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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.

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 13-lined ground squirrels (TLGS). Ureolytic gut microbes incorporate urea nitrogen into organic compounds that are absorbed by the host, with the nitrogen reincorporated into the TLGS 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 in hibernation and suggest mechanisms by which urea nitrogen recycling contributes to protein balance in other monogastric animals, including humans.One Sentence SummaryGround squirrels and their gut symbionts benefit from urea nitrogen recycling throughout hibernation.

Polyunsaturated fatty acid (PUFA)-rich diets are thought to provide beneficial effects toward metabolic health in part through their bioactive properties. We hypothesized that increasing PUFA intake in mice would increase peroxisome proliferator activated receptor delta (PPARδ) expression and activity, and we sought to examine the effect of different PUFA-enriched oils on muscle PPARδ expression. One of the oils we tested was cottonseed oil (CSO) which is primarily linoleic acid (53%) and palmitic acid (24%). Mice fed a CSO-enriched diet (50% energy from fat) displayed no change in muscle PPARδ expression; however, in the liver, it was consistently elevated along with its transcriptional coactivator Pgc-1. Male mice were fed chow or CSO-, saturated fat (SFA)-, or linoleic acid (18:2)-enriched diets that were matched for macronutrient content for 4 weeks. There were no differences in food intake, body weight, fasting glucose, glucose tolerance, or energy expenditure between chow- and CSO-fed mice, whereas SFA-fed mice had increased fat mass and 18:2-fed mice were less glucose tolerant. Metabolomic analyses revealed that the livers of CSO-fed mice closely matched those of chow-fed but significantly differed from SFA- and 18:2-enriched groups. Fatty acid composition of the diets and livers revealed an impairment in desaturase activity and the presence of dihydrosterculic acid (DHSA) in the CSO-fed mice. The effect of DHSA on PPARδ and stearoyl-CoA desaturase-1 expression mimicked that of the CSO-fed mice. Taken together, these data suggest that DHSA from CSO may be an effective means to increase PPARδ expression with concomitant suppression of liver stearoyl-CoA desaturase-1 activity.

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.

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 of13C to12C in the exhaled CO2of 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 O2use, carbohydrates might be an advantageous metabolic fuel during the early phase of arousal, when metabolic demands are high but ventilation rates and, thus, O2supply are relatively low.

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&rsquo, 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 &micro, M vs. 20 &micro, M, 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.

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non-carbohydrate sweeteners. In an investigation of sequence-dependent functional properties of the protein, we used NMR spectroscopy to determine the three-dimensional structures and dynamic properties of two Brz variants: one with a single-site substitution (D40K), which is three-fold sweeter than wild-type Brz, and one with a two-residue insertion between residues 18 and 19 (ins18 RI19 ), which is devoid of sweetness. Although the three-dimensional folds of the two variants were very similar to wild-type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter-stand H-bond between the side chains of residues Gln46 and Asp40 present in wild-type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G-protein coupled human sweet receptor. On the other hand, the Arg-Ile insertion within Loop9-19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.

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.

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 (GPRC) 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 inadequate methods for producing large homogeneous quantities of the taste-receptor protein and a lack of reliable in vitro assays to precisely measure productive ligand binding modes leading to activity upon their interactions with the receptor protein. We report a multimodal high throughput assays to monitor ligand binding, receptor stability and conformational changes to model the molecular interactions between ligand-receptor. 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.

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.

Polycystic ovary syndrome (PCOS), a common female endocrinopathy, is a complex metabolic syndrome of enhanced weight gain. The goal of this pilot study was to evaluate metabolic differences between normal (n=10) and PCOS (n=10) women via breath carbon isotope ratio, urinary nitrogen and nuclear magnetic resonance (NMR)-determined serum metabolites. Breath carbon stable isotopes measured by cavity ring down spectroscopy (CRDS) indicated diminished (p

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.

