Effect of Akkermansia muciniphila on pancreatic islet ß-cell function in rats with prediabetes mellitus induced by a high-fat diet.

Prediabetes is an important stage in the development of diabetes. It is necessary to find a safe, effective and sustainable way to delay and reverse the progression of prediabetes. Akkermansia muciniphila (A. muciniphila) is one of the key bacteria associated with glucose metabolism. Recent studies mainly focus on the effect of A. muciniphila on obesity and insulin resistance, but there is no research on the effect of A. muciniphila on pancreatic ß-cell function and its mechanism in prediabetes. In this study, we investigated the effects of A. muciniphila on ß-cell function, apoptosis and differentiation, as well as its effects on the gut microbiome, intestinal barrier, metaflammation and the expression of Toll-like receptors (TLRs) in a high-fat diet (HFD)-induced prediabetic rat model. The effect of A. muciniphila was compared with dietary intervention. The results showed both A. muciniphila treatment and dietary intervention can reduce metaflammation by repairing the intestinal barrier in rats with prediabetes induced by an HFD and improve ß-cell secretory function, apoptosis and differentiation through signaling pathways mediated by TLR2 and TLR4. Additionally, A. muciniphila can further elevate ß-cell secretion, attenuate apoptosis and improve differentiation and the TLR signaling pathway on the basis of diet.

Unraveling the rate-limiting step in microorganisms' mediation of denitrification and phosphorus absorption/transport processes in a highly regulated river-lake system.

River-lake ecosystems are indispensable hubs for water transfers and flow regulation engineering, which have frequent and complex artificial hydrological regulation processes, and the water quality is often unstable. Microorganisms usually affect these systems by driving the nutrient cycling process. Thus, understanding the key biochemical rate-limiting steps under highly regulated conditions was critical for the water quality stability of river-lake ecosystems. This study investigated how the key microorganisms and genes involving nitrogen and phosphorus cycling contributed to the stability of water by combining 16S rRNA and metagenomic sequencing using the Dongping river-lake system as the case study. The results showed that nitrogen and phosphorus concentrations were significantly lower in lake zones than in river inflow and outflow zones (p < 0.05). Pseudomonas, Acinetobacter, and Microbacterium were the key microorganisms associated with nitrate and phosphate removal. These microorganisms contributed to key genes that promote denitrification (nirB/narG/narH/nasA) and phosphorus absorption and transport (pstA/pstB/pstC/pstS). Partial least squares path modeling (PLS-PM) revealed that environmental factors (especially flow velocity and COD concentration) have a significant negative effect on the key microbial abundance (p < 0.001). Our study provides theoretical support for the effective management and protection of water transfer and the regulation function of the river-lake system.

Compatibilizing Biodegradable Poly(lactic acid)/polybutylene adipate-co-terephthalate Blends via Reactive Graphene Oxide for Screw-Based 3D Printing.

Vinyl-functionalized graphene oxide (VGO) was used as a reactive compatibilizer to prepare poly(lactic acid)/polybutylene adipate-co-terephthalate (PLA/PBAT) blends. The linear rheological and scanning electron microscopy results confirmed that the VGO nanosheets were quite efficient in compatibilizing PLA/PBAT blends. The size of the PBAT dispersed phase was remarkably decreased in the presence of VGO nanosheets. Moreover, the VGO nanosheets exhibited strong nucleating effects on the crystallization process of PLA. The crystallinity of PLA component in the compatibilized blend with various VGO nanosheets was higher than 40%, upon the cooling rate of 20 °C/min. The prepared PLA/PBAT pellets were applied to 3D printing, using a self-developed screw-based 3D printer. The results showed that all the prepared PLA/PBAT blend pellets can be 3D printed successfully. The notched Izod impact test results showed that, in the presence of VGO, an increase of at least 142% in impact strength was achieved for PLA/PBAT blend. This could be attributed to the compatibilizing effect of the VGO nanosheets. Thus, this work provides a novel way to prepare tough PLA-based materials for 3D printing.

Pinocembrin alleviates chronic morphine-induced analgesic tolerance and hyperalgesia by inhibiting microglial activation.

BACKGROUND: Chronic opioid induced analgesic tolerance is a major obstacle in pain management. Microglial activation is involved in morphine tolerance and pinocembrin suppresses microglial activation in several disease models. We aim to investigate whether and how pinocembrin alleviates morphine tolerance. METHODS: We induced chronic morphine tolerance in mice by daily morphine injection, with some mice receiving pinocembrin. Analgesic tolerance and hyperalgesia were determined by behavioral assays. The effects of pinocembrin on morphine induced microglial activation, neuroinflammation, and STAT3 activation were determined by Iba1 immunostaining, Il1b and Tnfa mRNA levels and STAT3 phosphorylation. Finally, the effects of STAT3 inhibition on chronic morphine tolerance were assessed. RESULTS: We show that pinocembrin not only suppressed but also reversed preexisting chronic morphine tolerance and hyperalgesia. We found that chronic administration of morphine lead to microglial activation and neuroinflammation, which were suppressed by pinocembrin. Our results reveal a strong connection of STAT3 with morphine tolerance and pinocembrin suppressed morphine-induced STAT3 activation both in vivo and in BV2 cells. Finally, we show that STAT3 inhibition is sufficient to suppress morphine tolerance and hyperalgesia. CONCLUSION: Our study suggests that pinocembrin effectively prevents and alleviates chronic morphine tolerance through inhibition of STAT3 mediated microglia activation and neuroinflammation.

Microstructure, Microhardness and Tribological Properties of Bronze-Steel Bimetallic Composite Produced by Vacuum Diffusion Welding.

In this paper, a lead-bronze/steel bimetal composite was produced by vacuum diffusion welding technology. The microstructure, hardness and tribological properties under the dry sliding condition of the bimetal structured material were investigated and compared with two reference samples, i.e., lead-bronze and Mn/Si-brass. The wear mechanism of the three materials was also analyzed in detail. It was found that the bimetallic structure possessed the best wear resistance among the three samples. When paired with the ball bearing steel, the wear rates of the lead-bronze and Mn/Si-brass were 13 and 54 times higher than that of the bimetal composite. When paired with bearing steel, the wear rates of the two materials were 13 and 54 times higher than the bimetallic composite, respectively. This is because the steel layer served as a bearing layer to decrease the plastic deformation of the bronze layer. Furthermore, the lead can accelerate the formation of a dense hardened layer at the sliding interfaces to avoid subsequent wear of the bronze surface. Nevertheless, this hardened layer caused severe scuffing on the steel balls. Therefore, lead-bronze/steel structured material is recommended to match with hard counterface material, such as cemented carbide.

Factors associated with progression of different prediabetic status to Diabetes: A Community-based cohort study.

AIMS: To examine the predictive factors associated with the progression of different prediabetic status to diabetes. METHODS: A two-year retrospective cohort study was conducted on 5741 participants aged 40 years or older. Finally, 1685 participants with prediabetes defined by IFG (impaired fasting glucose), IGT (impaired glucose tolerance) and CGI (combined IFG and IGT) were included. Logistic regression model was used to evaluate the risk of prediabetes progression to diabetes. RESULTS: Of the 1685 subjects with prediabetes at baseline, 212 (12.6%) subjects progressed to diabetes and 1473 (87.4%) subjects did not. Logistic regression analysis demonstrated that people with CGI were associated with an increased risk of progressing to diabetes compared to those with IFG (OR, 95% CI: 3.127, 2.047-4.776). Moreover, males, obese people, people with increased BMI and WHR (Waist/ Hip ratio), and hypertension were positively associated with the progression to diabetes, while HOMA-ß was negatively associated with the progression to diabetes. CONCLUSIONS: Subjects with CGI are prone to progressed to diabetes compared to those with IFG or IGT in middle-aged and older person in China. More attention should be paid to male and obese prediabetic subjects, and measures should be taken to control the increase in their BMI and WHR.

High Concentration of Dezocine Induces Immune Escape of Lung Cancer and Promotes Glucose Metabolism through Up-Regulating PD-L1 and Activating the NF-κB Pathway.

BACKGROUND: Dezocine is an opioid analgesic that can affect the immune system. Here, we explored the synergy of high concentration of Dezocine and programmed death-ligand 1 (PD-L1) with regards to immune escape and glucose metabolism in lung cancer (LC). METHODS: PD-L1 level in human LC cell lines was determined and the influence of Dezocine at different concentrations for the proliferation of LC cells was identified. Next, LC cells were transfected to alter PD-L1 level, and exposed to Dezocine at 8 µg/mL to explore their effects on cell proliferation, production of interferon-γ (IFN-γ), contents of glucose, lactate, and NADPH/NADP+, and activation of the nuclear factor-κB (NF-κB) pathway. RESULTS: PD-L1 level was increased in LC cells and Dezocine (8 µg/mL) impaired the proliferation of LC cells. Down-regulating PD-L1 inhibited cell proliferation, enhanced production of IFN-γ, and reduced the contents of glucose, lactate, and NADPH/NADP+, while up-regulating PD-L1 caused the opposite results. Dezocine (8 µg/mL) induced immune escape and glucose metabolism in LC, and Dezocine-induced effects were reversed by down-regulating PD-L1. Dezocine (8 µg/mL) up-regulated PD-L1 by activating the NF-κB pathway. CONCLUSION: Dezocine at 8 µg/mL promotes immune escape and glucose metabolism in LC through up-regulating PD-L1 and activating the NF-κB pathway.

