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Introduction

It is evident from the discussion in the preceding chapters on various diseases/disorders that low-grade systemic inflammation plays a significant role in the pathobiology of obesity, hypertension, type 2 diabetes mellitus, dyslipidemia, atherosclerosis, coronary heart disease, cancer, aging, Alzheimer’s disease, schizophrenia, depression, dementia and even stroke (though this disease was not discussed in details, in general, it occurs as a result of underlying hypertension, diabetes mellitus, hyperlipidemia and hence, could be considered as a consequence of these diseases rather than as a separate disease entity). The presence of low-grade systemic inflammation as evidenced by increased plasma levels of CRP, IL-6, TNF-α, HMGB-1, MIF, ROS, iNO, and a concomitant decrease in anti-inflammatory cytokines such as IL-4, IL-10, IL-12, TGF-β, and anti-oxidants, and decreased plasma and tissue levels of various PUFAs such as AA, EPA, DHA, GLA, DGLA and their anti-inflammatory products such as lipoxins, resolvins, protectins and maresins may underlie all these diseases. Thus, an imbalance between pro- and anti-inflammatory molecules seems to be a common feature in these diseases. Thus, the molecular events in all these diseases are similar but the target tissues are different. This implies that methods designed to suppress the production of pro-inflammatory molecules and/or increase the synthesis and secretion of anti-inflammatory molecules could be of benefit in their prevention and management (Fig. 16.1).

Fig. 16.1
figure 1

Scheme showing relationship between various mediators of tissue damage/resolution and clinical conditions and the role of PUFAs and their metabolites in these processes

Full size image

Based on the evidence that low-grade systemic inflammation is a common event in several adult diseases, it is reasonable to suggest that different features seen in specific conditions can be attributed to damage or dysfunction of specific tissues relevant to a given condition/disease. In other words, the underlying pathophysiology is similar but the clinical features of the diseases are different simply because the tissues/organs involved in the said disease processes are are different. For example, in hypertension endothelial cells are the target of the adverse action of the pro-inflammatory molecules that causes endothelial dysfunction; in schizophrenia it is the neuronal cells and various neurotransmitters; in atherosclerosis it is the endothelial and smooth muscle cells; and in type 2 diabetes mellitus it is the adipose tissue to start with but later muscle, endothelial cells, liver and hypothalamic neurons are also affected. Thus, the local imbalance between the pro- and anti-inflammatory molecules that is tilted more in favor of pro-inflammatory molecules in the specific cells/tissues/organs leads to damage or dysfunction of those specific tissues/organs that ultimately leads to the development of specific disease and the varied clinical features seen in those diseases. It is important to note that in some diseases the target tissues/organs could be more than one and yet times it may be difficult to determine which tissue/organ is the first to be affected by the inflammatory molecules. For instance, the target tissues in type 2 diabetes mellitus could be pancreatic β cells, endothelial cells, ventromedial hypothalamic neurons or adipose cells or a combination there of. In addition, in these diseases the initial trigger could be dietary or other environmental factors with or without underlying genetic predisposition or factors that initiate low-grade systemic inflammatory process. Once the initial trigger is cleared or abrogated by the innate and adaptive immune systems and/or anti-inflammatory homeostatic mechanisms that are set in motion to help in the resolution of the inflammation, healing of the tissue injury occurs that leads to restoration of the function of the involved tissues and organs and normalcy is restored and health is regained. But, when the target tissues/organ dysfunction persists for prolonged periods of time, collateral damage to others tissues/organs would occur that leads to major complications and so it is extremely difficult, if not impossible, to restore normalcy. For the repair process and restoration of normal physiological function to occur in several of the adult diseases, it is necessary for the stem cells to proliferate and differentiate to replace the damaged tissues and restore normal physiological function. In this context, it is important to note that cytokines, PUFAs, lipoxins, hormones and several vitamins and minerals have a significant role in the growth, differentiation and survival of stem cells [1].

When and How the Inflammatory Process is Initiated?

Despite the evidence that several adult diseases are low-grade systemic inflammatory conditions, it is still not clear whether inflammation is the cause or affect of the disease. It is not known when and how these diseases are initiated. In this context, the role of breast-feeding and perinatal feeding on fetal and childhood growth, the development of brain growth and development and programming of the hypothalamic centers that regulate blood pressure, insulin secretion, programming of body weight/appetite/satiety set point, and autonomic nervous system deserve special attention. Breast-fed children have decreased incidence of a number of low-grade systemic inflammatory conditions such as insulin resistance, obesity, hypertension, type 1 and type 2 diabetes, schizophrenia, metabolic syndrome and some types of cancer. But, the exact mechanism(s) for this beneficial action is not clear. Since hormonal signals and/or nutritional factors and infections to which the subject is exposed during the fetal and early childhood period may serve as programming stimuli that can have lifetime consequences, it is reasonable to propose that majority, if not all, of the adult diseases have their origin in the perinatal period. If this is true, it implies that even the low-grade systemic inflammation that participates in the pathophysiology of these diseases have their origins in the perinatal period.

Perinatal Programming of Adult Diseases

Stimuli or insults induced during the perinatal period can have lifetime consequences and is called as “programming”. Hormonal signals or nutritional factors may serve as programming stimuli. Smallness and thinness at birth, continued slow growth in early childhood, followed by acceleration of growth so that height and weight approach the population means is considered as the most unfavorable growth pattern that can result in fetal adaptations that may programme the development of insulin resistance, obesity, hypertension, diabetes mellitus, and ischemic heart disease (IHD) in later life [2–5]. This suggests that perinatal nutrition is an important determinant of adult diseases. One endogenous factor that has a negative feed-back control on TNF-α production that also plays an important role in the growth and development of brain is long chain polyunsaturated fatty acids (PUFAs). Since the development of brain occurs during the period between 2nd trimester to 5 years of age and again during the adolescence, it is reasonable to assume that perinatal nutrition and childhood nutrition plays a significant role in this process. Several studies showed that PUFAs and their long-chain metabolites, PUFAs such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential not only for brain growth and development but also regulate the synthesis of various cytokines; modulate insulin action, and concentrations of various neuropeptides. This suggests that various factors that influence the metabolism of PUFAs; action and levels of cytokines; synthesis, release and action of various neurotransmitters and the activity of autonomic nervous system; and the growth and development and function of various tissues and organs such as liver, adipose tissue, muscle, and the humoral and neural factors that influence the interaction and cross-talk among various organs and brain play a significant role in the pathobiology of adult diseases. In this context, the metabolism of PUFAs (see Chap. 4) and their products that modulate autonomic nervous system, neurotransmitters, and their ability to participate in the inflammation and resolution of inflammation is of particular interest.

Factors Influencing the Metabolism of EFAs

In Chap. 4, a detailed discussion of the metabolism of EFAs and the various factors that influence their metabolism and actions has been given. Here only a brief mention of factors that participate in the metabolism of EFAs is given.

Saturated fats, cholesterol, trans-fatty acids formed by vegetable oil processing, alcohol, adrenaline, and glucocorticoids inhibit Δ6 and Δ5 desaturases. Pyridoxine, zinc, and magnesium are necessary co-factors for normal Δ6 desaturase activity. Insulin activates Δ6 desaturase whereas diabetics have reduced Δ6 desaturase activity. The activity of Δ6 desaturase falls with age. Oncogenic viruses and radiation inhibit Δ6 desaturase activity. Total fasting and protein deficiency reduce the activity of Δ6 desaturase. A fat-free diet and partial caloric restriction enhances Δ6 desaturase activity. A glucose-rich diet inhibits Δ6 desaturase activity.

Peroxisome proliferator-activated receptor-α (PPAR-α) activates the transcription of hepatic Δ6 desaturase by more than 500%. Hepatic expression of Δ5 desaturase as well as Δ6 desaturase was highly activated in transgenic mice overexpressing nuclear SREBP-1a, -1c, and -2. Disruption of the SREBP-1 gene significantly reduced the expression of both desaturases in the livers of SREBP-1-deficient mice refed after fasting. The hepatic expression of both desaturases was downregulated by dietary PUFAs, which suppressed SREBP-1c gene expression. In contrast, sustained expression of hepatic nuclear SREBP-1c protein in the transgenic mice abolished the PUFA suppression of both desaturases. Fasting induced both the desaturases. These data suggest that both Δ6 and Δ5 desaturases are regulated by SREBP-1c and PPAR-α, two reciprocal transcription factors for fatty acid metabolism, and that some of their lipogenic actions are brought about by their ability to regulate the producing PUFAs [6].

