Bénédicte Langelier

Replication of Obesity and Associated Signaling Pathways Through Transfer of Microbiota From Obese-Prone Rats

Aberrations in gut microbiota are associated with metabolic disorders, including obesity. However, whether shifts in the microbiota profile during obesity are a characteristic of the phenotype or a consequence of obesogenic feeding remains elusive. Therefore, we aimed to determine differences in the gut microbiota of obese-prone (OP) and obese-resistant (OR) rats and examined the contribution of this microbiota to the behavioral and metabolic characteristics during obesity. We found that OP rats display a gut microbiota distinct from OR rats fed the same high-fat diet, with a higher Firmicutes-to-Bacteroidetes ratio and significant genera differences. Transfer of OP but not OR microbiota to germ-free (GF) mice replicated the characteristics of the OP phenotype, including reduced intestinal and hypothalamic satiation signaling, hyperphagia, increased weight gain and adiposity, and enhanced lipogenesis and adipogenesis. Furthermore, increased gut permeability through conventionalization resulted in inflammation by proinflammatory nuclear factor (NF)-κB/inhibitor of NF-κB kinase subunit signaling in adipose tissue, liver, and hypothalamus. OP donor and GF recipient animals harbored specific species from Oscillibacter and Clostridium clusters XIVa and IV that were completely absent from OR animals. In conclusion, susceptibility to obesity is characterized by an unfavorable microbiome predisposing the host to peripheral and central inflammation and promoting weight gain and adiposity during obesogenic feeding.

Comparative bioavailability of dietary α-linolenic and docosahexaenoic acids in the growing rat

Animal and human studies have indicated that developing mammals fed only α-linolenic acid (18∶3n−3) have lower docosahexaenoic acid (22∶6n−3) content in brain and tissue phospholipids when compared with mammals fed 18∶3n−3 plus 22∶6n−3. The aim of this study was to test the hypothesis that low bioavailability of dietary 18∶−3 to be converted to 22∶6n−3 could partly explain this difference in fatty acid accretion. For that purpose, we determined the partitioning of dietary 18∶3n−3 and 22∶6n−3 between total n−3 fatty acid body accumulation, excretion, and disappearance (difference between the intake and the sum of total n−3 fatty acids accumulated and excreted). This was assessed using the quantitative method of whole-body fatty acid balance in growing rats fed the same amount of a 5% fat diet supplying either 18∶3n−3 or 22∶6n−3 at a level of 0.45% of dietary energy (i.e., 200 mg/100 g diet). We found that 58.9% of the total amount of 18∶3n−3 ingested disappeared, 0.4% was excreted in feces, 21.2% accumulated as 18∶3n−3 (50% in total fats and 46% in the carcass-skin compartment), and 17.2% accumulated as long-chain derivatives (14% as 22∶6n−3 and 3.2% as 20∶5n−3+22∶5n−3). Similar results were obtained from the docosahexaenoate balance (as % of the total amount ingested): disappearance, 64.5%; excretion, 0.5%; total accumulation, 35% with 30.1% as 22∶6n−3. Thus, rats fed docosahexaenoate accumulated a twofold higher amount of 22∶6n−3, which was mainly deposited in the carcass-skin compartment (68%). Similar proportions of disappearance of dietary 18∶−3 and 22∶6n−3 lead us to speculate that these two n−3 polyunsaturated fatty acids were β-oxidized in the same amount.

Long chain-polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures.

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010.

Changes of the transcriptional and fatty acid profiles in response to n-3 fatty acids in SH-SY5Y neuroblastoma cells

Synthesis of docosahexaenoic acid (DHA) from its metabolic precursors contributes to membrane incorporation of this FA within the central nervous system. Although cultured neural cells are able to produce DHA, the membrane DHA contents resulting from metabolic conversion do not match the high values of those resulting from supplementation with preformed DHA. We have examined whether the DHA precursors downregulate the incorporation of newly formed DHA within human neuroblastoma cells. SH-SY5Y cells were incubated with gradual doses of α-linolenic acid (α-LNA), EPA, or docosapentaenoic acid (DPA), and the incorporation of DHA into ethanolamine glycerophospholipids was analyzed as a reflection of synthesizing activity. The incorporation of EPA, DPA, and preformed DHA followed a dose-response saturating curve, whereas that of DHA synthesized either from α-LNA, EPA, or DPA peaked at concentrations of precursors below 15–30 μM and sharply decreased with higher doses. The mRNA encoding for six FA metabolism genes were quantified using real-time PCR. Two enzymes of the peroxisomal β-oxidation, L-bifunctional protein and peroxisomal acyl-CoA oxidase, were expressed at lower levels than fatty acyl-CoA ligase 3 (FACL3) and Δ6-desaturase (Δ6-D). The Δ6-D mRNA slightly increased between 16 and 48 h of culture, and this effect was abolished in the presence of 70 μM EPA. In contrast, the EPA treatment resulted in a time-dependent increase of FACL3 mRNA. The terminal step of DHA synthesis seems to form a “metabolic bottleneck,” resulting in accretion of EPA and DPA when the precursor concentration exceeds a specific threshold value. We conclude that the critical precursor-concentration window of responsiveness may originate from the low basal expression level of peroxisomal enzymes.

