Adiponectin Enhances Fatty Acid Signaling in Human Taste Cells by Increasing Surface Expression of CD36.

Adiponectin, a key metabolic hormone, is secreted into the circulation by fat cells where it enhances insulin sensitivity and stimulates glucose and fatty acid metabolism. Adiponectin receptors are highly expressed in the taste system; however, their effects and mechanisms of action in the modulation of gustatory function remain unclear. We utilized an immortalized human fungiform taste cell line (HuFF) to investigate the effect of AdipoRon, an adiponectin receptor agonist, on fatty acid-induced calcium responses. We showed that the fat taste receptors (CD36 and GPR120) and taste signaling molecules (Gα-gust, PLCß2, and TRPM5) were expressed in HuFF cells. Calcium imaging studies showed that linoleic acid induced a dose-dependent calcium response in HuFF cells, and it was significantly reduced by the antagonists of CD36, GPR120, PLCß2, and TRPM5. AdipoRon administration enhanced HuFF cell responses to fatty acids but not to a mixture of sweet, bitter, and umami tastants. This enhancement was inhibited by an irreversible CD36 antagonist and by an AMPK inhibitor but was not affected by a GPR120 antagonist. AdipoRon increased the phosphorylation of AMPK and the translocation of CD36 to the cell surface, which was eliminated by blocking AMPK. These results indicate that AdipoRon acts to increase cell surface CD36 in HuFF cells to selectively enhance their responses to fatty acids. This, in turn, is consistent with the ability of adiponectin receptor activity to alter taste cues associated with dietary fat intake.

Recent development in zebrafish model for bioactivity and safety evaluation of natural products.

The zebrafish is a species of freshwater fish, popular in aquariums and laboratories. Several advantageous features have facilitated zebrafish to be extensively utilized as a valuable vertebrate model in the lab. It has been well-recognized that natural products possess multiple health benefits for humans. With the increasing demand for natural products in the development of functional foods, nutraceuticals, and natural cosmetics, the zebrafish has emerged as an unprecedented tool for rapidly and economically screening and identifying safe and effective substances from natural products. This review first summarized the key factors for the management of zebrafish in the laboratory, followed by highlighting the current progress on the establishment and applications of zebrafish models in the bioactivity evaluation of natural products. In addition, the zebrafish models used for assessing the potential toxicity or health risks of natural products were involved as well. Overall, this review indicates that zebrafish are promising animal models for the bioactivity and safety evaluation of natural products, and zebrafish models can accelerate the discovery of novel natural products with potential health functions.

Recent Advances in Bioactive Compounds, Health Functions, and Safety Concerns of Onion (Allium cepa L.).

Onion (Allium cepa L.) is a common vegetable, widely consumed all over the world. Onion contains diverse phytochemicals, including organosulfur compounds, phenolic compounds, polysaccharides, and saponins. The phenolic and sulfur-containing compounds, including onionin A, cysteine sulfoxides, quercetin, and quercetin glucosides, are the major bioactive constituents of onion. Accumulated studies have revealed that onion and its bioactive compounds possess various health functions, such as antioxidant, antimicrobial, anti-inflammatory, anti-obesity, anti-diabetic, anticancer, cardiovascular protective, neuroprotective, hepatorenal protective, respiratory protective, digestive system protective, reproductive protective, and immunomodulatory properties. Herein, the main bioactive compounds in onion are summarized, followed by intensively discussing its major health functions as well as relevant molecular mechanisms. Moreover, the potential safety concerns about onion contamination and the ways to mitigate these issues are also discussed. We hope that this paper can attract broader attention to onion and its bioactive compounds, which are promising ingredients in the development of functional foods and nutraceuticals for preventing and managing certain chronic diseases.

GPR84 Is Essential for the Taste of Medium Chain Saturated Fatty Acids.

