Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0011849 (diabetes)
277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prohormone convertase 2 (PC2) plays an essential role in the processing of proglucagon to mature active glucagon in pancreatic alpha-cells (J Biol Chem 276:27197-27202, 2001). Mice lacking PC2 demonstrate multiple defects, including chronic mild hypoglycemia and dramatic hyperplasia of the pancreatic alpha-cells. To define the contribution of mature glucagon deficiency to the hypoglycemia and alpha-cell hyperplasia, we have attempted to correct the defects by delivery of exogenous glucagon by micro-osmotic pumps. Intraperitoneal delivery of 0.5 microg glucagon/h in PC2(-/-) mice resulted in the normalization of blood glucose concentrations. Islet remodeling through the loss of hyperplastic alpha-cells was evident by day 11 after pump implantation; by 25 days postimplantation, PC2(-/-) islets were indistinguishable from wild-type islets. These rapid changes were brought about by induction of apoptosis in the alpha-cell population. Morphological normalization of islets was also accompanied by marked downregulation of endogenous preproglucagon gene expression, but with little or no change in the level of preproinsulin gene expression. Exogenous glucagon delivery also normalized hepatic expression of the gluconeogenic enzyme PEPCK. These results demonstrate that the lack of mature glucagon in PC2(-/-) mice is responsible for the aberrant blood glucose levels, islet morphology, and gene expression, and they confirm the role of glucagon as a tonic insulin antagonist in regulating glycemia.
Diabetes 2002 Feb
PMID:Glucagon replacement via micro-osmotic pump corrects hypoglycemia and alpha-cell hyperplasia in prohormone convertase 2 knockout mice. 1181 47

Cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) is a strong negative regulator of T cell activity. Like CD28 (a positive regulator) it binds to B7-1 and B7-2, and there is no known natural selective ligand. Monoclonal antibodies to CTLA-4 generally have a masking effect, enhancing rather than suppressing responses. However, a single amino acid substitution in B7-1 (W88 > A; denoted B7-1wa) abrogates binding to CD28 but not to CTLA-4. We constructed plasmids encoding B7-1 or B7-1wa, as cell-surface or Ig fusion proteins. In a bound state, B7-1-Ig enhanced CD3-mediated T cell activation, but B7-1wa-Ig was inhibitory, as expected of a CTLA-4 ligand. To alter immunity in vivo, we inoculated mice intramuscularly (i.m.) with a carcinoembryonic antigen (CEA) plasmid. Gene transfer was amplified by electroporation. Co-injection of a B7-1wa (membrane-bound form) plasmid blocked induction of anti-CEA immunity, whereas a B7-1 plasmid was stimulatory. We studied this DNA covaccination method in nonobese diabetic (NOD) mice with autoimmune diabetes. Delivery of either preproinsulin I (PPIns) or B7-1wa cDNA alone did not suppress the autoimmune anti-insulin response of spleen cells. However, co-delivery of B7-1wa and PPIns cDNA abrogated reactivity to insulin and ameliorated disease. Interferon-gamma and interleukin-4 were both depressed, arguing against a Th2 bias. Reactivity to glutamic acid decarboxylase 65, another major islet autoantigen, was not altered and suppressor cells were not identified, suggesting induction of tolerance to insulin by either T cell anergy or deletion. Selective engagement of CTLA-4 through gene transfer represents a novel and powerful way to block autoimmunity specifically.
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PMID:Immunoinhibitory DNA vaccine protects against autoimmune diabetes through cDNA encoding a selective CTLA-4 (CD152) ligand. 1186 Jul 6

