Characterization of the Intestinal Lactobacilli Community following Galactooligosaccharides and Polydextrose Supplementation in the Neonatal Piglet.

Recently, prebiotic supplementation of infant formula has become common practice; however the impact on the intestinal microbiota has not been completely elucidated. In this study, neonatal piglets were randomized to: formula (FORM, n = 8), formula supplemented with 2 g/L each galactooligosaccharides (GOS) and polydextrose (PDX, F+GP, n = 9) or a sow-reared (SOW, n = 12) reference group for 19 days. The ileal (IL) and ascending colon (AC) microbiota were characterized using culture-dependent and -independent methods. 16S amplicon sequencing identified no differences at the genera level in the IL. Interestingly, six genera in the AC were significantly different between FORM and F+GP (P<0.05): Lactobacillus, Ruminococcus, Parabacteroides, Oscillospira, Hydrogenoanaerobacterium and Catabacter. In particular, the relative abundance of AC Lactobacillus was higher (P = 0.04) in F+GP as compared to FORM. Culture-dependent analysis of the IL and AC lactobacilli communities of FORM and F+GP revealed a Lactobacillus spp. composition similar to 16S amplicon sequencing. Additional analysis demonstrated individual Lactobacillus isolates were unable to ferment PDX. Conversely, a majority of lactobacilli isolates could ferment GOS, regardless of piglet diet. In addition, the ability of lactobacilli isolates to ferment the longer chain GOS fragments (DP 3 or greater), which are expected to be present in the distal intestine, was not different between FORM and F+GP. In conclusion, prebiotic supplementation of formula impacted the AC microbiota; however, direct utilization of GOS or PDX does not lead to an increase in Lactobacillus spp.

Longitudinal Survey of Carotenoids in Human Milk from Urban Cohorts in China, Mexico, and the USA.

Emerging evidence indicates that carotenoids may have particular roles in infant nutrition and development, yet data on the profile and bioavailability of carotenoids from human milk remain sparse. Milk was longitudinally collected at 2, 4, 13, and 26 weeks postpartum from twenty mothers each in China, Mexico, and the USA in the Global Exploration of Human Milk Study (n = 60 donors, n = 240 samples). Maternal and neonatal plasma was analyzed for carotenoids from the USA cohort at 4 weeks postpartum. Carotenoids were analyzed by HPLC and total lipids by Creamatocrit. Across all countries and lactation stages, the top four carotenoids were lutein (median 114.4 nmol/L), ß-carotene (49.4 nmol/L), ß-cryptoxanthin (33.8 nmol/L), and lycopene (33.7 nmol/L). Non-provitamin A carotenoids (nmol/L) and total lipids (g/L) decreased (p<0.05) with increasing lactation stage, except the provitamin A carotenoids α- and ß-cryptoxanthin and ß-carotene did not significantly change (p>0.05) with lactation stage. Total carotenoid content and lutein content were greatest from China, yet lycopene was lowest from China (p<0.0001). Lutein, ß-cryptoxanthin, and ß-carotene, and lycopene concentrations in milk were significantly correlated to maternal plasma and neonatal plasma concentrations (p<0.05), with the exception that lycopene was not significantly associated between human milk and neonatal plasma (p>0.3). This enhanced understanding of neonatal exposure to carotenoids during development may help guide dietary recommendations and design of human milk mimetics.

Effect of perinatally supplemented flavonoids on brain structure, circulation, cognition, and metabolism in C57BL/6J mice.

Evidence suggests that flavanol consumption can beneficially affect cognition in adults, but little is known about the effect of flavanol intake early in life. The present study aims to assess the effect of dietary flavanol intake during the gestational and postnatal period on brain structure, cerebral blood flow (CBF), cognition, and brain metabolism in C57BL/6J mice. Female wild-type C57BL/6J mice were randomly assigned to either a flavanol supplemented diet or a control diet at gestational day 0. Male offspring remained on the corresponding diets throughout life and performed cognitive and behavioral tests during puberty and adulthood assessing locomotion and exploration (Phenotyper and open field), sensorimotor integration (Rotarod and prepulse inhibition), and spatial learning and memory (Morris water maze, MWM). Magnetic resonance spectroscopy and imaging at 11.7T measured brain metabolism, CBF, and white and gray matter integrity in adult mice. Biochemical and immunohistochemical analyses evaluated inflammation, synaptic plasticity, neurogenesis, and vascular density. Cognitive and behavioral tests demonstrated increased locomotion in Phenotypers during puberty after flavanol supplementation (p = 0.041) but not in adulthood. Rotarod and prepulse inhibition demonstrated no differences in sensorimotor integration. Flavanols altered spatial learning in the MWM in adulthood (p = 0.039), while spatial memory remained unaffected. Additionally, flavanols increased diffusion coherence in the visual cortex (p = 0.014) and possibly the corpus callosum (p = 0.066) in adulthood. Mean diffusion remained unaffected, a finding that corresponds with our immunohistochemical data showing no effect on neurogenesis, synaptic plasticity, and vascular density. However, flavanols decreased CBF in the cortex (p = 0.001) and thalamus (p = 0.009) in adulthood. Brain metabolite levels and neuroinflammation remained unaffected by flavanols. These data suggest that dietary flavanols results in subtle alterations in brain structure, locomotor activity and spatial learning. Comparison of these data to published findings in aging or neurodegeneration suggests that benefits of dietary flavanols may increase with advancing age and in disease.

Impact of dietary n-3 polyunsaturated fatty acids on cognition, motor skills and hippocampal neurogenesis in developing C57BL/6J mice.

Maternal intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) is critical during perinatal development of the brain. Docosahexaenoic acid (DHA) is the most abundant n-3 PUFA in the brain and influences neuronal membrane function and neuroprotection. The present study aims to assess the effect of dietary n-3 PUFA availability during the gestational and postnatal period on cognition, brain metabolism and neurohistology in C57BL/6J mice. Female wild-type C57BL/6J mice at day 0 of gestation were randomly assigned to either an n-3 PUFA deficient diet (0.05% of total fatty acids) or an n-3 PUFA adequate diet (3.83% of total fatty acids) containing preformed DHA and its precursor α-linolenic acid. Male offspring remained on diet and performed cognitive tests during puberty and adulthood. In adulthood, animals underwent (31)P magnetic resonance spectroscopy to assess brain energy metabolites. Thereafter, biochemical and immunohistochemical analyses were performed assessing inflammation, neurogenesis and synaptic plasticity. Compared to the n-3 PUFA deficient group, pubertal n-3 PUFA adequate fed mice demonstrated increased motor coordination. Adult n-3 PUFA adequate fed mice exhibited increased exploratory behavior, sensorimotor integration and spatial memory, while neurogenesis in the hippocampus was decreased. Selected brain regions of n-3 PUFA adequate fed mice contained significantly lower levels of arachidonic acid and higher levels of DHA and dihomo-γ-linolenic acid. Our data suggest that dietary n-3 PUFA can modify neural maturation and enhance brain functioning in healthy C57BL/6J mice. This indicates that availability of n-3 PUFA in infant diet during early development may have a significant impact on brain development.

Caco-2 accumulation of lutein is greater from human milk than from infant formula despite similar bioaccessibility.

SCOPE: Clinical evidence suggests that the bioavailability of lutein is lower from infant formula than from human milk. The purpose of this study was to assess characteristics of human milk and lutein-fortified infant formula that may impact carotenoid delivery. METHODS AND RESULTS: Carotenoid bioaccessibility and intestinal absorption were modeled by in vitro digestion coupled with Caco-2 human intestinal cell culture. Twelve human milk samples were assessed from 1-6 months postpartum, and 10 lutein-fortified infant formula samples from three lutein sources in both ready-to-use and reconstituted powder forms. The relative bioaccessibility of lutein was not different (p > 0.05) between human milk (29 ± 2%) and infant formula (36 ± 4%). However, lutein delivery was 4.5 times greater from human milk than infant formula when including Caco-2 accumulation efficiency. Caco-2 accumulation of lutein was increasingly efficient with decreasing concentration of lutein from milk. Carotenoid bioaccessibility and Caco-2 accumulation were not affected by lactation stage, total lipid content, lutein source, or form of infant formula (powder vs. liquid). CONCLUSION: These data suggest that the bioavailability of carotenoids is greater from human milk than infant formula primarily due to intestinal absorptive processes, and that absorption of lutein is potentiated by factors from human milk especially at low lutein concentration.

Dietary supplementation of Bifidobacterium longum strain AH1206 increases its cecal abundance and elevates intestinal interleukin-10 expression in the neonatal piglet.

