Eicosapentaenoic acid modulates the synergistic action of CREB1 and ID/E2A family members in the rat pup brain and mouse embryonic stem cells.

The aim of this study was to investigate the molecular mechanism by which eicosapentaenoic acid (EPA) may exert neuroprotective effects through an "EPA-cyclic AMP response element-binding protein (CREB)" signaling pathway. The current study reveals that EPA modulates the exquisite interplay of interaction of CREB1 with the inhibitor of DNA binding (ID) and E2A family members, thereby delivering mechanistic insights into specific neural differentiation program. In this scenario, our work provides evidence for the capability of CREB1 to sequester ID:E2A family members in brain tissues and neural differentiating mouse embryonic stem cells (mESCs) through formation of a [CREB1]2:ID2:E47 tetrameric complex.In essence, the molecular function of CREB1 is to dynamically regulate the location-specific assembly or disassembly of basic-helix-loop-helix (bHLH):HLH protein complexes to mediate the activation of neural/glial target genes. Together, these findings support the one-to-many binding mechanism of CREB1 and indicate that EPA treatment potentiates the integration of CREB dependent signaling with HLH/bHLH transcriptional network, adding specificity to the CREB1-mediated gene regulation during neural/glial differentiation. Our current research on the EPA-CREB axis could reveal new molecular targets for treating neurogenerative disease.

Ethyl-eicosapentaenoic acid ameliorates the clinical course of experimental allergic encephalomyelitis induced in dark agouti rats.

Eicosapentaenoic acid (EPA), a fatty acid present in high amount in fish, modulates immune response and stimulates myelin gene expression. In the present paper, we investigated the effects of EPA in an established animal model for multiple sclerosis (MS): experimental autoimmune encephalomyelitis (EAE) induced in dark agouti rats. Diets supplemented either with 0.2% or 0.4% of EPA were administrated daily from the day of induction until the end of experiment. One group of rats received diet supplemented with 0.2% of EPA 10 days before induction. The control group (immunized rats) was fed with chow diet. The animals were analyzed at two different stages of the disease: during the acute phase (14 d.p.i.) and during the recovery phase (32 d.p.i.). We showed a delayed onset of clinical severity of disease in all groups of rats fed EPA-supplemented diets. This effect was associated to an increased expression of myelin proteins and an improved integrity of the myelin sheath as well as an up-regulation of FoxP3 expression in the central nervous system during the acute phase of EAE. No significant changes in T cell subsets were noted at the periphery. On the contrary, during the recovery phase of EAE, in animals assuming EPA-supplemented diet, an increase of CD4(+)CD25(+) and CD4(+)CD25(+)FoxP3(+) in peripheral lymphocytes was noted. Our results indicate that EPA-supplemented diets may provide benefits to MS patients.

Optimized rapeseed oil enriched with healthy micronutrients: a relevant nutritional approach to prevent cardiovascular diseases. Results of the Optim'Oils randomized intervention trial.

Rapeseeds are naturally rich in cardioprotective micronutrients but refining leads to substantial losses or the production of undesirable compounds. The Optim'Oils European project proposed innovative refining conditions to produce an optimized rapeseed oil enriched in micronutrients and low in trans linolenic acid. We aimed to investigate cardioprotective properties of this Optimized oil. In a randomized, double-blind, controlled, cross-over study, 59 healthy normolipidaemic men consumed either Optimized or Standard rapeseed oils (20 g/d) and margarines (22 g/d) for 3 weeks. The Optimized oil reduced the trans FA concentration (p=0.009) and increased the contents of alpha-tocopherol (p=0.022) and coenzyme Q10 (p<0.001) in comparison with the Standard oil. Over the 3-week trial, Total-/HDL-cholesterol and LDL-/HDL-cholesterol were increased by 4% (p<0.05) with the Standard oil consumption whereas none of them rose with the Optimized rapeseed oil which increased the HDL-cholesterol and ApoA1 plasma content (+2%, NS and +3%, p<0.05 respectively). The effects observed on the plasma HDL-cholesterol levels (p=0.059), the Total-/HDL-cholesterol ratio (p=0.092), and on the ApoA1 concentrations (p=0.060) suggest an improvement of the cholesterol profile with the Optimized rapeseed oil. Finally, the Optimized oil reduced the plasma content of LDLox (-6%, NS), this effect being significantly different from the Standard oil (p=0.050). In conclusion, reasonable intake of an Optimized rapeseed oil resulting from innovative refining processes and enriched in cardioprotective micronutrients represent a relevant nutritional approach to prevent the risk of cardiovascular diseases by improving the cholesterol profile and reducing LDL oxidation.

