < 0.05), there were no differences in physiological parameters among the recovery animals compared with control. PDHC activity and severe insulin-induced hypoglycemia

In control brains, the PDH act. was 7.1 ± 1.3 units/mg protein, which constituted 35 % of the total PDHC activity. PDH tot. was 20.2 ± 0.9 units/mg protein. In animals exhibiting a burst suppression EEG pattern, the PDHC activity was unchanged com­ pared with control. After 10 min of hypoglycemic coma, as identified by an isoelectric EEG pattern, the active portion of PDHC increased significantly (p < 0.01) to 18.6 ± 2.9 units/mg protein, or 81% of the total enzyme activity (Fig. 1). This activation persisted throughout the isoelectric period up to 60 min. In the recovery period, 30 min after glucose administration, the PDH act. had decreased signif­ icantly (p < 0.01) to 4.7 ± 0.9 units/mg protein, a decrease by 33% compared to control levels (Fig. 1). This constituted 24% of the total enzyme activ­ ity. The depression was still evident at 3 h of recov­ ery, at which time PDH act. was 5 .2 ± 1.1 units/mg protein (p < 0.05). At 24 h of recovery, however,

25

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*

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301

601

,..

20

15

E

Statistics

We analyzed the data obtained using analysis of vari­ ance with a post hoc Dunnett's test. Values are expressed as means ± SD.

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10

5

RESULTS Physiological parameters

In control animals, the blood glucose concentra­ tion was 5.75 ± 0.97 mM/kg, P02 was 113 ± 6 mm Hg, Peo2 was 37 ± 3 mm Hg, pH was 7.37 ± 0.03, MABP was 129 ± 8 mm Hg, and body temperature was 37.2 ± 0.2°C. During the period of burst sup­ pression EEG, blood glucose was 0.84 ± 0.08

J Cereb Blood Flow Metab. Vol.11, No. I, 1991

c

bs

1 01

30r

3hr

24hr

FIG. 1. Active portion of PDHC activity in rat cerebral cortex

of control animals (c) and animals exhibiting burst suppres­ sion EEG (bs), after 10, 30, and 60 min of isoelectric EEG (101, 301, 601) and following 30 min, 3 h, and 24 h after 30 min of hypoglycemic coma (30r, 3 hr, 24 hr). PDHC activity is ex­ pressed as nmol of C4-C]pyruvate/min/mg of protein. Data are means ± SO. Significant differences compared to con­ trol: 'p < 0.05 and "p < 0.01 (Dunnett's test).

HYPOGLYCEMIA AND BRAIN PDHC ACTIVITY the PDH act. was back at the control level, being 5.8 ± 0.5 units/mg protein. The decrease in enzyme activity after hyperglycemic coma was not due to a loss of total enzyme activity, PDH tot. (Fig. 2).

24 h of recovery. Pyruvate concentrations were close to normal but there was a tendency towards elevation of the lactate concentration at 30 min of recovery.

Energy and glycolytic metabolites

The changes in energy and glycolytic metabolites in the cerebral cortex during burst suppression EEG and hypoglycemic coma confirm those previ­ ously reported (Lewis et aI., 1974; Norberg and Siesj6, 1975; Agardh et aI., 1978) and extended the observations to 60 min of coma. Thus, when blood glucose decreased and a burst suppression pattern EEG developed, tissue stores of glucose, glycogen, pyruvate, and lactate were depleted. With ensuing coma, there was extensive breakdown of ATP and PCr, with accumulation of ADP, AMP, and cre­ atine, in addition to further depletion of glycolytic substrates and metabolites (see Table 1). Table 2 shows energy and glycolytic metabolites as measured 30 min, 180 min, and 24 h following 30 min of hypoglycemic coma. These data are in ac­ cordance with earlier published data using this model of hypoglycemia (Agardh et aI. , 1978), al­ though here extended to 24 h of recovery. Hence, PCr concentrations were restored to normal, with reciprocal changes in creatine concentration. The ADP and AMP concentrations were normalized promptly but the ATP concentration was back at the control level first after 24 h of recovery, prob­ ably because of slow resynthesis of adenine nucle­ otides (Chapman et aI. , 1981). There was extensive restoration of tissue glucose content, whereas gly­ cogen resynthesis initially lagged; however, the gly­ cogen concentration reached supranormal values at

30

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e

a.

20

rn E

:3

§

10

I 0 0..

c

bs

1 01

301

601

30r

3hr

24hr

FIG. 2. Total PDHC activity in rat cerebral cortex of control animals (c) and animals exhibiting burst suppression EEG (bs), after 10, 30, and 60 min of isoelectric EEG (101, 301, 601) and following 30 min, 3 h, and 24 h after 30 min of hypogly­ cemic coma (30r, 3 hr, 24 hr). PDHC activity is expressed as nmol of [14-GJpyruvate/min/mg of protein. Data are means ± SO.

125

DISCUSSION

In the present investigation, we have demon­ strated an =80% increase in pyruvate decarboxy­ lase activity during hypoglycemic coma. We have also shown that during the first 3 h of recovery, there is a 30% decrease in pyruvate dehydrogenase activity compared with control. This depression is reversed by 24 h after glucose infusion. In the fol­ lowing discussion, we would like to focus on (a) the factors regulating PDHC in the brain during burst suppression EEG and hypoglycemic coma, and (b) the posthypoglycemic metabolic depression and re­ duced PDHC activity, with special emphasis on the hypothesis of the "stimulus-response-metab­ olism" coupling, and its relationship to the calcium hypothesis of cell damage. PDHC activity during burst suppression and isoelectric EEG

The regulation of PDHC activity is achieved through phosphorylation-dephosphorylation of its regulatory a-subunit, via the PDH kinase that inac­ tivates and the PDH phosphatase that activates PDHC (for review, see Reed, 1981; Randle, 1981). Brain PDH kinase and phosphatase have similar properties as those in other tissues (Booth and Clark, 1978; Sheu et aI., 1983, 1984), and are mod­ ulated by metabolites of intermediary metabolism and divalent ions (Wieland, 1983; Hansford, 1985). The PDH kinase requires Mg2 + or Mn2+, is stimu­ lated by acetyl-CoA and NADH, and is inhibited by ADP and pyruvate. The PDH phosphatase requires Mg2 + or Mn2 + and is activated by Ca2+. The poly­ amines spermine, spermidine, and putrescine have also been shown to activate PDH phosphatase (Damuni et aI. , 1984; for review, see Reed and Yea­ man, 1987). In brain tissue, PDH kinase activity is dependent on ATP, and the enzyme can be inhib­ ited by pyruvate and thiamine pyrophosphate (Sheu et al., 1984), while PDH phosphatase is activated by calcium and magnesium ions (Sheu et aI., 1983). During burst suppression EEG, the pyruvate de­ carboxylase activity is not significantly changed. This is concordant with an unaltered activity of PDH kinase, since the levels of ATP, ADP, and PCr are normal. It has been shown in hypoglycemic cats that the NAD/NADH ratio is significantly changed only when the EEG becomes isoelectric (Uematsu et aI. , 1989), which corresponds to the observed unchanged lactate/pyruvate ratio in our animals

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M. CARDELL ET AL.

TABLE 1. Concentrations (fLmollg) of labile organic phosphates and glycolytic metabolites in neocortex of control

animals, animals exhibiting burst suppression EEG (BS). and after 10. 30, and 60 min of isoelectric EEG (iso) Control (n 7) 2.79 0.28 0.067 4.32 6.30 2.47 2.92 1.57 0.101 10.05 15.57

ATP ADP AMP PCr Cr Glucose Glycogen Lactate Pyruvate ATP/ADP Lactate/pyruvate

± ± ± ± ± ± ± ± ± ± ±

10 min iso (n 8)

BS (n

=

0.08 0.04 0.018 0.19 0.45 0.80 0.57 0.37 0.02 1.17 2.60

2.72 0.34 0.070 4.30 6.34 0.14 0.31 0.36 0.032 8.01 10.90

=

± ± ± ± ± ± ± ± ± ± ±

4)

