Brain Research, 580 (1992) 331-333
331
(~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00 BRES 25136
Short Communications
Insulin-specific sensitization of cultured cerebrocortical neurons to glutamate excitotoxicity Martin Sch~ifer and Sandor L. Erd6 Laboratory of Neurochemistry, Department of Anatomy, Georg-August University, G6ttingen (FRG) (Accepted 21 January 1992)
Key words: Insulin; Insulin-like growth factor; b-Fibroblast growth factor; Cortical culture; Neurotoxicity; Glutamate; N-Methyl-D-aspartate; Kainate; Quisqualate
The effect of insulin on the sensitivity of neurons to excitatory amino acid-induced cytotoxic cell death was examined in primary cultures of the rat cerebral cortex. Cells developed for two weeks in serum supplemented medium in the presence or absence of insulin, insulin-like growth factor or b-fibroblast growth factor. Excitotoxic cell death was induced by 1 mmoFl glutamate, N-methyl-D-aspartate, kainate or quisqualate. The vulnerability of cells was evaluated by the measurement of lactate dehydrogenase release due to cytotoxic injury. In contrast to the moderate evaluation of protein content by all the 3 growth factors, only insulin increased the vulnerability of cells to the neurotoxic effects of glutamate and of the 3 excitatory amino acid receptor agonists examined. Our results show that the induction of vulnerability in cortical cultures is a specific action of insulin and not a general effect of growth factors. Moreover, the increased vulnerability to N-methylD-aspartate, quisqualate and kainate suggests that the effect of insulin is exerted through intracellular mechanisms other than a selective induction of one subpopulation of excitatory amino acid receptors. Long-term excitation of neurons by high concentrations of glutamate, a m a j o r excitatory amino acid ( E A A ) transmitter in the m a m m a l i a n central nervous system (CNS), is known to induce excitotoxic cell d e a t h both in vivo and in cell cultures 6. The cytotoxic effect of glutamate is m e d i a t e d by at least 3 distinct E A A r e c e p t o r populations n a m e d after their specific agonists N-methyl-D-aspartate ( N M D A ) , quisqualate and kainate receptors 6. G l u t a m a t e - r e l a t e d excitotoxic mechanisms have been shown to be involved in the pathogenesis of anoxic, ischemic and hypoglycemic brain d a m a g e , as well as in cerebral injuries due to status epilepticus and various n e u r o d e g e n e r a t i v e disorders such as H u n t i n g t o n ' s chorea 6. Recently, we have d e m o n s t r a t e d that cytotoxic vulnerability of cultured cerebrocortical neurons to glutamate d e p e n d s on the chemical composition of culture m e d i u m 1'1°. R a t cortical cultures m a i n t a i n e d in a serums u p p l e m e n t e d m e d i u m (SSM) r e s p o n d e d to a long-term exposure to 1 mmol/1 glutamate by a r e m a r k a b l e degree of cell death. The excitotoxic injury was even m o r e pronounced in sister cultures grown in the additional presence of 5/xg/ml insulin 1°. The present study was an a t t e m p t to elucidate whether the insulin induction of vulnerability to glutamate is a c o m m o n effect of growth factors or a specific feature of
insulin. Therefore, the effects of insulin, insulin-like growth factor (IGF1) and b-fibroblast growth factor ( b F G F ) were comparatively examined in terms of their influence on the vulnerability of our culture system to glutamate-induced neuronal death. M o r e o v e r , the selectivity of the insulin effect towards one or m o r e E A A receptor populations mediating glutamate toxicity was also examined using selective E A A r e c e p t o r agonists, i.e. N M D A , quisqualate and kainate. Pregnant CD rats were o b t a i n e d from Charles River Wiga G m b H . O n gestational days 16-18, rats were anesthesized with ether and the embryos dissected by ceserean section. Cells were o b t a i n e d from the embryonic cerebral cortices and m a i n t a i n e d in a serum supplem e n t e d medium. A f t e r isolation, cortices were put into D u l b e c c o ' s modified Eagles m e d i u m ( D M E M ) containing 6 g/1 glucose and 1.2 g/1 N a H C O 3. Cells were trypsinized (0.25%) for 2 min and rinsed with D M E M s u p p l e m e n t e d with 10% fetal calf serum (FCS), then dispersed and plated onto poly-D-lysine-coated Petri dishes in a density of 1.5 × 106 cells p e r 8 cm 2. Cortical cultures were subsequently maintained at 37°C in a humidified, 5% C O 2 a t m o s p h e r e for two weeks in serum containing D M E M (10% FCS), in the absence or presence of 5 pg/ml insulin, 5 ng/ml b F G F or 25 ng/ml IGF1. The above concentrations of growth factors are known to
Correspondence: S.L. Erd6, Laboratory of Cellular and Molecular Pharmacology, Gedeon Richter Co., P.O. Box 27, H-1475 Budapest 10, Hungary.
332 stimulate cell growth and differentiation in cultures 2'3. Cell death was caused by treatment of cultures with glutamate, N M D A , quisqualate or kainate (1 mmol/1 each) in a serum-flee N 2 medium containing transferrin (100 /~g/ml), sodium selenite (30 nmol/1), putrescine (100 pmol/l), progesterone (20 nmoi/1) and insulin (5 pg/ml) in D M E M for 18 h. The extent of cell damage was quantified by measuring the activity of lactate dehydrogenase ( L D H ) in 0.1 ml aliquots of culture media 18 h posttreatment in both treated and untreated (spontaneous cell death) cultures and in frozen-thawed suspensions of the same cultures (total activity in cells plus medium). The activity of L D H was estimated as described earlier L 10, in a phosphate buffer (0.1 mol/1, p H 7.5) containing 22.7 mmol/1 pyruvate as a substrate and 8 mg/100 ml N A D H as coenzyme, by following the decrease of extinction at 340 nm for 10 min. L D H activities, corrected for blank values (spontaneous cell death), were expressed as a percentage of total activities (cells plus medium) in the same cultures. The L D H release induced by glutamate (1 mmol/1) in serum s u p p l e m e n t e d cultures (SSM) grown in the absence of growth factors was taken as 100%. Protein content was estimated in frozen-thawed membranes of cultures previously rinsed and suspended in serum-free medium. The assay was p e r f o r m e d according to the m e t h o d of Lowry et al. 7 with human serum albumin as standard. Values obtained for protein were calculated as a percentage of the respective data for growth factor-free SSM cultures. D M E M dry m e d i u m was o b t a i n e d from Biochrom K G , FCS from Gibco, N M D A , kainate and quisqualate from Tocris Neuramin, F G F and I G F from Boehringer. All other chemicals were purchased from Sigma and were of analytical grade. Cell death caused by 1 mmol/1 glutamate in cultures grown in the absence of exogenous growth factors was taken as 100%. Cultures supplemented with b F G F or IGF1 did not show any alteration in excitotoxic vulnerability to glutamate, whereas in insulin-supplemented cultures the same glutamate concentrations caused a remarkable increase in cell death (Fig. 1). To verify the trophic action of growth factors in our cortical cultures, their effects on protein contents were also examined in the same series of sister cultures. As shown in Fig. 1, all growth factors p r o d u c e d a m o d e r a t e elevation of protein content (20-40%). Cultures grown in SSM in the presence or absence of 5 ktg/ml insulin were exposed for 18 h to glutamate, N M D A , kainate and quisqualate (each 1 mmol/1). Insulin increased the vulnerability of cells not only to glutamate but also to the other 3 E A A r e c e p t o r agonists (Fig. 2). Cell death caused by glutamate and quisqualate was
o/. c o n t r o l 200
~
150
100
50
0 LDN
PROTEIN
~ssM
E~INS
~IGF
~---~-~FGF
Fig. 1. Effect of growth factors on the development of excitotoxic vulnerability to glutamate in cortical cultures. Cells previously grown for 14 days in serum-supplemented medium (SSM) in the absence or presence of insulin, insulin-like growth factor (IGF1) or b-fibroblast growth factor (bFGF) were treated with 1 mmot/1 glutamate. Cell death was quantified by measuring excitotoxic lactate dehydrogenase (LDH) release 18 h after treatment. Protein contents were assayed in frozen-thawed suspensions of the very same cultures. Results obtained for SSM cultures were taken as 100%. Columns and vertical bars represent the mean and S.E.M. of values obtained in 3 experiments, each with 3-5 parallels. ** P < 0.01, * P < 0.05 vs. control, two-tailed Student's t-test.
increased in insulin cultures to a similar extent, although the later effect was not statistically significant. The induction of N M D A and kainate toxicity by insulin was even more pronounced. The role of insulin in the CNS has attracted increas°/°insulin induced increose in ceil deoth 160~ 140
-~
120 IO0
4() 2O 0
,
,
+ GLUTAMATE
,
, , , , , +NMDA + QUIS©UALATE Agonists
+KAINATE
SSM+INS I
Fig. 2. A comparison of the effect of glutamate, N-methyl-o-aspartate (NMDA), kainate and quisqualate in cortical cultures grown in serum supplemented medium in the presence or absence of insulin. Fourteen-day-old cultures were treated with one of the agonists (1 mmol/1 each). Cell death was quantified by LDH measurement 18 h after treatment. Results are expressed as a percent increase in LDH leakage in insulin-supplemented cultures vs. insulin-flee (SSM) controls. Columns and vertical bars represent the mean and S.E.M. of values obtained in 3 experiments, each with triplicate cultures. * P < 0.01 vs. the respective insulin-free controls, two-tailed Student's t-test.
333 ing interest in the last few years. Its receptors and trophic effects on nerve cells have been characterized tl. Nevertheless the physiological relevance of insulin in neuronal tissue remains to be elucidated. The induction of vulnerability to E A A s observed in cortical cultures seems to be a new specific action of insulin. The specificity is supported by the fact that IGF1 and b F G F did not sensitize cortical neurons to glutamate-induced cell death. Moreover, it has been demonstrated that insulin A and B chains alone, or a mixture of both, do not increase neuronal cell death caused by glutamate, indicating that this effect depends on an intact insulin molecular complex 1°. IGF1 is known to be about 100-times more potent than insulin in inducing hormonal effects, including those on cell growth and differentiation 2. Although in our cultures 25 ng/ml IGF1 and 5/~g/ml insulin evoked similar increases in protein content, IGF1 did not reproduce the insulin induction of vulnerability. The effect of insulin demonstrated in our cultures seems to be in virtual contrast to the beneficial action of insulin observed in ischemic brain damage in vivo 12. It has been proposed that the protection caused by acute insulin treatment in vivo is an indirect effect on carbohydrate homeostasis, since normoglycemia (80-120 mg/ 100 ml) or a moderate hypoglycemia ( > 65 mg/100 ml) are known to reduce hypoxic/ischemic brain damage and E A A - i n d u c e d cerebral lesions 5'12. On the other hand, strong hypoglycemia ( < 65 mg/100 ml), like hypoxia and ischemia, has been found to induce neuronal lesions in the brain 5"9. In contrast, the chronic effects of insulin observed in our cultures should be derived from a direct action of the hormone on cortical cells and cannot be explained by insulin-dependent changes in glucose homeostasis. In fact, the high concentration (6 g/l) of glucose in the fresh
culture medium was never reduced below 4.5 g/l, a level still representing hyperglycemia, and the decrease of glucose levels was similar in control (SSM) and insulin-supplemented cultures (unpublished data). It has also been shown that growth factors, such as FGF, exhibit neuroprotective effects against glutamate toxicity in hippocampal pyramidal cell cultures 8. However, a similar treatment of our cortical cultures with F G F failed to influence cell death under our experimental conditions, and chronic exposure to insulin did even increase the toxicity of glutamate. Although we cannot explain this discrepancy, the different cell types grown in hippocampal and cortical cultures may account for the dissimilar responses. These considerations raised the possibility that insulin may selectively increase the expression of one or more E A A receptor populations in our cultures. However, our results show that the insulin induction is not specific for one of the glutamate receptor subpopulations. This suggests that insulin might induce intracellular mechanisms (e.g. Ca 2+) being involved in cell death 6. Obviously, the effects of insulin observed in our cultures are not necessarily relevant in vivo. Nevertheless, elevated insulin levels during chronic treatment of diabetic patients 4 or due to insulinoma might contribute to peripheral neuropathies, even in patients with intact blood-brain barriers (BBB). Furthermore, the effect of insulin might be of relevance also for the brain, if the BBB is damaged or not yet developed, e.g. in stroke or in prenatal or perinatal age.
