Micronutrient deficits and their effects

Micronutrients, and in particular zinc and selenium, act as key regulators of metabolic and immune functions^{(Reference Elmadfa and Meyer31,Reference Kieliszek32)} . Zinc deficiency in human subjects is now known to be an important malnutrition problem worldwide. It is more prevalent in areas of high cereal and low animal food consumption not because the diet could be low in zinc and selenium but because phytic acid is the main known inhibitor of zinc absorption^{(Reference Oberleas and Harland33)}, while selenocysteine, the organic form of selenium most easily absorbed by human subjects, dominates in products of animal origin^{(Reference Kieliszek32)}. Compared to adults, infants, children, adolescents, pregnant and lactating women have increased requirements for zinc and selenium and are at increased risk of deficiencies. Zinc deficiency results in growth failure, while epidermal, gastrointestinal, central nervous, immune, skeletal and reproductive systems are the organs clinically most affected by zinc and selenium deficiencies^{(Reference Xu, Hao and Li34,Reference Roohani, Hurrell and Kelishadi35)} .

Hence, in a context characterised by significant maternal malnutrition, micronutrient deficiency during the periconceptional period, and in particular zinc and selenium deficiencies, is likely to result in widespread, low level but persistent maternal as well as foetal metabolic dysregulations with deleterious consequences upon placentation and embryogenesis, negatively affecting foetal development as a whole^{(Reference Cetin, Berti and Calabrese36)}. Indeed, an adequate supply of these trace elements is essential for healthy foetoplacental development. For instance, zinc plays major functional roles in zinc-dependent enzymes, zinc-binding factors and zinc transporters required in a variety of complex mechanisms during cell replication, maturation and adhesion, such as DNA and RNA metabolism, signal recognition and transduction, gene expression and hormone regulation^{(Reference Donangelo and King37)}. Among the proteins encoded in the human genome which require zinc for their physiological function are 397 hydrolases, followed by 302 ligases, 167 transferases, 43 oxidoreductases and 24 lyases/isomerases. Proteinases include carboxypeptidases, aminopeptidases, matrix metalloproteinases and peptide hormone processing enzymes/convertases, indicating a wide role of zinc in proteostasis. Hydrolases also include zinc-dependent phosphodiesterases, phospholipases, alkaline and acid phosphatases and pyrophosphatases with roles in regulating second messenger metabolism and signal transduction pathways. Zinc is used in DNA and protein (histone) modification in demethylases and deacetylases, in DNA and RNA metabolism and in DNA repair enzymes. Another significant group of zinc enzymes is involved in regulation: transferases such as geranyl and farnesyl transferase, palmitoyl transferases, ligases such as E3 ubiquitin-protein ligases, SUMO conjugating enzymes and the corresponding hydrolases^{(Reference Maret38,Reference Andreini, Banci and Bertini39)} .

In parallel, selenium is vital for efficient antioxidant defence in both mother and foetus. Weak placental antioxidant defence due to low maternal plasma selenium concentration increases the risk of small for gestational age infants^{(Reference Iqbal, Ali and Rust40)}. Many of the beneficial effects of selenium are attributable to its presence as selenocysteine in the selenoproteins, a small but vital group of proteins^{(Reference Pappas, Zoidis and Surai41,Reference Roman, Jitaru and Barbante42)} . Selenoproteins W and N (SELENOW and SELENON) are both required for muscle growth, differentiation and regeneration, as well as satellite cell maintenance in skeletal muscle^{(Reference Castets, Bertrand and Beuvin43–Reference Brown and Arthur46)}. Selenoprotein S (SELENOS) is involved in the degradation process of misfolded endoplasmic reticulum (ER) luminal proteins^{(Reference Williams and Dhoot47)}, while selenoprotein T (SELENOT) is involved in the control of glucose tolerance by contributing to prolonged adenylate cyclase-activating polypeptide 1 (ADCYAP1/PACAP)-induced insulin secretion^{(Reference Prevost, Arabo and Jian48)} while also contributing to increased quantitative insulin sensitivity^{(Reference Tabrizi, Akbari and Moosazadeh49)}. Growth retardation, poor appetite and mental lethargy are some of the manifestations of chronically zinc-deficient human subjects^{(Reference Prasad50)}. Furthermore, low serum zinc has been reported as a major predictor of anaemia mediating the effects of low selenium upon oxidative stress-dependent haemoglobin denaturation and erythrocytes osmotic fragility^{(Reference Houghton, Parnell and Thomson51)}, whereas vitamin B12 and folate deficiencies were found not to be associated with anaemia^{(Reference Wirth, Woodruff and Engle-Stone52)}. Table 2 lists the selected key enzymes that are either zinc-/selenium-dependent or the activities of which are controlled by zinc/selenium and are of prime relevance in the context of maternal malnutrition.

