A novel short-type peptidoglycan recognition protein with unique polysaccharide recognition specificity in sea cucumber, Apostichopus japonicus.

Pattern recognition receptors (PRRs) are the first line of immune defense in invertebrates against pathogen infection; they recognize pathogens and transmit signals to downstream immune pathways. Among these, peptidoglycan recognition proteins (PGRPs) are an important family in invertebrates that generally comprise of complicated isoforms. A comprehensive understanding of PGRPs in evolutionarily and economically important marine invertebrates, such as the sea cucumber, Apostichopus japonicus, is crucial. Previous studies have identified two PGRPs in sea cucumber, AjPGRP-S and AjPGRP-S1, and another novel short-type PGRP, AjPGRP-S3, was additionally identified here. The full-length cDNA sequence of AjPGRP-S3 was obtained here by PCR-RACE, followed by which showed its gene expression analyses by in situ hybridization that showed it to be relatively highly expressed in coelomocytes and tube feet. Based on an analysis of the recombinant protein, rAjPGRP-S3, a board-spectrum pathogen recognition ability was noted that covered diverse Gram-negative and -positive bacteria, and fungi. Moreover, according to the results of yeast two-hybridization, it was suggested that rAJPGRP-S3 interacted with multiple immune-related factors, including proteins involved in the complement system, extracellular matrix, vesicle trafficking, and antioxidant system. These findings prove the important functions of AjPGRP-S3 in the transduction of pathogen signals to downstream immune effectors and help explore the functional differences in the AjPGRP isoforms.

Exploiting the gut microbiota to predict the origins and quality traits of cultured sea cucumbers.

Nowadays, the true economic and nutritional value of food is underpinned by both origin and quality traits, more often expressed as increased quality benefits derived from the origin source. Gut microbiota contribute to food metabolism and host health, therefore, it may be suitable as a qualifying indicator of origin and quality of economic species. Here, we investigated relationships between the gut microbiota of the sea cucumber (Apostichopus japonicus), a valuable aquaculture species in Asia, with their origins and quality metrics. Based on data from 287 intestinal samples, we generated the first biogeographical patterns for A. japonicus gut microbiota from origins across China. Importantly, A. japonicus origins were predicted using the random forest model that was constructed using 20 key gut bacterial genera, with 97.6% accuracy. Furthermore, quality traits such as saponin, fat and taurine were also successfully predicted by random forest models based on gut microbiota, with approximately 80% consistency between predicted and true values. We showed that substantial variations existed in the gut microbiota and quality variables in A. japonicus across different origins, and we also demonstrated the great potential of gut microbiota to track A. japonicus origins and predict their quality traits.

Cloning and characterization of a phosphomevalonate kinase gene that is involved in saponin biosynthesis in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.

Functions of lysin motif (LysM)-containing protein in antibacterial responses of sea cucumbers, Apostichopus japonicus.

The lysin motif (LysM)-containing protein is one of widespread pattern-recognition receptors in prokaryotes and eukaryotes. Numerous LysM-containing gene sequences are present in gene databases; however, few have been well characterized, especially in echinoderms. In this study, the full-length cDNA of a novel LysM-containing gene was obtained from the sea cucumber Apostichopus japonicus, named AjLysM-1, using polymerase chain reaction (PCR) combined with rapid amplification of cDNA ends. We prepared and expressed recombinant AjLysM-1 protein (rAjLysM-1) and determined its pathogen-recognition ability by enzyme-linked immunosorbent and immunofluorescence assays. We also analyzed the tissue expression pattern and response to immune challenges of AjLysM-1 using quantitative real-time reverse transcription-PCR and in situ hybridization. The AjLysM-1 protein was predicted to be an intracellular non-secreted LysM-containing protein, highly homologous to the same protein in other marine echinoderms. AjLysM-1 transcripts were highest expressed in coelomocytes and were strikingly induced by challenge with representative bacterial and fungal polysaccharides. rAjLysM-1 showed weak binding to mannan, Pseudoalteromonas nigrifaciens, and Shewanella baltica, implying that AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. Notably, rAjLysM-1 also interacted with tyrosine protein kinase and filamin-B, indicating that it could be involved in focal adhesion in A. japonicus. These findings improve our understanding of the functions of LysM-containing proteins in marine echinoderms.

