Taurine Activates SIRT1/AMPK/FOXO1 Signaling Pathways to Favorably Regulate Lipid Metabolism in C57BL6 Obese Mice.

SCOPE: The identification of novel therapeutic agents capable of modulating lipid metabolism holds a promising potential in combating obesity and its associated complications. This study is conducted to evaluate the lipid lowering effect of dietary taurine administration on high-fat fed C57BL6 mice and to study the mechanism by which taurine impacts lipid metabolism. METHODS AND RESULTS: C57BL6 mice are grouped into four (n = 6): i) normal diet (ND), ii) a high-fat diet (HFD), iii) HFD + orlistat (STD), iv) HFD + taurine (TAU) group for 12 weeks. The results show that taurine administration for 12 weeks reduces high fat-induced weight gain, and liver weight when compared with HFD fed mice. It also improves serum biochemical parameters like total cholesterol and triglycerides. Sirtuin 1 (SIRT1) activity, Nicotinamide adenine dinucleotide (NAD+) levels, SIRT1 mRNA, and protein expression are increased in HFD + TAU diet group as compared to HFD group. Taurine treatment suppresses the expression of lipogenic genes (sterol regulatory element binding protein 1c [SREBP1c], fatty acid synthase [FAS], Peroxisome proliferator-activated receptor gamma [PPARγ]) and increases the expression of ß-oxidation (peroxisome proliferator-activated receptor alpha [PPARα], liver x receptor beta [LXRß], peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC1α], AMP-activated protein kinase [AMPK]) and lipolytic (forkhead box protein O1 [FOXO1]) genes. Further, taurine mitigates hepatic inflammation by suppressing nuclear factor kappa B (NF-κB) gene expression and pro-inflammatory cytokine markers (IL-6, IL-1ß, and TNFα). CONCLUSION: Taurine exerts lipid lowering effects through activating SIRT1/AMPK/FOXO1 signaling pathways and regulating their downstream targets.

Recent insights into the molecular regulators and mechanisms of taurine to modulate lipid metabolism: a review.

Lipid metabolism disorders such as hypertriglyceridemia and hypercholesterolemia are risk factors for cardiovascular diseases and atherosclerosis that are grave public health issues. Taurine, a sulfur-containing non-essential amino acid exerts a wide range of physiological effects that regulate lipid metabolic disorders. Although the effects of taurine on lipid-lowering have been reported in animals and humans, mechanisms elucidating the lipid-lowering action of taurine remain unclear. A series of molecular regulators associated with lipid metabolism have been identified in the past few decades. These include nuclear receptors, transcription factors, and enzymes that undergo important changes during taurine treatment. In this review, we focus on the role of taurine in lipid metabolism and discuss taurine-related interventions in combating lipid disorders.

Phytochemical and Functional Characterization of Different Parts of the Garcinia xanthochymus Fruit.

The fruit of Garcinia xanthochymus is consumed traditionally and is known to possess health-promoting effects. However, studies involving the characterization of phytochemicals of different parts of the fruit, and their biological activity were limited and hence warranted a comprehensive study. The proximate analyses reveal that fruit peel was rich in crude fiber. The levels of essential minerals, fatty acids, amino acids, carotenoids, organic acids, and polyphenols were significantly higher in the peel, followed by the rind, seed, and pulp. The in vitro antioxidant assays revealed that the polyphenolic extract of the peel possesses a high antioxidant effect compared to the extracts from other parts of theG. xanthochymus fruit. Furthermore, the in vitro assays reveal the antidiabetic potential of the methanol extract. This is the first comprehensive report involving the characterization and biological properties of different parts of the G. xanthochymus fruit. Hence, our study implicates the potential use of this fruit for the development of functional foods for diabetes.

Physicochemical Characterization and In Vitro Digestibility Study of an In Silico Designed Recombinant Protein Enriched with Large Neutral Amino Acids and Lacking Phenylalanine for Phenylketonuria.

In our previous study, a 3D structure of LNAA66 model protein containing 4-5 α-helices, high large neutral amino acids (LNAA) and lacking phenylalanine was designed, refined, expressed in Pichia pastoris and confirmed by Western blotting. Here the study is focused on the characterization of the expressed and purified recombinant LNAA66 protein. The results revealed that the expressed protein had 68.59% of LNAA enrichment, containing 41.6% of α-helix, 50.4% turns and 8% ß-sheet, which are as per the in silico designed protein. The LC-ESI-MS/MS results confirmed the recombinant protein by identifying the first 30 N-terminal amino acids with a sequence coverage of ~ 29%. The protein was digested entirely into smaller molecular weight fragments when treated with digestive enzymes mimicking the human GI tract digestion, which indicated complete digestibility of the protein. These results suggest that the protein can be utilized for the envisioned application of dietary treatment for phenylketonuria.

SIRT1 activation by Taurine: in vitro evaluation, molecular docking and molecular dynamics simulation studies.

Sirtuin1 (SIRT1) plays a major role in regulating different genes involved in several metabolic pathways. SIRT1 activators have protective role in several metabolic disorders. Taurine, a sulfur containing amino acid is involved in many physiological functions and has been reported as a beneficial molecule for regulating metabolic disorders. This study aims to investigate the effect of taurine on SIRT1 activation and its underlying mechanism. HepG2 cells were treated with different concentrations of Taurine. Subsequently, the cellular mRNA and protein expression and enzyme activity of SIRT1 were analyzed using quantitative real time and reverse transcriptase PCR, western blot and fluorescent assays. The effect of Taurine on key genes involved in lipid metabolism viz. PPAR-α, LXR-ß, SREBP-1c and PGC-1α were analyzed using mRNA and protein expression data. The results showed a significant increase in SIRT1 protein expression and its activity levels (P<.05) in the cells treated with Taurine. PPAR-α, LXR-ß, PGC-1α mRNA and protein levels were increased and SREBP-1c expression was decreased after treatment. Molecular docking and molecular dynamic simulation studies were carried out to understand the binding of taurine to SIRT1 protein with resveratrol as reference molecule. Molecular docking and simulation studies endorse the role of taurine in SIRT1 activation.  In conclusion, our data collectively establishes Taurine as a positive regulator of SIRT1.  The lipid lowering effect of Taurine via modulation of lipid metabolic genes may contribute to protection against metabolic disorders like obesity and age-related disease in humans.

Crocetin exerts hypocholesterolemic effect by inducing LDLR and inhibiting PCSK9 and Sortilin in HepG2 cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important post-transcriptional regulator of plasma levels of low-density lipoprotein cholesterol (LDL-C). Inhibition of PCSK9 has emerged as an attractive strategy in recent years to combat hypercholesterolemia stimulating the search for PCSK9 inhibitors. The carotenoid crocetin exhibits hypocholesterolemic effect. However, it is unknown whether the beneficial effect is mediated through PCSK9 modulation. We hypothesized that crocetin inhibits PCSK9 and therefore, in our quest for natural and safe PCSK9 inhibitors, we investigated crocetin on PCSK9 expression and other key molecular targets involved in hepatic cholesterol metabolism using the human hepatoma cell line HepG2 as a model system. We demonstrate for the first time that crocetin treatment significantly decreases PCSK9 and sterol regulatory element binding proteins (SREBP) expression in a dose-dependent manner, accompanied by a concomitant increase in the hepatic low-density lipoprotein receptor (LDLR) expression. Additionally, crocetin significantly downregulates the levels of both mRNA and protein expression of sortilin, a key sorting receptor that facilitates PCSK9 transport in the trans Golgi network in a dose dependent manner. Overall, our results suggest that crocetin is a LDLR inducer, and an inhibitor of PCSK9, sortilin and SREBPs, thus making it an effective natural anti-cholesterol agent.

Characterization of in silico modeled synthetic protein enriched with branched-chain amino acids expressed in Pichia pastoris.

We have designed earlier the 3-dimensional structure of protein enriched with 56% branched-chain amino acids (BCAA) based on an α-helical coiled-coil structure. The chemically synthesized DNA (BCAA51 gene) was expressed in Pichia pastoris and confirmed by SDS-PAGE and western blot analysis. In the present study, the purified recombinant protein was characterized using circular dichroism and data revealed that the secondary structure contained 53.5% α-helix, 3.2% ß-strand, and 43.3% turns, which is in concurrence with the overall structure predicted by in silico modeling. The LC-ESI-MS/MS spectra revealed that three peptide masses showed similarity to peptides like EQLTK, LEIVIR, and ILDK, of the modeled BCAA51 protein with the sequence coverage of ~16% from N-terminal region. The N-terminal sequence of the first seven amino acid residues (EQLTKLE) was exactly matching with the in silico designed protein. In vitro digestibility of the protein using SGF and SIF showed the disappearance of ~11 kDa band and appearance of low molecular weight peptides, which indicated that the protein was easily digestible and non-allergenic, which is the overall objective of this study. Further in vivo digestibility and toxicology studies are required to conclusively utilize this protein as a supplement for the treatment of chronic liver diseases.

Identification, isolation, and heterologous expression of Sunflower wax synthase for the synthesis of tailored wax esters.