We have chosen a vertebrate model accessible during neurulation, the chick, for analysis of endogenous insulin signaling and its contribution to early embryonic cell survival. Unlike rodents, humans and chickens have a single preproinsulin gene, facilitating its prepancreatic expression characterization. We show that in vivo interference with embryonic insulin signaling using antisense oligonucleotides against the insulin receptor increases apoptosis during neurulation. In contrast, high glucose administration does not increase the level of apoptosis in culture or in vivo. Exogenous insulin and, remarkably, proinsulin achieve similar survival protective effects at 10(-8) mol/l. The low abundant preproinsulin mRNA from the prepancreatic embryo is translated to a protein that remains as unprocessed proinsulin. This concurs with the absence of prohormone convertase 2 (PC2) in the embryo, whereas PC2 is present later in embryonic pancreas. A C-peptide--specific antibody stains proinsulin-containing neuroepithelial cells of the chick embryo in early neurulation, as well as other cells in mesoderm- and endoderm-derived structures in the 2.5-day embryo. We have determined by 5'-RACE (rapid amplification of cDNA ends), and confirmed by RNase protection assay, that prepancreatic and pancreatic proinsulin mRNA differ in their first exon, suggesting differential transcriptional regulation. All these data support the role of endogenous proinsulin in cell survival in the chick embryo during important pathophysiologic periods of early development.
Diabetes 2002 Mar
PMID:Unprocessed proinsulin promotes cell survival during neurulation in the chick embryo. 1187 78

The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.
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PMID:Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo. 1187 14

In human type 1 diabetes (T1D) autoantibodies to insulin precede clinical disease, while little is known about the contribution of insulin-specific T lymphocytes-in particular, T helper (Th) subsets. Here we have studied the in vivo primed cytokine response to preproinsulin in peripheral blood mononuclear cells (PBMCs) and two major Th cell subsets-CD45RO+ memory cells and CD45RA+ naive/resting cells-in 35 individuals with HLA-DRB1*04, DQB1*0302 diabetes risk marker: 12 patients with T1D, 12 autoantibody-positive (Ab+) individuals, and 11 healthy controls. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, and IL-10) was measured in the supernatants of the cultures stimulated with 21 overlapping preproinsulin peptides as well as proinsulin and insulin. In Ab+ individuals our results reveal higher IL-4 levels in CD45RO+ memory cells and higher IL-5 levels in CD45RA+ naive/resting cells, while higher IL-2 production was found in PBMCs. In contrast, in PBMCs of T1D patients higher IFN-gamma and IL-10 secretion was found. Our data delineate characteristic cytokine patterns in peripheral T lymphocytes from patients at different stages of the T1D development.
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PMID:Th2 dominance of T helper cell response to preproinsulin in individuals with preclinical type 1 diabetes. 1202 Nov 8

Cell therapy may have the potential for the treatment of Type I diabetes. To date, cells suitable for this purpose have not been developed. This study investigates the feasibility of modifying Vero, a cell line that may be considered safe to implant into humans, for this purpose. Stable Vero transfectants containing full-length human preproinsulin complementary deoxyribonucleic acid (cDNA) were generated using a liposomal transfection reagent. Reverse transcriptase-polymerase chain reaction, immunocytochemistry, Western blotting, and enzyme-linked immunosorbent assays were used to assess the resulting cells. Proinsulin was expressed but was not processed to insulin by these cells. Proinsulin cDNA was genetically modified, resulting in a form that is furin sensitive. The resulting stably transfected Vero clones constitutively release approximately 34%/h (32.68 +/- 2.21 to 35.62 +/- 3.14%) of the product formed, approximately 62% (59.99 +/- 6.45 to 64.64 +/- 4.57%) of which is mature insulin. These Vero transfectants did not exhibit glucose-stimulated insulin secretion. As GLUT2 and glucokinase (GCK) are not constitutively expressed by these cells, human GLUT2 cDNA and GCK cDNA were cotransfected with furin-sensitive preproinsulin cDNA into Vero cells. Insulin and GCK proteins were detected in the cytoplasmic region of the resulting cells, whereas GLUT2 was predominantly expressed in the nucleus. Coexpression of GLUT2 and GCK did not result in glucose-stimulated insulin secretion. The results from this study demonstrate the feasibility of engineering a relatively "safe" nonbeta cell line to produce human insulin. Coexpression of GLUT2 and GCK, at the levels achieved here, is not adequate enough to induce glucose-stimulated insulin secretion in such cells; the subcellular location of transfected components may be relevant.
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PMID:Engineering Vero cells to secrete human insulin. 1202 63