The ability of mammalian taste cells to respond to fatty acids (FAs) has garnered significant attention of late and has been proposed to represent a sixth primary taste. With few exceptions, studies on FA taste have centered exclusively on polyunsaturated FAs, most notably on linoleic acid. In the current study, we have identified an additional FA receptor, GPR84, in the gustatory system that responds to the medium-chain saturated FAs (MCFAs) in male mice. GPR84 ligands activate both Type II and Type III taste cells in calcium imaging and patch-clamp recording assays. MCFAs depolarize and lead to a rise in intracellular free [Ca2+] in mouse taste cells in a concentration-dependent fashion, and the relative ligand specificity in taste cells is consistent with the response profile of GPR84 expressed in a heterologous system. A systemic Gpr84-/- mouse model reveals a specific deficit in both the neural (via chorda tympani recording) and behavioral responses to administration of oral MCFAs compared with WT mice. Together, we show that the peripheral taste system can respond to an additional class of FAs, the saturated FAs, and that the cognate receptor necessary for this ability is GPR84.

Spatiotemporal dynamic monitoring of fatty acid-receptor interaction on single living cells by multiplexed Raman imaging.

Numerous fatty acid receptors have proven to play critical roles in normal physiology. Interactions among these receptor types and their subsequent membrane trafficking has not been fully elucidated, due in part to the lack of efficient tools to track these cellular events. In this study, we fabricated the surface-enhanced Raman scattering (SERS)-based molecular sensors for detection of two putative fatty acid receptors, G protein-coupled receptor 120 (GPR120) and cluster of differentiation 36 (CD36), in a spatiotemporal manner in single cells. These SERS probes allowed multiplex detection of GPR120 and CD36, as well as a peak that represented the cell. This multiplexed sensing system enabled the real-time monitoring of fatty acid-induced receptor activation and dynamic distributions on the cell surface, as well as tracking of the receptors' internalization processes on the addition of fatty acid. Increased SERS signals were seen in engineered HEK293 cells with higher fatty acid concentrations, while decreased responses were found in cell line TBDc1, suggesting that the endocytic process requires innate cellular components. SERS mapping results confirm that GPR120 is the primary receptor and may work synergistically with CD36 in sensing polyunsaturated fatty acids and promoting Ca2+ mobilization, further activating the process of fatty acid uptake. The ability to detect receptors' locations and monitor fatty acid-induced receptor redistribution demonstrates the specificity and potential of our multiplexed SERS imaging platform in the study of fatty acid-receptor interactions and might provide functional information for better understanding their roles in fat intake and development of fat-induced obesity.

Ya-fish (Schizothorax prenanti) spexin: identification, tissue distribution and mRNA expression responses to periprandial and fasting.

Spexin (SPX) is a novel peptide which was known for its role in physiological homeostasis. A recent study has confirmed that SPX plays an important role in the feeding regulation. However, the reports about SPX are very limited. In the present study, we characterized the structure, distribution and mRNA expression responses to feeding status of SPX in Ya-fish (Schizothorax prenanti). The full-length cDNA of Ya-fish SPX was 1330 base pairs (bp), which encoded 106 amino acid residues. These residues contained a 31-amino acid signal peptide region and a 14-amino acid mature peptide. The sequence alignment demonstrated that the Ya-fish SPX showed high conservation with other species. Our data revealed that SPX was widely expressed in all test tissues. The highest expression of SPX mRNA was observed in Ya-fish forebrain. Compared with the Ya-fish SPX mRNA expression in the forebrain between the preprandial and postprandial groups, the fed group was prominently increased than unfed groups after a meal, while the unfed group at 1 and 3 h substantially decreased than preprandial groups (P < 0.01). In addition, SPX mRNA expression in forebrain was significantly decreased (P < 0.01) during fasting for a week and sharply increased (P < 0.01) after refeeding on the 7th day, and then return to normal level on the 9th day. These results point toward that SPX mRNA expression is regulated by metabolic status or feeding conditions in Ya-fish.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

Appetite regulation in Schizothorax prenanti by three CART genes.