Prevalence and risk factors of diabetes mellitus: a community-based sectional survey.

BACKGROUND: The prevalence of diabetes in China has increased by nearly 18 times from 0.67% in 1980 to 12.8% in 2020. The incidence has occurred with regional diversity, following different climates, diet, and lifestyle. This study aimed to explore the glucose metabolism status and analyze the risk factors for diabetes among over 45-year-old inhabitants from the Shanghai Songjiang district. METHODS: A total of 1,213 subjects without diabetes history, thyroid dysfunction history, or other diagnosed diseases were enrolled in this cross-sectional study, all of whom were over 45 years old and from the Shanghai Songjiang district. All subjects participated in a complete physical examination and clinical history collection including name, gender, age, history of drinking and smoking, and presence of other disease. Fasting glucose, postprandial glucose, biochemical, and other metabolic parameters were measured in all subjects. RESULTS: According to the WHO [1990] Standard, the normal glucose regulation (NGR), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), combined glucose impaired (CGI), and newly diagnosis diabetes mellitus (NDDM) were defined and grouped. Among 1,213 subjects in this area, 50.2% had abnormal glucose, including 36.5% with pre-diabetes (PD) and 13.7% with NDDM. We substantiated that hyper-glucose was positively associated with age, body mass index, waist-hip ratio, fat mass, fat percentage, total triglycerides, total cholesterol, low-density lipoprotein, systolic blood pressure, and heart rate in this study. We also found the prevalence of hyper-glucose occurred mainly in women in the 56-60-year-old age group and in men in the 61-65-year-old age group in this area. The overweight rate of male subjects with abnormal glucose was 61.9%, while in females this was 56.3%, especially the central obesity ratio, which reached 91.4% among the female hyper-glucose subjects. CONCLUSIONS: In the Shanghai Songjiang district, 56-60-year-old female subjects who were overweight and with central obesity were apt to abnormal glucose regulation. Community-based diabetes management is an important approach for screening diabetes and high-risk factors to reduce the risk and financial burden of individuals and the society.

Preparation of high-expansion open-cell polylactic acid foam with superior oil-water separation performance.

Environmentally friendly and non-toxic polylactic acid (PLA) foam has shown great application prospects in heat preservation, adsorption and other fields. However, it is still challenging to prepare high-expansion PLA foam. Herein, a cooling batch foaming process under supercritical CO2 (sc-CO2), based on pre-melted non-crystalline state, was proposed to prepare PLA foams with high expansion ratio. The CO2 dissolved in the polymer melt will lower the crystallization temperature of PLA. Due to the lack of crystallization, the foaming temperature of PLA can be reduced, which increases the CO2 saturation and helps foam. When foaming is triggered before crystallization, ultrahigh expansion foam can be produced. Based on pre-melting treatment, the maximum expansion ratio of PLA has reached 59.7-fold. At the same time, an open-pore structure is produced by this method, which can selectively absorb oil from water. In addition, the adsorption capacity of CCl4 reaches 15 g/g, and there is no significant attenuation in 20 adsorption-desorption cycles. This work provides a green, solvent-free method to prepare biodegradable oil-adsorbing foam.

Circular RNA hsa_circ_0102231 sponges miR-145 to promote non-small cell lung cancer cell proliferation by up-regulating the expression of RBBP4.

Circular RNAs (circRNAs) are important regulators in various cancers. Previous studies have found that hsa_circ_0102231 is an oncogene in lung adenocarcinoma. Here, we investigated its mechanism in the development of non-small cell lung cancer (NSCLC). We detected the levels of hsa_circ_0102231 in five NSCLC cell lines and one normal bronchial epithelium cell line. The interaction between hsa_circ_0102231 and miR-145 was predicted and confirmed by pull-down and luciferase assays. The nuclear mass separation assay and fluorescence in situ hybridization were used to detect the distribution of hsa_circ_0102231. Cell Counting Kit-8 and Transwell assays were used to assess the cell proliferative and invasive ability. Western blot and RT-qPCR, respectively, detected the protein and mRNA levels of RBBP4. The RBBP4 promoter activity was detected with a luciferase assay. We found that hsa_circ_0102231 level was higher in NSCLC cells. hsa_circ_0102231 is mainly localized to the cytoplasm. hsa_circ_0102231 promotes NSCLC cell proliferation and invasion by sponge for miR-145. miR-145 significantly decreases the RBBP4 promoter activity, and its mRNA and protein levels. RBBP4 is an oncogene to promote proliferation and invasion ability. Our findings suggest that hsa_circ_0102231 promotes proliferation and invasion by mediating the miR-145/RBBP4 axis in NSCLC, indicating that it might be a potential target for NSCLC treatment.

