The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the beta cells of hypox compared with normal, age-matched rats whereas glucagon in alpha cells and somatostatin in delta cells showed increase. IGF-I, which localized to alpha cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a "low IGF-I state" affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I production

Structural basis for delta cell paracrine regulation in pancreatic islets

International audienceLittle is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.publishedVersio

Effects of insulin--like growth factor-I treatment on the endocrine pancreas of hypophysectomized rats: comparison with growth hormone replacement

BACKGROUND: In GH-deficient humans, GH and IGF-I treatment cause opposite effects on serum insulin concentrations and insulin sensitivity. This finding contrasts with the somatomedin hypothesis that IGF-I mediates GH action, as postulated for skeletal growth, and raises the question whether GH-induced IGF-I acts on the endocrine pancreas in the same way as administered IGF-I. OBJECTIVE: To compare the effects of the two hormones on the endocrine pancreas of hypophysectomized rats. METHODS: Animals were infused for 2 days, via miniosmotic pumps, with IGF-I (300 microg/day), GH (200 mU/day) or vehicle. We measured (i) glucose, IGF-I, insulin, C-peptide and glucagon in serum and (ii) IGF-I, insulin and glucagon mRNAs and peptides in the pancreas by radioimmunoassay, immunohistochemistry and northern analysis. RESULTS: Both GH and IGF-I treatment increased serum and pancreatic IGF-I but, unlike GH, IGF-I treatment strongly reduced serum insulin and C-peptide (and, to a lesser extent, serum glucagon). Nevertheless, the animals did not become hyperglycaemic. GH, but not IGF-I, increased pancreatic insulin and glucagon content, as also indicated by immunohistochemistry, and increased IGF-I mRNA. Neither GH nor IGF-I caused significant changes in insulin and glucagon mRNA. CONCLUSIONS: The decrease in serum insulin and C-peptide by IGF-I treatment without significant changes in insulin gene expression and pancreatic insulin content suggests inhibition of insulin secretion. Within this setting, the absence of hyperglycaemia points to enhanced insulin sensitivity, although an insulin-like action of infused IGF-I may have partially compensated for the decreased insulin concentrations. GH-induced circulating or pancreatic IGF-I, or both, does not mimic the pancreatic effects of infused IGF-I in the absence of GH, suggesting that GH may counteract the action of GH-induced IGF-I on the endocrine pancreas

Establishment of a protocol for large-scale gene expression analyses of laser capture microdissected bladder tissue

PURPOSE: Lower urinary tract symptoms (LUTS) can be caused by structural and functional changes in different compartments of the bladder. To enable extensive investigations of individual regions even in small bladder biopsies, we established a combination protocol consisting of three molecular techniques: laser capture microdissection microscopy (LCM), RNA preamplification and quantitative polymerase chain reaction (qPCR). METHODS: Urinary bladders of ten mice were resected and frozen immediately or after a delay of 15 min. Cryosections were obtained and smooth muscle was isolated using the LCM technique. Then, RNA was extracted, including protocols with and without DNase digestion as well as with and without the addition of carrier RNA. Extracted RNA was either used for reverse transcriptase (RT)-PCR plus qPCR or for a combination of RNA preamplification and qPCR. RESULTS: Our data showed that with RNA preamplification, 10 μg cDNA can be regularly generated from 2.5 ng RNA. Depending on expression levels, this is sufficient for hundreds of pPCR reactions. The efficiency of preamplification, however, was gene-dependent. DNase digestion before preamplification lead to lower threshold cycles in qPCR. The use of partly degraded RNA for RNA preamplification did not change the results of the following qPCR. CONCLUSIONS: RNA preamplification strongly enlarges the spectrum of genes to be analyzed in distinct bladder compartments by qPCR. It is an easy and reliable method that can be realized with standard laboratory equipment. Our protocol may lead in near future to a better understanding of the pathomechanisms in LUTS

Transcriptome profile of human colorectal adenomas

Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation