Regenerating 1 and 3b gene expression in the pancreas of type 2 diabetic Goto-Kakizaki (GK) rats.

Regenerating (REG) proteins are associated with islet development, β-cell damage, diabetes and pancreatitis. Particularly, REG-1 and REG-3-beta are involved in cell growth/survival and/or inflammation and the Reg1 promoter contains interleukin-6 (IL-6)-responsive elements. We showed by transcriptome analysis that islets of Goto-Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, overexpress Reg1, 3α, 3β and 3γ, vs Wistar islets.

Cell-type, allelic, and genetic signatures in the human pancreatic beta cell transcriptome.

Elucidating the pathophysiology and molecular attributes of common disorders as well as developing targeted and effective treatments hinges on the study of the relevant cell type and tissues. Pancreatic beta cells within the islets of Langerhans are centrally involved in the pathogenesis of both type 1 and type 2 diabetes. Describing the differentiated state of the human beta cell has been hampered so far by technical (low resolution microarrays) and biological limitations (whole islet preparations rather than isolated beta cells).

Hypercholesterolaemia, signs of islet microangiopathy and altered angiogenesis precede onset of type 2 diabetes in the Goto-Kakizaki (GK) rat.

Aims/hypothesis: The adult non-obese Goto-Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass.

Islet endothelial activation and oxidative stress gene expression is reduced by IL-1Ra treatment in the type 2 diabetic GK rat.

Background: Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra).

IL-1 antagonism reduces hyperglycemia and tissue inflammation in the type 2 diabetic GK rat.

Recent studies suggest an inflammatory process, characterized by local cytokine/chemokine production and immune cell infiltration, regulates islet dysfunction and insulin resistance in type 2 diabetes. However, the factor initiating this inflammatory response is not known. Here, we characterized tissue inflammation in the type 2 diabetic GK rat with a focus on the pancreatic islet and investigated a role for IL-1.

Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion.

Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not.

Induction of CXCL1 by extracellular matrix and autocrine enhancement by interleukin-1 in rat pancreatic beta-cells.

As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix.

Islet inflammation and fibrosis in a spontaneous model of type 2 diabetes, the GK rat.

The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families.

Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor.

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor.

Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of beta-cell muscarinic receptor activation on cAMP production.

Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose).

Activation of NF-kappaB by extracellular matrix is involved in spreading and glucose-stimulated insulin secretion of pancreatic beta cells.

Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) engages beta1 integrins to induce spreading, improve glucose-stimulated insulin secretion (GSIS), and increase survival of pancreatic beta cells. The present study examines whether 804G-ECM activates the transcriptional activity of NF-kappaB and the involvement of NF-kappaB in those effects of 804G-ECM on pancreatic beta cells. 804G-ECM induces nuclear translocation and the DNA binding activity of the p65 subunit of NF-kappaB.

Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines.

To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not.

Mutant proinsulin that cannot be converted is secreted efficiently from primary rat beta-cells via the regulated pathway.

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called "constitutive-like" pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus.

Trafficking/sorting and granule biogenesis in the beta-cell.

Proinsulin is packaged into nascent (immature, clathrin-coated) secretory granules in the trans-Golgi network (TGN) of the beta -cell along with other granular constituents including the proinsulin conversion enzymes. It is assumed that such packaging is dependent on an active sorting process, separating granular proteins from other secretory or membrane proteins, but the mechanism remains elusive. As granules mature, the clathrin coat is lost, the intragranular milieu is progressively acidified, and proinsulin is converted to insulin and C-peptide.

Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells.

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles.

Sorting human beta-cells consequent to targeted expression of green fluorescent protein.

Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells.

Role of the prohormone convertase PC3 in the processing of proglucagon to glucagon-like peptide 1.

Proglucagon is processed differentially in pancreatic alpha-cells and intestinal endocrine L cells to release either glucagon or glucagon-like peptide-1-(7-36amide) (tGLP-1), two peptide hormones with opposing biological actions. Previous studies have demonstrated that the prohormone convertase PC2 is responsible for the processing of proglucagon to glucagon, and have suggested that the related endoprotease PC3 is involved in the formation of tGLP-1. To understand better the biosynthetic pathway of tGLP-1, proglucagon processing was studied in the mouse pituitary cell line AtT-20, a cell line that mimics the intestinal pathway of proglucagon processing and in the rat insulinoma cell line INS-1.

Proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase E-deficient Cpefat/Cpefat mice.

Sorting of proinsulin from the trans-Golgi network to secretory granules is critical for its conversion to insulin as well as for regulated insulin secretion. The proinsulin sorting mechanism is unknown. Recently, carboxypeptidase E (CPE) was proposed as a sorting receptor for prohormones.

Role of the prohormone convertase PC2 in the processing of proglucagon to glucagon.

Proglucagon is alternatively processed to glucagon in pancreatic alpha-cells, or to glucagon-like peptide-1 in intestinal L cells. Here, the specificity of PC2, the major prohormone convertase of alpha-cells, was examined both in vivo and in vitro. Adenovirus-mediated co-expression of proglucagon and PC2 in GH4C1 cells resulted in a pattern of processing products very similar to that observed in alpha-cells.

Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3.

Proinsulin conversion to insulin occurs in secretory granules of pancreatic beta-cells. This processing has been suggested to require both the endoproteases PC2 and PC3 with each cleaving at only one of the two sites linking the insulin A- and B-chains with C-peptide. To evaluate this in an appropriate cellular setting, conversion of human proinsulin was followed in GH3 (rat pituitary) cells normally unable to convert this prohormone but equipped with the regulated secretory pathway.

Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus.

Proinsulin is converted to insulin by the two endoproteases PC2 and PC3. For complete conversion to insulin, cleavage must occur at both the B-chain/C-peptide and C-peptide/A-chain junctions of proinsulin. Studies in vitro have shown the specificity of PC3 for the B-chain/C-peptide junction and that of PC2 for the C-peptide/A-chain junction.

Des-(27-31)C-peptide. A novel secretory product of the rat pancreatic beta cell produced by truncation of proinsulin connecting peptide in secretory granules.

Insulin and connecting peptide (C-peptide) are produced in equimolar amounts during proinsulin conversion in the pancreatic beta cell secretory granule. To determine whether insulin and C-peptide are equally stable in beta cell granules (and thus secreted in equimolar amounts), neonatal and adult rat beta cells were pulse-chased, and radiolabeled insulin and C-peptide analyzed by high performance liquid chromatography. A novel truncated C-peptide was identified and shown by mass spectrometry to be des-(27-31)C-peptide (loss of 5 C-terminal amino acids).

Sequence requirements for proinsulin processing at the B-chain/C-peptide junction.

Proinsulin is converted into insulin by the action of two endoproteases. Type I (PC1/PC3) is thought to cleave between the B-chain and the connecting peptide (C-peptide) and type II (PC2) between the C-peptide and the A-chain. An acidic region immediately C-terminal to the point of cleavage at the B-chain/C-peptide junction is well conserved throughout evolution and has been suggested to be important for proinsulin conversion [Gross, Villa-Komaroff, Kahn, Weir and Halban (1989) J.

Processing of proinsulin by furin, PC2, and PC3 in (co) transfected COS (monkey kidney) cells.

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions.