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Chen-Hung Lee,1 Ming-Jer Hsieh,1 Shang-Hung Chang,1 Kuo-Chun Hung,1 Chao-Jan Wang,2 Ming-Yi Hsu,2 Jyuhn-Huarng Juang,3 I-Chang Hsieh,1 Ming-Shien Wen,1 Shih-Jung Liu4,5 1Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital-Linkou, Chang Gung University College of Medicine, Linkou, Taiwan; 2Department of Medical Imaging and Intervention, Chang Gung Memorial Hospital, Linkou, Tao-Yuan, Taiwan; 3Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University and Chang Gung Memorial Hospital, Tao-Yuan, Taiwan; 4Department of Mechanical Engineering, Chang Gung University, Tao-Yuan, Taiwan; 5Department of Orthopedic Surgery, Chang Gung Memorial Hospital-Linkou, Tao-Yuan 33305, TaiwanCorrespondence: Shih-Jung LiuBiomaterials Lab, Department of Mechanical Engineering, Chang Gung University, 259, Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, TaiwanTel +886 3 211 8166Fax +886 3 211 8558Email profsjliu5347@gmail.comBackground: The high lifetime risk of vascular disease is one of the important issues that plague patients with diabetes mellitus. Systemic oral vildagliptin administration favors endothelial recovery and inhibits smooth muscle cell (SMC) proliferation. However, the localized release of vildagliptin in the diabetic vessel damage has seldom been investigated.Research design and methods: In this work, nanofiber-eluting stents that loaded with vildagliptin, a dipeptidyl peptidase-4 enzyme (DPP-4) inhibitor, was fabricated to treat diabetic vascular disease. To prepare nanofibers, the poly (D,L)-lactide-co-glycolide (PLGA) and vildagliptin were mixed using hexafluoroisopropanol and electrospinning process. In vitro and in vivo release rates of the vildagliptin were characterized using high-performance liquid chromatography.Results: Effective vildagliptin concentrations were delivered for more than 28 days from the nanofibrous membranes coating on the surface of the stents in vitro and in vivo. The vildagliptin-eluting PLGA membranes greatly accelerated the recovery of diabetic endothelia and reduced SMC hyperplasia. The type I collagen content of the diabetic vascular intimal area that was treated by vildagliptin-eluting stents was lower than that of the non-vildagliptin-eluting group.Conclusion: The experimental results revealed that stenting with vildagliptin-eluting PLGA membranes could potentially promote healing for diabetic arterial diseases.Keywords: re-endothelialization, neointimal hyperplasia, vildagliptin, biodegradable drug-eluting nanofibers

Cheng-Hung Lee,1 Ming-Jer Hsieh,1 Kuo-Sheng Liu,2,3 Che-Wei Cheng,3 Shang-Hung Chang,1 Shih-Jung Liu,3,4 Chao-Jan Wang,5 Ming-Yi Hsu,5 Kuo-Chun Hung,1 Yung-Hsin Yeh,1 Wei-Jan Chen,1 I-Chang Hsieh,1 Jyuhn-Huarng Juang,6 Ming-Shien Wen1 1Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, Linkou, Chang Gung University College of Medicine, Tao-Yuan, Taiwan; 2Department of Cardiovascular Surgery, Chang Gung Memorial Hospital-Linkou, Tao-Yuan, Taiwan; 3Department of Mechanical Engineering, Chang Gung University, Tao-Yuan, Taiwan; 4Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan; 5Department of Medical Imaging and Intervention, Chang Gung Memorial Hospital, Linkou, Tao-Yuan, Taiwan; 6Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University and Chang Gung Memorial Hospital, Tao-Yuan, Taiwan Objective: The current treatment of atherosclerotic coronary heart disease with limus-eluting stents can lead to incomplete endothelialization and substantial impairment of arterial healing relative to treatment with bare-metal stents. The sustained and local delivery of ticagrelor, a reversibly binding P2Y12 receptor inhibitor, using hybrid biodegradable nanofibers/stents, was developed to reduce neointimal formation and endothelial dysfunction.Methods: In this investigation, a solution of ticagrelor, poly(D,L)-lactide-co-glycolide, and hexafluoro isopropanol was electrospun to fabricate ticagrelor-eluting nanofibrous drug-eluting stents. The in vitro and in vivo ticagrelor concentrations were measured using a high-performance liquid chromatography assay. The effectiveness of ticagrelor-eluting stents was examined relative to that of sirolimus-eluting stents. Results: Adequate ticagrelor levels were detected for four weeks in vitro. Less HES5-positive labeling was found near the ticagrelor-eluting stented vessels (0.33±0.12) than close to the sirolimus-eluting stented vessels (0.57±0.15) (p

Recently, we successfully utilized noninvasive magnetic resonance and bioluminescence imaging to track MIN6 cells subcutaneously transplanted in immunocompromised nude mice for up to 64 days. In this study, we further used bioluminescence imaging to investigate the immune rejection of MIN6 cells in immunocompetent C3H mice. A total of 5 × 10 6 luciferase-transfected MIN6 cells were implanted into the subcutaneous space of each nude or C3H mouse. After transplantation, hypoglycemia and persistent bioluminescence signals were observed in eight of eight (100%) nude mice and five of nine (56%) C3H mice ( p < 0.05). We then presensitized a group of C3H mice with C57BL/6 spleen cells just prior to transplantation (n = 14). Interestingly, none of them had hypoglycemia or persistent bioluminescence signals ( p < 0.01 vs. C3H mice without presensitization). Histological examination of the grafts revealed a lack or minimal presence of insulin-positive cells in recipients without hypoglycemia and persistent bioluminescence signals. In contrast, recipients with hypoglycemia and persistent bioluminescence signals showed a significant presence of insulin-positive cells in their grafts. Our results indicate that rejection of MIN6 cells occurred in C3H mice and could be enhanced by presensitization with C57BL/6 spleen cells and that bioluminescence imaging is a useful noninvasive tool for detecting rejection of subcutaneously transplanted MIN6 cells.

Previously, we have successfully used noninvasive magnetic resonance (MR) and bioluminescence imaging to detect and monitor mPEG-poly(Ala) hydrogel-embedded MIN6 cells at the subcutaneous space for up to 64 days. In this study, we further explored the histological evolution of MIN6 cell grafts and correlated it with image findings. MIN6 cells were incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) and then 5 × 10 6 cells in the 100 μL hydrogel solution were injected subcutaneously into each nude mouse. Grafts were removed and examined the vascularization, cell growth and proliferation with anti-CD31, SMA, insulin and ki67 antibodies, respectively, at 8, 14, 21, 29 and 36 days after transplantation. All grafts were well-vascularized with prominent CD31 and SMA staining at all time points. Interestingly, insulin-positive cells and iron-positive cells were scattered in the graft at 8 and 14 days; while clusters of insulin-positive cells without iron-positive cells appeared in the grafts at 21 days and persisted thereafter, indicating neogrowth of MIN6 cells. Moreover, proliferating MIN6 cells with strong ki67 staining was observed in 21-, 29- and 36-day grafts. Our results indicate that the originally transplanted MIN6 cells proliferated from 21 days that presented distinctive bioluminescence and MR images.