30 min iso 5) (n

=

0.12 0.05 0.024 0.23 0.34 0.13" O.Olh 0.17 0.007h 1.06" 3.45

0.82 0.77 0.64 0.77 10.01 0.20 0.10 0.47 0.033 1.11 12.78

± ± ± ± ± ± ± ± ± ± ±

60 min iso (n 4) =

=

0.16h 0.12h 0.088h 0.25h 1.061> 0.31h 0.12h 0.37" 0.011h 0.38h 8.88

0.50 0.57 0.518 0.54 9.55 0.10 0.08 0.33 0.035 0.88 9.63

± ± ± ± ± ± ± ± ± ± ±

0.13b 0.05h 0.132h 0.16h 0.85 0.061> 0.08h 0.12" O.Ollb 0.18b 1.34

0.56 0.56 0.353 0.69 8.54 0.13 0.09 0.54 0.030 1.01 17.82

± ± ± ± ± ± ± ± ± ± ±

0.03b 0.08h 0.073b 0.18h 1.311> O.13h 0.04b 0.21 0.002h 0.18h 6.63

All values are mean ± SD. a p < 0.05; bp < 0.01 compared to control (Dunnett's test).

(Lewis et aI., 1974). The PDH phosphatase is not expected to be activated since during this period the polarity of the plasma membrane is preserved (As­ trup and Norberg, 1976; Harris et aI., 1984), with no d ecrease in extracellular Ca 2 + concentration (Ca2 + e ) (Harris et aI., 1984), and no increase in in­ tracellular calcium ion concentration (Ca2 +) (Ue­ matsu et aI., 1989). Our data are in contrast to the findings of Jope and Blass (1976), showing that in­ sulin activates PDHC in mouse brain despite nor­ mal ATP levels. Changes in PDHC activity during hypoglycemic coma can be interpreted as follows: When an iso­ electric EEG heralds the onset of coma, there is an increased ADP/ATP ratio as well as an increased NAD/NADH ratio, leading to an allosteric activa­ tion of PDHC. The decreased levels of ATP sup­ presses the PDH kinase activity (Sheu et aI., 1984). In addition, the massive influx of calcium concom­ itant with membrane depolarization (Harris et aI., 1984; Uematsu et aI., 1989) activates PDHC via de­ phosphorylation by PDH phosphatase. This is sim-

ilar to the activation seen during ischemia (Jope and Blass, 1976; Ksiezak, 1976; Cardell et aI., 1989). PDHC activity in the recovery phase, metabolic depression, and Ca2 + i

In the recovery phase following glucose infusion, the lactate/pyruvate ratio is initially elevated but returns to control levels by 3 h. ATP levels are de­ pressed during the first 3 h. The observed sustained increase in ADP/ATP ratio and the normalized py­ ruvate and NADH levels thus favor a decrease in PDH kinase activity. Furthermore, polyamines, known to activate PDH phosphatase (Damuni et aI., 1984), are elevated in the posthypoglycemic period (Paschen et aI., 1989, personal communication). Despite this constellation of factors, the PDHC ac­ tivity is depressed by 30% of control levels for up to 3 h of recovery. This is not due to irreversible dam­ age to the PDH phosphatase since we can fully ac­ tivate PDHC in our assay. Since PDHC activity is the net result of the PDH kinase and PDH phos­ phatase activities, it is conceivable that a decrease

TABLE 2. Concentrations (fLmollg) of labile organic phosphates and glycolytic metabolites in neocortex of control animals and 30 min. 3 h, and 24 h following 30 min of hypoglycemic coma (rec) Control (n 7) =

ATP ADP AMP PCr Cr Glucose Glycogen Lactate Pyruvate ATP/ADP Lactate/pyruvate

2.79 0.28 0.067 4.32 6.30 2.47 2.92 1.57 0.101 10.05 15.57

± ± ± ± ± ± ± ± ± ± ±

0.08 0.04 0.018 0.19 0.45 0.80 0.57 0.37 0.02 1.17 2.60

30 min rec (n 5) =

1.96 0.25 0.052 4.36 5.88 4.09 0.21 4.38 0.155 8.09 27.58

± ± ± ± ± ± ± ± ± ± ±

O.17b 0.05 0.011 0.71 0.55 1.29 0.04b 1.63b 0.025h l.2Sa 6.96b

All values are mean ± SD. " p < 0.05; hp < 0.01 compared to control (Dunnett's test).

