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Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin's access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs., Introduction Interorgan communication is essential for maintaining physiological homeostasis. One way organs communicate with each other is by sending molecular signals through the circulation. Before these signals can stimulate biological [...]

Those suffering from Duchenne muscular dystrophy share with mdx mice a lack of dystrophin, which induces a loss of dystrophin-associated proteins (DAPs) in the sarcolemma. This loss could be mitigated by the dystrophin-related protein (DRP) utrophin, which is found in the sarcolemma in cardiac and skeletal muscles of adult mdx mice. Because DRP and DAP colocalize to the neuromuscular junction, DRP could help retain DAP in the sarcolemma.

Duchenne muscular dystrophy (DMD) is a disease of the muscles caused by a defective gene on the X chromosome; it affects only boys and usually results in death by age 20. Study of this defective DMD gene has revealed coding for dystrophin, an integral protein in the membrane of normal skeletal muscle fiber, but people with DMD are missing this protein. The study of other genetic diseases has revealed that when one protein is affected, others associated with it are affected also. In DMD patients, one of the four glycoproteins (carbohydrate and protein complexes) that make up the structure of this protein is deficient. It is hypothesized that the reduction of this dystrophin-associated glycoprotein may hold a clue to discovering the cause of this type of muscular dystrophy. Reduction of this glycoprotein can lead to a destabilization of the plasma membrane around the skeletal muscle cell, and the proteins associated with this membrane. This membrane destabilization can in turn lead to a deregulation of the intracellular calcium, which could cause abnormal activation of calcium-dependent protease (enzymes that breakdown proteins) activity. In DMD the muscle protein gradually degenerates, which could be explained by this proposed abnormal enzymatic activity. (Consumer Summary produced by Reliance Medical Information, Inc.

Abstract: The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a “testing funnel” of assays to identify genuine hits using high‐throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists. [ABSTRACT FROM AUTHOR]

Activation of free fatty acid receptor 1 (GPR40) by synthetic partial and full agonists occur via distinct allosteric sites. A crystal structure of GPR40-TAK-875 complex revealed the allosteric site for the partial agonist. Here we report the 2.76-Å crystal structure of human GPR40 in complex with a synthetic full agonist, compound 1, bound to the second allosteric site. Unlike TAK-875, which acts as a Gαq-coupled partial agonist, compound 1 is a dual Gαq and Gαs-coupled full agonist. compound 1 binds in the lipid-rich region of the receptor near intracellular loop 2 (ICL2), in which the stabilization of ICL2 by the ligand is likely the primary mechanism for the enhanced G protein activities. The endogenous free fatty acid (FFA), γ-linolenic acid, can be computationally modeled in this site. Both γ-linolenic acid and compound 1 exhibit positive cooperativity with TAK-875, suggesting that this site could also serve as a FFA binding site. [ABSTRACT FROM AUTHOR]

The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet β-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing. A clinical study with 3 administered to subjects with T2DM provided proof of concept of 3 as a potential glucose-lowering therapy. This manuscript summarizes the scientific rationale, medicinal chemistry, preclinical, and early development data of this new class of GPR40 agonists. [ABSTRACT FROM AUTHOR]

LY2881835 is a selective, potent, and efficacious GPR40 agonist. The objective of the studies described here was to examine the pharmacological properties of LY2881835 in preclinical models of T2D. Significant increases in insulin secretion were detected when LY2881835 was tested in primary islets from WT mice but not in islets from GPR40 KO mice. Furthermore, LY2881835 potentiated glucose stimulated insulin secretion in normal lean mice. Acute administration of LY2881835 lowered glucose during OGTTs in WT mice but not in GPR40 KO mice. These findings demonstrate that LY2881835 induces GPR40-mediated activity ex vivo and in vivo. LY2881835 was administered orally at 10 mg/kg to diet-induced obese ( DIO) mice (an early model of T2D due to insulin resistance) for 14 days. Statistically significant reductions in glucose were seen during OGTTs performed on days 1 and 15. When a study was done for 3 weeks in Zucker fa/fa rats, a rat model of insulin resistance, normalization of blood glucose levels equivalent to those seen in lean rats was observed. A similar study was performed in streptozotocin ( STZ)-treated DIO mice to explore glucose control in a late model of T2D. In this model, pancreatic insulin content was reduced ~80% due to STZ-treatment plus the mice were insulin resistant due to their high fat diet. Glucose AUCs were significantly reduced during OGTTs done on days 1, 7, and 14 compared to control mice. In conclusion, these results demonstrate that LY2881835 functions as a GPR40-specific insulin secretagogue mediating immediate and durable glucose control in rodent models of early- and late-stage T2D. [ABSTRACT FROM AUTHOR]

