Prenatal treatment with preimplantation factor improves early postnatal neurogenesis and cognitive impairments in a mouse model of Down syndrome.

Down syndrome (DS) is a genetic disease characterized by a supernumerary chromosome 21. Intellectual deficiency (ID) is one of the most prominent features of DS. Central nervous system defects lead to learning disabilities, motor and language delays, and memory impairments. At present, a prenatal treatment for the ID in DS is lacking. Subcutaneous administration of synthetic preimplantation factor (sPIF, a peptide with a range of biological functions) in a model of severe brain damage has shown neuroprotective and anti-inflammatory properties by directly targeting neurons and microglia. Here, we evaluated the effect of PIF administration during gestation and until weaning on Dp(16)1Yey mice (a mouse model of DS). Possible effects at the juvenile stage were assessed using behavioral tests and molecular and histological analyses of the brain. To test the influence of perinatal sPIF treatment at the adult stage, hippocampus-dependent memory was evaluated on postnatal day 90. Dp(16)1Yey pups showed significant behavioral impairment, with impaired neurogenesis, microglial cell activation and a low microglial cell count, and the deregulated expression of genes linked to neuroinflammation and cell cycle regulation. Treatment with sPIF restored early postnatal hippocampal neurogenesis, with beneficial effects on astrocytes, microglia, inflammation, and cell cycle markers. Moreover, treatment with sPIF restored the level of DYRK1A, a protein that is involved in cognitive impairments in DS. In line with the beneficial effects on neurogenesis, perinatal treatment with sPIF was associated with an improvement in working memory in adult Dp(16)1Yey mice. Perinatal treatment with sPIF might be an option for mitigating cognitive impairments in people with DS.

Spermatogonial depletion and a spermatogenesis defect in the Dp(16)1Yey mouse model of Down syndrome.

Down syndrome (DS), or Trisomy 21, is the most common chromosomal disorder in humans. Men with DS are infertile. The DYRK1A gene on Hsa21 is involved in several features of DS. Overexpression of the homolog dyrk1A disrupts primordial germ cell migration in zebrafish, and overexpression of Dyrk1A impairs gonadotropic axis function and the early stages of spermatogenesis in the mouse. Other genes on Hsa21 might be involved in the pathogenesis of infertility in DS. We investigated the Dp(16)1Yey mouse model of DS, which features segmental duplication of chromosome Mmu16 (orthologous to a large part of Hsa21 and carrying Dyrk1A and 112 other genes). Using an immunohistochemical assay for the spermatogonial marker STRA8, we observed spermatogonial depletion in the Dp(16)1Yey mouse. This was correlated with low mRNA expression of GFR1 (a marker of the self-renewal stem cell pool) in an RT-qPCR assay and low protein expression of PLZF (a marker of differentiating stem cells) in a slot-blot assay. Spermatogenesis was present but impaired, with a low sperm count, low protamine-1 expression, a low testis weight, and a low seminiferous tubule diameter. Low circulating luteinizing hormone and follicle-stimulating hormone levels and an elevated testis anti-Müllerian hormone level (as measured in ELISAs) revealed the presence of hypogonadotropic hypogonadism. The Dp(16)1Yey mouse model of DS recapitulates observations made in zebrafish and mice overexpressing DYRK1A homologs. The presence of an excess of Mmu16 material perturbs spermatogenesis and the gonadotropic axis. More generally, DYRK1A's role in human infertility (outside DS) remains to be characterized.

Hepatic deletion of serine palmitoyl transferase 2 impairs ceramide/sphingomyelin balance, bile acids homeostasis and leads to liver damage in mice.

