Toward more potent imidazopyridine inhibitors of Candida albicans Bdf1: Modeling the role of structural waters in selective ligand binding.

Novel agents to treat invasive fungal infections are urgently needed because the small number of established targets in pathogenic fungi makes the existing drug repertoire particularly vulnerable to the emergence of resistant strains. Recently, we reported that Candida albicans Bdf1, a bromodomain and extra-terminal domain (BET) bromodomain with paired acetyl-lysine (AcK) binding sites (BD1 and BD2) is essential for fungal cell growth and that an imidazopyridine (1) binds to BD2 with selectivity versus both BD1 and human BET bromodomains. Bromodomain binding pockets contain a conserved array of structural waters. Molecular dynamics simulations now reveal that one water molecule is less tightly bound to BD2 than to BD1, explaining the site selectivity of 1. This insight is useful in the performance of ligand docking studies to guide design of more effective Bdf1 inhibitors, as illustrated by the design of 10 new imidazopyridine BD2 ligands 1a-j, for which experimental binding and site selectivity data are presented.

The feasibility and safety of disposable endoscope vs. conventional endoscope for upper gastrointestinal tract examination: a multicenter, randomized, parallel, non-inferiority trial.

BACKGROUND: A disposable upper gastrointestinal endoscope can effectively decrease infectious outbreaks associated with endoscope reuse. In the present study, we aimed to evaluate the feasibility and safety of a disposable endoscope for upper gastrointestinal examination. METHODS: In a prospective, randomized trial, 144 upper endoscopic procedures were allocated to either the disposable endoscope group or the conventional endoscope group. The primary outcomes were rates of excellent and good image qualities and maneuverability satisfaction. The second outcome included procedure duration, endoscopic diagnosis, and adverse events. RESULTS: A total of 144 subjects were enrolled in the present analysis and prospectively randomized to 2 study groups. Finally, 70 and 69 subjects were enrolled in the novel disposable endoscope group and the conventional endoscope group, respectively, due to the schedule cancellation of 5 subjects. The baseline characteristics of the patients were similar in both groups. The excellent and good image quality rates and maneuverability satisfaction of the novel disposable endoscope were not inferior to the conventional endoscope (p = 0.99 and p = 0.99, respectively). Moreover, no significant between-group difference was observed in the endoscopic results and adverse events (p = 0.30 and p = 1, respectively). However, the procedure duration in the novel disposable endoscope was longer compared with the conventional endoscope (8.40 ± 4.28 min vs. 5.12 ± 2.65 min, p < 0.001). CONCLUSIONS: The novel disposable endoscope was as safe, effective, and maneuverable as a conventional endoscope. However, the novel disposable endoscope was associated with a longer procedure duration.

Construction and validation of a TP53-associated immune prognostic model for gastric cancer.

Increasing evidence indicates that TP53 mutation impacts the patients' prognosis by regulating the gastric cancer (GC) immunophenotype. An immune prognostic signature (IPS) was constructed based on TP53 status. The effects of the IPS on the immune microenvironment of GC were analyzed. We also constructed a nomogram integrating the IPS and other clinical factors. An IPS was constructed in the TCGA cohort and validated in the meta-GEO cohort. TP53 mutation resulted in the downregulation of the immune response in GC. Concretely, high-risk patients were characterized by increased monocyte, macrophage M0 and T cell follicular helper infiltration; increased stromal score, ESTIMATE score and immune score; higher TIM3 and BTLA expression; and decreased dendritic cell and T cell CD4 memory-activated infiltration and tumor purity. The nomogram also showed good predictive performance. These results suggest that the IPS is an effective prognostic indicator for GC patients, which might provide a theoretical foundation for immunotherapy.

[Relationship between serum thyroxin-stimulating hormone and papillary thyroid micrcarcinoma in nodular thyroid disease].

