Using recombinase-mediated cassette exchange to engineer MIN6 insulin-secreting cells based on a newly identified safe harbor locus.

AIMS/INTRODUCTION: Recent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin-secreting MIN6 cells. MATERIALS AND METHODS: MIN6 cells were first engineered to have a tetracycline-regulated expression system. Then, we used the recombination-mediated cassette exchange strategy to explore the silencing-resistant site in the genome and generated a master cell line based on this site. RESULTS: We identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt-based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over- or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively. CONCLUSIONS: We engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion.

Usefulness of Post-labeling Delay for the Assessment of Bright Vessel Appearance by Arterial Spin Labeling.

Objective: This study was performed to clarify the differences in blood flow strength, blood vessel diameter, and post-labeling delay (PLD) by physical experiments, and to examine whether bright vessel appearance (BVA) can be observed by arterial spin labeling (ASL). Methods: We introduced simulated blood flow (25 cm/sec, 12.5 cm/sec) using a specially made phantom of fixed tubes in a plastic container. At each speed, we scanned at several points of PLD using ASL imaging. We measured the signal in the tube to obtain a signal intensity (SI). We revised the T1 level from the SI and obtained SIblood. We used SItissue with normal perfusion measured from obtained clinical images by ASL and compared it with SIblood. Results: In tubes with a narrow inner diameter, the signal slightly decreased. SI also decreased under slow flow compared with fast flow. At each flow rate, SIblood significantly exceeded SItissue. Conclusion: PLD distinguishes spin in brain tissue from 1525 msec to 2525 msec, and it can be observed. As spin signal decreases when the flow rate is slow, attention is necessary for observation. Assessment at PLD1525-2525 msec where normal perfusion was obtained suggested that BVA can be observed.

Hemichorea after hypoglycemic episodes with negative MRI findings in an elderly woman with poorly controlled type 2 diabetes mellitus: a case report.

BACKGROUND: Diabetic chorea appears during the course of poorly-controlled diabetes. While chorea associated with diabetes mellitus usually occurs during hyperglycemic episodes, hypoglycemia can also cause diabetic chorea. Brain magnetic resonance imaging (MRI) is useful for evaluating the pathogenesis of diabetic chorea. However, several diabetic chorea cases have reportedly not shown abnormal high-intensity in the putamen and striatum on T1-weighted images. CASE PRESENTATION: We report a 74-year-old woman who was admitted to our hospital for treatment of poorly-controlled type 2 diabetes mellitus. Intensified insulin treatment gradually normalizeed blood glucose, but on the 19th hospital day, after a blood glucose measurement of 49 mg/dL, she showed hemichorea of the left face, shoulder, arm and leg. MRI revealed no abnormalities of either the putamen or the striatum on T1-weighted images. She was treated with dopamine receptor antagonists, which alleviated her hemichorea symptoms and allowed discharge from the hospital. 1 year after the first hospitalization, she had to be readmitted because her glycemic control had markedly deteriorated. Glycemic control improved rapidly, and, because hemichorea did not recur, the dopamine receptor antagonists were stopped. 1 month later, however, hemichorea recurred. She resumed taking the dopamine receptor antagonists, resulting in immediate disappearance of the hemichorea. CONCLUSIONS: We herein describe a rare case of diabetes-associated hemichorea occurring after hypoglycemic episodes without abnormal high-intensity findings in the basal ganglia on T1-weighted images. The hemichorea relapsed with cessation of dopamine receptor antagonists. This case also underscores the importance of longitudinal assessment and treatment for hemichorea after hypoglycemic episodes, even in the absence of MRI findings, in elderly diabetic patients.

Elsberg syndrome related to varicella zoster virus infection with painless skin lesions in an elderly woman with poorly controlled type 2 diabetes mellitus.

