A combination of lipidomics, MS imaging, and PET scan imaging reveals differences in cerebral activity in rat pups according to the lipid quality of infant formulas.

We evaluated the effect of adding docosahexaenoic:arachidonic acids (3:2) (DHA+ARA) to 2 representative commercial infant formulas on brain activity and brain and eye lipids in an artificially reared rat pup model. The formula lipid background was either a pure plant oil blend, or dairy fat with a plant oil blend (1:1). Results at weaning were compared to breast milk-fed pups. Brain functional activity was determined by positron emission tomography scan imaging, the brain and eye fatty acid and lipid composition by targeted and untargeted lipidomics, and DHA brain regional location by mass-spectrometry imaging. The brain functional activity was normalized to controls with DHA+ARA added to the formulas. DHA in both brain and eyes was influenced by formula intake, but more than two-thirds of tissue DHA-glycerolipids remained insensitive to the dietary challenge. However, the DHA lipidome correlated better with brain function than sole DHA content ( r = 0.70 vs. r = 0.48; P < 0.05). Brain DHA regional distribution was more affected by the formula lipid background than the provision of PUFAs. Adding DHA+ARA to formulas alters the DHA content and lipidome of nervous tissue in the neonate, making it closer to dam milk-fed controls, and normalizes brain functional activity.-Aidoud, N., Delplanque, B., Baudry, C., Garcia, C., Moyon, A., Balasse, L., Guillet, B., Antona, C., Darmaun, D., Fraser, K., Ndiaye, S., Leruyet, P., Martin, J.-C. A combination of lipidomics, MS imaging, and PET scan imaging reveals differences in cerebral activity in rat pups according to the lipid quality of infant formulas.

An infant formula containing dairy lipids increased red blood cell membrane Omega 3 fatty acids in 4 month-old healthy newborns: a randomized controlled trial.

BACKGROUND: When breastfeeding is not possible, infants are fed formulas (IF) in which lipids are usually of plant origin. However, the use of dairy fat in combination with plant oils enables a lipid profile closer to breast milk in terms of fatty acid (FA) composition, triglyceride structure, polar lipids and cholesterol contents. The objective of this study was to determine the effect of an IF containing a mix of dairy fat and plant oils on Omega-3 FA content in red blood cells (RBC). METHODS: This study was a monocentric, double-blind, controlled, randomized trial. Healthy term infants were fed formulas containing a mix of dairy fat and plant oils (D), plant oils (P) or plant oils supplemented with ARA and DHA (PDHA). Breastfed infants were enrolled as a reference group (BF). FA in RBC phosphatidylethanolamine was evaluated after 4 months and FA in whole blood were evaluated at enrollment and after 4 months by gas chromatography. Differences between groups were assessed using an analysis of covariance with sex and gestational age as covariates. RESULTS: Seventy IF-fed and nineteen BF infants completed the protocol. At 4 months, RBC total Omega-3 FA levels in infants fed formula D were significantly higher than in group P and similar to those in groups PDHA and BF. RBC DHA levels in group D were also higher than in group P but lower than in groups PDHA and BF. RBC n-3 DPA levels in group D were higher than in groups P, PDHA and BF. A decrease in proportions of Omega-3 FA in whole blood was observed in all groups. CONCLUSIONS: A formula containing a mix of dairy lipids and plant oils increased the endogenous conversion of Omega-3 long-chain FA from precursor, leading to higher total Omega-3, DPA and DHA status in RBC than a plant oil-based formula. Modifying lipid quality in IF by adding dairy lipids should be considered as an interesting method to improve Omega-3 FA status. TRIAL REGISTRATION: Identifier NCT01611649 , retrospectively registered on May 25, 2012.

No effect of adding dairy lipids or long chain polyunsaturated fatty acids on formula tolerance and growth in full term infants: a randomized controlled trial.

BACKGROUND: When breastfeeding is not possible, infants are fed formulas in which lipids are usually of plant origin. However, the use of dairy fat in combination with plant oils enables a lipid profile in formula closer to breast milk in terms of fatty acid composition, triglyceride structure and cholesterol content. The objectives of this study were to investigate the impact on growth and gastrointestinal tolerance of a formula containing a mix of dairy lipids and plant oils in healthy infants. METHODS: This study was a monocentric, double-blind, controlled, randomized trial. Healthy term infants aged less than 3 weeks whose mothers did not breastfeed were randomly allocated to formula containing either: a mix of plant oils and dairy fat (D), only plant oils (P) or plant oils supplemented with long-chain polyunsaturated fatty acids (PDHA). Breastfed infants were included in a reference group (BF). Anthropometric parameters and body composition were measured after 2 and 4 months. Gastrointestinal tolerance was evaluated during 2 day-periods after 1 and 3 months thanks to descriptive parameters reported by parents. Nonrandomized BF infants were not included in the statistical analysis. RESULTS: Eighty eight formula-fed and 29 BF infants were enrolled. Gains of weight, recumbent length, cranial circumference and fat mass were similar between the 3 formula-fed groups at 2 and 4 months and close to those of BF. Z-scores for weight, recumbent length and cranial circumference in all groups were within normal ranges for growth standards. No significant differences were noted among the 3 formula groups in gastrointestinal parameters (stool frequency/consistency/color), occurrence of gastrointestinal symptoms (abdominal pain, flatulence, regurgitation) or infant's behavior. CONCLUSIONS: A formula containing a mix of dairy lipids and plant oils enables a normal growth in healthy newborns. This formula is well tolerated and does not lead to abnormal gastrointestinal symptoms. Consequently, reintroduction of dairy lipids could represent an interesting strategy to improve lipid quality in infant formulas. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01611649 , retrospectively registered on May 25, 2012.

Properties of myenteric neurones and mucosal functions in the distal colon of diet-induced obese mice.

Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet-induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2-methyl-5-HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5-hydroxytryptamine (5-HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5-HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin-6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5-HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.

Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor.

Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

The influence of a formula supplemented with dairy lipids and plant oils on the erythrocyte membrane omega-3 fatty acid profile in healthy full-term infants: a double-blind randomized controlled trial.

BACKGROUND: Human milk is the optimal nutrition for infants. When breastfeeding is not possible, supplementation of infant formula with long chain polyunsaturated fatty acids appears to promote neurodevelopmental outcome and visual function. Plant oils, that are the only source of fat in most of infant formulas, do not contain specific fatty acids that are present in human and cow milk and do not encounter milk fat triglyceride structure. Experimental data suggest that a mix of dairy lipids and plant oils can potentiate endogenous synthesis of n-3 long chain polyunsaturated fatty acids. This trial aims to determine the effect of an infant formula supplemented with a mixture of dairy lipids and plant oils on the erythrocyte membrane omega-3 fatty acid profile in full-term infants (primary outcome). Erythrocyte membrane long chain polyunsaturated fatty acids and fatty acids content, the plasma lipid profile and the insulin-growth factor 1 level, the gastrointestinal tolerance, the changes throughout the study in blood fatty acids content, in growth and body composition are evaluated as secondary outcomes. METHODS/DESIGN: In a double-blind controlled randomized trial, 75 healthy full-term infants are randomly allocated to receive for four months a formula supplemented with a mixture of dairy lipids and plant oils or a formula containing only plant oils or a formula containing plant oils supplemented with arachidonic acid and docosahexaenoic acid. Twenty-five breast-fed infants constitute the reference group. Erythrocyte membrane omega-3 fatty acid profile, long chain polyunsaturated fatty acids and the other fatty acids content, the plasma lipid profile and the insulin-growth factor 1 level are measured after four months of intervention. Gastrointestinal tolerance, the changes in blood fatty acids content, in growth and body composition, assessed by means of an air displacement plethysmography system, are also evaluated throughout the study. DISCUSSION: The achievement of an appropriate long chain polyunsaturated fatty acids status represents an important goal in neonatal nutrition. Gaining further insight in the effects of the supplementation of a formula with dairy lipids and plant oils in healthy full-term infants could help to produce a formula whose fat content, composition and structure is more similar to human milk. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01611649.

Addition of Dairy Lipids and Probiotic Lactobacillus fermentum in Infant Formulas Modulates Proteolysis and Lipolysis With Moderate Consequences on Gut Physiology and Metabolism in Yucatan Piglets.