OBJECTIVE: To explore the relationship between serum level of thyroxin-stimulating hormone (TSH) and development and progression of papillary thyroid microcarcinoma (PTMC) in nodular thyroid disease. METHODS: A total of 365 eligible patients with thyroid nodules undergoing initial thyroidectomy were enrolled, including 113 patients with PTMC diagnosed by postoperative pathology (PTMC group) and 252 patients with benign thyroid nodules (BTN group). Their clinical data were retrospectively reviewed. The serum levels of TSH in two groups and the proportion of PTMC in different serum TSH level groups in all patients were compared respectively. The relationship of preoperative serum TSH levels with tumor size and lymphatic metastasis in patients with PTMC were analyzed. RESULTS: No significant difference existed in serum TSH levels between PTMC and BTN groups (P > 0.05). The median age was younger in PTMC group than that in BTN group (Z = -2.877, P = 0.004). And the TGAb levels were higher in PTMC group than those in BTN group (Z = -2.887, P = 0.004). They were divided into 6 groups according to the serum TSH levels, and there weren't significant difference in the proportion of PTMC among those group (P > 0.05). Binary logistic regression analysis showed age was the only risk factor of PTMC (OR = 0.971, 95%CI: 0.953-0.990, P = 0.003). The serum TSH levels were positively correlated with tumor size in patients with PTMC (r = 0.218, P = 0.025). However, the proportions of lymphatic metastasis were comparable among different TSH levels groups in patients with PTMC (P > 0.05). CONCLUSION: Serum TSH is probably associated with the de novo oncogenesis of PTMC. However, serum TSH may be involved in the growth of preexisting PTMC.

[The 10-year stroke risk in hypertensive outpatients combined with diabetes in cardiovascular clinics of 36 tertiary hospitals in China].

OBJECTIVE: To assess the estimated 10-year risk of stroke among hypertensive outpatients known with diabetes from cardiovascular clinics of 36 tertiary hospitals in China and to analyze the characteristics of the risk factors and the 10-year risk of stroke between the southern and the northern patients. METHODS: A multi-center prevalence survey was conducted from October 2011 to June 2012. Hypertensive outpatients known with diabetes were enrolled from cardiovascular clinics of 36 tertiary hospitals in China. A total of 15 914 outpatients were included in the final analysis. The 10-year probability of stroke was evaluated by the Framingham stroke risk profile. According to the 10-year probability of stroke, patients were divided into low risk ( ≤ 5%), medium risk (6%∼9%) and high risk ( ≥ 10%). RESULTS: (1) Of all the hypertensive outpatients known with diabetes, the mean age was (64.6 ± 10.1) years and the mean systolic pressure was (138.7 ± 19.3) mmHg (1 mmHg = 0.133 kPa). Among them, 7.4% with atrial fibrillation, 11.2% with left ventricular hypertrophy, 57.2% with cardiovascular diseases, 17.1% smokers and 37.0% using mono-hypoglycemic agent. The southern patients who were older with more smokers had higher proportions of men and left ventricular hypertrophy, lower levels of systolic blood pressure, and lower proportions of other cardiovascular diseases than those of the northern patients ( all P < 0.05). (2) The mean 10-year probability of stroke was (20.9 ± 16.2) %. The southern patients had a higher mean 10-year probability of stroke than that of the northern patients [(22.4 ± 17.1) % vs (19.7 ± 15.2) %] (P < 0.01) . After adjusted by age and sex, the southern patients still had a higher mean 10-year probability of stroke (P < 0.05) . (3) All the patients had 7.7% with low risk, 17.4% with medium risk, and 74.9% with high risk. The southern patients had lower proportions of low and medium risk than those of the northern patients (6.7% vs 8.4%, 15.5% vs 18.9%), but had a higher proportion of high risk than that of the northern patients (77.7% vs 72.7%, all P < 0.01). CONCLUSIONS: Among the hypertensive outpatients known with diabetes from the cardiovascular clinics of our study, most of them were at the 10-year high risk of stroke. The southern patients had a higher mean 10-year probability of stroke than that of the northern patients.

Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion.

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-ß-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

Association of Thyroid-Stimulating Hormone (TSH) Levels With the Prognosis of Patients Undergoing Heart Transplantation: A Retrospective Study.

