Growth factors such as insulin, the insulin-like growth factors (IGFs) and the neurotrophins have extensive effects on the physiology of neurones. Insulin, IGF-1, IGF-II and nerve growth factor (NGF) regulate the survival, differentiation and axonal outgrowth of embryonic and adult neurones. Such effects on neuronal phenotype are mediated via global and co-ordinated alterations in gene expression. For such profound changes in gene expression to occur the cellular protein synthetic machinery must be optimised and an efficient use and supply of energy resources is a prerequisite. The mitochondrion is the main source of ATP within the cell and, therefore, alterations in neuronal phenotype will depend on the functioning of this organelle.

This study tested the hypothesis that mitochondrial function is directly modulated by growth factors, such as insulin. Real time video microscopy of rhodamine 123 fluorescence was used to monitor mitochondrial inner membrane potential in cultured adult dorsal root ganglia (DRG) neurones. All animals were killed according to UK legislation. Acute insulin treatment (0.75 nM for 20 min) had no effect; however, 6 h and 3 day treatment significantly increased the mitochondrial membrane potential (as assesed by the normlized amplitude of CCCP-induced increase of rhodamine 123 fluorescence) of DRG neurones (control, 0.38 ± 0.05; 6 h insulin, 0.61 ± 0.12; 3 day insulin, 1.4 ± 0.19; mean ± S.E.M., P < 0.05 vs. control, one-way ANOVA, arbitrary units). The insulin effect was maximal at a dose of 0.75 nM, higher doses of 7.5 and 75 nM had no additional effect, suggesting that signalling via the IGF type I receptor was not involved. The availability of glucose (10Ð50 mM) was varied but had no effect, suggesting that changes in metabolic status had no impact on mitochondrial membrane potential. The redox state of the NADH/NADPH pool was also analysed using real-time fluorescence and insulin had no effect, implying that insulin was not directly modulating electron transport within the mitochondrion.

The signal transduction pathways downstream from the insulin receptor were investigated. Inhibition of phosphoinositide 3-kinase (PI 3-kinase) by 0.001Ð0.01 mM LY294002 prevented the insulin effect on mitochondrial membrane potential. However, blockage of mitogen-activated protein kinase (MAPK) using the MEK inhibitor, U0126, had no effect. Western blots showed that insulin activated the protein kinase, PKB or Akt, and the transcription factor, CREB (both downstream of PI 3-kinase), but had no effect on MAPK or the related kinases Ð c-jun N-terminal kinase and p38 kinase.

The results show for the first time that insulin can directly modulate the membrane potential of neuronal mitochondria. Studies are now focused on the molecular mechanism of this process. Of particular interest are targets downstream from Akt, such as the bcl-2 family of proteins that are known to regulate mitochondrial properties.

This work was funded by grants from Diabetes UK (P.F. and A.V.) and the Juvenile Diabetes Research Foundation (P.F.). Tze-Jen Huang was supported by an Overseas Research Scholarship.

All procedures accord with current UK legislation.