Recently, we have shown that manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) conjugated with exendin-4 (Ex4) act as a contrast agent that directly trace implanted mouse islet β-cells by magnetic resonance imaging (MRI). Here we further advanced this technology to track implanted porcine neonatal pancreatic cell clusters (NPCCs) containing ducts, endocrine, and exocrine cells. NPCCs from one-day-old neonatal pigs were isolated, cultured for three days, and then incubated overnight with MnMEIO-Ex4 NPs. Binding of NPCCs and MnMEIO-Ex4 NPs was confirmed with Prussian blue staining in vitro prior to the transplantation of 2000 MnMEIO-Ex4 NP-labeled NPCCs beneath the left renal capsule of six nondiabetic nude mice. The 7.0 T MRI on recipients revealed persistent hypointense areas at implantation sites for up to 54 days. The MR signal intensity of the graft on left kidney reduced 62-88% compared to the mirror areas on the contralateral kidney. Histological studies showed colocalization of insulin/iron and SOX9/iron staining in NPCC grafts, indicating that MnMEIO-Ex4 NPs were taken up by mature β-cells and pancreatic progenitors. We conclude that MnMEIO-Ex4 NPs are excellent contrast agents for detecting and long-term monitoring implanted NPCCs by MRI.

Background: Previous studies showed inconsistent Results of the effects of dipeptidyl peptidase (DPP)-IV inhibitors on syngeneic mouse islet transplantation. We hypothesized that the implanted islet numbers are critical for the effects of DPP-IV inhibitors on the outcomes of transplantation., Methods: One hundred and fifty or three hundred islets were syngeneically transplanted under the renal capsule of each streptozocin-diabetic C57BL/6 mouse and recipients were then treated without or with LAF237 (10 mg/kg/day, po) for 6 weeks. After transplantation, recipients' blood glucose, body weight and intraperitoneal glucose tolerance test (IPGTT) were followed-up periodically. The graft was removed for the measurement of β-cell mass at 6 weeks., Results: In recipients with 150 islets, it was not significantly different between the LAF237- treated group (n = 14) and control group (n = 14) in terms of the blood glucose, body weight, glucose tolerance at 2, 4 and 6 weeks or the graft β-cell mass at 6 weeks. In contrast, in recipients with 300 islets, the LAF237-treated group (n = 24) did have a lower area under the curve of the IPGTT at 4 weeks (p = 0.0237) and 6 weeks (p = 0.0113) as well as more graft β-cell mass at 6 weeks (0.655 ± 0.008 mg vs. 0.435 ± 0.006 mg, p = 0.0463) than controls (n = 24)., Conclusions: Our findings revealed 6-week treatment of LAF237 improves glucose tolerance and increases graft β-cell mass in diabetic mice transplanted with a sufficient number but not a marginal number of islets. These indicate that the effects of DPP-IV inhibitors are influenced by the implanted islet mass., Competing Interests: Conflicts of interest The authors declare no conflicts of interests., (Copyright © 2020 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

To specifically detect and trace transplanted islet β-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) with exendin-4 (Ex4) which specifically binds glucagon-like peptide-1 receptors on the surface of β-cells. The size distribution of MnMEIO and MnMEIO-Ex4 NPs were 67.8 ± 1.3 and 70.2 ± 2.3 nm and zeta potential 33.3 ± 0.5 and 0.6 ± 0.1 mV, respectively. MnMEIO and MnMEIO-Ex4 NPs with iron content ≤ 40 μg/mL did not affect MIN6 β-cell viability and insulin secretion. Positive iron staining was found in MIN6 β-cells loaded with MnMEIO-Ex4 NPs but not in those with MnMEIO NPs. A transmission electron microscope confirmed MnMEIO-Ex4 NPs were distributed in the cytoplasm of MIN6. In vitro MR images revealed a loss of signal intensity in MIN6 β-cells labeled with MnMEIO-Ex4 NPs but not with MnMEIO NPs. After transplantation of islets labeled with MnMEIO-Ex4, the graft under kidney capsule could be visualized on MRI as persistent hypointense areas up to 17 weeks. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted β-cells by MRI.

The purpose of this study was to evaluate the effects of a hypoglycemia problem-solving program (HPSP) on problem-solving ability and glycemic control in diabetics with hypoglycemia. This was a prospective, quasi-experimental study with two groups, using a pre- and post-repeated measures design. A total of 71 diabetic patients with hypoglycemia were purposively assigned to an experimental group ( n = 34) and a control group ( n = 37). The experimental group participated in an 8-week HPSP, and each weekly session lasted approximately 90 min, while the control group received usual care. Participants were assessed at baseline, 1, 3, and 6 months after intervention care. In the experimental group, 6 months after the HPSP intervention, HbA1c was superior to that before the intervention. In both groups, the score obtained using the hypoglycemia problem-solving scale (HPSS) was low before the intervention. In the experimental group, HPSS tracking improved at all stages after the intervention compared to before the intervention. In the control group, the HPSS score improved slightly in the first month and sixth months after usual care. There were significant differences between and within groups in HbA1c levels and HPSS score over time. The intervention based on the HPSP effectively improves HbA1c level and hypoglycemia problem-solving ability in patients with hypoglycemia.

Neonatal pancreatic cell clusters (NPCCs) are potential tissues for the treatment of diabetes. Different from adult cells, they continuously proliferate and differentiate after transplantation. In this study, we utilized magnetic resonance imaging (MRI) to detect and monitor implanted NPCCs. NPCCs were isolated from one-day-old neonatal pigs, cultured for three days, and then incubated overnight with the contrast agent chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles. In vitro, Prussian blue staining and MR scans of CSPIO-labeled NPCCs were performed. In vivo, we transplanted 2000 CSPIO-labeled NPCCs under the kidney capsule of nondiabetic nude mice. Recipients were scanned with 7.0T MRI. Grafts were removed for histology with insulin and Prussian blue staining. After being incubated overnight with CSPIO, NPCCs showed positive iron staining and appeared as dark spots on MR scans. After transplantation of CSPIO-labeled NPCCs, persistent hypointense areas were observed at recipients' implant sites for up to 54 days. Moreover, histology showed colocalization of the insulin and iron staining in 15-, 51- and 55-day NPCC grafts. Our results indicate that transplanted NPCCs survived and differentiated to β cells after transplantation, and that MRI is a useful tool for the detection and monitoring of CSPIO-labeled NPCC grafts.

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight; 5 × 10 6 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for β-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

More than half of diabetic wounds demonstrate clinical signs of infection at presentation and lead to poor outcomes. This work develops coaxial sheath-core nanofibrous poly(lactide- co -glycolide) (PLGA) scaffolds that are loaded with bioactive antibiotics and platelet-derived growth factor (PDGF) for the repair of diabetic infectious wounds. PDGF and PLGA/antibiotic solutions were pumped, respectively, into two independent capillary tubings for coaxial electrospinning to prepare biodegradable sheath-core nanofibers. Spun nanofibrous scaffolds sustainably released PDGF, vancomycin, and gentamicin for 3 weeks. The scaffolds also reduced the phosphatase and tensin homologue content, enhanced the amount of angiogenesis marker (CD31) around the wound area, and accelerated healing in the early stage of infected diabetic wound repair. Antibiotic/biomolecule-loaded PLGA nanofibers may provide a very effective way to aid tissue regeneration at the sites of infected diabetic wounds.

