The reactive pyruvate metabolite dimethylglyoxal mediates neurological consequences of diabetes.

Complications of diabetes are often attributed to glucose and reactive dicarbonyl metabolites derived from glycolysis or gluconeogenesis, such as methylglyoxal. However, in the CNS, neurons and endothelial cells use lactate as energy source in addition to glucose, which does not lead to the formation of methylglyoxal and has previously been considered a safer route of energy consumption than glycolysis. Nevertheless, neurons and endothelial cells are hotspots for the cellular pathology underlying neurological complications in diabetes, suggesting a cause that is distinct from other diabetes complications and independent of methylglyoxal. Here, we show that in clinical and experimental diabetes plasma concentrations of dimethylglyoxal are increased. In a mouse model of diabetes, ilvb acetolactate-synthase-like (ILVBL, HACL2) is the enzyme involved in formation of increased amounts of dimethylglyoxal from lactate-derived pyruvate. Dimethylglyoxal reacts with lysine residues, forms Nε-3-hydroxy-2-butanonelysine (HBL) as an adduct, induces oxidative stress more strongly than other dicarbonyls, causes blood-brain barrier disruption, and can mimic mild cognitive impairment in experimental diabetes. These data suggest dimethylglyoxal formation as a pathway leading to neurological complications in diabetes that is distinct from other complications. Importantly, dimethylglyoxal formation can be reduced using genetic, pharmacological and dietary interventions, offering new strategies for preventing CNS dysfunction in diabetes.

Estrogen receptor-α signaling in tanycytes lies at the crossroads of fertility and metabolism.

BACKGROUND: Estrogen secretion by the ovaries regulates the hypothalamic-pituitary-gonadal axis during the reproductive cycle, influencing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, and also plays a role in regulating metabolism. Here, we establish that hypothalamic tanycytes-specialized glia lining the floor and walls of the third ventricle-integrate estrogenic feedback signals from the gonads and couple reproduction with metabolism by relaying this information to orexigenic neuropeptide Y (NPY) neurons. METHODS: Using mouse models, including mice floxed for Esr1 (encoding estrogen receptor alpha, ERα) and those with Cre-dependent expression of designer receptors exclusively activated by designer drugs (DREADDs), along with virogenic, pharmacological and indirect calorimetric approaches, we evaluated the role of tanycytes and tanycytic estrogen signaling in pulsatile LH secretion, cFos expression in NPY neurons, estrous cyclicity, body-weight changes and metabolic parameters in adult females. RESULTS: In ovariectomized mice, chemogenetic activation of tanycytes significantly reduced LH pulsatile release, mimicking the effects of direct NPY neuron activation. In intact mice, tanycytes were crucial for the estrogen-mediated control of GnRH/LH release, with tanycytic ERα activation suppressing fasting-induced NPY neuron activation. Selective knockout of Esr1 in tanycytes altered estrous cyclicity and fertility in female mice and affected estrogen's ability to inhibit refeeding in fasting mice. The absence of ERα signaling in tanycytes increased Npy transcripts and body weight in intact mice and prevented the estrogen-mediated decrease in food intake as well as increase in energy expenditure and fatty acid oxidation in ovariectomized mice. CONCLUSIONS: Our findings underscore the pivotal role of tanycytes in the neuroendocrine coupling of reproduction and metabolism, with potential implications for its age-related deregulation after menopause. SIGNIFICANCE STATEMENT: Our investigation reveals that tanycytes, specialized glial cells in the brain, are key interpreters of estrogen signals for orexigenic NPY neurons in the hypothalamus. Disrupting tanycytic estrogen receptors not only alters fertility in female mice but also impairs the ability of estrogens to suppress appetite. This work thus sheds light on the critical role played by tanycytes in bridging the hormonal regulation of cyclic reproductive function and appetite/feeding behavior. This understanding may have potential implications for age-related metabolic deregulation after menopause.

Gene therapy targeting the blood-brain barrier.