Activities of Δ6 and Δ5 desaturases are decreased in diabetes mellitus, hypertension, hyperlipidemia and the metabolic syndrome. Trans-fats interfere with the metabolism of EFAs and promote inflammation, atherosclerosis and coronary heart disease. The pro-inflammatory action of trans-fats can be attributed to their ability to interfere with the metabolism of EFAs. Several PUFAs, especially EPA and DHA are known to inhibit the production of pro-inflammatory cytokines: interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1, and IL-2 (reviewed in [4, 5, 7, 8]). Saturated fatty acids and cholesterol also interfere with the metabolism of EFAs and thus, promote the production of pro-inflammatory cytokines, which explains their ability to cause atherosclerosis and coronary heart disease (CHD). This suggests that trans-fats, saturated fats, and cholesterol have pro-inflammatory actions whereas PUFAs such as GLA, DGLA, EPA and DHA and their products namely lipoxins, resolvins, protectins, maresins and nitrolipids possess anti-inflammatory properties. By interfering with the metabolism of EFAs, saturated fats, cholesterol and trans-fats could reduce the formation of their long-chain metabolites GLA, DGLA, AA, EPA, and DHA (PUFAs) that are essential for the formation of biologically active and beneficial prostacyclin (PGI2), PGI3, lipoxins, resolvins, and NPD1.

PUFAs Modulate Glucose and Glutamine Uptake and Their Metabolism

PUFAs modulate the fluidity of the cell membrane and thus, determine and influence the behaviour of membrane-bound enzymes and receptors. Such an action of PUFAs on neurons is particularly significant since, this suggests that PUFAs will be able to modulate the synthesis, release and binding of various neurotransmitters to their respective receptors and thus modulate their action. In this context, it is noteworthy that infants preferentially accumulate AA, EPA and DHA in the brain during the last trimester of pregnancy and the first months of life. Adequate amounts of AA and DHA are essential for optimal development and function of central nervous system (reviewed in [9–15]). Infants are capable of forming AA and DHA by elongation and desaturation of EFAs, LA and ALA, respectively. But, vegetable oil based infant feed formulas lead to sub-optimal neural development and performance due to decrease in brain PUFA content [16, 17].

It is generally believed that omega-3 and omega-6 PUFA are not only critical for infant and childhood brain development and somatic growth, but that their levels especially of EPA and DHA are often low in the Western diet. Both epidemiological and intervention studies, indicated that DHA and AA supplementation, during pregnancy, lactation, or childhood plays an important role in childhood neurodevelopment and for infant growth and development [18–21]. A positive association between blood DHA levels and improvement on tests of cognitive and visual function in healthy children was reported. Controlled trials showed that supplementation with DHA and EPA may help in the management of childhood psychiatric disorders, and improve visual and motor functions in children with phenylketonuria. In all studies, DHA and EPA supplementation was found to be well tolerated [18–21].

Human infants accumulate AA, EPA and DHA from maternal and/or placental transfer, consumption of human milk, and synthesis from LA and ALA. AA regulates energy metabolism in the cerebral cortex by stimulating glucose uptake in cerebral cortical astrocytes [22]. AA metabolites LTB4 and 5-HETE also stimulated the uptake of glucose in human leukocytes [23]. Exposure of adipocytes to AA rapidly enhanced basal 2-deoxyglucose uptake, reaching maximal effect at approximately 8 h, while insulin-stimulated 2-deoxyglucose uptake was not altered. AA increased the apparent Vmax of basal 2-deoxyglucose uptake was more than doubled, while the apparent Km for 2-deoxyglucose remained unchanged and enhanced the content of the ubiquitous glucose transporter (GLUT-1) in both total cellular and plasma membranes by a PKC-independent mechanism [24].

It is interesting to note that growth of the murine B-lympho-cyte cell line CC9C10 and the myeloma SP2/0 was enhanced significantly by the presence of the unsaturated fatty acids, oleic and linoleic acids in serum-free culture. The cellular content of linoleic and oleic acids gradually increased during continuous culture passage, with no evidence of regulatory control. Over 10 culture passages in the presence of these fatty acids, the unsaturated/saturated fatty acid ratio of all cellular lipid fractions increased substantially. Most of the fatty acid accumulated in the polar lipid fraction (more than 74%) and only a small proportion was oxidized to CO2 (0.5%). LA caused a decrease to one-eighth in the rate of metabolism of glutamine and a 1.4-fold increase in the rate of metabolism of glucose with no change in the relative flux of glucose through the pathways of glycolysis, pentose phosphate or the tricarboxylic acid cycle. The changes in energy metabolism were reversed when the cells were removed from fatty acid-supplemented medium. It appeared that LA decreased the rate of uptake of glutamine into cells as evidence by the observation that growth of the CC9C10 cells in the presence of LA caused the Km of glutamine uptake to increase from 2.7 to 23 mM, whereas glucose uptake was unaffected [25]. This change in glutamine uptake in the presence of LA and the resultant increase in the growth of the cells is understandable since, tumor cells use large amounts of glutamine to form glutamate and then to α-ketoglutarate that is fed into the Krebs cycle [26]. In addition, it was also reported that PGF2α may act with cAMP (cyclic AMP) in a synergistic way to increase glucose transport by enhanced GLUT1 expression by a PKC-dependent mechanism in adipose cells [27]. This evidence suggests that both PUFAs and their products such as PGs and LTs modulate glucose uptake and its metabolism by neuronal, adipose and tumor cells.

On the other hand, glucose enhances ACh release in the brain [28]. Since AA enhances glucose uptake and, in turn, glucose augments ACh release, it is likely that AA augments ACh release [29]. DHA, another PUFA, enhances cerebral ACh levels and improves learning ability in rats [30]. ACh modulates long-term potentiation and synaptic plasticity in neuronal circuits and interacts with dopamine receptor in the hippocampus [31]. In obesity, a decrease in the number of dopamine receptors or dopamine concentrations occurs [32] and obesity is common in type 2 diabetes. These results imply that PUFAs, glucose and glutamine uptake and their metabolism, Ach release and dopamine function and the synaptic plasticity are interrelated and function in a cohesive manner that may have relevance to several neurological conditions including schizophrenia, Alzheimer’s disease, depression, and the role of hypothalamic neurons in obesity, satiety and appetite control.

PUFAs, Insulin, and Acetylcholine Function as Endogenous Cyto- and Neuroprotectors

Insulin receptor tyrosine kinase substrate p58/53 and the insulin receptor are components of synapses in the CNS [33]. Insulin and calorie restriction augment the activities of desaturases (reviewed in [4, 5, 7, 8]) and this increases the formation of PUFAs from their precursors. Insulin-like growth factor-1 (IGF-1) and insulin antagonize neuronal death induced by TNF-α [34, 35]. AA, DHA, and EPA and other PUFAs have neuroprotective and cytoprotective actions [36–41] and are also potent inhibitors of IL-1, IL-2 and TNF-α production [42–44]. Insulin and PUFAs regulate superoxide anion generation and enhance the production of eNO [45–51]. NO is anti-inflammatory in nature [49] and quenches superoxide anion. IGF-I and, possibly, insulin enhance ACh release from rat cortical slices [52, 53]. ACh inhibits the synthesis and release of TNF-α both in vitro and in vivo and thus, has anti-inflammatory actions [54] and is also a potent stimulator of eNO synthesis [55]. These data suggest that insulin and IGF-I enhance the formation of PUFAs in the brain by their action on desaturases, and PUFAs, in turn, enhance ACh levels in the brain (this is in addition to the ability of insulin and IGF-I to directly enhance ACh levels in the brain) and inhibit the production of TNF-α. Thus, insulin, ACh, and PUFAs suppress TNF-α production and augment the synthesis of eNO. ACh and eNO are not only neuroprotective in nature but also interact with other neurotransmitters and regulate their secretion, release and action. Thus, insulin, IGF-I, ACh, and PUFAs protect brain from insults induced by TNF-α and other molecules.

