Low glibenclamide concentrations affect endoplasmic reticulum stress in INS-1 cells under glucotoxic or glucolipotoxic conditions

BACKGROUND/AIMS: beta-Cell apoptosis caused by increased endoplasmic reticulum (ER) stress is an important pathogenic component of type 2 diabetes mellitus. In theory, sulfonylureas, used for the treatment of diabetes, can contribute to ER stress. We assessed changes in ER stress in pancreatic beta-cells under glucotoxic or glucolipotoxic conditions using low concentrations of the sulfonylurea, glibenclamide (GB). METHODS: Low concentrations of GB (10 or 100 nM) were added to INS-1 cells cultured under glucotoxic or glucolipotoxic conditions. The degree of viability, level of apoptosis and levels of markers associated with ER stress were measured. RESULTS: Apoptosis decreased in response to low concentrations of GB under glucolipotoxic but not glucotoxic conditions. Most ER stress markers decreased upon the addition of GB. Under glucotoxic conditions, changes in the levels of ER stress markers were not consistent. However, all decreased significantly under glucolipotoxic conditions. CONCLUSIONS: Low concentrations of GB exerted antiapoptotic effects through the attenuation of ER stress under glucolipotoxic conditions.

The Effects of Glyburide on Apoptosis and Endoplasmic Reticulum Stress in INS-1 Cells in a Glucolipotoxic Condition

BACKGROUND: beta-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglycemic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study examined ER stress of beta-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes. METHODS: Apoptosis was induced by adding various concentrations of GB (0.001 to 200 microM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated. RESULTS: Addition of any concentration of GB in 150 microM palmitate and 33 mM glucose did not increase apoptosis. The expression of phosphorylated eukaryotic initiation factor (eIF-2alpha) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly. CONCLUSION: GB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotoxic condition. Increased phosphorylation of eIF-2alpha may attenuate ER stress for adaptation to increased ER protein load.

The Effect of Atorvastatin and Simvastatin on NIS Expression of the TPC-1 Cell under the Therapeutic Blood Concentrations

BACKGROUND: Although so many experimental trials have been done to improve the redifferentiation and responsiveness of radioiodide therapy, they have not yet yielded any satisfactory results. As statins inhibit both farnesylation and geranylgeranylation, they have been reported to have an antineoplastic and redifferentiation effect in experimental and clinical studies. In this study, we investigated the relationship between statins and the alteration of the NIS expression and, TPC-1 cell apotosis to evaluate the possibility of using statins as adjuvant therapeutic agents for papillary thyroid cancer. METHODS: We used the TPC-1 cell lines for our experiments. Cell viabilities were measured by CCK-8. The degrees of apoptosis and, the expressions of NIS mRNA and NIS protein were measured by flow cytometry, semi quantitative RT-PCR and Western blot assay. RESULTS: Increased levels of NIS mRNA and NIS protein were observed under therapeutic blood concentrations (concentrations of simvastatin: 20, 50, 80 nM, concentrations of atorvastatin: 50, 80,110 nM), but the dose-response relationship was only manifested within simvastatin. The TPC-1 cells showed a concentration dependent decrease of viability and an increase of apoptosis not under therapeutic blood concentrations, but under excessively high concentrations (after treatment with 10-50 microM of atorvastatin and with 1-10 microM of simvastatin). CONCLUSION: The results of this study show that effective therapeutic blood concentrations of simvastatin and atorvastatin can give a favorable effect on the NIS expression under effective therapeutic blood concentrations. Therefore, we demonstrated the possibility that simvastatin and atorvastatin might have an important role as adjuvant therapeutic agents to improve the responsiveness of radioiodide therapy for papillary thyroid cancer. Further studies are needed to clarify this issue.

The Effect of Glucose Fluctuation on Apoptosis and Function of INS-1 Pancreatic Beta Cells / 당뇨병