Endothelial cells are the building blocks of vessels in the central nervous system (CNS) and form the blood-brain barrier (BBB). An intact BBB limits permeation of large hydrophilic molecules into the CNS. Thus, the healthy BBB is a major obstacle for the treatment of CNS disorders with antibodies, recombinant proteins or viral vectors. Several strategies have been devised to overcome the barrier. A key principle often consists in attaching the therapeutic compound to a ligand of receptors expressed on the BBB, for example, the transferrin receptor (TfR). The fusion molecule will bind to TfR on the luminal side of brain endothelial cells, pass the endothelial layer by transcytosis and be delivered to the brain parenchyma. However, attempts to endow therapeutic compounds with the ability to cross the BBB can be difficult to implement. An alternative and possibly more straight-forward approach is to produce therapeutic proteins in the endothelial cells that form the barrier. These cells are accessible from blood circulation and have a large interface with the brain parenchyma. They may be an ideal production site for therapeutic protein and afford direct supply to the CNS.

Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκß pathway.

BACKGROUND AND AIMS: Mitochondrial antiviral signaling protein (MAVS) is a critical regulator that activates the host's innate immunity against RNA viruses, and its signaling pathway has been linked to the secretion of proinflammatory cytokines. However, the actions of MAVS on inflammatory pathways during the development of metabolic dysfunction-associated steatotic liver disease (MASLD) have been little studied. APPROACH AND RESULTS: Liver proteomic analysis of mice with genetically manipulated hepatic p63, a transcription factor that induces liver steatosis, revealed MAVS as a target downstream of p63. MAVS was thus further evaluated in liver samples from patients and in animal models with MASLD. Genetic inhibition of MAVS was performed in hepatocyte cell lines, primary hepatocytes, spheroids, and mice. MAVS expression is induced in the liver of both animal models and people with MASLD as compared with those without liver disease. Using genetic knockdown of MAVS in adult mice ameliorates diet-induced MASLD. In vitro, silencing MAVS blunts oleic and palmitic acid-induced lipid content, while its overexpression increases the lipid load in hepatocytes. Inhibiting hepatic MAVS reduces circulating levels of the proinflammatory cytokine TNFα and the hepatic expression of both TNFα and NFκß. Moreover, the inhibition of ERK abolished the activation of TNFα induced by MAVS. The posttranslational modification O -GlcNAcylation of MAVS is required to activate inflammation and to promote the high lipid content in hepatocytes. CONCLUSIONS: MAVS is involved in the development of steatosis, and its inhibition in previously damaged hepatocytes can ameliorate MASLD.

Partial Resistance to Thyroid Hormone-Induced Tachycardia and Cardiac Hypertrophy in Mice Lacking Thyroid Hormone Receptor ß.

Background: Thyroid hormones regulate cardiac functions mainly through direct actions in the heart and by binding to the thyroid hormone receptor (TR) isoforms α1 and ß. While the role of the most abundantly expressed isoform, TRα1, is widely studied and well characterized, the role of TRß in regulating heart functions is still poorly understood, primarily due to the accompanying elevation of circulating thyroid hormone in TRß knockout mice (TRß-KO). However, their hyperthyroidism is ameliorated at thermoneutrality, which allows studying the role of TRß without this confounding factor. Methods: Here, we noninvasively monitored heart rate in TRß-KO mice over several days using radiotelemetry at different housing temperatures (22°C and 30°C) and upon 3,3',5-triiodothyronine (T3) administration in comparison to wild-type animals. Results: TRß-KO mice displayed normal average heart rate at both 22°C and 30°C with only minor changes in heart rate frequency distribution, which was confirmed by independent electrocardiogram recordings in freely-moving conscious mice. Parasympathetic nerve activity was, however, impaired in TRß-KO mice at 22°C, and only partly rescued at 30°C. As expected, oral treatment with pharmacological doses of T3 at 30°C led to tachycardia in wild-types, accompanied by broader heart rate frequency distribution and increased heart weight. The TRß-KO mice, in contrast, showed blunted tachycardia, as well as resistance to changes in heart rate frequency distribution and heart weight. At the molecular level, these observations were paralleled by a blunted cardiac mRNA induction of several important genes, including the pacemaker channels Hcn2 and Hcn4, as well as Kcna7. Conclusions: The phenotyping of TRß-KO mice conducted at thermoneutrality allows novel insights on the role of TRß in cardiac functions in the absence of the usual confounding hyperthyroidism. Even though TRß is expressed at lower levels than TRα1 in the heart, our findings demonstrate an important role for this isoform in the cardiac response to thyroid hormones.

Incidence of microvascular dysfunction is increased in hyperlipidemic mice, reducing cerebral blood flow and impairing remote memory.