Islet transplantation has been demonstrated to be a promising therapy for type 1 diabetes mellitus. Although it is a minimally invasive operating procedure and provides easy access for graft monitoring, subcutaneous transplantation of the islet only has limited therapeutic outcomes, owing to the poor capacity of skin tissue to foster revascularization in a short period. Herein, 3D cell spheroids of clinically accessible umbilical cord blood mesenchymal stem cells and human umbilical vein endothelial cells are formed and employed for codelivery with β cells subcutaneously. The 3D stem cell spheroids, which can secrete multiple proangiogenic and prosurvival growth factors, induce robust angiogenesis and prevent β cell graft death, as indicated by the results of in vivo bioluminescent tracking and histological analysis. These experimental data highlight the efficacy of the 3D stem cell spheroids that are fabricated using translationally applicable cell types in promoting the survival and function of subcutaneously transplanted β cells., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Patients with diabetes mellitus have up to a 15% lifetime risk of non-healing and poorly healing wounds. This work develops core-shell nanofibrous bioactive insulin-loaded poly-D-L-lactide-glycolide (PLGA) scaffolds that release insulin in a sustained manner for repairing wounds in diabetic rats. To prepare the biodegradable core-shell nanofibers, PLGA and insulin solutions were fed into two capillary tubes of different sizes that were coaxially electrospun using two independent pumps. The scaffolds sustainably released insulin for four weeks. The hydrophilicity and water-containing capacity of core-shell nanofibrous insulin/PLGA scaffolds significantly exceeded those of blended nanofibrous scaffolds. The nanofibrous core-shell insulin-loaded scaffold reduced the amount of type I collagen in vitro, increased the transforming growth factor-beta content in vivo, and promoted diabetic would repair. The core-shell insulin-loaded nanofibrous scaffolds prolong the release of insulin and promote diabetic wound healing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Increasing the intestinal dissolution of orally administered poorly water-soluble drugs that have poor oral bioavailability to a therapeutically effective level has long been an elusive goal. In this work, an approach that can greatly enhance the oral bioavailability of a poorly water-soluble drug such as curcumin (CUR) is developed, using a "Transformers"-like nanocarrier system (TLNS) that can self-emulsify the drug molecules in the intestinal lumen to form nanoemulsions. Owing to its known anti-inflammation activity, the use of CUR in treating pancreatitis is evaluated herein. Structural changes of the TLNS in the intestinal environment to form the CUR-laden nanoemulsions are confirmed in vitro. The therapeutic efficacy of this TLNS is evaluated in rats with experimentally induced acute pancreatitis (AP). Notably, the CUR-laden nanoemulsions that are obtained using the proposed TLNS can passively target intestinal M cells, in which they are transcytosed and then transported into the pancreatic tissues via the intestinal lymphatic system. The pancreases in rats that are treated with the TLNS yield approximately 12 times stronger CUR signals than their counterparts receiving free CUR, potentially improving the recovery of AP. These findings demonstrate that the proposed TLNS can markedly increase the intestinal drug dissolution, making oral delivery a favorable noninvasive means of administering poorly water-soluble drugs.

Efficient nonviral oral gene delivery offers an attractive modality for chronic protein replacement therapy. Herein, the oral delivery of insulin gene is reported by a nonviral vector comprising a copolymer with a high degree of substitution of branched polyethylenimine on chitosan (CS-g-bPEI). Protecting the plasmid from gastric acidic degradation and facilitating transport across the gut epithelium, the CS-g-bPEI/insulin plasmid DNA nanoparticles (NPs) can achieve systemic transgene expression for days. A single dose of orally administered NPs (600 µg plasmid insulin (pINS)) to diabetic mice can protect the animals from hyperglycemia for more than 10 d. Three repeated administrations spaced over a 10 d interval produce similar glucose-lowering results with no hepatotoxicity detected. Positron-emission-tomography and computed-tomography images also confirm the glucose utilization by muscle cells. While this work suggests the feasibility of basal therapy for diabetes mellitus, its significance lies in the demonstration of a nonviral oral gene delivery system that can impact chronic protein replacement therapy and DNA vaccination.

Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1 + /insulin - and SOX9 + pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin + cells increased and PDX1 + and SOX9 + cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin + cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived β cells early post-Tx. A higher percentage of NPCC-derived β cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin + cells budding out from PDX1 + /SOX9 + epithelium. Interestingly, a drop in SOX9 + progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1 + cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new β cells post-Tx. In conclusion, PDX1 + /SOX9 + cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new β cell post-Tx.

Background: Hypoglycemia is recognized as a limiting factor in diabetes management. Fear of experiencing hypoglycemia may lead to lower quality of life, impaired glycemic control, and emotional distress, all of which impair the ability of patients to self-manage their diabetes effectively. Problem solving is central to diabetes self-management and may help patients achieve effective self-care of their disease., Purpose: The aim of this study was to investigate the ability of people with diabetes to avoid hypoglycemia and to explore associated factors., Methods: A cross-sectional, descriptive design was used for the study. Data were collected using a demographic and disease characteristics datasheet, the Hypoglycemic Problem Solving Scale, and the Disease-Associated Negative Mood Scale., Results: Three hundred thirteen participants were recruited, with a mean age of 55.49 years. The average item score for the questions on hypoglycemic problem-solving ability was 2.43 (SD = 0.75). In comparing Hypoglycemic Problem Solving Scale subscales, participants scored highest on the problem orientation subscales and lowest on the problem-solving skills subscales. Multiple regression analysis revealed that being younger and unmarried and having a higher level of education, a diagnosis of Type 1 diabetes, and a lower negative mood score were each significantly associated with greater problem-solving ability as regards hypoglycemic events., Conclusions/implications for Practice: We suggest that patients with diabetes, especially those who are older or with lower levels of education, receive disease-related psychological interventions and that healthcare professionals teach problem-solving abilities in conjunction with hypoglycemia management.

Sodium dodecyl sulfate (SDS) is generally regarded as a potent permeability enhancer in oral formulations; however, one concern related to the use of any permeation enhancer is its possible absorption of unwanted toxins during the period of epithelial permeability enhancement. In this work, the safety and efficacy of an SDS-containing bubble carrier system that is developed from an orally administered enteric-coated capsule are evaluated. The bubble carriers comprise diethylene triamine pentaacetic acid (DTPA) dianhydride, sodium bicarbonate (SBC), SDS, and insulin. Upon exposure to the intestinal fluid, DTPA dianhydride hydrolyzes to yield acids, and SBC rapidly reacts with these acids to generate CO 2 , producing bubble carriers, each containing a self-assembling water film. The hydrophilic insulin is entrapped in the self-assembled water film, which is stabilized by SDS. The SDS in the bubble carrier system can act as a dissolution enhancer in the dispersion of insulin molecules, as a surfactant that stabilizes the bubble carriers, as a protease inhibitor that protects the protein drug, and as a permeation enhancer that augments its oral bioavailability. Hence, a significant increase in the plasma insulin level and an excellent blood glucose-lowering response in diabetic rats are effectively achieved. Moreover, the enhancement of epithelial permeation by this SDS-containing formulation does not promote the absorption of intestinal endotoxins. The above facts indicate that the bubble carrier system that is stabilized by SDS can be used as a safe and potent carrier in the oral delivery of therapeutic proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To develop and psychometrically test a new instrument, the hypoglycaemia problem-solving scale (HPSS), which was designed to measure how well people with diabetes mellitus manage their hypoglycaemia-related problems., Methods: A cross-sectional survey design approach was used to validate the performance assessment instrument. Patients who had a diagnosis of type 1 or type 2 diabetes mellitus for at least 1 year, who were being treated with insulin and who had experienced at least one hypoglycaemic episode within the previous 6 months were eligible for inclusion in the study., Results: A total of 313 patients were included in the study. The initial draft of the HPSS included 28 items. After exploratory factor analysis, the 24-item HPSS consisted of seven factors: problem-solving perception, detection control, identifying problem attributes, setting problem-solving goals, seeking preventive strategies, evaluating strategies, and immediate management. The Cronbach's α for the total HPSS was 0.83., Conclusions: The HPSS was verified as being valid and reliable. Future studies should further test and improve the instrument to increase its effectiveness in helping people with diabetes manage their hypoglycaemia-related problems., (© The Author(s) 2016.)

