BACKGROUND:Women with a history of gestational diabetes mellitus (GDM) have a 7-fold higher risk of developing type 2 diabetes (T2D) during midlife and an elevated risk of developing hypertension and cardiovascular disease. Glucose tolerance reclassification after delivery is recommended, but fewer than 40% of women with GDM are tested. Thus, improved risk stratification methods are needed, as is a deeper understanding of the pathology underlying the transition from GDM to T2D. We hypothesize that metabolites during the early postpartum period accurately distinguish risk of progression from GDM to T2D and that metabolite changes signify underlying pathophysiology for future disease development.METHODS AND FINDINGS:The study utilized fasting plasma samples collected from a well-characterized prospective research study of 1,035 women diagnosed with GDM. The cohort included racially/ethnically diverse pregnant women (aged 20-45 years-33% primiparous, 37% biparous, 30% multiparous) who delivered at Kaiser Permanente Northern California hospitals from 2008 to 2011. Participants attended in-person research visits including 2-hour 75-g oral glucose tolerance tests (OGTTs) at study baseline (6-9 weeks postpartum) and annually thereafter for 2 years, and we retrieved diabetes diagnoses from electronic medical records for 8 years. In a nested case-control study design, we collected fasting plasma samples among women without diabetes at baseline (n = 1,010) to measure metabolites among those who later progressed to incident T2D or did not develop T2D (non-T2D). We studied 173 incident T2D cases and 485 controls (pair-matched on BMI, age, and race/ethnicity) to discover metabolites associated with new onset of T2D. Up to 2 years post-baseline, we analyzed samples from 98 T2D cases with 239 controls to reveal T2D-associated metabolic changes. The longitudinal analysis tracked metabolic changes within individuals from baseline to 2 years of follow-up as the trajectory of T2D progression. By building prediction models, we discovered a distinct metabolic signature in the early postpartum period that predicted future T2D with a median discriminating power area under the receiver operating characteristic curve of 0.883 (95% CI 0.820-0.945, p < 0.001). At baseline, the most striking finding was an overall increase in amino acids (AAs) as well as diacyl-glycerophospholipids and a decrease in sphingolipids and acyl-alkyl-glycerophospholipids among women with incident T2D. Pathway analysis revealed up-regulated AA metabolism, arginine/proline metabolism, and branched-chain AA (BCAA) metabolism at baseline. At follow-up after the onset of T2D, up-regulation of AAs and down-regulation of sphingolipids and acyl-alkyl-glycerophospholipids were sustained or strengthened. Notably, longitudinal analyses revealed only 10 metabolites associated with progression to T2D, implicating AA and phospholipid metabolism. A study limitation is that all of the analyses were performed with the same cohort. It would be ideal to validate our findings in an independent longitudinal cohort of women with GDM who had glucose tolerance tested during the early postpartum period.CONCLUSIONS:In this study, we discovered a metabolic signature predicting the transition from GDM to T2D in the early postpartum period that was superior to clinical parameters (fasting plasma glucose, 2-hour plasma glucose). The findings suggest that metabolic dysregulation, particularly AA dysmetabolism, is present years prior to diabetes onset, and is revealed during the early postpartum period, preceding progression to T2D, among women with GDM.TRIAL REGISTRATION:ClinicalTrials.gov Identifier: NCT01967030.

Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events in vivo in the hippocampus of MeCP2 mutant mice. While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.

Mammalian genomes are well established, and highly conserved regions within odorant receptors that are unique from other G-protein coupled receptors have been identified. Numerous functional studies have focused on specific conserved amino acids motifs; however, not all conserved motifs have been sufficiently characterized. Here, we identified a highly conserved 18 amino acid sequence motif within transmembrane domain seven (CAS-TM7) which was identified by aligning odorant receptor sequences. Next, we investigated the expression pattern and distribution of this conserved amino acid motif among a broad range of odorant receptors. To examine the localization of odorant receptor proteins, we used a sequence-specific peptide antibody against CAS-TM7 which is specific to odorant receptors across species. The specificity of this peptide antibody in recognizing odorant receptors has been confirmed in a heterologous in vitro system and a rat-based in vivo system. The CAS-TM7 odorant receptors localized with distinct patterns at each region of the olfactory epithelium; septum, endoturbinate and ectoturbinate. To our great interests, we found that the CAS-TM7 odorant receptors are primarily localized to the dorsal region of the olfactory bulb, coinciding with olfactory epithelium-based patterns. Also, these odorant receptors were ectopically expressed in the various non-olfactory tissues in an evolutionary constrained manner between human and rats. This study has characterized the expression patterns of odorant receptors containing particular amino acid motif in transmembrane domain 7, and which led to an intriguing possibility that the conserved motif of odorant receptors can play critical roles in other physiological functions as well as olfaction.

Cyclic AMP (cAMP) and protein kinase A (PKA), classical examples of spatially compartmentalized signaling molecules, are critical axon determinants that regulate neuronal polarity and axon formation, yet little is known about micro-compartmentalization of cAMP and PKA signaling and its role in developing neurons. Here, we revealed that cAMP forms a gradient in developing hippocampal neurons, with higher cAMP levels in more distal regions of the axon compared to other regions of the cell. Interestingly, this cAMP gradient changed according to the developmental stage and depended on proper anchoring of PKA by A-kinase anchoring proteins (AKAPs). Disrupting PKA anchoring to AKAPs increased the cAMP gradient in early-stage neurons and led to enhanced axon elongation. Our results provide new evidence for a local negative-feedback loop, assembled by AKAPs, for the precise control of a growth-stage-dependent cAMP gradient to ensure proper axon growth.

The regulation of food intake is important for body energy homeostasis. Hypothalamic insulin signaling decreases food intake by upregulating the expression of anorexigenic neuropeptides and downregulating the expression of orexigenic neuropeptides. INS-2, a Mn(2+) chelate of 4-O-(2-amino-2-deoxy-β-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol, acts as an insulin mimetic and sensitizer. We found that intracerebroventricular injection of INS-2 decreased body weight and food intake in mice. In hypothalamic neuronal cell lines, INS-2 downregulated the expression of neuropeptide Y (NPY), an orexigenic neuropeptide, but upregulated the expression of proopiomelanocortin (POMC), an anorexigenic neuropeptide, via modulation of the AKT-forkhead box-containing protein-O1 (FoxO1) pathway. Pretreatment of these cells with INS-2 enhanced the action of insulin on downstream signaling, leading to a further decrease in NPY expression and increase in POMC expression. These data indicate that INS-2 reduces food intake by regulating the expression of the hypothalamic neuropeptide genes through the AKT-FoxO1 pathway downstream of insulin.

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose- and time-dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK-Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation-dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat-shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant-induced activation of the MEK-Erk pathway. These findings provide important insights into the mechanisms of activity-driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation-mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure. We investigated the role of odorant stimulation in generating cellular stress and the molecular mechanisms mitigating such stress and promoting neuronal survival. Odorant stimulation promoted olfactory sensory neuron (OSN) survival and also induced intracellular oxidative stress, which was exacerbated when MEK/Erks pathway was inhibited. Sensory stimulation simultaneously activated at least two parallel pathways, the AC/cAMP cascade responsible for odorant detection, and phosphoinositide hydrolysis to promote odorant stimulation-dependent neuronal survival odorants may activate parallel signaling cascades to mediate sensory detection and sensory stimulation-dependent survival. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; Erk, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor (LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master metabolic regulator that has been shown to inhibit the establishment of neuronal polarity/axogenesis under energy stress conditions, whereas brain-specific kinase (BRSK) promotes the establishment of axon-dendrite polarity and synaptic development. However, little information exists regarding the localized activity and regulation of these kinases in developing neurons. In this study, using a fluorescence resonance energy transfer (FRET)-based activity reporter that responds to both AMPK and BRSK, we found that BRSK activity is elevated in the distal region of axons in polarized hippocampal neurons before any stimulation and does not respond to either Ca(2+) or 2-deoxyglucose (2-DG) stimulation. In contrast, AMPK activity is stimulated by either Ca(2+) or 2-DG in the soma, dendrites, and axons of hippocampal neurons, with maximal stimulated activity observed in the distal region of the axon. Our study shows that the activities of both AMPK and BRSK are polarized in developing hippocampal neurons, with high levels in the distal region of extended axons.

