Hye Seung Jung

Loss of Autophagy Diminishes Pancreatic β Cell Mass and Function with Resultant Hyperglycemia

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.

Diabetes-free survival in patients who underwent islet autotransplantation after 50% to 60% distal partial pancreatectomy for benign pancreatic tumors.

Background Several retrospective studies with short-term follow-up have demonstrated a low rate of new-onset diabetes after distal pancreatectomy for benign pancreatic tumors. We sought to determine the long-term diabetes-free survival of patients who underwent islet autotransplantation (IAT) after distal pancreatectomy and to identify any associations between the isolation parameters of autologous islets and diabetes-free survival. Methods Among the 37 nondiabetic patients who underwent 50% to 60% partial pancreatectomy, 20 underwent IAT (IAT group; median follow-up period, 61 months). In the IAT group, diabetes-free survival was determined based on annual oral glucose tolerance tests, fasting blood glucose, and hemoglobin A1C. Results The 7-year diabetes-free survival rate was 51% in the IAT group (median follow-up period, 61 months) and 45% in the 37 study subjects. Diabetes-free survival was significantly prolonged when islet yield per gram of pancreas weight was more than 5154 islet equivalents (IEQ)/g, even in patients with prediabetes and high insulin resistance who had a markedly high rate of diabetes development. The proportion of patients with impaired glucose tolerance at 2 years after distal pancreatectomy was 12 of 16 in the control group, 6 of 7 in patients with islet yields of less than 5154 IEQ/g, and 3 of 11 in patients with islet yields of more than 5154 IEQ/g (P=0.019). Conclusions Partial (50%–60%) pancreatectomy for benign pancreatic tumors had a major metabolic consequence, especially in patients with prediabetes and high insulin resistance. In this setting, prolonged diabetes-free survival was observed in patients who underwent IAT when a high islet yield per gram of pancreas was achieved.

A better yield of islet cell mass from living pancreatic donors compared with cadaveric donors

Abstract:u2002 Studies in rats have shown that brain death decreases β‐cell function and causes islet cell death during islet isolation and transplantation. Because a direct comparison of human islet cells between living and cadaveric donors has not been reported to date, we studied the effects of brain death on islet cell yield. A total of 36 pancreas specimens from 20 living donors and 16 cadaveric donors were used for analysis. Islets were isolated with a Ricordi chamber, and counted as equivalent islet numbers (EIN). Living donors were predominantly female, and cadaveric donors were mainly male. Although the cold ischemic time, pancreas distensibility and digestion time were not different, islet yield was observed to be higher in living donors compared with cadaveric donors (5800u2003±u20033500 vs. 1900u2003±u20032000u2003EIN/g pancreas). Islet isolation success rates (when defined as more than 2000u2003EIN/g) were 94.1% and 42.9%, respectively. Post‐Ficoll islet recovery rates and purity were also better in living donors. However, islet viability and in vitro function of isolated islets showed no significant differences between the groups. These results suggested that brain death negatively affected the processes of islet isolation from the pancreas.

Macroautophagy in homeostasis of pancreatic β-cell

Diabetes mellitus is characterized by decreased insulin secretion and action. Decreased insulin secretion results from a reduction in pancreatic β-cell mass and function. Apoptosis, oxidative stress, mitochondrial dysfunction and ER stress responses including JNK activation have been suggested as mechanisms of the changes of pancreatic β-cells in type 2 diabetes, however, the underlying causes were not clearly elucidated. Autophagy is an intracellular process that plays a crucial role in cellular homeostasis through degradation and recycling of organelles constitutively or in response to the environmental condition. We studied the role of autophagy in pancreatic β-cells using mice with β-cell-specific deletion of the Atg7 (autophagy-related 7) gene. Atg7-mutant mice showed increased apoptosis and decreased proliferation of β-cells with resultant reduction in β-cell mass. Pancreatic insulin content was decreased due to the decreased β-cell mass and reduced number of insulin granules. Morphological analysis of β-cells revealed accumulation of ubiquitinated proteins, swollen mitochondria, and distended ER. Insulin secretory function ex vivo was also impaired. As a result, autophagy-deficient mice showed hypoinsulinemia and hyperglycemia. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic β-cells. Here we discuss the significance of autophagy in pancreatic β-cells with its potential relevance to the development of diabetes.