BACKGROUND: Blood glucose level continuously fluctuates within a certain range in the human body. In diabetes patients, the extent of such fluctuation is large, despite the strict control of blood glucose. Blood glucose fluctuation has been shown to mediate more adverse effects on vascular endothelial cells and diabetes complications than chronic hyperglycemia, which has been explained as due to oxidative stress. As few previous studies have reported the effects of chronic and intermittent hyperglycemia on the apoptosis and function of pancreatic beta cells, this study reported herein was performed to investigate such effects on these cells. METHODS: For chronic hyperglycemia, INS-1 cells were cultured for 5 days with changes of RPMI 1640 medium containing 33 mM glucose every 12 hours. For intermittent hyperglycemia, the medium containing 11 mM glucose was exchanged with the medium containing 33 mM glucose every 12 hours. Apoptosis was assessed by TUNEL assay Hoechst staining and cleaved caspase 3. Insulin secretory capacity was assessed, and the expression of Mn-SOD and Bcl-2 was measured by Western blotting. RESULTS: In comparison to the control group, INS-1 cells exposed to chronic hyperglycemia and intermittent hyperglycemia showed an increase in apoptosis. The apoptosis of INS-1 cells exposed to intermittent hyperglycemia increased significantly more than the apoptosis of INS-1 cells exposed to chronic hyperglycemia. In comparison to the control group, the insulin secretory capacity in the two hyperglycemic states was decreased, and more with intermittent hyperglycemia than with chronic hyperglycemia. The expression of Mn-SOD and Bcl-2 increased more with chronic hyperglycemia than with intermittent hyperglycemia. CONCLUSION: Intermittent hyperglycemia induced a higher degree of apoptosis and decreased the insulin secretory capacity more in pancreatic beta cells than chronic hyperglycemia. This activity may be mediated by the anti-oxidative enzyme Mn-SOD and the anti-apoptotic signal Bcl-2.

The Protective Effect of EGCG on INS-1 Cell in the Oxidative Stress and Mechanism / 당뇨병

BACKGROUND: Oxidative stress is important in both diabetic complications and the development and the progression of type 2 diabetes via the effects on the pancreatic beta-cells. EGCG (epigallocatechin galleate), a major constituent of green tea, has been known to have beneficial effects on various diseases through the mechanisms of antioxidant and cell signaling modulation. But, very small numbers of studies were published about the direct effects of EGCG on the pancreatic beta cell lines. We performed this study to see the protective effect of EGCG on pancreatic beta cell line under H2O2 and the mechanisms of this phenomenon. METHODS: We used INS-1 cells and hydrogen peroxide as an oxidative stressor. Their viabilities were verified by MTT assay and FACS. The activity of glutathione peroxidase was assessed by total glutathione quantification kit. Western blot and semi-quantitative RT-PCR for the catalase, SOD (superoxide dismutase), PI3K and Akt were performed. Functional status of INS-1 cells was tested by GSIS (glucose stimulated insulin secretion). RESULTS: The biological effects of EGCG were different according to its concentrations. 10 micrometer EGCG effectively protected hydrogen peroxide induced damage in INS-1 cells. The expression and the activity of SOD, catalase and the glutathione peroxidase were significantly increased by EGCG. EGCG significantly increased PI3K and Akt activity and its effect was inhibited partially by wortmannin. GSIS was well preserved by EGCG. CONCLUSION: EGCG in low concentration effectively protected INS-1 cells from the oxidative stress through the activation of both antioxidant systems and anti-apoptosis signaling. Further studies will be necessary for the more detailed mechanisms and the clinical implications.

Cloning of Novel Epidermal Growth Factor (EGF) Plasmid for Gene Therapy on Diabetic Foot Ulcer / 당뇨병

BACKGROUND: Epidermal Growth Factor (EGF) is one of the important growth factors involved in the epithelialization during cutaneous wound healing. Peptide EGF has been used for the treatment of diabetic foot ulcer. But the inferiority of cost-effectiveness and the inconvenience of daily application might have restricted its wide clinical usage. EGF gene therapy could dramatically improve the efficacy and inconvenience through long-term expression and bypassing the EGF degradation by hostile non-specific proteinases expressed in the wound bed. METHODS: EGF DNAs were amplified via PCR. For the more effective secretion from the transfected cell, we inserted furin cleavage site into EGF plasmids. The efficacy of novel plasmid pbeta-EGF was verified by transfection into the various animal cell lines, and the biologic potency of expressed EGF was confirmed via phosphorylation of PI3K and GSK3beta by Western blotting. RESULTS: We tested various kinds of human EGFs. One of the human EGF isoforms, EGF(828) including a membrane-anchoring domain was successfully released as the mature EGF protein in the cell culture media. Also EGF plasmid including furin cleavage site showed more than 2-fold increased EGF expression compared with the sequence without furin cleavage site. CONCLUSION: In conclusion, these findings suggest that mature EGF could be released easily out of cells by modifying EGF DNA sequence. Our novel EGF plasmid DNA could markedly increase the efficiency of non-viral gene therapy for diabetic foot ulcer.

The Changes of Bone and Soft Tissue after Maxillary Anterior Segmental Osteotomy and Advancement Genioplasty

PURPOSE: Mid and lower facial convexity is more common in Oriental people than in Caucasian. Bimaxillary dentoalveolar protrusion is characterized by procumbent teeth, protruding lips, acute nasolabial angle, gummy smile, receding chin, facial convexity. Especially, pure maxillary dentoalveolar protrusion is less frequent than bimaxillary dentoalveolar protrusion. Therefore, it is important to make an accurate decision for the operation throughout the history taking, cephalogram, dental cast to arrive at accurate diagnosis and surgical plan. METHODS: From December 2002 to June 2004, ten patients with maxillary dentoalveolar protrusion and microgenia were corrected by maxillary anterior segmental osteotomy and advancement genioplasty. 10 patients were analyzed by preoperative and postoperative clinical photography, posteroanterior and lateral cephalograms. RESULTS: No major complications were occurred throughout the follow-up period except one of the over- recessed, otherwise most of the patients were satisfied with the result. CONCLUSION: We could correct the occulusal relationship with teeth and improve lower facial profile, asthetically and functionally, by maxillary anterior segmental osteotomy and advancement genioplasty.

Correction of the plunging nasal tip: 7CASES

The primary factors determining nasal tip outline are shape and position of the alar cartilages. The relation of the alar cartilages to the upper lateral cartilages, septum, and soft tissue envelope defines the conformation of the tip-lobule complex. Plunging tip is a condition as long nose, tip drooping and becomes more accentuated with aging. Hanging columella is a prominent downward bowing of the columella. Plunging tip and hanging columella are common causes of acute nasolabial angle.Correction of the plunging tip in the long nose is usually achieved satisfactorily through shortening of the lateral walls by excising an adequate amount of cartilage from the septal, lateral, and alar cartilage. Tip rotation in a cephalic direction can be achieved commonly by resection of the cephalic portion of the lateral crura, excision of a triangular segment of the caudal margin of the septum and a cartilaginous septal transfixion incision involving excision of a superiorly based triangle of septum with cephalic rotation of the entire nasal lobule.Modification of the caudal margin of the septum is done to achieve three goals: (1) cephalic rotation of the tip, (2) shortening of nasal length, and (3) alterations in the nasolabial angle. We have elevated and rotated in a cephalic direction nasal tip by means of resection of cephalic portion of lateral crura, resection of the caudal margin of the septum and mucous membrane, and /or invagination technique for correction of the plunging tip and hanging columella. From March 1996 to February 1998, we have performed the tip-plasty in 7 patients of the plunging tip and hanging columella. We have found good cosmetic results with improved nasolabial angle.

Study on powdered eggshell as new bone substitute for use in craniofacial surgery

The first successful bone transplantation carried out in 1688 by Van Meek'ren, a Dutch surgeon. The ideal bone substitute should be biocompatible, osteoinductive or at least osteoconductive, available in unlimited quantity, low cost and have satisfactory mechanical properties. To accomplish these goals, autografts are still preferred material. However, bone harvesting procedures have been focused to solve the following problems; necessity of a second surgical site, morbidity and potential deformity of the donor site, an increased operative time, donor availability limitations, and resorption of autogenic bone grafts.Ten adult white New Zealand rabbits, three, 10 mm in diameter, full layer skull defects were made in the frontoparietal bone. Two bone defects were filled with hydroxyapatite and powdered eggshell, the other defect was not filled(control). All animals were sacrified at 10 weeks, the specimens were examined macroscopically to test for graft mobility. The graft with surrounding bone was then harvested and studied by histology. The results were as follows: 1. Control: Bone regeneration occurred nearly complete. 2. Hydroxyapatite: Macroscopically - No encapsulation. Despite a gross delineation between implant and bone, imlant was firmly united to bone. Histology - Bony trabeculae surrounded by proliferated connective tissue are observed in the defect site and implant. Osteoblastic rimming is noted along the bone fragments 3. Powdered eggshell:Macroscopically - Grafted site was encapsulated by proliferated connective tissue and palpable softer than the surrounding bone. Histology - The presence of eggshell particles encapsulated by fibrous connective tissue. Partial bone regeneration from the defect margin was noticed, but the bone healing was never complete. In conclusion, the use of safe and inexpensive material is recommended for filling limited bone defects in non-weight bearing areas. The use of powdered eggshell for bone substitute may also be considered, after further studies, to access its long term stability, porosity and biocompatibility.