Methyl CpG binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Alterations in the levels of MeCP2 have been related to neurodevelopmental disorders. Studies in mouse models of MeCP2 deficiency have demonstrated that this protein is important for neuronal maturation, neurite complexity, synaptogenesis, and synaptic plasticity. However, the mechanisms by which MeCP2 dysfunction leads to neurodevelopmental defects, and the role of activity, remain unclear, as most studies examine the adult nervous system, which may obfuscate the primary consequences of MeCP2 mutation. We hypothesize that MeCP2 plays a role during the formation and activity-driven maturation of neural circuits at early postnatal stages. To test this hypothesis, we use the olfactory system as a neurodevelopmental model. This system undergoes postnatal neurogenesis; axons from olfactory neurons form highly stereotyped projections to higher-order neurons, facilitating the detection of possible defects in the establishment of connectivity. In vivo olfactory stimulation paradigms were used to produce physiological synaptic activity in gene-targeted mice in which specific olfactory circuits are visualized. Our results reveal defective postnatal refinement of olfactory circuits in Mecp2 knock out (KO) mice after sensory (odorant) stimulation. This failure in refinement was associated with deficits in the normal responses to odorants, including brain-derived neurotrophic factor (BDNF) production, as well as changes in adhesion molecules known to regulate axonal convergence. The defective refinement observed in Mecp2 KO mice was prevented by daily treatment with ampakine beginning after the first postnatal week. These observations indicate that increasing synaptic activity at early postnatal stage might circumvent the detrimental effect of MeCP2 deficiency on circuitry maturation. The present results provide in vivo evidence in real time for the role of MeCP2 in activity-dependent maturation of olfactory circuitry, with implications for understanding the mechanism of MeCP2 mutations in the development of neural connectivity.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a cellular and whole organism energy sensor to regulate ATP-consuming (anabolic) and ATP-generating (catabolic) pathways. The heterotrimeric AMPK complex consists of a catalytic α-subunit, regulatory β-subunit, and an AMP/ATP-binding γ-subunit. Several alternate isoforms exist for each subunit (α1, α2, β1, β2, γ1, γ2 and γ3). However, little is known of the expression pattern or function of the individual catalytic complexes in regulating neuronal structure. In this study, we examined the role of AMPK subunits in differentiating hippocampal neurons. We found that during development, the expression of AMPK subunits increase and that activation of AMPK by energetic stress inhibits neuronal development at multiple stages, not only during axon outgrowth, but also during dendrite growth and arborization. The presence of a single functional AMPK catalytic complex was sufficient to mediate these inhibitory effects of energetic stress. Activation of AMPK mediates these effects by suppressing both the mTOR and Akt signaling pathways. These findings demonstrate that the energy-sensing AMPK pathway regulates neuronal structure in distinct regions of developing neurons at multiple stages of development.

Modification of hypothalamic fatty acid (FA) metabolism can improve energy homeostasis and prevent hyperphagia and excessive weight gain in diet-induced obesity (DIO) from a diet high in saturated fatty acids. We have shown previously that C75, a stimulator of carnitine palmitoyl transferase-1 (CPT-1) and fatty acid oxidation (FAOx), exerts at least some of its hypophagic effects via neuronal mechanisms in the hypothalamus. In the present work, we characterized the effects of C75 and another anorexigenic compound, the glycerol-3-phosphate acyltransferase (GPAT) inhibitor FSG67, on FA metabolism, metabolomics profiles, and metabolic stress responses in cultured hypothalamic neurons and hypothalamic neuronal cell lines during lipid excess with palmitate. Both compounds enhanced palmitate oxidation, increased ATP, and inactivated AMP-activated protein kinase (AMPK) in hypothalamic neurons in vitro. Lipidomics and untargeted metabolomics revealed that enhanced catabolism of FA decreased palmitate availability and prevented the production of fatty acylglycerols, ceramides, and cholesterol esters, lipids that are associated with lipotoxicity-provoked metabolic stress. This improved metabolic signature was accompanied by increased levels of reactive oxygen species (ROS), and yet favorable changes in oxidative stress, overt ER stress, and inflammation. We propose that enhancing FAOx in hypothalamic neurons exposed to excess lipids promotes metabolic remodeling that reduces local inflammatory and cell stress responses. This shift would restore mitochondrial function such that increased FAOx can produce hypothalamic neuronal ATP and lead to decreased food intake and body weight to improve systemic metabolism.

