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Diet Pill Horror StoryChitosan for metabolism -
Microarray analysis of chitosan-targeted organs was further applied to globally elucidate gene expression profiles of chitosan and to find the novel mechanisms of chitosan.
Our data showed that chitosan activated PPAR activities in brain and stomach. Additionally, chitosan regulated several pathways involved in lipid and glucose metabolism, which was in agreement with the well-known hypocholesterolemic and hypoglycemic effects of chitosan.
The PPRE tandem repeats were then filled in and inserted into the blunted Xba I site of the pBKS-P tk vector to generate pBKS-PPRE2x-P tk , pBKS-PPRE3x-P tk , pBKS-PPRE4x-P tk , pBKS-PPRE5x-P tk , pBKS-PPRE6x-P tk , pBKS-PPRE7x-P tk , and pBKS-PPRE8x-P tk. A The schematic diagram of PPRE reporter constructs.
Two PPRE oligonucleotides were annealed and ligated to form various tandem repeats of PPRE. Eight reporter constructs containing various numbers of PPREs were shown on the right. B Effect of rosiglitazone on the inducibility of PPRE reporter constructs. HepG2 cells were transiently transfected with PPRE constructs and pcDNA3.
Luciferase and β-galactosidase activities were determined 24 hours later. Luciferase activities are expressed as induction fold, which is presented as comparison with RLU related to untreated cells. β-Galactosidase activities are expressed as OD Values are mean ± standard error of three independent assays.
C In vitro imaging. HepG2 cells were transiently transfected with PPRE constructs containing 5 tandem repeats of PPRE and treated without or with 0.
Luciferase activity was imaged at 24 h by IVIS system. Quantification of photon emission from the cells was shown at the bottom. Photos are representative images. Cells were transfected with PPRE reporter constructs and pcDNA3. Twenty-four hours later, transfected cells were treated with 0.
Luciferase assay and β-galactosidase assay were performed as described previously [24] , [25]. Induction fold was calculated by dividing the relative luciferase unit RLU of rosiglitazone-treated cells by the RLU of untreated cells. Plasmid DNA pGL-PPRE5x-P tk was linearized with Not I and Sal I to generate a 3.
All transgenic mice were crossed with wild-type F1 mice to yield PPRE heterozygous mice with the FVB genetic background. Louis, MO, USA and dissolved in DDW.
Mice were then imaged for the luciferase activity or sacrificed for microarray analysis at indicated periods. Mouse experiments were conducted under ethics approval from the China Medical University Animal Ethics Committee permit number N.
In vivo and ex vivo imaging of luciferase activity was performed as described previously [24] , [26]. Five minutes later, mice were placed in the chamber and imaged for 1 min with the camera set at the highest sensitivity by IVIS Imaging System® Series Xenogen, Hopkinton, MA, USA.
Photons emitted from tissues were quantified using Living Image® software Xenogen. For ex vivo imaging, mice were anesthetized and injected with luciferase intraperitoneally. Five minutes later, mice were sacrificed and tissues were rapidly removed. Tissues were placed in the IVIS system and imaged with the same setting used for in vivo studies.
Total RNAs were extracted from brain and stomach as described previously [25]. The RNA sample with a RNA integrity number greater than 7.
Microarray analysis was performed as described previously [25]. Three replicates from three independent mice were performed. The Cy5 fluorescent intensity of each spot was analyzed by genepix 4. The signal intensity of each spot was corrected by subtracting background signals in the surrounding.
We filtered out spots that signal-to-noise ratio was less than 0 or control probes. We used the WebGestalt tool to test significant KEGG pathways. Microarray data are MIAME compliant and the raw data have been deposited in a MIAME compliant database Gene Expression Omnibus, accession number GSE RNA samples were reverse-transcribed for 2 h at 37°C with High Capacity cDNA Reverse Transcription Kit Applied Biosystems, Foster City, CA, USA.
qPCR was performed by using 1 µl of cDNA, 2× SYBR Green PCR Master Mix Applied Biosystems , and nM of forward and reverse primers. The reaction condition was followed: 10 min at 95°C, and 40 cycles of 15 sec at 95°C, 1 min at 60°C. Each assay was run on an Applied Biosystems Real-Time PCR system in triplicates.
