24 results on '"Sloop, Kyle W."'
Search Results
2. Discovery of an Orally Efficacious Positive Allosteric Modulator of the Glucagon-like Peptide-1 Receptor
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Willard, Francis S., Wainscott, David B., Showalter, Aaron D., Stutsman, Cynthia, Ma, Wenzhen, Cardona, Guemalli R., Zink, Richard W., Corkins, Christopher M., Chen, Qi, Yumibe, Nathan, Agejas, Javier, Cumming, Graham R., Minguez, José Miguel, Jiménez, Alma, Mateo, Ana I., Castaño, Ana M., Briere, Daniel A., Sloop, Kyle W., and Bueno, Ana B.
- Abstract
The identification of LSN3318839, a positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), is described. LSN3318839 increases the potency and efficacy of the weak metabolite GLP-1(9-36)NH2to become a full agonist at the GLP-1R and modestly potentiates the activity of the highly potent full-length ligand, GLP-1(7-36)NH2. LSN3318839 preferentially enhances G protein-coupled signaling by the GLP-1R over β-arrestin recruitment. Ex vivoexperiments show that the combination of GLP-1(9-36)NH2and LSN3318839 produces glucose-dependent insulin secretion similar to that of GLP-1(7-36)NH2. Under nutrient-stimulated conditions that release GLP-1, LSN3318839 demonstrates robust glucose lowering in animal models alone or in treatment combination with sitagliptin. From a therapeutic perspective, the biological properties of LSN3318839 support the concept that GLP-1R potentiation is sufficient for reducing hyperglycemia.
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- 2021
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3. Structural insights into probe-dependent positive allosterism of the GLP-1 receptor
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Bueno, Ana B., Sun, Bingfa, Willard, Francis S., Feng, Dan, Ho, Joseph D., Wainscott, David B., Showalter, Aaron D., Vieth, Michal, Chen, Qi, Stutsman, Cynthia, Chau, Betty, Ficorilli, James, Agejas, Francisco J., Cumming, Graham R., Jiménez, Alma, Rojo, Isabel, Kobilka, Tong Sun, Kobilka, Brian K., and Sloop, Kyle W.
- Abstract
Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a ‘molecular glue’. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein–protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.
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- 2020
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4. Activation of the GLP-1 receptor by a non-peptidic agonist
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Zhao, Peishen, Liang, Yi-Lynn, Belousoff, Matthew J., Deganutti, Giuseppe, Fletcher, Madeleine M., Willard, Francis S., Bell, Michael G., Christe, Michael E., Sloop, Kyle W., Inoue, Asuka, Truong, Tin T., Clydesdale, Lachlan, Furness, Sebastian G. B., Christopoulos, Arthur, Wang, Ming-Wei, Miller, Laurence J., Reynolds, Christopher A., Danev, Radostin, Sexton, Patrick M., and Wootten, Denise
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Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2–6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
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- 2020
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5. LY3298176, a novel dual GIP and GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus: From discovery to clinical proof of concept.
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Coskun, Tamer, Sloop, Kyle W., Loghin, Corina, Alsina-Fernandez, Jorge, Urva, Shweta, Bokvist, Krister B., Cui, Xuewei, Briere, Daniel A., Cabrera, Over, Roell, William C., Kuchibhotla, Uma, Moyers, Julie S., Benson, Charles T., Gimeno, Ruth E., D'Alessio, David A., and Haupt, Axel
- Abstract
Abstract Objective A novel dual GIP and GLP-1 receptor agonist, LY3298176, was developed to determine whether the metabolic action of GIP adds to the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM). Methods LY3298176 is a fatty acid modified peptide with dual GIP and GLP-1 receptor agonist activity designed for once-weekly subcutaneous administration. LY3298176 was characterised in vitro , using signaling and functional assays in cell lines expressing recombinant or endogenous incretin receptors, and in vivo using body weight, food intake, insulin secretion and glycemic profiles in mice. A Phase 1, randomised, placebo-controlled, double-blind study was comprised of three parts: a single-ascending dose (SAD; doses 0.25–8 mg) and 4-week multiple-ascending dose (MAD; doses 0.5–10 mg) studies in healthy subjects (HS), followed by a 4-week multiple-dose Phase 1 b proof-of-concept (POC; doses 0.5–15 mg) in patients with T2DM (ClinicalTrials.gov no. NCT02759107). Doses higher than 5 mg were attained by titration, dulaglutide (DU) was used as a positive control. The primary objective was to investigate safety and tolerability of LY3298176. Results LY3298176 activated both GIP and GLP-1 receptor signaling in vitro and showed glucose-dependent insulin secretion and improved glucose tolerance by acting on both GIP and GLP-1 receptors in mice. With chronic administration to mice, LY3298176 potently decreased body weight and food intake; these effects were significantly greater than the effects of a GLP-1 receptor agonist. A total of 142 human subjects received at least 1 dose of LY3298176, dulaglutide, or placebo. The PK profile of LY3298176 was investigated over a wide dose range (0.25–15 mg) and supports once-weekly administration. In the Phase 1 b trial of diabetic subjects, LY3298176 doses of 10 mg and 15 mg significantly reduced fasting serum glucose compared to placebo (least square mean [LSM] difference [95% CI]: −49.12 mg/dL [−78.14, −20.12] and −43.15 mg/dL [−73.06, −13.21], respectively). Reductions in body weight were significantly greater with the LY3298176 1.5 mg, 4.5 mg and 10 mg doses versus placebo in MAD HS (LSM difference [95% CI]: −1.75 kg [−3.38, −0.12], −5.09 kg [−6.72, −3.46] and −4.61 kg [−6.21, −3.01], respectively) and doses of 10 mg and 15 mg had a relevant effect in T2DM patients (LSM difference [95% CI]: −2.62 kg [−3.79, −1.45] and −2.07 kg [−3.25, −0.88], respectively. The most frequent side effects reported with LY3298176 were gastrointestinal (vomiting, nausea, decreased appetite, diarrhoea, and abdominal distension) in both HS and patients with T2DM; all were dose-dependent and considered mild to moderate in severity. Conclusions Based on these results, the pharmacology of LY3298176 translates from preclinical to clinical studies. LY3298176 has the potential to deliver clinically meaningful improvement in glycaemic control and body weight. The data warrant further clinical evaluation of LY3298176 for the treatment of T2DM and potentially obesity. Highlights • LY3298176 activates both GIP and GLP-1 receptor signaling in vitro. • LY3298176 lowers blood glucose in mice through actions on both incretin receptors. • LY3298176 reduced fasting glucose in humans with type 2 diabetes. • Weight loss was greater with LY3298176 than the selective GLP-1 receptor agonist, dulaglutide in healthy humans. • Tolerability of LY3298176 was comparable to GLP-1 receptor agonists. [ABSTRACT FROM AUTHOR]
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- 2018
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6. Synthesis and Characterization of Urofuranoic Acids: In Vivo Metabolism of 2-(2-Carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic Acid (CMPF) and Effects on in Vitro Insulin Secretion.
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Nagy, Edith, Ying Liu, Prentice, Kacey J., Sloop, Kyle W., Sanders, Phillip E., Batchuluun, Battsetseg, Hammond, Craig D., Wheeler, Michael B., and Durham, Timothy B.
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- 2017
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7. β Cell GLP-1R Signaling Alters α Cell Proglucagon Processing after Vertical Sleeve Gastrectomy in Mice
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Garibay, Darline, Lou, Jon, Lee, Seon A., Zaborska, Karolina E., Weissman, Margot H., Sloma, Erica, Donahue, Leanne, Miller, Andrew D., White, Andrew C., Michael, M. Dodson, Sloop, Kyle W., and Cummings, Bethany P.
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Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible β cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the β cell GLP-1R in islet function. Our data show that loss of β cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a β cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the β cell GLP-1R as a regulator of α cell proglucagon processing.
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- 2018
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8. Tirzepatide induces a thermogenic-like amino acid signature in brown adipose tissue.
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Samms, Ricardo J., Zhang, GuoFang, He, Wentao, Ilkayeva, Olga, Droz, Brian A., Bauer, Steven M., Stutsman, Cynthia, Pirro, Valentina, Collins, Kyla A., Furber, Ellen C., Coskun, Tamer, Sloop, Kyle W., Brozinick, Joseph T., and Newgard, Christopher B.