Table 2. List of the key enzymes that are either zinc-/selenium-dependent or the activities of which are controlled by zinc/selenium and are of prime relevance in the context of maternal malnutrition

In selenium deficiency, there is a strict hierarchy of selenium supply to specific tissues and also to different selenoenzymes within a tissue. Concentrations of selenium and selenoenzymes are greatly decreased in liver, kidney and muscle, whereas those in the brain and endocrine organs such as the thyroid gland are less affected. Within different organs, specific selenoproteins are retained at the expense of others, presumably to preserve the most important aspects of metabolism in selenium deficiency. For example, in the selenium-deficient rat, DIO1 is better retained than cytoplasmic glutathione peroxidase in thyroid, liver and kidney, presumably in order to preserve thyroid function and iodothyronine de-iodination and to limit changes in plasma T4, T3 and TSH^{(Reference Arthur and Beckett76)}. Many of the beneficial effects of selenium are attributable to its presence as selenocysteine in the selenoproteins, a small but vital group of proteins^{(Reference Pappas, Zoidis and Surai41,Reference Roman, Jitaru and Barbante42)} . Selenoproteins W and N (SELENOW and SELENON) are both required for muscle growth, differentiation and regeneration, as well as satellite cell maintenance in skeletal muscle^{(Reference Castets, Bertrand and Beuvin43–Reference Brown and Arthur46)}. Selenoprotein S (SELENOS) is involved in the degradation process of misfolded endoplasmic reticulum luminal proteins^{(Reference Williams and Dhoot47)}, while selenoprotein T (SELENOT) is involved in the control of glucose tolerance by contributing to prolonged adenylate cyclase-activating polypeptide 1 (ADCYAP1/PACAP)-induced insulin secretion^{(Reference Prevost, Arabo and Jian48)} while also contributing to increased quantitative insulin sensitivity^{(Reference Tabrizi, Akbari and Moosazadeh49)}.

Growth retardation, poor appetite and mental lethargy are some of the manifestations of chronically zinc-deficient human subjects^{(Reference Prasad50)}. Furthermore, low serum zinc has been reported as a major predictor of anaemia mediating the effects of low selenium upon oxidative stress-dependent haemoglobin denaturation and erythrocytes osmotic fragility^{(Reference Houghton, Parnell and Thomson51)}, whereas vitamin B12 and folate deficiencies were found not to be associated with anaemia^{(Reference Wirth, Woodruff and Engle-Stone52)}. The foods with the highest zinc contents include meat, shellfish, eggs, nuts and seeds such as hemp, flax, pumpkin or squash. Legumes, such as chickpeas, lentils and beans, all contain substantial amounts of zinc, while whole grains like wheat, quinoa, rice and oats contain some zinc (USDA Food Composition Databases). The foods with the highest selenium content include Brazil nuts, fish, meat, dairy products, eggs and bananas (USDA Food Composition Databases). However, low-quality and less varied plant-based diets in low-income communities have a high content of phytic acid [myo-inositol hexaphosphate (InsP6)] and associated magnesium, potassium and calcium salts, termed ‘phytate’, which together constitute potent inhibitors of iron and zinc absorption and bioavailability^{(Reference Gupta, Gangoliya and Singh79–Reference Gibson, Raboy and King81)}.

Hence, the origin of high to severe anaemia in Indian women and children stands to be a direct consequence of malnutrition compounded by low micronutrient intake, and in particular low availability of zinc and selenium resulting in suboptimal haemoglobin synthesis associated with oxidative stress-dependent haemoglobin denaturation and osmotic fragility of erythrocytes (see earlier). Furthermore, maternal micronutrient deficiency in association with chronic malnutrition will most probably provoke significant maternal metabolic skewing.