Fold changes were calculated using the comparative C T method. Data were presented as mean ± standard error. Student's t -test was used for comparisons between two experiments. Multiple tandem repeats of PPRE were constructed and cloned upstream the tk promoter.
The resulting PPRE- tk constructs were then inserted upstream the luciferase gene and droven the expression of luciferase gene Figure 1 A. To test which report constructs were significantly induced by rosiglitazone a PPARγ agonist , we transiently transfected HepG2 cells with various reporter constructs and treated cells with rosiglitazone.
As shown in Figure 1 B , rosiglitazone significantly induced the luciferase activity driven by five, six, seven, or eight tandem repeats of PPRE. The maximal induction was observed in HepG2 cells transfected with five or six tandem repeats of PPRE construct.
The β-galactosidase activities were consistent, suggesting that the transfection efficacies were similar in various reporter constructs.
Moreover, in vitro imaging showed that bioluminescent signal was significantly induced by rosiglitazone in pGL-PPRE5x-P tk -transfected HepG2 cells Figure 1 C. Ciani et al. constructed the luciferase reporter plasmids containing one, two, three, and five tandem repeats of PPRE, and found that the induced luciferase activity is directly proportional to the number of PPREs present in the promoter region [28].
However, the maximal induction of luciferase activity was observed in HepG2 cells transfected with five or six tandem repeats of PPRE construct in this study. Therefore, we selected the construct containing five tandem repeats of PPRE for further experiment, and these findings suggested that induced luciferase activity might not be proportional to the number of PPREs.
Plasmid DNA pGL-PPRE5x-P tk was selected for the generation of transgenic mice following pronuclear microinjection of FVB oocytes. Because the transgene contained a luciferase gene driven by PPRE, the luciferase activity reflected the PPAR trans -activity.
To monitor the constitutive and induced PPAR activity, transgenic mice were treated without or with rosiglitazone and imaged 6 hours later. As shown in Figure 2 A , the diffuse luminescence was detected throughout the body and the intense signals were emitted in the head and abdominal region.
Administration of rosiglitazone significantly induced the PPAR-dependent luminescent signals in mice. Ex vivo imaging showed that the maximal intensity was observed in the brain, moderate luminescent signals were observed in liver and stomach, and slight intensity was observed in heart, lung, spleen, kidney, and intestine Figure 2 B.
Administration of rosiglitazone increased the PPAR-dependent luminescent intensity in brain and stomach. These data indicated that endogenous PPAR activities were widely present in most organs and the greater endogenous PPAR activities were observed in brain, liver, and stomach.
Moreover, rosiglitazone activated the PPAR activities in the brain and stomach, which was consistent with previous studies [28] , suggesting that PPRE transgenic mice could be applied to report the PPAR activity in vivo.
A In vivo imaging. Quantification of photon emission from the mice was shown at the bottom. B Ex vivo imaging. Six hours later, mice were sacrificed and organs were subjected to image. Quantification of photon emission from the organs was shown at the bottom. Previous studies have shown that chitosan activates the PPAR activity in adipocytes [13].
We further analyzed the in vivo PPAR activity after chitosan administration and the targeted organs that chitosan acted on by bioluminescent imaging. Figure 3 A shows that chitosan gradually increased the luminescence intensity, reached a maximal intensity on 3 d, and gradually decreased the luminescent signals.
We further sacrificed mice on 3 d after administration, and ex vivo imaging showed that chitosan significantly induced PPAR-dependent bioluminescent signals in brain 1. These findings indicated that administration of chitosan activated PPAR activities in brain and stomach.