In addition, there is evidence to suggest that PUFAs and insulin have cytoprotective actions as well. For example, we showed that PUFAs prevent radiation and chemical-induced cytotoxicity and genotoxic actions both in vitro and in vivo [41, 56, 57]. Alloxan-induced cytotoxicity to pancreatic β cells was prevented by AA, EPA and DHA both in vitro and in vivo [58–61], suggesting that PUFAs may have anti-diabetic actions. PUFAs, especially n-3 PUFAs, were found to prevent status epilepticus-associated neuropathological changes in the hippocampal formation of rats with epilepsy [62]. In our studies, we noted that both cyclo-oxygenase and lipoxygenase inhibitors did not prevent the cytoprotective actions of PUFAs against alloxan-induced damage to pancreatic β cells, suggesting that either the fatty acids themselves are active or other products are formed that have cytoprotective actions. Recent studies suggested that AA, EPA and DHA form precursors to anti-inflammatory and potent cytoprotective products such as lipoxins, resolvins, protectins and maresins [63–70] that could be responsible for the cytoprotective actions of various PUFAs. It is likely that normal cells when exposed to PUFAs produce significant amounts of lipoxins, resolvins and protectins that protect them from the cytotoxic actions of various chemicals and radiation, while tumor cells when supplemented with the same fatty acids do not produce these cytoprotective lipid molecules but produce cytotoxic molecules such as 17-hydroxydocosahexaenoic acid (17-HDHA) via 17-hydroperoxydocosahexaenoic acid (17-HpDHA) through 15-lipoxygenase and autoxidation. In contrast to normal neural cells, neuroblastoma cells did not produce the anti-inflammatory and protective lipid mediators, resolvins and protectins. The cytotoxic effect of DHA in neuroblastoma seems to be mediated through production of hydroperoxy fatty acids that accumulate to toxic intracellular levels. These evidences suggest that normal and tumor cells metabolize PUFAs in a differential fashion that seems to underlie the cytoprotective action in normal cells and at the same time PUFAs are directed to form toxic hydroperoxy fatty acids to kill tumor cells.

In addition, PUFAs and insulin interact with each other to bring about some of their actions. Incorporation of significant amounts of PUFAs into the cell membranes increase their fluidity that, in turn, enhances the number of insulin receptors on the membranes and the affinity of insulin to its receptors. Thus PUFAs attenuate insulin resistance [71–78]. Hereditary hypertriglyceridemic (hHTg) rats have reduced activity of the Δ6 desaturase in liver without any changes in gene expression for this enzyme; and the concentration of AA was significantly decreased in hHTg rat liver suggesting that impaired insulin action in hHTg rat is due to a deficiency of PUFAs. Feeding these animals with fish oil, a rich source of EPA and DHA, not only reduced plasma levels of triglycerides but also restored insulin sensitivity [79, 80]. These results were supported by the observation that supplementation of fish oil to high fat diet fed experimental animals improved in vivo insulin action; and this insulin sensitizing effect of fish oil was accompanied by a decrease of circulating triglycerides, free fatty acids and glycerol levels in the postprandial state and by a lower lipid content in liver and skeletal muscle [80]. These results are interesting since it is known that increase in IMCL is associated with insulin resistance and increased expression of perilipins, whereas EPA/DHA reduce IMCL and possibly that of perilipins. Thus, one mechanism by which EPA/DHA are beneficial in the metabolic syndrome could be by reducing IMCL and the expression of perilipins.

Since brain is rich in PUFAs, especially AA, EPA, and DHA, one important function of PUFAs in the brain could be to ensure the presence of adequate number of insulin receptors. Thus a defect in the metabolism of PUFAs or when adequate amounts of PUFAs are not incorporated into the neuronal cell membranes during the fetal development and infancy, it may cause a defect in the expression or function of insulin receptors in the brain. This may lead to the development of type 2 diabetes as seen in NIRKO mice [81]. Furthermore, systemic injections of either glucose or insulin in ad libitum fed rats resulted in an increase in extracellular acetylcholine in the amygdala [82]. Acetylcholine (ACh) modulates dopamine release that, in turn, regulates appetite [83]. ACh inhibits the production of pro-inflammatory cytokines (IL-1, IL-2 and TNF-α) in the brain and thus, may also protect the neurons.

The cytoprotective actions of insulin, which is similar to that of PUFAs and acetylcholine, is further evident from our previous study wherein it was noted that insulin infusion protected cardiac tissue from ischemia-reperfusion induced injury by inhibiting ischemia/reperfusion-induced TNF-α production through the Akt-activated and eNOS-NO-dependent pathway in cardiomyocytes. The antiinflammatory property elicited by insulin may contribute to its cardioprotective and prosurvival effects both in vitro and in vivo [84]. These results are in support of the previous proposal that insulin has anti-inflammatory actions, shows cytoprotective and cardioprotective actions [85–90].

In addition to their ability to possess cytoprotective actions, PUFAs also have a significant role in the growth and development of brain.

PUFAs in Brain Growth and Development

Brain is rich in AA, EPA and DHA which constitute as much as 30–50% of the total fatty acids in the brain, where they are predominantly associated with membrane phospholipids. Hence, when the concentrations of these fatty acids are inadequate, especially, during the critical period of brain growth, which is from third trimester to 2 years post-term and adolescence; the development, maturation, synaptic connections of hypothalamic neurons (especially in the VMH), the synthesis, release and functionality of various neurotransmitters is expected to be inappropriate or inadequate. Such a developmental aberration of the hypothalamic neurons will lead to a defect in the expression or function of insulin receptors in the brain, various neurotransmitters and their receptors that, in turn, predisposes to defective blood glucose sensing both in the brain and periphery resulting in failure of pancreatic β cells to produce adequate amounts of insulin. These events could eventually result in the development of the metabolic syndrome. In this context, it is noteworthy that PUFAs to have a critical and direct regulatory role in the growth and development of brain. PUFAs also have a regulatory role in the synthesis, release and function of various neurotransmitters and hypothalamic peptides.

Syntaxin, SNARE Complex and PUFAs

Increase in cell membrane surface area and growth of neurite processes from the cell body are critical for proper neuronal development and synapse formation [91]. Nerve growth cones are highly enriched with AA-releasing phospholipases, which have been implicated in neurite outgrowth [92, 93]. The fusion of transport organelles with plasma membrane leads to cell membrane expansion [94]. Syntaxin 3, a plasma membrane protein that has an important role in the growth of neurites has been shown to be a direct target for AA, DHA and other PUFAs [95]. It was reported that AA, DHA, and other PUFAs but not saturated and monounsaturated fatty acids activated syntaxin 3. Of all the fatty acids tested, AA and DHA were found to be the most potent compared to LA and ALA. Even syntaxin-1 that is specifically involved in fast calcium-triggered exocytosis of neurotransmitters is sensitive to AA [96]. These results suggest that AA is involved both in exocytosis of neurotransmitters and neurite outgrowth. SNAP-25 (synaptosomal-associated protein of 25 kDa), a syntaxin partner, implicated in neurite outgrowth interacted with syntaxin 3 only in the presence of AA that allowed the formation of the binary syntaxin 3-SNAP 25 complex. AA stimulated syntaxin 3 to form the ternary SNARE complex (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), which is needed for the fusion of plasmalemmal precursor vesicles into the cell surface membrane that leads to membrane fusion. The intrinsic tyrosine fluorescence of syntaxin 3 showed marked changes upon addition of AA, DHA, LA, and ALA, whereas saturated and monounsaturated (oleic acid) fatty acids were ineffective. These results indicate that AA and DHA change the α-helical syntaxin structure to expose SNARE motif for immediate SNAP 25 engagement and thus, facilitate neurite outgrowth.