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Tirzepatide, a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to selective GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes (T2D). These results have fueled mechanistic studies focused on understanding how tirzepatide achieves its therapeutic efficacy. Recently, we found that treatment with tirzepatide improves insulin sensitivity in humans with T2D and obese mice in concert with a reduction in circulating levels of branched-chain amino (BCAAs) and keto (BCKAs) acids, metabolites associated with development of systemic insulin resistance (IR) and T2D. Importantly, these systemic effects were found to be coupled to increased expression of BCAA catabolic genes in thermogenic brown adipose tissue (BAT) in mice. These findings led us to hypothesize that tirzepatide may lower circulating BCAAs/BCKAs by promoting their catabolism in BAT. To address this question, we utilized a murine model of diet-induced obesity and employed stable-isotope tracer studies in combination with metabolomic analyses in BAT and other tissues. Treatment with tirzepatide stimulated catabolism of BCAAs/BCKAs in BAT, as demonstrated by increased labeling of BCKA-derived metabolites, and increases in levels of byproducts of BCAA breakdown, including glutamate, alanine, and 3-hydroxyisobutyric acid (3-HIB). Further, chronic administration of tirzepatide increased levels of multiple amino acids in BAT that have previously been shown to be elevated in response to cold exposure. Finally, chronic treatment with tirzepatide led to a substantial increase in several TCA cycle intermediates (α-ketoglutarate, fumarate, and malate) in BAT. These findings suggest that tirzepatide induces a thermogenic-like amino acid profile in BAT, an effect that may account for reduced systemic levels of BCAAs in obese IR mice. [Display omitted] • Tirzepatide augments the catabolism of BCAA in brown adipose tissue (BAT) of obese mice. • Tirzepatide promotes BCAA catabolism in BAT, despite its lower potency to activate the mouse GIPR relative to mouse GIP. • Tirzepatide increases amino acids and TCA cycle intermediates in BAT, as also observed in BAT thermogenesis. [ABSTRACT FROM AUTHOR]
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- 2022
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9. Insulin and Glucagon: Partners for Life.
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Holst, Jens Juul, Holland, William, Gromada, Jesper, Lee, Young, Unger, Roger H, Yan, Hai, Sloop, Kyle W, Kieffer, Timothy J, Damond, Nicolas, and Herrera, Pedro L
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In August 2016, several leaders in glucagon biology gathered for the European Association for the Study of Diabetes Hagedorn Workshop in Oxford, England. A key point of discussion focused on the need for basal insulin to allow for the therapeutic benefit of glucagon blockade in the treatment of diabetes. Among the most enlightening experimental results presented were findings from studies in which glucagon receptor-deficient mice were administered streptozotocin to destroy pancreatic β cells or had undergone diphtheria toxin-induced β cell ablation. This article summarizes key features of the discussion as a consensus was reached. Agents that antagonize glucagon may be of great benefit for the treatment of diabetes; however, sufficient levels of basal insulin are required for their therapeutic efficacy.
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- 2017
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10. LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept.
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Coskun, Tamer, Urva, Shweta, Roell, William C., Qu, Hongchang, Loghin, Corina, Moyers, Julie S., O'Farrell, Libbey S., Briere, Daniel A., Sloop, Kyle W., Thomas, Melissa K., Pirro, Valentina, Wainscott, David B., Willard, Francis S., Abernathy, Matthew, Morford, LaRonda, Du, Yu, Benson, Charles, Gimeno, Ruth E., Haupt, Axel, and Milicevic, Zvonko
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With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro , LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943. [Display omitted] • LY3437943 has triple agonist activity at the glucagon, GIP, and GLP-1 receptors • LY3437943 caused greater body weight loss in obese mice than tirzepatide • LY3437943 increased energy expenditure through glucagon receptor activation • Safety and tolerability of LY3437943 were similar to other incretin-based drugs Coskun et al. demonstrate that LY3437943, a triple glucagon, GIP, and GLP-1 receptor agonist for the treatment of obesity and type 2 diabetes, can reduce body weight through increased energy expenditure and reduced calorie intake in obese mice. Its safety and tolerability in healthy participants were similar to other incretin-based therapies. [ABSTRACT FROM AUTHOR]
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- 2022
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11. β-Cell Glucagon-Like Peptide-1 Receptor Contributes to Improved Glucose Tolerance After Vertical Sleeve Gastrectomy
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Garibay, Darline, McGavigan, Anne K., Lee, Seon A., Ficorilli, James V., Cox, Amy L., Michael, M. Dodson, Sloop, Kyle W., and Cummings, Bethany P.