Primary study on the toxic mechanism of vanadyl trehalose in Kunming mice.

It has been shown that vanadyl trehalose could lower blood glucose but show mild toxicity to the stomach and intestine in diabetic Kunming mice. We analysed antioxidant levels, pro-inflammatory cytokine expression, apoptosis factors and intestinal microflora alteration to explore the mechanism of vanadyl trehalose toxicity in Kunming mice. The results revealed that oral administration of vanadyl trehalose at tested dose caused significant changes in oxidative stress factor (MDA levels elevated but SOD and T-AOC decreased), expression of inflammatory factor (IL-1ß, COX-2, TNF-α and iNOS increased), and apoptosis factor (Bcl-2/Bax decreased and caspase-3 increased), and intestinal microflora dysbiosis (the number of Enterobacteriaceae and Enterococcus increased and Lactobacillus and Bifidobacterium decreased) relative to the control of Kunming mice. These results suggest that the toxic mechanisms of vanadyl trehalose on the stomach and intestine likely involve activation of the oxidative stress system, increased inflammatory response, promotion of apoptosis and the disruption of the normal intestinal microflora.

Acute toxicity, twenty-eight days repeated dose toxicity and genotoxicity of vanadyl trehalose in kunming mice.

A new trend has been developed using vanadium and organic ligands to form novel compounds in order to improve the beneficial actions and reduce the toxicity of vanadium compounds. In present study, vanadyl trehalose was explored the oral acute toxicity, 28 days repeated dose toxicity and genotoxicity in Kunming mice. The Median Lethal Dose (LD50) of vanadyl trehalose was revealed to be 1000 mg/kg body weight in fasted Kunming mice. Stomach and intestine were demonstrated to be the main target organs of vanadyl trehalose through 28 days repeated dose toxicity study. And vanadyl trehalose also showed particular genotoxicity through mouse bone marrow micronucleus and mouse sperm malformation assay. In brief, vanadyl trehalose presented certain, but finite toxicity, which may provide experimental basis for the clinical application.

[Construction of yeast strains expressing long-acting glucagon-like peptide-1 (GLP-1) and their therapeutic effects on type 2 diabetes mellitus mouse model].

Probiotics, i.e., bacteria expressing therapeutic peptides (protein), are used as a new type of orally administrated biologic drugs to treat diseases. To develop yeast strains which could effectively prevent and treat type 2 diabetes mellitus, we firstly constructed the yeast integrating plasmid pNK1-PGK which could successfully express green fluorescent protein (GFP) in Saccharomyces cerevisiae. The gene encoding ten tandem repeats of glucagon-like peptide-1(10 × GLP-1) was cloned into the vector pNK1-PGK and the resulting plasmids were then transformed into the S. cerevisiae INVSc1. The long-acting GLP-1 hypoglycemic yeast (LHY) which grows rapidly and expresses 10 × GLP-1 stably was selected by nutrition screening and Western blotting. The amount of 10 × GLP-1 produced by LHY reached 1.56 mg per gram of wet cells. Moreover, the oral administration of LHY significantly reduced blood glucose level in type 2 diabetic mice induced by streptozotocin plus high fat and high sugar diet.

Proteomics reveals gender differences in physiological characteristics of the gonads and tube feet of the sea cucumber, Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is an important aquaculture species in China because of its high nutritional and medicinal values. Gender, as a factor affecting the physiology of organisms, is always considered when improving the breeding efficiency of economically important animals. In the present study, protein expression profiles of the gonads and tube feet of male and female A. japonicus were investigated using a comparative proteomics approach. A set of 7499 proteins were identified, which covered a broad range of functions based on function annotations. A significant difference in protein expression profiles was observed between the gonads and tube feet of A. japonicus; gonads showed more apparent gender differences than tube feet. Moreover, the findings revealed that male A. japonicus had more specific functions and most of these functions were associated with energy consumption. Further analyses suggested that the regulation of ERK activity and the capacity of tyrosine production and virus immunity might be more powerful in male and female A. japonicus, respectively. Some candidate proteins were also recognized as potential targets for gender identification of A. japonicus. Overall, our study provides new insights into the understanding of molecular mechanisms underlying gender-based physiological differences in A. japonicus. SIGNIFICANCE: The current study aimed to reveal gender differences in the physiological characteristics of gonads and tube feet of the sea cucumber A. japonicus. To the best of our knowledge, this is the first proteomics study to analyze the differences in the protein expression profiles of external organs between male and female A. japonicus. The analysis revealed gender differences in the protein expression profiles of both gonads and tube feet of A. japonicus, and the gender differences in gonads were quite apparent. Moreover, according to the recognition of differentially expressed proteins and the enrichment analyses based on Kyoto Encyclopedia of Genes and Genomes, a draft view of how the physiological functions of A. japonicus were affected by gender was obtained. Male A. japonicus could have more specific functions related to energy consumption than females. The regulation of ERK activity and virus immunity might be more robust in male and female A. japonicus, respectively. Some candidate proteins were also recognized as potential targets for gender identification of A. japonicus. The findings presented here will improve the understanding of researchers about the molecular mechanisms underlying gender-based differences in A. japonicus and contribute to the meticulous breeding of A. japonicus.