n-3 long-chain fatty acids and regulation of glucose transport in two models of rat brain endothelial cells.

Several in vivo studies suggest that docosahexaenoic acid (22:6 n-3), the main n-3 long-chain polyunsaturated fatty acids (LC-PUFA) of brain membranes, could be an important regulator of brain energy metabolism by affecting glucose utilization and the density of the two isoforms of the glucose transporter-1 (GLUT1) (endothelial and astrocytic). This study was conducted to test the hypothesis that 22:6 n-3 in membranes may modulate glucose metabolism in brain endothelial cells. It compared the impact of 22:6 n-3 and the other two main LC-PUFA, arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3), on fatty acid composition of membrane phospholipids, glucose uptake and expression of 55-kDa GLUT1 isoform in two models of rat brain endothelial cells (RBEC), in primary culture and in the immortalized rat brain endothelial cell line RBE4. Without PUFA supplementation, both types of cerebral endothelial cells were depleted in 22:6 n-3, RBE4 being also particularly low in 20:4 n-6. After exposure to supplemental 20:4 n-6, 20:5 n-3 or 22:6 n-3 (15microM, i.e. a physiological dose), RBEC and RBE4 avidly incorporated these PUFA into their membrane phospholipids thereby resembling physiological conditions, i.e. the PUFA content of rat cerebral microvessels. However, RBE4 were unable to incorporate physiological level of 20:4 n-6. Basal glucose transport in RBEC (rate of [(3)H]-3-o-methylglucose uptake) was increased after 20:5 n-3 or 22:6 n-3 supplementation by 50% and 35%, respectively, whereas it was unchanged with 20:4 n-6. This increase of glucose transport was associated with an increased GLUT1 protein, while GLUT1 mRNA was not affected. The different PUFA did not impact on glucose uptake in RBE4. Due to alterations in n-6 PUFA metabolism and weak expression of GLUT1, RBE4 seems to be less adequate than RBEC to study PUFA metabolism and glucose transport in brain endothelial cells. Physiological doses of n-3 LC-PUFA have a direct and positive effect on glucose transport and GLUT1 density in RBEC that could partly explain decreased brain glucose utilization in n-3 PUFA-deprived rats.

Ovariectomy and 17β-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 17β-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 β, 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.

Perinatal high-fat diet increases hippocampal vulnerability to the adverse effects of subsequent high-fat feeding

Epidemiological observations report an increase in fat consumption associated with low intake of n-3 relative to n-6 polyunsaturated fatty acids (PUFAs) in women of childbearing age. However, the impact of these maternal feeding habits on cognitive function in the offspring is unknown. This study aims to investigate the impact of early exposure to a high-fat diet (HFD) with an unbalanced n-6/n-3 PUFAs ratio on hippocampal function in adult rats. Furthermore, we explored the effects of perinatal HFD combined with exposure to HFD after weaning. Dams were fed a control diet (C, 12% of energy from lipids, n-6/n-3 PUFAs ratio: 5) or HFD (HF, 39% of energy from lipids, n-6/n-3 PUFAs ratio: 39) throughout gestation and lactation. At weaning, offspring were placed either on control (C-C, HF-C) or high-fat (HF-HF) diets. In adulthood, hippocampus-dependent memory was assessed using the water-maze task and potential hippocampal alterations were determined by studying PUFA levels, gene expression, neurogenesis and astrocyte morphology. Perinatal HFD induced long-lasting metabolic alterations and some changes in gene expression in the hippocampus, but had no effect on memory. In contrast, spatial memory was impaired in animals exposed to HFD during the perinatal period and maintained on this diet. HF-HF rats also exhibited low n-3 and high n-6 PUFA levels, decreased neurogenesis and downregulated expression of several plasticity-related genes in the hippocampus. To determine the contribution of the perinatal diet to the memory deficits reported in HF-HF animals, an additional experiment was conducted in which rats were only exposed to HFD starting at weaning (C-HF). Interestingly, memory performance in this group was similar to controls. Overall, our results suggest that perinatal exposure to HFD with an unbalanced n-6/n-3 ratio sensitizes the offspring to the adverse effects of subsequent high-fat intake on hippocampal function.