Micronutrient-enriched rapeseed oils improve the brain oxidant/antioxidant system in rats fed a high-fat diet.

The main proposal of this study was to evaluate in vivo whether micronutrient-enriched rapeseed oils obtained using different crushing and refining procedures and characterized by different quantities and qualities of micronutrients (optimized oils) could have any beneficial effect on the antioxidant status of the brain. Sprague-Dawley rats were fed a high-fat diet for 4 weeks. The lipid source consisted of 20% optimized rapeseed oils with different quantities and qualities of micronutrients. The control group received traditional refined rapeseed oil. The experimental optimized oils decreased lipid peroxidation and increased endogenous antioxidant status in parallel with the enhancement of micronutrients. No alteration in acetylcholinesterase activity was induced by the high-fat diet in any experimental group. These results indicate that a regular intake of optimized rapeseed oils can prevent oxidative stress, providing evidence that optimized rapeseed oils could be a functional food with potentially important neuroprotective properties.

Micronutrient-enriched rapeseed oils reduce cardiovascular disease risk factors in rats fed a high-fat diet.

Many epidemiological studies have demonstrated that vegetable food consumption is associated with a reduced risk of cardiovascular diseases. The beneficial effects have been attributed to the content of bioactive molecules present in large quantities in plant food. The main proposal of this study was to evaluate in vivo whether micronutrient-enriched rapeseed oils (optimised oils) obtained using different crushing and refining procedures and characterised by different quantities and qualities of micronutrients, could have any beneficial effect on lipid profile and antioxidant status of plasma and liver. Sprague-Dawley rats were fed a high-fat diet for 4 weeks. The lipid source consisted of 20% optimised rapeseed oils with different quantities and qualities of micronutrients. The control group received traditional refined rapeseed oil. The experimental optimised oils all had a hypolipidaemic effect. In the group fed the highest levels of micronutrients, the reduction in plasma and hepatic triglycerides reached 25% and 17%, respectively, that of cholesterol 20% and 14%, respectively. In plasma, the ferric antioxidant capacity, superoxide dismutase, glutathione peroxidase and reduced glutathione significantly increased and lipid peroxidation decreased in parallel with the enhancement of micronutrients. The same trend was observed in the liver, except for glutathione peroxidase which was not affected by optimised oils. These results indicate that a regular intake of optimised rapeseed oils can help to improve lipid status and prevent oxidative stress, providing evidence that optimised oils could be a functional food with potentially important cardioprotective properties.

Sexual dimorphic evolution of metabolic programming in non-genetic non-alimentary mild metabolic syndrome model in mice depends on feed-back mechanisms integrity for pro-opiomelanocortin-derived endogenous substances.

Previously, we showed that our post-natal handling model induces pro-opiomelanocortin-derived (POMC) endogenous systems alterations in male mice at weaning. These alterations last up to adult age, and are at the basis of adult hormonal and metabolic conditions similar to mild metabolic syndrome/type-2 diabetes. Here, we evaluate how sex influences post-natal programming in these metabolic conditions. Subjects are adult control (non-handled) female (NHF) and male (NHM) CD-1 mice; adult post-natal handled female (HF) and male (HM) mice. Handling consists of daily maternal separation (10 min) plus sham injection, from birth to weaning (21 days). In adult handled males (90-days old) we find not only POMC-derived hormones alterations (enhanced basal plasma corticosterone (+91%) and ACTH (+109%)) but also overweight (+5.4%), fasting hyperglycemia (+40%), hypertriglyceridemia (+21%), enhanced brain mRNA expression of hydroxysteroid(11-beta)dehydrogenase type-1 (HSD11B1) (+49%), and decreased mRNA-HSD11B2 (-39%). Conversely, uric acid, creatinine, HDL(C), total cholesterol, glucose and insulin incremental area under-the-curve are not affected. In females, post-natal handling does not produce both hormonal and dysmetabolic diabetes-like changes; but handling enhances n3- and n6-poly-unsaturated, and decreases saturated fatty acids content in erythrocyte membrane composition in HF versus NHF. In conclusion, for the first time we show that female sex in mice exerts effective protection against the hypothalamus-pituitary-adrenal homeostasis disruption induced by our post-natal handling model on POMC cleavage products; endocrine disruption is in turn responsible for altered metabolic programming in male mice. The role of sex hormones is still to be elucidated.

PMP70 knock-down generates oxidative stress and pro-inflammatory cytokine production in C6 glial cells.

By using RNA interference (RNAi) in rat C6 glial cells, we previously generated the cell line abcd3kd in which the peroxisomal half-transporter PMP70 was stably knocked-down. The observations that abcd3kd cells had peroxisomal beta-oxidation impairment and an increase of hexacosenoic acid in cholesterol ester fraction, indicated an overlapping function of PMP70 with adrenoleukodystrophy protein (ALDP), the peroxisomal half-transporters involved in X-linked adrenoleukodystrophy (X-ALD). The objective of the present study was to investigate whether PMP70 could affect some oxidative and inflammatory parameters, since many findings indicate oxidative damage in the brain of ALD patients and inflammation is a hallmark of the cerebral forms of X-ALD. We thus measured parameters indicative of oxidative stress, the expression or activity of antioxidant enzymes, and the production of some pro-inflammatory cytokines. Our results show that, due to inducible nitric oxide synthase up-regulation, abcd3kd cell line produces higher levels of nitrites than native C6 cells. The enhanced production of superoxide and thiobarbituric acid-reactive substances, the increased expression of mitochondrial superoxide dismutase, and the reduction of catalase and glutathione peroxidase activities confirm the presence of an oxidative process. We then measured the concentrations of TNFalpha, IFNgamma, and IL-12 and we observed that abcd3kd cells produce higher amounts of pro-inflammatory cytokines compared to native C6 cells. By using neutralizing antibodies against IL-12, not only inflammatory parameters significantly decrease, but nitrite and superoxide production is also affected. This demonstrates that oxidative status of abcd3kd cells is not a direct PMP70 knock-down consequence, but depends on IL-12 release. The scenery induced by the knock-down of PMP70 in C6 cells recall the oxidative and inflammatory status observed in human X-ALD and thus reinforce the idea that PMP70 could affect the clinical course of the disease.

RNAi-mediated silencing of ABCD3 gene expression in rat C6 glial cells: a model system to study PMP70 function.

The function of PMP70, one of the four ABC half-transporters of mammalian peroxisomes, encoded by ABCD3 gene, is still unclear. The finding that PMP70 over-expression partially corrected very long-chain fatty acid oxidation defects in fibroblasts of X-linked adrenoleukodystrophy patients, has unveiled its potential clinical relevance, prompting us to set up a model system to study PMP70 function. We used the RNA interference technique, a powerful approach to loss-of-function gene expression analysis, to knockdown the ABCD3 gene in the rat glial C6 cell line, since glia could represent the target tissue of X-linked adrenoleukodystrophy disease. Cells were transfected with a vector for RNA interference generating small interfering RNAs that specifically target the ABCD3 mRNA. By using a puromycin-selectable version of the plasmid, we generated a stable cell line (abcd3kd), in which we observed a stable decrease of PMP70 protein expression greater than 70%. We thus examined the effect of ABCD3 knockdown on lignoceric and palmitic acids beta-oxidation and we found that in abcd3kd cells the rate of peroxisomal and mitochondrial beta-oxidation activities were both reduced about one-third compared with control cells. The mitochondrial membrane potential, determined by cytofluorometric analysis, was also affected. Lipid and fatty acid analyses of abcd3kd cells showed an increase of hexacosenoic acid (C26:0) in the cholesteryl-ester fraction. These results add another clue about the overlapping function of PMP70 and ALDP, the peroxisomal protein involved in X-linked adrenoleukodystrophy, since C26:0 is the biochemical marker of the disease and in the brain lesions it is accumulated in the cholesteryl-ester fraction. Considered as a whole, our results indicate that the abcd3kd cell line is a valuable tool to further study the function of PMP70 and eventually its role in X-linked adrenoleukodystrophy.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP-mediated pathways. In this study, we investigated whether n-3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2-day-old rats, which were then killed after 3 days post-injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin-specific mRNAs was analyzed by real-time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA- and EPA-treated animals, but the effect was more pronounced in EPA-treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA-treated rats. A further indicator of accelerated myelination was the increase in 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) protein levels. In EPA-treated rats, the increased expression of myelin genes coincided with a decrease of cAMP-response element-binding protein (CREB)-DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA-treated rats was less pronounced than in controls. The down-regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n-3 PUFA beneficial effects on several demyelinating diseases.

Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells.

Glial cells play a pivotal role in brain fatty acid metabolism and membrane biogenesis. However, the potential regulation of lipogenesis and cholesterologenesis by fatty acids in glial cells has been barely investigated. Here, we show that physiologically relevant concentrations of various saturated, monounsaturated, and polyunsaturated fatty acids significantly reduce [1-(14)C]acetate incorporation into fatty acids and cholesterol in C6 cells. Oleic acid was the most effective at depressing lipogenesis and cholesterologenesis; a decreased label incorporation into cellular palmitic, stearic, and oleic acids was detected, suggesting that an enzymatic step(s) of de novo fatty acid biosynthesis was affected. To clarify this issue, the activities of acetyl-coenzyme A carboxylase (ACC) and FAS were determined with an in situ digitonin-permeabilized cell assay after incubation of C6 cells with fatty acids. ACC activity was strongly reduced ( approximately 80%) by oleic acid, whereas no significant change in FAS activity was observed. Oleic acid also reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). The inhibition of ACC and HMGCR activities is corroborated by the decreases in ACC and HMGCR mRNA abundance and protein levels. The downregulation of ACC and HMGCR activities and expression by oleic acid could contribute to the reduced lipogenesis and cholesterologenesis.

Docosahexaenoic acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells.

In view of the promising use of n-3 polyunsaturated fatty acids (PUFAs) in the prevention and treatment of neurological diseases, it is necessary to ascertain the lack of detrimental oxidative effects. We evaluated short- and long-term effects of 25, 50 and 75 muM docosahexaenoic acid (DHA) supplementation on the oxidative status of C6 glial cells. DHA was incorporated into cells dose and time dependently without any cytotoxic effect. Reactive oxygen species (ROS) level was related to DHA dose and supplementation time. At the lowest dose no significant increase in ROS values was observed at hour 24. Low doses of DHA strengthened the cellular antioxidant defence system as highlighted by a raise in both GPX and catalase activity, and the decreased levels of lipid peroxidation. This effect was pronounced at 24 h of supplementation, almost disappeared at hour 48, while after 72 h an opposite effect was observed: lipid peroxidation increased concomitantly with DHA doses. Therefore, the final effect of DHA on cellular redox status is dependent on dose and time supplementation.

Eicosapentaenoic acid stimulates leptin receptor gene expression in the hypothalamus of newborn rats.