High Throughput Screening (HTS) assays are used to rapidly identify promising drug candidates or leads from hundreds of thousands of compounds. Hence, it is important that the assay should be designed to discriminate responses from the active and inactive compounds and the background noise. We introduce the idea of a signal window which provides a degree of separation between signals. This allows one to correctly identify new molecular entities with desired level of activity (called "hits") in the presence of variability. The statistical criteria for setting and calculating signal windows are presented along with illustrative examples. Results show that the ideal signal window should be 2 standard deviations (SD) of the largest signal in screening assays, although a 1 SD window size is the minimum acceptable limit. When signal windows are set below 2 SD, the probability of missing "hits" increases significantly. [ABSTRACT FROM PUBLISHER]

Membranes prepared from stable transfected cells expressing the human gene encoding a functional 5-hydroxytryptaminelE (5HT1E) receptor were used to investigate high-affinity [3H]serotonin ([3H]5-HT) binding with scintillation proximity assay (SPA) technology. In this nonseparation format, membranes are captured by WGA-coated SPA fluoromicrospheres for detection of receptor-bound radioligand. Total binding observed was approximately 2000 cpm compared to a nonspecific signal of 100 cpm determined in the presence of 10,uM unlabeled 5-HT. Non-proximity effects (NPE) for the radiolabel and SPA beads averaged less than 100 cpm. Saturation binding analysis yielded an equilibrium dissociation constant (Kd) of 5.38 ± 0.43 nM and a maximum binding capacity (Bmax) of 6.42 + 0.15 pmol/mg protein. Competition with unlabeled serotonergic compounds demonstrated a specificity of the assay with rank potencies (5-HT > a-Me-5-HT > 2-Me-5-HT > 5-CT) similar to those observed using traditional fitration techniques. The variability of the assay and the stability of all reagents were investigated to validate the assay for extended use throughout a typical high throughput screen operation lasting several months. [ABSTRACT FROM PUBLISHER]

The syntheses of seven novel synthetic analogues of the naturally occurring insecticide ryanodine are described. These, and other synthetic and naturally occurring analogues, have been used to characterize the selectivity of a monoclonal antibody which has been produced by immunization with 9-hydroxy-21-(4-azidobenzyloxy)-9-epiryanodine photo-conjugated to keyhole limpet haemocyanin. The antibody binds [3H]ryanodine with a dissociation constant of 0.37 nM. The specificity of this antibody in terms of its ability to recognise 11 natural and synthetic analogues of ryanodine has been determined by [3H]ryanodine displacement and shown to be similar (for a partially overlapping set of analogues) to that determined earlier for a rabbit polyclonal antibody (Kahl, S. D., et al., Anal. Biochem., 218 (1994) 55-62). The selectivity of the antibodies is shown to be related to that of the sarcoplasmic reticulum Ca2+ release channelfrom rabbit skeletal muscle, both for this set of ryanodine analogues and for three structurally dissimilar, low-molecular-weight compounds identified by high-throughput screening. The advantages of these antibody models of the Ca2+ release channel for screening are illustrated by their superior performance in a homogeneous bindingassay. [ABSTRACT FROM AUTHOR]

Abstract: Extensive structure–activity relationship studies utilizing a β-MSH-derived cyclic nonapeptide, Ac-Tyr-Arg-[Cys-Glu-His-d-Phe-Arg-Trp-Cys]-NH2 (3), led to identification of a series of novel MC-4R selective disulfide-constrained hexapeptide analogs including Ac-[hCys-His-d-Phe-Arg-Trp-Cys]-NH2 (12). The structural modifications associated with profound influence on MC-4R potency and selectivity were ring size, ring conformation, and the aromatic substitution of the d-Phe7. These cyclic peptide analogs provide novel and enhanced reagents for use in the elucidation of melanocortin-4 receptor-related physiology, and may additionally find application in the treatment of obesity and related metabolic disorders. [Copyright &y& Elsevier]