Impact of high-fat feeding on basic helix-loop-helix transcription factors controlling enteroendocrine cell differentiation.

Impact of high-fat feeding on basic helix–loop–helix transcription factors controlling enteroendocrine cell differentiation

Docosahexaenoic acid membrane content and mRNA expression of acyl-CoA oxidase and of peroxisome proliferator-activated receptor-δ are modulated in Y79 retinoblastoma cells differently by low and high doses of α-linolenic acid

The mRNA expression levels of acyl‐CoA oxidase (AOX), a key enzyme in very‐long‐chain fatty acid peroxisomal oxidation, and of peroxisome proliferator‐activated receptor‐δ (PPAR‐δ), a nuclear receptor possibly involved in the gene regulation of brain lipid metabolism, were determined in human Y79 retinoblastoma cells by using real‐time quantitative polymerase chain reaction. Cells were dosed with α‐linolenic acid (18:3n‐3), the essential metabolic precursor of the n‐3 polyunsaturated fatty acid series that normally gives rise through terminal peroxisomal oxidation to the synthesis of membrane docosahexaenoic acid (22:6n‐3, or DHA). The AOX and PPAR‐δ relative expression levels increased 2.3 and 3.4 times, respectively, upon dosing of cells with 7 μM 18:3n‐3, whereas AOX cDNA abundance decreased by 50% upon dosing with 70 μM 18:3n‐3. Concurrently, the DHA content increased by 23% in the membrane ethanolamine‐phosphoglycerides from cells dosed with 7 μM 18:3n‐3, whereas it decreased by 38% upon dosing with 70 μM 18:3n‐3. The DHAs upstream precursors (20:5n‐3 and 22:5n‐3) both accumulated in cells dosed with 7 or 70 μM 18:3n‐3. The 18:3n‐3‐induced changes in membrane phospholipid fatty acid composition support the hypothesis that the terminal peroxisomal step of n‐3 conversion is rate limiting in the Y79 line. The concurrent 7 μM 18:3n‐3‐induced increase of mRNAs encoding for AOX and for PPAR‐δ suggests that 18:3n‐3 (or its metabolites) at low concentration could trigger its proper conversion to DHA, possibly through activation of PPAR‐δ‐mediated transcription of AOX. Decreased membrane DHA content and mRNA expression level of AOX in 70‐μM 18:3n‐3‐dosed cells corroborated the relationship between AOX expression and DHA synthesis and suggested that simultaneous down‐regulating events occurred at high concentrations of 18:3n‐3.

Gene expression of fatty acid transport and binding proteins in the blood-brain barrier and the cerebral cortex of the rat: differences across development and with different DHA brain status.

Specific mechanisms for maintaining docosahexaenoic acid (DHA) concentration in brain cells but also transporting DHA from the blood across the blood-brain barrier (BBB) are not agreed upon. Our main objective was therefore to evaluate the level of gene expression of fatty acid transport and fatty acid binding proteins in the cerebral cortex and at the BBB level during the perinatal period of active brain DHA accretion, at weaning, and until the adult age. We measured by real time RT-PCR the mRNA expression of different isoforms of fatty acid transport proteins (FATPs), long-chain acyl-CoA synthetases (ACSLs), fatty acid binding proteins (FABPs) and the fatty acid transporter (FAT)/CD36 in cerebral cortex and isolated microvessels at embryonic day 18 (E18) and postnatal days 14, 21 and 60 (P14, P21 and P60, respectively) in rats receiving different n-3 PUFA dietary supplies (control, totally deficient or DHA-supplemented). In control rats, all the genes were expressed at the BBB level (P14 to P60), the mRNA levels of FABP5 and ACSL3 having the highest values. Age-dependent differences included a systematic decrease in the mRNA expressions between P14-P21 and P60 (2 to 3-fold), with FABP7 mRNA abundance being the most affected (10-fold). In the cerebral cortex, mRNA levels varied differently since FATP4, ACSL3 and ACSL6 and the three FABPs genes were highly expressed. There were no significant differences in the expression of the 10 genes studied in n-3 deficient or DHA-supplemented rats despite significant differences in their brain DHA content, suggesting that brain DHA uptake from the blood does not necessarily require specific transporters within cerebral endothelial cells and could, under these experimental conditions, be a simple passive diffusion process.