Maternal metabolic skewing and its consequences

Transmethylation cycle (one-carbon metabolism) alterations

Maternal dietary methyl donor intake (methionine, folate and choline) and cofactor (zinc and vitamins B2, B6 and B12) play crucial roles in one-carbon metabolism and DNA methylation in the foetus and placenta, impacting foetal growth and lifelong health outcomes^{(Reference McGee, Bainbridge and Fontaine-Bisson82)}. However, in a context characterised by significant maternal malnutrition, not only such dietary intakes will be highly restricted, but the deficiencies in cofactors intake apparently promote down-regulation of the THF-transmethylation cycle. The resulting elevation in Hcy and 5mTHF concurrently with low GSH circulating levels observed in the populations studied probably does not arise from low vitamin B12 availability but from attenuation of zinc-dependent methionine synthase and betaine–homocysteine S-methyltransferase enzymatic activity. Here, high Hyc circulating levels lead to GSH depletion through uncoupling of the activities of NOX (NADPH-dependent) from those of XO [S-adenosylhomocysteine (SAH)-dependent] and NOS (BH4-dependent), both of which are negatively affected by dysregulation of the transmethylation pathway^{(Reference Topal, Brunet and Millanvoye83)}. This dysregulation, resulting in S-adenosyl methionine (SAM) deficiency, besides reducing methylation potential, also has an effect upon choline biosynthesis from phosphatidylethanolamine (PE), each step of which requires methyl groups donated by SAM^{(Reference Obeid, Awwad and Knell84)} (Fig. 1). In pregnancy, increased maternal Hcy levels are associated with increased risk of adverse pregnancy outcomes such as intrauterine growth restriction leading to small size for gestational age at birth and LBW^{(Reference Azzini, Ruggeri and Polito85)}. Dietary protein restriction in animals and marginal protein intake in human subjects cause characteristic changes in one-carbon metabolism that are further exacerbated by micronutrient deficiency, negatively impacting the health of the mother, impairing growth and reprogramming metabolism of the foetus, and causing long-term morbidity in the offspring^{(Reference McGee, Bainbridge and Fontaine-Bisson82,Reference Kalhan86)} .

Fig. 1. Schematic representation of the interconnected transmethylation cycle/transsulphuration pathway, a critical branching point in metabolism connecting sulphur metabolism, cofactor chemistries, biological methylations and redox homoeostasis. In these interconnected pathways, methionine synthase (MS) and betaine–homocysteine S-methyltransferase (BHMT) are zinc-dependent enzymes. In parallel, methionine adenosyltransferase (MAT), N-glycine methyltransferase (GNMT), SAH hydrolase, cystathionine β-synthetase (CBS), γ-glutamylcysteine synthetase (glutamate-cysteine ligase, GCL), methylenetetrahydrofolate reductase (MTHFR) and serine hydroxymethyltransferase (SHMT) are all regulated by zinc-dependent transcription factors. Additional zinc enzymes are histone and DNA methyltransferases and adenosine deaminase^{(Reference Maret38,Reference Andreini, Banci and Bertini39)} . DHFR, dihydrofolate reductase; THF, tetrahydrofolate; MTHF, methylenetetrahydrofolate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; SAM, S-adenosyl methionine; SAH, S-adenosylHcy; CSE, cystathionase; GSH, glutathione; H2S, hydrogen sulphide. Figure adapted from Azzini et al. ^{(Reference Azzini, Ruggeri and Polito85)}.

Amino acid metabolism alterations

Four amino acids (aspartic acid, serine, threonine and histidine) show elevated serum levels in malnourished pregnant women, and in particular aspartic acid is extremely elevated (see Table 1). Under protein deficiency, the NEAA aspartate becomes a significant metabolic hub, a major product of the oxidative glutaminolysis pathway and a required substrate for other anabolic pathways, including the synthesis of purines and pyrimidines^{(Reference Vettore, Westbrook and Tennant87)}. Besides playing a key role in the urea cycle as well as in pyrimidine synthesis, aspartate carries reducing equivalents in the malate–aspartate shuttle, which utilises the ready interconversion of aspartate and oxaloacetate in order to maintain mitochondrial oxidative phosphorylation (Fig. 2). Hence, extremely elevated serum aspartate levels could be a consequence of high biosynthesis and interconversion rates. Aspartate can be synthesised by the transamination of oxaloacetate using either alanine or glutamine, yielding aspartate and an α-keto acid^{(Reference Levin88–Reference Nielsen, Stottrup and Lofgren90)}. Interestingly, in malnourished pregnant women alanine, and even more so glutamine, show low circulating levels, possibly comforting hypothetically increased aspartate de novo synthesis and interconversion levels.