A method for simultaneously analysis of inorganic elements in rare and endangered plant spinulose tree fern was established. Alsophila Spinulosa samples were pretreated by using the hermetic microwave digestion. Six elements Na, Mg, Al, P, K and Ca were determined by using the inductively coupled plasma optical emission spectrometry (ICP-OES) and ten elements V, Cr, Mn, Fe, Ni, Cu, Zn, Cd, Hg and Pb were determined by using inductively coupled plasma mass spectrometry (ICP-MS). The working conditions were optimized as well. In order to keep the stability of the experiment, low concentration of HCl was added into the sample during testing. Multicomponent Spectral Fitting (MSF) and dynamic reaction cell (DRC) were used to correct the spectral interference and mass interference. Under optimized conditions, the standard linear relationships were satisfactory and the detection limit ranged from 1.87 ng·L-1～12.31 μg·L-1. Finally, the standard reference material NIST SRM 1 547 (peach leaves) was used to verify the proposed method. The obtained results were consistent with the reference method with the sameaccuracy and precision. Two Alsophila Spinulosa samples analysis results showed that Alsophila Spinulosa have higher concentrations of Na, Mg, K, Ca, Al, and P, and concentration of K is the highest. These results indicated that Alsophila Spinulosa have strong capability in K-enrichment. Alsophila Spinulosa have low concentrations of V, Cr, Mn, Fe, Cu, and Zn. Concentrations of Ni, Cd, Hg, and Pb are at very low trace levels, which indicated that the wild living environment is very healthy. This method has features of fast analyzing, high accuracy and convenience in operation, which can provide scientific basis for Alsophila Spinulosa inorganic elements contents measure.

Background: Obesity and insulin resistance lead to islet hyperplasia. However, how the islet remodeling influences the pancreatic environment and the associated neurovascular networks is largely unknown. The lack of information is primarily due to the difficulty of global visualization of the hyperplasic islet (>200 μm) and the neurovascular environment with high definition., Methods: We modulated the pancreatic optical property to achieve 3-dimensional (3-D) whole-islet histology and to integrate transmitted light microscopy (which provides the ground-truth tissue information) with confocal fluorescence imaging. The new optical and imaging conditions were used to globally examine the hyperplastic islets of the young (2 months) obese db/db and ob/ob mice, which otherwise cannot be easily portrayed by the standard microtome-based histology. The voxel-based islet micrographs were digitally processed for stereo projection and qualitative and quantitative analyses of the islet tissue networks., Results: Paired staining and imaging of the pancreatic islets, ducts and neurovascular networks reveal the unexpected formation of the 'neuro-insular-ductal complex' in the young obese mice. The complex consists of the peri- and/or intra-islet ducts and prominent peri-ductal sympathetic nerves; the latter contributes to a marked increase in islet sympathetic innervation. In vascular characterization, we identify a decreased perivascular density of the ob/ob islet pericytes, which adapt to ensheathing the dilated microvessels with hypertrophic processes., Conclusions: Modulation of pancreatic optical property enables 3-D panoramic examination of islets in the young hyperphagic mice to reveal the formation of the islet-duct complex and neurovascular remodeling. On the basis of the morphological proximity of the remodeled tissue networks, we propose a reactive islet microenvironment consisting of the endocrine cells, ductal epithelium and neurovascular tissues in response to the metabolic challenge that is experienced early in life.

The nanofibrous biodegradable drug-loaded membranes that sustainably released recombinant human platelet-derived growth factor (rhPDGF-BB) to repair diabetic wounds were developed in this work.rhPDGF-BB and poly(lactic-co-glycolic acid) (PLGA) were mixed in hexafluoroisopropyl alcohol, followed by the electrospinning of the solutions into biodegradable membranes to equip the nanofibrous membranes. An elution technique and an enzyme-linked immunosorbent assay kit were used to determine the rhPDGF-BB release rates in vitro and in vivo from this membrane. Eighteen Sprague-Dawley streptozotocin-induced diabetic rats were randomized into 3 groups: rhPDGF-BB-loaded nanofibrous membrane group, PLGA only membrane group, and conventional gauze sponge group for the wound associated with diabetes of rat in each group.The nanofibrous biodegradable membranes released effective concentrations of rhPDGF-BB for over 21 days. The nanofibrous rhPDGF-BB-loaded PLGA membranes contained more water and were further hydrophilic than PLGA only fibers. The rhPDGF-BB-loaded PLGA membranes considerably helped the diabetic wounds repairing. Furthermore, the proliferative cells and angiogenesis of rats associated with diabetes by rhPDGF-BB-loaded nanofibrous membranes were greater than those of other groups, owing to the increased matrix metalloproteinase 9.These biodegradable rhPDGF-BB-loaded membranes were effective in treating diabetic wounds as very advanced accelerators during the initial phases of wound-healing process., Competing Interests: The authors have no conflicts of interest to disclose.

This work developed nanofibrous drug-loaded collagen/poly-D-L-lactide-glycolide (PLGA) scaffold membranes that provided the sustained release of glucophage for the wounds associated with diabetes. PLGA, glucophage, and collagen were firstly dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol and were spun into nanofibrous membranes by electrospinning. High-performance liquid chromatography assay was used to characterize the in vivo and in vitro release rates of the pharmaceuticals from the membranes. High concentrations of glucophage were released for over three weeks from the nanofibrous membranes. The nanofibrous glucophage-loaded collagen/PLGA membranes were more hydrophilic than collagen/PLGA membranes and exhibited a greater water-containing capacity. The glucophage-loaded collagen/PLGA membranes markedly promoted the healing of diabetic wounds. Moreover, the collagen content of diabetic rats using drug-eluting membranes was higher than that of the control rats, because of the down-regulation of matrix metalloproteinase 9. The experimental results herein suggest that the nanofibrous glucophage-loaded collagen/PLGA membranes had effect for increasing collagen content in treating diabetic wounds and very effective promoters of the healing of such wounds in the early stages., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