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3 h rec (n 6) =

2.22 0.28 0.071 4.82 5.34 6.67 1.82 1.60 0.099 8.20 16.47

± ± ± ± ±

± ± ±

± ± ±

O.lOb 0.05 0.035 0.23 0.19h 2.13h 0.74h 0.34 0.021 1.60" 3.48

24 h rec (n 5) =

2.69 0.29 0.060 4.15 5.75 3.48 7.91 2.26 0.122 9.17 18.28

± ± ± ± ± ± ± ± ± ± ±

0.23 0.03 0.012 0.51 0.40 0.97 0.89b 0.66 0.019 0.52 2.50

HYPOGLYCEMIA AND BRAIN PDHC ACTIVITY in PDH phosphatase activity causes the observed deranged PDHC activity. This implies that posthy­ poglycemia, similarly as postischemia (Cardell et aI., 1989; Lundgren et aI., 1990), the mitochondrial and, possibly, cytosolic calcium levels are de­ creased. This is in accord with the finding that the cytosolic calcium concentration is normalized within 6 min after glucose administration following 10 min of isoelectric EEG (Uematsu et aI., 1989), and the fact that the total calcium content in the cortex 1.5-24 h following a 30 min period of hypo­ glycemia is unchanged (Siesjo and Deshpande, 1987). Calcium ions have recently been suggested to play a pivotal role in "stimulus-response-metabo­ lism" coupling through its ability to stimulate intra­ mitochondrial oxidative metabolism and promote synthesis of ATP (Hansford, 1985; McCormack and Denton, 1986). This could be achieved through the activation of three key oxidative enzymes in the mitochondrial matrix, namely PDHC, NAD­ isocitrate dehydrogenase, and 2-oxyglutarate dehy­ drogenase by intramitochondrial Ca2 + . If this hy­ pothesis is valid also in brain tissue, a reduced met­ abolic rate observed both in the posthypoglycemic (Abdul-Rahman and Siesjo, 1980; Ghajar et aI., 1982) and postischemic phase (Pulsinelli et aI., 1982; Kozuka et aI., 1989) would comply with the concept of " stimulus-response-metabolism" cou­ pling. Recovery after hypoglycemic coma is char­ acterized by severe neurological depression and de­ layed restoration of ATP levels (Agardh et aI., 1978; Ghajar et aI., 1982). This parallels a decreased glu­ cose consumption to 50% of control, 90 min after 30 min of hypoglycemic coma, found by Abdul­ Rahman and Siesjo (1980), and a reduced rate of cerebral oxygen consumption measured up to 20 min after 2-5 min of hypoglycemic coma by Ghajar et al. (1982). Cerebral blood flow has also been shown to be reduced in the recovery phase (Abdul­ Rahman et aI., 1980; Ghajar et aI., 1982), which lends further support to the concept of metabolic depression in the recovery phase (Sokoloff, 1981). The decrease in metabolism in the posthypoglyce­ mic period thus correlates to the depressed PDHC activity, indicating that the intramitochondrial Ca2 + levels are decreased. It has previously been suggested that neuronal necrosis induced by hypoglycemia is caused by cel­ lular calcium accumulation (Siesjo, 1981), partly as a consequence of glutamate receptor activation (Wie\och, 1985; Simon et aI., 1986; Zhang et aI., 1990). In view of the present results and those of Siesjo and Deshpande (1987) and Uematsu et al. (1989), it appears likely that the adverse effects of

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calcium accumulation are confined to the period of hypoglycemia, and are not extended into the recov­ ery period. Acknowledgment: The authors gratefully acknowledge the skilled technical assistance of Karin Hansson, Helene Sjostrom, and Lena Sjoberg. This work has been sup­ ported by the Swedish Medical Research Council (4X08644) and the United States Public Health Services (NS25302).

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