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Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible for this remain incompletely defined. Glucagon-like peptide-1 (GLP-1) is a gut hormone that contributes to the maintenance of glucose homeostasis and is elevated after VSG. VSG-induced increases in postprandial GLP-1 secretion have been proposed to contribute to the glucoregulatory benefits of VSG; however, previous work has been equivocal. In order to test the contribution of enhanced β-cell GLP-1 receptor (GLP-1R) signaling we used a β-cell-specific tamoxifen-inducible GLP-1R knockout mouse model. Male β-cell-specific Glp-1rβ-cell+/+wild type (WT) and Glp-1rβ-cell−/−knockout (KO) littermates were placed on a high-fat diet for 6 weeks and then switched to high-fat diet supplemented with tamoxifen for the rest of the study. Mice underwent sham or VSG surgery after 2 weeks of tamoxifen diet and were fed ad libitum postoperatively. Mice underwent oral glucose tolerance testing at 3 weeks and were euthanized at 6 weeks after surgery. VSG reduced body weight and food intake independent of genotype. However, glucose tolerance was only improved in VSG WT compared with sham WT, whereas VSG KO had impaired glucose tolerance relative to VSG WT. Augmentation of glucose-stimulated insulin secretion during the oral glucose tolerance test was blunted in VSG KO compared with VSG WT. Therefore, our data suggest that enhanced β-cell GLP-1R signaling contributes to improved glucose regulation after VSG by promoting increased glucose-stimulated insulin secretion.
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- 2016
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12. The Furan Fatty Acid Metabolite CMPF Is Elevated in Diabetes and Induces β Cell Dysfunction.
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Prentice, Kacey J., Luu, Lemieux, Allister, Emma M., Liu, Ying, Jun, Lucy S., Sloop, Kyle W., Hardy, Alexandre B., Wei, Li, Jia, Weiping, Fantus, I. George, Sweet, Douglas H., Sweeney, Gary, Retnakaran, Ravi, Dai, Feihan F., and Wheeler, Michael B.
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Summary: Gestational diabetes (GDM) results from failure of the β cells to adapt to increased metabolic demands; however, the cause of GDM and the extremely high rate of progression to type 2 diabetes (T2D) remains unknown. Using metabolomics, we show that the furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is elevated in the plasma of humans with GDM, as well as impaired glucose-tolerant and T2D patients. In mice, diabetic levels of plasma CMPF induced glucose intolerance, impaired glucose-stimulated insulin secretion, and decreased glucose utilization. Mechanistically, we show that CMPF acts directly on the β cell, causing impaired mitochondrial function, decreasing glucose-induced ATP accumulation, and inducing oxidative stress, resulting in dysregulation of key transcription factors and ultimately reduced insulin biosynthesis. Importantly, specifically blocking its transport through OAT3 or antioxidant treatment could prevent CMPF-induced β cell dysfunction. Thus, CMPF provides a link between β cell dysfunction and GDM/T2D that could be targeted therapeutically. [Copyright &y& Elsevier]
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- 2014
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13. Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells*
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Zhang, Ming, Robitaille, Mélanie, Showalter, Aaron D., Huang, Xinyi, Liu, Ying, Bhattacharjee, Alpana, Willard, Francis S., Han, Junfeng, Froese, Sean, Wei, Li, Gaisano, Herbert Y., Angers, Stéphane, Sloop, Kyle W., Dai, Feihan F., and Wheeler, Michael B.
- Abstract
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.