Enhancement of β-Cell Regeneration by Islet Transplantation After Partial Pancreatectomy in Mice

Background. Pancreatic &bgr; cells are known to regenerate, especially when insulin requirements are increased. Islet transplantation (ITx) is one strategy for insulin replacement in patients with diabetes. ITx can provide not only insulin in a physiological manner but also can exert additional effects such as &bgr;-cell regeneration. This study examined the effects of ITx on endogenous &bgr;-cell mass in mice. Methods. Male Balb/c mice were 70% pancreatectomized and transplanted with syngeneic islets, then compared with pancreatectomized mice with or without insulin treatment. Blood glucose levels and weight were evaluated for 10 days, with remnant pancreas obtained for evaluation of &bgr;-cell mass and turnover. Results. Hyperglycemia and weight loss were induced after pancreatectomy (Px). After ITx or insulin treatment, blood glucose levels recovered to normal, and body weight started to increase. At 10 days after Px, fasting serum insulin levels, &bgr;-cell mass, and insulin content in the remnant pancreas were higher in the ITx group than they were in the Px group. Insulin treatment after Px also increased &bgr;-cell mass, but did not increase pancreatic insulin content compared with those in the Px group. The enhanced &bgr;-cell mass and insulin content in the remnant pancreas of the ITx group resulted from increased &bgr;-cell proliferation/neogenesis and from prevention of &bgr;-cell apoptosis. Conclusions. These findings suggest that ITx after partial Px in mice enhances endogenous &bgr;-cell regeneration and survival, rendering &bgr;-cell mass increased and glucose homeostasis improved.

Delayed improvement of insulin secretion after autologous islet transplantation in partially pancreatectomized patients

The purpose of this study was to evaluate the effects of autologous islet transplantation (ITx) on glucose homeostasis and insulin secretory function after partial pancreatectomy (Px). Fourteen nondiabetic patients who underwent distal Px and autologous ITx for benign pancreatic tumors were enrolled in the study (Px + ITx group). Fourteen normal glucose-tolerant controls and 6 Px without ITx controls were recruited, and all groups were followed over a 24-month period. They performed the 75-g oral glucose tolerance test and the 1-mg glucagon stimulation test. Hemoglobin A(1c) was measured, and indices of insulin secretion were calculated. In the Px + ITx group, insulin secretion increased after a nadir at 6 months. Glucose tolerance, which had been abruptly impaired immediately after Px, recovered until 6 months and stabilized thereafter. As a result, differences in glucose intolerance emerged between the subjects in the Px group and those in the Px + ITx group at 24 months after Px. Characteristic variables in the better insulin secretory subjects in the Px + ITx group included younger age, less extensive pancreas resection, and a greater number of total islets. In summary, delayed amelioration of glucose intolerance was induced by autologous ITx after partial Px, even with a small number of islets.

Improved outcome of islet transplantation in partially pancreatectomized diabetic mice by inhibition of dipeptidyl peptidase-4 with sitagliptin.

Objective: Glucagon-like peptide-1 (GLP-1) is known to promote beta cell proliferation, and dipeptidyl peptidase-4 (DPP-4) inhibitor increases GLP-1 levels by preventing its degradation. This study was designed to evaluate the effects of sitagliptin (sita), a DPP-4 inhibitor, on the outcome of islet transplantation (ITx) in diabetic mice after partial pancreatectomy (Px). Methods: A diabetic mouse model was prepared by performing 70% Px in C57BL/6 mice. The diabetic mice were treated with sita, subjected to ITx, or both treated with sita and subjected to ITx. After 12 days of sita treatment, the pancreatic remnants and transplanted islets were histologically examined. Results: Dipeptidyl peptidase-4 inhibitor increased the concentration of plasma active GLP-1 regardless of ITx and improved glycemic control in the ITx group. The beta cell mass of the pancreatic remnants increased in the ITx group, and mice that received combined treatment with ITx and sita showed a greater increase in the beta cell mass. Dipeptidyl peptidase-4 inhibitor seems to induce proliferation and inhibit apoptosis of beta cells in pancreatic remnants. Conclusions: The DPP-4 inhibitor favorably affects ITx in partially pancreatectomized diabetic mice by increasing the beta cell mass through cell proliferation and inhibition of beta cell apoptosis.

