Fasting/refeeding: an experimental model to study the impact of early thermal manipulation on hepatic metabolism in mule ducks.

An increase in egg incubation temperature was previously shown to enhance the metabolism of mule ducks and increase liver fattening after overfeeding, through a metabolic programming mechanism. Here, we examined whether fasting (F) followed by refeeding (RF) in 11-wk-old mule ducks could become an accelerated model to study the mechanisms of metabolic programming following embryonic thermal manipulation. This study investigated the hepatic response of mule ducks subjected to 23 h of fasting and 1 h of refeeding, in control or thermally programmed animals (with an increase of 1°C, 16 h per day from days 13 to 27 of embryogenesis). Liver weight and energy composition, hepatocyte structure, plasma parameters, and gene expression levels were measured at 1, 2, and 4 h after RF. All these parameters were strongly affected by RF, whereas significant impacts of embryonic programming were measured in cell size (+1 µm on average), lipid composition (+4.2% of saturated fatty acids 4 h after the meal), and relative gene expressions (including HK1, SCD1, ELOVL6, and FASN). In addition to confirming previously identified molecular targets of thermal manipulation, this study revealed new ones, thanks to kinetic sampling after RF. Finally, the detailed description of the impact of the F/RF challenge on the liver structure, composition, and gene expression, but also on plasma parameters allowed us to draw a parallel with these same traits measured during overfeeding. This comparative analysis suggests that this protocol could become a pertinent model to study the mechanisms involved in embryonic liver thermal programming, without overfeeding.

Ontogeny of hepatic metabolism in mule ducks highlights different gene expression profiles between carbohydrate and lipid metabolic pathways.

BACKGROUND: The production of foie gras involves different metabolic pathways in the liver of overfed ducks such as lipid synthesis and carbohydrates catabolism, but the establishment of these pathways has not yet been described with precision during embryogenesis. The early environment can have short- and long-term impacts on the physiology of many animal species and can be used to influence physiological responses that is called programming. This study proposes to describe the basal hepatic metabolism at the level of mRNA in mule duck embryos in order to reveal potential interesting programming windows in the context of foie gras production. To this end, a kinetic study was designed to determine the level of expression of selected genes involved in steatosis-related liver functions throughout embryogenesis. The livers of 20 mule duck embryos were collected every 4 days from the 12th day of embryogenesis (E12) until 4 days after hatching (D4), and gene expression analysis was performed. The expression levels of 50 mRNAs were quantified for these 7 sampling points and classified into 4 major cellular pathways. RESULTS: Interestingly, most mRNAs involved in lipid metabolism are overexpressed after hatching (FASN, SCD1, ACOX1), whereas genes implicated in carbohydrate metabolism (HK1, GAPDH, GLUT1) and development (HGF, IGF, FGFR2) are predominantly overexpressed from E12 to E20. Finally, regarding cellular stress, gene expression appears quite stable throughout development, contrasting with strong expression after hatching (CYP2E1, HSBP1, HSP90AA1). CONCLUSION: For the first time we described the kinetics of hepatic ontogenesis at mRNA level in mule ducks and highlighted different expression patterns depending on the cellular pathway. These results could be particularly useful in the design of embryonic programming for the production of foie gras.

Kinetic study of the expression of genes related to hepatic steatosis, glucose and lipid metabolism, and cellular stress during overfeeding in mule ducks.

Induced by overfeeding, hepatic steatosis is a process exploited for the "foie gras" production in mule ducks. To better understand the mechanisms underlying its development, the physiological responses of mule ducks overfed with corn for a duration of 11 days were analyzed. A kinetic analysis of glucose and lipid metabolism and cell protection mechanisms was performed on 96 male mule ducks during overfeeding with three sampling times (after the 4th, the 12th, and the 22nd meal). Gene expression and protein analysis realized on the liver, muscle, and abdominal fat showed an activation of a cholesterol biosynthetic pathway during the complete overfeeding period mainly in livers with significant correlations between its weight and its cholesterolemia (r = 0.88; P < 0.0001) and between the liver weight and the hmgcr and soat1 expression (r = 0.4, P < 0.0001 and r = 0.67; P < 0.0001, respectively). Results also revealed an activation of insulin and amino acid cells signaling a pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and use via glycolysis and lipogenesis. Cellular stress analysis revealed an upregulation of key autophagy-related gene expression atg8 and sqstm1(P < 0.0001) during the complete overfeeding period, mainly in the liver, in contrast to an induction of cyp2e1(P < 0.0001), suggesting that autophagy could be suppressed during steatosis development. This study has highlighted different mechanisms enabling mule ducks to efficiently handle the starch overload by keeping its liver in a nonpathological state. Moreover, it has revealed potential biomarker candidates of hepatic steatosis as plasma cholesterol for the liver weight.