Proteomics reveals the gender differences in humoral immunity and physiological characteristics associated with reproduction in the sea cucumber Apostichopus japonicus.

Due to the importance of characteristics associated with the immunity and physiology of organisms, gender is always taken into account when improving the breeding efficiency of economically important animals. Protein variations in the cell-free ceolomic fluid (CCF) of male and female Apostichopus japonicus before and after spawning were investigated using a comparative proteomic approach, for the purpose of understanding the influence of gender and gender associated reproductive processes on humoral immunity and physiological characteristics of the sea cucumber, A. japonicus. A total of 6839 peptides were obtained and a set of 1466 proteins were identified in the CCF of male and female A. japonicus before and after spawning. Partial least squares discrimination analysis indicated that protein expression profiles in the CCF of A. japonicus were significantly affected by both gender and reproductive processes. In addition, the differentially expressed proteins (DEPs) in the CCF of different genders and reproductive stages of A. japonicus were determined and immune-related proteins in these DEPs were screened. Findings revealed that the alternative pathway of complement activation in both male and female A. japonicus before spawning might take precedence over that after spawning. Further KEGG enrichment analyses of DEPs suggested that the capacity of muscle contraction in A. japonicus females, and food digestion, nutrition absorption, epithelial differentiation as well as growth in A. japonicus males might be enhanced after spawning. Further, compared with A. japonicus females, males may have an advantage in growth in the stages following spawning. Besides, according to KEGG annotation related to metabolism, arginine biosynthesis and purine metabolism were dominant in the CCF of male and female A. japonicus, respectively, suggesting notable metabolic differences in female and male A. japonicus. Overall, our study provides new insight into the understanding of molecular mechanisms underlying gender-determined immune and physiological differences in A. japonicus. SIGNIFICANCE: The current study aimed to reveal the gender differences in humoral immunity and physiological characteristics associated with reproduction in the sea cucumber Apostichopus japonicus. To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile between male and female A. japonicus. By analyzing the expression differences of the proteome via label-free proteomic technology, we revealed that both gender and reproduction could alter the protein composition and abundance in the cell-free colemic fluid (CCF) of A. japonicus. Based on the recognition of differentially expressed proteins (DEPs) related to immune function among the CCFs, shifts in humoral immunity of male and female A. japonicus that underwent reproduction were obtained. Moreover, according to the enrichment analyses of DEPs based on Kyoto Encyclopedia of Genes and Genomes, a draft view of how the male and female A. japonicus affected by reproduction physiologically was drawn. The results suggested that the alternative pathway of complement activation in both male and female A. japonicus were weakened after spawning. In addition, after reproduction, the capacity of muscle contraction might be enhanced in female A. japonicus, and the nutrients consumption, epithelial differentiation and growth might be strengthened in males. Further, ipath analysis indicated that A. japonicus with different genders also had notable differences in metabolism that related to arginine biosynthesis and purine metabolism. The findings presented here will improve the cognition of researchers in the molecular mechanism underlying the gender-determined immune and phyisological differences of A. japonicus and contribute for the meticulous breeding of A. japonicus.

Construction of a dietary-cure Saccharomyces cerevisiae expressing long-acting glucagon-like peptide-1 and investigation of its hypoglycemic activity in type 2 diabetes mellitus mouse model.

