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.

[Transduction of heme oxygenase-1 gene to human Islet cells via adenovirus vector enhances anti-apoptosis function and insulin release].

OBJECTIVE: To investigate the effects of heme oxygenase-1 (HO-1) gene on human islets in vitro, and to explore the potential value of gene therapy in clinical islet transplantation. METHODS: Adenovirus vector carrying human HO-1 gene (Ad-HO-1) or EGPF (Ad-EGFP) were established respectively. Human cadaveric pancreases were isolated, purified, cultured, and divided into 3 groups to be transfected with Ad-HO-1, Ad-EGFP or blank vector. Human tumor necrosis factor and cyclohexamide (CHX) were added into the culture fluid of the pancreatic islets. 48 hours later the pancreatic islets were digested into single cells. Flow cytometry was used to detect the apoptosis. Glucose of the concentration of 16.7 mmol/L was added into the culture fluid of the 3 groups of islet cells. After 1-hour co-incubation radioimmunochemistry was used to detect the level of insulin in the supernatant. RESULTS: After stimulation of glucose the insulin concentration in the supernatant of the Ad-HO-1 group was 270 mIU/L +/- 89 mIU/L, significantly higher than those of the Ad-EGFP group (189 mIU/L +/- 88 mIU/L) and control group (182 mIU/L +/- 59 mIU/L, both P < 0.05). The apoptotic ratio of the Ad-HO-1 group was 63.1% +/- 10.9%, significantly lower than that of the control group (90.9% +/- 11.3%, P < 0.01) after treatment with TNFalpha and CHX. CONCLUSION: Transfection of Ad-HO-1 into human islets improves anti-apoptotic function in cultured human islets and promotes insulin release of human pancreatic islets.

[Study of the effects of immunosuppressive agents on human islet cells in vitro].

OBJECTIVE: To investigate the effects of immunosuppressive agents on insulin secretion of human islet cells in vitro. METHODS: Human islet cells were isolated by the solution of the liberase and purified by Ficoll's density gradient centrifugation and then were exposed to various concentrations of four immunosuppressive agents for 24 hr respectively. Glucose-stimulated insulin secretion during subsequent static incubation was measured using the human insulin ELISA kit. RESULTS: Glucose-stimulated insulin secretion from human islet cells was significantly reduced after exposed to high concentrations of MMF and FK506 (both P < 0.05). No significant reduction in insulin secretion was observed from human islet cells after exposed to FTY720 and rapamycin (both P > 0.05). CONCLUSION: (1) High concentrations of MMF and FK506 have deleterious effects on insulin secretion in human islet cells. Low-dose FK506 + MMF is available for clinical use. (2) FTY720 and rapamycin have no adverse effects on insulin secretion in human islet cells. FTY720 and rapamycin may become useful immunosupressants for future clinical islet allotransplantation.

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.

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.