Type 2 diabetes risk gene Dusp8 regulates hypothalamic Jnk signaling and insulin sensitivity.

Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic-pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D.

Towards frailty biomarkers: Candidates from genes and pathways regulated in aging and age-related diseases.

OBJECTIVE: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel. METHODS: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers. RESULTS: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified. CONCLUSION: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFß (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Acute dietary fat intake initiates alterations in energy metabolism and insulin resistance.

BACKGROUND: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice. METHODS: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses. RESULTS: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK). CONCLUSION: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202. FUNDING: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

Functional inactivation of the genome-wide association study obesity gene neuronal growth regulator 1 in mice causes a body mass phenotype.

To date, genome-wide association studies (GWAS) have identified at least 32 novel loci for obesity and body mass-related traits. However, the causal genetic variant and molecular mechanisms of specific susceptibility genes in relation to obesity are yet to be fully confirmed and characterised. Here, we examined whether the candidate gene NEGR1 encoding the neuronal growth regulator 1, also termed neurotractin or Kilon, accounts for the obesity association. To characterise the function of NEGR1 for body weight control in vivo, we generated two novel mutant mouse lines, including a constitutive NEGR1-deficient mouse line as well as an ENU-mutagenised line carrying a loss-of-function mutation (Negr1-I87N) and performed metabolic phenotypic analyses. Ablation of NEGR1 results in a small but steady reduction of body mass in both mutant lines, accompanied with a small reduction in body length in the Negr1-I87N mutants. Magnetic resonance scanning reveals that the reduction of body mass in Negr1-I87N mice is due to a reduced proportion of lean mass. Negr1-I87N mutants display reduced food intake and physical activity while normalised energy expenditure remains unchanged. Expression analyses confirmed the brain-specific distribution of NEGR1 including strong expression in the hypothalamus. In vitro assays show that NEGR1 promotes cell-cell adhesion and neurite growth of hypothalamic neurons. Our results indicate a role of NEGR1 in the control of body weight and food intake. This study provides evidence that supports the link of the GWAS candidate gene NEGR1 with body weight control.

A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia.

Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.

Neurobeachin, a regulator of synaptic protein targeting, is associated with body fat mass and feeding behavior in mice and body-mass index in humans.

Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/- mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.

Identification of a potential biomarker for FABP4 inhibition: the power of lipidomics in preclinical drug testing.

The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms.

A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model.

OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Urocortin 3 modulates social discrimination abilities via corticotropin-releasing hormone receptor type 2.

Urocortin 3 (UCN3) is strongly expressed in specific nuclei of the rodent brain, at sites distinct from those expressing urocortin 1 and urocortin 2, the other endogenous ligands of corticotropin-releasing hormone receptor type 2 (CRH-R2). To determine the physiological role of UCN3, we generated UCN3-deficient mice, in which the UCN3 open reading frame was replaced by a tau-lacZ reporter gene. By means of this reporter gene, the nucleus parabrachialis and the premammillary nucleus were identified as previously unknown sites of UCN3 expression. Additionally, the introduced reporter gene enabled the visualization of axonal projections of UCN3-expressing neurons from the superior paraolivary nucleus to the inferior colliculus and from the posterodorsal part of the medial amygdala to the principal nucleus of the bed nucleus of the stria terminalis, respectively. The examination of tau-lacZ reporter gene activity throughout the brain underscored a predominant expression of UCN3 in nuclei functionally connected to the accessory olfactory system. Male and female mice were comprehensively phenotyped but none of the applied tests provided indications for a role of UCN3 in the context of hypothalamic-pituitary-adrenocortical axis regulation, anxiety- or depression-related behavior. However, inspired by the prevalent expression throughout the accessory olfactory system, we identified alterations in social discrimination abilities of male and female UCN3 knock-out mice that were also present in male CRH-R2 knock-out mice. In conclusion, our results suggest a novel role for UCN3 and CRH-R2 related to the processing of social cues and to the establishment of social memories.

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry.

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.