Bio-drug is a new type of beneficial biology expressing therapeutic peptides (protein) as orally administrated medicine to treat diseases, in particular, chronic diseases like diabetes. In order to develop recombinant yeast strains as a bio-drug which could effectively ameliorate type 2 diabetes, an integrating expression vector pNK1-PGK that could successfully express green fluorescent protein (GFP) in Saccharomyces cerevisiae was constructed to demonstrate the normality of the function. A pNK1-PGK vector, which expresses 10 tandem repeats of long-acting glucagon-like peptide-1(10laGLP-1), was cloned and then transformed into the S. cerevisiae INVSc1. The long-acting GLP-1 hypoglycemic yeast (LHY168) that grew rapidly and expressed 10laGLP-1 stably was screened by uracil-deficient plates and Western blot. The expression quantity of 10laGLP-1 reached 1.56 mg/g cell wet weight. Moreover, the oral administration of LHY168 significantly declined the blood glucose in type 2 diabetic mice that were constructed through co-induction of streptozotocin (STZ) and high-fat and high-sugar diet.

Pharmacological Effects and Pharmacokinetic Properties of a Dual-Function Peptide 5rolGLP-HV.

In order to better understand the therapeutic mechanism of dual-function peptide 5rolGLP-HV in treatment of treat diabetes and its complication of thrombosis, the pharmacological effects and pharmacokinetic properties of 5rolGLP-HV were conducted in this study. 5rolGLP-HV was orally administered to diabetic mice, and the hypoglycemic mechanism was investigated. Thrombotic mice were applied to study the thrombus dissolving ability of 5rolGLP-HV. The concentration of rolGLP and rHV in rat plasma following single oral dose or intravenous injection of 5rolGLP-HV was measured. Treatment with 5rolGLP-HV decreased insulin resistance (2.96 ± 1.43 vs. 9.35 ± 1.51, p < 0.05) of diabetic mice. 5rolGLP-HV shortened the length of thrombus in thrombosis mice (2.92 ± 0.74 vs. 5.92 ± 1.16 cm, p < 0.01) and extended the thrombin time (15.35 ± 1.22 vs. 8.67 ± 0.89 s, p < 0.01) of normal mice. Meanwhile, 5rolGLP-HV restored the damage of pancreatic, liver, kidney, and adipose tissues induced in the diabetic mice. 5rolGLP-HV exhibited a fast absorption and slow elimination phase after digested into rolGLP-1 and rHV in vivo. These results suggested that 5rolGLP-HV had an ideal therapeutic potential in the prevention of ß cell dysfunction in type 2 diabetes and delay of the thrombus.

Effect of Vanadyl Rosiglitazone, a New Insulin-Mimetic Vanadium Complexes, on Glucose Homeostasis of Diabetic Mice.

Diabetes has been cited as the most challenging health problem in the twenty-first century. Accordingly, it is urgent to develop a new type of efficient and low-toxic antidiabetic medication. Since vanadium compounds have insulin-mimetic and potential hypoglycemic activities for type 1 and type 2 diabetes, a new trend has been developed using vanadium and organic ligands to form a new compound in order to increase the intestinal absorption and reduce the toxicity of vanadium compound. In the current investigation, a new organic vanadium compounds, vanadyl rosiglitazone, was synthesized and determined by infrared spectra. Vanadyl rosiglitazone and three other organic vanadium compounds were administered to the diabetic mice through oral administration for 5 weeks. The results of mouse model test indicated that vanadyl rosiglitazone could regulate the blood glucose level and relieve the symptoms of polydipsia, polyphagia, polyuria, and weight loss without side effects and was more effective than the other three organic vanadium compounds including vanadyl trehalose, vanadyl metformin, and vanadyl quercetin. The study indicated that vanadyl rosiglitazone presents insulin-mimetic activities, and it will be a good potential candidate for the development of a new type of oral drug for type 2 diabetes.

Anti-diabetic activity of recombinant irisin in STZ-induced insulin-deficient diabetic mice.

In order to investigate the hypoglycemic effects and potential mechanism of recombinant irisin on diabetes, STZ-induced diabetic mice were established and treated with irisin. The results showed that daily water and food intake, and blood glucose significantly decreased after various concentrations of recombinant irisin treatment by intraperitoneal injection, of which 1.0 mg/kg was the optimal dose for lowering blood glucose. However, the body weight exhibited no significant difference during the treatment within groups, although the 0.9% NaCl treated group showed a trend of decreased body weight and the irisin treated groups showed a tendency of increasing weight. The oral glucose tolerance was improved, and serum insulin and circulating irisin content were significantly elevated in diabetic mice after 1.0 mg/kg irisin-injection treatment, compared to diabetic mice treated with 0.9% NaCl. 1.0 mg/kg irisin-injection also significantly increased the expression of energy and metabolism-related genes. In addition, oral administration of irisin lowered the blood glucose in diabetic mice. Our data suggested that irisin could lower blood glucose in insulin-deficient diabetic mice, to some extent, through irisin-mediated induction of energy and metabolic genes expression. These observations laid a foundation for the development of irisin-based therapy.

Toxicology Assessment of a Dual-Function Peptide 5rolGLP-HV in Mice.

5rolGLP-HV had an ideal therapeutic potential in the prevention of hyperglycemia in type 2 diabetes and delay of the thrombosis. The objective of the study was to investigate the toxicology effects of 5rolGLP-HV and guarantee its safety. In acute toxicity test, the mice were orally receiving 5rolGLP-HV at a single dose of 300 mg/kg or 2000 mg/kg. For sub-chronic toxicity study, the mice received 5rolGLP-HV at doses of 800 mg/kg or 1600 mg/kg for 9 weeks. No significant adverse effects were evident in acute and sub-chronic toxicity tests, indicating that the LD50 value is greater than 2000 mg/kg. Although the liver and kidney exhibited a little abnormal in sub-chronic toxicity study, they could recovery to normal after withdrawal 5rolGLP-HV for 2 weeks. In micronucleus assay, the mice received 5rolGLP-HV at doses of 250, 500, or 1000 mg/kg for two consecutive days. The micronucleus numbers and the polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) ratios among 5rolGLP-HV groups were within the normal range. Similarly, sperm aberration test demonstrated that 5rolGLP-HV had no teratogenic effect on the mice sperm. In conclusion, the combined results clearly demonstrated the safety of 5rolGLP-HV and support its use as a drug to treat diabetes and thrombosis.

Construction of a Fusion Peptide 5rolGLP-HV and Analysis of its Therapeutic Effect on Type 2 Diabetes Mellitus and Thrombosis in Mice.

Glucagon-like peptide-1 (GLP-1), is currently used to treat type 2 diabetes mellitus and hirudin (HV), plays an important role in controlling thrombosis and cardiovascular diseases. This investigation aimed to develop a fusion peptide 5rolGLP-HV which combined functions of rolGLP-1 and rHV to treat diabetes and thrombosis. In this study, we constructed a fusion gene including five copies of rolGLP-1 and one copy of rHV (5rolGLP-HV). The optimum expression conditions of 5rolGLP-HV in a soluble form were 0.8 mM IPTG induction when OD600 reached 0.6-0.8 and further growing at 25 °C for 9 h. Isolated rolGLP-1 and rHV were acquired by trypsin digestion in vitro, and the concentration of them was determined by HPLC in vivo. Oral administration of 5rolGLP-HV significantly decreased the levels of blood glucose, GHbA1C, TC, and TG in diabetic mice at the time of 3 weeks compared to the saline-treated group (p < 0.05), while the insulin level was reversed significantly (p < 0.05). 5rolGLP-HV treatment significantly shortened the length of thrombus in thrombosis mice compared to the saline-treated group (p < 0.01). These results indicated that 5rolGLP-HV had dual-function in treating diabetes and preventing thrombosis.

Liver histone H3 methylation and acetylation may associate with type 2 diabetes development.

Type 2 diabetes (T2D) is a complicated systemic disease, and the exact pathogenetic molecular mechanism is unclear. Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in diabetes. In the current study, C57BL/6 J mice were used to build T2D model, then treated with exendin-4 (10 µg/kg). Histone H3K9 and H3K23 acetylation, H3K4 monomethylation and H3K9 dimethylation were explored by Western blotting of liver histone extracts. Real-time polymerase chain reaction (PCR) was used to examine expression levels of diabetes-related genes, while chromatin immunoprecipitation (ChIP) was applied to analyze H3 and H3K9 acetylation, H3K4 monomethylation, and H3K9 dimethylation in the promoter of facilitated glucose transporter member 2 (Glut2) gene. The results showed that liver's total H3K4 monomethylation and H3K9 dimethylation was increased in diabetic mice, which was abrogated with the treatment of exendin-4. In contrast, H3K9 and H3K23 acetylation were reduced in diabetic mice, while exendin-4 only alleviated the reduction of H3K9 acetylation. Our data indicated that the progression of type 2 diabetes mellitus (T2D) is associated with global liver histone H3K9 and H3K23 acetylation, H3K4 monomethylation, and H3K9 dimethylation. Exploiting exact histone modify enzyme inhibitors, which may represent a novel strategy to prevent T2D.