A Time course. Transgenic mice were subcutaneously injected saline mock or chitosan, and images at indicated periods. Results are expressed as relative intensity, which is presented as comparison with the luminescent intensity relative to mock.
B Ex vivo imaging and quantification of photon emission from individual organs. Transgenic mice were subcutaneously injected saline mock or chitosan, and sacrificed 3 days later for organ imaging. In order to understand the chitosan-induced biological events in brain and stomach, we extracted RNA samples from brain and stomach on 3 d and performed microarray analysis.
In a total of 29, genes, the transcripts of and genes in the stomach and brain, respectively, passed the aforementioned criteria Table S1 and Table S2 and selected for KEGG classification. Tables 1 and 2 show the pathways significantly regulated by chitosan in the stomach and brain, respectively.
Five pathways, including oxidative phosphorylation, ribosome, GnRH signaling pathway, tumor necrosis factor-α TNF-α signaling pathway and insulin signaling pathway, were affected commonly by chitosan in both organs, and oxidative phosphorylation and ribosome pathways were the top two pathways affected by chitosan.
These data showed that chitosan might alter several pathways involved in lipid and glucose metabolism in brain and stomach. To elucidate which PPAR subtype contributed to the chitosan-affected gene expression profile, we performed Pscan to analyze the PPRE in the promoter regions of chitosan-regulated genes.
Pscan is a software that scans promoter sequences of genes with motifs describing the binding specificity of known transcription factors [29]. PPAR-α and PPAR-γ-regulated genes were further validated by qPCR. The expression levels of PPAR-γ-regulated genes, xpo4 and penk1, and PPAR-α-regulated genes, pin1 and prdx2, were upregulated by chitosan in the stomach and brain, which were in agreement with microarray data Table 3.
Ghrelin and apoB are expressed in the stomach and have been shown to be involved in energy and lipid metabolism [30] , [31]. We further applied qPCR to validate the transcriptional expression levels of these genes. As shown in Table 3 , the expression levels of ghrelin and apoB genes were down-regulated by chitosan, which was consistent with the microarray data.
In this study, we found that chitosan significantly activated PPAR activity in brain and stomach. Microarray analysis of brain and stomach further showed that several pathways involved in glucose and lipid metabolism were affected by chitosan.
PPARs are ligand-activated nuclear receptors and key regulators of fatty acid and glucose homeostasis [19] , [20]. In vivo and ex vivo imaging showed that maximal luciferase activities were detected in brain and gastrointestinal tract. These data suggested that PPARs were highly expressed in these organs.
Previous studies have shown that PPAR activities are activated in brain and gastrointestinal tract, and their activation play important roles in these organs. In this study, we found that PPAR-driven luminescent intensity was strong in the brain and stomach, which was in agreement with previous study.
Therefore, these findings suggested that bioluminescent imaging of PPAR transgenic mice was capable of reflecting the real-time PPAR activity in living animals. Chitosan is a nontoxic, antibacterial, biodegradable, and biocompatible biopolymer. It has been widely used in food and biomaterial industries as weight-loss aids, cholesterol-lowering agents, and medical devices, such as bio-scaffolds for tissue engineering, wound healing products, and haemostatic bandages [1] , [3] — [8].
Although chitosan is usually administered by an oral route as a dietary supplement, food additive, or oral drug delivery, chitosan would be degraded by gut microflora or influence the distribution and number of gut microflora by oral administration [1] , [2] , [34].
Therefore, we administered transgenic mice with chitosan by a parenteral route to avoid the influence of gut microflora.
Chitosan induced a maximal intensity on 3 d, and the signal gradually decreased after three days, suggesting that subcutaneous administration of chitosan evoked the PPAR activity, and the induced PPAR activity was decreased to the basal level after 3 days.
Administration of chitosan evoked PPAR activations in brain and stomach. These findings suggested that chitosan might affect the biological events in brain and stomach. It has been shown that PPARs play important roles in the pathogenesis of various disorders of central nervous system.