PUFAs Modulate RAR-RXR and Other Nuclear Receptors and are Essential for Brain Growth and Development

Retinoic acid (RA) has profound effects on the development of vertebrate limb and nervous system, and in epithelial cell differentiation that are transduced by its binding to a nuclear retinoic acid receptor (RAR) which, in the presence of ligand, is transformed into a transcription factor. The differential expression of RAR gene family receptors: RAR-α, RAR-β, and RAR-γ, is important for correct transduction of the RA signal in various tissues. The other subtype of retinoid receptor is the retinoid X receptor (RXR), which also could be α, β, and γ. RXRs are also transcription factors that can act as ligand-dependent and -independent partners for RARs and other nuclear receptors. There is evidence to suggest that RAR-RXR dimmers act on the β-catenin signaling pathway to produce some of their actions. RAR-RXR nuclear receptors are essential for the development brain and other neural structures [97]. AA, DHA, and possibly, EPA are known to serve as endogenous ligands of RAR-RXR and activate them [98–100]. Several RXR heterodimerization partners such as peroxisome proliferator-activated receptors (PPARs), the liver X receptors (LXR) and farnesoid X receptor (FXR) are essential for regulating energy and nutritional homeostasis and in the development of brain and other neural structures. This suggests that AA, DHA, and EPA participate in the growth and development of brain and other neuronal structures by their ability to bind to RAR-RXR, LXR, FXR and other nuclear receptor heterodimers. This is supported by the observation that EPA/DHA and other fatty acids alter gene expression in the developing brain [101]. Myelin-specific mRNA levels were found to be developmentally regulated and influenced by dietary fat. Neonatal brain stearoyl CoA desaturase and LDL receptor mRNA levels were altered by neonatal fat intake. The neonatal response to dietary fat is tissue-specific at the mRNA level. [101].

Since PUFAs are structural components of all tissues and are indispensable for cell membrane synthesis, especially of the brain, retina and other neural tissues; and serve as precursors for eicosanoids, which regulate numerous cell and organ functions, it is reasonable to expect that n-3 and n-6 fatty acids are essential for the growth and development of human brain, particularly in early life. It is known that light sensitivity of retinal rod photoreceptors is significantly reduced in newborns with n-3 fatty acid deficiency, and that DHA significantly enhanced visual acuity maturation and cognitive functions. Clinical studies revealed that dietary supplementation with EPA/DHA-rich oils resulted in increased blood levels of DHA and AA, as well as an associated improvement in visual function in formula-fed infants matching that of human breast-fed infants. These beneficial effects are not only due to the known effects of these fatty acids on membrane biophysical properties, neurotransmitter content, and the corresponding electrophysiological correlates but also because of their ability to alter gene expression of the developing retina and brain. Intracellular fatty acids or their metabolites regulate transcriptional activation of gene expression during adipocyte differentiation and retinal and nervous system development. Regulation of gene expression by PUFAs occurs at the transcriptional level and is probably mediated by nuclear transcription factors activated by fatty acids and by modulating micro-RNAs. These nuclear receptors are part of the family of steroid hormone receptors. AA/EPA/DHA have significant effects on photoreceptor membranes and neurotransmitters involved in the signal transduction process; rhodopsin activation, rod and cone development, neuronal dendritic connectivity, and functional maturation of the central nervous system [102].

For example, PGD2-synthesizing enzyme that is expressed in antigen-presenting cells, mast cells, and other immunocompetent cells is also present in microglia and the migration pathway of microglia in the developing mouse brain. The expression of PGD2 synthase enzyme mRNA peaked at postnatal day 10, decreased gradually thereafter, and reached a plateau at postnatal day 20. Most of the PGD2 synthase positive cells at postnatal day 10 had morphological characteristics of ameboid microglia and gave positive immunostaining with microglia-specific markers, which became less detectable later on, but PGD2 synthase was still expressed even in resting microglia. These evidences suggest that PGD2 synthase enzyme is a useful marker for microglial development. In addition, spaciotemporal evaluation of microglial development and migration with PGD2 synthase immunostaining revealed that the migration pathways of microglia in the postnatal brain could be: from the lateral ventricle via subventricular zones to brain parenchyma; from the leptomeninges around the cerebellopontine angle to the cerebellar white matter; and from the overlying leptomeninges to the hippocampus, basal forebrain, and brainstem [103]. This suggests that one can use PGD2 synthase marker to trace the growth and migration pathways of microglia.

In this context, it is noteworthy that albumin, a serum protein present in the developing brain, could serve as a stimulator of fatty acid synthesis and thus, may aid in brain growth and development. It is known that albumin binds tightly to PUFAs and could carry various fatty acids from place to place. For instance, when albumin is infused it could mobilize DHA and, possibly, other PUFAs from liver to the target tissues [104]. It was shown that albumin stimulates the synthesis of oleic acid by cultured astrocytes by inducing stearoyl-CoA 9-desaturase, the rate-limiting enzyme in oleic acid synthesis, through activation of the sterol regulatory element-binding protein-1. In experimental animals, albumin reaches maximal brain level by day 1 after birth, coinciding with activation of the sterol response element binding protein-1, which is responsible for the transcription of the enzymes required for oleic acid synthesis. The developmental profile of stearoyl-CoA 9-desaturase-1 mRNA expression follows that of sterol regulatory element-binding protein-1 activation, indicating that these phenomena are tightly linked. Since, oleic acid induces neuronal differentiation, as indicated by the expression of growth associated protein-43 and the expression of growth associated protein-43 mRNA peaks at about day 7 after birth, following the maximal expression of stearoyl-CoA 9-desaturase-1 mRNA that occurs between days 3 and 5 postnatally, it is reasonable to conclude that the synthesis of oleic acid is linked to neuronal differentiation during rat brain development [105]. It is possible that similar function could be attributed to other fatty acids such as AA/EPA/DHA. Furthermore, DHA/EPA/AA appears to be versatile molecules with a wide range of actions spanning from participation in cellular oxidative processes and intracellular signaling to modulatory roles in gene expression and growth regulation [106].

The essentiality of PUFAs in brain and growth development is further evident from the fact that maternal α-linolenic acid (ALA, 18:3 n-3) dietary deficiency in postnatal rat brain showed a marked decrease of the dopamine-synthesizing enzyme tyrosine hydroxylase accompanied by a down-regulation of the vesicular monoamine transporter (VMAT-2) and a depletion of VMAT-associated vesicles in the hippocampus compared with adequately fed controls. The dopamine transporter (DAT) was not affected by the ALA deficiency indicative of a DAT/VMAT-2 ratio increase that may enhance the risk of damage of the dopaminergic terminal. A robust increase in dopamine receptor (DAR1 and DAR2) levels was noticed in the cortex and striatum structures possibly to compensate for the low levels of DA in synaptic clefts. Microglia activation was noticed following ALA deficiency. Since ALA deficiency could lead to decreased DHA synthesis, it has been proposed that reduced levels of anti-oxidants in the developing brain might be responsible for microglial activation and enhanced oxidative stress that increased the risk of dopamine-associated neurological disorders [107, 108] that include obesity, schizophrenia, depression and anxiety.

It is important to note that DHA/EPA supplementation significantly reduced DNA fragmentation and caspase-3 activation in developing cerebellum of hypothyroid pups. This anti-apoptotic actions of EPA/DHA is due to their ability to decrease the levels of pro-apoptotic basal cell lymphoma protein-2 (Bcl-2)-associated X protein (Bax) and increase the levels of anti-apoptotic proteins like Bcl-2 and Bcl-extra large (Bcl-x(L)). Furthermore, EPA/DHA restored levels of cerebellar phospho (p)-AKT, phospho-extracellular regulated kinase (p-ERK) and phospho-c-Jun N-terminal kinase (p-JNK). These results suggest that some of the beneficial actions of EPA/DHA in brain growth and development include their ability to regulate apoptotic signaling pathways under stress [109].

Furthermore, the vital role of PUFAs particularly during embryonic development became evident from the observation that the expression of genes encoding enzymes involved in PUFA biosynthesis, namely fatty acyl desaturase (Fad) and Elovl5- and Elovl2-like elongases, showed temporal expression of all three genes from the beginning of embryogenesis (zygote), suggesting maternal mRNA transfer to the embryo. When spatial expression was studied by whole-mount in situ hybridisation in 24 embryos, both fad and elovl2 were found to be highly expressed in the head area where neuronal tissues are developing. Of all, elovl5 showed specific expression in the pronephric ducts, suggesting an as yet unknown role in fatty acid metabolism during zebrafish early embryonic development. The yolk syncytial layer also expressed all three genes, suggesting an important role in remodelling of yolk fatty acids during zebrafish early embryogenesis. Tissue distribution in zebrafish adults demonstrates that the target genes are expressed in all tissues but more particularly in liver, intestine and brain showing the highest expression [110]. These results suggest that PUFAs are essential during embryo development and more so for brain growth and development and zebrafish could be used as a model organism to delve more deeply into the role of PUFAs in the development of various organs.