Codon optimization and expression of irisin in Pichia pastoris GS115.

Irisin is a novel hormone which is related to many metabolic diseases. In order to illuminate the function and therapeutic effect of irisin, gaining active irisin is necessary. In this work, a codon-optimized irisin gene was designed according to Pichia pastoris synonymous codon usage bias and cloned into the pPIC9K expression vector. Sequencing result indicating that the sequence of irisin was consistent with the modified irisin and the irisin was in frame with α-factor secretion signal ATG. The plasmid pPIC9K-irisin was transformed into GS115 P. pastoris cells through electroporation. The positive transformants were screened on MD medium and analyzed by PCR. Five recombinant GS115/pPIC9K-irisin strains were obtained, but only one strain expressed irisin successfully. SDS-PAGE and Western blot were used to assess the expression level and purity of irisin. The irisin was also simply purified and the effect of pH value, methanol concentration and induction time on the production of irisin was investigated. The results showed that the best conditions of irisin expression were as follows: pH 6.0, 2.0% methanol and induction for 96 h. This work laid the basis for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.

Expression of CTB-10×rolGLP-1 in E. coli and its therapeutic effect on type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is a short peptide that can significantly reduce blood glucose level. Recombination oral long-acting glucagon-like peptide-1 (rolGLP-1), is a GLP-1 analog generated from site-specific mutation of GLP-1. CTB is a non-toxic portion of the cholera toxin and an ideal protein antigen carrier. In this study, we firstly constructed a vector pET-22b (+)-CTB-10×rolGLP-1 to express a fusion protein composed of CTB and ten tandem repeated rolGLP-1 in BL21 (DE3) line of E. coli. The CTB-10×rolGLP-1 was expressed efficiently in the inclusion bodies. The expression product was analyzed by SDS-PAGE electrophoresis and Western blotting. The inclusion bodies were then denatured, refolded and purified by ion exchange chromatography to obtain a high-purity CTB- 10×rolGLP-1. The therapeutic effect of CTB-10×rolGLP-1 was assessed in comparison with 10×rolGLP-1 alone by daily oral-gavage administration up to 10 days in streptozotocin-induced type 2 diabetic mice. The results showed that the level of blood glucose was reduced more effectively and the oral glucose tolerance of mice was improved more significantly with the administration of CTB-10×rolGLP-1. Our results provided a potentially promising oral biological drug for the treatment of type 2 diabetes.

Liver histone H3 methylation and acetylation may associate with type 2 diabetes development

Type 2 diabetes (T2D) is a complicated systemic disease, and the exact pathogenetic molecular mechanism is unclear. Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in diabetes. In the current study, C57BL/6 J mice were used to build T2D model, then treated with exendin-4 (10 μg/kg). Histone H3K9 and H3K23 acetylation, H3K4 monomethylation and H3K9 dimethylation were explored by Western blotting of liver histone extracts. Real-time polymerase chain reaction (PCR) was used to examine expression levels of diabetes-related genes, while chromatin immunoprecipitation (ChIP) was applied to analyze H3 and H3K9 acetylation, H3K4 monomethylation, and H3K9 dimethylation in the promoter of facilitated glucose transporter member 2 (Glut2) gene. The results showed that liver’s total H3K4 monomethylation and H3K9 dimethylation was increased in diabetic mice, which was abrogated with the treatment of exendin-4. In contrast, H3K9 and H3K23 acetylation were reduced in diabetic mice, while exendin-4 only alleviated the reduction of H3K9 acetylation. Our data indicated that the progression of type 2 diabetes mellitus (T2D) is associated with global liver histone H3K9 and H3K23 acetylation, H3K4 monomethylation, and H3K9 dimethylation. Exploiting exact histone modify enzyme inhibitors, which may represent a novel strategy to prevent T2D