Inter genotype differences in expression of genes involved in glucose metabolism in the establishment of hepatic steatosis in Muscovy, Pekin and mule ducks.

In waterfowls, overfeeding leads to a hepatic steatosis, also called "foie gras". Our main objectives were to determine what is the share of genes involvement of glucose metabolism in the establishment of fatty liver in three genotypes of waterfowls: Muscovy (Cairina moschata), Pekin ducks (Anas platyrhynchos) and their crossbreed, the mule duck. 288 male ducks of Pekin, Muscovy and mule genotypes were reared until weeks 12 and overfed between weeks 12 and 14. We analysed gene expression at the beginning, the middle and the end of the overfeeding period in different tissues. We have shown an upregulation of glucose transporters (GLUT) in peripheral tissues (pectoralis major or adipose tissue) in Pekin ducks. In addition, GLUT2 was not found in jejunal mucosa and another GLUT seems to replace it 3 h after the meal: GLUT3. Mule ducks upregulating GLUT3 earlier compared to Pekin ducks. However, these results need further investigations. In liver, globally, Pekin ducks exhibit the highest expression of GLUT or enzymes implicated in glycolysis. The few significant variations of gene expressions in glucose metabolism between these three genotypes and the momentary specific overexpression of GLUT do not allow us to detect a lot of specific genotype differences. To conclude, the differences in response to overfeeding of Pekin, Muscovy and mule ducks, for the establishment of hepatic steatosis, cannot be only explained by the glucose metabolism at transcriptomic level.

α-Lipoic acid treatment increases mitochondrial biogenesis and promotes beige adipose features in subcutaneous adipocytes from overweight/obese subjects.

α-Lipoic acid (α-Lip) is a natural occurring antioxidant with beneficial anti-obesity properties. The aim of this study was to investigate the putative effects of α-Lip on mitochondrial biogenesis and the acquirement of brown-like characteristics by subcutaneous adipocytes from overweight/obese subjects. Thus, fully differentiated human subcutaneous adipocytes were treated with α-Lip (100 and 250µM) for 24h for studies on mitochondrial content and morphology, mitochondrial DNA (mtDNA) copy number, fatty acid oxidation enzymes and brown/beige characteristic genes. The involvement of the Sirtuin1/Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (SIRT1/PGC-1α) pathway was also evaluated. Our results showed that α-Lip increased mitochondrial content in cultured human adipocytes as revealed by electron microscopy and by mitotracker green labeling. Moreover, an enhancement in mtDNA content was observed. This increase was accompanied by an up-regulation of SIRT1 protein levels, a decrease in PGC-1α acetylation and up-regulation of Nuclear respiratory factor 1 (Nrf1) and Mitochondrial transcription factor (Tfam) transcription factors. Enhanced oxygen consumption and fatty acid oxidation enzymes, Carnitine palmitoyl transferase 1 and Acyl-coenzyme A oxidase (CPT-1 and ACOX) were also observed. Mitochondria from α-Lip-treated adipocytes exhibited some morphological characteristics of brown mitochondria, and α-Lip also induced up-regulation of some brown/beige adipocytes markers such as cell death-inducing DFFA-like effector a (Cidea) and T-box 1 (Tbx1). Moreover, α-Lip up-regulated PR domain containing 16 (Prdm16) mRNA levels in treated adipocytes. Therefore, our study suggests the ability of α-Lip to promote mitochondrial biogenesis and brown-like remodeling in cultured white subcutaneous adipocytes from overweight/obese donors.