In recent years, cocaine- and amphetamine-regulated transcript (CART) has received much attention as mediators of appetite regulation in mammals. However, the involvement of CART in the feeding behavior of teleosts has not been well understood. In this study, three distinct CARTs were cloned from the Schizothorax prenanti (S. prenanti). Real-time quantitative PCR were applied to characterize the tissue distribution and appetite regulatory effects of CARTs in S. prenanti. The S. prenanti CART-1, CART-2 and CART-3 full-length cDNA sequences were 597 bp, 694 bp and 749 bp in length, encoding the peptides of 125, 120 and 104 amino acid residues, respectively. All the S. prenanti CARTs consisted of three exons and two introns. Tissue distribution analysis showed that the high mRNA levels of S. prenanti CART-1 were observed in the telencephalon and eye, followed by the hypothalamus, myelencephalon, and mesencephalon. The S. prenanti CART-2 mRNA was mainly found in the mesencephalon, hypothalamus, telencephalon and myelencephalon. The S. prenanti CART-3 mRNA was widely distributed among the tissues, with the high levels in the hypothalamus and foregut. In the periprandial experiment, all three CARTs mRNA expressions in the hypothalamus were highly elevated after a meal, suggesting that CARTs are postprandial satiety signals. In the fasting experiment, all three CARTs mRNA expressions decreased after fasting and increased after refeeding, suggesting that CARTs might be involved in regulation of appetite in the S. prenanti.

Molecular cloning, expression analysis, and appetite regulatory effect of peptide YY in Siberian sturgeon (Acipenser baerii).

Peptide YY (PYY) is an anorectic brain-gut peptide involved in feeding regulation and well characterized in mammals. However, the functional role of PYY in the appetite regulatory of fish is not clear. In this study, we characterized a high conservation of PYY cDNA and found high expression levels of PYY mRNA in the brain and digestive tract of Siberian sturgeon. Then, we examined preprandial (pre- and post-feeding) changes of PYY mRNA expression in the brain that showed a significantly increased in 3h post-feeding, suggesting an anorectic possible function of PYY in Siberian sturgeon. Next, we examined the expression of PYY mRNA during 15 days fasting and refeed after fasting. The SsPYY mRNA expression of unfed fish had a significant 2.4, 1.7, 2.0, 2.2, and 2.1-fold decrease compared to 1-, 3-, 6-, 10- and 15-day ad libitum fed animals, respectively. After refeed, SsPYY mRNA significantly increased 1.9 and 4.1-fold above that of the 15-day fed and unfed fish control group (P<0.01). Furthermore, a single intraperitoneal injection of 10, 100 and 200 ng/g BW SsPYY1-36 caused a reduction in the next feeding and no significant reduction in food intake was observed in fish injected with a 1 ng/g BW. Overall, PYY has a potentially role in food intake attenuation of Siberian sturgeon.

Molecular and physiological evidences for the role in appetite regulation of apelin and its receptor APJ in Ya-fish (Schizothorax prenanti).

Apelin is a recently discovered peptide produced by several tissues with diverse physiological actions mediated by its receptor APJ. In order to better understand the role of apelin in the regulation of appetite in fish, we cloned the cDNAs encoding apelin and APJ, and investigated their mRNA distributions in Ya-fish (Schizothorax prenanti) tissues. We also assessed the effects of different nutritional status on apelin and APJ mRNAs abundance. Apelin and APJ mRNAs were ubiquitously expressed in all tissues tested, relatively high expression levels were detected in the heart, spleen, hypothalamus and kidney. Short-term fasting significant increased APJ mRNA expression, but no significant difference between fasted fish and fed control on 5- and 7-day. Meanwhile, apelin mRNA expression consistently increased during the 7-day food deprivation. In order to further characterize apelin in fish, we performed intraperitoneal (i.p.) injection of apelin-13 and examined food intake of the injected fish. Apelin injected at a dose of 100 ng/g body weight induced a significant increase in food intake compared to saline injected fish. Our results suggest that apelin acts as an orexigenic factor in Ya-fish. Their widespread distributions also suggest that apelin and APJ might play multiple physiological regulating roles in fish.

Leptin and cholecystokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and fasting.

