Actividad de la diacilglicerol quinasa en terminales sinápticos de áreas del sistema nervioso central : rol en el mecanismo de señalización de la insulina

Abstract

Several studies have demonstrated through the activation of its central nervous system (CNS) receptor, that insulin has a key role in important functions related to neuronal plasticity, thus modulat ing the cascade activat ion of intracellular signaling. In addit ion, research f rom our laboratory demonstrates that insulin regulates lipid signal transduction pathways in the SNC, which are altered in the process of normal aging. The purpose of the present research is to study a lipid signaling pathway associated to the CNS insulin signaling transduct ion mechanism and its role in normal aging. Particular emphasis is put on the action of insulin as activator of the diacylglycerol kinase (DAGK), a key enzyme in the regulation of two important lipid messengers, namely phosphat idic acid (PA) and diacylglycerol (DAG), both of which are essential molecules in the regulation of cellular responses. The f irst stage of this research focused on the character izat ion of DAGK activity in cerebral cortex synaptosomes of adult rats in order to evaluate its activity in endogenous and exogenous DAGs by means of the use of detergents such as the carrier of lipid substrate DAG. The kinetic parameters of DAGK activity f rom endogenous DAG and exogenous DAG with dif ferent acyl composition were also studied. We analyzed dipalmitoylglycerol (DPG), 1-stearoyl, 2-arachidonoylglycerol (SAG) and dioleoylglycerol (DOG) as exogenous lipid substrates. Results show that the enzymatic activity preferentially uses DAG containing arachydonic acid in position 2 of the chain of glycerol (SAG) as substrate, thus indicating the presence of épsilon type of DAGK (DAGKe) in CC synaptosomes, which is the only type with substrate preferences. The analysis of DAGK act ivat ion by insulin revealed that hormonal action stimulates DAGK act ivity by means of two mechanisms: 1) by increasing the levels of DAG in the plasmatic membrane f rom the stimulus of PI-PLC and PC-PLD/PAP2 pathways, thus producing an increase of DAGK action; and 2) by stimulat ing DAGK act ivity in a direct and independent form of DAG generation in the membrane. It was also found that the stimulus that exerts insulin independently of DAG generation is associated to an epsilon DAGK act ivity which is possibly regulated by a decrease in phosphoinosit ide levels and which could be associated to polyphosphoinositides resynthesis. In CC synaptosomal membranes of senile animals, an increase in DAG content was observed without a signif icant modif ication in acyl composition. This can be correlated with the simultaneous f inding of a decreased DAGK activity in endogenous DAGs. Whereas the use of saturated exogenous DAG (DPG) showed no modif icat ions by aging, an important decrease in DAGK ability to transform SAG into PA was observed. A similar result was observed in synaptosomes f rom hippocampus. It was also observed that insulin act ing in CC synapt ic terminals exerted a minor st imulatory ef fect with respect to adult animals in DPG transformation, whereas it showed the same ef fectiveness to increase SAG transformation. Interestingly, insulin strongly st imulated DAGK activity in SAG in hippocampus synaptosomes of senile rats with respect to adults. As to the recovery of a PA with the potential ability to be phosphoinositide precursor, our f indings suggest that this hormonal action is a compensatory mechanism in the regulation of the synthesis of these phospholipids. This partly explains the hormone role as neuroprotector agent.