Introduction: The development of cognitive dysfunction is not necessarily associated with diet-induced obesity. We hypothesized that cognitive dysfunction might require additional vascular damage, for example, in atherosclerotic mice. Methods: We induced atherosclerosis in male C57BL/6N mice by injecting AAV-PCSK9DY (2x1011 VG) and feeding them a cholesterol-rich Western diet. After 3 months, mice were examined for cognition using Barnes maze procedure and for cerebral blood flow. Cerebral vascular morphology was examined by immunehistology. Results: In AAV-PCSK9DY-treated mice, plaque burden, plasma cholesterol, and triglycerides are elevated. RNAseq analyses followed by KEGG annotation show increased expression of genes linked to inflammatory processes in the aortas of these mice. In AAV-PCSK9DY-treated mice learning was delayed and long-term memory impaired. Blood flow was reduced in the cingulate cortex (-17%), caudate putamen (-15%), and hippocampus (-10%). Immunohistological studies also show an increased incidence of string vessels and pericytes (CD31/Col IV staining) in the hippocampus accompanied by patchy blood-brain barrier leaks (IgG staining) and increased macrophage infiltrations (CD68 staining). Discussion: We conclude that the hyperlipidemic PCSK9DY mouse model can serve as an appropriate approach to induce microvascular dysfunction that leads to reduced blood flow in the hippocampus, which could explain the cognitive dysfunction in these mice.

Seasonal biology: Tanycytes give the hypothalamus a spring makeover.

In many species, metabolic and reproductive functions are coupled to the seasons. Tanycytes, specialized glial cells in the hypothalamus, play an important function in these physiological changes. A new study now shows that light exposure drastically alters the formation of sensory cilia on tanycytes.

p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway.

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage. In mice, genetic depletion of TAp63 in HSCs reduces the diet-induced liver fibrosis. In vitro silencing of p63 blunts TGF-ß1-induced HSCs activation by reducing mitochondrial respiration and glycolysis, as well as decreasing acetyl CoA carboxylase 1 (ACC1). Ectopic expression of TAp63 induces the activation of HSCs and increases the expression and activity of ACC1 by promoting the transcriptional activity of HER2. Genetic inhibition of both HER2 and ACC1 blunt TAp63-induced activation of HSCs. Thus, TAp63 induces HSC activation by stimulating the HER2-ACC1 axis and participates in the development of liver fibrosis.

Endothelial cells and macrophages as allies in the healthy and diseased brain.

Diseases of the central nervous system (CNS) are often associated with vascular disturbances or inflammation and frequently both. Consequently, endothelial cells and macrophages are key cellular players that mediate pathology in many CNS diseases. Macrophages in the brain consist of the CNS-associated macrophages (CAMs) [also referred to as border-associated macrophages (BAMs)] and microglia, both of which are close neighbours or even form direct contacts with endothelial cells in microvessels. Recent progress has revealed that different macrophage populations in the CNS and a subset of brain endothelial cells are derived from the same erythromyeloid progenitor cells. Macrophages and endothelial cells share several common features in their life cycle-from invasion into the CNS early during embryonic development and proliferation in the CNS, to their demise. In adults, microglia and CAMs have been implicated in regulating the patency and diameter of vessels, blood flow, the tightness of the blood-brain barrier, the removal of vascular calcification, and the life-time of brain endothelial cells. Conversely, CNS endothelial cells may affect the polarization and activation state of myeloid populations. The molecular mechanisms governing the pas de deux of brain macrophages and endothelial cells are beginning to be deciphered and will be reviewed here.

Phenotypic and genome-wide studies on dicarbonyls: major associations to glomerular filtration rate and gamma-glutamyltransferase activity.