Objective 3-Iodothyronamine (T1AM), an analog of thyroid hormone, is a recently discovered fast-acting endogenous metabolite. Single high-dose treatments of T1AM have produced rapid short-term effects, including a reduction of body temperature, bradycardia, and hyperglycemia in mice. Design and Methods The effect of daily low doses of T1AM (10 mg/kg) for 8 days on weight loss and metabolism in spontaneously overweight mice was monitored. The experiments were repeated twice (n = 4). Nuclear magnetic resonance (NMR) spectroscopy of plasma and real-time analysis of exhaled 13CO2 in breath by cavity ring down spectroscopy (CRDS) were used to detect T1AM-induced lipolysis. Results CRDS detected increased lipolysis in breath shortly after T1AM administration that was associated with a significant weight loss but independent of food consumption. NMR spectroscopy revealed alterations in key metabolites in serum: valine, glycine, and 3-hydroxybutyrate, suggesting that the subchronic effects of T1AM include both lipolysis and protein breakdown. After discontinuation of T1AM treatment, mice regained only 1.8% of the lost weight in the following 2 weeks, indicating lasting effects of T1AM on weight maintenance. Conclusions CRDS in combination with NMR and 13C-metabolic tracing constitute a powerful method of investigation in obesity studies for identifying in vivo biochemical pathway shifts and unanticipated debilitating side effects.

The sweet protein brazzein, a member of the Csβα 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.

3-Iodothyronamine (T1AM) is a structural analog of thyroid hormone that has been demonstrated to have potent affects on numerous physiological systems. Most studies on T1AM have explored its effects in healthy functional systems; while its potential therapeutic uses and safety, and efficacy in pathological conditions are largely unknown. We sought to evaluate the effects of T1AM and its structural analog SG-2 on cancer cell growth and viability. We analyzed the cytotoxicity of these analogs on MCF7 breast cancer cells, HepG2 hepatocellular cancer cells as well as normal control cells using primary human foreskin fibroblasts and mouse preadipocytes control cells. The cytotoxicity of T1AM and SG-2 was determined by cell growth curves, and validated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assays. Cellular uptake analysis was conducted using confocal microscopy. Real-time (RT)-PCR was conducted to identify gene pathways affected by SG-2 in cancer cells. The IC 50 of T1AM was approximately double the concentration of its analog SG-2 in cancer cells. Cytotoxicity studies on normal cells revealed that IC 50 concentrations of SG-2 in cancer cells had no significant impact on cell viability in these cell types. Cell-imaging experiments demonstrated rapid uptake and localization to the mitochondrial membrane. T1AM and SG-2 are able to reduce cancer cell growth and viability. These findings support the potential for use of these compounds and related analogs for their antiproliferation properties in cancer cells.

Complex diseases such as polycystic ovary syndrome (PCOS) are associated with intricate pathophysiological, hormonal, and metabolic feedbacks that make their early diagnosis challenging, thus increasing the prevalence risks for obesity, cardiovascular, and fatty liver diseases. To explore the crosstalk between endocrine and lipid metabolic pathways, we administered 3‐iodothyronamine (T1AM), a natural analog of thyroid hormone, in a mouse model of PCOS and analyzed plasma and tissue extracts using multidisciplinary omics and biochemical approaches. T1AM administration induces a profound tissue‐specific antilipogenic effect in liver and muscle by lowering gene expression of key regulators of lipid metabolism, PTP1B and PLIN2, significantly increasing metabolites (glucogenic, amino acids, carnitine, and citrate) levels, while enhancing protection against oxidative stress. In contrast, T1AM has an opposing effect on the regulation of estrogenic pathways in the ovary by upregulating STAR , CYP11A1, and CYP17A1. Biochemical measurements provide further evidence of significant reduction in liver cholesterol and triglycerides in post‐T1AM treatment. Our results shed light onto tissue‐specific metabolic vs. hormonal pathway interactions, thus illuminating the intricacies within the pathophysiology of PCOS. This study opens up new avenues to design drugs for targeted therapeutics to improve quality of life in complex metabolic diseases.

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 2H, 13C, and 15N 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.

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. Copyright © 2009 John Wiley & Sons, Ltd.

Brazzein protein comes from an edible fruit, which has a long history of being a staple in the local human diet in Africa. The attractive features of brazzein as a potential commercial sweetener include its small size (53 amino acid residues), its stability over wide ranges of temperature and pH, and the similarity of its sweetness to sucrose. Heterologous production of brazzein is complicated by the fact that the protein contains four disulfide bridges and requires a specific N-terminal sequence. Our previous protocol for producing the protein from Escherichia coli involved several steps with low overall yield: expression as a fusion protein, denaturation and renaturation, oxidation of the cysteines, and cleavage by cyanogen bromide at an engineered methionine adjacent to the desired N-terminus. The new protocol described here, which is much faster and leads to a higher yield of native protein, involves the production of brazzein in E. coli as a fusion with SUMO. The isolated protein product contains the brazzein domain folded with correct disulfide bonds formed and is then cleaved with a specific SUMO protease to liberate native brazzein. This protocol represents an important advancement that will enable more efficient research into the interaction between brazzein and the receptor as well as investigations to test the potential of brazzein as a commercially viable natural low calorie sweetener.

Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non-carbohydrate sweeteners. In an investigation of sequence-dependent functional properties of the protein, we used NMR spectroscopy to determine the three-dimensional structures and dynamic properties of two Brz variants: one with a single-site substitution (D40K), which is three-fold sweeter than wild-type Brz, and one with a two-residue insertion between residues 18 and 19 (ins18 RI19 ), which is devoid of sweetness. Although the three-dimensional folds of the two variants were very similar to wild-type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter-stand H-bond between the side chains of residues Gln46 and Asp40 present in wild-type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G-protein coupled human sweet receptor. On the other hand, the Arg-Ile insertion within Loop9-19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.

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.

Metabolomics is an important component of system biology complementing genomics, transcriptomis, and proteomics. The project is to determine the effects of 6-month green tea polyphenols (GTP) suppl...

Host diet influences the gut microbiota in species that regularly consume food. We showed that winter fasting in hibernating ground squirrels increases relative abundance of bacterial taxa that can degrade host glycans (e.g., mucins) and reduces abundance of taxa that prefer plant glycans. Here we determined the functional significance of seasonally changing microbiotas by gavaging summer squirrels and aroused hibernators in early and late winter with 13C-inulin (25 mg/kg), a plant-derived fiber resistant to digestion by mammalian enzymes. Subsequent measurement of δ13CO2 in breath over 2-4 h was used an index of bacterial degradation of 13C-inulin. Compared with summer, peak changes in δ13CO2 in hibernators were delayed and/or reduced by 60-90% after 1 month of hibernation (early winter) and nearly abolished after 4 months (late winter). Intraperitoneal injection of 13C-inulin in summer squirrels produced minimal changes in breath δ13CO2, confirming the bacterial nature of the δ13C signal. The results su...

Iodothyronamide (T1AM) is a naturally produced endogenous hormone like molecule. Only recently it has been discovered in the past decade, the proceeding body of research emerging from its initial discovery has revealed a substantial capacity of T1AM as a new potential hormone that affects numerous physiological processes and organs. Initially, it was hypothesized to be a byproduct of Thyroid Hormone (TH) metabolism; however, the current body of evidence suggests that its production and physiological function appear to be uncoupled and dramatically divergent from that of TH. This review summarizes the physiological and biochemical effects of T1AM reported in the literature along with its proposed mechanisms of action and production pathways. The physiological effects of T1AM appear to be dose specific, in some cases exerting opposing effects in the same biological processes. Uptake, storage, and degree of effect appear to be tissue specific as well. From the current body of literature, potential therapeutic applications with T1AM are quite apparent, ranging from sleep/torpidity induction, conferring protection against ischemic injury, and anti-obesogenic by inducing increased metabolic reliance on lipid oxidation. Future research is needed to understand the specific mechanisms of its dose dependent and tissue dependent effects, its mechanism of entry into the cell, its cellular targets, and primary site of production.

In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (transhydrogen bond couplings). We have applied this approach to screen a series of five single site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long range, three-dimensional data correlating connectivities among backbone 1HN, 15N, and 13C′ atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three “sweet” variants exhibited the same pattern of hydrogen bonds, whereas all three “non-sweet” variants lacked one hydrogen bond at the middle of the α-helix, where it is kinked, and one hydrogen bond in the middle of β-strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.

Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all sweetness. The same pattern occurred again at the beta-turn region, where Glu41Lys gave the highest sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey sweet taste in primates.

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.

