Paul A. Insel

Experimental design and analysis and their reporting: new guidance for publication in BJP

This Editorial is part of a series. To view the other Editorials in this series, visit: http://onlinelibrary.wiley.com/doi/10.1111/bph.12956/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12954/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12955/abstract and http://onlinelibrary.wiley.com/doi/10.1111/bph.13112/abstract

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.

A Single Amino Acid Mutation Contributes to Adaptive Beach Mouse Color Pattern

Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor (Mc1r) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Floridas Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.

A Model of the Kinetics of Insulin in Man

The design of the present study of the kinetics of insulin in man combines experimental features which obviate two of the major problems in previous insulin studies. (a) The use of radioiodinated insulin as a tracer has been shown to be inappropriate since its metabolism differs markedly from that of the native hormone. Therefore porcine insulin was administered by procedures which raised insulin levels in arterial plasma into the upper physiologic range. Hypoglycemia was prevented by adjusting the rate of an intravenous infusion of glucose in order to control the blood glucose concentration (the glucose-clamp technique). (b) Estimation of a single biological half-time of insulin after pulse injection of the hormone has been shown to be inappropriate since plasma insulin disappearance curves are multiexponential. Therefore the SAAM 25 computer program was used in order to define the parameters of a three compartment insulin model. The combined insulin mass of the three compartments (expressed as plasma equivalent volume) is equal to inulin space (15.7% body wt). Compartment 1 is apparently the plasma space (4.5%). The other two compartments are extra-vascular; compartment 2 is small (1.7%) and equilibrates rapidly with plasma, and compartment 3 is large (9.5%) and equilibrates slowly with plasma. The SAAM 25 program can simulate the buildup and decay of insulin in compartments 2 and 3 which cannot be assayed directly. Insulin in compartment 3 was found to correlate remarkably with the time-course of the servo-controlled glucose infusion. Under conditions of a steady-state arterial glucose level, glucose infusion is a measure of glucose utilization. We conclude that compartment 3 insulin (rather than plasma insulin) is a more direct determinant of glucose utilization. We suggest that the combined use of glucose-clamp and kinetic-modeling techniques should aid in the delineation of pathophysiologic states affecting glucose and insulin metabolism.

Adrenergic receptors in man

THE catecholamines norepinephrine and epinephrine are key regulators of many physiologic events in human beings; norepinephrine acts primarily as a neurotransmitter released from sympathetic-nerve ...

The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology.

The many components of G‐protein‐coupled receptor (GPCR) signal transduction provide cells with numerous combinations with which to customize their responses to hormones, neurotransmitters, and pharmacologic agonists. GPCRs function as guanine nucleotide exchange factors for heterotrimeric (α, β, γ) G proteins, thereby promoting exchange of GTP for GDP and, in turn, the activation of ‘downstream’ signaling components. Recent data indicate that individual cells express mRNA for perhaps over 100 different GPCRs (out of a total of nearly a thousand GPCR genes), several different combinations of G‐protein subunits, multiple regulators of G‐protein signaling proteins (which function as GTPase activating proteins), and various isoforms of downstream effector molecules. The differential expression of such protein combinations allows for modulation of signals that are customized for a specific cell type, perhaps at different states of maturation or differentiation. In addition, in the linear arrangement of molecular interactions involved in a given GPCR–G‐protein–effector pathway, one needs to consider the localization of receptors and post‐receptor components in subcellular compartments, microdomains, and molecular complexes, and to understand the movement of proteins between these compartments. Co‐localization of signaling components, many of which are expressed at low overall concentrations, allows cells to tailor their responses by arranging, or spatially organizing in unique and kinetically favorable ways, the molecules involved in GPCR signal transduction. This review focuses on the role of lipid rafts and a subpopulation of such rafts, caveolae, as a key spatial compartment enriched in components of GPCR signal transduction. Recent data suggest cell‐specific patterns for expression of those components in lipid rafts and caveolae. Such domains likely define functionally important, cell‐specific regions of signaling by GPCRs and drugs active at those GPCRs.

Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2–4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling.

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Insulin Control of Glucose Metabolism in Man: A New Kinetic Analysis

Analyses of the control of glucose metabolism by insulin have been hampered by changes in bloog glucose concentration induced by insulin administration with resultant activation of hypoglycemic counterregulatory mechanisms. To eliminate such mechanisms, we have employed the glucose clamp technique which allows maintenance of fasting blood glucose concentration during and after the administration of insulin. Analyses of six studies performed in young healthy men in the postabsorptive state utilizing the concurrent administration of [14C]glucose and 1 mU/kg per min (40 mU/m2 per min) porcine insulin led to the development of kinetic models for insulin and for glucose. These models account quantitatively for the control of insulin on glucose utilization and on endogenous glucose production during nonsteady states. The glucose model, a parallel three-compartment model, has a central compartment (mass = 68 +/- 7 mg/kg; space of distribution = blood water volume) in rapid equilibrium with a smaller compartment (50 +/- 17 mg/kg) and in slow equilibrium with a larger compartment (96 +/-21 mg/kg). The total plasma equivalent space for the glucose system averaged 15.8 liters or 20.3% body weight. Two modes of glucose loss are introduced in the model. One is a zero-order loss (insulin and glucose independent) from blood to the central nervous system; its magnitude was estimated from published data. The other is an insulin-dependent loss, occurring from the rapidly equilibrating compartment and, in the basal period, is smaller than the insulin-independent loss. Endogenous glucose production averaged 1.74 mg/kg per min in the basal state and enters the central compartment directly. During the glucose clamp experiments plasma insulin levels reached a plateau of 95 +/-8 microU/ml. Over the entire range of insulin levels studied, glucose losses were best correlated with levels of insulin in a slowly equilibrating insulin compartment of a three-compartment insulin model. A proportional control by this compartment on glucose utilization was adequate to satisfy the observed data. Insulin also rapidly decreased the endogenous glucose production to 33% of its basal level (0.58 mg/kg per min), this suppression being maintained for at least 40 min after exogenous insulin infusion was terminated and after plasma insulin concentrations had returned to basal levels. The change in glucose utilization per unit change in insulin in the slowly equilibrating insulin compartment is proposed as a new measure for insulin sensitivity. This defines insulin effects more precisely than previously used measures, such as plasma glucose/plasma insulin concentration ratios. Glucose clamp studies and the modeling of the coupled kinetics of glucose and insulin offers a new and potentially valuable tool to the study of altered states of carbohydrate metabolism.

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, β1-adrenergic receptors (β1-AR), β2-AR, Gαs, and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (β-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and α-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/μm of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Gαs from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-Gs-AC in lipid rafts/caveolae.