Hepatic oleate regulates one-carbon metabolism during high carbohydrate feeding.

Obesity is a major risk factor for type 2 diabetes, coronary heart disease, and strok. These diseases are associated with profound alterations in gene expression in metabolic tissues. Epigenetic-mediated regulation of gene expression is one mechanism through which environmental factors, such as diet, modify gene expression and disease predisposition. However, epigenetic control of gene expression in obesity and insulin resistance is not fully characterized. We discovered that liver-specific stearoyl-CoA desaturase-1 (Scd1) knockout mice (LKO) fed a high-carbohydrate low-fat diet exhibit dramatic changes in hepatic gene expression and metabolites of the folate cycle and one-carbon metabolism respectively for the synthesis of S-adenosylmethionine (SAM). LKO mice show an increased ratio of S-adenosylmethionine to S-adenosylhomocysteine, a marker for increased cellular methylation capacity. Furthermore, expression of DNA and histone methyltransferase genes is up-regulated while the mRNA and protein levels of the non-DNA methyltransferases including phosphatidylethanolamine methyltransferase (PEMT), Betaine homocysteine methyltransferase (Bhmt), and the SAM-utilizing enzymes such as glycine-N-methyltransferase (Gnmt) and guanidinoacetate methyltransferase (Gamt) are generally down-regulated. Feeding LKO mice a high carbohydrate diet supplemented with triolein, but not tristearin, and increased endogenous hepatic synthesis of oleate but not palmitoleate in Scd1 global knockout mice normalized one carbon gene expression and metabolite levels. Additionally, changes in one carbon gene expression are independent of the PGC-1α-mediated ER stress response previously reported in the LKO mice. Together, these results highlight the important role of oleate in maintaining one-carbon cycle homeostasis and point to observed changes in one-carbon metabolism as a novel mediator of the Scd1 deficiency-induced liver phenotype.

Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome.

Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Shifts in metabolic fuel use coincide with maximal rates of ventilation and body surface rewarming in an arousing hibernator.

It is well established that hibernating mammals rely predominantly on lipid stores to fuel metabolism throughout the hibernation season. However, it is unclear if other endogenous fuels contribute to the rapid, ~400-fold increase in metabolic rate during the early phase of arousal from torpor. To investigate this issue, we used cavity ring-down spectroscopy, a technique that provides a real-time indication of fuel use by measuring the ratio of 13C to 12C in the exhaled CO2 of arousing 13-lined ground squirrels (Ictidomys tridecemlineatus). We used infrared thermography to simultaneously measure ventilation and surface temperature change in various body regions, and we interpreted these data in light of changing plasma metabolite abundances at multiple stages of arousal from torpor. We found that hibernating squirrels use a combination of lipids and, likely, carbohydrates to fuel the initial ~60 min of arousal before switching to predominantly lipid oxidation. This fuel switch coincided with times of maximal rates of ventilation and rewarming of different body surface regions, including brown adipose tissue. Infrared thermography revealed zonal rewarming, whereby the brown adipose tissue region was the first to warm, followed by the thoracic and head regions and, finally, the posterior half of the body. Consistent with the results from cavity ring-down spectroscopy, plasma metabolite dynamics during early arousal suggested a large reliance on fatty acids, with a contribution from carbohydrates and glycerol. Because of their high oxidative flux rates and efficient O2 use, carbohydrates might be an advantageous metabolic fuel during the early phase of arousal, when metabolic demands are high but ventilation rates and, thus, O2 supply are relatively low.

Lipolytic Effects of 3-Iodothyronamine (T1AM) and a Novel Thyronamine-Like Analog SG-2 through the AMPK Pathway.

3-Iodothyronamine (T1AM) and its synthetic analog SG-2 are rapidly emerging as promising drivers of cellular metabolic reprogramming. Our recent research indicates that in obese mice a sub-chronic low dose T1AM treatment increased lipolysis, associated with significant weight loss independent of food consumption. The specific cellular mechanism of T1AM's lipolytic effect and its site of action remains unknown. First, to study the mechanism used by T1AM to gain entry into cells, we synthesized a fluoro-labeled version of T1AM (FL-T1AM) by conjugating it to rhodamine (TRITC) and analyzed its cellular uptake and localization in 3T3-L1 mouse adipocytes. Cell imaging using confocal microscopy revealed a rapid intercellular uptake of FL-T1AM into mitochondria without localization to the lipid droplet or nucleus of mature adipocytes. Treatment of 3T3-L1 adipocytes with T1AM and SG-2 resulted in decreased lipid accumulation, the latter showing a significantly higher potency than T1AM (10 µM vs. 20 µM, respectively). We further examined the effects of T1AM and SG-2 on liver HepG2 cells. A significant decrease in lipid accumulation was observed in HepG2 cells treated with T1AM or SG-2, due to increased lipolytic activity. This was confirmed by accumulation of glycerol in the culture media and through activation of the AMPK/ACC signaling pathways.