Ceramides (Cer) have been shown as lipotoxic inducers, which disturb numerous cell-signaling pathways, leading to metabolic disorders such as type 2 diabetes. In this study, we aimed to determine the role of de novo hepatic ceramide synthesis in energy and liver homeostasis in mice. We generated mice lacking serine palmitoyltransferase 2 (Sptlc2), the rate limiting enzyme of ceramide de novo synthesis, in liver under albumin promoter. Liver function, glucose homeostasis, bile acid (BA) metabolism and hepatic sphingolipids content were assessed using metabolic tests and LC-MS. Despite lower expression of hepatic Sptlc2, we observed an increased concentration of hepatic Cer, associated with a 10-fold increase in neutral sphingomyelinase 2 (nSMase2) expression, and a decreased sphingomyelin content in the liver. Sptlc2ΔLiv mice were protected against obesity induced by high fat diet and displayed a defect in lipid absorption. In addition, an important increase in tauro-muricholic acid was associated with a downregulation of the nuclear BA receptor FXR target genes. Sptlc2 deficiency also enhanced glucose tolerance and attenuated hepatic glucose production, while the latter effect was dampened in presence of nSMase2 inhibitor. Finally, Sptlc2 disruption promoted apoptosis, inflammation and progressive development of hepatic fibrosis, worsening with age. Our data suggest a compensatory mechanism to regulate hepatic ceramides content from sphingomyelin hydrolysis, with deleterious impact on liver homeostasis. In addition, our results show the involvement of hepatic sphingolipid modulation in BA metabolism and hepatic glucose production in an insulin-independent manner, which highlight the still under-researched role of ceramides in many metabolic functions.

Homocysteine Metabolism Pathway Is Involved in the Control of Glucose Homeostasis: A Cystathionine Beta Synthase Deficiency Study in Mouse.

Cystathionine beta synthase (CBS) catalyzes the first step of the transsulfuration pathway from homocysteine to cystathionine, and its deficiency leads to hyperhomocysteinemia (HHcy) in humans and rodents. To date, scarce information is available about the HHcy effect on insulin secretion, and the link between CBS activity and the setting of type 2 diabetes is still unknown. We aimed to decipher the consequences of an inborn defect in CBS on glucose homeostasis in mice. We used a mouse model heterozygous for CBS (CBS+/-) that presented a mild HHcy. Other groups were supplemented with methionine in drinking water to increase the mild to intermediate HHcy, and were submitted to a high-fat diet (HFD). We measured the food intake, body weight gain, body composition, glucose homeostasis, plasma homocysteine level, and CBS activity. We evidenced a defect in the stimulated insulin secretion in CBS+/- mice with mild and intermediate HHcy, while mice with intermediate HHcy under HFD presented an improvement in insulin sensitivity that compensated for the decreased insulin secretion and permitted them to maintain a glucose tolerance similar to the CBS+/+ mice. Islets isolated from CBS+/- mice maintained their ability to respond to the elevated glucose levels, and we showed that a lower parasympathetic tone could, at least in part, be responsible for the insulin secretion defect. Our results emphasize the important role of Hcy metabolic enzymes in insulin secretion and overall glucose homeostasis.

Disruption of Pituitary Gonadotrope Activity in Male Rats After Short- or Long-Term High-Fat Diets Is Not Associated With Pituitary Inflammation.

Overnutrition is associated with the activation of inflammatory pathways in metabolically linked organs and an early hypothalamic inflammation is now known to disrupt the central control of metabolic function. Because we demonstrated that fatty acids (FA) target the pituitary and affect gonadotropin synthesis, we asked whether overnutrition induces pituitary inflammation that may contribute to obesity-associated disorders in the control of reproduction. We analyzed pituitary inflammation and hypothalamic-pituitary-testicular axis in male rats fed a short- (4 weeks) or long-term (20 weeks) high-fat diet. The effect of diet enrichment with the ω3 polyunsaturated FA, DHA, was also analyzed. After only 4 weeks and before weight gain of rats, high-fat diet caused a significant decrease in pituitary gonadotropin and hypothalamic GnRH transcript levels despite unchanged testosterone and inhibin B levels. Contrasting with the hypothalamus, there was no concomitant increases in gene expression of pituitary inflammatory mediators and even a reduction of prototypical cytokines such as interleukin-1ß and TNF-α. No inflammation was still detected in the pituitary after 20 weeks although gonadotropin transcripts and circulating levels were still altered. Gonadotropins were the only pituitary hormones remaining affected at this stage of the regimen, underlying a differential susceptibility of pituitary lineages to metabolic disorders. DHA enrichment of the diet did not prevent alterations of gonadotrope activity due to either a long- or a short-term high-fat diet although it blocked early hypothalamic inflammation and attenuated several metabolic effects. Taken together, our findings suggest that high-fat diet-induced defects in gonadotrope activity in male rats occurred despite a lack of pituitary inflammation.