Objectives: T1AM subchronic low doses cause rapid lipolysis and weight loss with no change in food consumption. We investigated tissue specific effects of T1AM, a natural thyroid hormone analog, on metabolic pathways and associated transcriptional gene signaling and protein profiles. Methods: Three groups (n=5), randomly selected spontaneously obese female CD -1 mice were injected once daily for 7 days with saline, 10 mg/kg T1AM, or 25 mg/kg T1AM. Blood drawn on days -3, 4, and 7, was analyzed by 1H-NMR. Mice were sacrificed on day 7 and the organs collected for real-time PCR and protein profiles. Results: Multivariate analysis of the 1H-NMR data revealed increased 3-hydroxybutyrate and acetate and no differences in plasma glucose levels in T1AM-treated vs. control mice, . Mice treated with high dose T1AM show significant changes in tissue specific gene expression. The livers had increased expression of SIRT6 and GCK, and decreased expression of SIRT4, but in adipose tissue only SIRT6 increased. Protein e...

This study used an optical technique to measure the effects of treating low (10 mg/kg) and high (25 mg/kg) doses of 3-iodothyronamine (T1AM) on the metabolism in the kidney and heart of mice. The ratio of two intrinsic fluorophores in tissue, (NADH/FAD), called the NADH redox ratio (NADH RR), is a marker of the metabolic state of the tissue. A cryofluorescence imaging instrument was used to provide a quantitative assessment of NADH RR in both kidneys and hearts in mice treated with 3-iodothyronamine. We compared those results to corresponding tissues in control mice. In the kidneys of mice treated with a high dose T1AM, the mean values of the maximum projection of NADH RR were 2.6 ± 0.6 compared to 3.20 ± 0.03 in control mice, indicating a 19% ( ± 0.4) significant increase in oxidative stress (OS) in the high dose-treated kidneys ( P = 0.047). However, kidneys treated with a low dose of T1AM showed no difference in NADH RR compared to the kidneys of control mice. Furthermore, low versus high dose treatment of T1AM showed different responses in the heart than in the kidneys. The mean value of the maximum projection of NADH RR in the heart changed from 3.0 ± 0.3 to 3.2 ± 0.6 for the low dose and the high dose T1AM-treated mice, respectively, as compared to 2.8 ± 0.7 in control mice. These values correspond to a 9% (±0.5) ( P = 0.045) and 14% (±0.5) ( P = 0.008) significant increase in NADH RR in the T1AM-treated hearts, indicating that the high dose T1AM-treated tissues have reduced OS compared to the low dose-treated tissues or the control tissues. These results suggest that while T1AM at a high dose increases oxidative response in kidneys, it has a protective effect in the heart and may exert its effect through alternative pathways at different doses and at tissue specific levels.

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.

Subunit c of the H(+)-transporting F1Fo ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha helices with a conserved Asp/Glu residue, centered in the second membrane-spanning helix, which is thought to function in H+ translocation. NMR studies indicate that the purified subunit c from Escherichia coli is also folded as a hairpin in a chloroform/methanol/H2O (4:4:1) solvent mixture [Girvin, M. E., & Fillingame, R. H. (1993) Biochemistry 32, 12167-12177] and that the conserved Asp remains uniquely reactive in this solvent mixture [Girvin, M. E., & Fillingame, R. H. (1994) Biochemistry 33, 665-674]. The pKa of Asp61 is of interest because of its unique reactivity and because it is thought to protonate and deprotonate during each proton translocation cycle. We have determined the pKa value of the carboxyl group of the functional Asp in wild type and two functional, mutant subunit c proteins, i.e. the Ala24-->Asp (D24D61) and the Ala24-->Asp/Asp61-->Asn (D24N61) mutant proteins. The pKa values were determined by 1H NMR spectroscopy by measuring changes in the alpha and beta proton chemical shifts by constant time two-dimensional (2D) correlated spectroscopy. The pKa of Asp61 in the purified wild type protein was 7.1. This pKa was significantly higher than the pKa of the other two Asp residues, i.e. Asp7 and Asp44 which were 5.4 and 5.6, respectively. The pKa of the two Glu residues in the protein were determined by 2D total correlation spectroscopy and found to be approximately 5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary 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.

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.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.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.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.

Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, type 2 diabetes mellitus, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. We used a well-defined prenatally exposed glucocorticoid mousemodel that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy and cavity ring-down spectroscopy to analyze serial plasma samples and real-time breath measurements following selective 13 C-isotope–assisted labeling. These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. This novel diagnostics approach is fast, noninvasive, and sensitive for determining specific pathway utilization, and provides a direct translational application in the health care field.

Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.

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.

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 Vmax 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 A ˚ ). Comparing the structure of CKR-brazzein with that of WT-brazzein revealed that the terminal b-strands of the variant display extended b-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.