In the present study, full-length cDNA sequences of leptin and cholecystokinin (CCK) were cloned from Schizothorax prenanti (S. prenanti), and applied real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin and CCK in S. prenanti. The S. prenanti leptin and CCK full-length cDNA sequences were 1121 bp and 776 bp in length, encoding the peptide of 171 and 123 amino acid residues, respectively. Tissue distribution analysis showed that leptin mRNA was mainly expressed in the liver of S. prenanti. CCK was widely expressed, with the highest levels of expression in the hypothalamus, myelencephalon, telencephalon and foregut of S. prenanti. The CCK mRNA expression was highly elevated after feeding, whereas the leptin mRNA expression was not affected by single meal. These results suggested that CCK is a postprandial satiety signal in S. prenanti, but leptin might not be. In present study, leptin and CCK gene expression were both decreased after fasting and increased after refeeding, which suggested leptin and CCK might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin and CCK in S. prenanti as well as in other teleosts.

Molecular characterization and tissue expression of peptide YY in Schizothorax prenanti: effects of periprandial changes and fasting on expression in the hypothalamus.

Peptide YY (PYY) is a potent anorectic neuropeptide implicated in feeding regulation in mammals. However, the involvement of PYY in the feeding behavior of teleosts has not been well understood. In this study, we employed molecular, real-time quantitative PCR and physiological studies to characterize the structure, distribution, and appetite regulatory effects of PYY in Schizothorax prenanti (S. prenanti). A very high conservation in PYY sequences was found in teleosts. PYY is widely expressed, with the highest levels of expression in telencephalon, medulla oblongata, pituitary and hypothalamus of S. prenanti. The PYY mRNA expression in the hypothalamus was highly elevated after a meal, suggesting a satiety signal role for PYY in S. prenanti. In addition, PYY gene expression in the hypothalamus was decreased after fasting and increased sharply after refeeding, which suggested that PYY might be involved in the central regulation of appetite in S. prenanti. Overall, our result provides basis for further investigation into the regulation of feeding in S. prenanti.

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti).

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg-Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.

Schizothorax davidi ghrelin: cDNA cloning, tissue distribution and indication for its stimulatory character in food intake.

Ghrelin is a gut/brain hormone with a unique acyl modification and various biological functions in fish and mammals. The objectives of this project were to identify ghrelin gene organization, study tissue specific ghrelin mRNA expression and investigate the short- (0, 0.5, 1.5, 3, 6, 9, 12h post-fasting) and long- (1, 3, 5, 7 days) term fasting as well as refeeding after a 7 day fasting induced changes in the expression of ghrelin mRNA in Schizothorax davidi. Our reverse transcription polymerase chain reaction analysis confirmed the predicted ghrelin sequence available in the GenBank and identified ghrelin mRNA expression in several tissues including the gut, liver, brain, heart, spleen, head kidney, gill and muscle. Quantitative PCR studies indicated that the expression level of ghrelin mRNA presented ascendant trend in short-term fasting group compared to the fed group, but it did not reach the significant level on statistics, while there is a significant increase in ghrelin mRNA expression in the gut of Schizothorax davidi fasted for 3, 5 and 7 days when compared to the expression in ad libitum fed fish. Refeeding after a 7 day fasting caused a significant and dramatic decrease in ghrelin mRNA expression in the gut of Schizothorax davidi. An increase in the expression of ghrelin mRNA during fasting, and its decrease following refeeding suggests an orexigenic role for ghrelin in Schizothorax davidi. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin during evolution.

Cloning, distribution and effects of fasting status of melanocortin 4 receptor (MC4R) in Schizothorax prenanti.

Melanocortin 4 receptor (MC4R) has an important role in the regulation of energy homeostasis in both mammals and fish. In this study, MC4R was characterized in S. prenanti (Schizothorax prenanti) and designated as SpMC4R. SpMC4R cDNA is composed of 1004 nucleotides with a 978 nucleotide open reading frame encoding a protein of 326 amino acids. The SpMC4R contained predicted regions that were structural features of MCR subtypes of vertebrates. In addition, phylogenetic analyses suggested that S. prenanti MC4R was closely related to fish MC4Rs. The SpMC4R mRNA was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. Using real-time RT-PCR, MC4R is widely expressed, with highest levels of expression in brain and ovary. An experiment was conducted to determine the expression profile of MC4R during short-term and long-term fasting of the brain. The expression level of MC4R in unfed fish was significantly increased at 6, 9 and 24h post-fasting (hpf) and 14days fasting than in fed fish, this suggests that MC4R is conserved peptide that might be involved in the regulation of food intake and other physiological function in S. prenanti.