Purpose: To investigate the impact of TSH levels using a more stringent cutoff of subclinical hypothyroidism (i.e., TSH > 2.5 mIU/L) on the short-term complications and long-term prognosis in patients who underwent heart transplantation (HTx). Methods: This is a retrospective study of consecutive patients with end-stage heart failure (HF) who underwent HTx. They were divided into three groups: thyroid-stimulating hormone (TSH) ≤ 2.50 mIU/L (L-TSH), 2.50 < TSH ≤ 4.91 mIU/L (M-TSH), and TSH > 4.91 mIU/L (H-TSH). The outcomes are all-cause death and cardiogenic death. Results: There are 63 (70%) males and 27 (30%) females. Nine (10%) patients died within 1 month after surgery, including five cardiogenic deaths. By 1 year, a total of 19 patients total were dead. The survival rate in the M-TSH group was significantly higher than that of the L-TSH group (P = 0.017). After adjusted by variables of sex, age, BMI, diabetes history, hypertension history, the multivariable Cox analysis showed that body mass index (HR = 0.804, 95%CI: 0.680-0.951, P = 0.011), and L-TSH (HR = 8.757, 95%CI: 1.786-42.948, P = 0.007 vs. M-TSH), and H-TSH (HR = 6.427, 95%CI: 1.137-36.327, P = 0.035 vs. M-TSH) were independently associated with all-cause death. The multivariable Cox analysis showed that body mass index (HR = 0.703, 95%CI: 0.564-0.878, P = 0.002), and L-TSH (HR = 17.717, 95%CI: 1.907-164.607, P = 0.011 vs. M-TSH) were independently associated with cardiogenic death. Conclusion: For patients with end-stage HF undergoing HTx, low and high baseline TSH levels are independently associated with 1-year all-cause death and low baseline TSH levels with cardiogenic death.

[Sensory organization test results for maintaining an upright balance in patients with type 2 diabetes mellitus and patients with peripheral vestibular disorders].

Objective:To research the differences of sensory organization testing in maintaining postural stability between patients with type 2 diabetes mellitus and patients with peripheral vertigo using computerized posturography. Methods:Participants were divided into the control group （52 cases）, the type 2 diabetes mellitus group （T2DM） （45 cases）, and the peripheral vertigo group （PV） （47 cases）. All participants were examined under six conditions by computerized posturography: The sensory organization test, a part of computerized dynamic posturography, was used to assess the abilities of vision, somatosensory and vestibular systems in maintaining postural stability. Results:The scores of statokinesiogram （SKG） of the T2DM group in condition 1 （standing on static platform with eye open）, condition 4（standing on foam platform with eyes open） and condition 6（standing on foam platform with servo-controlled vision） were significantly greater than that in the vertigo group （P<0.01）. The visual scores in the T2DM group were lower than those of the PV group（P<0.01） in the anteroposterior and lateral directions. Conclusion:Patients with type 2 diabetes and peripheral vertigo have a decreased ability to maintain balance in the upright position. Patients with type 2 diabetes have a poorer ability to maintain balance with visual systems than patients with peripheral vertigo.

[Improvement of the function of islet beta and alpha cells by intervention against glucotoxicity: experiment with rats].

