Characterization of the Modes of Binding between Human Sweet Taste Receptor and Low-Molecular-Weight Sweet Compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2–hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein–coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling–based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands

Bitter Taste Receptors Influence Glucose Homeostasis

TAS1R- and TAS2R-type taste receptors are expressed in the gustatory system, where they detect sweet- and bitter-tasting stimuli, respectively. These receptors are also expressed in subsets of cells within the mammalian gastrointestinal tract, where they mediate nutrient assimilation and endocrine responses. For example, sweeteners stimulate taste receptors on the surface of gut enteroendocrine L cells to elicit an increase in intracellular Ca2+ and secretion of the incretin hormone glucagon-like peptide-1 (GLP-1), an important modulator of insulin biosynthesis and secretion. Because of the importance of taste receptors in the regulation of food intake and the alimentary responses to chemostimuli, we hypothesized that differences in taste receptor efficacy may impact glucose homeostasis. To address this issue, we initiated a candidate gene study within the Amish Family Diabetes Study and assessed the association of taste receptor variants with indicators of glucose dysregulation, including a diagnosis of type 2 diabetes mellitus and high levels of blood glucose and insulin during an oral glucose tolerance test. We report that a TAS2R haplotype is associated with altered glucose and insulin homeostasis. We also found that one SNP within this haplotype disrupts normal responses of a single receptor, TAS2R9, to its cognate ligands ofloxacin, procainamide and pirenzapine. Together, these findings suggest that a functionally compromised TAS2R receptor negatively impacts glucose homeostasis, providing an important link between alimentary chemosensation and metabolic disease

Metabolome dynamics of T1AM, an endogenous thyroid hormone derivative: Effects on lipid metabolism, weight loss, and appetite in mice.

Objectives: T1AM is an endogenous natural compound that has opposing physiological effects to those of thyroid hormone. T1AM is thought to switch metabolism from carbohydrate to lipid. Due to its recent discovery, detailed actions of T1AM on affected metabolic pathways are still unknown. In a previous pilot study, we observed that subchronic low doses of T1AM could significantly increase lipolysis in mice. The present work is focused on studying the effects of T1AM on carbohydrate and lipid metabolism in obese mice using Nuclear Magnetic Resonance (NMR) spectroscopy by dynamic metabolomics. This novel way of monitoring all serum metabolites simultaneously in individual animals in response to T1AM could potentially identify pathways through which T1AM acts. Methods: To distinguish carbohydrate from lipid pathways, we used 13C-glucose labeling in three groups of spontaneously overweight mice: Group1: five obese control mice injected with saline once daily for seven days; Group 2: five obese mice injected with T1AM (10 mg/kg/day) once daily for seven days; Group 3: same as in Group 2 but using an higher dose of T1AM (25 m/kg/day). The mice were monitored prior to and after the week of injections. In particular we examined small molecules intermediates in carbohydrate and lipid metabolism, appetite through food intake, and weight measurements over the course of four weeks. Results: We observed that exogenous T1AM administration was associated with a body weight loss trend. After T1AM withdrawal, mice regained only part of lost weight in the following 2 weeks, indicating long-lasting effects of this natural molecule. No difference in food intake was observed at any time. NMR metabolic profiling experiments are currently underway to better identify key changes in fuel and energy metabolism. Conclusions: T1AM produced beneficial effects in obese mice and might become an effective human weight-loss drug

WEIGHT LOSS WITH PRESERVED GLUCOSE HOMEOSTASIS DURING CHRONIC TREATMENT OF OBESE MICE WITH 3-IODOTHYRONAMINE (T1AM): ROLE OF THE SIRTUIN FAMILY

Objectives: T1AM, an endogenous thyroid hormone derivative, is regarded as a rapid modulator of metabolism. In a recent study we provided the first evidence that subchronic low doses of T1AM increased lipolysis determining significant weight loss without changes in food consumption. In the present work we investigated the effects of subchronic T1AM treat- ment on metabolism at the molecular level. To this aim, we performed Nuclear Magnetic Resonance (NMR)-based metabolomics on plasma samples from control and T1AM treated mice in conjunction with organ specific (liver and adipose tissue) gene and protein expression profiling. Methods: Three groups of spontaneously obese female CD-1 mice were injected once daily with saline (n = 5), 10 mg/kg T1AM (n = 5), or 25 mg/kg T1AM (n = 5). Blood was drawn on study days -3, 4, and 7 and analyzed by 1H-NMR. On day 7, animals were cervically dislocated and their organs were collected for real-time PCR and WB analyses. Results: Multivariate statistical analysis of the 1H-NMR data sets revealed increased plasma 3-hydroxybutyrate and acetate concentrations in T1AM-treated mice compared to control mice, whereas no differences were observed in plasma glucose levels. Mice treated with T1AM (25mg/kg/day) showed significant changes in gene expression. In liver we observed increased expression of SIRT6 and glucokinase (GCK), and decreased expression of SIRT4, whereas in adipose tissue only SIRT6 was increased. Protein expres- sion studies confirmed over-expression of SIRT6 and GCK in liver. Conclusions: Since SIRT6 functions as a master gene regulator of glucose levels by maintaining the normal processes by which cells convert glucose into energy, whereas SIRT4 function as a negative regulator of fatty acids oxi- dative metabolism, our results suggest that T1AM can act as a master regulator of both glucose and fat metabolism in obese mice

The recent development of a sweet-tasting brazzein and its potential industrial applications

Brazzein is a small heat- and pH-stable sweet-tasting protein isolated from the West African plant, Pentadiplandra brazzeana. Brazzein combines a highly sweet potency, a long history of human consumption, and a remarkable stability, giving it great potential as a natural sweetener. Due to the difficulties of obtaining brazzein from its natural source, several efforts have been made to express brazzein using various heterologous expression systems. This chapter describes the biochemical, structural, sensory, and physiological properties of brazzein. We will summarize the current knowledge of the structure-activity relationship of brazzein. The biotechnological production of brazzein using various expression systems will also be reviewed. Furthermore, the emerging application of brazzein in the food industry to replace traditional sugars by acting as a natural, good, low-calorie sweetener will be discussed

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt’s lymphoma (BL) Daudi cells by NMR spectroscopy

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cellderived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenylcarboxamido- Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt’s lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt’s lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.Griffith Sciences, School of Natural SciencesFull Tex