Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet ...transplantation has been proposed to be a potential treatment for type 1 diabetes. More recently, advances in human islet transplantation have further strengthened this view. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation. Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.
Increases in adiposity trigger metabolic and inflammatory changes that interfere with insulin action in peripheral tissues, culminating in beta cell failure and overt diabetes. We found that the cAMP ...Response Element Binding protein (CREB) is activated in adipose cells under obese conditions, where it promotes insulin resistance by triggering expression of the transcriptional repressor ATF3 and thereby downregulating expression of the adipokine hormone adiponectin as well as the insulin-sensitive glucose transporter 4 (GLUT4). Transgenic mice expressing a dominant-negative CREB transgene in adipocytes displayed increased whole-body insulin sensitivity in the contexts of diet-induced and genetic obesity, and they were protected from the development of hepatic steatosis and adipose tissue inflammation. These results indicate that adipocyte CREB provides an early signal in the progression to type 2 diabetes.
Most people with type 2 diabetes (T2D) have reduced beta-cell mass, and apoptosis is a key factor for this reduction. Previously, we showed that ATF3, an adaptive-response gene, is induced by various ...stress signals relevant to T2D, such as high glucose and high fatty acid. Because ATF3 is proapoptotic in beta-cells, we tested the hypothesis that ATF3 plays a detrimental role and contributes to the development of T2D. We compared wild-type (WT) and ATF3 knockout (KO) mice in an animal model for T2D, high-fat diet-induced diabetes. We also used INS-1 beta-cells and primary islets to analyze the roles of ATF3 in beta-cell function, including insulin gene expression and glucose-induced insulin secretion. Surprisingly, WT mice performed better in glucose tolerance test than KO mice, suggesting a protective, rather than detrimental, role of ATF3. At 12 wk on high-fat diet, no beta-cell apoptosis was observed, and the WT and KO mice had comparable beta-cell areas. However, ATF3 deficiency significantly reduced serum insulin levels in the KO mice without affecting insulin sensitivity, suggesting reduced beta-cell function in the KO mice. Analyses using INS-1 cells and primary islets support the notion that this defect is due, at least partly, to reduced insulin gene transcription in the KO islets without detectable reduction in glucose-induced calcium influx, a critical step for insulin secretion. In conclusion, our results support a model in which, before apoptosis becomes obvious, expression of ATF3 can be beneficial by helping beta-cells to cope with higher metabolic demand.
Pulmonary hypertension (PH) in patients with bronchopulmonary dysplasia (BPD) results from vasoconstriction and/or vascular remodeling, which can be regulated by mitogen-activated protein kinases ...(MAPKs). MAPKs are deactivated by dual-specificity phosphatases (DUSPs). We hypothesized that single-nucleotide polymorphisms (SNPs) in DUSP genes could be used to predict PH in BPD.
Preterm infants diagnosed with BPD (n = 188) were studied. PH was defined by echocardiographic criteria. Genomic DNA isolated from patient blood samples was analyzed for 31 SNPs in DUSP genes. Clinical characteristics and minor allele frequencies were compared between BPD-PH (cases) and BPD-without PH (control) groups. Biomarker models to predict PH in BPD using clinical and SNP data were tested by calculations of area under the ROC curve.
In our BPD cohort, 32% (n = 61) had PH. Of the DUSP SNPs evaluated, DUSP1 SNP rs322351 was less common, and DUSP5 SNPs rs1042606 and rs3793892 were more common in cases than in controls. The best fit biomarker model combines clinical and DUSP genetic data with an area under the ROC curve of 0.76.
We identified three DUSP SNPs as potential BPD-PH biomarkers. Combining clinical and DUSP genetic data yields the most robust predictor for PH in BPD.
Identified over a dozen years ago in the brain and pancreatic islet, β IV-spectrin is critical for the local organization of protein complexes throughout the nervous system. β IV-Spectrin targets ion ...channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and β IV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in β IV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that β IV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that β IV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, β IV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, β IV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKIIδ. β IV-Spectrin–targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of K ATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of K ATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in K ATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant K ATP channel phosphorylation in β IV-spectrin mutant mice. In summary, our findings establish a broader role for β IV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of K ATP channels.
