Elsevier

Neuroscience

Volume 79, Issue 2, 12 May 1997, Pages 525-534
Neuroscience

Insulin-like growth factor-I is an osmoprotectant in human neuroblastoma cells

https://doi.org/10.1016/S0306-4522(96)00611-2Get rights and content

Abstract

A role in neuronal homeostasis is suggested by the persistent expression of the insulin-like growth factors in the adult nervous system. SH-SY5Y human neuroblastoma cells, a well-characterized in vitro model of human neurons, were used to investigate the effects of hyperosmotic stress on neurons. Neuronal DNA fragmentation was detected within 1 h and pyknotic nuclei were apparent in attached cells after 12 h of hyperosmotic stress. In parallel, flow cytometry measurements revealed a sudden increase in the rate of cells irreversibly undergoing programmed cell death after 12 h of hyperosmotic exposure. Insulin-like growth factor-I delayed the onset of a laddered DNA fragmentation pattern for 24 h and provided continuing protection against hyperosmotic exposure for 72 h. Amino acid uptake was decreased in hyperosmotic medium even in the presence of insulin-like growth factor-I; the protein synthesis inhibitor cycloheximide neither prevented the induction of programmed cell death nor interfered with the ability of insulin-like growth factor-I to act as an osmoprotectant in hyperosmotic medium. Cysteine and serine protease inhibitors each prevented DNA fragmentation under hyperosmotic conditions, suggesting that the osmoprotectant activity of insulin-like growth factor-I involves the suppression of protease activity.

Collectively, these results indicate that insulin-like growth factor-I limits the death of neurons under stressful environmental conditions, suggesting that it may provide a candidate therapy in the treatment of hyperosmolar coupled neurological injury.

Section snippets

Materials

Tissue culture flasks and plates were purchased from Corning (Corning, NY, U.S.A.). Dulbecco's modified Eagle's medium (DMEM), Hank's balanced salt solution (HBSS) and calf serum (CS) were from Gibco BRL (Gaithersburg, MD, U.S.A.). IGF-I was stored in 10 mM acetic acid at −80°C. Solutions containing IGF-I were stored at 4°C and used within three days. RNase A, proteinase K, tosyl-l-chloromethylketone and tosyl-l-phenylmethylketone were from Boehringer Mannheim (Germany). [3H]Leucine (1 mCi/ml)

Appearance of cell nuclei after hyperosmotic exposure

The nuclei of cells undergoing programmed cell death often experience a progressive condensation that results in the splitting of the nucleus into individual membrane-bound spheres. Our previous results demonstrated that approximately 40% of unprotected cells become detached within the first 24 h of exposure to hyperosmotic medium.[21]To determine the temporal relationship between changes in nuclear morphology and the state of cell attachment we used the fluorescent DNA-binding dye bisbenzimide

Discussion

Plasma hyperosmolarity is a common systemic feature of diseases with serious neurological consequences.[60]We have previously reported that SH-SY5Y cells, a well-characterized model of human neuronal growth and differentation,37, 38, 64show arrested growth and die when exposed to media made hyperosmotic by the addition of glucose, NaCl or mannitol.[40]This in vitro paradigm of hyperosmolar-coupled neuronal dysfunction was used to screen for agents which could serve as neural osmoprotectants. In

Conclusions

In summary, IGF-I can protect SH-SY5Y cells from hyperosmotic-coupled programmed cell death. The neuroprotective effect of IGF-I was not due to the traditional mitogenic or growth-promoting activities of IGF-I[21]and did not appear to require active protein synthesis. Addition of IGF-I up to the point of commitment to death markedly increased neuronal survival, implying a rapid onset of IGF-I action. Inhibitors of proteolytic activity prevented neuronal DNA fragmentation, suggesting that IGF-I

Acknowledgements

This study was supported by R29NS32843 (ELF), NIH NINDS T32 NS07222 (CCM) and grants from the Juvenile Diabetes Foundation no. 194130 (ELF) and the American Diabetes Association (ELF). The authors wish to thank Dr Kelli Sullivan for expert editorial assistance, James Beals for figure preparation and Judith Boldt for manuscript preparation.

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