DYRK1A Overexpression in Mice Downregulates the Gonadotropic Axis and Disturbs Early Stages of Spermatogenesis.

Down syndrome (DS) is the most common chromosomal disorder. It is responsible for intellectual disability (ID) and several medical conditions. Although men with DS are thought to be infertile, some spontaneous paternities have been reported. The few studies of the mechanism of infertility in men with DS are now dated. Recent research in zebrafish has indicated that overexpression of DYRK1A (the protein primarily responsible for ID in DS) impairs gonadogenesis at the embryonic stage. To better ascertain DYRK1A's role in infertility in DS, we investigated the effect of DYRK1A overexpression in a transgenic mouse model. We found that overexpression of DYRK1A impairs fertility in transgenic male mice. Interestingly, the mechanism in mice differs slightly from that observed in zebrafish but, with disruption of the early stages of spermatogenesis, is similar to that seen in humans. Unexpectedly, we observed hypogonadotropic hypogonadism in the transgenic mice.

Plasma triacylglycerols are biomarkers of ß-cell function in mice and humans.

OBJECTIVES: To find plasma biomarkers prognostic of type 2 diabetes, which could also inform on pancreatic ß-cell deregulations or defects in the function of insulin target tissues. METHODS: We conducted a systems biology approach to characterize the plasma lipidomes of C57Bl/6J, DBA/2J, and BALB/cJ mice under different nutritional conditions, as well as their pancreatic islet and liver transcriptomes. We searched for correlations between plasma lipids and tissue gene expression modules. RESULTS: We identified strong correlation between plasma triacylglycerols (TAGs) and islet gene modules that comprise key regulators of glucose- and lipid-regulated insulin secretion and of the insulin signaling pathway, the two top hits were Gck and Abhd6 for negative and positive correlations, respectively. Correlations were also found between sphingomyelins and islet gene modules that overlapped in part with the gene modules correlated with TAGs. In the liver, the gene module most strongly correlated with plasma TAGs was enriched in mRNAs encoding fatty acid and carnitine transporters as well as multiple enzymes of the ß-oxidation pathway. In humans, plasma TAGs also correlated with the expression of several of the same key regulators of insulin secretion and the insulin signaling pathway identified in mice. This cross-species comparative analysis further led to the identification of PITPNC1 as a candidate regulator of glucose-stimulated insulin secretion. CONCLUSION: TAGs emerge as biomarkers of a liver-to-ß-cell axis that links hepatic ß-oxidation to ß-cell functional mass and insulin secretion.

Obesity in Midlife Hampers Resting and Sensory-Evoked Cerebral Blood Flow in Mice.