Partial inhibition of adipose tissue lipolysis improves glucose metabolism and insulin sensitivity without alteration of fat mass.

When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet-fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.

Fatty acids from fat cell lipolysis do not activate an inflammatory response but are stored as triacylglycerols in adipose tissue macrophages.

AIMS/HYPOTHESIS: Activation of macrophages by fatty acids (FAs) is a potential mechanism linking obesity to adipose tissue (AT) inflammation and insulin resistance. Here, we investigated the effects of FAs released during adipocyte lipolysis on AT macrophages (ATMs). METHODS: Human THP-1 macrophages were treated with media from human multipotent adipose-derived stem (hMADS) adipocytes stimulated with lipolytic drugs. Macrophages were also treated with mixtures of FAs and an inhibitor of Toll-like receptor 4, since this receptor is activated by saturated FAs. Levels of mRNA and the secretion of inflammation-related molecules were measured in macrophages. FA composition was determined in adipocytes, conditioned media and macrophages. The effect of chronic inhibition or acute activation of fat cell lipolysis on ATM response was investigated in vivo in mice. RESULTS: Whereas palmitic acid alone activates THP-1, conditioned media from hMADS adipocyte lipolysis had no effect on IL, chemokine and cytokine gene expression, and secretion by macrophages. Mixtures of FAs representing de novo lipogenesis or habitual dietary conditions also had no effect. FAs derived from adipocyte lipolysis were taken up by macrophages and stored as triacylglycerol droplets. In vivo, chronic treatment with an antilipolytic drug did not modify gene expression and number of ATMs in mice with intact or defective Tlr4. Stimulation of adipocyte lipolysis increased storage of neutral lipids by macrophages without change in number and phenotype. CONCLUSIONS/INTERPRETATION: Our data suggest that adipocyte lipolysis does not activate inflammatory pathways in ATMs, which instead may act as scavengers of FAs.

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1ß expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1ß expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.

Interleukin-1ß inhibition prevents choroidal neovascularization and does not exacerbate photoreceptor degeneration.

The pro-inflammatory cytokine IL-1ß has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1ß in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1ß expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1ß was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1ß significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1ß on choroidal endothelial cell proliferation. Inhibition of IL-1ß in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1ß inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.

Impacts of Embryonic Thermal Programming on the Expression of Genes Involved in Foie gras Production in Mule Ducks.

Embryonic thermal programming has been shown to improve foie gras production in overfed mule ducks. However, the mechanisms at the origin of this programming have not yet been characterized. In this study, we investigated the effect of embryonic thermal manipulation (+1°C, 16 h/24 h from embryonic (E) day 13 to E27) on the hepatic expression of genes involved in lipid and carbohydrate metabolisms, stress, cell proliferation and thyroid hormone pathways at the end of thermal manipulation and before and after overfeeding (OF) in mule ducks. Gene expression analyses were performed by classic or high throughput real-time qPCR. First, we confirmed well-known results with strong impact of OF on the expression of genes involved in lipid and carbohydrates metabolisms. Then we observed an impact of OF on the hepatic expression of genes involved in the thyroid pathway, stress and cell proliferation. Only a small number of genes showed modulation of expression related to thermal programming at the time of OF, and only one was also impacted at the end of the thermal manipulation. For the first time, we explored the molecular mechanisms of embryonic thermal programming from the end of heat treatment to the programmed adult phenotype with optimized liver metabolism.

CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.

The role of retinal microglial cells (MCs) in age-related macular degeneration (AMD) is unclear. Here we demonstrated that all retinal MCs express CX3C chemokine receptor 1 (CX3CR1) and that homozygosity for the CX3CR1 M280 allele, which is associated with impaired cell migration, increases the risk of AMD. In humans with AMD, MCs accumulated in the subretinal space at sites of retinal degeneration and choroidal neovascularization (CNV). In CX3CR1-deficient mice, MCs accumulated subretinally with age and albino background and after laser impact preceding retinal degeneration. Raising the albino mice in the dark prevented both events. The appearance of lipid-bloated subretinal MCs was drusen-like on funduscopy of senescent mice, and CX3CR1-dependent MC accumulation was associated with an exacerbation of experimental CNV. These results show that CX3CR1-dependent accumulation of subretinal MCs evokes cardinal features of AMD. These findings reveal what we believe to be a novel pathogenic process with important implications for the development of new therapies for AMD.

Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.

PURPOSE: This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo. METHODS: Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA. RESULTS: The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction. CONCLUSIONS: This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

Positive Impact of Thermal Manipulation During Embryogenesis on Foie Gras Production in Mule Ducks.

Animal studies have shown that very early life events may have programing effects on adult metabolism and health. In this study, we aim, for the first, time to elucidate the effects of embryonic thermal manipulation (TM) on the performance of overfed mule ducks, in particular for the production of foie gras (fatty liver). We designed three embryonic TMs with different protocols for increasing the incubation temperature during the second part of embryogenesis, to determine whether hepatic metabolism could be "programed" to improve its fattening response to overfeeding at the age of three months. Initial results confirm that an increase in the incubation temperature leads to faster development (observed for all treated groups compared to the control group), and a decrease in the body surface temperature at birth. Thereafter, in a very innovative way, we showed that the three TM conditions specifically increased liver weights, as well as liver lipid content after overfeeding compared to the non-TM control group. These results demonstrate that embryonic TM effectively "programs" the metabolic response to the challenge of force-feeding, resulting in increased hepatic steatosis. Finally, our goal of improving foie gras production has been achieved with three different embryonic thermal stimuli, demonstrating the high reproducibility of the method. However, this repeatability was also perceptible in the adverse effects observed on two groups treated with exactly the same cumulative temperature rise leading to a reduction in hatchability (75 and 76% vs. 82% in control), in addition to an increase in the melting rate after cooking. These results suggest that embryonic thermal programing could be an innovative and inexpensive technique for improving foie gras production, although the specific protocol (duration, level or period of temperature increase), remains to be elucidated in order to avoid adverse effects.

CD36 Deficiency Inhibits Retinal Inflammation and Retinal Degeneration in Cx3cr1 Knockout Mice.

Background: CD36, a member of the class B scavenger receptor family, participates in Toll-like receptor signaling on mononuclear phagocytes (MP) and can promote sterile pathogenic inflammation. We here analyzed the effect of CD36 deficiency on retinal inflammation and photoreceptor degeneration, the hallmarks of age-related macular degeneration (AMD), that characterize Cx3cr1-/-mice. Methods: We analyzed subretinal MP accumulation, and cone- and rod-degeneration in light-challenged and aged, CD36 competent or deficient, hyper-inflammatory Cx3cr1-/- mice, using histology and immune-stained retinal flatmounts. Monocytes (Mo) were subretinally adoptively transferred to evaluate their elimination rate from the subretinal space and Interleukin 6 (IL-6) secretion from cultured Mo-derived cells (MdCs) of the different mouse strains were analyzed. Results: CD36 deficient Cx3cr1-/- mice were protected against age- and light-induced subretinal inflammation and associated cone and rod degeneration. CD36 deficiency in Cx3cr1-/- MPs inhibited their prolonged survival in the immune-suppressive subretinal space and reduced the exaggerated IL-6 secretion observed in Cx3cr1-/- MPs that we previously showed leads to increased subretinal MP survival. Conclusion:Cd36 deficiency significantly protected hyperinflammatory Cx3cr1-/- mice against subretinal MP accumulation and associated photoreceptor degeneration. The observed CD36-dependent induction of pro-inflammatory IL-6 might be at least partially responsible for the prolonged MP survival in the immune-suppressive environment and its pathological consequences on photoreceptor homeostasis.

Interaction between hormone-sensitive lipase and ChREBP in fat cells controls insulin sensitivity.