Enteroendocrine (EE) cells represent complex, rare, and diffusely-distributed intestinal epithelial cells making them difficult to study in vivo. A specific sub-population of EE cells called Gut K-cells produces and secretes glucose-dependent insulinotropic peptide (GIP), a hormone important for glucose homeostasis. The factors that regulate hormone production and secretion, as well as the timing of peptide release, are remarkably similar for K-cells and islet beta-cells suggesting engineering insulin production by K-cells is a potential gene therapeutic strategy to treat diabetes. K-cell lines could be used to study the feasibility of this potential therapy and to understand Gut K-cell physiology in general. Heterogeneous STC-1 cells were transfected with a plasmid (pGIP/Neo) encoding neomycin phosphotransferase, driven by the GIP promoter-only cells in which the GIP promoter was active survived genetic selection. Additional clones expressing pGIP/Neo plus a GIP promoter/insulin transgene were isolated-only doubly transfected cells produced preproinsulin mRNA. Bioactive insulin was stored and then released following stimulation with arginine, peptones, and bombesin-physiological GIP secretagogues. Like K-cells in vivo, the GIP/insulin-producing cells express the critical glucose sensing enzyme, glucokinase. However, glucose did not regulate insulin or GIP secretion or mRNA levels. Conversely, glyceraldehyde and methyl-pyruvate were secretagogues, indicating cells depolarized in response to changes in intracellular metabolite levels. Potassium channel opening drugs and sulphonylureas had little effect on insulin secretion by K-cells. The K-cell lines also express relatively low levels of Kir 6.1, Kir 6.2, SUR1, and SUR2 suggesting secretion is independent of K(ATP) channels. These results provided unexpected insights into K-cell physiology and our experimental strategy could be easily modified to isolate/characterize additional EE cell populations.
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PMID:Novel insulin/GIP co-producing cell lines provide unexpected insights into Gut K-cell function in vivo. 1212 79

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.
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PMID:T cell response to preproinsulin I and II in the nonobese diabetic mouse. 1219 12

GAD2 maps to chromosome 10p11.23 and encodes the 65-kDa isoform of GAD65, a major autoantigen in type 1 diabetes. The genetic variation that influences expression of preproinsulin mRNA, encoding another major autoantigen in type 1 diabetes, has already been shown to be genetically associated with disease. Previous reports that have assessed the association of GAD2 with type 1 diabetes have not used a dense map of markers surrounding the gene and have relied on very small clinical sample sizes. Consequently, no definite conclusions can be drawn from their negative results. We have therefore systematically searched all exons, the 3' untranslated region (UTR), the 5' UTR, and the 5' upstream region of GAD2, for polymorphisms in 32 white European individuals. We have genotyped these polymorphisms in a maximum of 472 U.K. type 1 diabetic affected sib pair families exhibiting linkage to type 1 diabetes on chromosome 10p and have tested both single variants and haplotypes in the GAD2 region for association with disease. We subsequently followed up our results by genotyping a subset of these single-nucleotide polymorphisms in a maximum of 873 Finnish families with at least one affected child. Our results suggest that GAD2 does not play a major role in type 1 diabetes in these two European populations.
Diabetes 2002 Sep
PMID:A comprehensive, statistically powered analysis of GAD2 in type 1 diabetes. 1219 83

Using the Adeno-associated virus (AAV) as a gene delivery vehicle, we have constructed a recombinant vector containing the full length rat preproinsulin gene (vLP-1). Utilizing the well described non-obese diabetic (NOD) mouse model, an experimental group (n = 10) of animals were intramuscularly (i.m.) injected with 10(7) rAAV virions containing the insulin gene and compared to a mock-injected control group (n = 10). Blood glucose (glc) was then measured weekly for 16 weeks. Data showed that the experimental group contained 70% euglycemic animals (defined as glc<200 mg/dL) versus 10% of the control animals (P < .05) at 14 weeks. Mean weight in the treated group was greater than the untreated group. Insulin mRNA was detected at the injection site of all of the treated animals, but not controls. Complete destruction of islets was confirmed by histology ruling out the possibility of spontaneous reversal of insulinitis. We conclude that i.m. delivery of the insulin gene in the NOD mouse was able to prevent clinical DM up to 14 weeks in a majority of treated animals. Our experimental data suggests that gene therapy may be an alternative treatment for IDDM in the future.
Int J Exp Diabetes Res 2001
PMID:Prevention of diabetes in the NOD mouse by intra-muscular injection of recombinant adeno-associated virus containing the preproinsulin II gene. 1236 16


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