Type 1 diabetes usually develops due to autoimmune destruction of β-cells in the pancreas. It has been shown that all-trans retinoid acid (ATRA), a potent derivative of vitamin A, hinders the development of autoimmune diabetes by inducing immune tolerance status. In addition, exendin-4, a glucagon-like peptide-1 receptor agonist, stimulates growth and differentiation of β-cells and exerts anti-apoptotic effect on β-cells. Thus, we hypothesized that the ATRA and exendin-4 therapy may improve the outcome of islet transplantation in non-obese diabetic (NOD) mice. After the onset of diabetes, each NOD mouse was transplanted with 300 or 600 islets isolated from NOD/severe combined immunodeficient (SCID) mice with or without treatment of ATRA (0.5 mg intraperitoneally every other day) and/or exendin-4 (3 μg/kg subcutaneously twice daily) for 6 weeks. After 300 or 600 NOD/SCID islet transplantation without any other treatment, all NOD recipients remained diabetic. However, the lowest blood glucose level in mice transplanted with 600 but not 300 islets was significantly lower than those without islet transplantation (P < .05), although their survival time was comparable. Among recipients treated with ATRA, exendin-4, ATRA and exendin-4, and without treatment, their lowest blood glucose levels and survival time were not different. However, one recipient treated with ATRA survived for 223 days with intermittent hyperglycemia and the other who was treated with ATRA and exendin-4 achieved normoglycemia. In conclusion, islet transplantation lowered blood glucose levels in diabetic NOD mice. With a few exceptions, treatment with ATRA and exendin-4 alone or in combination in islet recipients could not reverse diabetes or prolong survival., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This work developed biodegradable nanofibrous drug-eluting membranes that provided sustained release of metformin for repairing wounds associated with diabetes. To prepare the biodegradable membranes, poly-d-l-lactide-glycolide (PLGA) and metformin were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and were spun into nanofibrous membranes by electrospinning. An elution method and an HPLC assay were utilized to characterize the in vivo and in vitro release rates of the pharmaceuticals from the membranes. The biodegradable nanofibrous membranes released high concentrations of metformin for more than three weeks. Moreover, nanofibrous metformin-eluting PLGA membranes were more hydrophilic and had a greater water-containing capacity than virgin PLGA fibers. The membranes also improved wound healing and re-epithelialization in diabetic rats relative to the control. The experimental results in this work suggest that nanofibrous metformin-eluting membranes were functionally active in the treatment of diabetic wounds and very effective as accelerators in the early stage of healing of such wounds.

Microscopic examination of transplanted islets in an ectopic environment provides information to evaluate islet engraftment, including revascularization and reinnervation. However, because of the dispersed nature of blood vessels and nerves, global visualization of the graft neurovascular network has been difficult. In this research we revealed the neurovascular network by preparing transparent mouse islet grafts under the kidney capsule with optical clearing to investigate the sympathetic reinnervation via three-dimensional confocal microscopy. Normoglycemic and streptozotocin-induced diabetic mice were used in syngeneic islet transplantation, with both groups maintaining euglycemia after transplantation. Triple staining of insulin/glucagon, blood vessels, and tyrosine hydroxylase (sympathetic marker) was used to reveal the graft microstructure, vasculature, and sympathetic innervation. Three weeks after transplantation, we observed perigraft sympathetic innervation similar to the peri-islet sympathetic innervation in the pancreas. Six weeks after transplantation, prominent intragraft, perivascular sympathetic innervation was achieved, resembling the pancreatic intraislet, perivascular sympathetic innervation in situ. Meanwhile, in diabetic recipients, a higher graft sympathetic nerve density was found compared with grafts in normoglycemic recipients, indicating the graft neural plasticity in response to the physiological difference of the recipients and the resolving power of this imaging approach. Overall, this new graft imaging method provides a useful tool to identify the islet neurovascular complex in an ectopic environment to study islet engraftment.

It has been shown that all-trans retinoid acid (ATRA) hinders the development of autoimmune diabetes by inducing immune tolerance status. Meanwhile, exendin-4 increases beta-cell function and mass. Thus, we hypothesized that ATRA and exendin-4 combination therapy would prevent and reverse autoimmune diabetes. NOD/scid mice were intravenously transferred with splenocytes isolated from 12-week-old female NOD mice. After adoptive transfer, mice were treated with vehicle, ATRA (0.5 mg/mouse intraperitoneally every other day), exendin-4 (3  μ g/kg subcutaneously twice daily), or combination for 6 weeks. Compared with vehicle, ATRA (P = 0.022) and ATRA plus exendin-4 (P = 0.013) treatment delayed the onset of diabetes. The pancreatic insulin content in mice treated with ATRA (P = 0.013) and exendin-4 (P < 0.02) was significantly higher than that of control mice. All but one spontaneous diabetic NOD mouse treated with ATRA and/or exendin-4 remained persistent hyperglycemic. ATRA and/or exendin-4 treatment did not alter their blood glucose levels and survival. Our results indicate that, before the onset of autoimmune diabetes, ATRA and exendin-4 treatment alone preserves pancreatic beta cells; ATRA and ATRA plus exendin-4 treatment delays the onset of autoimmune diabetes. However, after the onset of autoimmune diabetes, ATRA and/or exendin-4 treatment is unable to reverse hyperglycemia or improve survival.

Dipeptidyl peptidase (DPP)-4 inhibitors increase circulating levels of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide which may promote β-cell proliferation and survival. This study tested if DPP-4 inhibition with MK-0431 is beneficial for diabetic mice syngeneically transplanted with a marginal number of islets. We syngeneically transplanted 150 C57BL/6 mouse islets under the kidney capsule of each streptozotocin-diabetic mouse and then treated recipients with (n = 21) or without (n = 17) MK-0431 (30 mg/kg/day, po) for 6 weeks. After islet transplantation, blood glucose levels decreased in both MK-0431-treated and control groups. However, the blood glucose and area under the curve of the intraperitoneal glucose tolerance test at 2, 4, and 6 weeks were not significantly different between MK-0431-treated mice and controls. During 6 weeks, both groups exhibited increased body weights over time. However, the weight between two groups did not differ throughout the study period. At 6 weeks after transplantation, the graft beta-cell mass (0.024 ± 0.005 versus 0.023 ± 0.007 mg, P = 0.8793) and insulin content (140 ± 48 versus 231 ± 63 ng, P = 0.2939) were comparable in the MK-0431-treated group and controls. Our results indicate posttransplant DPP-4 inhibition with MK-0431 in the diabetic recipient with a marginal number of islets is not beneficial to transplantation outcome or islet grafts.

