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Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30–50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20–40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.
A large proportion of membrane phospholipids in the CNS are composed of long-chain polyunsaturated fatty acids, namely arachidonic (20:4 n-6) and docosahexaenoic acid (22:6 n-3). These fatty acids must either be provided by diet or may be synthesized from linoleic (LA 18:2 n-6) and α-linolenic acid (LnA 18:3 n-3), respectively. In neural and retinal tissues, membrane phospholipids are particularly rich in 22:6 n-3 (Sinclair 1975; Bourre et al. 1990; McGee et al. 1994). Several studies have shown that an α-linolenic acid deficient diet lowers membrane 22:6 n-3 levels (Connor et al. 1990; Anderson and Connor 1994; Guesnet et al. 1997; Carriéet al. 2000), leading to behavioural alterations such as poor performance in learning tasks and brightness discrimination (Yamamoto et al. 1988; Bourre et al. 1989), as well as changes in the physiological properties of receptors, transporters and enzymes in neuronal membranes (Viani et al. 1991; Delion et al. 1994; Youdim et al. 2000). Moreover, Tsukada et al. (2000) reported that ‘dairy 22:6 n-3 supply restores the age-related impairment of the coupling between neuronal activation and regional cerebral blood flow (CBF) response to vibrotactile stimulation in the somatory cortex in monkey brain’.
As high levels of 22:6 n-3 have been reported in ;endothelial cells (Selivonchick and Roots 1976), mitochondrial and neuronal membranes (Youdim et al. 2000), we speculated that a modification in membrane polyunsaturated fatty acid (PUFA) composition could affect the function of transporters and of enzymes implicated in the coupling mechanism between CBF, metabolism and neuronal activity.
Glucose, the primary source of energy for mammalian brain, is transported across the endothelial cells of the blood–brain barrier (BBB) to neurones and astrocytes. In the brain, the most important glucose transporters are two isoforms of GLUT1, the 55-kDa and 45-kDa isoforms primarily detected in endothelial cells of the BBB and in glial cells, respectively (Harik et al. 1990; Bondy et al. 1992), and GLUT3 40 kDa, a neuronal glucose transporter (Nagamatsu et al. 1993). Brain regions with a high density of glucose transporters also possess high rates of utilization of cerebral glucose (e.g. cerebral cortex and hippocampus) (Maher et al. 1994). Sokoloff (1981) showed a linear relationship between local cerebral glucose utilization and CBF in the rat. The coupling between CBF and cerebral metabolism is used as an index of metabolic activation underlying the increase in neuronal activity (Gsell et al. 2000). According to physiological conditions, mitochondrial oxidative phosphorylation can vary widely in order to match ATP synthesis to energy demand. Cytochrome oxidase (CO), the last enzyme of the respiratory chain components inserted at the inner mitochondrial membrane, is regarded as a metabolic marker for neuronal functional activity (Wong-Riley 1989).
The purpose of this study was to investigate the effect of n-3 PUFA deficiency on glucose uptake and oxidative metabolism in the brain of rats fed on a diet deficient in LnA compared with rats fed on a diet with appropriate LnA supply. Three different brain regions were chosen: the fronto-parietal cortex, the hippocampus and the suprachiasmatic nucleus (SCN), which show a high rate of energy metabolism. The fatty acid composition of the two main phospholipid classes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was studied in the three cerebral areas. The local expression of GLUT1 55 kDa and GLUT3 40 kDa in the three brain regions was investigated on control and n-3 fatty acid deficient rats. To find out whether the changes observed in immunoreactivity of the glucose transporters could be related to a modified utilization of glucose, experiments using autoradiographic 2-deoxyglucose method (Sokoloff et al. 1977) and an histochemical study (Wong-Riley 1979) were performed to evaluate the local rates of glucose uptake and of CO activity.
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The effect of LnA dietary deficiency on the phospholipid fatty acid composition in cerebral membranes is well established (Bourre et al. 1984). Several studies have reported that this alteration is associated with learning and behavioural deficits (Bourre et al. 1989; Wainwright 1992; Moriguchi et al. 2000), changes in local neurotransmission (Delion et al. 1994; Zimmer et al. 2000) or hormonal release (Gazzah et al. 1993). These effects were observed in animals maintained for two or three generations on an LnA-deficient diet. We show here that feeding rats an LnA-deficient diet for ;one generation is sufficient to induce disturbances in membrane PUFA composition, and consequently significant and important alterations in brain metabolic activity. In our ;study, rats showed a significant reduction of 22:6 n-3 fatty acid (30–50%) within brain membrane phospholipids, ;and a remarkable decrease in glucose uptake (30–35%) and ;CO activity (20–30%) in the three brain regions. We assume that these effects can be attributed primarily to the ;alteration of 22:6 n-3 in membrane phospholipid composition.
Though we did not measure PUFA phospholipid levels in the mitochondrial membrane, we suppose that our experimental procedure induced a notable reduction of 22:6 n-3 mitochondrial phospholipids because several studies have shown dietary fat induced changes in mitochondrial phospholipid fatty acid composition (Tahin et al. 1981; Barzanti et al. 1994; Marteinsdottir et al. 1998). It has been proposed that phospholipids containing 22:6 n-3 could be necessary for the assembly of mitochondrial enzyme complexes at inner membrane, and that a reduction of 22:6 n-3 level would reduce oxidative phosphorylation (Infante 1987) and the activity of oxidative enzymes such as succinate dehydrogenase and cytochrome oxidase (Thomas et al. 1993). Here, in n-3 fatty acid deficient animals, we showed that a significant decrease in CO activity and also in glucose uptake could be explained as a consequence of the depressed oxidative phosphorylation.
Although glucose uptake decreased, GLUT3 immunoreactive cells increased, and immunoreactivity of GLUT1 declined. Similar results were reported in rats after one week of moderate hypoglycemia where glucose utilization was decreased, the mean density of glucose transporters GLUT1 remained unchanged whereas the mean density of GLUT3 increased slightly (Duelli et al. 1999). In our study, n-3 fatty acid deficient animals had normal blood glucose levels, which means that glycemia did not contribute to the decrease in BBB GLUT1-Ir during n-3 deficiency.
In n-3 fatty acid deficient rats, the reduced oxidative phosphorylation could function as a signal to neuronal cells to mobilize GLUT3 at the neuronal membrane surface. Thus, GLUT3 translocation could represent a mechanism for sustaining glucose uptake into neurones to ensure cellular brain metabolism and functionality. Nevertheless, the higher immunoreactivity demonstrated for GLUT3 in neuronal cells of n-3 fatty acid deficient rats did not result in normal glucose uptake into the cells.
In contrast to GLUT3, GLUT1-Ir levels were lower in endothelial cells of the cortex, hippocampus and SCN of n-3 fatty acid deficient rats. It has been demonstrated that endothelial cell GLUT1 has an asymmetric distribution across the luminal (blood side) and abluminal (brain side) membranes (Gerhart et al. 1989). An electron microscopic immunogold study in rat brain has reported that 12% is present at the luminal and 48% at the abluminal membrane, with 40% in the cytoplasmic compartment, suggesting that recruitment of the cytoplasmic pool could be involved in regulation of GLUT1 at the BBB (Farrell and Pardridge 1991). More recently, Simpson et al. (2001) suggested that the lower distribution of GLUT1 described at the luminal membrane could be explained by a conformational change in GLUT1 protein that makes immunodetection difficult. Nugent et al. (2001) reported a significant increase in basal glucose uptake in 3T3-L1 adipocyte-cultured cells supplemented with 20:4 n-6 or 22:6 n-3, and no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 at the plasma membrane. They hypothesized, either a translocation of glucose transporter to ;the plasma membrane, or a reduction of the rate of internalization. In our study, because we showed modifications in GLUT1 and GLUT3-Ir but no change in protein levels, we can address an equivalent hypothesis. A conformational change in GLUT1 protein or an alteration in the translocation mechanism for the regulation of transporter concentration in the luminal or abluminal membranes caused by membrane phospholipid modifications could make glucose transport through the BBB difficult.
Altogether, the decrease in immunoreactivity of GLUT1 at the BBB associated with enhanced GLUT3-Ir in neurones shows that there is a differential regulation of these different forms of glucose transporters for brain glucose uptake and utilization by neuronal cells in n-3 fatty acid deficient animals. GLUT3 glucose transporter is reported to possess a higher affinity to glucose than GLUT1. Therefore, it is thought that the higher GLUT3 expression in the neurones might improve glucose uptake from the extracellular space at low glucose concentrations (Gould and Seatter 1997).
In conclusion, we suggest that neurones tend to balance energy deficiency, and that structural changes in membranes do not allow glucose transporters to furnish the supply of glucose to the brain cells, and consequently changes observed in CO would not be a direct result of the changes in membrane composition, but would be secondary to change in glucose transport.
Although we did not evaluate the effects of the n-3-deficient diet on the glucose transporter or cellular metabolism in glial cells, astrocytes certainly play an important role in the metabolic changes observed as they participate in metabolic coupling with neurones (Magistretti et al. 1999) and in the delivery of essential fatty acids to the BBB and brain (Bernoud et al. 1998).