Breast milk is the gold standard in neonatal nutrition, but most infants are fed infant formulas in which lipids are usually of plant origin. The addition of dairy lipids and/or milk fat globule membrane extracts in formulas improves their composition with beneficial consequences on protein and lipid digestion. The probiotic Lactobacillus fermentum (Lf) was reported to reduce transit time in rat pups, which may also improve digestion. This study aimed to investigate the effects of the addition of dairy lipids in formulas, with or without Lf, on protein and lipid digestion and on gut physiology and metabolism. Piglets were suckled from postnatal days 2 to 28, with formulas containing either plant lipids (PL), a half-half mixture of plant and dairy lipids (DL), or this mixture supplemented with Lf (DL+Lf). At day 28, piglets were euthanized 90 min after their last feeding. Microstructure of digesta did not differ among formulas. Gastric proteolysis was increased (P < 0.01) in DL and DL+Lf (21.9 ± 2.1 and 22.6 ± 1.3%, respectively) compared with PL (17.3 ± 0.6%) and the residual proportion of gastric intact caseins decreased (p < 0.01) in DL+Lf (5.4 ± 2.5%) compared with PL and DL (10.6 ± 3.1% and 21.8 ± 6.8%, respectively). Peptide diversity in ileum and colon digesta was lower in PL compared to DL and DL+Lf. DL and DL+Lf displayed an increased (p < 0.01) proportion of diacylglycerol/cholesterol in jejunum and ileum digesta compared to PL and tended (p = 0.07) to have lower triglyceride/total lipid ratio in ileum DL+Lf (0.019 ± 0.003) as compared to PL (0.045 ± 0.011). The percentage of endocrine tissue and the number of islets in the pancreas were decreased (p < 0.05) in DL+Lf compared with DL. DL+Lf displayed a beneficial effect on host defenses [increased goblet cell density in jejunum (p < 0.05)] and a trophic effect [increased duodenal (p = 0.09) and jejunal (p < 0.05) weights]. Altogether, our results demonstrate that the addition of dairy lipids and probiotic Lf in infant formula modulated protein and lipid digestion, with consequences on lipid profile and with beneficial, although moderate, physiological effects.

Comparative effects of dietary n-3 docosapentaenoic acid (DPA), DHA and EPA on plasma lipid parameters, oxidative status and fatty acid tissue composition.

The specific and shared physiologic and metabolic effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and even more of n-3 docosapentaenoic acid (DPA) are poorly known. We investigated the physiological effects and the overall fatty acid tissue composition of a nutritional supplementation of DPA compared both to EPA and DHA in healthy adult rats. Rats (n=32) were fed with semisynthetic diets supplemented or not with 1% of total lipids as EPA, DPA or DHA in ethyl esters form from weaning for 6 weeks. Fatty acid tissue composition was determined by gas chromatography-mass spectrometry, and blood assays were performed. The DPA supplementation was the only one that led to a decrease in plasma triglycerides, total cholesterol, non-high-density lipoprotein (HDL)-cholesterol, cholesterol esters and total cholesterol/HDL-cholesterol ratio compared to the nonsupplemented control group. The three supplemented groups had increased plasma total antioxidant status and superoxide dismutase activity. In all supplemented groups, the n-3 polyunsaturated fatty acid level increased in all studied tissues (liver, heart, lung, spleen, kidney, red blood cells, splenocytes, peripheral mononucleated cells) except in the brain. We showed that the DPA supplementation affected the overall fatty acid composition and increased DPA, EPA and DHA tissue contents in a similar way than with EPA. However, liver and heart DHA contents increased in DPA-fed rats at the same levels than in DHA-fed rats. Moreover, a large part of DPA seemed to be retroconverted into EPA in the liver (38.5%) and in the kidney (68.6%). In addition, the digestibility of DPA was lower than that of DHA and EPA.

Impact of n-3 Docosapentaenoic Acid Supplementation on Fatty Acid Composition in Rat Differs Depending upon Tissues and Is Influenced by the Presence of Dairy Lipids in the Diet.

The n-3 docosapentaenoic acid (n-3 DPA) could be a novel source of n-3 long-chain polyunsaturated fatty acids (LCPUFA) with beneficial physiological effects. Following the supplementation of 0.5% purified n-3 DPA for 3 weeks from weaning, the n-3 DPA content increased in one-half of the 18 studied tissues (from +50% to +110%, p < 0.05) and mostly affected the spleen, lung, heart, liver, and bone marrow. The n-3 DPA was slightly converted into DHA (+20% in affected tissues, p < 0.05) and mostly retroconverted into EPA (35-46% of n-3 DPA intake in liver and kidney) showing an increased content of these LCPUFA in specific tissues. The partial incorporation of dairy lipids in the diet for 6 weeks increased overall n-3 PUFA status and brain DHA status. Furthermore, the n-3 DPA supplementation and dairy lipids had an additive effect on the increase of n-3 PUFA tissue contents. Moreover, n-3 DPA supplementation decreased plasma cholesterol.