Identification, tissue distribution and regulation of preproghrelin in the brain and gut of Schizothorax prenanti.

Ghrelin is an important gastrointestinal hormone involved in the regulation of feeding in both mammals and fish. In this study, the preproghrelin cDNA sequence was cloning in the gut of Schizothorax prenanti (S. prenanti). The preproghrelin gene, encoding 103-amino acids, was strongly expressed in the gut and brain using real-time quantitative RT-PCR (qPCR). The S. prenanti preproghrelin was detected in embryonic developmental stages. Further, it was detectable in unfertilized eggs, suggesting that ghrelin could be classified as maternal mRNA. An experiment was conducted to determine the expression profile of ghrelin during post-feeding and fasting status of the brain and gut. The results revealed a significant postprandial decrease in ghrelin mRNA expression in the gut 6h post-feeding (hpf) and brain (1.5 and 9hpf) compared to an unfed control group, indicating that food intake and processing affect the regulation of expression of ghrelin in S. prenanti. The constructed recombinant plasmid pMD-19T-ghrelin was transformed to Escherichia coli BL21 and induced with IPTG, and the expressed product was identified by SDS-PAGE. The prokaryotic expression vector for ghrelin was constructed successfully, and fusion protein was expressed in E. coli BL21, which laid the foundation for the further study on the function of this protein and its mechanism. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin in S. prenanti. Further studies are required to identify the tissue specific functions of ghrelin in S. prenanti.

Characterization, tissue distribution and regulation of agouti-related protein (AgRP) in a cyprinid fish (Schizothorax prenanti).

Agouti-related protein (AgRP) is an important neuropeptide involved in the regulation of feeding in both mammals and fish. In this study, we have cloned the full-length cDNA sequence for AgRP in a cyprinid fish (Schizothorax prenanti). The AgRP gene, encoding 126-amino acids, was strongly expressed in the brain. The AgRP gene was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. An experiment was conducted to determine the expression profile of AgRP during short-term and long-term fasting of the hypothalamus. The expression level of AgRP in unfed fish was significantly increased at 3 and 4h post-fasting than in fed fish but did not affect AgRP mRNA expression after 14 days fasting. Overall, our results suggest that AgRP is a conserved peptide that might be involved in the regulation of short-term feeding and other physiological function in Schizothorax prenanti.

[Genetic analysis of microsatellite polymorphism in the Elliot's Pheasant (Syrmaticus ellioti) in China].

In this study, we reported the population genetic analyses in the Elliot's Pheasant(Syrnaticus ellioti) using seven polymorphism microsatellite loci based on 105 individuals from 4 geographical populations. Departures from Hardy-Weinberg equilibrium were found in four geographical populations. The average number of alleles was 8.86, with a total of 62 alleles across 7 loci; observed heterozygosity (HO) was generally low and the average number was 0.504. For the seven microsatellite loci, the polymorphism information content ranged from 0.549 to 0.860, with an average number 0.712. Population bottlenecks of the four geographical populations were tested by infinite allele mutation model, step-wise mutation model and two-phase mutation model, which found that each population had experienced bottleneck effect during the recent period. Fst analysis across all geographical populations indicated that the genetic differentiaton between the Guizhou geographical population and the Hunan geographical population was highly significant (P<0.001), a finding supported by the far genetic relationship showed by the neighbor-joining tree of four geographical populations based on Nei's unbiased genetic distances. Using hierarchical analysis of molecular variance (Guizhou geographical population relative to all others pooled), we found a low level of the genetic variation among geographical populations and that between groups. However, differences among populations relative to the total sample explained most of the genetic variance (92.84%), which was significant.