Wax esters (WE) are neutral lipids formed by condensation of fatty alcohol with fatty acyl-CoA by wax synthases. They serve as carbon and energy reserves and are potential substrates for various commercial applications. Sunflower (Helianthus annuus) an edible oil seed is a source of WE, however, the gene responsible for WE formation has hitherto remained unidentified. Using an in silico approach we identified, isolated putative Sunflower wax synthase (HaWS) gene and investigated it's potential for WE production in yeast. Heterologous expression of HaWS in Saccharomyces cerevisiae H1246 exhibited 57 kDa protein which was confirmed by immunoblotting. Recombinant yeast expressing HaWS were fed with combinations of C16, C18 fatty alcohols with 16:0, 18:0 fatty acyl CoA's as potential substrates to validate WE formation in vivo. The yeast cells accumulated C-32 to C-36 WE. Our study reveals identification, isolation, and heterologous functional expression of WS gene from Sunflower for the first time. PRACTICAL APPLICATIONS: Wax synthases (WSs) are critical enzymes for wax ester (WE) biosynthesis. WEs are high value products having several industrial applications. WE serve as substrates for lubricants, food coatings, cosmetics, and pharmaceuticals. There is a demand for alternate renewable resource of WEs. In this study, we have successfully isolated a putative wax synthase gene from Sunflower and submitted its sequence data to the GenBank (Accession number MH460820). Conserved sequence search analysis showed presence of condensation superfamily motif‒HHXXXDG, critical for WE biosynthesis. Heterologous expression of HaWS in yeast revealed synthesis of C-32 to C-36 WE. Our study demonstrates the efficacy of HaWS to accumulate specific WE of desired lengths in yeast, and thus represents an alternate source of WE for commercial applications and for biotechnological production of tailored WE in eukaryotic expression systems.

Recovery, physicochemical and functional characteristics of proteins from different meat processing wastewater streams.

Recovery, physicochemical and functional characteristics of proteins recovered from different meat processing wastewater streams were revealed in the present study. Wastewaters from surimi processing (SPW) and slaughterhouses, namely fish (FSW), cattle (CSW), poultry (PSW), and goat (GSW), exhibited protein, fat, ash, moisture, and microbial load in the range of 1.28-7.04%, 0.86-2.34%, 0.02-0.80%, 89.81-97.44%, and 5.33-5.81 CFU/mL, respectively. Among the wastewaters, SPW presented slightly higher protein (7.04%), fat (2.34%), and ash (0.80%) contents (P < 0.05). Furthermore, proteins recovered from SPW (SPWP) and FSW (FSWP), CSW (CSWP), PSW (PSWP), and GSW (GSWP) presented yield, protein, fat, ash, and moisture content in the range of 55.54-76.81%, 65.86-78.22%, 7.26-11.45%, 4.58-11.75%, and 5.67-14.79%. All protein samples displayed higher essential amino acid (EAA) content with leucine (8.47-14.52 g/100 g) as a predominant amino acid. GSWP and SPWP scored the highest and lowest EAA contents, respectively. SPWP displayed myofibrillar proteins as dominant proteins, while slaughterhouses' wastewater proteins showed blood proteins as major proteins. ß-Sheet is the major secondary structure presented by all protein samples. SPWP showed the highest lightness value as compared to other protein counterparts (P < 0.05). All protein samples from slaughterhouse wastewaters had the lowest protein solubility at pH 4.5. However, SPWP presented minimum solubility at pH 5.5. Among all protein samples, SPWP presented slightly higher water holding capacity and foaming property (P < 0.05), whereas FSWP displayed slightly higher emulsion property (P < 0.05). Overall, all meat processing wastewater streams served as good sources of high-quality proteins, which could be used as protein ingredients in animal feed formulation.

Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal.

A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810).

Real-time PCR (RT-PCR) is the preferred method for the quantification of genetically modified organisms (GMOs) and implementation of labeling regulations. The precision, sensitivity, and reproducibility of RT-PCR data depend on the use of external calibrators. In this investigation, a dual target plasmid designated pRSETMON-02 comprising of MON 810 maize event specific and endogenous zein gene sequences in 1:1 ratio in tandem was constructed and validated. Commutability of plasmid DNA (pDNA) and genomic DNA (gDNA) calibrators for the quantification of MON 810 maize was assessed by employing a TaqMan RT-PCR targeting the P-35S and zein gene. Higher PCR efficiencies, good linearity and lower relative standard deviation (RSD) values were associated with pRSETMON-02 as opposed to gDNA calibrants. pDNA calibrants exhibited better performance characteristic in terms of closeness to the expected value of unknown samples than their genomic counterparts. Short term stability study of the pRSETMON-02 plasmid stored at different temperatures showed that pDNA is stable for 45 days at -20, and 4 °C. The results demonstrated that the developed dual target plasmid pRSETMON-02 is fit for the intended use of quantifying MON 810 maize and is a better alternative to conventional seed powder calibrants.

Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR® real-time PCR.

BACKGROUND: Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect-resistant EE-1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties. RESULTS: End-point and real-time polymerase chain reaction (PCR) methods were used to detect EE-1 brinjal. In end-point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3' transgene integration sequence, primers specific for the event EE-1 brinjal were designed. These primers were used for end-point single, multiplex and SYBR-based real-time PCR. End-point single PCR showed that the designed primers were highly specific to event EE-1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE-1 brinjal genomic DNA. The limits of detection and quantification for SYBR-based real-time PCR assay were 10 and 100 copies respectively. CONCLUSION: The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations.

Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection.

Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.