The Hibernator Microbiome: Host-Bacterial Interactions in an Extreme Nutritional Symbiosis.

Animals that undergo seasonal cycles of feeding and fasting have adaptations that maintain integrity of organ systems when dietary nutrients are lacking. Food deprivation also challenges the gut microbiota, which relies heavily on host diet for metabolic substrates and the gastrointestinal tract, which is influenced by enteral nutrients and microbial activity. Winter fasting in hibernators shifts the microbiota to favor taxa with the capacity to degrade and utilize host-derived substrates and disfavor taxa that prefer complex plant polysaccharides. Microbiome alterations may contribute to hibernation-induced changes in the intestinal immune system, epithelial barrier function, and other host features that are affected by microbial short-chain fatty acids and other metabolites. Understanding mechanisms by which the hibernator host and its gut symbionts adapt to the altered nutritional landscape during winter fasting may provide insights into protective mechanisms that are compromised when nonhibernating species, such as humans, undergo long periods of enteral nutrient deprivation.

Metabolic Reprogramming by 3-Iodothyronamine (T1AM): A New Perspective to Reverse Obesity through Co-Regulation of Sirtuin 4 and 6 Expression.

Obesity is a complex disease associated with environmental and genetic factors. 3-Iodothyronamine (T1AM) has revealed great potential as an effective weight loss drug. We used metabolomics and associated transcriptional gene and protein expression analysis to investigate the tissue specific metabolic reprogramming effects of subchronic T1AM treatment at two pharmacological daily doses (10 and 25 mg/kg) on targeted metabolic pathways. Multi-analytical results indicated that T1AM at 25 mg/kg can act as a novel master regulator of both glucose and lipid metabolism in mice through sirtuin-mediated pathways. In liver, we observed an increased gene and protein expression of Sirt6 (a master gene regulator of glucose) and Gck (glucose kinase) and a decreased expression of Sirt4 (a negative regulator of fatty acids oxidation (FAO)), whereas in white adipose tissue only Sirt6 was increased. Metabolomics analysis supported physiological changes at both doses with most increases in FAO, glycolysis indicators and the mitochondrial substrate, at the highest dose of T1AM. Together our results suggest that T1AM acts through sirtuin-mediated pathways to metabolically reprogram fatty acid and glucose metabolism possibly through small molecules signaling. Our novel mechanistic findings indicate that T1AM has a great potential as a drug for the treatment of obesity and possibly diabetes.

Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties.

Taste signaling is a complex process that is linked to obesity and its associated metabolic syndromes. The sweet taste is mediated through a heterodimeric G protein coupled receptor (GPCR) in a species-specific manner and at multi-tissue specific levels. The sweet receptor recognizes a large number of ligands with structural and functional diversities to modulate different amplitudes of downstream signaling pathway(s). The human sweet-taste receptor has been extremely difficult to study by biophysical methods due to the difficulty in producing large homogeneous quantities of the taste-receptor protein and the lack of reliable in vitro assays to precisely measure productive ligand binding modes that lead to activation of the receptor protein. We report here a multimodal high throughput assay to monitor ligand binding, receptor stability and conformational changes to model the molecular ligand-receptor interactions. We applied saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) complemented by differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and intrinsic fluorescence spectroscopy (IF) to characterize binding interactions. Our method using complementary NMR and biophysical analysis is advantageous to study the mechanism of ligand binding and signaling processes in other GPCRs.