OBJECTIVE: This study aimed to investigate the effects of a high-fat diet (HFD) and aging on resting and activity-dependent cerebral blood flow (CBF). METHODS: To run a comparison between obese and age-matched control animals, 6-week-old mice were fed either with regular chow or an HFD for 3 months or 8 months. Glucose tolerance and insulin sensitivity were assessed for metabolic phenotyping. Resting and odor-evoked CBF at the microvascular scale in the olfactory bulb (OB) was investigated by multiexposure speckle imaging. Immunolabeling-enabled imaging of solvent-cleared organs was used to analyze vascular density. The ejection fraction was studied by using cardioechography. Olfactory sensitivity was tested by using a buried-food test. RESULTS: Glucose intolerance and compromised odor-evoked CBF were observed in obese mice in the younger group. Prolonged HFD feeding triggered insulin resistance and stronger impairment in activity-dependent CBF. Aging had a specific negative impact on resting CBF. There was no decrease in vascular density in the OB of obese mice, although cardiac function was impaired at both ages. In addition, decreased olfactory sensitivity was observed only in the older, middle-aged obese mice. CONCLUSIONS: OB microvasculature in obese mice showed a specific functional feature characterized by impaired sensory-evoked CBF and a specific deleterious effect of aging on resting CBF.

Ethanol-Induced Changes in Brain of Transgenic Mice Overexpressing DYRK1A.

Alcoholism is a chronic relapsing disorder defined by loss of control over excessive consumption of ethanol despite damaging effects on the liver and brain. We previously showed that the overexpression in mice of Dyrk1A (TgDyrk1A, for dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A) reduces the severity of alcohol mediated liver injury. Ethanol consumption has also been associated with increased brain glutamate concentration that led to therapies targeting glutamatergic receptors and normalization of glutamatergic neurotransmission. Interestingly, mice overexpressing Dyrk1A (TgDyrk1A mice) present a reduction of glutamatergic brain transmission, which we propose could be protective against alcohol intake. To answer this question, we investigated the ethanol preference in TgDyrk1A mice using a two-bottle choice paradigm. TgDyrk1A mice showed a non-significant decrease of voluntary ethanol intake and ethanol preference compared with wild-type mice. At the peripheral level, mice overexpressing Dyrk1A show lower ethanol plasma levels, indicating a faster ethanol metabolism. At the end of the protocol, lasting 21 days, brains were extracted for protein analysis. Ethanol reduced levels of the synaptic protein PSD-95 and increased the glutamate decarboxylase GAD65, specifically in the cortex of TgDyrk1A mice. Our results suggest that overexpression of DYRK1A may cause different ethanol-induced changes in the brain.

New roles for prokineticin 2 in feeding behavior, insulin resistance and type 2 diabetes: Studies in mice and humans.

OBJECTIVE: Prokineticin 2 (PROK2) is a hypothalamic neuropeptide that plays a critical role in the rhythmicity of physiological functions and inhibits food intake. PROK2 is also expressed in the main olfactory bulb (MOB) as an essential factor for neuro-and morphogenesis. Since the MOB was shown to be strongly involved in eating behavior, we hypothesized that PROK2 could be a new target in the regulation of food intake and energy homeostasis, through its effects in the MOB. We also asked whether PROK2 could be associated with the pathophysiology of obesity, the metabolic syndrome (MetS), and type 2 diabetes (T2D) in humans. METHODS: We assessed in wild type mice whether the expression of Prok2 in the MOB is dependent on the nutritional status. We measured the effect of human recombinant PROK2 (rPROK2) acute injection in the MOB on food intake and olfactory behavior. Then, using a lentivirus expressing Prok2-shRNA, we studied the effects of Prok2 underexpression in the MOB on feeding behavior and glucose metabolism. Metabolic parameters and meal pattern were determined using calorimetric cages. In vivo 2-deoxyglucose uptake measurements were performed in mice after intraperitoneally insulin injection. Plasmatic PROK2 dosages and genetic associations studies were carried out respectively on 148 and more than 4000 participants from the D.E.S.I.R. (Data from an Epidemiologic Study on the Insulin Resistance Syndrome) cohort. RESULTS: Our findings showed that fasting in mice reduced Prok2 expression in the MOB. Acute injection of rPROK2 in the MOB significantly decreased food intake whereas Prok2-shRNA injection resulted in a higher dietary consumption characterized by increased feeding frequency and decreased meal size. Additionally, Prok2 underexpression in the MOB induced insulin resistance compared to scrambled shRNA-injected mice. In the human D.E.S.I.R. cohort, we found a significantly lower mean concentration of plasma PROK2 in people with T2D than in those with normoglycemia. Interestingly, this decrease was no longer significant when adjusted for Body Mass Index (BMI) or calorie intake, suggesting that the association between plasma PROK2 and diabetes is mediated, at least partly, by BMI and feeding behavior in humans. Moreover, common Single Nucleotide Polymorphisms (SNPs) in PROK2 gene were genotyped and associated with incident T2D or impaired fasting glycemia (IFG), MetS, and obesity. CONCLUSIONS: Our data highlight PROK2 as a new target in the MOB that links olfaction with eating behavior and energy homeostasis. In humans, plasma PROK2 is negatively correlated with T2D, BMI, and energy intake, and PROK2 genetic variants are associated with incident hyperglycemia (T2D/IFG), the MetS and obesity.

