BRAIN INSULIN

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Insulin and IR are found throughout the central nervous system (CNS) (83). Since the main feature of the insulin resistant brain state is disorder of the IR signaling cascade (47), the review is focused primarily to this aspect and therefore, brain insulin and IR distribution, regulation, function and dissimilarities to the peripheral insulin system will be briefly summarized.

Insulin origin. The source of insulin in the brain might be either peripheral or local in origin, or both (3,83). Insulin from the blood crosses the blood brain barrier (BBB) by a saturable transport mechanism, but all regions of the BBB are not equally permeable to insulin. Namely, BBB transports insulin into the pons-medulla and hypothalamus twice time (4), and into the olfactory bulb two to eight times (5) faster than into the whole brain. This is consistent with finding of regionally specific distribution of insulin in the CNS with the olfactory bulb and hypothalamus containing the highest concentration of insulin (6). There are also several lines of evidence consistent with a local synthesis of insulin in the brain. Insulin mRNA is distributed in a highly specific pattern with the highest density in pyramidal cells of the hippocampus and a high density in the medial prefrontal cortex, the enthorinal cortex, perirhinal cortex, thalamus, granule cell layer of the olfactory bulb, and hypothalamus (24,104). Synthesized insulin was shown to be secreted into the extracellular space specifically by neurones (35).

Insulin receptor and its signalling. Two different types of IR have been found in adult mammalian brain; a peripheral type, detected in lower density on glia cells, and a neuron-specific type of IR that has been widely distributed in the CNS on the regionally specific basis (6). High concentrations of this neuronal IR mRNA (the following text refers to this type of IR) were found in the chorioid plexus, olfactory bulb, piryform cortex, amygdaloid nucleus, hippocampus, hypothalamic nucleus and cerebellar cortex (60). Expression of IR protein levels is in accordance with its mRNA levels in most areas, but significant discrepancy is found in the hippocampus and cerebellar cortex, suggesting a higher stability of IR in the hippocampus and probably a rapid turnover of the receptor in the cerebellar cortex neurons (106). The mayor molecular structure and most of the biochemical properties of the neuronal IR are indistinguishable from those found in the periphery, but some structural and functional differences between the CNS and the peripheral IR have been suggested; the molecular mass of alpha and beta subunits of the brain IR is slightly lower than that in the peripheral tissue (42), and high concentrations of insulin downregulate the peripheral IR, but have no effect on the brain IR (12). It has to be mentioned that two insulin receptor isoforms (IR-A and IR-B) generated by alternative mRNA splicing have been identified at the periphery, and their expression found to be highly regulated in tissue specific fashion (7). Namely, IR exists in two isoforms differing by the absence (Ex11-) or presence (Ex11+) of 12 amino acids in the C-terminus of the alpha-subunit due to alternative splicing of exon 11.Ex11- binds insulin with two-fold higher affinity than Ex11+. This difference is paralleled by a decreased sensitivity for metabolic actions of insulin. However, data reported so far suggest that heterogeneity of IR in different tissues including brain is unrelated to alternative splicing of IR gene (13,51). Also, inconsistent data have been reported regarding the abundance of the two isoforms in relation to insulin resistance in type 2 diabetes in humans (84).

Insulin signal transduction in the brain is similar to that at the periphery. Insulin receptor belongs to the receptor tyrosine kinase superfamily. Briefly, activated IR recruits insulin receptor substrate (IRS-1/2) adapter protein (also regionally specifically distributed in the CNS and co-expressed with IR in certain population of neurones /29/) to its phosphorylated docking site, which then becomes phosphorylated on tyrosine residues and capable to recruit various SH2 domain-containing signaling molecules, among which are PI3 kinase and adapter proteins for MAPK pathway (Fig. 1) (50). Activation of MAPK pathway leads to insulin-induced mitogenic effects. Activation of PI3 kinase pathway transduces the signal to protein kinase B (PKB/Act), that in the peripheral insulin-sensitive tissue and as also shown in some brain regions, triggers glucose transporter GLUT4 translocation and consequently cellular glucose uptake (50,98). In addition, PKB/Act can modulate the glycogen synthase kinase (GSK-3) pathway that in the peripheral insulin-sensitive tissue leads to glycogen synthesis (50). In the brain, beside other roles, GSK-3 has been related to the regulation of amyloid-β peptides (Aβs) and tau-protein phosphorylation (47). Therefore, metabolism of amyloid precursor protein (APP), the intracellular formation of secreted APPs and Aβs, and the release of APPs and Aβs into the extracellular space, as well as the balanced phosphorylation of tau-protein, are under control of insulin/IR signal transduction (47). Factors that affect phosphorylation/dephosphorylation homeostasis of elements in insulin signal transduction are capable of modifying this cascade causing its dysfunction, e.g., phosphorylation of particular serine and threonine residues of IRS protein reduces insulin-stimulated IRS-1 associated PI3 kinase activity that at the periphery has been causative of insulin resistance and type 2 diabetes (27).

Role of insulin in the brain. The biological effects of brain insulin depend on the availability of the hormone in the brain, its binding and activating specific brain IR with related signaling pathways, and to a minor extent also on regulation of the brain insulin that is synthesized within the CNS. Regional and state-dependent differences in transport across the BBB and in its distribution within the CNS have been suggested as possible factors that may explain how brain insulin can have so diverse effects (3). Namely, while the insulin/IR associated with the hypothalamus plays important role in regulation of the body energy homeostasis and feeding, the hippocampus- and cerebral cortex-distributed insulin/IR has been shown to be involved in brain cognitive function, including learning and memory (75,105).