Incorporation of Dairy Lipids in the Diet Increased Long-Chain Omega-3 Fatty Acids Status in Post-weaning Rats.

In human nutrition, optimized the status of n-3 long-chain polyunsaturated fatty acids (LCPUFA) and especially docosahexaenoic acid (DHA) during growth appears to be one of the most important goal. We investigated the potential impact of a partial incorporation of dairy lipids (DL) in the diet to increase the n-3 LCPUFA content in tissues, compared to a mixture of vegetable oils. Rats were fed with vegetable oil diet or DL diet, supplemented or not supplemented with DHA, from weaning for 6 weeks. All diets provided the same quantity of 2.3% of total fatty acids of precursor α-linolenic acid. LCPUFA levels in brain, retina, liver, heart, red blood cells and epididymal adipose tissue, Δ-6 desaturase activity and mRNA expression in liver, and plasma cholesterol were measured. Rats fed a DL diet increased their DHA content in brain and retina compared with rats fed a vegetable oil diet and reached the same level than rats directly supplemented with DHA. The status of n-3 docosapentaenoic acid increased with DL diet in heart, red blood cells and liver. The n-3 docosapentaenoic acid specifically discriminated DL diets in the heart. DL diet increased α-linolenic acid content in liver and epididymal adipose tissue, provided specific fatty acids as short- and medium-chain fatty acids and myristic acid, and increased plasma cholesterol. We hypothesized that dairy lipids may increase the n-3 LCPUFA enrichment in tissues by preserving precursor α-linolenic acid from ß-mitochondrial oxidation, associated with the presence of short- and medium-chain fatty acids in DL diets. In conclusion, a partial incorporation of dairy lipids in the diet with an adequate α-linolenic acid content improved the n-3 LCPUFA status, especially DHA in brain and retina.

Addition of dairy lipids and probiotic Lactobacillus fermentum in infant formula programs gut microbiota and entero-insular axis in adult minipigs.

Clinical and animal studies have demonstrated beneficial effects of early consumption of dairy lipids and a probiotic, Lactobacillus fermentum (Lf), on infant gut physiology. The objective of this study was to investigate their long-term effects on gut microbiota and host entero-insular axis and metabolism. Piglets were suckled with a milk formula containing only plant lipids (PL), a half-half mixture of plant lipids and dairy lipids (DL), or this mixture supplemented with Lf (DL + Lf). They were weaned on a standard diet and challenged with a high-energy diet until postnatal day 140. DL and DL + Lf modulated gut microbiota composition and metabolism, increasing abundance of several Clostridia genera. Moreover, DL + Lf specifically decreased the faecal content of 2-oxoglutarate and lysine compared to PL and 5-aminovalerate compared to PL and DL. It also increased short-chain fatty acid concentrations like propionate compared to DL. Furthermore, DL + Lf had a beneficial effect on the endocrine function, enhancing caecal GLP-1 and GLP-1 meal-stimulated secretion. Correlations highlighted the consistent relationship between microbiota and gut physiology. Together, our results evidence a beneficial programming effect of DL + Lf in infant formula composition on faecal microbiota and entero-insular axis function.

Soluble Milk Protein Supplementation with Moderate Physical Activity Improves Locomotion Function in Aging Rats.

Aging is associated with a loss of muscle mass and functional capacity. Present study was designed to compare the impact of specific dairy proteins on muscular function with or without a low-intensity physical activity program on a treadmill in an aged rat model. We investigated the effects of nutritional supplementation, five days a week over a 2-month period with a slow digestible protein, casein or fast digestible proteins, whey or soluble milk protein, on strength and locomotor parameters in sedentary or active aged Wistar RjHan rats (17-19 months of age). An extensive gait analysis was performed before and after protein supplementation. After two months of protein administration and activity program, muscle force was evaluated using a grip test, spontaneous activity using an open-field and muscular mass by specific muscle sampling. When aged rats were supplemented with proteins without exercise, only minor effects of different diets on muscle mass and locomotion were observed: higher muscle mass in the casein group and improvement of stride frequencies with soluble milk protein. By contrast, supplementation with soluble milk protein just after physical activity was more effective at improving overall skeletal muscle function in old rats compared to casein. For active old rats supplemented with soluble milk protein, an increase in locomotor activity in the open field and an enhancement of static and dynamic gait parameters compared to active groups supplemented with casein or whey were observed without any differences in muscle mass and forelimb strength. These results suggest that consumption of soluble milk protein as a bolus immediately after a low intensity physical activity may be a suitable nutritional intervention to prevent decline in locomotion in aged rats and strengthen the interest to analyze the longitudinal aspect of locomotion in aged rodents.