Deciphering the Link Between Hyperhomocysteinemia and Ceramide Metabolism in Alzheimer-Type Neurodegeneration.

Aging is one of the strongest risk factor for Alzheimer's disease (AD). However, several data suggest that dyslipidemia can either contribute or serve as co-factors in AD appearance. AD could be examined as a metabolic disorder mediated by peripheral insulin resistance. Insulin resistance is associated with dyslipidemia, which results in increased hepatic ceramide generation. Hepatic steatosis induces pro-inflammatory cytokine activation which is mediated by the increased ceramides production. Ceramides levels increased in cells due to perturbation in sphingolipid metabolism and upregulated expression of enzymes involved in ceramide synthesis. Cytotoxic ceramides and related molecules generated in liver promote insulin resistance, traffic through the circulation due to injury or cell death caused by local liver inflammation, and because of their hydrophobic nature, they can cross the blood-brain barrier and thereby exert neurotoxic responses as reducing insulin signaling and increasing pro-inflammatory cytokines. These abnormalities propagate a cascade of neurodegeneration associated with oxidative stress and ceramide generation, which potentiate brain insulin resistance, apoptosis, myelin degeneration, and neuro-inflammation. Therefore, excess of toxic lipids generated in liver can cause neurodegeneration. Elevated homocysteine level is also a risk factor for AD pathology and is narrowly associated with metabolic diseases and non-alcoholic fatty liver disease. The existence of a homocysteine/ceramides signaling pathway suggests that homocysteine toxicity could be partly mediated by intracellular ceramide accumulation due to stimulation of ceramide synthase. In this article, we briefly examined the role of homocysteine and ceramide metabolism linking metabolic diseases and non-alcoholic fatty liver disease to AD. We therefore analyzed the expression of mainly enzymes implicated in ceramide and sphingolipid metabolism and demonstrated deregulation of de novo ceramide biosynthesis and S1P metabolism in liver and brain of hyperhomocysteinemic mice.

LPS-Induced Inflammation Abolishes the Effect of DYRK1A on IkB Stability in the Brain of Mice.

Down syndrome is characterized by premature aging and dementia with neurological features that mimic those found in Alzheimer's disease. This pathology in Down syndrome could be related to inflammation, which plays a role in other neurodegenerative diseases. We previously found a link between the NFkB pathway, long considered a prototypical proinflammatory signaling pathway, and the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). DYRK1A is associated with early onset of Alzheimer's disease in Down syndrome patients. Here, we sought to determine the role of DYRK1A on regulation of the NFkB pathway in the mouse brain. We found that over-expression of Dyrk1A (on a C57BL/6J background) stabilizes IκBα protein levels by inhibition of calpain activity and increases cytoplasmic p65 sequestration in the mouse brain. In contrast, Dyrk1A-deficient mice (on a CD1 background) have decreased IκBα protein levels with an increased calpain activity and decreased cytoplasmic p65 sequestration in the brain. Taken together, our results demonstrate a role of DYRK1A in regulation of the NFkB pathway. However, decreased IκBα and DYRK1A protein levels associated with an increased calpain activity were found in the brains of mice over-expressing Dyrk1A after lipopolysaccharide treatment. Although inflammation induced by lipopolysaccharide treatment has a positive effect on calpastatin and a negative effect on DYRK1A protein level, a positive effect on microglial activation is maintained in the brains of mice over-expressing Dyrk1A.