PUFAs Modulate Gene Expression and Interact with Cytokine TNF-α and Insulin to Influence Neuronal Growth and Synapse Formation

It was reported that mRNA level of genes involved in myelination were affected by a diet lacking essential fatty acids [101]. The expression level of 102 cDNAs, representing 3.4% of the total 3200 DNA elements on the microarray, were significantly altered (either upregulated or downregulated) in brains of rats fed with ω-3 DHA/ALA diets [111–114]. Of all the genes examined, 55 genes were upregulated and 47 were downregulated relative to controls. The altered genes included those involved in synaptic plasticity, cytoskeleton, signal transduction, ion channel formation, energy metabolism, and regulatory proteins. Of all, the 15 genes that responded more intensely to the ALA/DHA diet include those that encode a clathrin-associated adaptor protein, farnesyl pyrophosphatase synthetase, Sec24 protein, NADH dehydrogenase/cytochrome c oxidase, cytochrome b, cytochrome c oxidase subunit II, ubiquitin-protein ligase Nedd42, and transcription factor-like protein. In addition, several genes that participate in signal transduction, like RAB6B, small GTPase and calmodulins were also upregulated. α- and γ-synuclein and D-cadherin genes were upregulated in response to ALA/DHA-rich diet, which are specifically enriched at synaptic contacts and are known to play a significant role in neural plasticity, development and maturation of neurons [115]. The overexpression of mitochondrial enzymes observed in ALA/DHA diet supplemented rats suggests that the brain was in an elevated metabolic state. Perinatal supply of ω-3 fatty acids influences brain gene expression later in life and is critical to the development and maturation of several brain centers that are specifically involved in the regulation of appetite and satiety. It is possible that the quality and quantity of PUFAs available during the perinatal period may determine the expression level of various genes, their response to the environmental agents, and determine the quality and levels of expression of various pro-oxidant and anti-oxidant enzymes, cytokines, pro-resolution and wound healing molecules, etc., timing of their expression, duration of expression and their interaction(s) with other concerned genes. Thus, in essence PUFAs might be the master regulators of gene expression and they may be able to regulate and determine gene expression at various stages of growth and development and at different periods of age and the response of genes to different environmental and endogenous stimuli and molecules.

For example, it was reported that perinatal supplementation of ω-3 fatty acids (especially DHA) induces overexpression of genes coding for cytochrome c and TNF receptor (TNFRSF1A), while omega-3 lipids decreased TNF-α and PGE2 production in LPS-stimulated macrophages [116], probably, through decreasing NF-kappaB activation. It should also be noted that PUFAs may have a direct effect on the expression of genes and/or may bring about their actions through their products such as eicosanoids, lipoxins, resolvins, protectins and nitrolipids. It was reported that aspirin-treated enterocytes generated 15R-HETE, a precursor of 15-epi-LXA4 biosynthesis, which sharply inhibited TNF-α-induced IL-8 and thus, downregulated mucosal inflammatory events [117]. Similarly, eicosanoids derived from PUFAs may inhibit proinflammatory gene expression by acting on the PPAR-γ expression to bring about their biological actions [118]. Hence, the actions of PUFAs may occur at several stages and brought about by several of their products. It is also important to note that sometimes the actions of products of various PUFAs may have antagonistic actions (for example: some eicosanoids have pro-inflammatory actions whereas lipoxins have anti-inflammatory actions). Hence, the final outcome of the actions of various PUFAs and their products on a physiological function in a given tissue or organ will depend on the balance between these mutually antagonistic molecules. Similarly, even in a given pathological process the balance between mutually antagonistic actions of various PUFAs and their very many metabolites will determine the continuation of the diseases process or recovery from the same.

The actions of PUFAs on the expression of neurotransmitter genes is particularly relevant while considering the role of PUFAs in brain growth and development and their involvement in various neurological diseases. For instance, it was reported that supplementation of AA and EPA/DHA increased the expression of serotonin receptor in hypothalamus [119]. 5-HT4 receptor increases in expression have been shown to augment hippocampal acetylcholine outflow. It was also reported that AA and EPA/DHA feeding enhanced the expression of POMC in hippocampus suggesting that AA/EPA/DHA can influence appetite and satiety and thus, control energy metabolism. Changes in the expression of acetylcholine is of particular interest since, it has a regulatory role in the release and action of various other neurotransmitters such as serotonin, dopamine and catecholamines and is also a potent anti-inflammatory molecule [120–124], while catecholamines have pro-inflammatory actions [125]. Thus, yet another mechanism by which PUFAs regulate inflammation and immune response is by altering the levels of acetylcholine in the brain. Thus, PUFAs, its anti-inflammatory products such as lipoxins, resolvins, protectins, nitrolipids and acetylcholine are essential to prevent inflammation in the brain to ensure its proper growth and development in perinatal period.

These results are interesting since; there is now evidence to suggest that TNF-α produced by glial cells enhances synaptic efficacy by increasing surface expression of AMPA receptors. Continued presence of TNF-α is required for preservation of synaptic strength at excitatory synapses [126, 127]. TNF-α production is suppressed by EPA/DHA and acetylcholine, whereas excess TNF-α induces apoptosis of neurons. On the other hand, hepatic vagus nerve stimulation attenuated Fas-induced hepatocyte apoptosis through alpha7 nicotinic AChR by causing the Kupffer cells to reduce their generation of an excessive amount of reactive oxygen species [128] and there it is likely that similar anti-apoptotic function could be served by acetylcholine in the brain also. Thus, there appears to be a balance maintained between pro-apoptotic action of TNF-α and anti-apoptotic function of acetylcholine (and incidentally acetylcholine suppresses the synthesis and release of TNF-α) that are essential to regulate the neuronal generation, their synaptic formation and the strength of the synapses that are formed. PUFAs and their metabolites by regulating the acetylcholine outflow and TNF-α synthesis may play a pivotal role in the neurogenesis. Since, during the development of brain in the perinatal period there is a constant cycles and waves of neuronal death and generation and regeneration from the neuronal stem cells, it is plausible that the levels of PUFAs and their metabolites, acetylcholine and TNF-α and possibly, other cytokines change (increase and decrease) in a wave form to modulate the brain growth during its various phases of development. It is likely that there is a sort of a celestial dance among the levels of various PUFAs, their metabolites, acetylcholine, TNF-α and other cytokines and a close interaction(s) that ultimately moulds the growth and development of the brain.

Neurogenesis and Neuronal Movement During the Growth and Development of Brain and PUFAs

The most important event(s) that happen in the growth and development of brain is a massive rearrangement of the neuronal cells that transforms a relatively uniform ball of cells into a multilayered organ with innumerable connections (synapses) at the right time and at the right place. This has to occur in a precise choreography that is strikingly similar among organisms from flies to fish to people. Although neuronal cell movements are crucial for the development of brain and sometimes involve longer journeys [129], if the intricate dance of neuronal cells goes awry, the resulting defects are usually catastrophic.

Just what causes the neuronal cells to move and guides them to their designated places is fascinating. There could be a link between genetic signaling cascades to molecules that actually affect the movements of neuronal cells, including those that cause cells to stick together and those that promote movement. It is likely that rearrangements of neuronal cells arose from cell division—that certain cells divide faster than others and change the architecture of the brain. It is also likely that cells constantly shift places in a specified pattern. Before cells can move they must first loosen the adhesives holding them together called cadherins, which protrude from the cell surface allowing cells to stick to each other. Reelin and mDab1 are two proteins that are involved in neuronal migration [130] though; there could be many more such proteins. These proteins might interact with the intracellular signaling enzymes tyrosine kinases and have the ability to bind to Src and thus, can link a tyrosine kinase like Src to another protein in a signaling pathway.

Fetal stem cells can multiply and differentiate to neurons and glia. The adult nervous system contains multipotential precursors for neurons, astrocytes, and oligodendrocytes. Cultured cells from both the fetal and adult CNS that have proliferated in vitro can differentiate to show morphological and electrophysiological features characteristic of neurons: regenerative action potentials and synaptic structures, suggesting the multipotential nature of cells derived from the CNS. Sonic hedgehog and members of the transforming growth factor-β (TGF-β), basal-fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), neurotrophins, epidermal growth factor (EGF), BMPs (bone morphogenetic factors), angiopoietin are some of the factors that seem to be involved in the growth, differentiation and proliferation of neural stem cells [131]. PTEN is also involved in the control of neural cell size, and in the proliferation and self-renewal of neural stem cells [132]. Wnt signaling has recently emerged as a key factor in controlling stem cell expansion. There is now evidence to suggest that many of these factors involved in neuronal stem cell proliferation and differentiation interact with PUFAs as discussed below.