This work examined the feasibility of preparing a pH-responsive nanoparticle (NP) system composed of chitosan and poly(γ-glutamic acid) conjugated with ethylene glycol tetraacetic acid (γPGA-EGTA) for oral insulin delivery in diabetic rats during an oral glucose tolerance test (OGTT). OGTT has been used largely as a model to mimic the period that comprises and follows a meal, which is often associated with postprandial hyperglycemia. Based on Förster resonance energy transfer (FRET), this work also demonstrated the ability of γPGA-EGTA to protect insulin from an intestinal proteolytic attack in living rats, owing to its ability to deprive the environmental calcium. Additionally, EGTA-conjugated NPs were effective in disrupting the epithelial tight junctions, consequently facilitating the paracellular permeation of insulin throughout the entire small intestine. Moreover, results of positron emission tomography and computer tomography demonstrated the effective absorption of the permeated insulin into the systemic circulation as well as promotion of the glucose utilization in the myocardium, and skeletal muscles of the chest wall, forelimbs and hindlimbs, resulting in a significant glucose-lowering effect. Above results indicate that as-prepared EGTA-conjugated NPs are a promising oral insulin delivery system to control postprandial hyperglycemia and thus may potentially prevent the related diabetic complications., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Current insulin therapy via subcutaneous administration can lead to occasional hypoglycemia and peripheral hyperinsulinemia, due to its nonphysiological route. This study evaluates the feasibility of using bovine insulin and exendin-4 in a form of combination therapy, as orally delivered by nanoparticles composed of chitosan and poly(γ-glutamic acid) (CS/γPGA NPs), to control blood glucose levels in rats with type 2 diabetes mellitus (T2DM) undergoing the oral glucose tolerance test. Experimental results indicate that CS/γPGA NPs could enhance the intestinal paracellular permeation; consequently, the exogenous bovine insulin and exendin-4 could be delivered into the liver and pancreas, where they could elicit their glucoregulatory activities. In response to the stimulus of exogenously delivered bovine insulin and the endogenously secreted rat insulin stimulated by the ingested exendin-4, significant glucose utilizations were found in the cardiac and skeletal muscles, resulting in the glucose-lowering effect. Owing to its synergic stimulation effects, the hypoglycemic effect of oral ingestion of NPs containing bovine insulin and exendin-4 was significantly greater than that of the group solely treated with insulin NPs. Above results demonstrate that oral combination therapy with bovine insulin and exendin-4 improves the modulation of blood glucose levels in T2DM rats, making it highly promising for treating those T2DM patients not adequately controlled by the current insulin therapy., (2013 Published by Elsevier Ltd.)

Object: To better understand the fate of islet isografts and allografts, we utilized a magnetic resonance (MR) imaging technique to monitor mouse islets labeled with a novel MR contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles., Materials and Methods: After being incubated with and without CSPIO (10 µg/ml), C57BL/6 mouse islets were examined under transmission electron microscope (TEM) and their insulin secretion was measured. Cytotoxicity was examined in α (αTC1) and β (NIT-1 and βTC) cell lines as well as islets. C57BL/6 mice were used as donors and inbred C57BL/6 and Balb/c mice were used as recipients of islet transplantation. Three hundred islets were transplanted under the left kidney capsule of each mouse and then MR was performed in the recipients periodically. At the end of study, the islet graft was removed for histology and TEM studies., Results: After incubation of mouse islets with CSPIO (10 µg/mL), TEM showed CSPIO in endocytotic vesicles of α- and β-cells at 8 h. Incubation with CSPIO did not affect insulin secretion from islets and death rates of αTC1, NIT-1 and βTC cell lines as well as islets. After syngeneic and allogeneic transplantation, grafts of CSPIO-labeled islets were visualized on MR scans as persistent hypointense areas. At 8 weeks after syngeneic transplantation and 31 days after allogeneic transplantation, histology of CSPIO-labeled islet grafts showed colocalized insulin and iron staining in the same areas but the size of allografts decreased with time. TEM with elementary iron mapping demonstrated CSPIO distributed in the cytoplasm of islet cells, which maintained intact ultrastructure., Conclusion: Our results indicate that after syngeneic and allogeneic transplantation, islets labeled with CSPIO nanoparticles can be effectively and safely imaged by MR.

Chitosan (CS) is a potential paracellular permeation enhancer for trans-epithelial drug delivery; however, its ability to enhance epithelial permeability in a pH-dependent manner remains unclear. This study was designed to explore the underlying molecular mechanisms with regard to the effect of CS on tight junction (TJ) disruption at different pH environments in Caco-2 cell monolayers. The experimental results revealed that the direct interaction between CS and integrin α(V)β(3) on cell surfaces has a crucial role in CS-induced TJ opening, an indication of receptor activation. The mechanism of action appeared to be the electrostatic interaction between the positively-charged CS and the negatively-charged integrin α(V)β(3). This electrostatic interaction led to the conformation change of integrin α(V)β(3) and its clustering along the cell border, F-actin reorganization, and CLDN4 down-regulation, eventually resulting in the disruption of TJs and an increase in paracellular permeability. The above observations were all in a pH-dependent manner. As pH increased, CS became less positively charged, thereby losing its capability to interact with integrin α(V)β(3) and failing to induce the TJ opening. These consequences might help to better understand the molecular mechanism of TJ opening mediated by CS, thereby facilitating the use of CS for trans-epithelial drug delivery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chitosan (CS) and its derivatives have been investigated as paracellular permeation enhancers for facilitating the oral bioavailability of hydrophilic macromolecules. As is well known, CS can transiently open the tight junctions (TJs) between epithelial cells, thus enhancing the paracellular permeability. However, the signaling mechanism that is related to the effect of CS on TJs remains unclear. Therefore, this study elucidates the potential transduction cascade of TJ opening in Caco-2 cell monolayers subsequent to CS exposure. Experimental results indicate that activation of integrin receptors on cell membranes significantly contributes to CS-mediated TJ disruption, initiating the cascade of TJ opening. Additionally, treatment of Caco-2 cell monolayers with CS leads to the clustering of integrins along the cell border, phosphorylation of FAK and Src tyrosine kinases, and results in the regulation of TJ permeability via the redistribution of TJ protein CLDN4 from the cell membrane to the cytosol. Elucidating the signaling mechanism of CS-induced TJ opening in intestinal cells significantly contributes to efforts to use CS and its derivatives as paracellular permeation enhancers., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bioartificial liver support (BAL) systems are potential new therapeutic approaches for use as liver support to prevent nutrient deficiencies, hypoxia, or ischemia before the acquisition of donated organs. To investigate whether islets are beneficial for hepatocyte function and survival, we cocultured BALB/c mouse islets with C57BL/6J hepatocytes to assess hepatocyte viability, function, and apoptosis. We observe cell viability to decrease progressively by 50% from day 0 to day 3 among isolated hepatocytes (group A) and hepatocytes cocultured with islets (group B). However, group A was prone to necrosis and reduced albumin secretion during culture. In contrast, at day 7 group B maintained albumin secretion (0.3351 ± 0.0581 vs 0.1451 ± 0.0329 μg/h/mL; P < .05). Early apoptosis was observed at day 3 among group A but at day 7 in group B. In addition, quantitative analysis of the apoptotic cells revealed group B to show a delayed phenotype of both early and late apoptosis compared with group A. Our results indicated that islets could retain hepatocyte function and delay apoptosis, suggesting that the coculture system is potentially applicable to develop a high-performance BAL., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Interactions of Fas with its ligand (FasL) play an important role in the maintenance of immunologic homeostasis and peripheral tolerance. Heme oxygenase-1 (HO-1) is a protein capable of cytoprotection via radical scavenging and apoptosis prevention. The aim of this study was to test whether overexpression of FasL and HO-1 in murine islets resulted in cell protection and improved functional performance after transplantation. We first generated FasL and HO-1 double transgenic mice to investigate the protective effect of transgenic islets on transplantation. Islets were isolated from FasL and HO-1 double transgenic and nontransgenic Balb/c mice, for transplantation of 300 islets under the left kidney capsule of each streptozotocin-diabetic Balb/c mouse. During 6 weeks after transplantation, the blood glucose gradually decreased in recipients of double transgenic and nontransgenic islets. However, the decrease in blood glucose was more pronounced in the former (450 ± 16 mg/dL at day 0 to 302 ± 55 mg/dL at day 42; P = .01) than the latter (468 ± 17 mg/dL at day 0 to 379 ± 71 mg/dL at day 42; P = .24). The areas under the curve of intraperitoneal glucose tolerance tests at 2, 4, and 6 weeks were not significantly different between recipients of double transgenic and nontransgenic islets. The body weight increased in recipients of double transgenic islets (21.1 ± 1.4 g at day 0 to 26.2 ± 0.8 g at day 42; P = .0002) and nontransgenic islets (21.0 ± 1.4 g at day 0 to 25.1 ± 0.4 g at day 42; P = .0448). Our data suggested modest beneficial effects of transgenic islets with FasL and HO-1 overexpression for transplantation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aims: To examine the impact of the threat of hypoglycaemic episodes on people with diabetes in Taiwan., Background: Intensive diabetes treatment in people with diabetes helps them to achieve better glycaemic control. However, it also causes more frequent hypoglycaemic episodes and has an impact on their overall quality of life. Hypoglycaemia is accompanied by various distressing symptoms which may cause excessive fear, affecting decision making in hypoglycaemic management., Design: Purposive sampling and in-depth, face-to-face interviews were used to collect data., Methods: Semi-structured interviews were conducted from July 2008-January 2009 with 17 individuals treated with insulin who had previous hypoglycaemic episodes. Data were analysed using qualitative content analysis., Results: Four themes were generated from the analysis, 'inability to control fluctuations in health', 'challenges to interpersonal relationships', 'facing the disease alone' and 'finding a balance between competing symptoms'., Conclusions: Hypoglycaemia is a major health issue for many people with diabetes. Understanding individuals' experiences with hypoglycaemic episodes should help practitioners become more fully involved in promoting self-management. We identified key areas that health care providers should address, including concerns about patient education and professional support for people with diabetes experiencing hypoglycaemia, to enhance problem solving skills for them and their families., Relevance to Clinical Practice: We recommend that health care providers make proper use of support groups for family caregivers or other important individuals in the lives of people with diabetes to provide education, clarification, support and guidance. In addition, health care providers also need to provide clients with hypoglycaemia-related emotional support, while enhancing diabetes self-management and problem-solving skills., (© 2011 Blackwell Publishing Ltd.)