Patients with diabetes mellitus (DM) are at increased risk of infections, with the urinary tract being the most frequent infection site. Incomplete bladder emptying, frequent urination and abdominal distension are typical symptoms of urinary tract infections (UTIs). A 68-year-old female with a long history of poorly controlled type 2 DM (T2DM) visited our hospital complaining of urinary retention, which was initially diagnosed as cystitis by another doctor. The urologist at our hospital identified a skin rash extending from the left hip to her genital area. A dermatologist was consulted. She was clinically diagnosed with herpes zoster (HZ) involving the left sacral dermatome area. As Elsberg syndrome (ES) was suspected, a lumbar puncture was performed, revealing aseptic meningitis associated with varicella zoster virus (VZV) infection. Intravenous acyclovir with urinary catheterization in combination with methylprednisolone pulse therapy resulted in a good clinical course. HZ very uncommonly involves sacral dermatomes, but it can develop in patients with prolonged poorly controlled DM. Furthermore, early diagnosis can be difficult when patients have diabetic peripheral neuropathy, which may mask symptoms related to skin lesions. Because this disease is potentially severe, detailed examination is important for clinicians managing patients with DM who have complaints indicative of urinary tract disorders.

Hemodynamic ischemia of the anterior choroidal artery and reversal by carotid artery stenting 10 years after ipsilateral superficial temporal artery-middle cerebral artery bypass for symptomatic left middle cerebral artery stenosis.

The pathogenesis of anterior choroidal artery (AChA) territory infarction includes various mechanisms, but hemodynamic causes are rare and difficult to diagnose. 77- year-old man, who had moderate left ICA stenosis and he had treated with STA-MCA bypass surgery for severe symptomatic left MCA stenosis 10 years earlier, was admitted with right hemiparesis and confused state. On admission, magnetic resonance imaging and angiography demonstrated patent bypass, but severe stenosis of left ICA with no opacification of the left AChA and A1 portion of the left ACA. Diffusionweighted imaging demonstrated ischemic lesion in the left corona radiata. Together with clinical findings, hemodynamic ischemia of the AChA region was suspected and left carotid artery stenting resulted in prompt improvement of symptoms. Hemodynamic ischemia of the AChA territory is rare, however, should be considered as a potential target of treatment when the ipsilateral ICA, A1 and M1 show stenoocclusive lesions.

The prolyl isomerase Pin1 increases ß-cell proliferation and enhances insulin secretion.

The prolyl isomerase Pin1 binds to the phosphorylated Ser/Thr-Pro motif of target proteins and enhances their cis-trans conversion. This report is the first to show that Pin1 expression in pancreatic ß cells is markedly elevated by high-fat diet feeding and in ob/ob mice. To elucidate the role of Pin1 in pancreatic ß cells, we generated ß-cell-specific Pin1 KO (ßPin1 KO) mice. These mutant mice showed exacerbation of glucose intolerance but had normal insulin sensitivity. We identified two independent factors underlying impaired insulin secretion in the ßPin1 KO mice. Pin1 enhanced pancreatic ß-cell proliferation, as indicated by a reduced ß-cell mass in ßPin1 KO mice compared with control mice. Moreover, a diet high in fat and sucrose failed to increase pancreatic ß-cell growth in the ßPin1 KO mice, an observation to which up-regulation of the cell cycle protein cyclin D appeared to contribute. The other role of Pin1 was to activate the insulin-secretory step: Pin1 KO ß cells showed impairments in glucose- and KCl-induced elevation of the intracellular Ca2+ concentration and insulin secretion. We also identified salt-inducible kinase 2 (SIK2) as a Pin1-binding protein that affected the regulation of Ca2+ influx and found Pin1 to enhance SIK2 kinase activity, resulting in a decrease in p35 protein, a negative regulator of Ca2+ influx. Taken together, our observations demonstrate critical roles of Pin1 in pancreatic ß cells and that Pin1 both promotes ß-cell proliferation and activates insulin secretion.