Homocysteine-lowering gene therapy rescues signaling pathways in brain of mice with intermediate hyperhomocysteinemia.

Hyperhomocysteinemia due to cystathionine beta synthase (CBS) deficiency is associated with diverse cognitive dysfunction. Considering the role of the serine/threonine kinase DYRK1A, not only in developmental defects with life-long structural and functional consequences, but also in multiple neurodegenerative diseases, its protein expression and kinase activity has been analyzed in brain of heterozygous CBS deficient mice and found to be increased. We previously demonstrated that specific liver treatment with an adenovirus expressing Dyrk1A normalizes hepatic DYRK1A level and decreases hyperhomocysteinemia in mice with moderate to intermediate hyperhomocysteinemia. We here use a hepatocyte-specific recombinant adeno-associated viral (AAV) serotype 8-mediated DYRK1A gene therapy (AAV2/8-DYRK1A) to analyze the effect of hepatic Dyrk1A gene transfer on some altered molecular mechanisms in brain of mice with intermediate hyperhomocysteinemia. Our selective hepatic treatment alleviates altered DYRK1A protein level and signaling pathways in brain of mice, the MAPK/ERK and PI3K/Akt pathways initiated by receptor tyrosine kinase, the BDNF dependent TrkB pathway, and NFkB pathway. These results demonstrate the positive effect of AAV2/8-DYRK1A gene transfer on neuropathological and inflammatory processes in brain of mice with intermediate hyperhomocysteinemia.

Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets.

AIMS/HYPOTHESIS: Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells. METHODS: We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling. RESULTS: We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid ß-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated. CONCLUSIONS/INTERPRETATION: Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

Lipoprotein Lipase Expression in Hypothalamus Is Involved in the Central Regulation of Thermogenesis and the Response to Cold Exposure.

Lipoprotein lipase (LPL) is expressed in different areas of the brain, including the hypothalamus and plays an important role in neural control of the energy balance, including feeding behavior and metabolic fluxes. This study tested the hypothesis that hypothalamic LPL participates in the control of body temperature. We first showed that cold exposure induces decreased activity and expression of LPL in the mouse hypothalamus. We then selectively deleted LPL in the mediobasal hypothalamus (MBH) through an adeno-associated virus approach in LPL-floxed mice and generated MBHΔ Lpl mice with 30-35% decrease in hypothalamic LPL activity. Results showed a decrease in body temperature in MBHΔ Lpl mice when compared with controls at 22°C. Exposure to cold (4°C for 4 h) decreased the body temperature of the control mice while that of the MBHΔ Lpl mice remained similar to that observed at 22°C. MBHΔ Lpl mice also showed increased energy expenditure during cold exposure, when compared to controls. Finally, the selective MBH deletion of LPL also increased the expression of the thermogenic PRMD16 and Dio2 in subcutaneous and perigonadal adipose tissues. Thus, the MBH LPL deletion seems to favor thermogenesis. These data demonstrate that for the first time hypothalamic LPL appears to function as a regulator of body temperature and cold-induced thermogenesis.

Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances ß-cell function of obese Zucker rats.

OBJECTIVES: Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity. METHODS: Hypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin) or molecular (si-Serine Palmitoyl Transferase 2, siSPT2) approaches. Obese Zucker rats (OZR) were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and ß-cell mass was also determined. RESULTS: We show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC) inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin secretion and an increase in ß-cell mass of OZR. Electrophysiological recordings also showed an improvement of glucose-stimulated parasympathetic nerve activity in OZR centrally treated with myriocin. CONCLUSION: Our results highlight a key role of hypothalamic de novo ceramide synthesis in central insulin resistance installation and glucose homeostasis dysregulation associated with obesity.