1 Erd6, S.L., Michler, A., Wolff, J.R. and Tytko, H., Lack of excitotoxic cell death in serum-free cultures of rat cerebral cortex, Brain Res., 526 (1990) 328-332. 2 Froesch, E.R. and Zapf, J., Insulin-like growth factors and insulin: comparative aspects, Diabetologia, 28 (1985) 485-493. 3 Gospoddarowicz, D., Ferrara, N., Schweigerer, L. and Neufeld, G., Structural characterization and biological functions of fibroblast growth factor, Endocr. Rev., 8 (1987) 95-114. 4 Heding, L.G., Determination of total serum insulin (IRI) in insulin-treated diabetic patients, Diabetologia, 8 (1972) 260-266. 5 LeMay, D.R., Gehua, L., Zelenock, G.B. and D'Alecy, L.G., Insulin administration protects neurologic function in cerebral ischemia in rats, Stroke, 19 (1988) 1411-1419. 6 Lodge, D., (Ed.), Excitatory Amino Acids in Health and Disease, Wiley and Sons, Chichester, 1988. 7 Lowry, O.H., Rosenbrough, N.J, Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
8 Manson, M.P., Murrain, M., Guthrie, P.B. and Kater, S.B., Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture, J. Neurosci., 9 (1989) 3728-3740. 9 Sandberg, M., Butcher, S.T. and Hagberg, H., Extracellular overflow of neuroactive amino acids during severe insulin-induced hypoglycemia: in vivo dialysis of rat hippocampus, J. Neurochem., 47 (1986) 178-184. 10 Sch~ifer, M. and Erd6, S.L., Development of glutamate neurotoxicity in cortical cultures: induction by insulin, Dev. Brain Res., 62 (1991) 293-296. 11 Szabo, A.J., CNS regulation of carbohydrate metabolism. In R. Levine and R. Luft (Eds.), Advances in Metabolic Disorders, VoL 10, Academic Press, New York, 1983. 12 Voll, C.L. and Auer, R.N., The effect of postischemic blood glucose levels on ischemic brain damage in the rat, Ann. Neurol., 24 (1988) 638-646.
Special thanks are due to Mrs. Helga Tytko for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Wo 279/10-2).
331
(~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00 BRES 25136
Short Communications
Insulin-specific sensitization of cultured cerebrocortical neurons to glutamate excitotoxicity Martin Sch~ifer and Sandor L. Erd6 Laboratory of Neurochemistry, Department of Anatomy, Georg-August University, G6ttingen (FRG) (Accepted 21 January 1992)
Key words: Insulin; Insulin-like growth factor; b-Fibroblast growth factor; Cortical culture; Neurotoxicity; Glutamate; N-Methyl-D-aspartate; Kainate; Quisqualate
The effect of insulin on the sensitivity of neurons to excitatory amino acid-induced cytotoxic cell death was examined in primary cultures of the rat cerebral cortex. Cells developed for two weeks in serum supplemented medium in the presence or absence of insulin, insulin-like growth factor or b-fibroblast growth factor. Excitotoxic cell death was induced by 1 mmoFl glutamate, N-methyl-D-aspartate, kainate or quisqualate. The vulnerability of cells was evaluated by the measurement of lactate dehydrogenase release due to cytotoxic injury. In contrast to the moderate evaluation of protein content by all the 3 growth factors, only insulin increased the vulnerability of cells to the neurotoxic effects of glutamate and of the 3 excitatory amino acid receptor agonists examined. Our results show that the induction of vulnerability in cortical cultures is a specific action of insulin and not a general effect of growth factors. Moreover, the increased vulnerability to N-methylD-aspartate, quisqualate and kainate suggests that the effect of insulin is exerted through intracellular mechanisms other than a selective induction of one subpopulation of excitatory amino acid receptors. Long-term excitation of neurons by high concentrations of glutamate, a m a j o r excitatory amino acid ( E A A ) transmitter in the m a m m a l i a n central nervous system (CNS), is known to induce excitotoxic cell d e a t h both in vivo and in cell cultures 6. The cytotoxic effect of glutamate is m e d i a t e d by at least 3 distinct E A A r e c e p t o r populations n a m e d after their specific agonists N-methyl-D-aspartate ( N M D A ) , quisqualate and kainate receptors 6. G l u t a m a t e - r e l a t e d excitotoxic mechanisms have been shown to be involved in the pathogenesis of anoxic, ischemic and hypoglycemic brain d a m a g e , as well as in cerebral injuries due to status epilepticus and various n e u r o d e g e n e r a t i v e disorders such as H u n t i n g t o n ' s chorea 6. Recently, we have d e m o n s t r a t e d that cytotoxic vulnerability of cultured cerebrocortical neurons to glutamate d e p e n d s on the chemical composition of culture m e d i u m 1'1°. R a t cortical cultures m a i n t a i n e d in a serums u p p l e m e n t e d m e d i u m (SSM) r e s p o n d e d to a long-term exposure to 1 mmol/1 glutamate by a r e m a r k a b l e degree of cell death. The excitotoxic injury was even m o r e pronounced in sister cultures grown in the additional presence of 5/xg/ml insulin 1°. The present study was an a t t e m p t to elucidate whether the insulin induction of vulnerability to glutamate is a c o m m o n effect of growth factors or a specific feature of
insulin. Therefore, the effects of insulin, insulin-like growth factor (IGF1) and b-fibroblast growth factor ( b F G F ) were comparatively examined in terms of their influence on the vulnerability of our culture system to glutamate-induced neuronal death. M o r e o v e r , the selectivity of the insulin effect towards one or m o r e E A A receptor populations mediating glutamate toxicity was also examined using selective E A A r e c e p t o r agonists, i.e. N M D A , quisqualate and kainate. Pregnant CD rats were o b t a i n e d from Charles River Wiga G m b H . O n gestational days 16-18, rats were anesthesized with ether and the embryos dissected by ceserean section. Cells were o b t a i n e d from the embryonic cerebral cortices and m a i n t a i n e d in a serum supplem e n t e d medium. A f t e r isolation, cortices were put into D u l b e c c o ' s modified Eagles m e d i u m ( D M E M ) containing 6 g/1 glucose and 1.2 g/1 N a H C O 3. Cells were trypsinized (0.25%) for 2 min and rinsed with D M E M s u p p l e m e n t e d with 10% fetal calf serum (FCS), then dispersed and plated onto poly-D-lysine-coated Petri dishes in a density of 1.5 × 106 cells p e r 8 cm 2. Cortical cultures were subsequently maintained at 37°C in a humidified, 5% C O 2 a t m o s p h e r e for two weeks in serum containing D M E M (10% FCS), in the absence or presence of 5 pg/ml insulin, 5 ng/ml b F G F or 25 ng/ml IGF1. The above concentrations of growth factors are known to
Correspondence: S.L. Erd6, Laboratory of Cellular and Molecular Pharmacology, Gedeon Richter Co., P.O. Box 27, H-1475 Budapest 10, Hungary.
332 stimulate cell growth and differentiation in cultures 2'3. Cell death was caused by treatment of cultures with glutamate, N M D A , quisqualate or kainate (1 mmol/1 each) in a serum-flee N 2 medium containing transferrin (100 /~g/ml), sodium selenite (30 nmol/1), putrescine (100 pmol/l), progesterone (20 nmoi/1) and insulin (5 pg/ml) in D M E M for 18 h. The extent of cell damage was quantified by measuring the activity of lactate dehydrogenase ( L D H ) in 0.1 ml aliquots of culture media 18 h posttreatment in both treated and untreated (spontaneous cell death) cultures and in frozen-thawed suspensions of the same cultures (total activity in cells plus medium). The activity of L D H was estimated as described earlier L 10, in a phosphate buffer (0.1 mol/1, p H 7.5) containing 22.7 mmol/1 pyruvate as a substrate and 8 mg/100 ml N A D H as coenzyme, by following the decrease of extinction at 340 nm for 