Intestinal microbiota of infants differ in response to gestational age, delivery mode and feeding regimen. Dietary supplementation of probiotic bacteria is one method of promoting healthy populations. We examined the impact of a novel probiotic strain of Bifidobacterium longum (AH1206) on the health, growth and development of neonatal pigs as a model for infants. Day-old pigs were fed milk-based formula containing AH1206 at 0, 109, or 10¹¹ CFU/d for 18 d (n=10/treatment). Differences were not detected in growth, organ weights or body temperatures (P>0.1); however pigs fed the high dose showed a small (2%) reduction in feed intake. Bacterial translocation was not affected as indicated by total anaerobic and aerobic counts (CFU) in samples of spleen, liver and mesenteric lymph nodes (P>0.1). Feeding AH1206 had no effects on fecal consistency, but increased the density of B. longum in the cecum. Ileal TNF expression tended to increase (P=0.08) while IL-10 expression increased linearly (P=0.01) with supplementation. Based upon findings in the suckling piglet model, we suggest that dietary supplementation with B. longum (AH1206) may be safe for human infants based on a lack of growth, development or deleterious immune-related effects observed in piglets.

Assessment of phytochemical content in human milk during different stages of lactation.

OBJECTIVE: The present study reports the presence of several carotenoids and flavonoids in human milk samples. METHODS: Samples were collected from 17 women who delivered healthy term babies (≥ 37 wk of gestation) at 1-, 4-, and 13-wk postpartum intervals. RESULTS: Epicatechin (63.7-828.5 nmol/L), epicatechin gallate (55.7-645.6 nmol/L), epigallocatechin gallate (215.1-2364.7 nmol/L), naringenin (64.1-722.0 nmol/L), kaempferol (7.8-71.4 nmol/L), hesperetin (74.8-1603.1 nmol/L), and quercetin (32.5-108.6 nmol/L) were present in human milk samples with high inter-/intraindividual variability. With the exception of kaempferol, the mean flavonoid content in human milk was not statistically different among lactation stages. In contrast, carotenoids α-carotene (59.0-23.2 nmol/L), ß-carotene (164.3-88.0 nmol/L), α-cryptoxanthin (30.6-13.5 nmol/L), ß-cryptoxanthin (57.4-24.8 nmol/L), zeaxanthin (46.3-21.4 nmol/L), lutein (121.2-56.4 nmol/L), and lycopene (119.9-49.5 nmol/L) significantly decreased from weeks 1 to 13 of lactation. CONCLUSION: The observed differences in the relative concentrations of the two phytochemical classes in human milk may be a result of several factors, including dietary exposure, stability in the milk matrix, efficiency of absorption/metabolism, and transfer from plasma to human milk. These data support the notion that flavonoids, as with carotenoids, are dietary phytochemicals present in human milk and potentially available to breast-fed infants.

Pomegranate seed oil reduces intestinal damage in a rat model of necrotizing enterocolitis.

Pomegranate seed oil (PSO), which is the major source of conjugated linolenic acids such as punicic acid (PuA), exhibits strong anti-inflammatory properties. Necrotizing enterocolitis (NEC) is a devastating disease associated with severe and excessive intestinal inflammation. The aim of this study was to evaluate the effects of orally administered PSO on the development of NEC, intestinal epithelial proliferation, and cytokine regulation in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), formula-fed rats (FF), or rats fed with formula supplemented with 1.5% of PSO (FF + PSO). All groups were exposed to asphyxia/cold stress to induce NEC. Intestinal injury, epithelial cell proliferation, cytokine production, and trefoil factor 3 (Tff3) production were evaluated in the terminal ileum. Oral administration of PSO (FF+PSO) decreased the incidence of NEC from 61 to 26%. Feeding formula with PSO improved enterocyte proliferation in the site of injury. Increased levels of proinflammatory IL-6, IL-8, IL-12, IL-23, and TNF-α in the ileum of FF rats were normalized in PSO-treated animals. Tff3 production in the FF rats was reduced compared with DF but not further affected by the PSO. In conclusion, administration of PSO protects against NEC in the neonatal rat model. This protective effect is associated with an improvement of intestinal epithelial homeostasis and a strong anti-inflammatory effect of PSO on the developing intestinal mucosa.

Arachidonic acid/docosahexaenoic acid-supplemented diet in early life reduces body weight gain, plasma lipids, and adiposity in later life in ApoE*3Leiden mice.