Overexpression of USF Increases TGF-β1 Protein Levels, But G1 Phase Arrest was not Induced in FRTL-5 Cells

Transforming growth factor-β1 (TGF-β1) is a potent inhibitor of cellular growth and proliferation by G1 phase arrest or apoptosis. We investigated the association of TGF-β1 with the anti-proliferative effect of upstream stimulatory factor (USF) in Fischer rat thyroid cell line (FRTL-5) cells. [Methyl-3H] thymidine uptake was measured after treatment of FRTL-5 cells with TGF-β1 to identify its anti-proliferative effect. USF-1 and USF-2 proteins were in vitro translated, and an electrophoretic mobility shift assay was performed to identify the interaction between USF and the TGF-β1 promoter. FRTL-5 cells were transfected with USF cDNA, and then the expression of TGF-β1 was examined with Northern and Western blotting. The cell cycle-regulating proteins associated with TGF-β1 were also measured. TGF-β1 significantly inhibited [methyl-3H] thymidine uptake in FRTL-5 cells. Two specific binding sites for USF were found in the TGF-β1 promoter: -1,846~-1,841 (CACATG) and -621~-616 (CATGTG). Overexpression of USF increased both the mRNA levels and protein levels of TGF-β1. However, the expression of cyclin D1, CDK4, cyclin E, and CDK2, and the phosphorylation of retinoblastoma protein remained unchanged. Overexpression of USF in FRTL-5 cells increased the expression of TGF-β10 through specific binding to TGF-β1 promoter. However, the USF-induced expression of TGF-β1 did not cause G1 arrest.

Pharmacokinetics of mycophenolic acid in living donor liver transplantation.

PURPOSEnThis study sought to define the pharmacokinetics of mycophenolic acid (MPA) in Korean living donor liver transplant recipients.nnnMETHODSnThirty-two liver transplant recipients (29 males, 3 females) were administered 750 mg mycophenolate mofetil (MMF) twice daily with concomitant tacrolimus. Plasma MPA concentrations were measured by liquid chromatography with tandem mass spectrometry detection assay from samples drawn before dosing (C0) and after dosing at 0.5 hours (C1/2) and 2 hours (C2), providing a total of 114 pharmacokinetic profiles at various periods from 3 days to 6 months posttransplantation (D3, D7, D14, M1, M3, and M6).nnnRESULTSnThe mean area-under-the-curve from 0 to 2 hours (AUC0-2) was 30.0+/-11.6 microg.h/mL (range, 7.8-60.7). Of 114 pharmacokinetic profiles, 40 (35%) AUC and 7 (6.1%) trough values were within the target value (30-60 microg.h/mL and 1.7-4.0 microg/mL, respectively). The C0, C1/2, and C2 concentrations showed large interindividual variability: C0 (0.01-4.46 microg/mL), C1/2 (0.14-36.86 microg/mL), and C2 (0.79-18.19 microg/mL). A positive correlation was observed between AUC and C0 (r=.6374; P<.0001), and C2 (r=.7460; P<.0001). When analyzed according to the date posttransplant, a positive correlation between AUC and C0 was shown on day 7, day 14, and at month 1. There was no difference in any pharmacokinetic parameter relative to age, weight, or albumin level.nnnCONCLUSIONnThis study demonstrated that C0 values on day 7, day 14, and at month 1 provided valuable information for MPA monitoring. C0 was shown to be the most reliable monitoring time in relation to AUC. However, results from a larger randomized trial with more time intervals are eagerly awaited.

SU-F-BRD-11: A Virtual Simulator Designed for Collision Prevention in Proton Therapy

Purpose: nIn proton therapy, collisions between patient and nozzle potentially occur in attaining minimal air gap due to the large nozzle structure. Thus, we developed software predicting the collisions of the nozzle and patient by simulating treatments. n nMethods: n3D modeling of a gantry inner-floor, nozzle and robotic-couch was done by using the SolidWorks based on the manufacturers machine data. To obtain patient body information, a 3D-scanner was utilized to scan a patient right before CT scanning. From the acquired images, a 3D-image of the patients body contour was reconstructed. The accuracy of the image was confirmed against the CT image for a humanoid phantom. The machine components and the virtual patient were combined on the treatment-room coordinate system, resulting in a virtual simulator. The simulator simulated the motion of its components such as rotation and translation of gantry, nozzle and couch, in real scale. Collision, if any, was examined both in static mode and dynamic mode. The static mode checks only at fixed positions of the machines components while dynamic mode examines while one component is in motion. Collision was notified if any voxel of two components, for example a nozzle and a patient or couch, overlapped when calculating volume locations. The event and collision point are visualized and colliding volumes are reported. n nResults: nAll components were successfully assembled and the motions could be accurately controlled. The 3D-shape of a phantom agreed with CT images within a deviation of 2 mm. Collision situations can be simulated within minutes and the results are displayed and reported. n nConclusion: nThe developed software will be useful in improving patient safety and clinical efficiency for proton therapy. n nThis work was supported by the National Research Foundation of Korea funded by Ministry of Science, ICT & Future Planning (2012M3A9B6055201, 2013M2A2A7043507), and Samsung Medical Center grant (GFO1130081).