Rett syndrome (RTT) is an autism spectrum disorder (ASD) caused by mutations in the X-linked MECP2 gene that encodes methyl-CpG binding protein 2 (MeCP2). Symptoms range in severity and include psychomotor disabilities, seizures, ataxia, and intellectual disability. Symptom onset is between 6-18 months of age, a critical period of brain development that is highly energy-dependent. Notably, patients with RTT have evidence of mitochondrial dysfunction, as well as abnormal levels of the adipokines leptin and adiponectin, suggesting overall metabolic imbalance. We hypothesized that one contributor to RTT symptoms is energy deficiency due to defective nutrient substrate utilization by the TCA cycle. This energy deficit would lead to a metabolic imbalance, but would be treatable by providing anaplerotic substrates to the TCA cycle to enhance energy production. We show that dietary therapy with triheptanoin significantly increased longevity and improved motor function and social interaction in male mice hemizygous for Mecp2 knockout. Anaplerotic therapy in Mecp2 knockout mice also improved indicators of impaired substrate utilization, decreased adiposity, increased glucose tolerance and insulin sensitivity, decreased serum leptin and insulin, and improved mitochondrial morphology in skeletal muscle. Untargeted metabolomics of liver and skeletal muscle revealed increases in levels of TCA cycle intermediates with triheptanoin diet, as well as normalizations of glucose and fatty acid biochemical pathways consistent with the improved metabolic phenotype in Mecp2 knockout mice on triheptanoin. These results suggest that an approach using dietary supplementation with anaplerotic substrate is effective in improving symptoms and metabolic health in RTT.

BACKGROUND:Reperfusion in ischemia is believed to generate cytotoxic oxidative stress, which mediates reperfusion injury. These stress conditions can initiate lipid peroxidation and damage to proteins, as well as promote DNA strand breaks. As biliverdin and bilirubin produced by heme oxygenase isoform 1 (HO-1) have antioxidant properties, the production of both antioxidants by HO-1 may help increase the resistance of the ischemic brain to oxidative stress. In the present study, the survival effect of HO-1 was confirmed using hemin.METHODS:To confirm the roles of HO-1, carbon monoxide, and cyclic guanosine monophosphate further in the antioxidant effect of HO-1 and bilirubin, cells were treated with cycloheximide, desferoxamine, and zinc deuteroporphyrin IX 2,4 bis glycol, respectively.RESULTS:HO-1 itself acted as an antioxidant. Furthermore, iron, rather than carbon monoxide, was involved in the HO-1-mediated survival effect. HO-1 activity was also important in providing bilirubin as an antioxidant.CONCLUSION:Our results suggested that HO-1 helped to increase the resistance of the ischemic brain to oxidative stress.

Osteoporosis and osteonecrosis are associated with corticosteroid treatment, but the pathophysiologies are unclear. We hypothesized that mature adipocytes present within the bone marrow compartment play a key role in the development of both diseases. Adipocytes have recognized regulatory effects on bone viability and healing by releasing signaling molecules called adipokines. Our purpose was to evaluate whether dexamethasone alters adipokine expression in differentiated bone-marrow-derived adipocytes. Adipocytes differentiated from mouse D1 mesenchymal stromal cells were treated with dexamethasone (10(-5), 10(-6), 10(-7), or 10(-8) M) or with diluent alone (controls) for up to 6 days. Using real-time polymerase chain reaction and enzyme-linked immunosorbent assay analyses, six key adipokines and the transcription factor HIF-1 alpha were evaluated. Dexamethasone treatment increased PAI-1 protein expression with increased mRNA expression at 4 days, while decreasing HIF-1 alpha mRNA expression and protein concentrations. VEGF A mRNA expression was increased at 4 days for most dexamethasone concentrations, with minimal changes in protein levels. Dexamethasone increased adiponectin mRNA expression and protein levels at 4 and 6 days and decreased leptin, interleukin-6, and tumor necrosis factor a mRNA expression at all time periods. Dexamethasone treatment of bone-marrow-derived adipocytes resulted in detectable changes in mRNA expression and protein levels of adipokines and HIF-1 alpha. The detected adipokine alterations could be important early events in the pathogenesis of steroid-induced osteonecrosis and osteoporosis. (C) 2014 Elsevier Inc. All rights reserved.