Body weight and obesity are controlled by the binding leptin (Ob) receptor, but in newborn rats, despite high Ob levels, hypothalamic leptin receptors (Ob-Rb) are only weakly expressed. In this study we have attempted to stimulate expression of the Ob-Rb gene by administering 2 polyunsaturated fatty acids (PUFAs) recommended for the maternal diet and known as gene regulators: docosahexaenoic acid and eicosapentaenoic acid (EPA). We studied the effects of a single dose injected into a cerebral ventricle of newborn rats on postnatal day 2. On days 1, 2, and 3 after administration, we dissected the hypothalamus and analyzed Ob-Rb and Ob messenger RNAs by polymerase chain reaction and protein expression by Western blot immunoassay. Our results demonstrate that EPA, but not docosahexaenoic acid, caused an early messenger RNA expression of the gene, 24 hours earlier than in the controls, and the protein was also detected earlier. Our data corroborate the observations regarding the role of PUFAs, EPA in particular, in the regulation of gene expression. In addition, they support the recommendation to enrich the maternal diet with fish and seafood rich in n-3 PUFA, because the concentrations of n-6 and n-3 PUFA in human milk reflect the composition of fat in the mother's diet.

Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells.

n-3 polyunsaturated fatty acids (PUFAs) have been described to have beneficial effects on brain development and in the prevention and treatment of brain damage. C6 glioma cells were incubated with 100 microM of either C20:4n-6 (ARA), or C20:5n-3 (EPA), or C22:6n-3 (DHA) for different time periods to assess whether these acids altered the cellular oxidative state. The ARA and EPA were promptly metabolised to C22:4n-6 and C22:5n-3, respectively, whereas DHA treatment simply increased the amount of DHA in the cells. Cell viability was not affected by ARA, while a cytotoxic effect was observed 72 h after n-3 PUFAs supplementation. The levels of reactive oxygen species and thiobarbituric acid-reactive substances were significantly higher in DHA-treated cells than in EPA- and ARA-treated groups. This modification in the oxidative cellular status was also highlighted by a significant increase in catalase activity and a decrease in glutathione content in DHA-supplemented cells. Glucose-6-phosphate dehydrogenase activity, an enzyme involved in redox regulation, and O2*- release were significantly increased both in EPA and DHA groups. The effect of DHA was more severe than that of EPA. No significant changes were observed in the ARA group with respect to untreated cells. These data show that EPA and DHA induce alterations in the oxidative status that could affect the glial function.

Effects of L-mono methyl-arginine, N-acetyl-cysteine and diphenyleniodonium on free radical release in C6 glial cells enriched in hexacosenoic acid.

Previously, we have shown that C6 glial cells enriched in hexacosenoic acid (HA) incubated with oxidative stressors released higher amounts of nitric oxide (NO) products and superoxide (O2(-)), compared to native C6 cells. In the present study, we examined the effects of pretreatment with some of free radical release inhibitors. The aim was to determine the origin of the enhanced generation of NO and superoxide, and to test the possibility of preventing it. Pre-treatment with L-mono-methyl-arginine and N-acetyl-cysteine in oxidized low-density lipoprotein (ox-LDL) exposed HA cells, inhibited not only nitrite but also superoxide production suggesting that O2(-) anion could partially derive from inducible NO synthase. We also observed that ox-LDL treatment of HA cells reduced the intracellular glutathione levels and activated extracellular signal-related kinases. Since this signalling is related to neurotoxic effect, our data substantiate the role of the free radicals in X-linked adrenoleukodystrophy pathogenesis, as HA cells have been used as an in vitro model for this disease.

Identification of seven novel mutations in ABCD1 by a DHPLC-based assay in Italian patients with X-linked adrenoleukodystrophy.

We report the molecular findings in 14 patients (12 families) with X-linked adrenoleukodystrophy (X-ALD, MIM# 300100), a well-defined peroxisomal disorder attributed to mutations in the ABCD1 gene on chromosome Xq28. With the aims of determining the spectrum of mutations and developing an efficient molecular genetic test for analysis of at-risk women whose carrier status is unknown, and to offer molecular confirmation of their status to obligate heterozygotes, regardless of their clinical status, we carried out molecular screening by setting up a denaturing high-performance liquid chromatography (DHPLC)-based protocol. We identified eleven hemizygous base changes in ABCD1, including seven new mutations (c.145underscore;146ins4, c.264C>G, c.919C>T, c.994C>T, c.1027G>A, c.1508T>C, and c.1540A>C, resulting in the p.Pro193fs, p.Cys88Trp, p.Gln307X, p.Gln332X, p.Gly343Ser, p.Leu503Pro, and p.Ser514Arg changes, respectively). Adding new variants to the repertoire of ABCD1 mutations in X-ALD, our data provide an efficient, cost-effective, and reliable DHPLC detection protocol for mutation screening of X-ALD families.