Exendin-4 is a potent insulinotropic agent in diabetes patients; however, its therapeutic utility is limited due to the frequent injections required. In this study, an orally available exendin-4 formulation, using an enteric-coated capsule containing pH-responsive NPs, was developed. Following oral administration of (123)I-labeled-exendin-4 loaded NPs in rats, the biodistribution of the administered drug was investigated using a dual isotope dynamic SPECT/CT scanner. The results showed that the radioactivity of (123)I-exendin-4 propagated from the esophagus, stomach, and small intestine and then was absorbed into the systemic circulation; with time progressing, (123)I-exendin-4 was metabolized and excreted into the urinary bladder. In the in vivo dissolution study, it was found that the enteric-coated capsule remained intact while in the stomach; the capsule was completely dissolved in the proximal segment of the small intestine and the loaded contents were then released. Oral administration of the capsule containing exendin-4 loaded NPs showed a maximum plasma concentration at 5 h after treatment; the bioavailability, relative to its subcutaneous counterpart, was found to be 14.0 ± 1.8%. The absorbed exendin-4 could then stimulate the insulin secretion and provide a prolonged glucose-lowering effect. The aforementioned results suggest that the orally available exendin-4 formulation developed warrants further exploration as a potential therapy for diabetic patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: To examine the prevalence of microalbuminuria (MAU) and chronic kidney disease as well as the correlation between MAU and renal and cardiovascular risks of Type 2 diabetes mellitus (T2DM) patients for public health policy making in Taiwan., Methods: This was a multicenter, hospital-based, randomly selected, and cross-sectional study. T2DM patients aged 18-80 years without a known diagnosis of proteinuria were eligible. MAU was defined as urinary albumin-to-creatinine ratio (ACR) within 30-299 mg/g, and macroalbuminuria as that greater than or equal to 300 mg/g. Two positive out of three urinary screening results were required to make the diagnosis of MAU. The adjusted prevalence of MAU was calculated by conditional probability approach., Results: 51.1% of the analyzed population (n=1,827) were women, with a mean (standard deviation) age of 59.16 years (11.19 years) and mean hemoglobin A1c (HbA1c) of 8.15% (1.83%). Median duration of DM history was 6 years (interquartile range, 3-11 years). The adjusted prevalence of MAU was 26.9%. Overall prevalence of chronic kidney disease Stage 3 or higher (estimated Glomerular filtration rate (eGFR) less than 60/mL/min/1.73 m²) was 13.8%. Only 4.7% of the T2DM patients had serum albumin test recorded and 68.7% with serum creatinine test recorded within the last 6 months. After adjustment for center and gender, the odds ratios for MAU or macroalbuminuria were 1.73 (95% CI, 1.27-2.36) for age greater than or equal to 60 years, 1.54 (1.13-2.10) for abnormal waist circumference, 1.10 (1.02-1.19) for every 1% increase in hemoglobin A1c, 1.91 (1.38-2.65) for higher systolic blood pressure, and 1.92 (1.19-3.07) for abnormal serum creatinine level., Conclusion: This study demonstrates the application of "conditional probability" method to justify the rationale of adopting two positive out of three urinary screening tests for the diagnosis of MAU. An adjusted prevalence rate of MAU as 26.9% is reported. These results may provide a basis for cost-benefit consideration in designing preventive and interventional policies in public health. Furthermore, the awareness and practice of early monitoring of MAU for DM patients should be strengthened., (Copyright © 2011. Published by Elsevier B.V.)