OBJECTIVE: To investigate the effects of intervention against glucotoxicity on improvement of the function and pathological changes of islet beta and alpha cells. METHODS: Thirty-six male Sprague Dawley rats were randomly divided into four equal groups: normal control (NC) group, fed with standard chow, high-fat (HF) group, fed with extra high-fat chow; diabetes mellitus (DM) control group, fed with high-fat chow for 8 weeks followed by 30 mg/kg streptozotocin injection to establish DM models; and insulin (INS) group, treated with subcutaneous injection of long-acting insulin (glargine, 0.5 U x kg(-1) x d(-1)) for 4 weeks after the establishment of DM models. 48 h, 2 weeks, and 4 weeks after the STZ injection to the 2 DM groups oral glucose tolerance test (OGTT) was performed to all rats. Peripheral blood samples were collected from the caudal vein. Serum insulin level was assayed by radioimmunoassay. Total serum cholesterol (TC) and triglyceride (TG) were measured by enzyme-colorimetric method. By the end of experiment the rats were killed with their pancreases taken out. Immunohistochemistry was used to observe the morphological changes of the islet beta and alpha cells. Beta cell and alpha cell masses were calculated by the proportions of positive area in the islet. Proinsulin mRNA level was detected by RT-PCR. Insulin protein content in islets was detected by Western blotting. RESULTS: Four weeks after the insulin intervention against glucotoxicity, the fasting blood glucose and blood glucose 2 h after sugar-taking of the INS group were both significantly lower than those of the DM group (both P < 0.01). The relative beta cell mass of the INS group was 0.38 +/- 0.08, significantly bigger, 2.45 times, that of the DM group (0.11 +/- 0.05, P < 0.01). The relative alpha cells mass in islets of the INS group was 0.16 +/- 0.04, significantly lower, by 43%, than that of the DM group (0.28 +/- 0.15, P < 0.01). The insulin contents in beta cells of the INS group was 0.58 +/- 0.03, significantly higher, by 70.6%, than that of the DM group (0.34 +/- 0.14, P < 0.01). The proinsulin mRNA level of the INS group was 1.52 +/- 0.14, significantly higher, by 20.6%, than that of the DM group. CONCLUSION: The morphology of islet beta, alpha cells in diabetic rats was improved by four weeks of Intervention against glucotoxicity improves the pathology of islet beta and alpha cells in diabetic and insulin synthesis.

[The medical cost of diabetic patients with foot problems due to peripheral artery disease].

OBJECTIVE: To investigate the medical cost of diabetic patients with foot problems and peripheral artery disease. METHODS: Type 2 diabetic patients with foot problems admitted into the endocrinology departments of 14 teaching hospitals from Jan. 1 to Dec. 31, 2004 were surveyed for their type and phase of foot ulcers, diabetic complications, medical cost and general personal characteristics. RESULTS: The average medical cost of the hospitalization of these patients was RMB yen 14,906 +/- 7072 (about US $ 1640 +/- 873); medication and examination cost was separately 56% and 19% of the total cost. There was obviously higher medical cost for these patients with longer diabetes duration of over 20 years and with the occupation of laborer and retired worker. Patients with kidney disease had significantly higher medical cost than those without (RMB yen 11 690.7 vs yen 9493.0; P = 0.0013), even if the hospital stay was nearly the same (21 days vs 20 days). The medical cost increase with the severity of diabetic foot problems based on the classification of Wagner System or Texas System. Patients with infection, ischemic foot and gangrene foot stayed in the hospitals longer and had much higher medical cost. CONCLUSION: The medical cost is higher for diabetic patients with foot problems and is related with the presence of complicating kidney disease, infection and ischemia as well as the severity of foot ulcers.

[A multicenter survey on the diabetic foot and its neuropathy in China].

OBJECTIVE: To investigate the characteristics of diabetic foot with neuropathy and its related factors. METHODS: 530 out- and in-patients in 14 grade A class 3 comprehensive hospitals in China with foot problems were surveyed. 337 of the 500 patients (63.58%) suffered from neuropathy, 172 (32.45%) with diabetic foot with simple neuropathy and 165 (31.13%) with simple neuropathy combined with peripheral artery disease (PAD). 193 of the 500 patients (36.42%) suffered from peripheral artery disease (PAD). 77.7% of ulcer were caused by physical factors. Questionnaire survey was conducted to collect the demographic data, present and past history, history of the hyperglycemia and lipid disorders, classification and phases of the foot ulcers based on Wagner' system and Texas system, characteristics of neuropathy and other diabetic complications, and relative risk factors. Detailed physical examination was performed, including 10 g nylon filament sensation examination. RESULTS: The duration of diabetic foot of the patients with simple neuropathy was 3 (1, 60) months, significantly shorter than that of the diabetic foot patients with PAD [5 (1, 96) months, P < 0.001]. The Wagner degree of ulcer was related to the duration of diabetes, economic income, foot deformity, nerve reflection, diapason vibration sensation of foot, sensation point of 10 g nylon filament, ankle/brachial index (ABI), foot artery pulse, fasting blood sugar (FBS) and glycated hemoglobin (HbA1c). Stepwise regression analysis revealed that ABI of left posterior tibial artery, vibration detection threshold and economic income were the most significant influencing factors of the degree of ulcer. CONCLUSION: Neuropathy ulcer is common in diabetic foot patients. The prognosis of healing in diabetic foot with neuropathy is prior to that of diabetic foot with PAD. The neuropathy and PAD of foot influence each other and aggravate the condition of diabetic foot. The examinations of diapason vibration sensation of foot, sensation point of 10 g nylon filament, and Achilles tendon reflex are simple and practical, and are worth recommending.