Gut bacteria selectively promoted by dietary fibers alleviate type 2 diabetes.

The gut microbiota benefits humans via short-chain fatty acid (SCFA) production from carbohydrate fermentation, and deficiency in SCFA production is associated with type 2 diabetes mellitus (T2DM). We conducted a randomized clinical study of specifically designed isoenergetic diets, together with fecal shotgun metagenomics, to show that a select group of SCFA-producing strains was promoted by dietary fibers and that most other potential producers were either diminished or unchanged in patients with T2DM. When the fiber-promoted SCFA producers were present in greater diversity and abundance, participants had better improvement in hemoglobin A1c levels, partly via increased glucagon-like peptide-1 production. Promotion of these positive responders diminished producers of metabolically detrimental compounds such as indole and hydrogen sulfide. Targeted restoration of these SCFA producers may present a novel ecological approach for managing T2DM.

Dysbiosis of Gut Microbiota Associated with Clinical Parameters in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. Gut microbiota has been implicated to play a critical role in metabolic diseases and may modulate the secretion of mediators of the brain-gut axis. Interaction between gut microbiota and the endocrine and biochemical disturbances in PCOS still remains elusive. Here, we showed an altered gut microbiota significantly correlated with PCOS phenotype. There were 33 patients with PCOS (non-obese PCOS individuals, PN, n = 12; obese PCOS individuals, PO, n = 21) as well as 15 control subjects (non-obese control individuals, CN, n = 9; obese control individuals, CO, n = 6) enrolled in our study. The plasma levels of serotonin, ghrelin, and peptide YY (PYY) were significantly decreased in patients with PCOS compared with controls, and have a significantly negative correlation with waist circumference and testosterone. Sequencing of the V3-V4 region of the 16S rRNA gene in fecal samples revealed the substantial differences of gut microbial species between the PCOS and non-obese controls. Bacterial species were clustered into 23 co-abundance groups (CAGs) based on the SparCC correlation coefficients of their relative abundance. The CAGs increased in PCOS, including the bacteria belonging to Bacteroides, Escherichia/Shigella and Streptococcus, were negatively correlated with ghrelin, and positively correlated with testosterone and BMI. Furthermore, the CAGs that were decreased in PCOS, including the bacteria from Akkermansia and Ruminococcaceae, showed opposite relationship with body-weight, sex-hormone, and brain-gut peptides. In conclusion, gut microbial dysbiosis in women with PCOS is associated with the disease phenotypes.

Effects of yoga training in patients with chronic obstructive pulmonary disease: a systematic review and meta-analysis.

INTRODUCTION: Currently, several studies have assessed the effect of yoga training on the management of chronic obstructive pulmonary disease (COPD), but these studies involved a wide variation of sample and convey inconclusive results. Hence, the present study was performed a systematic review and meta-analysis to investigate the efficacy of yoga training in COPD patients. METHODS: PubMed, EMBASE, the Cochrane Library, Google Scholar, and ClinicalTrials.gov databases were searched for relevant studies. The primary outcomes were forced expiratory volume in one second (FEV1), FEV1% predicted (% pred). Secondary outcomes included 6-min walking distance (6 MWD), arterial oxygen tension (PaO2), and arterial carbon dioxide tension (PaCO2). Weighted mean differences (WMDs) and 95% confidence intervals (CIs) were calculated, and heterogeneity was assessed with the I(2) test. RESULTS: Five randomized controlled trials (RCTs) involving 233 patients fulfilled the inclusion criteria. Yoga training significantly improved FEV1 (WMD: 123.57 mL, 95% CI: 4.12-243, P=0.04), FEV1% pred (WMD: 3.90%, 95% CI: 2.27-5.54, P<0.00001), and 6 MWD (WMD: 38.84 m, 95% CI: 15.52-62.16, P=0.001). However, yoga training had no significant effects on PaO2 (WMD: 1.29 mmHg, 95% CI: -1.21-3.78, P=0.31) and PaCO2 (WMD: -0.76 mmHg, 95% CI: -2.06-0.53, P=0.25). CONCLUSIONS: The current limited evidence suggested that yoga training has a positive effect on improving lung function and exercise capacity and could be used as an adjunct pulmonary rehabilitation program in COPD patients. However, further studies are needed to substantiate our preliminary findings and to investigate the long-term effects of yoga training.

Construction of the N1-C3 linkage stereogenic centers by catalytic asymmetric amination reaction of 3-bromooxindoles with indolines.

The catalytic asymmetric amination reaction of 3-bromooxindoles with indolines for the construction of the N1-C3 linkage stereogenic centers has been realized for the first time. Moreover, the racemic substrates (3-substituted indolines) were also applicable under the same chiral conditions. The newly developed method conveniently led to a formal synthesis of (+)-psychotrimine.

Efficient construction of highly functionalized spiro[γ-butyrolactone-pyrrolidin-3,3'-oxindole] tricyclic skeletons via an organocatalytic 1,3-dipolar cycloaddition.

Highly functionalized spiro[γ-butyrolactone-pyrrolidin-3,3'-oxindole] tricyclic skeletons were delivered successfully with high optical purity using an effective yet simple procedure.

Protective effect of α-lipoic acid on islet cells co-cultured with 3T3L1 adipocytes.

Obesity and ß-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant α-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-ß and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.

Brain-selective kinase 2 (BRSK2) phosphorylation on PCTAIRE1 negatively regulates glucose-stimulated insulin secretion in pancreatic ß-cells.

Brain-selective kinase 2 (BRSK2) has been shown to play an essential role in neuronal polarization. In the present study, we show that BRSK2 is also abundantly expressed in pancreatic islets and MIN6 ß-cell line. Yeast two-hybrid screening, GST fusion protein pull-down, and co-immunoprecipitation assays reveal that BRSK2 interacts with CDK-related protein kinase PCTAIRE1, a kinase involved in neurite outgrowth and neurotransmitter release. In MIN6 cells, BRSK2 co-localizes with PCTAIRE1 in the cytoplasm and phosphorylates one of its serine residues, Ser-12. Phosphorylation of PCTAIRE1 by BRSK2 reduces glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Conversely, knockdown of BRSK2 by siRNA increases serum insulin levels in mice. Our results reveal a novel function of BRSK2 in the regulation of GSIS in ß-cells via a PCTAIRE1-dependent mechanism and suggest that BRSK2 is an attractive target for developing novel diabetic drugs.

[3T3L1 adipocytes induce rat beta-cell dysfunction].

OBJECTIVE: To investigate the integrated effects of adipocytes on rat beta-cells, differentiated 3T3L1 adipocytes and rat islet cells co-culture system was established. METHODS: There were two groups: control group (SD rat islet cells) and co-culture group (islet cells and 3T3L1 adipocytes coculture system). Islet cells were obtained for determination of (1) insulin secretion and insulin content; (2) mRNA expressions of GLUT2, GCK and Kir6.2; (3) protein expressions of IR-beta, IRS-1 and their tyrosine phosphorylation level. RESULTS: (1) At low glucose, insulin secretion of co-culture group increased compared with that of control group (0.79 +/- 0.35) ng x h(-1) x ml(-1) islet vs. (0.38 +/- 0.09) ng x h(-1) x ml(-1) x islet, P = 0.028. At high glucose, insulin secretion of those two groups was almost at the same level (P = 0.760). Compared with control group (2.84 +/- 0.92), stimulation index (SI, insulin release at high glucose/ low glucose) of co-culture system decreased to (1.57 +/- 0.61, P = 0.04). And the insulin content of the both groups was almost at the same level (P = 0.102). (2) The mRNA of GCK, GLUT2 and Kir6.2 in co-culture group downregulated to (0.27 +/- 0.11, P = 0.01), (0.34 +/- 0.24, P = 0.009) and (0.41 +/- 0.09, P = 0.003) compared with control group (mRNA = 1). (3) The protein levels of IR-beta, IRS-1 and their tyrosine phosphorylation decreased in co-culture system. CONCLUSIONS: 3T3L1 adipocytes are involved in beta-cell dysfunction, which may facilitate the development of type 2 diabetes. The effects may be mediated by multiple pathways, which include downregulation of GSIS related gene expressions and suppression of islet cell insulin signaling.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture.

AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05). CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Protection of human islets from induction of apoptosis and improved islet function with HO-1 gene transduction.

BACKGROUND: Islet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation. METHODS: Cadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours. RESULTS: Adenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05). CONCLUSIONS: Transduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.