Increased Hepatic Insulin Action in Diet-Induced Obese Mice Following Inhibition of Glucosylceramide Synthase

Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder.DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity.These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity

Superoxide dismutase deficiency impairs olfactory sexual signaling and alters bioenergetic function in mice

Oxidative stress (an overproduction of reactive oxygen species in relation to defense mechanisms) may restrict investment in life history traits, such as growth, reproduction, lifespan, and the production of sexual signals to attract mates. The constraint on sexual signaling by oxidative stress is of particular interest because it has been proposed as a mechanism ensuring that only good-quality males produce the most attractive sexual signals. Despite these predictions, evidence supporting this theory is, at best, equivocal. We used a superoxide dismutase knockout mouse to demonstrate that oxidative stress directly impairs investment in morphological (preputial glands) and molecular (major urinary proteins) components of olfactory signaling essential for mate attraction. By maintaining males in a much more competitive environment than usual for mouse laboratory experiments, we also revealed a range of phenotypes of superoxide dismutase deficiency not observed in previous studies of this mouse model. This range included impaired bioenergetic function, which was undetectable in the control environment of this study. We urge further examination of model organisms in seminatural conditions and more competitive laboratory environments, as important phenotypes can be exposed under these more demanding conditions

Accumulation of serum lipids by vascular smooth muscle cells involves a macropinocytosis-like uptake pathway and is associated with the downregulation of the ATP-binding cassette transporter A1

Vascular smooth muscle cells (VSMC) are present in arterial intima before atherosclerotic plaques develop and are likely to be exposed to unmodified serum lipids as they enter the vessel wall. We examined the effects of sera from mice on the morphology and function of mouse VSMC. Incubation of a mouse VSMC line (MOVAS) with sera from normocholesterolemic (C57BL/6J) or hypercholesterolemic (APOE(-/-)) mice caused concentration-dependent increases in lipid accumulation as measured by AdipoRed, with the extent of lipid uptake significantly greater with the latter sera type. Inhibition of c-Jun N-terminal kinases (SP600125), Src kinases (AG1879), and clathrin-dependent endocytosis (monodansylcadaverine) to disrupt scavenger receptor-mediated uptake of lipids had no effect on serum-induced lipid accumulation by VSMC. By contrast, inhibition of macropinocytosis with antagonists of PI-3 kinase (LY294002) and actin (cytochalasin D) markedly reduced lipid accumulation. Serum exposure reduced the expression of the ATP-binding cassette transporter A1, consistent with impaired cholesterol efflux, but had no effect on the expression of markers of VSMC differentiation. Moreover, the expression of several inflammation and foam cell markers was unchanged (CCL2, CCL5, and CD68) by mouse sera. The accumulation of unmodified serum lipids by VSMC involves a macropinocytosis-like uptake pathway and is associated with the downregulation of the ATP-binding cassette transporter. We speculate that VSMC may play an atheroprotective role in arterial intima by acting as a "sink" for unmodified lipids

Endogenous progesterone levels and frontotemporal dementia: Modulation of TDP-43 and Tau levels in vitro and treatment of the A315T TARDBP mouse model

Frontotemporal dementia (FTD) is associated with motor neurone disease (FTD-MND), corticobasal syndrome (CBS) and progressive supranuclear palsy syndrome (PSPS). Together, this group of disorders constitutes a major cause of young-onset dementia. One of the three clinical variants of FTD is progressive nonfluent aphasia (PNFA), which is focused on in this study. The steroid hormone progesterone (PROG) is known to have an important role as a neurosteroid with potent neuroprotective and promyelination properties. In a case-control study of serum samples (39 FTD, 91 controls), low serum PROG was associated with FTD overall. In subgroup analysis, low PROG levels were significantly associated with FTD-MND and CBS, but not with PSPS or PNFA. PROG levels of >195 pg/ml were significantly correlated with lower disease severity (frontotemporal dementia rating scale) for individuals with CBS. In the human neuroblastoma SK-N-MC cell line, exogenous PROG (9300-93,000 pg/ml) had a significant effect on overall Tau and nuclear TDP-43 levels, reducing total Tau levels by ~1.5-fold and increasing nuclear TDP-43 by 1.7- to 2.0-fold. Finally, elevation of plasma PROG to a mean concentration of 5870 pg/ml in an Ala315Thr (A315T) TARDBP transgenic mouse model significantly reduced the rate of loss of locomotor control in PROG-treated, compared with placebo, mice. The PROG treatment did not significantly increase survival of the mice, which might be due to the limitation of the transgenic mouse to accurately model TDP-43-mediated neurodegeneration. Together, our clinical, cellular and animal data provide strong evidence that PROG could be a valid therapy for specific related disorders of FTD

Identification of Native and Posttranslationally Modified HLA-B*57:01-Restricted HIV Envelope Derived Epitopes Using Immunoproteomics

The recognition of pathogen‐derived peptides by T lymphocytes is the cornerstone of adaptive immunity, whereby intracellular antigens are degraded in the cytosol and short peptides assemble with class I human leukocyte antigen (HLA) molecules in the ER. These peptide‐HLA complexes egress to the cell surface and are scrutinized by cytotoxic CD8+ T‐cells leading to the eradication of the infected cell. Here, naturally presented HLA‐B*57:01 bound peptides derived from the envelope protein of the human immunodeficiency virus (HIVenv) are identified. HIVenv peptides are present at a very small percentage of the overall HLA‐B*57:01 peptidome (<0.1%) and both native and posttranslationally modified forms of two distinct HIV peptides are identified. Notably, a peptide bearing a natively encoded C‐terminal tryptophan residue is also present in a modified form containing a kynurenine residue. Kynurenine is a major product of tryptophan catabolism and is abundant during inflammation and infection. Binding of these peptides at a molecular level and their immunogenicity in preliminary functional studies are examined. Modest immune responses are observed to the modified HIVenv peptide, highlighting a potential role for kynurenine‐modified peptides in the immune response to HIV and other viral infections

MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

© 2016 Macmillan Publishers Limited. Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia

Arrested hematopoiesis and vascular relaxation defects in mice with a mutation in Dhfr

© 2016, American Society for Microbiology. All Rights Reserved. Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo. In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling