John F. Morris

ISLET AMYLOID FORMED FROM DIABETES-ASSOCIATED PEPTIDE MAY BE PATHOGENIC IN TYPE-2 DIABETES

Pancreatic islet amyloid deposits were found in 22 of 24 type-2 diabetic subjects (aged 48-68 years) and were not present in 10 age-matched controls. A novel peptide, 37 aminoacids long, termed diabetes-associated peptide (DAP), has been identified in amyloid-containing pancreatic extracts from 3 type-2 diabetic patients but not in extracts from 6 non-diabetic subjects. DAP has major homology with calcitonin-gene related peptide (CGRP) and the islet amyloid of all 22 diabetics showed CGRP immunoreactivity. The immunoreactivity was inhibited by preabsorption of three different CGRP antisera either with CGRP carboxyterminal peptide 28-37 or with extracted DAP. Both diabetic and non-diabetic subjects had CGRP/DAP immunoreactivity in islet B-cells. Electron microscopy of islets containing amyloid indicated fibrillar amyloid between the endocrine cells and capillaries, usually penetrating into deep invaginations of the plasma membrane of the B-cells. These results suggest that islet amyloid contains DAP, which may originate from B-cells. Accumulation of amyloid in islets is likely to impair islet function and may be a causal factor in the development of type-2 diabetes.

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

The 37‐kDa protein annexin 1 (Anx‐1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx‐1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX‐2 and cPLA2. In carrageenin‐ or zymosan‐induced inflammation, Anx‐1−/− mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL‐1β generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx‐1−/− polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx‐1−/− mice had reduced cell surface CD 11b (MAC‐1) but enhanced CD62L (L‐selectin) expression and Anx‐1−/− macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx‐1−/−mice that are not seen in the wild‐type animals, suggesting an interaction between sex hormones and inflammation in Anx‐1−/− animals.

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

SummaryIslet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.

Bone formation in organ cultures of bone marrow

SummaryBone formation in organ cultures of intact marrow fragments from mouse is described. Marrow explants were cultured on the top surface of a millipore filter at a gas-liquid interface. Observations with both light- and electron microscopes demonstrated the formation of a well-organised trabecular matrix lined with osteoblast-like cells. The tissue and cells were positive for alkaline-phosphatase activity. Large amounts of thick, well-banded collagen fibrils and matrix vesicles typical of those found in bone were present. The tissue became mineralised in the presence of 10 mM Na-β-glycerophosphate; in its absence a similar trabecular matrix developed but mineralisation did not take place.

Morphological heterogeneity of the GABAergic network in the suprachiasmatic nucleus, the brain's circadian pacemaker.

GABA (gamma‐amino‐butyric acid) is the predominant neurotransmitter in the mammalian suprachiasmatic nucleus (SCN), with a central role in circadian time‐keeping. We therefore undertook an ultrastructural analysis of the GABA‐containing innervation in the SCN of mice and rats using immunoperoxidase and immunogold procedures. GABA‐immunoreactive (GABA‐ir) neurons were identified by use of anti‐GABA and anti‐GAD (glutamic acid decarboxylase) antisera. The relationship between GABA‐ir elements and the most prominent peptidergic neurons in the SCN, containing vasopressin‐neurophysin (VP‐NP) or vasoactive intestinal polypeptide (VIP), was also studied. Within any given field in the SCN, approximately 40–70% of the neuronal profiles were GABA‐ir. In GABA‐ir somata, immunogold particles were prominent over mitochondria, sparse over cytoplasm, and scattered as aggregates over nucleoplasm. In axonal boutons, gold particles were concentrated over electron‐lucent synaptic vesicles (diameter 40–60 nm) and mitochondria, and in some instances over dense‐cored vesicles (DCVs, diameter 90–110 nm). GABA‐ir boutons formed either symmetric or asymmetric synaptic contacts with somata, dendritic shafts and spines, and occasionally with other terminals (axo‐axonic). Homologous or autaptic connections (GABA on GABA, or GAD on GAD) were common. Although GABA appeared to predominate in most neuronal profiles, colocalisation of GABA within neurons that were predominantly neuropeptide‐containing was also evident. About 66% of the VIP‐containing boutons and 32% of the vasopressinergic boutons contained GABA. The dense and complex GABAergic network that pervades the SCN is therefore comprised of multiple neuronal phenotypes containing GABA, including a wide variety of axonal boutons that impinge on heterologous and homologous postsynaptic sites.

Annexin I is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis.

Annexin I, a member of the calcium‐ and phospholipid‐binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co‐localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.

CENP-C is necessary but not sufficient to induce formation of a functional centromere

CENP‐C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP‐C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP‐C with those of inducibly overexpressing the protein. Removing CENP‐C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase–anaphase junction. Overexpressed CENP‐C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP‐C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP‐C changes during the cell cycle. In metaphase, the excess CENP‐C coats the chromosome arms. At the metaphase–anaphase transition, the excess CENP‐C clusters, and during interphase it is present in large bodies which form around pre‐existing centromeres which are also clustered. These results indicate that CENP‐C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP‐C may be regulated during the cell cycle.

Chronic overproduction of islet amyloid polypeptide/amylin in transgenic mice : lysosomal localization of human islet amyloid polypeptide and lack of marked hyperglycaemia or hyperinsulinaemia

SummaryType 2 (non-insulin-dependent) diabetes mellitus is characterised by hyperglycaemia, peripheral insulin resistance, impaired insulin secretion and pancreatic islet amyloid formation. The major constituent of islet amyloid is islet amyloid polypeptide (amylin). Islet amyloid polypeptide is synthesized by islet beta cells and co-secreted with insulin. The ability of islet amyloid polypeptide to form amyloid fibrils is related to its species-specific amino acid sequence. Islet amyloid associated with diabetes is only found in man, monkeys, cats and racoons. Pharmacological doses of islet amyloid polypeptide have been shown to inhibit insulin secretion as well as insulin action on peripheral tissues (insulin resistance). To examine the role of islet amyloid polypeptide in the pathogenesis of Type 2 diabetes, we have generated transgenic mice with the gene encoding either human islet amyloid polypeptide (which can form amyloid) or rat islet amyloid polypeptide, under control of an insulin promoter. Transgenic islet amyloid polypeptide mRNA was detected in the pancreas in all transgenic mice. Plasma islet amyloid polypeptide levels were significantly elevated (up to 15-fold) in three out of five transgenic lines, but elevated glucose levels, hyperinsulinaemia and obesity were not observed. This suggests that insulin resistance is not induced by chronic hypersecretion of islet amyloid polypeptide. Islet amyloid polypeptide immunoreactivity was localized to beta-cell secretory granules in all mice. Islet amyloid polypeptide immunoreactivity in beta-cell lysosomes was seen only in mice with the human islet amyloid polypeptide gene, as in human beta cells, and might represent an initial step in intracellular formation of amyloid fibrils. These transgenic mice provide a unique model with which to examine the physiological function of islet amyloid polypeptide and to study intracellular and extracellular handling of human islet amyloid polypeptide in pancreatic islets.

Annexin 1, Glucocorticoids, and the Neuroendocrine–Immune Interface

Abstract:  Annexin 1 (ANXA1) was originally identified as a mediator of the anti‐inflammatory actions of glucocorticoids (GCs) in the host defense system. Subsequent work confirmed and extended these findings and also showed that the protein fulfills a wider brief and serves as a signaling intermediate in a number of systems. ANXA1 thus contributes to the regulation of processes as diverse as cell migration, cell growth and differentiation, apoptosis, vesicle fusion, lipid metabolism, and cytokine expression. Here we consider the role of ANXA1 in the neuroendocrine system, particularly the hypothalamo‐pituitary‐adrenocortical (HPA) axis. Evidence is presented that ANXA1 plays a critical role in effecting the negative feedback effects of GCs on the release of corticotrophin (ACTH) and its hypothalamic‐releasing hormones and that it is particularly pertinent to the early‐onset actions of the steroids that are mediated via a nongenomic mechanism. The paracrine/juxtacrine mode of ANXA1 action is discussed in detail, with particular reference to the significance of the secondary processing of ANXA1, the processes that control the intracellular and transmembrane trafficking of the protein of the molecule and the mechanism of ANXA1 action on its target cells. In addition, the role of ANXA1 in the perinatal programming of the HPA axis is discussed.

Annexin 1 and the regulation of endocrine function

Annexin 1 (ANXA1) was first identified as a mediator of the anti-inflammatory actions of glucocorticoids in the host defence system. Subsequent work revealed that this protein fulfils a wider brief and it is now recognized as an important signalling intermediate in a variety of other systems. Here, we consider the role of ANXA1 in the endocrine system, placing particular emphasis on new insights into the mechanisms and functional significance of the secondary processing of ANXA1, the processes that control the intracellular and transmembrane trafficking of the molecule and the molecular mechanisms of ANXA1 action that have identified a novel role for the protein as a paracrine/juxtacrine mediator of the non-genomic actions of glucocorticoids in the neuroendocrine system.