Insulin, PUFAs and Neuronal Proliferation

Insulin is needed for neuronal growth and differentiation and synaptic plasticity in the CNS [133, 134] but also stimulates the formation of AA/EPA/DHA by activating of Δ6 and Δ5 desaturases, and suppresses TNF-α production. Insulin has been shown to determine final size of the cells and body possibly, by regulating metabolism [134]. Calorie restriction activates Δ6 and Δ5 desaturases; partly, by enhancing insulin action, and promotes the formation of AA/EPA/DHA. Calorie restriction also promotes mitochondrial biogenesis by inducing the expression of eNOS [134] and the enhanced formation of NO that occurs as a result, is a neurotransmitter and vasodilator that may aid the rapidly growing brain during perinatal period. Furthermore, as already described above, both insulin and AA/EPA/DHA stimulate eNO formation. This close interaction and feed-back regulation between TNF-α, EPA/DHA, insulin, Δ6 and Δ5 desaturases, and neuronal growth and synapse formation, and the fact that TNF-α is needed for synaptic strength whereas AA/EPA/DHA is needed for the activation of syntaxin 3 and neurite outgrowth suggests that growth of neurons and synaptic formation will be optimum only when all these factors are present in physiological concentrations. In contrast, when AA/EPA/DHA concentrations are sub-optimal, TNF-α levels tend to be high. High TNF-α concentrations have neurotoxic actions and hence, could cause damage to VMH neurons. This will lead to hyperphagia, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and IGT. Thus, TNF-α may participate in the pathogenesis of metabolic syndrome by two mechanisms: (a) inducing peripheral and central insulin resistance, and (b) damage or interfere with the action of VMH neurons.

Catenin, wnt and Hedgehog Signaling Pathway in Brain Growth and Development and PUFAs

It is well recognized that during brain development, proliferation of neural progenitor cells is tightly controlled to produce the organ of predetermined size. To achieve this objective, cell-cell communication is essential so that information concerning the density of their cellular neighborhood is provided. Adherens junctions, which contain cadherins, β-catenins, and α-catenins, mediate intercellular adhesion in neural progenitors [135]. In a recent study, it was observed that mice with a conditional αE-catenin allele (αE-catenin loxP/loxP) crossed with mice carrying nestin-promoter driven Cre recombinase (Nestin-Cre +/−), which is expressed in CNS stem/neural progenitors starting at embryonic day 10.5 (E10.5) resulted in αE-catenin loxP/loxP/Nestin-Cre +/− animals that displayed loss of αE-catenin in neural progenitor cells [136]. The knockout αE-catenin loxP/loxP/Nestin-Cre +/− mice were born with bodies similar to their littermates, but with enlarged heads due to shortening of the cell cycle, decreased apoptosis, and cortical hyperplasia as a result of abnormal activation of hedgehog pathway. Hedgehog pathway plays a critical role in mammalian CNS development and brain cancer. Hedgehog pathway promotes survival and blocks apoptosis of neuroepithelial cells and hence, its activation may produce cortical hyperplasia in αE-catenin loxP/loxP/Nestin-Cre +/− mice. These results suggest that the increase in cell density is sensed by an increase in the per cell area occupied by adherens junctions that is transmitted to the hedgehog pathway. This, in turn, provides a negative feedback loop resulting in a decrease in cell proliferation that ultimately controls the size of developing brain [136]. It is interesting to note that β-catenin is required for the mitogenic activity of PGE2 in colon cancer cells [137], whereas GLA, the precursor of AA (from which PGE2 is formed) inhibits the expression of catenin both in vascular endothelial cells and human cancer cells [138, 139]. This suggests that PUFAs have a negative feedback control on catenin expression and thus, they may regulate brain size, development and growth. Thus, one of the functions of AA, EPA and DHA in the brain could be not only to regulate synapse formation and neurite growth but also to control brain growth and size. It was reported that TNF-α also induced a significant decrease of E-cadherin and β-catenin expression [140] suggesting that cytokines play a role in brain growth and development. This is especially interesting in the light of the known fact that at high concentrations TNF-α induces apoptosis of neuronal cells [34, 35]. Thus, there seems to be a close interaction(s) between the expression of catenins, and their modulation by TNF-α and possibly, other cytokines, and PUFAs are crucial to neurite growth, synapse formation, and brain growth and development. Proper development of neurons and synaptic connections between different neurons ultimately determines the response of various neurons, especially those of hypothalamic neurons, to various neurotransmitters and plasma glucose that, in turn, regulates insulin secretion by pancreatic β cells and glucose production by liver. This is supported by the observation that an increase in circulating glucose and a primary increase in hypothalamic glucose levels inhibits glucose production in the liver and thus, lowers blood glucose [141]. Activation of neuronal pyruvate flux is required for hypothalamic (especially the arcuate nucleus) glucose sensing and for control of blood glucose and liver glucose metabolism through the activation of ATP-sensitive potassium channels in the glucose sensing hypothalamic neurons [141]. These results suggest that specific hypothalamic neurons play a significant role in the control of blood glucose levels, glucose production by liver and insulin secretion by pancreatic β cells. The ability of these specific hypothalamic neurons to control glucose homeostasis may, in turn, depend on the health of these neurons and their synaptic connections with other neurons and their ability to respond to various neurotransmitters in an appropriate manner. Impairment in the biochemical sensing of carbohydrates (especially glucose) by the hypothalamic neurons may represent a basic underpinning for defects in the regulation of food intake [142, 143], β-cell function [144], and liver glucose homeostasis [145]. Both type 2 diabetes mellitus and metabolic syndrome are typical examples of diseases the prevalence of which is dependent on environmental, nutritional factors operating on genetic susceptibility.

One important regulatory factor that controls β-catenin-dependent transcription of target genes is Wnt proteins that signal through seven-pass transmembrane receptors of the frizzled family to activate β-catenin. The Wnt family of secreted glycoproteins regulates a large number of developmental processes including cell growth, cell polarity, cell-fate determination, tissue patterning, tissue specification, and tumorigenesis. Wnts are crucial cell signaling molecules during development and in adult life. In the absence of Wnt receptor activation, the modular protein Axin provides a scaffold for the binding of glycogen synthase kinase-3β (GSK3β), Adenomatous polyposis coli protein (APC) and β-catenin. This, in turn, facilitates β-catenin phosphorylation by GSK3β [146, 147] and leads to the degradation of β-catenin via the ubiquitin pathway [148]. Upon Wnt’s binding of the frizzled receptor, the Axin-GSK3β-APC-β-catenin complex is disrupted. As a result, β-catenin is no longer targeted for ubiquitin degradation and so accumulates in the nuclei [149], where it interacts with the members of the lymphoid enhancer factor/T-cell factor classes of transcription factors to regulate the expression of target genes. Overexpression of GSK3β and Axin or depletion of maternal β-catenin RNA causes deficiencies in dorsal structures [150–152]. β-catenin induces growth of cardiomyocytes in vitro and is necessary for hypertrophic stimulus-induced growth of cardiomyocytes in vivo [153]. β-catenin is stabilized in cardiomyocytes on exposure to hypertrophic stimuli. But, in this instance, the stabilization of β-catenin was independent of Wnt signaling though inhibition of GSK3β remained central to hypertrophic stimulus-induced stabilization of β-catenin.