SCOPE: This study addresses whether early life arachidonic acid (ARA)/docosahexaenoic acid (DHA) supplementation or eicosapentaenoic acid (EPA)/DHA (Omacor) supplementation affects body weight gain, lipid metabolism, and adipose tissue quantity and quality in later life in ApoE*3Leiden-transgenic mice, a humanized model for hyperlipidemia and mild obesity. METHODS AND RESULTS: Four-week-old male ApoE*3Leiden mice were fed chow diet with or without a mixture of ARA (0.129 wt%) and DHA (0.088 wt%) or Omacor (0.30 wt% EPA, 0.25 wt% DHA). At age 12 weeks, mice were fed high-fat/high-carbohydrate (HFHC) diet without above supplements until age 20 weeks. Control mice received chow diet throughout the study. Mice receiving ARA/DHA gained less body weight compared to control and this effect was sustained when fed HFHC. Omacor had no significant effect on body weight gain. Plasma cholesterol and triglycerides were significantly lowered by both supplementations. At 20 weeks, epididymal fat mass was less in ARA/DHA-supplemented mice, while Omacor had no significant effect on fat mass. Both ARA/DHA and Omacor reduced inguinal adipocyte cell size; only ARA/DHA significantly reduced epididymal macrophage infiltration. CONCLUSION: This study shows that early life ARA/DHA, but not Omacor supplementation improves body weight gain later in life. ARA/DHA and to a lesser extent Omacor both improved adipose tissue quality.

Effect of phytochemicals on phase II enzyme expression in infant human primary skin fibroblast cells.

Phase II metabolising enzymes enable the metabolism and excretion of potentially harmful substances in adults, but to date it is unclear whether dietary phytochemicals can induce phase II enzymes differently between adults and infants. We investigated the expression of phase II enzymes in an in vitro model of primary skin fibroblasts at three different developmental stages, 1 month, 2 years and adult, to examine potential differences in age-related phase II enzymes in response to different phytochemicals (5-20 µm) including sulphoraphane, quercetin and catechin. Following phytochemical treatment, a significant increase in mRNA of glutathione S-transferase A1 (GSTA1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) was observed, with the most marked increases seen in response to sulphoraphane (3-10-fold for GSTA1, P = 0·001, and 6-35-fold for NQO1, P = 0·001-0·017). Catechin also induced 3-5-fold changes in NQO1 transcription, whereas quercetin had less effect on NQO1 mRNA induction in infant cells. Moreover, NQO1 protein levels were significantly increased in 2-year-old and adult cell models in response to sulphoraphane treatment. These results suggest that metabolic plasticity and response to xenobiotics may be different in infants and adults; and therefore the inclusion of phytochemicals in the infant diet may modulate their induction of phase II metabolism, thereby providing increased protection from potentially harmful xenobiotics in later life.

Branched chain fatty acids reduce the incidence of necrotizing enterocolitis and alter gastrointestinal microbial ecology in a neonatal rat model.

INTRODUCTION: Branched chain fatty acids (BCFA) are found in the normal term human newborn's gut, deposited as major components of vernix caseosa ingested during late fetal life. We tested the hypothesis that premature infants' lack of exposure to gastrointestinal (GI) BCFA is associated with their microbiota and risk for necrotizing enterocolitis (NEC) using a neonatal rat model. METHODS: Pups were collected one day before scheduled birth. The pups were exposed to asphyxia and cold stress to induce NEC. Pups were assigned to one of three experimental treatments. DF (dam-fed); Control, hand-fed rat milk substitute; BCFA, hand-fed rat milk substitute with 20%w/w BCFA. Total fat was equivalent (11%wt) for both the Control and BCFA groups. Cecal microbiota were characterized by 16S rRNA gene pyrosequencing, and intestinal injury, ileal cytokine and mucin gene expression, interleukin-10 (IL-10) peptide immunohistochemistry, and BCFA uptake in ileum phospholipids, serum and liver were assessed. RESULTS: NEC incidence was reduced by over 50% in the BCFA group compared to the Control group as assessed in ileal tissue; microbiota differed among all groups. BCFA-fed pups harbored greater levels of BCFA-associated Bacillus subtilis and Pseudomonas aeruginosa compared to Controls. Bacillus subtilis levels were five-fold greater in healthy pups compared to pups with NEC. BCFA were selectively incorporated into ileal phospholipids, serum and liver tissue. IL-10 expression increased three-fold in the BCFA group versus Controls and no other inflammatory or mucosal mRNA markers changed. CONCLUSION: At constant dietary fat level, BCFA reduce NEC incidence and alter microbiota composition. BCFA are also incorporated into pup ileum where they are associated with enhanced IL-10 and may exert other specific effects.