10 min. L D H activities, corrected for blank values (spontaneous cell death), were expressed as a percentage of total activities (cells plus medium) in the same cultures. The L D H release induced by glutamate (1 mmol/1) in serum s u p p l e m e n t e d cultures (SSM) grown in the absence of growth factors was taken as 100%. Protein content was estimated in frozen-thawed membranes of cultures previously rinsed and suspended in serum-free medium. The assay was p e r f o r m e d according to the m e t h o d of Lowry et al. 7 with human serum albumin as standard. Values obtained for protein were calculated as a percentage of the respective data for growth factor-free SSM cultures. D M E M dry m e d i u m was o b t a i n e d from Biochrom K G , FCS from Gibco, N M D A , kainate and quisqualate from Tocris Neuramin, F G F and I G F from Boehringer. All other chemicals were purchased from Sigma and were of analytical grade. Cell death caused by 1 mmol/1 glutamate in cultures grown in the absence of exogenous growth factors was taken as 100%. Cultures supplemented with b F G F or IGF1 did not show any alteration in excitotoxic vulnerability to glutamate, whereas in insulin-supplemented cultures the same glutamate concentrations caused a remarkable increase in cell death (Fig. 1). To verify the trophic action of growth factors in our cortical cultures, their effects on protein contents were also examined in the same series of sister cultures. As shown in Fig. 1, all growth factors p r o d u c e d a m o d e r a t e elevation of protein content (20-40%). Cultures grown in SSM in the presence or absence of 5 ktg/ml insulin were exposed for 18 h to glutamate, N M D A , kainate and quisqualate (each 1 mmol/1). Insulin increased the vulnerability of cells not only to glutamate but also to the other 3 E A A r e c e p t o r agonists (Fig. 2). Cell death caused by glutamate and quisqualate was
o/. c o n t r o l 200
~
150
100
50
0 LDN
PROTEIN
~ssM
E~INS
~IGF
~---~-~FGF
Fig. 1. Effect of growth factors on the development of excitotoxic vulnerability to glutamate in cortical cultures. Cells previously grown for 14 days in serum-supplemented medium (SSM) in the absence or presence of insulin, insulin-like growth factor (IGF1) or b-fibroblast growth factor (bFGF) were treated with 1 mmot/1 glutamate. Cell death was quantified by measuring excitotoxic lactate dehydrogenase (LDH) release 18 h after treatment. Protein contents were assayed in frozen-thawed suspensions of the very same cultures. Results obtained for SSM cultures were taken as 100%. Columns and vertical bars represent the mean and S.E.M. of values obtained in 3 experiments, each with 3-5 parallels. ** P < 0.01, * P < 0.05 vs. control, two-tailed Student's t-test.
increased in insulin cultures to a similar extent, although the later effect was not statistically significant. The induction of N M D A and kainate toxicity by insulin was even more pronounced. The role of insulin in the CNS has attracted increas°/°insulin induced increose in ceil deoth 160~ 140
-~
120 IO0
4() 2O 0
,
,
+ GLUTAMATE
,
, , , , , +NMDA + QUIS©UALATE Agonists
+KAINATE
SSM+INS I
Fig. 2. A comparison of the effect of glutamate, N-methyl-o-aspartate (NMDA), kainate and quisqualate in cortical cultures grown in serum supplemented medium in the presence or absence of insulin. Fourteen-day-old cultures were treated with one of the agonists (1 mmol/1 each). Cell death was quantified by LDH measurement 18 h after treatment. Results are expressed as a percent increase in LDH leakage in insulin-supplemented cultures vs. insulin-flee (SSM) controls. Columns and vertical bars represent the mean and S.E.M. of values obtained in 3 experiments, each with triplicate cultures. * P < 0.01 vs. the respective insulin-free controls, two-tailed Student's t-test.