Glucose is the primary fuel for the brain and cerebral metabolism of glucose requires its transport across the BBB by insulin-insensitive glucose transport GLUT1 (64), that has been found downregulated in uncontrolled diabetes (69), and upregulated in chronic hypoglycaemia (63). Majority of glucose utilization within the CNS appears to be mediated through glucose transporters GLUT1 and GLUT3, both insulin-insensitive (28). However, rapid and transient changes in cerebral glucose utilization may occur via insulin-sensitive glucose transporters GLUT4 and GLUT8, found in the brain (76,77,98). Glucose transporter GLUT4 is the main transporter type responsible for glucose uptake in the peripheral insulin target cell, and in the brain  neuronal localization of GLUT4 mRNA and protein in hypothalamus, cerebellum, cortex and hippocampus exhibits overlapping distribution with IR, as well (57,65). Activation of brain IR leads to stimulation of the glycolytic key enzymes and pyruvate oxidation in the brain (48,78).

Growing evidence suggests that insulin interacts with both orexigenic and anorexigenic peptides in the brain in the control of feeding behaviour, maintenance of body weight and energy homeostasis, but most of the research has been focused on insulin “cross-talk” with leptin in the hypothalamus, as reviewed elsewhere (36,72). Both insulin and leptin acutely regulate the membrane potential and firing rates of a specific subset of hypothalamic neurones, the effect being dependent on signaling through PI3 kinase (89,90). Genetic studies demonstrated that brain-specific knockouts of the IR and animals lacking IRS-2 show a phenotype of obesity and reproductive dysfunction (14,16), and knockdown of IR expression locally in the hypothalamus resulted in cumulative food intake of 152% and fat mass of 186% relative to controls (73). These findings support the conclusion that insulin action in the hypothalamus is essential in the regulation of energy homeostasis. It has been suggested that central insulin has fundamentally catabolic (i.e. reducing food intake and body weight), whereas peripheral insulin has anabolic (i.e. increasing energy storage and potentially increasing body weight if an individual has too much insulin) activity (72). Like most physiological systems, the peripheral and central actions of insulin regarding this issue are balanced. Therefore, the lack of catabolic insulin action in the brain (coupled with disturbances like the decrease in serum leptin) leads to marked increases in food intake and obesity that is often associated with insulin resistance and hyperinsulinemia (72).

Insulin receptor signaling plays a role in synaptic plasticity in hippocampus by modulating activity of excitatory and inhibitory receptors and consequently affecting cognitive functions like learning and memory (105). During learning insulin binds to its receptors, and activated IR may be involved in memory formation via potentiation of glutamatergic NMDA receptor channel activity leading to increase in Ca ²⁺ influx and long-term potentiation (85). Via PI3 kinase it may be involved in long-term depression by internalisation of glutamatergic AMPA receptors (59), or via recruiting of functional GABA receptors to the postsynaptic membrane (99). Additionally, IR induced activation of MAPK pathway after learning may lead to regulation of gene expression that is required for long-term memory storage, while IR interaction with G-protein coupled receptor may activate protein kinase C leading to facilitation of short-term memory encoding (105). Also, IR-IRS-PI3 kinase pathway may trigger endothelial nitric oxide synthase (eNOS) activity and generation of nitric oxide that acts as a retrograde messenger for release of neurotransmitters involved in learning and synaptic plasticity  (26,68).

Regulation of brain insulin. It is still a controversial issue if the effects of insulin on the brain should mainly be regarded as an extension of its peripheral action, or insulin effects on the brain that are clearly independent from its peripheral metabolic effects. If the latter is the case, how can these peripheral and central effects of insulin be regulated independent from each other, assuming that circulating insulin is the major source of insulin in the brain. This suggests not only the complexity of brain insulin regulation, but also the potential significance of the centrally originated portion of brain insulin.

Brain insulin is subject to a multifactorial control exerted at various levels. It can be regulated both peripherally and centrally; biosynthesis and secretion in the pancreas along with transport in the brain, internalisation, storage, stability, as well as its local synthesis and release within the CNS may be affected by numerous metabolites, circulating hormones, regulatory peptides and neurotransmitters (36). The molecular mechanisms involved in the production and release of insulin in the CNS seem to share similarities to that in the periphery: structures specific for glucose sensing and metabolizing at the level of beta cells, such as glucose transporter GLUT2 and glucokinase,  have been demonstrated co-localized in particular hypothalamic cells (49); both beta cells and hypothalamic neurons contain ATP-sensitive-K⁺ channels, a key protein in the glucose response mechanism and insulin secretion (2,97). Furthermore, insulin released from adult rat brain synaptosomes under depolarising conditions depends on calcium influx, suggesting that insulin is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association to neural activity (101).

Regulation of brain insulin, in particular its release, has been investigated at the level of hypothalamus. Increased insulin release in hypothalamus has been found in relation to carbohydrate meals (37), and local glucose (38,82) and serotonin (74) increament, respectively. This regulation showed regional specificity, since the modifications induced in the hypothalamus were not observed in the cerebellum, and neither insulinemia nor glycemia were affected. Leptin may interact with insulin directly or via other neuromodulators (serotonin, melanocortin, etc.) and there is also a cross-talk of insulin and leptin receptors in the CNS (72). The potential effects of glucocorticoids on brain insulin could be a result of both peripheral and central action, where local regulation has been suggested to involve effects via glucose and serotonin (72). Participation of other hormones and regulatory peptides in regulation of brain insulin is also being investigated.