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- 2014
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14. Differential Activation and Modulation of the Glucagon-Like Peptide-1 Receptor by Small Molecule Ligands
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Wootten, Denise, Savage, Emilia E., Willard, Francis S., Bueno, Ana B., Sloop, Kyle W., Christopoulos, Arthur, and Sexton, Patrick M.
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The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target for the treatment of type 2 diabetes due to its role in glucose homeostasis. Despite the availability of peptide-based GLP-1R drugs for treatment of this disease, there is great interest in developing small molecules that can be administered orally. The GLP-1R system is complex, with multiple endogenous and clinically used peptide ligands that exhibit different signaling biases at this receptor. This study revealed that small molecule ligands acting at this receptor are differentially biased to peptide ligands and also from each other with respect to the signaling pathways that they activate. Furthermore, allosteric small molecule ligands were also able to induce bias in signaling mediated by orthosteric ligands. This was dependent on both the orthosteric and allosteric ligand as no two allosteric-orthosteric ligand pairs could induce the same signaling profile. We highlight the need to profile compounds across multiple signaling pathways and in combination with multiple orthosteric ligands in systems such as the GLP-1R where more than one endogenous ligand exists. In the context of pleiotropical coupling of receptors and the interplay of multiple pathways leading to physiologic responses, profiling of small molecules in this manner may lead to a better understanding of the physiologic consequences of biased signaling at this receptor. This could enable the design and development of improved therapeutics that have the ability to fine-tune receptor signaling, leading to beneficial therapeutic outcomes while reducing side effect profiles.
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- 2013
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15. Small Molecule Allosteric Modulation of the Glucagon-Like Peptide-1 Receptor Enhances the Insulinotropic Effect of Oxyntomodulin
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Willard, Francis S., Wootten, Denise, Showalter, Aaron D., Savage, Emilia E., Ficorilli, James, Farb, Thomas B., Bokvist, Krister, Alsina-Fernandez, Jorge, Furness, Sebastian G. B., Christopoulos, Arthur, Sexton, Patrick M., and Sloop, Kyle W.
- Abstract
Identifying novel mechanisms to enhance glucagon-like peptide-1 (GLP-1) receptor signaling may enable nascent medicinal chemistry strategies with the aim of developing new orally available therapeutic agents for the treatment of type 2 diabetes mellitus. Therefore, we tested the hypothesis that selectively modulating the low-affinity GLP-1 receptor agonist, oxyntomodulin, would improve the insulin secretory properties of this naturally occurring hormone to provide a rationale for pursuing an unexplored therapeutic approach. Signal transduction and competition binding studies were used to investigate oxyntomodulin activity on the GLP-1 receptor in the presence of the small molecule GLP-1 receptor modulator, 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP). In vivo, the intravenous glucose tolerance test characterized oxyntomodulin-induced insulin secretion in animals administered the small molecule. BETP increased oxyntomodulin binding affinity for the GLP-1 receptor and enhanced oxyntomodulin-mediated GLP-1 receptor signaling as measured by activation of the α subunit of heterotrimeric G protein and cAMP accumulation. In addition, oxyntomodulin-induced insulin secretion was enhanced in the presence of the compound. BETP was pharmacologically characterized to induce biased signaling by oxyntomodulin. These studies demonstrate that small molecules targeting the GLP-1 receptor can increase binding and receptor activation of the endogenous peptide oxyntomodulin. The biased signaling engendered by BETP suggests that GLP-1 receptor mobilization of cAMP is the critical insulinotropic signaling event. Because of the unique metabolic properties of oxyntomodulin, identifying molecules that enhance its activity should be pursued to assess the efficacy and safety of this novel mechanism.
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- 2012
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16. Allosteric Modulation of Endogenous Metabolites as an Avenue for Drug Discovery
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Wootten, Denise, Savage, Emilia E., Valant, Celine, May, Lauren T., Sloop, Kyle W., Ficorilli, James, Showalter, Aaron D., Willard, Francis S., Christopoulos, Arthur, and Sexton, Patrick M.