333 ing interest in the last few years. Its receptors and trophic effects on nerve cells have been characterized tl. Nevertheless the physiological relevance of insulin in neuronal tissue remains to be elucidated. The induction of vulnerability to E A A s observed in cortical cultures seems to be a new specific action of insulin. The specificity is supported by the fact that IGF1 and b F G F did not sensitize cortical neurons to glutamate-induced cell death. Moreover, it has been demonstrated that insulin A and B chains alone, or a mixture of both, do not increase neuronal cell death caused by glutamate, indicating that this effect depends on an intact insulin molecular complex 1°. IGF1 is known to be about 100-times more potent than insulin in inducing hormonal effects, including those on cell growth and differentiation 2. Although in our cultures 25 ng/ml IGF1 and 5/~g/ml insulin evoked similar increases in protein content, IGF1 did not reproduce the insulin induction of vulnerability. The effect of insulin demonstrated in our cultures seems to be in virtual contrast to the beneficial action of insulin observed in ischemic brain damage in vivo 12. It has been proposed that the protection caused by acute insulin treatment in vivo is an indirect effect on carbohydrate homeostasis, since normoglycemia (80-120 mg/ 100 ml) or a moderate hypoglycemia ( > 65 mg/100 ml) are known to reduce hypoxic/ischemic brain damage and E A A - i n d u c e d cerebral lesions 5'12. On the other hand, strong hypoglycemia ( < 65 mg/100 ml), like hypoxia and ischemia, has been found to induce neuronal lesions in the brain 5"9. In contrast, the chronic effects of insulin observed in our cultures should be derived from a direct action of the hormone on cortical cells and cannot be explained by insulin-dependent changes in glucose homeostasis. In fact, the high concentration (6 g/l) of glucose in the fresh
culture medium was never reduced below 4.5 g/l, a level still representing hyperglycemia, and the decrease of glucose levels was similar in control (SSM) and insulin-supplemented cultures (unpublished data). It has also been shown that growth factors, such as FGF, exhibit neuroprotective effects against glutamate toxicity in hippocampal pyramidal cell cultures 8. However, a similar treatment of our cortical cultures with F G F failed to influence cell death under our experimental conditions, and chronic exposure to insulin did even increase the toxicity of glutamate. Although we cannot explain this discrepancy, the different cell types grown in hippocampal and cortical cultures may account for the dissimilar responses. These considerations raised the possibility that insulin may selectively increase the expression of one or more E A A receptor populations in our cultures. However, our results show that the insulin induction is not specific for one of the glutamate receptor subpopulations. This suggests that insulin might induce intracellular mechanisms (e.g. Ca 2+) being involved in cell death 6. Obviously, the effects of insulin observed in our cultures are not necessarily relevant in vivo. Nevertheless, elevated insulin levels during chronic treatment of diabetic patients 4 or due to insulinoma might contribute to peripheral neuropathies, even in patients with intact blood-brain barriers (BBB). Furthermore, the effect of insulin might be of relevance also for the brain, if the BBB is damaged or not yet developed, e.g. in stroke or in prenatal or perinatal age.
1 Erd6, S.L., Michler, A., Wolff, J.R. and Tytko, H., Lack of excitotoxic cell death in serum-free cultures of rat cerebral cortex, Brain Res., 526 (1990) 328-332. 2 Froesch, E.R. and Zapf, J., Insulin-like growth factors and insulin: comparative aspects, Diabetologia, 28 (1985) 485-493. 3 Gospoddarowicz, D., Ferrara, N., Schweigerer, L. and Neufeld, G., Structural characterization and biological functions of fibroblast growth factor, Endocr. Rev., 8 (1987) 95-114. 4 Heding, L.G., Determination of total serum insulin (IRI) in insulin-treated diabetic patients, Diabetologia, 8 (1972) 260-266. 5 LeMay, D.R., Gehua, L., Zelenock, G.B. and D'Alecy, L.G., Insulin administration protects neurologic function in cerebral ischemia in rats, Stroke, 19 (1988) 1411-1419. 6 Lodge, D., (Ed.), Excitatory Amino Acids in Health and Disease, Wiley and Sons, Chichester, 1988. 7 Lowry, O.H., Rosenbrough, N.J, Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
8 Manson, M.P., Murrain, M., Guthrie, P.B. and Kater, S.B., Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture, J. Neurosci., 9 (1989) 3728-3740. 9 Sandberg, M., Butcher, S.T. and Hagberg, H., Extracellular overflow of neuroactive amino acids during severe insulin-induced hypoglycemia: in vivo dialysis of rat hippocampus, J. Neurochem., 47 (1986) 178-184. 10 Sch~ifer, M. and Erd6, S.L., Development of glutamate neurotoxicity in cortical cultures: induction by insulin, Dev. Brain Res., 62 (1991) 293-296. 11 Szabo, A.J., CNS regulation of carbohydrate metabolism. In R. Levine and R. Luft (Eds.), Advances in Metabolic Disorders, VoL 10, Academic Press, New York, 1983. 12 Voll, C.L. and Auer, R.N., The effect of postischemic blood glucose levels on ischemic brain damage in the rat, Ann. Neurol., 24 (1988) 638-646.
Special thanks are due to Mrs. Helga Tytko for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Wo 279/10-2).