BACKGROUND: The dicarbonyl compounds methylglyoxal (MG), glyoxal (GO) and 3-deoxyglucosone (3-DG) have been linked to various diseases. However, disease-independent phenotypic and genotypic association studies with phenome-wide and genome-wide reach, respectively, have not been provided. METHODS: MG, GO and 3-DG were measured by LC-MS in 1304 serum samples of two populations (KORA, n = 482; BiDirect, n = 822) and assessed for associations with genome-wide SNPs (GWAS) and with phenome-wide traits. Redundancy analysis (RDA) was used to identify major independent trait associations. FINDINGS: Mutual correlations of dicarbonyls were highly significant, being stronger between MG and GO (ρ = 0.6) than between 3-DG and MG or GO (ρ = 0.4). Significant phenotypic results included associations of all dicarbonyls with sex, waist-to-hip ratio, glomerular filtration rate (GFR), gamma-glutamyltransferase (GGT), and hypertension, of MG and GO with age and C-reactive protein, of GO and 3-DG with glucose and antidiabetics, of MG with contraceptives, of GO with ferritin, and of 3-DG with smoking. RDA revealed GFR, GGT and, in case of 3-DG, glucose as major contributors to dicarbonyl variance. GWAS did not identify genome-wide significant loci. SNPs previously associated with glyoxalase activity did not reach nominal significance. When multiple testing was restricted to the lead SNPs of GWASs on the traits selected by RDA, 3-DG was found to be associated (p = 2.3 × 10-5) with rs1741177, an eQTL of NF-κB inhibitor NFKBIA. INTERPRETATION: This large-scale, population-based study has identified numerous associations, with GFR and GGT being of pivotal importance, providing unbiased perspectives on dicarbonyls beyond the current state. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Munich, German Centre for Cardiovascular Research (DZHK), German Federal Ministry of Research and Education (BMBF).

Liver Metabolism in Ischemic Stroke.

Focal brain damage and neurological deficits are the direct consequences of acute ischemic stroke (AIS). In addition, cerebral ischemia causes systemic alterations across peripheral organs. Dysregulation of the autonomic and endocrine systems as well as the release of brain-derived pro-inflammatory mediators trigger a peripheral immune response and systemic inflammation. As a key metabolic organ, the liver contributes not only to post-stroke immunosuppression but also to stress-induced hyperglycemia. At the same time, increased ketogenesis and glutathione production in the liver are likely to combat inflammation and oxidative stress after AIS. The closely linked lipid metabolism could regulate both glucose and glutathione homeostasis. In addition, increased hepatic very low-density lipoprotein (VLDL) secretion may improve the availability of phospholipids, polyunsaturated fatty acids (PUFAs) and glutathione after AIS. This review provides an overview of recent findings concerning ischemic stroke and the liver and discusses the therapeutic potential of targeting the hepatic metabolism to improve patient outcome after stroke.

Unraveling the brain's response to hypoglycemia: Neurovascular coupling.

Functional magnetic resonance imaging has suggested the possibility that hypoglycemia could interfere with neurovascular coupling. Here we discuss the implications of a study by Nippert and colleagues showing that hypoglycemia does not impair neurovascular coupling.

Regulation of Thyroid Hormone Gatekeepers by Thyrotropin in Tanycytes.

Background: Tanycytes are specialized glial cells within the mediobasal hypothalamus that have multiple functions, including hormone sensing and regulation of hypophysiotropic hormone secretion. There are ongoing discussions about the role of tanycytes in regulating the supply of hypothalamic thyroid hormones (THs) through the expression of TH transporters (Slc16a2, Slco1c1) and deiodinases (Dio2, Dio3). In this study, we investigated the potential feedback effect of thyrotropin (TSH) on the transcription of these gatekeeper genes on tanycytes. Methods: We analyzed the changes in the expression of TH-gatekeeper genes, in TSH-stimulated primary tanycytes, using quantitative polymerase chain reaction (qPCR). We also used RNAScope® in brain slices to further reveal the local distribution of the transcripts. In addition, we blocked intracellular pathways and used small-interfering RNA (siRNA) to elucidate differences in the regulation of the gatekeeper genes. Results: TSH elevated messenger RNA (mRNA) levels of Slco1c1, Dio2, and Dio3 in tanycytes, while Slc16a2 was mostly unaffected. Blockade and knockdown of the TSH receptor (TSHR) and antagonization of cAMP response element-binding protein (CREB) clearly abolished the increased expression induced by TSH, indicating PKA-dependent regulation through the TSHR. The TSH-dependent expression of Dio3 and Slco1c1 was also regulated by protein kinase C (PKC), and in case of Dio3, also by extracellular signal-regulated kinase (ERK) activity. Importantly, these gene regulations were specifically found in different subpopulations of tanycytes. Conclusions: This study demonstrates that TSH induces transcriptional regulation of TH-gatekeeper genes in tanycytes through the Tshr/Gαq/PKC pathway, in parallel to the Tshr/Gαs/PKA/CREB pathway. These differential actions of TSH on tanycytic subpopulations appear to be important for coordinating the supply of TH to the hypothalamus and aid its functions.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Long-COVID cognitive impairments and reproductive hormone deficits in men may stem from GnRH neuronal death.