Ovariectomy and 17ß-estradiol alter transcription of lipid metabolism genes and proportions of neo-formed n-3 and n-6 long-chain polyunsaturated fatty acids differently in brain and liver.

Hormonal and nutritional factors regulate the metabolism of long-chain polyunsaturated fatty acids (LC-PUFA). We aimed to determine whether ovarian hormones influence the capacity of rats to synthesize the end-products 22:6n-3 (DHA) and 22:5n-6 (n-6DPA) from their respective dietary precursors (18:3n-3 and 18:2n-6), and can regulate PUFA conversion enzymes gene transcription in brain and/or liver. Females born with a low DHA status were fed from weaning to 8 weeks of age a diet providing both essential precursors, and were concurrently submitted to sham-operated control (SOC) or ovariectomy (OVX) in combination with or without 17ß-estradiol (E2) dosed at 8 or 16 µg/day. Relative to SOC, OVX increased the hepatic Δ9-, Δ6- and Δ5-desaturase transcripts and cognate transcription factors (PPARα, PPARγ, RXRα, RARα), but it did not affect LC-PUFA contents in phospholipids. In comparison with SOC and OVX groups, both E2 doses prevented the increase of transcripts, while paradoxically augmenting DHA and n-6DPA in liver phospholipids. Thus, in the liver of rats undergoing ovariectomy, changes of LC-PUFA synthesizing enzyme transcripts and of LC-PUFA proportions were not correlated. In brain, ovariectomy did not modify the transcripts of lipid metabolism genes, but it decreased DHA (-15%) and n-6DPA (-28%). In comparison with SOC and OVX groups, ovariectomized females treated with E2 preserved their status of both LC-PUFA in brain and had increased transcripts of E2 receptor ß, PPARδ, RARα and LC-PUFA synthesizing enzymes. In conclusion, E2 sustained the transcription of lipid metabolism genes and proportions of neo-formed DHA and n-6DPA differently in brain and liver.

Differential effects of steroids on the synthesis of polyunsaturated fatty acids by human neuroblastoma cells.

Polyunsaturated fatty acids (PUFA) are crucial for proper functioning of cell membranes, particularly in brain. Biologically important PUFA include docosahexaenoic acid (n-3 series) and arachidonic acid (n-6 series) which can be formed from their respective dietary essential precursors, alpha-linolenic acid (ALA) and linoleic acid (LA). Steroid hormones are thought to modulate PUFA synthesis in humans but whether they regulate PUFA status in brain and/or in neural membranes is unknown. In human neuroblastoma SH-SY5Y cells, we compared the effect of estradiol, testosterone, and progesterone on PUFA synthesis. Cells were incubated with ALA and/or LA 7 microM in combination with estradiol, testosterone, or progesterone at 10 nM without serum. The fatty acid composition was determined by gas chromatography and the mRNA expression of genes involved in PUFA metabolism by real-time RT-PCR. Estradiol affected both the n-3 and the n-6 PUFA conversion, the n-3 PUFA pathway being more sensitive to the estradiol treatment. In ALA-supplemented cells, estradiol increased while testosterone decreased the long-chain n-3 PUFA content (+17% and -15%, respectively) and the mRNA expression of the Delta5-desaturase (+11% and -9%), these two events being strongly correlated. Progesterone did not affect the PUFA composition. The positive effect of estradiol was blocked by the estrogen receptor antagonist ICI-182,780. We conclude that steroids have differential effects on PUFA synthesis and that their mode of action could involve the modulation of the Delta5-desaturase mRNA expression in neuroblastoma cells. These results help our understanding of the regulation of brain PUFA metabolism by steroid hormones.