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G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a key drug target class. Recently, allosteric drugs that can cobind with and modulate the activity of the endogenous ligand(s) for the receptor have become a major focus of the pharmaceutical and biotechnology industry for the development of novel GPCR therapeutic agents. This class of drugs has distinct properties compared with drugs targeting the endogenous (orthosteric) ligand-binding site that include the ability to sculpt cellular signaling and to respond differently in the presence of discrete orthosteric ligands, a behavior termed “probe dependence.” Here, using cell signaling assays combined with ex vivo and in vivo studies of insulin secretion, we demonstrate that allosteric ligands can cause marked potentiation of previously “inert” metabolic products of neurotransmitters and peptide hormones, a novel consequence of the phenomenon of probe dependence. Indeed, at the muscarinic M2receptor and glucagon-like peptide 1 (GLP-1) receptor, allosteric potentiation of the metabolites, choline and GLP-1(9–36)NH2, respectively, was ∼100-fold and up to 200-fold greater than that seen with the physiological signaling molecules acetylcholine and GLP-1(7–36)NH2. Modulation of GLP-1(9–36)NH2was also demonstrated in ex vivo and in vivo assays of insulin secretion. This work opens up new avenues for allosteric drug discovery by directly targeting modulation of metabolites, but it also identifies a behavior that could contribute to unexpected clinical outcomes if interaction of allosteric drugs with metabolites is not part of their preclinical assessment.
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- 2012
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17. Glucagon as a target for the treatment of Type 2 diabetes
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Sloop, Kyle W, Michael, M Dodson, and Moyers, Julie S
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Glucagon is the key counter-regulatory hormone that opposes the action of insulin. In states of relative hypoglycaemia, glucagon acts to increase blood glucose by stimulating hepatic glycogen breakdown and gluconeogenesis to achieve euglycaemia. Type 2 diabetes is characterised by inappropriate regulation of hepatic glucose production, which is due, at least in part, to an imbalance in the bihormonal relationship between plasma levels of glucagon and insulin. The glucose-lowering effects of glucagon peptide antagonists and antiglucagon neutralising antibodies first demonstrated the potential of glucagon receptor (GCGR) antagonism as a treatment for hyperglycaemia. In recent years, the development of GCGR antisense oligonucleotides and small molecular weight GCGR antagonists have been pursued as possible therapeu-tic agents to target glucagon action as a treatment for Type 2 diabe-tes.
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- 2005
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18. Transcriptional Regulation in Mammalian Pituitary Development and Disease
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Sloop, Kyle W., Parker, Gretchen E., and Rhodes, Simon J.
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The pituitary gland is a complex endocrine organ secreting hormones that regulate a wide array of vertebrate physiological processes, including growth, lactation, metabolic homeostasis, reproduction, water balance, and the stress response. In the mature organ, specialized cells that have a common origin in the early ectoderm release their characteristic products into the bloodstream. Together, the embryological processes that commit the hormone-secreting cells to their specific fates and the clinical and agricultural relevance of understanding pituitary function have defined pituitary development as an excellent model system for the study of the genetic cascades that guide cell determination and differentiation. Recently, many genes that regulate pituitary development have been identified. These genes encode transcription factors and signaling proteins and often are expressed in temporally controlled, pituitary-specific or pituitary-restricted patterns. Further, dominant and recessive mutations in these genes are associated with compound pituitary diseases in human patients and animal models. The advance of genome projects will facilitate approaches to understand the genetic mechanisms that regulate the activation of pituitary regulatory genes and to discover how the function of the encoded transcription factors is precisely regulated by intrinsic and extrapituitary signals. Gene array and differential screening approaches will enable the identification of direct and downstream target genes of pituitary transcription factors. Protein interaction screens will identify regulatory proteins required for pituitary gene control. Characterization of the pathways that coordinate pituitary development will direct treatment of pediatric and adult pituitary diseases, guide genetic counseling of families with hereditary conditions affecting the pituitary, and may allow embryonic manipulation to improve productivity in the meat industry.
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- 2001
19. An Isoform-specific Inhibitory Domain Regulates the LHX3 LIM Homeodomain Factor Holoprotein and the Production of a Functional Alternate Translation Form*
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Sloop, Kyle W., Dwyer, Conor J., and Rhodes, Simon J.