BACKGROUND: We have recently demonstrated a causal link between loss of gonadotropin-releasing hormone (GnRH), the master molecule regulating reproduction, and cognitive deficits during pathological aging, including Down syndrome and Alzheimer's disease. Olfactory and cognitive alterations, which persist in some COVID-19 patients, and long-term hypotestosteronaemia in SARS-CoV-2-infected men are also reminiscent of the consequences of deficient GnRH, suggesting that GnRH system neuroinvasion could underlie certain post-COVID symptoms and thus lead to accelerated or exacerbated cognitive decline. METHODS: We explored the hormonal profile of COVID-19 patients and targets of SARS-CoV-2 infection in post-mortem patient brains and human fetal tissue. FINDINGS: We found that persistent hypotestosteronaemia in some men could indeed be of hypothalamic origin, favouring post-COVID cognitive or neurological symptoms, and that changes in testosterone levels and body weight over time were inversely correlated. Infection of olfactory sensory neurons and multifunctional hypothalamic glia called tanycytes highlighted at least two viable neuroinvasion routes. Furthermore, GnRH neurons themselves were dying in all patient brains studied, dramatically reducing GnRH expression. Human fetal olfactory and vomeronasal epithelia, from which GnRH neurons arise, and fetal GnRH neurons also appeared susceptible to infection. INTERPRETATION: Putative GnRH neuron and tanycyte dysfunction following SARS-CoV-2 neuroinvasion could be responsible for serious reproductive, metabolic, and mental health consequences in long-COVID and lead to an increased risk of neurodevelopmental and neurodegenerative pathologies over time in all age groups. FUNDING: European Research Council (ERC) grant agreements No 810331, No 725149, No 804236, the European Union Horizon 2020 research and innovation program No 847941, the Fondation pour la Recherche Médicale (FRM) and the Agence Nationale de la Recherche en Santé (ANRS) No ECTZ200878 Long Covid 2021 ANRS0167 SIGNAL, Agence Nationale de la recherche (ANR) grant agreements No ANR-19-CE16-0021-02, No ANR-11-LABEX-0009, No. ANR-10-LABEX-0046, No. ANR-16-IDEX-0004, Inserm Cross-Cutting Scientific Program HuDeCA, the CHU Lille Bonus H, the UK Medical Research Council (MRC) and National Institute of Health and care Research (NIHR).

Neddylation of phosphoenolpyruvate carboxykinase 1 controls glucose metabolism.

Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues-K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.

Metabolic Fingerprints of Effective Fluoxetine Treatment in the Prefrontal Cortex of Chronically Socially Isolated Rats: Marker Candidates and Predictive Metabolites.

The increasing prevalence of depression requires more effective therapy and the understanding of antidepressants' mode of action. We carried out untargeted metabolomics of the prefrontal cortex of rats exposed to chronic social isolation (CSIS), a rat model of depression, and/or fluoxetine treatment using liquid chromatography-high resolution mass spectrometry. The behavioral phenotype was assessed by the forced swim test. To analyze the metabolomics data, we employed univariate and multivariate analysis and biomarker capacity assessment using the receiver operating characteristic (ROC) curve. We also identified the most predictive biomarkers using a support vector machine with linear kernel (SVM-LK). Upregulated myo-inositol following CSIS may represent a potential marker of depressive phenotype. Effective fluoxetine treatment reversed depressive-like behavior and increased sedoheptulose 7-phosphate, hypotaurine, and acetyl-L-carnitine contents, which were identified as marker candidates for fluoxetine efficacy. ROC analysis revealed 4 significant marker candidates for CSIS group discrimination, and 10 for fluoxetine efficacy. SVM-LK with accuracies of 61.50% or 93.30% identified a panel of 7 or 25 predictive metabolites for depressive-like behavior or fluoxetine effectiveness, respectively. Overall, metabolic fingerprints combined with the ROC curve and SVM-LK may represent a new approach to identifying marker candidates or predictive metabolites for ongoing disease or disease risk and treatment outcome.