Fig. 2. Interplay between amino acid metabolism and redox homoeostasis. The synthesis and catabolism of amino acids is interwoven into redox homoeostasis. The malate–aspartate shuttle, besides transporting NADH between the cytosol and the mitochondrial matrix, also moves the amino acids glutamate and aspartate between the two compartments and is functionally connected to the TCA cycle. When aspartate is removed from this cycle to synthesise asparagine, arginine or nucleosides, this would disrupt the cycle, requiring additional carbon input. Coupling oxaloacetate (OAA) production in oxidative TCA cycle activity with glutamate production by mitochondrial glutaminase maintains flux of α-ketoglutarate into the TCA cycle while removing OAA to permit continued activity, producing reducing potential to generate ATP. In this way, two metabolites central to homoeostasis, ATP and aspartate, are synthesised in parallel. These links go further, given that aspartate, glutamate, α-ketoglutarate and malate are functionally coupled through the malate–aspartate shuttle, which is important for moving reducing potential between the matrix and the cytosol. NADH oxidation reactions and NAD+ reduction reactions, which affect the connectivity of this network, are shown in green and red, respectively. Amino acids are represented in blue, while proteins (transporters and electron carriers) are in orange. αKG, α-ketoglutarate; 1,3 BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 3PP, 3-phosphopyruvate; AcCoA, acetyl CoA; Ala, alanine; Asn, asparagine; Asp, aspartate; Cit, citrate; G3P, glyceraldehyde 3-phosphate; Gln, glutamine; Glu, glutamate; Gly, glycine; Lac, lactate; Mal, malate; NAD+, nicotinamide adenine dinucleotide; NADH, reduced NAD+; OAA, oxaloacetate; P5C, pyrroline 5-carboxylate; Pro, proline; Pyr, pyruvate; Ser, serine. Figure adapted from Vettore et al. ^{(Reference Vettore, Westbrook and Tennant87)}.

However, such mechanisms would be at the expense of amino acid supply and cannot be sustained indefinitely. Furthermore, glutamate dehydrogenase, which, in this scheme, catalyses the oxidative deamination of glutamate to α-ketoglutarate and ammonia, is zinc-dependent and its activity would be down-regulated by zinc deficiency. This could be alleviated by increased FA oxidative catabolism, provided that sufficient L-carnitine is available. Since diets low in meat and dairy products lead to low L-carnitine uptake, this could be compensated by de novo L-carnitine biosynthesis, which takes place mainly in skeletal muscle, kidney and liver, using L-lysine, an EAA, as primary substrate^{(Reference Bjorndal, Brattelid and Strand91)} (Fig. 3). In malnourished pregnant women, serum lysine and methionine levels are lower than those of most other EAAs, indicative of active de novo L-carnitine biosynthesis. However, this methylation-dependent mechanism implicates SAM as methyl group donor^{(Reference Paik and Kim92)}, hence placing further demands on the transmethylation cycle.

Fig. 3. De novo L-carnitine biosynthesis. (A) Carnitine is synthesised from the amino acids’ lysine and methionine. Lysine provides the carbon backbone of carnitine, and the 4-N-methyl groups originate from methionine via the transmethylation pathway (see earlier). (B) In mammals, certain proteins such as calmodulin, myosin, actin, cytochrome c and histones contain N'-trimethyl-lysine (TML) residues. N-methylation of these lysine residues occurs as a post-translational event. This reaction is catalysed by specific methyltransferases, which use S-adenosyl methionine as a methyl donor. Lysosomal hydrolysis of these proteins results in the release of TML, which is the first metabolite of carnitine biosynthesis. TML is first hydroxylated on the three-position by TML dioxygenase (TMLD) to yield 3-hydroxy TML (HTML). Aldolytic cleavage of HTML yields 4-trimethylaminobutyraldehyde (TMABA) and glycine, a reaction catalysed by HTML aldolase (HTMLA). Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH) results in the formation of 4-Ntrimethylaminobutyrate (butyrobetaine). In the last step, butyrobetaine is hydroxylated on the three-position by γ-butyrobetaine dioxygenase (BBD) to yield carnitine. Figure adapted from Vaz and Wanders^{(Reference Vaz and Wanders93)}.