Impaired adipose tissue insulin signalling is a critical feature of insulin resistance. Here we identify a pathway linking the lipolytic enzyme hormone-sensitive lipase (HSL) to insulin action via the glucose-responsive transcription factor ChREBP and its target, the fatty acid elongase ELOVL6. Genetic inhibition of HSL in human adipocytes and mouse adipose tissue results in enhanced insulin sensitivity and induction of ELOVL6. ELOVL6 promotes an increase in phospholipid oleic acid, which modifies plasma membrane fluidity and enhances insulin signalling. HSL deficiency-mediated effects are suppressed by gene silencing of ChREBP and ELOVL6. Mechanistically, physical interaction between HSL, independent of lipase activity, and the isoform activated by glucose metabolism ChREBPα impairs ChREBPα translocation into the nucleus and induction of ChREBPß, the isoform with high transcriptional activity that is strongly associated with whole-body insulin sensitivity. Targeting the HSL-ChREBP interaction may allow therapeutic strategies for the restoration of insulin sensitivity.

CD36 deficiency leads to choroidal involution via COX2 down-regulation in rodents.

BACKGROUND: In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the "wet" form of AMD. In contrast, very little is known about the mechanisms of the predominant, "dry" form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD. METHODS AND FINDINGS: We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36-/- mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%-300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36-/- mice express reduced levels of COX2 and VEGF in vivo, and COX2-/- mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency. CONCLUSIONS: CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.

Th1- and Th2-related chemokine and chemokine receptor expression on the ocular surface in endotoxin-induced uveitis.

PURPOSE: To determine whether the ocular surface inflammation in uveitis mimics or counteracts intraocular inflammatory pathways by directly comparing T-helper (Th) lymphocytes Th1 and Th2 markers in conjunctival and ciliary body expression in endotoxin-induced uveitis (EIU). This study used the following inflammatory markers: chemokine receptor, CC chemokine receptor 4 (CCR4), and its ligand, macrophage-derived chemokine (MDC), to evaluate Th2 participation; chemokine receptor, CCR5, to evaluate the Th1 system; and its ligand, regulated on activation normal T cell expressed and secreted (RANTES), to evaluate both Th1 and Th2 systems. METHODS: Immunohistochemistry and real-time polymerase chain reaction (RT-PCR) were used to compare protein and RNA expression of CCR4, MDC, CCR5, and RANTES in the conjunctiva and ciliary body in EIU 6 h and 24 h after the lipopolysaccharide (LPS) injection and in control (without injection) Lewis rats. RESULTS: Immunohistochemistry with CCR5, RANTES, and MDC showed an increase in fluorescent staining in the conjunctiva and ciliary body in the rats with uveitis compared to the control rats. For CCR4, immunostaining was comparable in the conjunctiva and ciliary body and did not show any clear differences between control rats and rats with EIU. For RANTES, MDC, and CCR5, RT-PCR showed a significantly higher RNA expression in conjunctiva and in ciliary body at 6 h compared to 24 h and controls. For CCR4, RT-PCR did not illustrate any significant differences in conjunctiva and in ciliary body between all groups of animals. CONCLUSIONS: Protein and RNA expressions of RANTES, MDC, and CCR5 were higher in EIU rats than in control rats in the conjunctiva and ciliary body whereas the CCR4 level was not modified in the conjunctiva and ciliary body of EIU rats when compared to controls. Th1 activation seemed to predominate in this model with high levels of CCR5 expression and no increased expression of CCR4, but Th2 participation with MDC was noted. The expression of RANTES, MDC, CCR4 and CCR5 in EIU was quite similar between the conjunctiva and the ciliary body, so conjunctival inflammation might reproduce the intraocular inflammation, probably generated by local extension and diffusion in this model. If the ocular surface mimics intraocular inflammatory pathways, the conjunctiva may provide a new and easier access for uveitis studies.

Probiotics Strains Modulate Gut Microbiota and Lipid Metabolism in Mule Ducks.