In the initial days after transplantation, islet grafts may be attacked by cytokines via cyclooxygenase-2 (COX-2), producing primary nonfunction. In addition, chronic overstimulation of β-cells may impair insulin secretion. To enhance the function of transplanted islets, the present study investigated the effects of rofecoxib, a COX-2 inhibitor, and NN414 (6-chloro-3-[1-methylcyclopropyl]amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), an adenosine triphosphate-sensitive potassium channel opener, on islet transplantation. Male inbred C57BL/6 mice were used as donors and recipients. One hundred fifty islets were isolated via collagenase digestion and density gradient, and syngeneically transplanted under the kidney capsule in mice with streptozotocin-induced diabetes. Recipients were treated with or without rofecoxib, 10 mg/kg/d orally, or with or without NN414, 3 mg/kg/d orally, for 4 weeks. After transplantation, recipient body weight, blood glucose concentration, and intraperitoneal glucose tolerance were measured. The grafted kidney was extracted for determination of insulin content at 4 weeks. In the rofecoxib-treated and NN414-treated groups and both control groups, body weight remained stable, and the blood glucose concentration decreased progressively. However, at 4 weeks after transplantation in the groups treated or not treated with rofecoxib or NN414, no significant difference was observed in recipient body weight, blood glucose concentration, and glucose tolerance or in insulin content of the graft. These data indicate that posttransplantation treatment with rofecoxib or NN414 has no beneficial effect on transplantation outcome in diabetic mouse recipients engrafted with a marginal islet mass., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Although only 10% of islet transplant recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect superparamagnetic iron oxide (SPIO)-labeled transplanted islets. Recently, we successfully used a novel MRI contrast agent, chitosan-coated SPIO (CSPIO) nanoparticles, to monitor mouse islet isografts for 18 weeks after transplantation. In the present study, we tested whether CSPIO could be applied to monitor islet allografts, which are supposedly rejected without immune interventions. Male C57BL/6 and Balb/c mice were used as donors and recipients of islet transplantation, respectively. After overnight incubation with or without CSPIO (10 μg/mL), 300 C57BL/6 islets were transplanted under the left kidney capsule of each Balb/c mouse. Starting from day 10 after transplantation, 3.0-Tesla MRI of the recipients was performed weekly. Four mice were followed for ≥38 days. At 38 and 45 days, 1 islet graft was removed for insulin and Prussian blue staining, respectively. From days 10 to 45 after transplantation, CSPIO-labeled islet grafts were visualized on MRI scans as sustained distinct hypointense spots homogeneously located at the upper pole of left kidney, the site of transplantation. At days 38 and 45, the histology of CSPIO-labeled islet grafts revealed insulin and iron staining colocalized in the same areas. Our results in a mouse allotransplantation model indicated that CSPIO-labeled islets survived as long as 45 days with positive MRI., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In this study, we report the biodistribution of aspart-insulin, a rapid-acting insulin analogue, following oral or subcutaneous (SC) administration to rats using the single-photon emission computed tomography (SPECT)/computed tomography (CT). Oral delivery of aspart-insulin was achieved using a pH-responsive nanoparticle (NP) system composed of chitosan (CS) and poly(gamma-glutamic acid). The results obtained in the SPECT/CT study indicate that the orally administered aspart-insulin was absorbed into the systemic circulation, while the drug carrier (CS) was mainly retained in the gastrointestinal tract.Via the SC route, the peak aspart-insulin concentration in the peripheral tissue/plasma was observed at 20 min after injection. Within 3 h, half of the initial dose (ID) of aspart-insulin was degraded and excreted into the urinary bladder. In contrast, via oral delivery, there was constantly circulating aspart-insulin in the peripheral tissue/plasma during the course of the study, while 20% of the ID of aspart-insulin was metabolized and excreted into the urinary bladder. In the pharmacodynamic (PD) and pharmacokinetic (PK) evaluation in a diabetic rat model, the orally administered aspart-insulin loaded NPs produced a slower hypoglycemic response for a prolonged period of time, whereas the SC injection of aspart-insulin produced a more pronounced hypoglycemic effect for a relatively shorter duration. Finally, comparison of the PD/PK profiles of the orally administered aspart-insulin with those of the SC injection of NPH-insulin, an intermediate-acting insulin preparation, suggests the suitability of our NP system to be used as a non-invasive alternative for the basal insulin therapy., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%-50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.

Unlabelled: The vesicular monoamine transporter type 2 (VMAT2) is highly expressed in pancreatic beta-cells and thus has been proposed to be a potential target for measuring beta-cell mass (BCM) by molecular imaging. C-11- and F-18-labeled tetrabenazine derivatives targeting VMAT2 have shown some promising results as potential biomarkers for BCM. In the present study, we examined the binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine ([(18)F]AV-133), a potential PET tracer for BCM imaging, in rat pancreas and rat brain., Methods: Pancreatic exocrine cells and pancreatic islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum and hypothalamus regions, were used for in vitro binding studies. In vitro and ex vivo autoradiography studies with [(18)F]AV-133 were performed on rat brain and rat pancreas sections. Immunohistochemistry studies were performed to confirm the distribution of VMAT2 on islet beta-cells., Results: Excellent binding affinities of [(18)F]AV-133 were observed in rat striatum and hypothalamus homogenates with K(d) values of 0.19 and 0.25 nM, respectively. In contrast to single-site binding observed in rat striatum homogenates, rat islet cell homogenates showed two saturable binding sites (site A: K(d)=6.76 nM, B(max)=60 fmol/mg protein; site B: K(d)=241 nM, B(max)=1500 fmol/mg protein). Rat exocrine pancreas homogenates showed only a single low-affinity binding site (K(d)=209 nM), which was similar to site B in islet cells. In vitro autoradiography of [(18)F]AV-133 using frozen sections of rat pancreas showed specific labeling of islets, as evidenced by co-localization with anti-insulin antibody. Ex vivo VMAT2 pancreatic autoradiography in the rat, however, was not successful, in contrast to the excellent ex vivo autoradiography of VMAT2 binding sites in the brain. In vivo/ex vivo islet labeling may be complicated by the presence of the low-affinity/high-capacity site B binding in rat pancreas., Conclusions: [(18)F]AV-133 is an excellent imaging agent for mapping VMAT2 sites in rat brain and specifically binds rat islet cells in vitro and postmortem. Additional optimization may be required to achieve ex vivo islet beta-cell labeling in rats., ((c) 2010 Elsevier Inc. All rights reserved.)

A pH-sensitive nanoparticle (NP) system composed of chitosan and poly(gamma-glutamic acid) was prepared for the oral delivery of insulin. The biodistribution study in a rat model showed that some of the orally administered NPs were retained in the stomach for a long duration, which might lead to the disintegration of NPs and degradation of insulin. To overcome these problems, we freeze-dried NPs and filled them in an enteric-coated capsule. The small angle X-ray scattering (SAXS) profiles indicated that the freeze-drying process did not significantly disrupt the internal structure of NPs; additionally, their pH-sensitivity was preserved and the insulin release was pH-dependent. The results obtained in the native PAGE analysis indicated that the released insulin molecules were neither fragmented nor aggregated. Upon oral administration, the enteric-coated capsule remained intact in the acidic environment of the stomach, but dissolved rapidly in the proximal segment of the small intestine. Consequently, all the NPs loaded in the capsule were brought into the small intestine, thus enhancing the intestinal absorption of insulin and providing a prolonged reduction in blood glucose levels. The relative bioavailability of insulin was found to be approximately 20%. These results suggest that the formulation developed in the study might be employed as a potential approach for the oral delivery of insulin., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Porcine neonatal pancreatic cell clusters (NPCCs) isolated from 1- to 3-day-old pigs cured diabetic nude mice more than 14 weeks after transplantation. To shorten the latent period between transplantation and reversal of hyperglycemia, we investigated the effects of insulin-like growth factor-1 (IGF-1) and NPCCs isolated from 1-month-old pigs after transplantation. Pig pancreata were cut into fragments, collagenase digested, and then cultured. Three hundred and 2000 NPCCs were transplanted under the kidney capsule of nondiabetic and diabetic nude mice, respectively. After transplantation, the graft-bearing kidneys were removed to measure insulin content. NPCCs isolated from 1- to 3-day-old pigs were cultured with or without IGF-1 for 6 days. The stimulation index was not significantly different between the 2 groups at 1, 2, or 4 weeks. Moreover, at 4 weeks after transplantation of 300 NPCCs to nondiabetic nude mice yielded comparable graft insulin content as the recipients of NPCCs precultured with or without IGF-1. Two thousand cultured NPCCs isolated from 1-to 3-day-old pigs or 1-month-old pigs were transplanted into diabetic nude mice. The blood glucose levels of diabetic recipients in both groups decreased at the same rate after transplantation, achieving normoglycemia at 8 weeks. The graft insulin content at 12 weeks was not different between the 2 groups. Our data indicated that isolated NPCCs cultured with IGF-1 showed no beneficial effects on insulin secretion and transplantation; NPCCs isolated from 1-to 3-day-old and 1-month-old pigs displayed similar effects on transplantation.