In malnourished pregnant women, serum serine levels also are elevated, while glycine levels are not, suggesting down-modulated activity in serine hydroxymethyltransferase-mediated interconversion to glycine (synthesis of 5,10-methylene tetrahydrofolate from tetrahydrofolate) for the cytoplasmic synthesis of thymidylate, purines and methionine regeneration^{(Reference Zheng, Gupta and Patterson-Fortin94–Reference Giardina, Brunotti and Fiascarelli96)}. Furthermore, high circulating levels of serine might also be indicative of down-regulated PE biosynthesis^{(Reference Pacana, Cazanave and Verdianelli97)} which, together with SAM deficiency, could lead to low phosphatidylcholine synthesis. This may take particular importance in a context where the availability of choline-rich food items such as fish, crustaceans, meat and eggs are highly limited, since, in pregnancy, choline deficiency could worsen placental dysfunctions while promoting foetal slow growth as gestation progresses.

In human subjects, choline and phosphatidylcholine are synthesised de novo via the PE N-methyltransferase (PEMT) pathway^{(Reference Zeisel98)} but biosynthesis is not enough to meet physiological requirements^{(Reference Zeisel and da Costa99)}. In the hepatic PEMT pathway, 3-phosphoglycerate (3PG) receives two acyl groups from acyl-CoA forming a phosphatidic acid. It reacts with cytidine triphosphate to form cytidine diphosphate-diacylglycerol. Its hydroxyl group reacts with serine to form phosphatidylserine which decarboxylates to ethanolamine and PE forms. A PEMT enzyme moves three methyl groups from three SAM donors to the ethanolamine group of the PE to form choline in the form of a phosphatidylcholine. Three SAHs are formed as a by-product^{(Reference Zeisel and da Costa99)}. Most of the physiological requirements for phosphatidylcholine are met by channelling dietary choline through the CDP pathway which utilises adenosine triphosphate (ATP), cytidine triphosphate (CTP) and diacylglycerol to generate phosphatidylcholine (Fig. 4).

Fig. 4. Intersection between pathways of choline and methionine metabolism in the transmethylation cycle and key enzymes that control methyl group transfer: methionine adenosyltransferase 1A (MAT1A), betaine–homocysteine S-methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), methylenetetrahydrofolate reductase (MTHFR), microsomal TG transfer protein (MTTP), S-adenosylhomocysteine hydrolase (SAHH), glycine-N-methyltransferase (GNMT), guanidinoacetate N-methyltransferase (GAMT), phosphatidylethanolamine N-methyltransferase (PEMT), vitamin B12 (B12), dimethylglycine (DMG), glutathione (GSH), homocysteine (HCY), S-adenosyl methionine (SAM), S-adenosylhomocysteine (SAH), tetrahydrofolate (THF), phosphatidylethanolamine (PE), phosphatidylcholine (PC), very low-density lipoprotein (VLDL). Figure adapted from Chandler and White^{(Reference Chandler and White100)}.

In a context dominated by maternal malnutrition, low protein diet will lead to inhibition of mammalian target of rapamycin complex 1 (mTORC1), thereby promoting autophagy as a mechanism maintaining EAA availability for protein synthesis and protective mechanisms (see later) while supplying ketogenic and glucogenic precursors, such as glutamine and alanine, for ATP generating pathways^{(Reference Panchaud, Peli-Gulli and De Virgilio101–Reference Henagan, Laeger and Navard103)}. These effects would be amplified during pregnancy, triggering the placental mammalian amino acid response pathway and thereby programming the growth capacity of offspring not only in utero but also long after gestational protein restriction^{(Reference Wang, Wilson and Zhou104,Reference Strakovsky, Zhou and Pan105)} . Elevated plasma threonine, a member of the EAA group, could reflect zinc and/or vitamin B6 deficiencies, since the initial step in threonine catabolism to Kreb's cycle precursors requires vitamin B6, the activation of which, via pyridoxal kinase, is zinc-dependent^{(Reference Navarro, Ramirez-Sarmiento and Guixe106,Reference Chiba, Terada and Kameya107)} . However, this would also suggest sufficient dietary supply in EAAs which, in the context addressed here, is highly improbable. Hence, it appears more likely that elevated plasma threonine, arising from maternal autophagy, could supply the developing foetus with an immunostimulant which promotes thymus growth while concurrently promoting maternal innate immune defence functions^{(Reference Okabe, Sano and Nagata108)}. Elevated plasma histidine, another member of the EAA group, might also play significant protective roles benefiting both the mother and the foetus, particularly in a context dominated by chronic anaemia. Histidine is essential in globin synthesis and erythropoiesis and has also been implicated in the enhancement of iron absorption from human diets. Histidine-deficient diets predispose healthy subjects to anaemia and accentuate anaemia in chronic uraemic patients^{(Reference Vera-Aviles, Vantana and Kardinasari109)}. Furthermore, histidine plays key roles in the detoxification of cytotoxic oxidative stress metabolites such as reactive carbonyls^{(Reference Song, Joo and Aldini110)}.

Roles of adipokines in the inception of the LBW ‘thin-fat’ phenotype

Adiponectin is an adipocyte-derived plasma protein with insulin-sensitizing and anti-atherosclerotic properties. There is no correlation between cord adiponectin levels and maternal body mass index, cord leptin or insulin levels, and there is no correlation between cord and maternal adiponectin levels. However, high cord blood adiponectin levels, compared with serum levels in children and adults, positively correlate with foetal birth weights. Taking fat mass-related parameters such as the birth weight/birth length ratio into consideration, plasma adiponectin concentrations exhibit a significant inverse correlation with insulin concentrations. The high adiponectin levels in newborns may be due to lack of negative feedback on adiponectin production resulting from lack of adipocyte hypertrophy, low percentage of body fat or a different distribution of fat depots in the newborns as compared to children and adults^{(Reference Sivan, Mazaki-Tovi and Pariente128–Reference Tsai, Yu and Hsu131)}. This indicates that adiponectin in cord blood is derived from foetal and not from placental or maternal tissues.

During pregnancy, leptin and adiponectin seem to act in an autocrine/paracrine fashion on the placenta and adipose tissue, playing a role in the maternal–foetal interface and contributing to glucose metabolism and foetal development^{(Reference Lecke, Morsch and Spritzer132)}. Hence, the low cord blood adiponectin levels observed in LBW births and its correlation with hyperinsulinaemia and differential distribution of fat depots giving rise to the newborn's thin-fat phenotype clearly suggest that the metabolic skewing resulting from maternal malnutrition and anaemia induce significant changes in foetal metabolism.

Cumulative effects of maternal malnutrition and placental alterations in the development of the LBW ‘thin-fat’ phenotype

Maternal nutrition, particularly micronutrients, vitamins and omega-3 FAs play a role in modulating the activity of peroxisome proliferator-activated receptors (PPARs) during placentation and angiogenesis, which affects placental and foetal growth^{(Reference Meher, Sundrani and Joshi133)}. In placental angiogenesis, PPARγ signalling causes increased vascular endothelial growth factor receptor 2 (VEGFR2) expression. VEGF binding to VEGFR2 then mediates angiogenic signalling involving increases in NOS activity^{(Reference McCarthy, Drewlo and English134,Reference Viita, Markkanen and Eriksson135)} . Hcy suppresses PPARγ signalling and expression^{(Reference Wang, Dou and Yao136)} while impeding NO production. Hence, during pregnancy, the multiple methylation network dysregulations resulting from micronutrient and vitamin deficiencies are likely to primarily result in placental vascularisation defects, while endothelial ER-stress resulting from chronically elevated Hcy levels^{(Reference Wang, Sun and Yu137)} is in turn likely to result in elevated placental leptin production. These factors, together with the low circulating levels of vitamins, micronutrients and amino acids together with high TGs, are now likely to have serious consequences upon foetal development, predisposing the newborn to insulin resistance, dyslipidaemia^{(Reference Houde, Guay and Desgagne138)} and preferential subcutaneous adipocyte patterning, whereas imbalance in the availability of amino acids and low T3 hormone production, resulting from selenium deficiency, will promote growth retardation^{(Reference van Gucht, Meima and Moran73)}.

Differential adipocyte patterning and dyslipidaemia mechanisms in the development of the LBW ‘thin-fat’ phenotype

In mammals, individual white and brown adipocyte tissues (WAT and BAT, respectively) depots appear at different times in development and have unique functional characteristics. The distinction between subcutaneous and visceral fat may be oversimplified because evidence suggests that metabolic properties vary between some visceral fat depots, while heterogeneity exists even within a single fat depot^{(Reference Lee, Wu and Fried139)}. Furthermore, metabolic and environmental challenges highlight the extraordinary plasticity of the mammalian adipose organ. Two distinct subtypes of preadipocytes have been characterised in human fat (Myf5⁺ and Myf5⁻), the proportions of which vary among depot locations^{(Reference Cinti140,Reference Giralt and Villarroya141)} . Despite the heterogeneity in the adipocyte precursor cell compartment, it appears that the Myf5⁺ lineage may selectively differentiate in the BAT, subcutaneous and retroperitoneal WAT (sWAT and rWAT, respectively), while Myf5⁻ lineages selectively give rise to most adipocytes in the inguinal and visceral WAT (ingWAT and vWAT, respectively)^{(Reference Sanchez-Gurmaches, Hung and Sparks142)}. Up-regulation of the PI3K-Akt-mTORC1 pathway (PTEN silencing) dramatically redistributes body fat such that interscapular WAT (iWAT), sWAT and rWAT (the Myf5⁺ lineage depots) expand, while the ingWAT and vWAT (the Myf5⁻ lineage depots) disappear^{(Reference Sanchez-Gurmaches, Hung and Sparks142)}. In other words, the adipocytes of Myf5⁺ lineage expand (causing lipohypertrophy of BAT, sWATs and rWAT) at the expense of Myf5⁻ lineage (i.e. inguinal and visceral WAT).

Hence, the thin-fat phenotype of LBW infants, characterised by increased subcutaneous fat but decreased intra-abdominal fat, is clearly indicative of metabolic skewing towards AKT-mediated mTORC1 up-regulation, probably as a result of elevated Hcy and insulin supply, concurrently with foetal hypoxia and oxidative stress, as a result of placental dysfunction cumulatively with significant maternal anaemia, during in utero development^{(Reference Yates, Zafar and Hubbard143)}. As a direct consequence, adipogenesis (lineage commitment, clonal expansion and terminal differentiation of preadipocytes) and lipogenesis in adipose tissue will be promoted, while β-oxidation and ketogenesis will be attenuated, leading to dyslipidaemia and insulin resistance^{(Reference Ricoult and Manning144)}, a situation which shall remain dominant after birth. Should the affected individual be then exposed to chronic malnutrition, these developmental phenomena will then have predisposing effects towards stunting/wasting and the development of cardiovascular diseases later in life.

Post-birth nutrition and stunting/wasting in ‘thin-fat’ phenotype individuals

Following weaning, LBW infants are subjected to the same restrictive dietary conditions experienced by their parents, namely malnutrition characterised by diets low in proteins, vitamins and micronutrients but rich in carbohydrates and saturated fats, leading to metabolic dysfunctions, including significant anaemia, which will be considerably worsened by increased consumption of low-quality processed products rich in saturated fats, salt and sugars and low in vitamins and micronutrients (ill-nutrition, or so-called junk food). In this context, the deficiencies in micronutrients and metabolic cofactors, and in particular selenium and vitamins, appear to play a key role. Recurrent nightly leg muscle cramps, muscle weakness and fatigue, are indicative of significant L-carnitine deficiency^{(Reference Nakanishi, Kurosaki and Tsuchiya145)} and subsequent dysregulation of FA metabolism (see earlier), non-ketotic hypoglycaemia and muscle wasting^{(Reference Engel and Angelini146)}. The latter effect stands to be further reinforced by selenium deficiency. Selenium bioavailability, which, in muscles, plays a key role in oxidative stress defence and calcium transport control, and the development of nutritional muscular dystrophies affecting cardiac and skeletal muscles have long been established both in human subjects and livestock. Skeletal muscle degeneration leads to muscle weakness or stiffness, postural instability or walking disability, while cardiac muscle degeneration is associated with respiratory distress, cardiogenic shock, enlarged heart, cardiac arrhythmias, congestive heart failure and ultimately sudden death^{(Reference Oropeza-Moe, Wisloff and Bernhoft147)}. Concurrently, selenium deficiency will also result in deficient thyroid hormone supply^{(Reference van Gucht, Meima and Moran73)} which, conjunctly with malnutrition, will promote stunting.

It is important to note that the results of malnutrition, such as deficiencies in amino acids, vitamins and micronutrients together with an oversupply of saturated fats, do not lead to the full inhibition or full activation of metabolic reactions or pathways. These deficiencies and/or oversupplies are not absolute but only relative and act as ‘rheostats’, slowing down or facilitating particular metabolic reactions or pathways. These will in turn have discrete metabolic skewing effects, the cumulative result of which will swing development in a particular direction while opening the door to predisposing effects towards context-dependent pathologies over longer period.