Calibration of Smartphone-Based Weather Measurements Using Pairwise Gossip.

Accurate and reliable daily global weather reports are necessary for weather forecasting and climate analysis. However, the availability of these reports continues to decline due to the lack of economic support and policies in maintaining ground weather measurement systems from where these reports are obtained. Thus, to mitigate data scarcity, it is required to utilize weather information from existing sensors and built-in smartphone sensors. However, as smartphone usage often varies according to human activity, it is difficult to obtain accurate measurement data. In this paper, we present a heuristic-based pairwise gossip algorithm that will calibrate smartphone-based pressure sensors with respect to fixed weather stations as our referential ground truth. Based on actual measurements, we have verified that smartphone-based readings are unstable when observed during movement. Using our calibration algorithm on actual smartphone-based pressure readings, the updated values were significantly closer to the ground truth values.

Octreotide for hypoglycemia caused by sulfonylurea and DPP-4 inhibitor.

We describe a type 2 diabetes patient with persistent hypoglycemia caused by sulfonylurea misuse on top of a DPP-4 inhibitor. Hyperinsulinemia was exaggerated by dextrose administration, but was successfully treated with octreotide. Since many patients are currently treated with DPP-4 inhibitors, the importance of octreotide has been increasing. For refractory sulfonylurea-induced hypoglycemia, especially when the patient is also being given an incretin-based therapy, octreotide should be considered.

Addition of sitagliptin or metformin to insulin monotherapy improves blood glucose control via different effects on insulin and glucagon secretion in hyperglycemic Japanese patients with type 2 diabetes.

This study aimed to explore the effects of the dipeptidyl peptidase-4 inhibitor sitagliptin and the biguanide metformin on the secretion of insulin and glucagon, as well as incretin levels, in Japanese subjects with type 2 diabetes mellitus poorly controlled with insulin monotherapy. This was a single-center, randomized, open-label, parallel group study, enrolling 25 subjects. Eleven patients (hemoglobin A1c [HbA1c] 8.40 ± 0.96%) and 10 patients (8.10 ± 0.54%) on insulin monotherapy completed 12-week treatment with sitagliptin (50 mg) and metformin (750 mg), respectively. Before and after treatment, each subject underwent a meal tolerance test. The plasma glucose, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), C-peptide, and glucagon responses to a meal challenge were measured. HbA1c reductions were similar in patients treated with sitagliptin (0.76 ± 0.18%) and metformin (0.77 ± 0.17%). In the sitagliptin group, glucose excursion during a meal tolerance test was reduced and accompanied by elevations in active GLP-1 and active GIP concentrations. C-peptide levels were unaltered despite reduced glucose responses, while glucagon responses were significantly suppressed (-7.93 ± 1.95% of baseline). In the metformin group, glucose excursion and incretin responses were unaltered. C-peptide levels were slightly increased but glucagon responses were unchanged. Our data indicate that sitagliptin and metformin exert different effects on islet hormone secretion in Japanese type 2 diabetic patients on insulin monotherapy. A glucagon suppressing effect of sitagliptin could be one of the factors improving blood glucose control in patients inadequately controlled with insulin therapy.

Data delivery method based on neighbor nodes' information in a mobile ad hoc network.

This paper proposes a data delivery method based on neighbor nodes' information to achieve reliable communication in a mobile ad hoc network (MANET). In a MANET, it is difficult to deliver data reliably due to instabilities in network topology and wireless network condition which result from node movement. To overcome such unstable communication, opportunistic routing and network coding schemes have lately attracted considerable attention. Although an existing method that employs such schemes, MAC-independent opportunistic routing and encoding (MORE), Chachulski et al. (2007), improves the efficiency of data delivery in an unstable wireless mesh network, it does not address node movement. To efficiently deliver data in a MANET, the method proposed in this paper thus first employs the same opportunistic routing and network coding used in MORE and also uses the location information and transmission probabilities of neighbor nodes to adapt to changeable network topology and wireless network condition. The simulation experiments showed that the proposed method can achieve efficient data delivery with low network load when the movement speed is relatively slow.

Atf6α-null mice are glucose intolerant due to pancreatic ß-cell failure on a high-fat diet but partially resistant to diet-induced insulin resistance.

Activating transcription factor 6α (ATF6α) is essential for the endoplasmic reticulum (ER) stress response. Since recent studies suggested that ER stress is involved in the pathogenesis of type 2 diabetes mellitus, we have analyzed Atf6α-null (Atf6α(-/-)) mice challenged with metabolic overload or genetic manipulations. Atf6α(-/-) mice were fed a high-fat diet to create diet-induced obese (DO) mice, and were subjected to examination of glucose homeostasis with biochemical and morphological analysis of the pancreatic ß-cell and liver tissues. Atf6α-null mice were also crossed with genetic models of diabetes caused either by insulin resistance (Agouti obese mice) or by impaired insulin secretion (Ins2(WT/C96Y) mice). Atf6α(-/-) DO mice were less glucose tolerant with blunted insulin secretion compared to littermates on a high-fat diet. Pancreatic insulin content was lower in Atf6α(-/-) DO mice with the swollen ß-cell ER, a typical feature of cells with ER stress. In the liver of Atf6α(-/-) DO mice, XBP-1 splicing was increased, suggesting that higher ER stress was present. ATF6-deficient mice showed increased mRNA expressions of glucose-6-phosphatase and SREBP1c associated with a tendency for a higher degree of steatosis in the liver. However, Atf6α(-/-) DO mice exhibited higher insulin sensitivity with lower serum triglyceride levels. Similar phenotypes were observed in ATF6α-deficient Agouti mice. In addition, ATF6α-deficiency accelerated reduction in pancreatic insulin content in Ins2(WT/C96Y) mice. These data suggested that ATF6α contributes to both prevention and promotion of diabetes; it protects ß-cells from ER stress and suppresses hepatosteatosis, but plays a role in the development of hyperlipidemia and insulin resistance.

Involvement of endoplasmic stress protein C/EBP homologous protein in arteriosclerosis acceleration with augmented biological stress responses.

BACKGROUND: The processes of arteriosclerosis, including atherosclerosis and vascular remodeling, are affected by interactions among numerous biological pathways such as responses to inflammation, oxidative stress, and endoplasmic reticulum stress. C/EBP homologous protein (CHOP), which is well known to induce cellular apoptosis in response to severe endoplasmic reticulum stress, is reportedly upregulated in plaque lesions. METHODS AND RESULTS: We examined the effects of CHOP deficiency on 2 types of arteriosclerosis: cuff injury-induced neointimal formation and hypercholesterolemia-induced atherosclerosis. Cuff injury-induced neointimal formation was markedly inhibited in CHOP(-/-) mice with suppressed aortic expression of inflammatory factors and smooth muscle cell proliferation-related proteins. A CHOP deficiency also inhibited aortic plaque formation in hypercholesterolemic apolipoprotein E(-/-) mice with suppressed aortic expression of inflammatory factors and oxidative stress markers. Bone marrow transplantation experiments revealed that recipient CHOP deficiency significantly suppressed both cuff injury-induced neointimal formation and hypercholesterolemia-induced atherosclerotic plaque formation to a greater extent than donor CHOP deficiency, suggesting the importance of CHOP in vascular cells for arteriosclerosis progression. Furthermore, in our in vitro experiments, in not only macrophages but also endothelial and smooth muscle cell lines, endoplasmic reticulum stress inducers upregulated inflammation-, adhesion-, or smooth muscle cell proliferation-related proteins, whereas decreased CHOP expression remarkably suppressed endoplasmic reticulum stress-induced upregulation of these proteins. CONCLUSIONS: In addition to the well-known signaling for apoptosis induction, CHOP may play important roles in augmenting potentially pathological biological stress responses. This noncanonical role of CHOP, especially that expressed in vascular cells, may contribute to the progression of vascular remodeling and atherosclerosis.