For examples, activation of PPARs suppresses inflammation in peripheral macrophages and in models of human autoimmune diseases [35]. Activation of all PPAR isoforms has been found to be protective in murine models of multiple sclerosis, Alzheimer's disease, and Parkinson's diseases [36] , [37].
These findings suggested that chitosan might exhibit the beneficial effect on the neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, and Parkinson's diseases. In addition to brain, chitosan also evoked the PPAR activity in the stomach. KEGG pathway analysis further revealed that the half of chitosan-regulated pathways in the stomach was related to glucose or lipid metabolism.
It has been shown that chitosan and its derivatives markedly prevent the time course-related rise of serum glucose levels in diabetic mice [38]. Moreover, chitosan is well known for its hypotriglyceridemic and hypocholesterolemic effects [39] , and exhibits anti-obesity and anti-diabetic effects [1] , [9] — [11].
Previous studies showed that chitosan and its derivatives may bind to bile salt components and free fatty acids, resulting in the disrupted lipid absorption in the gut and the increased faecal fat excretion [40].
Our data showed that chitosan significantly regulated the IL-6 and TNF-α signaling pathways in the guts, which were consistent with previous findings. Microarray data showed that chitosan downregulated the expressions of apoB and ghrelin genes in the stomach. ApoB, a large amphipathic protein, is mainly expressed in the liver and is present on very-low density lipoproteins VLDL , intermediate density lipoproteins, and low-density lipoproteins.
ApoB is required for the formation of VLDL in the liver. Binding of apoB to the microsomal transport protein results in the incorporation of lipids into the apoB molecule and leads to the formation of VLDL particles [30] , [41].
In clinical practice, apoB can be used as a marker to estimate the total number of atherogenic lipoprotein particles [42].
Elevated apoB is a hallmark of several inherited disorders associated with atherosclerosis [43]. However, patients with extremely low levels of apoB seem to be protected against cardiovascular diseases [44]. Because apoB is an essential component of lipoprotein, the down-regulated expression of apoB gene by chitosan might contribute to the hypotriglyceridemic and hypocholesterolemic effects of chitosan.
Ghrelin is a peptide hormone mainly produced by the stomach. Ghrelin is a potent stimulator of growth hormone secretion [45]. Moreover, it is the only circulatory hormone that potently enhances the feeding and weight gain, increases the gastrointestinal mobility, and regulates the energy homeostasis [30] , [45].
Furthermore, ghrelin-based components may have therapeutic effects in treating malnutrition [31]. Because ghrelin has a great impact on the food intake or body weight, the down-regulated expression of ghrelin gene by chitosan might explain why chitosan exhibited the anti-obestic effect.
In conclusion, we applied PPAR bioluminescent imaging-guided transcriptomic analysis to evaluate the organs that chitosan acted on and to analyze the molecular mechanisms of chitosan in this study.
We found that administration of chitosan induced the PPAR-driven bioluminescent signals in brain and stomach.
Microarray analysis showed that several pathways associated with lipid and glucose metabolism were regulated by chitosan.
Moreover, we newly identified that chitosan may exhibit hypocholestemic and anti-obestic effects via downregulated expression of apoB and ghrelin genes. These findings suggested the feasibility of PPAR bioluminescent imaging-guided transcriptomic analysis on the evaluation of chitosan-affected metabolic responses in vivo.
Moreover, we newly identified that downregulated expression of apoB and ghrelin genes were the novel mechanisms for chitosan-affected metabolic responses in vivo. Expression levels of chitosan-regulated genes in the stomach.
Expression levels of chitosan-regulated genes in the brain. Conceived and designed the experiments: CHK TYH. Performed the experiments: CYH. Analyzed the data: CYH TYH. Wrote the paper: CHK CYH TYH. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Chitosan has been widely used in food industry as a weight-loss aid and a cholesterol-lowering agent. Aguila, State University of Rio de Janeiro, Biomedical Center, Institute of Biology, Brazil Received: November 15, ; Accepted: March 8, ; Published: April 4, Copyright: © Kao et al.