[Differences in risk factors of diabetic foot in the patients in South and North China].

OBJECTIVE: To investigate the differences on the diabetic foot problems and its risk factors in south and north of China. METHODS: Patients with foot problems were surveyed from January 1 to December 31, 2004 in 14 teaching hospitals located in different cities in China, including demographic data, present and past history of the foot problems and peripheral artery disease (PAD), the classification of the foot ulcers based on the Wagner' system, control of the hyperglycemia and lipids disorder, medical cost in hospital and the diabetic complications. All the patients were divided into two groups due to their geographical data, south and north. RESULTS: There were 285 and 349 patients for the group south and group north. No significant differences were found for duration of diabetes or foot problems, fasting or post-meal glucose, total cholesterol, triglycerides, HDL-C, and the numbers of patients with smoke, hypertension, nephropathy or neuropathy between the two groups. There were significant differences for the age (70 yrs vs 66 yrs), percentage of the patients with average person income with over RMB 1000 per month (57.7% vs 45.6%), coronary heart disease (42.6% vs 61.0%) and retinopathy (35.7% vs 49.5%), HbA1c (7.90% vs 8.80 %), LDL-C (2.75 mmol/L vs 2.98 mmol/L), WBC (6.70 x 10(9) vs 7.40 x 10(9)/L), HCT (0.37 vs 0.38), creatinine (87 micromol/L vs 76 micromol/L) and uric acid (333 mmol/L vs 271 mmol/L), and amputation rate (2.6% vs 9.7%) between south and north groups. Logistic analysis showed that severity of the foot problems was associated with ABI and WBC in south group, and with ABI, PLT and HCT in north group. CONCLUSION: Diabetic foot problems were more severe, with more risk factors and with more medical cost in north patients.

Selective BET bromodomain inhibition as an antifungal therapeutic strategy.

Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.

Acute intravenous glucose load impairs early insulin secretion and insulin content in islet ß cells in mice.

AIMS: To investigate insulin secretion and content in islet ß cells after intravenous glucose load in mice. MATERIALS AND METHODS: Acute hyperglycemia (≥16.7 mmol/L) in C57BL/J6 mice was achieved by hyperglycemic clamp. Mice were divided into four groups: a 2-hour and a 4-hour high glucose-infusion (2 h-HG and 4 h-HG) with 25% dextrose groups and control groups with saline infusion of the same duration. Insulin levels and response were measured using intraperitoneal glucose tolerance test (IPGTT) in mice and glucose-stimulated insulin secretion (GSIS) for isolated islets after overnight culture. Immunohistochemistry and electron microscopy (EM) for islet ß cells were used after the hyperglycemic clamp to study morphologic changes of insulin granules and to assess the impact of acute glucose load on islet histology. KEY FINDINGS: Blood glucose at 15, 30, 60 and 120 min was significantly higher in 4 h-HG compared with the other groups. Serum plasma insulin significantly decreased only at 15 min as a first-phase insulin response (FPIR). Insulin secretion at 2.8 and 16.7 mmol/L glucose stimulus in 4 h-HG group decreased 77% and 64% more than those in 2 h-HG, respectively (P<0.05). Similarly, residual insulin content in islet ß cells after 2.8 and 16.7 mmol/L glucose challenge decreased 30% and 43% more than those in 2 h-HG, respectively (P<0.05). EM showed decreased insulin granules in islet cells and swollen mitochondria only in 4 h-HG. SIGNIFICANCE: Short time intravenous glucose load blunted FPIRs and decreased insulin content of islet ß cells.