NMR Metabolomics Show Evidence for Mitochondrial Oxidative Stress in a Mouse Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is associated with metabolic and endocrine disorders in women of reproductive age. The etiology of PCOS is still unknown. Mice prenatally treated with glucocorticoids exhibit metabolic disturbances that are similar to those seen in women with PCOS. We used an untargeted nuclear magnetic resonance (NMR)-based metabolomics approach to understand the metabolic changes occurring in the plasma and kidney over time in female glucocorticoid-treated (GC-treated) mice. There are significant changes in plasma amino acid levels (valine, tyrosine, and proline) and their intermediates (2-hydroxybutyrate, 4-aminobutyrate, and taurine), whereas in kidneys, the TCA cycle metabolism (citrate, fumarate, and succinate) and the pentose phosphate (PP) pathway products (inosine and uracil) are significantly altered (p < 0.05) from 8 to 16 weeks of age. Levels of NADH, NAD(+), NAD(+)/NADH, and NADH redox in kidneys indicate increased mitochondrial oxidative stress from 8 to 16 weeks in GC-treated mice. These results indicate that altered metabolic substrates in the plasma and kidneys of treated mice are associated with altered amino acid metabolism, increased cytoplasmic PP, and increased mitochondrial activity, leading to a more oxidized state. This study identifies biomarkers associated with metabolic dysfunction in kidney mitochondria of a prenatal gluococorticoid-treated mouse model of PCOS that may be used as early predictive biomarkers of oxidative stress in the PCOS metabolic disorder in women.

NMRFAM-SDF: a protein structure determination framework.

The computationally demanding nature of automated NMR structure determination necessitates a delicate balancing of factors that include the time complexity of data collection, the computational complexity of chemical shift assignments, and selection of proper optimization steps. During the past two decades the computational and algorithmic aspects of several discrete steps of the process have been addressed. Although no single comprehensive solution has emerged, the incorporation of a validation protocol has gained recognition as a necessary step for a robust automated approach. The need for validation becomes even more pronounced in cases of proteins with higher structural complexity, where potentially larger errors generated at each step can propagate and accumulate in the process of structure calculation, thereby significantly degrading the efficacy of any software framework. This paper introduces a complete framework for protein structure determination with NMR--from data acquisition to the structure determination. The aim is twofold: to simplify the structure determination process for non-NMR experts whenever feasible, while maintaining flexibility by providing a set of modules that validate each step, and to enable the assessment of error propagations. This framework, called NMRFAM-SDF (NMRFAM-Structure Determination Framework), and its various components are available for download from the NMRFAM website (http://nmrfam.wisc.edu/software.htm).

Artificial sweeteners stimulate adipogenesis and suppress lipolysis independently of sweet taste receptors.

G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3.

Temperature-dependent conformational change affecting Tyr11 and sweetness loops of brazzein.

The sweet protein brazzein, a member of the Csßα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side-chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. METHODS: We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. RESULTS: Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.

Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies.

Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from Escherichia coli inclusion bodies. The heterologously expressed protein constructs retained full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with (2)H, (13)C, and (15)N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed.

Nitrogen recycling via gut symbionts increases in ground squirrels over the hibernation season.

Hibernation is a mammalian strategy that uses metabolic plasticity to reduce energy demands and enable long-term fasting. Fasting mitigates winter food scarcity but eliminates dietary nitrogen, jeopardizing body protein balance. Here, we reveal gut microbiome-mediated urea nitrogen recycling in hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Ureolytic gut microbes incorporate urea nitrogen into metabolites that are absorbed by the host, with the nitrogen reincorporated into the squirrel's protein pool. Urea nitrogen recycling is greatest after prolonged fasting in late winter, when urea transporter abundance in gut tissue and urease gene abundance in the microbiome are highest. These results reveal a functional role for the gut microbiome during hibernation and suggest mechanisms by which urea nitrogen recycling may contribute to protein balance in other monogastric animals.

Ligand-specific structural changes in the vitamin D receptor in solution.

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D3 [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.

Interactions between the human sweet-sensing T1R2-T1R3 receptor and sweeteners detected by saturation transfer difference NMR spectroscopy.