Polydextrose enrichment of infant formula demonstrates prebiotic characteristics by altering intestinal microbiota, organic acid concentrations, and cytokine expression in suckling piglets.

Oligosaccharides, the 3rd-most abundant component in human milk, are virtually absent from infant formulas and from the cow milk on which most are based. In breast-fed infants, human milk oligosaccharides (HMO) act as both receptor analogs, interfering with pathogen adhesion, and as prebiotics, stimulating the growth of certain commensal bacteria (e.g. bifidobacteria) and supporting the innate immunity. To further align the functional properties of infant formula with those of human milk, polydextrose (PDX) is proposed as a substitute for HMO. To determine the prebiotic functionality of PDX, 1-d-old pigs were fed a cow milk-based formula supplemented with increasing concentrations of PDX (0, 1.7, 4.3, 8.5, or 17 g/L) for 18 d (n = 13). Additional reference groups included pigs sampled at d 0 and sow-reared pigs sampled at d 18 (n = 12). Ileal Lactobacilli CFU, but not Bifidobacteria, increased linearly with increasing PDX (P = 0.02). The propionic acid concentration in digesta linearly increased with the PDX level (P = 0.045) and lactic acid increased linearly by 5-fold with increasing PDX (P = 0.001). Accordingly, digesta pH decreased linearly (P < 0.05) as PDX increased, with a maximal reduction approaching 0.5 pH units in pigs fed 17 g/L. Expression of TNFα, IL-1ß, and IL-8 showed a negative quadratic pattern in response to PDX supplementation, declining at intermediate concentrations and rebounding at higher concentrations of PDX. In summary, PDX enrichment of infant formula resulted in a prebiotic effect by increasing ileal lactobacilli and propionic and lactic acid concentrations and decreasing pH with associated alterations in ileal cytokine expression.

Activation of PPARγ and δ by dietary punicic acid ameliorates intestinal inflammation in mice.

The goal of the present study was to elucidate the mechanisms of immunoregulation by which dietary punicic acid (PUA) prevents or ameliorates experimental inflammatory bowel disease (IBD). The expression of PPARγ and δ, their responsive genes and pro-inflammatory cytokines was assayed in the colonic mucosa. Immune cell-specific PPARγ null, PPARδ knockout and wild-type mice were treated with PUA and challenged with 2·5 % dextran sodium sulphate (DSS). The prophylactic efficacy of PUA was examined in an IL-10(-/-) model of IBD. The effect of PUA on the regulatory T-cell (Treg) compartment was also examined in mice with experimental IBD. PUA ameliorated spontaneous pan-enteritis in IL-10(-/-) mice and DSS colitis, up-regulated Foxp3 expression in Treg and suppressed TNF-α, but the loss of functional PPARγ or δ impaired these anti-inflammatory effects. At the cellular level, the macrophage-specific deletion of PPARγ caused a complete abrogation of the protective effect of PUA, whereas the deletion of PPARδ or intestinal epithelial cell-specific PPARγ decreased its anti-inflammatory efficacy. We provide in vivo molecular evidence demonstrating that PUA ameliorates experimental IBD by regulating macrophage and T-cell function through PPARγ- and δ-dependent mechanisms.

Prenatal fatty acid status and child adiposity at age 3 y: results from a US pregnancy cohort.

BACKGROUND: Exposure to polyunsaturated fatty acids (PUFAs) in early life may influence adiposity development. OBJECTIVE: We examined the extent to which prenatal n-3 (omega-3) and n-6 (omega-6) PUFA concentrations were associated with childhood adiposity. DESIGN: In mother-child pairs in the Project Viva cohort, we assessed midpregnancy fatty acid intakes (n = 1120), maternal plasma PUFA concentrations (n = 227), and umbilical cord plasma PUFA concentrations (n = 302). We performed multivariable regression analyses to examine independent associations of n-3 PUFAs, including docosahexaenoic and eicosapentaenoic acids (DHA + EPA), n-6 PUFAs, and the ratio of n-6:n-3 PUFAs, with child adiposity at age 3 y measured by the sum of subscapular and triceps skinfold thicknesses (SS + TR) and risk of obesity (body mass index ≥95th percentile for age and sex). RESULTS: Mean (±SD) DHA + EPA intake was 0.15 ± 0.14 g DHA + EPA/d, maternal plasma concentration was 1.9 ± 0.6%, and umbilical plasma concentration was 4.6 ± 1.2%. In children, SS + TR was 16.7 ± 4.3 mm, and 9.4% of children were obese. In the adjusted analysis, there was an association between each SD increase in DHA + EPA and lower child SS + TR [-0.31 mm (95% CI: -0.58, -0.04 mm) for maternal diet and -0.91 mm (95% CI: -1.63, -0.20 mm) for cord plasma] and lower odds of obesity [odds ratio (95% CI): 0.68 (0.50, 0.92) for maternal diet and 0.09 (0.02, 0.52) for cord plasma]. Maternal plasma DHA + EPA concentration was not significantly associated with child adiposity. A higher ratio of cord plasma n-6:n-3 PUFAs was associated with higher SS + TR and odds of obesity. CONCLUSION: An enhanced maternal-fetal n-3 PUFA status was associated with lower childhood adiposity.