Wnt signaling leads to stabilization of β-catenin [154] and inappropriate activation of Wnt signaling has been described in many tumors. Transcriptional targets directly activated by β-catenin include: cyclin D1, c-myc, matrilysin, PPAR-δ, and upregulation of COX-2 [155–159]. Wnt expressing mammary epithelial cells and under conditions of nuclear β-catenin accumulation showed transcriptional upregulation of COX-2 [160, 161], and there is evidence to suggest that β-catenin causes upregulation of COX-2, whereas EPA suppresses COX-2 and catenin expression [138, 139, 214, 215] and also functions as an endogenous ligand of PPARs [162]. But it is not known whether PUFAs can directly influence the expression of Wnt. In this context, it is noteworthy that Wnt pathway plays a major role in cardiac myogenesis, myocardial hypertrophy, and heart failure, possibly, by inhibiting GSK-3β activity [163, 164], which leads to stabilization of β-catenin complex. This leads to β-catenin translocation to the nucleus where it participates in the transcription processes. In obesity, there is an overexpression of SRP4, an endogenous antagonist of Wnt protein and a repressor of Wnt receptors, FDZ6 and FDZ4, and also of Dsh3, a direct inhibitor of GSK-3β activity. These changes favor β-catenin ubiquitination and degradation in proteasomes and direct repression of several factors that favor a role in cardiac hypertrophy such as c-myc, GATA4, and MEF2B [165]. This regulation of the Wnt/β-catenin pathway noted in the obese heart has the potential to prevent the development of cardiac hypertrophy since the volume overload observed in obesity-related hypertension decreases the expression of β-catenin and connexin 43, whereas hearts from hypertensive patients showed decreased GSK-3β activity, nuclear accumulation of β-catenin that could lead to myocardial hypertrophy [166]. Thus, Wnt/β-catenin/GSK3β and hedgehog signaling pathway is not only involved in the growth and development of brain but also in cardiac hypertrophy. Since PUFAs have a negative feedback control on catenin expression and TNF-α synthesis [41–43, 138, 139] and TNF-α also induced a significant decrease of E-cadherin and β-catenin expression [140], it implies that in an indirect fashion PUFAs play a regulatory role in the expression and action of Wnt/β-catenin/GSK3β and hedgehog signaling pathway and thus, in brain growth and development (Fig. 16.2).

Fig. 16.2
figure 2

Scheme showing possible relationship among various molecules/molecular events and PUFAs, brain growth and development, neuropeptides, inflammation and diseases/disorders. For further details see text

Full size image

PUFAs Modulate NMDA, γ-Aminobutyric Acid (GABA), Serotonin and Dopamine in the Brain

Since PUFAs play a significant role in the growth and development of brain, it is possible that they (PUFAs) also regulate the fetal brain nerve growth cone membranes and monoaminergic neurotransmitters. This is especially so since, it is known that AA, DHA and other PUFAs but not saturated and monounsaturated fatty acids activate syntaxin 3, a plasma membrane protein that has an important role in the growth of neurites [95]. Further, syntaxin1 that is involved in fast calcium-triggered exocytosis of neurotransmitters is modulated by AA [96], implying that AA is involved both in exocytosis of neurotransmitters and neurite outgrowth. SNAP25 (synaptosomal-associated protein of 25 kDa), a syntaxin partner implicated in neurite outgrowth, interacted with syntaxin 3 only in the presence of AA, DHA, LA, and ALA, whereas saturated and monounsaturated fatty acids were ineffective, to form the ternary SNARE complex (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), which is needed for the fusion of plasmalemmal precursor vesicles into the cell surface membrane that leads to membrane fusion, an event that facilitates neurite outgrowth.

Rats fed purified diets containing safflower oil, a rich source of LA, soybean oil as a source of LA and ALA, and high fish oil, rich in DHA, through gestation showed that offspring of rats fed fish oil had significantly higher DHA in their brain nerve growth cone membrane phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) than the soybean oil group. The growth cone membrane phosphatidylcholine (PC), PE and PS AA was significantly lower in the fish oil than in the soybean or safflower oil groups. Serotonin concentration was significantly higher in brain of offspring in the safflower oil compared with the soybean oil group. The newborn brain dopamine was inversely related to PE DHA and PS DHA, but positively related to PC AA. These results suggest that maternal dietary fatty acids alter fetal brain growth cone fatty acid content and neurotransmitters involved in neurite extension, target finding and synaptogenesis [167].

In a study that investigated the effect of feeding formula from birth to 18 days with different PUFAs on the concentrations of monoaminergic neurotransmitters in various regions of the brain, it was observed that animals that received LA + ALA in formula had a significant effect on frontal cortex dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindolacetic acid; striatum serotonin and inferior colliculus serotonin, resulting in lower concentrations in piglets fed the low compared with adequate LA + ALA formula. Inclusion of AA and DHA in the low, but not in the adequate LA + ALA formula, resulted in increased concentrations of all monoamines in the frontal cortex, and in striatum and inferior colliculus serotonin, increased dopamine and 5-hydroxyindolacetic acid in superior and inferior colliculus, areas related to processing and integration of visual and auditory information. Higher dopamine and 5-hydroxyindolacetic acid were found in superior and inferior colliculus regions even when AA and DHA were added to the LA + ALA adequate formula [168]. Thus, it can be said that functional changes among animals and infants fed diets varying in ω-6 and ω-3 fatty acids could involve altered neurotransmitter metabolism that may explain the improvements in visual, auditory, and learning tasks reported for infants and animals given diets rich in ω-3 fatty acids [169–173]. In addition, piglets fed diets deficient in LA and ALA from birth to 18 days not only had lower amounts of AA in frontal cortex PC and PI and lower DHA in PC and PE but also had significantly lower frontal cortex dopamine, 3,4-dihydroxyphenylacetic (DOPAC), homovanillic acid (HVA), serotonin and 5-hydroxyindoleacetic acid (5-HIAA) concentrations. These indices were restored to normal or were even higher in piglets that received AA and DHA suggesting that dietary PUFAs fed for 18 days from birth affects frontal cortex neurotransmitters in rapidly growing piglets and that these changes are specifically due to AA and/or DHA [174]. These results coupled with the observation that both AA and DHA influence the expression of dopamine receptor genes and their products [175], modify monoaminergic neurotransmitters in frontal cortex and hippocampus [176, 177], and facilitate release and actions of GABA [178–181] and acetylcholine [182–185] lends support to the concept that PUFAs have a modulatory influence on the release, action and properties of various neurotransmitters in the brain. Exogenously added AA (20–160 μM) stimulated dopamine uptake when pre-incubated for short times (15–30 min); whereas at 160 μM AA inhibited following longer pre-exposures (45–60 min) in glioma cells [186]; markedly stimulated, in a dose-dependent manner, the spontaneous release of dopamine, inhibited in a dose-dependent manner dopamine uptake into synaptosomes, but still stimulated dopamine spontaneous release in the presence of dopamine uptake inhibitors in purified synaptosomes from the rat striatum indicating that AA both inhibits dopamine reuptake and facilitates its release process [187].

In Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced AA release that has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In contrast, CHO cells expressing D1 receptors, D1 agonists exerted no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of AA release. In view of the numerous actions of AA and its metabolites in neuronal signal transduction, these results suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism [188]. In this context, it is interesting to note that in obesity, a decrease in the number of dopamine receptors or dopamine concentrations occurs [32] and obesity is common in type 2 diabetes. Both in obesity and type 2 diabetes mellitus, plasma concentrations of PUFAs especially AA, EPA, and DHA are decreased [189–193]. Numerous studies showed an association between poor fetal growth and adult insulin resistance and increased incidence of type 2 diabetes mellitus and metabolic syndrome. Early growth retardation, as a result of maternal protein restriction, could lead to alterations in desaturase activities similar to those observed in human insulin resistance. This is supported by the observation that in both muscle and liver the ratio of DHA to docosapentaenoic acid (DPA) was reduced in low protein offspring. Δ5 desaturase activity in hepatic microsomes was reduced in the low protein offspring that was negatively correlated (r = −0.855) with fasting plasma insulin. No such correlation was observed in controls. These results suggest that it is possible to programme the activity of key enzymes involved in the desaturation of PUFAs by perinatal factors such as maternal protein intake [194]. Since, the PUFA composition of skeletal muscle membranes and insulin sensitivity are closely related [189–193] it is suggested that maternal protein restriction decreases Δ5 desaturase activity such that fetal tissue content of PUFAs are decreased (including muscle) that, in turn, programmes the development of insulin resistance and metabolic syndrome during their adult life, a mechanism linking fetal growth retardation to insulin resistance. Maternal factors (such as maternal protein restriction) could also influence PUFA content in the brain. Since PUFAs such as AA and DHA have profound influence on the secretion and actions of various neurotransmitters, it is reasonable to propose that alterations in the concentrations of various LCPUFAs in the brain (especially in the hypothalamus) during the perinatal period could lead to changes in the levels and actions of dopamine, serotonin, acetylcholine and other neurotransmitters that, in turn, lead to the development of insulin resistance and metabolic syndrome in adult life. This is so, since VMH-lesioned rats that develop all features of type 2 DM showed selectively decreased concentrations of norepinephrine and dopamine in the hypothalamus, long-term infusion of norepinephrine plus serotonin into the VMH impairs pancreatic islet function in as much as VMH norepinephrine and serotonin levels are elevated in hyperinsulinemic and insulin-resistant animals [195–197], suggesting that dysfunction of VMH, impaired pancreatic β cell function, insulin resistance, tissue concentrations of PUFAs, alterations in the actions and levels of various neurotransmitters, and the development of metabolic syndrome are closely related to each other (see Fig. 16.2). It is not only that perturbations in the concentrations of PUFAs in the brain as a result of maternal protein restriction induce changes in the concentrations and actions of various neurotransmitters serotonin, dopamine, acetylcholine, and food intake regulating peptides such as NPY, AgRP (agouti related peptide), POMC (pro-opiomelanocortin) and the number of their receptors and insulin action in the brain (as discussed above), neurotransmitters are also known to influence the metabolism and actions of PUFAs. For instance, it was reported that in the intact rat brain, D2 but not D1 receptors are coupled to the activation of PLA2 and the release of AA [198]. This suggests that there is both positive and negative feedback control between PUFAs and various neurotransmitters and their actions. In a similar fashion, various perinatal and maternal factors including PUFAs may regulate the expression, release and function of various other neurotransmitters and hypothalamic peptides such as leptin, NPY, AgRP and melanocortins. Such an interaction between PUFAs and hypothalamic peptides and neurotransmitters may program the hypothalamic bodyweight/appetite/satiety set point that could influence the development of obesity, metabolic syndrome, type 2 diabetes mellitus and hypertension in adult life. Such an influence of PUFAs in brain growth and development may also set the tone for the development of various neurological conditions such as schizophrenia, depression and Alzheimer’s disease.