[Analysis and evaluation of the balance function in patients with type 2 diabetes].

OBJECTIVE: Evaluation and analyze the characteristics of balance function in patients with type 2 diabetes, and to find out the importance of proprioception, vision and vestibular in postural control. METHOD: All subjects were divided into two groups, 37 normal individuals, 33 patients with type 2 diabetes mellitus. All were assessed by computerized posturography under six upright stance.conditions: including standing on the firm surface and foam with eyes open and closed. RESULT: (1) On anteroposterior,the scores of proprioception, vision and vestibular were 93.96 ± 7.95, 80.22 ± 16.24, 70.87 ± 20.99, the normal were 98.00 ± 2.18, 91.44 ± 6.01, 80.44 ± 7.81. There were significances between diabetes mellitus group and normal control group (P < 0.05) respectively. (2) On lateral, the scores of vision and vestibular were 80.39 ± 12.60, 73.96 ± 16.04, and the normal were 92.11 ± 4.50, 83.18 ± 9.45. There were significances with P < 0.05 between diabetes mellitus group and normal control group. However, there was no obvious difference in proprioception scores between the two groups. (3) The limit of stability of normal group were (176.47 ± 44.13) mm²; diabetic group was (143.13 ± 62.30) mm². There was statistical significance between the group with P < 0.05. (In diabetic patients, there was no significant difference between the no dizziness group and the dizziness group of the scores of proprioceptive, visual, vestibular as well as stable limits, P > 0.05. CONCLUSION: The balance function of patients with type 2 diabetes decreased. It is the main characteristic that the vision and vestibular decreased more significantly in the postural control.

Moderate calorie restriction to achieve normal weight reverses ß-cell dysfunction in diet-induced obese mice: involvement of autophagy.

BACKGROUND: Severe calorie restriction (CR) is shown to improve or even reverse ß-cell dysfunction in patients with obesity and type 2 diabetes mellitus. However, whether mild to moderate CR can reverse ß-cell dysfunction induced by obesity and the underlying mechanism remain unclear. Autophagy plays an important role in maintaining mass, architecture and function of ß-cells. While the impact of CR on ß-cell autophagy is unknown. This study aims to investigate the effects of moderate CR on ß-cell function and autophagy activity in diet-induced obese (DIO) mice. METHODS: DIO C57BL/6 mice were subjected to 3 weeks of switching to normal chow (HF → NC group) or normal chow with 40 % CR (HF → NC CR group). Then hematoxylin-eosin and immunohistochemistry staining were performed to observe ß-cell morphology. ß-cell function was evaluated by intraperitoneal glucose tolerance test in vivo and static GSIS (glucose-stimulated insulin secretion) in isolated islets. ß-cell autophagy activity was determined by transmission electron microscope and western blot. RESULTS: In the HF → NC CR group, CR normalized body weights, completely restored glucose tolerance, early-phase and second-phase insulin secretion, insulin sensitivity, and islet size. CR also normalized insulin content and glucose-stimulated insulin secretion in isolated islets in vitro. Furthermore, ß-cell autophagy level was increased in the HF → NC CR group, but AMPK phosphorylation remained unchanged. Although HF → NC mice achieved moderate weight loss and normal glucose tolerance, their insulin secretion was not improved compared with obese control mice, and additionally, ß-cell autophagy was not activated in these mice. CONCLUSIONS: Moderate (40 %) CR to achieve normal weight reversed ß-cell dysfunction and insulin resistance, and restored glucose homeostasis in DIO mice. Furthermore, the up-regulation of ß-cell autophagy may play a role in this process, independent of AMPK activation.

Effects of timely insulin treatment on protection of beta cells in a rat model of type 2 diabetes mellitus.

BACKGROUND: Insulin treatment plays a key role in management of diabetes mellitus. Clinical researches showed that extra improvements in restoration of insulin secretion of pancreatic beta cells were found in patients with newly diagnosed type 2 diabetes. The purpose of this study was to investigate the effects of early insulin treatment on insulin mRNA expression and morphological alterations of beta cells in a Sprague Dawley (SD) rat model of type 2 diabetes. METHODS: A rat model of type 2 diabetes mellitus (T2DM) was induced by a high fat diet (high energy, HE) and low doses of streptozotoxin (STZ, 40 mg/kg). A group of diabetic rats was then injected with protamine zinc insulin [PZI, 1 - 2 U x kg(-1) x d(-1)] for one week. Insulin mRNA expression, morphological features of pancreatic islets, and metabolic parameters were examined in rats using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and other techniques. RESULTS: In insulin-treated diabetic rats, insulin mRNA levels prominently increased by 81.3% (P < 0.05), as compared with untreated diabetic rats. Moreover, timely insulin treatment noticeably improved the insulin content of beta cells, with an increase of 10.2% (P < 0.05), despite a slight reduction in fasting blood glucose (FBG), triglyceride (TG), and free fatty acid (FFA) levels, as compared to an untreated diabetic group. CONCLUSION: Insulin treatment at the onset of T2DM effectively improves insulin synthesis, as confirmed by morphological changes to beta cells in a rat model of type 2 diabetes.

Inhibition effect of enteropeptidase on RANKL-RANK signalling by cleavage of RANK.

Enteropeptidase can cleave trypsinogen on the sequence of Asp-Asp-Asp-Asp-Lys and plays an important role in food digestion. The RANKL-RANK signalling pathway plays a pivotal role in bone remodelling. In this study, we reported that enteropeptidase can inhibit the RANKL-RANK signalling pathway through the cleavage of RANK. A surrogate peptide blocking assay indicated that enteropeptidase could specifically cleave RANK on the sequence NEEDK. Osteoclast differentiation assay and NF-κB activity assay confirmed that enteropeptidase could inhibit osteoclastogenesis in vitro through the cleavage of RANK. This is the first study to prove that the RANKL-RANK signalling pathway can be inhibited by cleavage of RANK instead of targeting RANKL.

Suppression of NADPH oxidase 2 substantially restores glucose-induced dysfunction of pancreatic NIT-1 cells.

Defects in insulin secretion by pancreatic cells and/or decreased sensitivity of target tissues to insulin action are the key features of type 2 diabetes. It has been shown that excessive generation of reactive oxygen species (ROS) is linked to glucose-induced ß-cell dysfunction. However, cellular mechanisms involved in ROS generation in ß-cells and the link between ROS and glucose-induced ß-cell dysfunction are poorly understood. Here, we demonstrate a key role of NADPH oxidase 2 (NOX2)-derived ROS in the deterioration of ß-cell function induced by a high concentration of glucose. Sprague-Dawley rats were fed a high-fat diet for 24 weeks to induce diabetes. Diabetic rats showed increased glucose levels and elevated ROS generation in blood, but decreased insulin content in pancreatic ß-cells. In vitro, increased ROS levels in pancreatic NIT-1 cells exposed to high concentrations of glucose (33.3 mmol·L(-1)) were associated with elevated expression of NOX2. Importantly, decreased glucose-induced insulin expression and secretion in NIT-1 cells could be rescued via siRNA-mediated NOX2 reduction. Furthermore, high glucose concentrations led to apoptosis of ß-cells by activation of p38MAPK and p53, and dysfunction of ß-cells through phosphatase and tensih homolog (PTEN)-dependent Jun N-terminal kinase (JNK) activation and protein kinase B (AKT/PKB) inhibition, which induced the translocation of forkhead box O1 and pancreatic duodenal homeobox-1, followed by reduced insulin expression and secretion. In conclusion, NOX2-derived ROS could play a critical role in high glucose-induced ß-cell dysfunction through PTEN-dependent JNK activation and AKT inhibition.