Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

BACKGROUND: Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. METHODS: PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. RESULTS: Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. CONCLUSION: Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.

Co-Culture of alpha TC-6 Cells and beta TC-1 Cells: Morphology and Function

BACKGROUND: In vitro experiments using only beta-cell lines instead of islets are limited because pancreatic islets are composed of four different types of endocrine cells. Several recent studies have focused on cellular interactions among these cell types, especially alpha- and beta-cells. Because islet isolation needs time and experience, we tested a simple co-culture system with alpha- and beta-cells. Their morphology and function were assessed by comparison to each single cell culture and pancreatic islets. METHODS: alpha TC-6 cells and beta TC-1 cells were maintained in Dulbecco's Minimal Essential Medium containing 5 mM glucose and 10% fetal bovine serum. Cells were mixed at a 1:1 ratio (5x10(5)) in 6-well plates and cultured for 24, 48, and 72 hours. After culture, cells were used for insulin and glucagon immunoassays and tested for glucose-stimulated insulin secretion (GSIS). RESULTS: alpha TC-6 and beta TC-1 cells became condensed by 24 hours and were more strongly compacted after 48 hours. beta TC-1 cells showed both beta-beta and beta-alpha cell contacts. GSIS increased with increasing glucose concentration in co-cultured cells, which showed lower secreted insulin levels than beta TC-1 cells alone. The increase in the secreted insulin/insulin content ratio was significantly lower for co-cultured cells than for beta-cells alone (P=0.04). Compared to islets, the alpha-/beta-cell co-culture showed a higher ratio of GSIS to insulin content, but the difference was not statistically significant (P=0.09). CONCLUSION: alpha TC-6 and beta TC-1 cells in the co-culture system showed cell-to-cell contacts and a similar stimulated insulin secretion pattern to islets. The co-culture system may be used to better mimic pancreatic islets in in vitro assessments.

The value of the mean peak systolic velocity of the superior thyroidal artery in the differential diagnosis of thyrotoxicosis

PURPOSE: The aim of this study was to validate the superior thyroidal artery mean peak systolic velocity (STA-mPSV) as an alternative to other diagnostic parameters in the differentiation of the causes of thyrotoxicosis in Korean patients. METHODS: This study was conducted with newly diagnosed and untreated thyrotoxic patients. Forty patients were diagnosed with Graves disease (GD) and 20 patients with destructive thyroiditis (DT). Another 60 healthy subjects without thyroid disease participated as the control group. Blood samples were taken to evaluate the thyroid function and thyroid autoantibodies (TRAb). Twenty-four hour radioactive iodine uptake (RAIU) scanning was performed to confirm GD or DT. The STA-mPSV was measured using color Doppler ultrasonography. RESULTS: The STA-mPSV was significantly higher in the untreated GD group than in the DT group (GD, 78.96+/-29.04 cm/sec; DT, 29.97+/-14.67 cm/sec; control, 17.55+/-4.99 cm/sec; P<0.001). The area under the curve (AUC) of the STA-mPSV for the differential diagnosis of untreated GD and DT was 0.9506 (optimal cutoff value, 41.3 cm/sec; sensitivity, 95%, 38/40; specificity, 85%, 17/20) in the receiver operating characteristic analysis. The AUC values of the STA-mPSV, RAIU, and TRAb were 0.9506, 1, and 0.9988, respectively (P=0.159). CONCLUSION: In clinical practice, the STA-mPSV has a diagnostic value similar to that of the TRAb and 24-hour RAIU in the differential diagnosis of newly diagnosed Korean thyrotoxic patients.

Thyrotoxic Periodic Paralysis Induced by Dexamethasone

Thyrotoxic periodic paralysis (TPP) is a disease characterized by sudden onset and muscle paralysis. It occurs in the setting of hypokalemia of thyrotoxicosis. Cases of TPP induced by a glucocorticoid such as prednisolone or methylprednisolone have been reported. We report on two patients, each of whom received a dexamethasone injection and subsequently developed TPP. Both patients experienced sudden, flaccid paralysis of both extremities after the injection but recovered completely after receiving a potassium replacement. Laboratory results revealed thyrotoxicosis. The patients were diagnosed with Graves' disease and discharged after receiving treatment with methimazole and propranolol. This report provides the clinical description of TPP induced by dexamethasone injection. These cases suggest that clinicians must consider the presence of hyperthyroid disease in patients who develop acute paralysis after treatment with a glucocorticoid, even in the absence thyrotoxic symptoms. Furthermore, physicians should be aware that TPP can occur even in response to dexamethasone used for treatment of thyrotoxic crisis or Graves' ophthalmopathy.

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.

Effect of High Dose External Irradiation on the Matrix Metalloprotease-2 Expression in a Rat Carotid Artery Injury Model

BACKGROUND AND OBJECTIVES: Remodeling of the injured arterial wall is dependent on the action of several extracellular proteases, including matrix metalloprotease-2 (MMP-2), and this protein is associated with the migration of vascular smooth muscle cells. The effect of a high dose of external irradiation (20 Gy) on the MMP-2 expression in neointimal hyperplasia is not known. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to balloon injury to the common carotid artery. At 24 hours after injury, 20 Gy external irradiation was done for the irradiated group (n=25) and this was not done for the control group (n=25). The percent area stenosis, the maximal intimal thickness, the intima/media area ratio on H-E staining and the MMP-2 positivity on the immunohistochemical staining were measured. Western blotting and a gelatin zymogram for determining the MMP-2 protein expression were also performed after the injury. RESULTS: The parameters of neointimal hyperplasia such as the percent area stenosis, the maximal intimal thickness and the intima/media area ratio were 40.2+/-12.1%, 0.30+/-0.12 mm and 1.27+/-0.32, respectively, at 14 days after injury, and these parameters were maintained as a hyperplastic state at 28 days after injury in the control group. There was undetectable neointimal hyperplasia in the irradiated group compared with the control group (p<0.01). Western blotting demonstrated an increase in the MMP-2 protein level beginning 2 to 4 days after injury in the control group, but there was only a transient increase in the MMP-2 level at day 2 after injury in the irradiated group. The gelatin zymogram and immunohistochemical staining also showed the expression of MMP-2 in the control group, but not in the irradiated group. CONCLUSION: These findings suggest the suppressed expression of MMP-2 is associated with reduced neointimal hyperplasia in the balloon injury-rat model.

Male Polythelia

Supernumerary nipples or polythelia are developmental abnormalities located along the embryonic mammary lines. It occurs sporadically but familial aggregation was been reported. Polythelia has been reported in association with congenital malformations, in particular with renal anomalies. We report a case of polythelia.