The JNK pathway modulates expression and phosphorylation of 4E-BP1 in MIN6 pancreatic beta-cells under oxidative stress conditions.

Stress-mediated apoptosis may play a crucial role in loss of pancreatic beta-cell mass, contributing to the development of diabetes. We have recently identified that translational control involving the translational suppressor eIF4E binding protein-1 (4E-BP1) which is important for beta-cell survival under endoplasmic reticulum (ER) stress. The Eif4ebp1 gene, encoding 4E-BP1, is a direct target of a transcription factor activating transcription factor-4 (ATF4), a master regulator of gene expression in stress responses. In the current study, we investigated 4E-BP1 expression in mouse insulinoma line 6 (MIN6) cells treated with arsenite, an inducer of oxidative stress which is another contributor of beta-cell loss. We found that arsenite-induced 4E-BP1 expression level was lower than that induced by thapsigargin, an ER stress inducer, although ATF4 was similarly induced by these agents. The ratio of the dephosphorylated form of 4E-BP1, which has the highest activity, to phosphorylated forms was, however, greater in MIN6 cells treated with arsenite as compared to that in thapsigargin-treated cells. Arsenite-induced 4E-BP1 mRNA and protein expressions were augmented by simultaneous treatment with a c-Jun N-terminal kinase (JNK) specific inhibitor, SP600125. The agent also suppressed the level of the dephosphrylated form of 4E-BP1 in arsenite-treated MIN6 cells. Thus, JNK activated by oxidative stress is involved in the modulation of 4E-BP1 expression and phosphorylation in MIN6 cells, which may contribute to fine tuning of translational control under stress conditions.

ATF4-mediated induction of 4E-BP1 contributes to pancreatic beta cell survival under endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress-mediated apoptosis may play a crucial role in loss of pancreatic beta cell mass, contributing to the development of diabetes. Here we show that induction of 4E-BP1, the suppressor of the mRNA 5' cap-binding protein eukaryotic initiation factor 4E (eIF4E), is involved in beta cell survival under ER stress. 4E-BP1 expression was increased in islets under ER stress in several mouse models of diabetes. The Eif4ebp1 gene encoding 4E-BP1 was revealed to be a direct target of the transcription factor ATF4. Deletion of the Eif4ebp1 gene increased susceptibility to ER stress-mediated apoptosis in MIN6 beta cells and mouse islets, which was accompanied by deregulated translational control. Furthermore, Eif4ebp1 deletion accelerated beta cell loss and exacerbated hyperglycemia in mouse models of diabetes. Thus, 4E-BP1 induction contributes to the maintenance of beta cell homeostasis during ER stress and is a potential therapeutic target for diabetes.

Direct correlation between ischemic injury and extracellular glycine concentration in mice with genetically altered activities of the glycine cleavage multienzyme system.

BACKGROUND AND PURPOSE: Ischemia elicits the rapid release of various amino acid neurotransmitters. A glutamate surge activates N-methyl-d-aspartate (NMDA) glutamate receptors, triggering deleterious processes in neurons. Although glycine is a coagonist of the NMDA receptor, the effect of extracellular glycine concentration on ischemic injury remains controversial. To approach this issue, we examined ischemic injury in mice with genetically altered activities of the glycine cleavage multienzyme system (GCS), which plays a fundamental role in maintaining extracellular glycine concentration. METHODS: A mouse line with increased GCS activity (340% of C57BL/6 control mice) was generated by transgenic expression of glycine decarboxylase, a key GCS component (high-GCS mice). Another mouse line with reduced GCS activity (29% of controls) was established by transgenic expression of a dominant-negative mutant of glycine decarboxylase (low-GCS mice). We examined neuronal injury after transient occlusion of the middle cerebral artery in these mice by measuring extracellular amino acid concentrations in microdialysates. RESULTS: High-GCS and low-GCS mice had significantly lower and higher basal concentrations of extracellular glycine than did controls, respectively. In low-GCS mice, the extracellular glycine concentration reached 2-fold of control levels during ischemia, and infarct volume was significantly increased by 69% with respect to controls. In contrast, high-GCS mice had a significantly smaller infarct volume (by 21%). No significant difference was observed in extracellular glutamate concentrations throughout the experiments. An antagonist for the NMDA glycine site, SM-31900, attenuated infarct size, suggesting that glycine operated via the NMDA receptor. CONCLUSIONS: There is a direct correlation between ischemic injury and extracellular glycine concentration maintained by the GCS.

Efficient and controlled gene expression in mouse pancreatic islets by arterial delivery of tetracycline-inducible adenoviral vectors.

Gene transfer with adenovirus vectors has been used extensively for pancreatic islet research. However, infection efficiency varies among reports. We reevaluated the infection efficiency, defined here as the percentage of islet cells expressing transgenes, in mouse islets. When the isolated islets were infected with adenoviruses, the infection efficiency was found to be 30-40% and the transduced cells were distributed in the islet periphery. Collagenase treatment of isolated islets before infection increased the infection efficiency to 70%, but with suppression of glucose-stimulated insulin secretion. To explore more efficient strategies, we employed arterial delivery of virus particles to islets in situ. Delivery of adenovirus (approximately 10(8) particles per pancreas) through the celiac and superior mesenteric arteries is highly efficient, resulting in more than 90% transduction without impairing glucose-stimulated insulin secretion. Arterial delivery of an adenovirus harboring glycerol kinase cDNA allowed us to observe glycerol-stimulated insulin secretion from mouse islets, which was not observed when we employed the conventional method. Furthermore, the arterial delivery method combined with a tetracycline-inducible adenovirus system induced efficient and controlled transgene expression. Our data provide new insights into gene transduction methods using recombinant adenoviruses in mouse islets, and are therefore anticipated to contribute to future basic and clinical islet research applications.

WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum.

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.

WFS1-deficiency increases endoplasmic reticulum stress, impairs cell cycle progression and triggers the apoptotic pathway specifically in pancreatic beta-cells.

Wolfram syndrome, an autosomal recessive disorder associated with diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein. Herein, we report that pancreatic islets of wfs1-deficient mice exhibit increases in phosphorylation of RNA-dependent protein kinase-like ER kinase, chaperone gene expressions and active XBP1 protein levels, indicating an enhanced ER stress response. We established wfs1-deficient MIN6 clonal beta-cells by crossing wfs1-deficient mice with mice expressing simian virus 40 large T antigen in beta-cells. These cells show essentially the same alterations in ER stress responses as wfs1-deficient islets, which were reversed by re-expression of WFS1 protein or overexpression of GRP78, a master regulator of the ER stress response. In contrast, these changes are not observed in heart, skeletal muscle or brown adipose tissues with WFS1-deficiency. The increased ER stress response was accompanied by reduced BrdU incorporation and increased caspase-3 cleavage, indicating impaired cell cycle progression and accelerated apoptotic processes in the mutant islets. These changes are associated with increased expression of the cell cycle regulator p21(CIP1) in wfs1-deficient islets and clonal beta-cells. Treatment of islets with thapsigargin, an ER stress inducer, caused upregulation of p21(CIP1). In addition, forced expression of p21(CIP1) resulted in reduced MIN6 beta-cell numbers, suggesting the ER stress-induced increase in p21(CIP1) expression to be involved in beta-cell loss in the mutant islets. These data indicate that WFS1-deficiency activates the ER stress response specifically in beta-cells, causing beta-cell loss through impaired cell cycle progression and increased apoptosis.