BACKGROUND: Livestock production should respond to societal, environmental and economic changes. Since 2006 and the ban on antibiotics as growth factors in European Union, the use of probiotics has become widespread and has demonstrated the effect of intestinal microbiota on the performance of farm animals. OBJECTIVE: The aim of this study was to investigate the effect of supplementation with Lactobacillus salivarius (as a probiotics strain or combined with other strains) on zootechnical performance, metabolic and immune gene expression and intestinal microbiota diversity in mule ducks using high-throughput sequencing and real-time PCR. METHOD: The mule ducks were reared for 79 days and overfed for 12 days with or without probiotics. Samples were collected at 14 (starting period) and 91 days (end of overfeeding period), 3 hours post feeding. RESULTS: Irrespective of digestive content, age, level of feed intake or supplementation with probiotics, Firmicutes, Proteobacteria and Bacteroidetes were the dominant phyla in the bacterial community in mule ducks. At 14 days, both the ileal and cecal samples were dominated by Firmicutes (in particular the Clostridiales order). Overfeeding induced a shift between Clostridiales and Lactobacillales in the ileal samples whereas in the cecal samples, the relative abundance of Firmicutes decreased. Overfeeding also induced hepatic over-expression of Fatty Acid Synthase (FAS) and of the lipid transporter gene Fatty Acid Binding Protein 4 (FABP4). This increase in lipid metabolism genes is associated with a decrease in inflammatory response. CONCLUSION: Finally, probiotic supplementation had only a slight impact on gene expression and microbiota diversity, both at 14 days and after overfeeding.

Adipocyte lipolysis and insulin resistance.

Obesity-induced insulin resistance is a major risk factor for the development of type 2 diabetes. Basal fat cell lipolysis (i.e., fat cell triacylglycerol breakdown into fatty acids and glycerol in the absence of stimulatory factors) is elevated during obesity and is closely associated with insulin resistance. Inhibition of adipocyte lipolysis may therefore be a promising therapeutic strategy for treating insulin resistance and preventing obesity-associated type 2 diabetes. In this review, we explore the relationship between adipose lipolysis and insulin sensitivity. After providing an overview of the components of fat cell lipolytic machinery, we describe the hypotheses that may support the causality between lipolysis and insulin resistance. Excessive circulating fatty acids may ectopically accumulate in insulin-sensitive tissues and impair insulin action. Increased basal lipolysis may also modify the secretory profile of adipose tissue, influencing whole body insulin sensitivity. Finally, excessive fatty acid release may also worsen adipose tissue inflammation, a well-known parameter contributing to insulin resistance. Partial genetic or pharmacologic inhibition of fat cell lipases in mice as well as short term clinical trials using antilipolytic drugs in humans support the benefit of fat cell lipolysis inhibition on systemic insulin sensitivity and glucose metabolism, which occurs without an increase of fat mass. Modulation of fatty acid fluxes and, putatively, of fat cell secretory pattern may explain the amelioration of insulin sensitivity whereas changes in adipose tissue immune response do not seem involved.

α-lipoic acid reduces fatty acid esterification and lipogenesis in adipocytes from overweight/obese subjects.

OBJECTIVE: α-Lipoic acid (α-LA) is a natural occurring antioxidant with beneficial effects on obesity. The aim of this study was to investigate the putative effects of α-LA on triglyceride accumulation and lipogenesis in subcutaneous adipocytes from overweight/obese subjects and to determine the potential mechanisms involved. METHODS: Fully differentiated human subcutaneous adipocytes were treated with α-LA (100 and 250 µM) during 24 h for studying triglyceride content, de novo lipogenesis, and levels of key lipogenic enzymes. The involvement of AMP-activated protein kinase (AMPK) activation was also evaluated. RESULTS: α-LA down-regulated triglyceride content by inhibiting fatty acid esterification and de novo lipogenesis. These effects were mediated by reduction in fatty acid synthase (FAS), stearoyl-coenzyme A desaturase 1, and diacylglycerol O-acyltransferase 1 protein levels. Interestingly, α-LA increased AMPK and acetyl CoA carboxylase phosphorylation, while the presence of the AMPK inhibitor Compound C reversed the inhibition observed on FAS protein levels. CONCLUSIONS: α-LA down-regulates key lipogenic enzymes, inhibiting lipogenesis and reducing triglyceride accumulation through the activation of AMPK signaling pathway in human subcutaneous adipocytes from overweight/obese subjects.