Introduction Chitosan is a polysaccharide comprising copolymers of glucosamine and N -acetylglucosamine. Download: PPT. Figure 1. Construction and optimization of PPRE reporter constructs. Generation of transgenic mice Plasmid DNA pGL-PPRE5x-P tk was linearized with Not I and Sal I to generate a 3.
In the industry of weight management and dietary supplements, chitosan has been used to bind ingested dietary fat and cholesterol, reducing their absorption and facilitating weight management.
Chitosan is a multifunctional biopolymer derived from chitin. Chitin is the second-most abundant polysaccharide on Earth, topped only by cellulose. Its main source is the hard exoskeleton of crustaceans, but it can also be found in invertebrates, insects, algae, and fungi.
Chitosan is a naturally fibrous polymer with fat-binding qualities and can actively absorb oils, fats, and lipids. Viscous soluble fibers, and chitosan in particular, are known to interfere with dietary fat metabolism, decreasing the absorption of fats.
Unlike other viscous soluble fibers, chitosan becomes positively charged once solubilized in the stomach acid. This positive charge is also the key element for the higher fat-binding capacity of chitosan versus other fibers. Chitosan may be considered a valuable prebiotic, promoting healthy gut flora and colonic conditions.
Accumulating evidence indicates that prebiotics have a diverse range of health benefits, particularly by influencing microbial gut ecology, mineral absorption, laxation, potential anticancer properties, and lipid metabolism, together with anti-inflammatory and other immune effects, including atopic disease.
In addition, chitosan has excellent emulsifying properties and is used to stabilize various oil-in-water emulsions to avoid the use of synthetic surfactants. The intrinsic properties vary with the molecular weight MW and degree of deacetylation DDA , which are the most important characteristics of chitosan.
LipoSan Ultra® is a unique and proprietary dietary fiber formulation, shown to significantly reduce body weight in a human clinical study. The main constituent in LipoSan Ultra® is chitosan, which has been optimized to enhance its solubility in stomach acid and fat binding performance.
The resulting enhanced performance of LipoSan Ultra® is characterized by rapid solubility in stomach acid, high density, and molecular weight. These factors all contribute to the greater fat-binding capacity of LipoSan Ultra®.
A randomized, double-blind, placebo-controlled study examined the effects of a rapidly soluble chitosan dietary supplement on weight loss and body composition in overweight and mildly obese individuals. The results showed that a daily dose 3 g of LipoSan Ultra® led to a significant weight loss 1 kg and reduced body mass index BMI in treated subjects.
For more information about Chitosan for metabolism Subject Areas, Chitosan for metabolism here. Chitosan Wound healing catechins been widely used Refillable first aid supplies ofr industry as Refillable first aid supplies weight-loss aid metaboljsm a cholesterol-lowering mtabolism. Previous studies have shown that chitosan affects metabolic responses Chitosab contributes metabolizm anti-diabetic, hypocholesteremic, and blood glucose-lowering effects; however, the in vivo targeting sites and mechanisms of chitosan remain to be clarified. In this study, we constructed transgenic mice, which carried the luciferase genes driven by peroxisome proliferator-activated receptor PPARa key regulator of fatty acid and glucose metabolism. Bioluminescent imaging of PPAR transgenic mice was applied to report the organs that chitosan acted on, and gene expression profiles of chitosan-targeted organs were further analyzed to elucidate the mechanisms of chitosan. Bioluminescent imaging showed that constitutive PPAR activities were detected in brain and gastrointestinal tract. Thank metabolksm for visiting nature. You Injury prevention through healthy eating using a browser version mdtabolism limited metabolis, for CSS. Chitosan for metabolism obtain the best experience, we recommend mrtabolism use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Background : Overweight and obesity is a prevalent and costly threat to public health. Compelling evidence links overweight and obesity with serious disorders such as cardiovascular diseases and diabetes.
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