The sweet receptor is a member of the G-protein coupled receptor family C that detects a wide variety of chemically and structurally diverse sweet-tasting molecules. We recently used saturation transfer difference spectroscopy (STD) to monitor the direct binding of a set of sweet agonists and antagonists to the human taste receptor in membranes prepared from human embryonic kidney (HEK293) cells transfected with and expressing the sweet receptor [F.M. Assadi-Porter, M. Tonelli, E. Maillet, K. Hallenga, O. Benard, M. Max, J.L. Markley, J. Am. Chem. Soc. 130 (2008) 7212-7213]. Here we review this work and related studies, discuss the procedures involved, and expand on their potential for identifying specific binding interactions of ligands to the membrane spanning and extracellular regions of the full heterodimeric sweet taste receptor. Whereas activity assays are unable to distinguish mutations that alter ligand-binding sites from those that alter signal transduction downstream of the binding site, STD NMR now allows us to make this distinction.

Structural role of the terminal disulfide bond in the sweetness of brazzein.

Brazzein, a 54 residue sweet-tasting protein, is thought to participate in a multipoint binding interaction with the sweet taste receptor. Proposed sites for interaction with the receptor include 2 surface loops and the disulfide bond that connects the N- and C-termini. However, the importance of each site is not well understood. To characterize the structural role of the termini in the sweetness of brazzein, the position of the disulfide bond connecting the N- and C-termini was shifted by substituting K3-C4-K5 with C3-K4-R5. The apparent affinity and V(max) of the C3-K4-R5-brazzein (CKR-brazzein) variant were only modestly decreased compared with the wild-type (WT) brazzein. We determined a high-resolution structure of CKR-brazzein by nuclear magnetic resonance spectroscopy (backbone root mean square deviation of 0.39 Å). Comparing the structure of CKR-brazzein with that of WT-brazzein revealed that the terminal ß-strands of the variant display extended ß-structure and increased dynamics relative to WT-brazzein. These results support previous mutagenesis studies and further suggest that, whereas interactions involving the termini are necessary for full function of brazzein, the termini do not constitute the primary site of interaction between brazzein and the sweet taste receptor.

Changes in the natural abundance of 13CO2/12CO2 in breath due to lipopolysacchride-induced acute phase response.

The natural abundance of carbon-13 in blood proteins increases during the cachectic state and may be a biomarker for disease status. We hypothesized a corresponding drop in the relative abundance of 13C in breath CO2. Using the lipopolysacchride (LPS)-induced endotoxemia model of the acute cachectic state, we demonstrated that the acute phase response causes shifts in the stable isotopes of carbon in exhaled CO2 (13CO2/12CO2 delta value) shortly after administration of LPS while glucocorticoid treatment does not. Mice were injected with LPS and stable isotopes of blood amino acids and carbon in exhaled CO2 were monitored. An increase in the relative isotopic mass of serum alanine, proline and threonine was observed at 3 h after LPS injection. Breath delta values began dropping immediately after administration of LPS, and were 4-5 delta values lower than those of the control animals by 2.5 h after injection. A corresponding drop in delta value was not observed with dexamethasone treatment. Thus protein synthesis during the acute phase response probably caused the fractionation of stable isotopes observed in the plasma amino acids and in exhaled breath 13CO2 delta values. The exhaled breath 13CO2 delta value may be a valuable real-time biomarker of cachexia associated with an acute phase response due to endotoxemia.

Effects of Repeated Sublethal External Exposure to Deep Water Horizon Oil on the Avian Metabolome.

We assessed adverse effects of external sublethal exposure of Deepwater Horizon, Mississippi Canyon 252 oil on plasma and liver metabolome profiles of the double-crested cormorant (Phalacrocorax auritus), a large (1.5 to 3.0 kg) diving waterbird common in the Gulf of Mexico. Metabolomics analysis of avian plasma showed significant negative effects on avian metabolic profiles, in some cases after only two external exposures (26 g cumulative) to oil. We observed significant (p < 0.05) changes in intermediate metabolites of energy metabolism and fatty acid and amino acid metabolic pathways in cormorants after repeated exposure to oil. Exposure to oil increased several metabolites (glycine, betaine, serine and methionine) that are essential to the one-carbon metabolism pathway. Lipid metabolism was affected, causing an increase in production of ketone bodies, suggesting lipids were used as an alternative energy source for energy production in oil exposed birds. In addition, metabolites associated with hepatic bile acid metabolism were affected by oil exposure which was correlated with changes observed in bile acids in exposed birds. These changes at the most basic level of phenotypic expression caused by sublethal exposure to oil can have effects that would be detrimental to reproduction, migration, and survival in avian species.

Direct NMR detection of the binding of functional ligands to the human sweet receptor, a heterodimeric family 3 GPCR.

We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.