Addition of polydextrose and galactooligosaccharide to formula does not affect bacterial translocation in the neonatal piglet.

OBJECTIVES: The aim of the study was to determine the effect of polydextrose (PDX) and galactooligosaccharide (GOS) on bacterial translocation (BT) in neonatal piglets. MATERIALS AND METHODS: Piglets (n = 36) were randomized 12 hours after birth to receive total enteral nutrition (TEN) as formula; TEN + GOS (4 g/L), TEN + PDX (4 g/L), or TEN + GOS + PDX (2 g/L each) for 7 days or were supported by total parenteral nutrition (TPN) as a positive control for BT (n = 8). Blood, spleen, liver, and mesenteric lymph node (MLN) samples were cultured for aerobic and anaerobic bacteria. Colon microbiota 16S rDNA was measured by polymerase chain reaction. Myeloperoxidase activity and tumor necrosis factor-α expression were measured in ileum and ascending colon. RESULTS: Among the enterally fed groups, no difference was seen in the Lactobacillus and Bacteroides 16S rDNA copies per gram of colonic contents, yet total bacterial levels were lower (P < 0.05) in the TEN + GOS group compared with TEN alone. Bacteria were detected in the blood, liver spleen, and MLN of TPN piglets. In contrast, bacterial counts were predominantly detected in the MLN of TEN piglets, at much lower levels than in TPN, and levels were not affected by GOS and PDX addition. TPN piglets had elevated (P < 0.05) ileal myeloperoxidase activity and a trend in elevated ascending colon tumor necrosis factor-α expression (P = 0.1). CONCLUSIONS: PDX and GOS added to formula do not induce BT in healthy piglets. Low levels of bacteria in MLN of healthy neonatal piglets may reflect mucosal sampling rather than pathological BT.

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids.

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori.

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.

Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta.

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.

Characterization of cholesterol transport from midgut to fat body in Manduca sexta larvae.

Using in vitro methods, we investigated the transfer of cholesterol from larval Manduca sexta midgut to the hemolymph lipoprotein, lipophorin, and the transfer of cholesterol from lipophorin to larval fat body. In the midgut, transfer of free cholesterol shows saturation kinetics, but the apparent Km is higher than the measured Kd for the midgut lipophorin-receptor complex. In addition, the transfer is unaffected by suramin, which binds to the receptor and inhibits lipophorin binding, and by antibodies to the lipid transfer particle, which is required for export of diacylglycerol from the midgut to lipophorin. In the fat body, transfer of free cholesterol also shows saturation kinetics, and the apparent Km is higher than the measured Kd for the fat body lipophorin-receptor complex. Suramin and anti-lipid transfer particle antibodies exert only a small (20%) inhibitory effect. In both tissues it seems that the most likely mode of cholesterol transfer is via aqueous diffusion, which is also an important mechanism in vertebrate cells. Based on these results, we propose that cholesterol homeostasis in larval M. sexta is maintained by a mass action mechanism in which cholesterol is freely transferred between lipophorin and tissues depending on the needs of the tissues. This simple mechanism is ideally suited to insects, which can neither make cholesterol nor internalize lipophorin, the two mechanisms that vertebrate cells use to control their cholesterol content.

Beta-cyclodextrin facilitates cholesterol efflux from larval Manduca sexta fat body and midgut in vitro.

The ability of 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) and methyl-beta-cyclodextrin (MbetaCD) to promote cholesterol efflux from [3H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both beta-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MbetaCD than for HPbetaCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.