Stimulation of myelin proteolipid protein gene expression by eicosapentaenoic acid in C6 glioma cells.

In this study, the role of exogenous fatty acids in the regulation of proteolipid protein (PLP) gene expression was investigated using the following model culture system: C6 glioma cells expressing the green-fluorescent protein (eGFP) driven by different segments of PLP promoter. Eicosapentanoic acid (EPA; 20:5 n-3), but not arachidonic acid (AA; 20:4 n-6), induced a significant increase in medium fluorescence intensity (MFI) determined by fluorescence-activated cell sorting (FACS). The induction of PLP promoter was time-dependent showing maximal activity between 24 and 48 h after EPA exposure. PLP promoter activation was dependent on fatty acid concentration, with maximum activation at 200 microM. Northern blot analysis confirmed the fluorescence data in C6 cells incubated with EPA. Furthermore, this treatment increased the adenylyl cyclase-cyclic AMP (cAMP) levels and the mitogen-activated protein kinase (MAPK) activation in C6 cells. PLP promoter activity was inhibited by pre-treatment with H89 (protein kinase A (PKA) inhibitor), but not with PD98059 (MAPK inhibitor), suggesting that EPA stimulates the expression of PLP via cAMP-mediated pathways.

Free radical release in C6 glial cells enriched in hexacosanoic acid: implication for X-linked adrenoleukodystrophy pathogenesis.

Free radicals have been implicated in the etiopathology of some neurological and demyelinating diseases. To evaluate their involvement in the cerebral form of X-linked adrenoleukodystrophy (cerALD) disorder, characterised by very long chain fatty acid (VLCFA) accumulation, we utilised an in vitro model using rat C6 glial cells, enriched in hexacosenoic acid (C26:0, HA). Modified cells were incubated in presence of oxidative stressors, such as bacterial endotoxin lipopolisaccharides (LPS) and human oxidised low-density lipoprotein (ox-LDL), and the production of proinflammatory cytokines, nitrite, nitrate and superoxide was determined in the supernatants. The results show that modified cells produce higher amounts of nitric oxide (NO) products and superoxide compared to native C6 cells, supporting the role of free radicals as important pathophysiological modulator of the neuroinflammatory response in ALD. This hypothesis suggests that the cerebral damage in ALD could be due to intracellular signalling activated by interaction of exogenous factors with the particular membrane fatty acid composition.

Dietary prenatal lipids affect myelin gene expression in postnatal undernourished rats.

The effects on myelin gene expression of prenatal diets with a different lipid content and fatty acid composition were investigated in undernourished pups. Three groups of rats were fed ad libitum during gestation diets containing either 10% of margarine or 10% of microbial lipids or a standard diet containing 3.5% of lipids. After birth, all groups were switched to a reduced intake (60%) of the standard diet. A control group received ad libitum, the standard diet during pregnancy and throughout lactation. At birth no difference in body and brain weight was observed among the groups and the only significant difference in the brain fatty acid composition was the presence of odd-chain fatty acids in the group fed microbial lipids. Milk was removed from the stomach of pups at 1, 5 and 9 days of lactation for fatty acid analysis. During undernourishment, the monoenoic fatty acid and polyenoic fatty acid percentage was always higher and lower, respectively, in the groups fed 10% than in the group fed 3.5% of lipids during pregnancy. The expression of myelin genes at 11 days of undernutrition was determined in different brain regions by Northern analysis. In the standard group, the proteolipid protein and myelin oligodendrocyte glycoprotein transcripts were well detected only in the medulla whereas in groups fed 10% of lipids the transcripts were also visible in the cerebellum. These data suggest that the high lipid content of the prenatal diet independently from its fatty acid composition affects the myelin gene expression decreasing myelin susceptibility to postnatal undernutrition.