An early, reversible cholesterolgenic etiology of diet-induced insulin resistance.

Objective: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1.

Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion.

G protein-coupled receptors (GPCRs) in intestinal enteroendocrine cells (EECs) respond to nutritional, neural, and microbial cues and modulate the release of gut hormones. Here we show that Gpr17, an orphan GPCR, is co-expressed in glucagon-like peptide-1 (GLP-1)-expressing EECs in human and rodent intestinal epithelium. Acute genetic ablation of Gpr17 in intestinal epithelium improves glucose tolerance and glucose-stimulated insulin secretion (GSIS).

A high-fat diet catalyzes progression to hyperglycemia in mice with selective impairment of insulin action in Glut4-expressing tissues.

Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes.

Phenotypic sexual dimorphism in response to dietary fat manipulation in C57BL/6J mice.

Background: Obesity and the metabolic syndrome are increasingly prevalent in society and their complications and response to treatment exhibit sexual dimorphism. Mouse models of high fat diet-induced obesity are commonly used for both mechanistic and therapeutic studies of metabolic disease and diabetes. However, the inclusion of female mammals in obesity research has not been a common practice, and has resulted in a paucity of data regarding the effect of sex on metabolic parameters and its applicability to humans.

On the theoretical limitations in estimating thickness of a plate-like structure from a full-field single-tone response Lamb wave measurement.

A persistent question in wavenumber analysis in the estimation of thickness from a steady-state wave field is identifying the theoretical sensitivity of the system. A well-known trade-off between spatial frequency/wavenumber resolution and thickness resolution exists. The current work presents a calculation of the Cramer-Rao Lower bound (CRLB), specifically as applied to thickness estimates, for a 2-dimensional multi-mode waveform in Gaussian noise.

On a Method For Reconstructing Computed Tomography Datasets from an Unstable Source.

As work continues in neutron computed tomography, at Los Alamos Neutron Science Center (LANSCE) and other locations, source reliability over the long imaging times is an issue of increasing importance. Moreover, given the time commitment involved in a single neutron image, it is impractical to simply discard a scan and restart in the event of beam instability. In order to mitigate the cost and time associated with these options, strategies are presented in the current work to produce a successful reconstruction of computed tomography data from an unstable source.

Adiponectin receptor fragmentation in mouse models of type 1 and type 2 diabetes.

The protein hormone adiponectin regulates glucose and fatty acid metabolism by binding to two PAQR-family receptors (AdipoR1 and AdipoR2). Both receptors feature a C-terminal segment which is released by proteolysis to form a freely circulating C-terminal fragment (CTF) found in the plasma of normal individuals but not in some undefined diabetes patients. The AdipoR1-CTF is a competitive inhibitor of tumor necrosis factor α cleavage enzyme (TACE) but it contains a shorter peptide domain (AdipoR1 CTF) that is a strong non-competitive inhibitor of insulin-degrading enzyme (IDE).

Syntaxin 4 up-regulation increases efficiency of insulin release in pancreatic islets from humans with and without type 2 diabetes mellitus.

Context: Evidence suggests that dysfunctional β-cell insulin release precedes type 1 and type 2 diabetes (T1D and T2D, respectively) and that enhancing the efficiency of insulin release from pancreatic islet β-cells may delay/prevent these diseases. We took advantage of the rare opportunity to test this paradigm using islets from human type 2 diabetic individuals.

Objectives: Insulin release capacity is limited by the abundance of fusogenic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins.

Divergent compensatory responses to high-fat diet between C57BL6/J and C57BLKS/J inbred mouse strains.

Impaired glucose tolerance (IGT) and type 2 diabetes (T2DM) are polygenic disorders with complex pathophysiologies; recapitulating them with mouse models is challenging. Despite 70% genetic homology, C57BL/6J (BL6) and C57BLKS/J (BLKS) inbred mouse strains differ in response to diet- and genetic-induced obesity. We hypothesized these differences would yield insight into IGT and T2DM susceptibility and response to pharmacological therapies.

Mouse and human islets survive and function after coating by biosilicification.

Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes.

Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease.

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories(1-4), variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies.

Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model.

Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation.

p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model.

The unique hypusine modification of eIF5A promotes islet beta cell inflammation and dysfunction in mice.