- Abstract
The LHX3 LIM homeodomain transcription factor is required for pituitary development and motor neuron specification. TheLhx3gene encodes two isoforms, LHX3a and LHX3b, that differ in their amino-terminal sequences. Humans and mice with defective Lhx3genes are deficient in gonadotrope, lactotrope, somatotrope, and thyrotrope pituitary cells. We show that, whereas Lhx3bis highly expressed in theseLhx3-dependent cell types, high levels ofLhx3aexpression are restricted to α glycoprotein subunit-expressing thyrotropes and gonadotropes. Cross-species comparison reveals the LHX3b-specific domain is more conserved than the LHX3a-specific domain. We demonstrate that the LHX3b-specific domain is a transferable inhibitor that reduces gene activation and DNA binding by homeodomain proteins. In addition, we identify a novel LHX3 protein (M2-LHX3) and determine that this molecule is generated by an internal translation initiation codon. The LHX3a- and LHX3b-specific coding sequences regulate differential usage of this internal start codon. Further, we identify the major activation domain of LHX3 in the carboxyl terminus of the molecule. M2-LHX3 is active because it retains this domain and binds DNA better than LHX3a or LHX3b. Other LIM homeodomain genes, including Lhx4, generate similar truncated proteins. These studies describe how transcriptional regulatory genes can generate multiple functional proteins.
- Published
- 2001
- Full Text
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20. LHX3 transcription factor mutations associated with combined pituitary hormone deficiency impair the activation of pituitary target genes
- Author
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Sloop, Kyle W., Parker, Gretchen E., Hanna, Kimberly R., Wright, Heather A., and Rhodes, Simon J.
- Abstract
The Lhx3 LIM homeodomain transcription factor is critical for pituitary gland formation and specification of the anterior pituitary hormone-secreting cell types. Two mutations in LHX3, a missense mutation changing a tyrosine to a cysteine and an intragenic deletion that results in a truncated protein lacking the DNA-binding homeodomain, have been identified in humans. These mutations were identified in patients with retarded growth and combined pituitary hormone deficiency and also abnormal neck and cervical spine development. For both the LHX3a and LHX3b isoforms, we compared the ability of wild type and mutant LHX3 proteins to trans-activate pituitary genes, bind DNA recognition elements, and interact with partner proteins. The tyrosine missense mutation inhibits the ability of LHX3 to induce transcription from selected target genes but does not prevent DNA binding and interaction with partner proteins such as NLI and Pit-1. Mutant LHX3 proteins lacking a homeodomain do not bind DNA and do not induce transcription from pituitary genes. These studies demonstrate that mutations in the LHX3 isoforms impair their gene regulatory functions and support the hypothesis that defects in the LHX3gene cause complex pituitary disease in humans.
- Published
- 2001
- Full Text
- View/download PDF
21. Differentiation Method-Dependent Expression of Leptin in Adipocyte Cell Lines
- Author
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Slieker, Lawrence J., Sloop, Kyle W., and Surface, Peggy L.
- Abstract
Leptin, the product of theobgene, is expressed exclusively in adipose tissue. However, adipocyte cell lines, such as 3T3-L1 adipocytes, have generally been reported to express extremely low levels of leptin mRNA. We compared 3T3-L1's to the closely related line 3T3-F442A, and to another murine adipocyte line, TA1. TA1 cells, when differentiated by indomethacin/insulin treatment, express leptin at levels greater than those of 3T3-L1 adipocytes differentiated by the traditional methylisobutylxanthine/dexamethasone/insulin protocol. However, when 3T3-L1's are differentiated in the presence of indomethacin/insulin their expression levels of leptin increase dramatically. 3T3-F442A preadipocytes also express high levels of leptin when differentiated in the presence of T3and insulin, but when differentiated in the presence of indomethacin/insulin, expression levels drop precipitously. These changes in leptin mRNA and protein expression are not reflected by changes in CCAAT/enhancer binding protein-α (c/EBPα), peroxizomal proliferator activated receptor-γ (PPARγ), lipoprotein lipase (LPL), fatty-acid binding protein aP2 or uncoupling protein-2 (UCP2) mRNA levels, and suggest a mechanism unique to the leptin gene.
- Published
- 1998
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22. Extension of Neuroendocrine Axes to Include Leptin
- Author
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Heiman, Mark L., Sloop, Kyle W., Chen, Yanyun, and Caro, José F.
- Abstract
An adipocyte factor transducing the quantity of adipose to a hormonal message that targets the hypothalamus has been postulated for many years. These findings indicate that adipose tissue performs important biological functions beyond storage of fat. In fact, data indicate that adipose tissue functions very much like other endocrine tissues, releasing a hormone named leptin into the circulatory system to relay a message to its targets. Although there may be more than one target tissue for leptin, the best is the hypothalamus. Because most types of human and murine obesity are accompanied by elevated leptin levels and the concentrations of this hormone correlate well with degree of adiposity, it is thought that leptin may serve as a fuel gage for the hypothalamus. When sufficient fuel is present or when lipid is being synthesized and stored in adipose, leptin levels rise and reduce further feeding behavior. In addition, a signal of abundant fuel reserve serves as a message that energy can be expended for anabolism of protein and other biological processes such as growth, repair, and reproduction. Our data, together with those reported by others, indicate that leptin inhibits the hypothalamic-pituitary-adrenal axis and thus antagonizes the catabolic glucocorticoids. A review of data available also supports that leptin stimulates the hypothalamic-growth hormone axis and, hence, promotes protein synthesis. Energy must be expended to support this anabolic state and data indicate the energy is derived from oxidative metabolism of fuel that is stimulated by the hypothalamic-pituitary-thyroid axis. In conclusion, each of these neuroendocrine axes require information of the fuel supply to function efficiently and this data can be at least partially supplied by leptin.
- Published
- 1999
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23. Changes in Leptin Expression Are Not Associated with Corresponding Changes in CCAAT/Enhancer Binding Protein-α
- Author
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Sloop, Kyle W., Surface, Peggy L., Heiman, Mark L., and Slieker, Lawrence J.
- Abstract
C/EBP-α binds a C/EBP consensus site in the leptin promoter and activates transcriptionin vitro.We assessed adipose tissue expression of C/EBP-α, leptin and β-actin in Sprague Dawley rats under conditions that modulate leptin mRNA abundance in order to study the relationship between leptin and C/EBP-α expression patterns. During acute fasting, which decreased the level of leptin and β-actin mRNA, C/EBP-α mRNA expression was unaltered. In leptin-treated and pair-fed animals, C/EBP-α mRNA was unaltered compared toad libitumfed controls, while leptin and β-actin mRNA expression was again decreased. These results indicate that changes in the level of leptin gene expression are not directly associated with changes in the level of C/EBP-α abundance.
- Published
- 1998
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24. Regulation of Expression of obmRNA and Protein by Glucocorticoids and cAMP (∗)
- Author
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Slieker, Lawrence J., Sloop, Kyle W., Surface, Peggy L., Kriauciunas, Aidas, LaQuier, Frank, Manetta, Joseph, Bue-Valleskey, Julie, and Stephens, Thomas W.
- Abstract
Regulation of obesegene (ob) expression in ob/oband db/dbmice and in cultured rat adipocytes was examined. It has been demonstrated that exogenous human OB protein (leptin) treatment reduces food intake and weight gain, as well as insulin, glucose, and corticosterone levels in ob/obmice. In the present report we show that leptin treatment down-regulates endogenous adipose obmRNA. However, treatment of isolated rat adipocytes with 100 ng/ml human or murine leptin had no direct effect on expression of endogenous obmRNA, suggesting that leptin may be able to down-regulate its own expression by an indirect, non-autocrine mechanism. Glucocorticoids increased both obmRNA levels and secreted leptin levels in vitro. Conversely, agents that increase intracellular cAMP, such as β-adrenergic agonists or Bt2cAMP itself, decreased obmRNA expression and leptin secretion. Therefore, increased glucocorticoid levels and decreased sympathetic neural activity may contribute to the elevated obmRNA expression observed in genetically obese, hyperglucocorticoid rodents. Furthermore, leptin might regulate its own expression through a feedback mechanism involving the hypothalamic pituitary axis.
- Published
- 1996
- Full Text
- View/download PDF
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