Aim
Pulmonary hypertension (PH) develops in 25–40% of bronchopulmonary dysplasia (BPD) patients, substantially increasing mortality. We have previously found that asymmetric dimethylarginine (ADMA), ...an endogenous inhibitor of nitric oxide (NO) production, is elevated in patients with BPD‐associated PH. ADMA is metabolised by Nᴳ,Nᴳ‐dimethylarginine dimethylaminohydrolase (DDAH). Presently, we test the hypothesis that there are single nucleotide polymorphisms (SNPs) in DDAH1 and/or DDAH2 associated with the development of PH in BPD patients.
Methods
BPD patients were enrolled (n = 98) at Nationwide Children's Hospital. Clinical characteristics and 36 SNPs in DDAH1 and DDAH2 were compared between BPD‐associated PH patients (cases) and BPD‐alone patients (controls).
Results
In BPD patients, 25 (26%) had echocardiographic evidence of PH (cases). In this cohort, DDAH1 wild‐type rs480414 was 92% sensitive and 53% specific for PH in BPD, and the DDAH1 SNP rs480414 decreased the risk of PH in an additive model of inheritance (OR = 0.39; 95% CI 0.18–0.88, p = 0.01).
Conclusion
The rs480414 SNP in DDAH1 may be protective against the development of PH in patients with BPD. Furthermore, the DDAH1 rs480414 may be a useful biomarker in developing predictive models for PH in patients with BPD.
beta-Cell failure is an essential component of all types of diabetes, and the insulin receptor substrate 2 (IRS2) branch of signaling plays a key role in beta-cell survival and function. We tested ...the hypothesis that activating transcription factor 3 (ATF3), a stress-inducible proapoptotic gene, downregulates the expression of IRS2 in beta-cells.
We used both the gain- and loss-of-function approaches to test the effects of ATF3 on IRS2 gene expression. We also analyzed the binding of ATF3 to the IRS2 promoter by chromatin immunoprecipitation assay and the transcription of the IRS2 gene by polymerase II occupancy assay. Furthermore, we tested the ability of IRS2 to alleviate the proapoptotic effects of ATF3 in cultured beta-cells and in transgenic mice using the rat insulin promoter to drive the transgenes.
Expression of ATF3 is sufficient to reduce IRS2 gene expression; in contrast, knockdown or knockout of ATF3 reduces the ability of stress signals to downregulate IRS2 expression. ATF3 binds to the IRS2 promoter in vivo, and the binding of ATF3 correlates with decreased IRS2 gene transcription. Functionally, expression of IRS2 protects beta-cells from ATF3-induced apoptosis.
IRS2 is a target gene of ATF3, and its repression by ATF3 contributes, at least partly, to the apoptosis induced by ATF3. Because ATF3 is a stress-inducible gene, our work provides a direct link to explain how environmental stress factors can modulate IRS2 gene transcription.
► Similarities and difference in the phenotypes of genetic deletion ghrelin and GOAT knockout mouse lines are reported. ► Overlapping expression patterns are discussed. ► Expression pattern of ...ghrelin and GOAT suggests that additional peptides may be acylated by GOAT.
Ghrelin is a gastric hormone that has been shown to regulate food intake and energy metabolism. One unique feature of ghrelin is that its activity is regulated post transcriptionally by ghrelin O-acyltransferase (GOAT) through the addition of fatty acid to the serine residue in the N terminal region. Despite much biochemical characterization, to date no other proteins have been shown to be specifically octonylated by GOAT, suggesting a unique matching of the acyl transferase for a single ligand, ghrelin. If this is indeed correct, then genetic deletion of ghrelin or GOAT should produce near identical phenotypes and there should be extensive overlap in expression patterns. This review summarizes the similarities and differences in the phenotypes with the genetic deletion of ghrelin and GOAT in the various knockout mouse lines reported to date. While there is considerable overlap in expression pattern between ghrelin and GOAT, the latter does exhibit some unique tissue expression that could suggest that additional peptides may be acylated and await discovery and characterization.
Wolf-Hirschhorn syndrome (WHS) is a rare but recurrent microdeletion syndrome associated with hemizygosity of an interstitial segment of Chromosome 4 (4p16.3). Consistent with historical reports in ...which overlapping deletions defined a minimal critical region in WHS patients, recent reports from exome sequence analysis have provided further evidence that haploinsufficiency of a specific gene within this critical region,
(
), is causal for many features of the syndrome. In this report, we describe three unrelated patients with loss-of-function alterations in
who presented clinically with WHS features including intrauterine growth retardation and global developmental delay. Two of the three patients also had overlapping features of failure to thrive, short stature, constipation, and hypotonia. This series adds additional cases to expand the phenotypic spectrum of WHS and reports novel
variants.
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