In both type 1 and type 2 diabetes, pancreatic islet dysfunction results in part from cytokine-mediated inflammation. The ubiquitous eukaryotic translation initiation factor 5A (eIF5A), which is the only protein to contain the amino acid hypusine, contributes to the production of proinflammatory cytokines. We therefore investigated whether eIF5A participates in the inflammatory cascade leading to islet dysfunction during the development of diabetes.

Phosphorylation of p22phox on threonine 147 enhances NADPH oxidase activity by promoting p47phox binding.

NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events.

A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity.

Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91(phox) and p22(phox), which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47(phox) and p67(phox). A fifth subunit, p40(phox), plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P).

A fluorescently tagged C-terminal fragment of p47phox detects NADPH oxidase dynamics during phagocytosis.

The assembly of cytosolic p47(phox) and p67(phox) with flavocytochrome b(558) at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47(phox) and p67(phox) are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67(phox) translocation in neutrophils, but is not required for oxidase activity in model systems.

Fc gamma R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome.

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b(558) and cytosolic p67(phox), p47(phox), and p40(phox) subunits that undergo membrane translocation upon cellular activation. The function of p40(phox), which binds p67(phox) in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40(phox) and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes.

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity.

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox.

The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis.

Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67(phox), p47(phox), and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47(phox), p67(phox), and the FcgammaIIA receptor were expressed from stable transgenes in COS7 cells.

Neural stem cells spontaneously express dopaminergic traits after transplantation into the intact or 6-hydroxydopamine-lesioned rat.

The ability to differentiate neural stem cells (NSCs) into dopamine neurons is fundamental to their role in cell replacement therapies for neurodegenerative disorders such as Parkinson's disease. We show here that when a clonal line (C17.2) of undifferentiated NSCs is transplanted into the intact or 6-hydroxydopamine-lesioned striatum, cells withdraw from the cell cycle (BrdU(-)), migrate extensively in the host striatum, and express markers associated with neuronal (beta-tubulin III(+), NSE(+), NeuN(+)) but not glial (GFAP(-), MBP(-), A2B5(-)) differentiation.

Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function.

The NADPH oxidase is a multicomponent enzyme that transfers electrons from NADPH to O2 to generate superoxide (O2*-), the precursor of microbicidal oxygen species that play an important role in host defense. Flavocytochrome b558, a heterodimeric oxidoreductase comprised of gp91(phox) and p22(phox) subunits, contains two nonidentical, bis-histidine-ligated heme groups imbedded within the membrane. Four histidine residues that appear to serve as noncovalent axial heme ligands reside within the hydrophobic N terminus of gp91(phox), but the role of p22(phox) in heme binding is unclear.

Antioxidant compounds protect dopamine neurons from death due to oxidative stress in vitro.

Using tissue culture models of oxidative stress caused by serum deprivation or MPTP/MPP+ toxicity, the present study establishes that the antioxidants epigallocatechin gallate, lazaroids U74389G and U83836E, reservatrol, MnTBAP, MCI 186, trolox, and melatonin protect 68-100% of dopamine (DA) neurons from cell death. In contrast, the nitric oxide inhibitor LY83583, the caspase inhibitors Z-VAD-FMK, Ac-DQMD-CHO and Z-DEVD-FMK, and the CDK-5 inhibitor, roscovotine were not neuroprotective, although death was often delayed by 1 day in vitro. We conclude that antioxidants are more effective at preventing cell death in vitro than are inhibitors at later stages in the death cascade.

Induction of a dopaminergic phenotype in cultured striatal neurons by bone morphogenetic proteins.

In the present study, we examined whether the bone morphogenetic proteins (BMPs), which are important in the developmental specification of transmitter type in certain classes of neurons, might also play a role in signaling the differentiation of a dopaminergic (DA) phenotype. We found that BMP-2, -4 and -6 were each capable of inducing, in a dose and time dependent manner, moderate levels of the DA enzyme tyrosine hydroxylase (TH) in cultured neurons from the mouse embryonic striatum. In contradistinction to other TH-inducing agents, BMPs initiated de novo TH expression without the required synergy of exogenous growth factors or co-activating substances and in neurons presumably aged (E16) beyond the critical period for induction.

Differentiation of human dopamine neurons from an embryonic carcinomal stem cell line.

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine, TPA, IBMX/forskolin), will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study, we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media.