Such a concept may explain the relationship between perinatal and in utero nutrition and its long-term effects into adulthood. The excitatory and inhibitory inputs/outputs onto the NPY/AgRP and POMC/CART neurons reported [199–205] also suggests that leptin affects not only the transcription and release of neuropeptides but also the functional activity of neurotransmitters such as GABA (inhibitory) and glutamine (excitatory) that are ultimately the mediators of the metabolic signals of leptin, ghrelin, and other neuropeptides. If this concept is true, it suggests that maternal diet could influence EFA metabolism and leptin expression and action in the fetus and the newborn [205].

Maternal Diet Influences EFA Metabolism and Leptin Levels

Low birth weight is associated with high prevalence of metabolic syndrome in later life [206, 207]. Babies with low birth weights have 10 times greater chance of developing metabolic syndrome compared to those whose birth weight were normal. In addition, postnatal nutrition and growth also play a role in the development of metabolic syndrome in later life [208]. Though, the exact cause for this is not known, at least, in part, this could be attributed to the maternal and perinatal factors especially their diet. Maternal protein restriction or increased consumption of saturated and/or trans-fatty acids and energy rich diets (maternal over-nutrition) during pregnancy decrease the activity of Δ6 and Δ5 desaturase enzymes that are essential for the metabolism of dietary essential fatty acids LA and ALA and the formation of their long-chain metabolites such as AA, EPA and DHA. Perinatal protein depletion leads to almost complete absence of activities of Δ6 and Δ5 desaturases in fetal liver and placenta [209–212]. Thus, both protein deficiency and high-energy diet decreases the activities of Δ6 and Δ5 desaturases that, in turn, leads to maternal and fetal deficiency of EPA, DHA and AA.

Dietary quantity and quality has been shown to affect serum leptin levels [213–215]. A diet rich in PUFAs increases leptin levels in diet-induced obese adult rats [213], suggesting that variation in the type of diet during pregnancy and lactation significantly modulate fetal and neonatal growth and development by leptin-associated mechanisms since leptin influences NPY/AgRP and POMC/CART neurons and their connections [199–204]. Plasma leptin levels were found to be low in the lactating dams fed the EFA-deficient diet and their suckling pups compared with controls [216]. The suckling pups showed decreased concentrations of leptin even in their adipose tissue [217], suggesting that maternal EFA deficiency can produce a decrease in leptin levels in several tissues, possibly, even in the hypothalamus. These low leptin levels during the perinatal period alters NPY/AgRP and POMC/CART homeostasis [199–204] that may lead to the hypothalamic “body weight/ appetite/ satiety set point” set at a higher level that is long-lasting and potentially irreversible onto adulthood. Thus, maternal malnutrition, low perinatal PUFAs and consequent low leptin concentrations could lead to the development of metabolic syndrome in adulthood.

EPA, DHA, and AA inhibit TNF-α and IL-6 synthesis. Hence, PUFAs deficiency due to maternal malnutrition increases the generation of TNF-α and IL-6 both in the maternal and fetal tissues that, in turn, induces insulin resistance. Prenatal exposure to TNF-α produces obesity [218], and obese children and adults have high levels of TNF-α and IL-6 [219, 220]. Low plasma and tissue concentrations of EPA, DHA, and AA also decrease adiponectin levels that further aggravate insulin resistance. TNF-α and IL-6 increase the activity of 11β-HSD-1 that causes abdominal obesity, a characteristic feature of metabolic syndrome [221]. Since a close positive and negative feedback regulation between perilipins, TNF-α, adipocyte size, PPAR-γ, exercise and insulin resistance exists, low plasma and tissue concentrations of PUFAs and leptin due to maternal malnutrition will also explain abnormalities of perilipins and IMCL seen in obese subjects who are prone to develop metabolic syndrome.

In addition, AA and DHA enhance cerebral ACh levels and improve learning ability in rats [29, 30] and ACh modulates long-term potentiation and synaptic plasticity in neuronal circuits and interacts with dopamine receptor in the hippocampus [31]. ACh also inhibits synthesis and release of TNF-α and thus, has anti-inflammatory actions [54] and is a potent stimulator of eNO synthesis [55].

Perinatal PUFA Deficiency May Initiate Low-Grade Systemic Inflammation and Adult Diseases

It is evident from the preceding discussion that a deficiency of PUFAs during the critical period of brain growth and development and somatic growth leads to a deficiency of leptin, ACh, and an imbalance in the NPY/AgRP and POMC/CART homeostasis, changes in the concentrations of dopamine, serotonin, GABA and other neuropeptides, and an increase in the levels of TNF-α, an inflammatory cytokine that has neurotoxic actions. All these adverse events as a result of perinatal PUFA inadequacy, could lead to the initiation of low-grade systemic inflammation (due to enhanced TNF-α production) and neuronal damage may predispose to the development of various neurological conditions such as Alzheimer’s disease, schizophrenia and depression and obesity, hypertension, osteoporosis and type 2 diabetes mellitus later in life. Thus, various adult diseases may have their origins in perinatal period. These evidences imply that metabolic syndrome, Alzheimer’s disease, depression schizophrenia, hypertension, type 2 diabetes mellitus and obesity could be due to perinatal deficiency of EPA, DHA and AA and their metabolites such as lipoxins, resolvins, protectins and nitrolipids (Figs. 16.1 and 16.2). Thus, it is proposed that adult diseases enumerated above have their origins in the perinatal period [4, 5]. This also implies that the low-grade systemic inflammation starts in the perinatal period of life itself and that these diseases/disorders are disorders of the brain as discussed in previous chapters. In view of this, PUFAs and their metabolites play a significant role in all these diseases as already discussed in previous chapters.

In this context, the significance of breast-feeding lies in the fact that human breast milk is rich in AA, EPA, DHA, GLA, DGLA, LA and AA. It is likely that when the child is adequately breast fed, the tissue and plasma concentrations of various PUFAs will be optimal that leads to formation of optimal amounts of lipoxins, resolvins, protectins, maresins and nitrolipids so that (a) inflammatory processes are under control; (b) brain growth and development is adequate; (c) neuronal synaptic connections are perfect; (d) neurotransmitters are produced in adequate amounts and at the right time and right place; and (e) various tissues and organs are able to meet the endogenous and external challenges in a favorable fashion so that tissue damage is minimal and the repair process and wound healing is normal and restoration of target organs to normal is easily reestablished. This implies that supplementation of various PUFAs and their anti-inflammatory products and other endogenous molecules involved in the restoration of homeostasis are provided in optimal amounts when homeostatic mechanisms are disturbed, so that health can be restored.