Lipoprotein lipase in hypothalamus is a key regulator of body weight gain and glucose homeostasis in mice.

AIMS/HYPOTHESIS: Regulation of energy balance involves the participation of many factors, including nutrients, among which are circulating lipids, acting as peripheral signals informing the central nervous system of the energy status of the organism. It has been shown that neuronal lipoprotein lipase (LPL) participates in the control of energy balance by hydrolysing lipid particles enriched in triacylglycerols. Here, we tested the hypothesis that LPL in the mediobasal hypothalamus (MBH), a well-known nucleus implicated in the regulation of metabolic homeostasis, could also contribute to the regulation of body weight and glucose homeostasis. METHODS: We injected an adeno-associated virus (AAV) expressing Cre-green fluorescent protein into the MBH of Lpl-floxed mice (and wild-type mice) to specifically decrease LPL activity in the MBH. In parallel, we injected an AAV overexpressing Lpl into the MBH of wild-type mice. We then studied energy homeostasis and hypothalamic ceramide content. RESULTS: The partial deletion of Lpl in the MBH in mice led to an increase in body weight compared with controls (37.72 ± 0.7 g vs 28.46 ± 0.12, p < 0.001) associated with a decrease in locomotor activity. These mice developed hyperinsulinaemia and glucose intolerance. This phenotype also displayed reduced expression of Cers1 in the hypothalamus as well as decreased concentration of several C18 species of ceramides and a 3-fold decrease in total ceramide intensity. Conversely, overexpression of Lpl specifically in the MBH induced a decrease in body weight. CONCLUSIONS/INTERPRETATION: Our study shows that LPL in the MBH is an important regulator of body weight and glucose homeostasis.

Molecular phenotyping of multiple mouse strains under metabolic challenge uncovers a role for Elovl2 in glucose-induced insulin secretion.

OBJECTIVE: In type 2 diabetes (T2D), pancreatic ß cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress. METHODS: Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis. RESULTS: A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2, encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human ß cell lines. CONCLUSION: Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of ß cell failure under metabolic stress.

The iron component of particulate matter is antiapoptotic: A clue to the development of lung cancer after exposure to atmospheric pollutants?

The classification of outdoor air pollution as carcinogenic for humans strengthens the increasing concern about particulate matter (PM). We previously demonstrated that PM exposure produces an antiapoptotic effect resulting from polycyclic aromatic hydrocarbons (PAH) and water-soluble components. In this study, we investigated transition metallic compounds, particularly iron, in order to decipher their underlying molecular mechanisms that prevent apoptosis. Human bronchial epithelial cells were exposed for 4 h to different PM samples with established antiapoptotic effect (e.g. PM-AW) or not (e.g. PM-VS) or to their metallic components (Fe, Mn, Zn and Al) before apoptosis induction by the calcium ionophore A23187 or Staurosporine. PM-AW, Fe, Mn and Al significantly reduced induced apoptosis. The antiapoptotic effect of Fe was enhanced by benzo(a)pyrene, a typical PAH compound, but was totally reversed by the iron chelator, deferiprone. Furthermore, particles and iron triggered cellular ROS generation and prevented the depletion in glutathione levels observed during A23187-induced apoptosis. In contrast to benzo(a)pyrene, PM-AW and Fe rapidly activated NRF2, subsequently upregulated several target genes (HO1, NQO1 and GPX1) and modulated some genes which control cell death (BCL2, BAX and p53). The key role of the NRF2 pathway in the antiapoptotic effect mediated by Fe and PM was demonstrated using siRNA technology. Our results suggest that the iron component participates in the antiapoptotic effect of PM by activating a NRF2-dependent antioxidant process. As resisting to cell death is one of the hallmarks of cancer cells, these findings provide additional clues for understanding the development of lung cancer after atmospheric pollution exposure.