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

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

To test whether hepatocytes engineered in vivo can serve as surrogate beta cells by similarly secreting mature insulin in a glucose-sensitive manner, we prepared adenoviral vectors encoding wild-type proinsulin (hIns-wt), a modified proinsulin cleavable by the ubiquitously expressed protease furin (hIns-M3), or each of the two beta cell specific pro-insulin convertases PC2 and PC3. Following a detailed in vitro characterization of the proteins produced by our vectors, we infected the liver and, for comparison, the muscle of a chemically induced murine model of type I diabetes. Insulin expression from the transduced tissues was extensively characterized and showed to be constitutive rather than regulated. To obtain regulated expression, we placed expression of hIns-M3 under the control of the dimerizer-inducible transcription system. Hormone secretion from mouse liver was negligible in the absence of the dimerizer drug rapamycin, was inducible in a dose-dependent manner upon its administration, and reversible following drug withdrawal. These data confirm liver as a promising target for in vivo expression of processed insulin. While suggesting that hepatocytes cannot provide authentic glucose-responsive regulation, these results demonstrate that pharmacological regulation is a promising alternative route to the controlled delivery of insulin following hepatic gene transfer.
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PMID:Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer. 1208 45

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic beta cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for beta cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic beta cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic beta cells and become a therapy for the treatment of type I diabetes.
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PMID:Function of a genetically modified human liver cell line that stores, processes and secretes insulin. 1262 53

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.
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PMID:Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line. 1279 Aug 3

Cell engineering or gene therapy may represent an alternative to current methods of treating diabetes mellitus. Cells could be engineered to secrete insulin ex vivo for transplantation or the insulin gene could be administered directly by injection into muscle. A problem has been that non-neuroendocrine cells lack the endoproteases (PC3/1 and PC2) that are responsible for the processing of proinsulin to insulin. This can be surmounted by engineering the paired basic amino acid processing sites within proinsulin to sites that would be recognized by the ubiquitously expressed protease, furin. However, in every study to date, the expression of the furin-cleavable construct was greatly reduced relative to that of the unmodified proinsulin construct. We investigated possible causes for this, including mRNA stability, the presence of additional CpG islands, and the amino acid substitutions within furin-cleavable proinsulin. Several furin-cleavable rat proinsulin I cDNAs were engineered and used to transfect human HEK293, rat L6 and mouse C(2)C(12) cell lines. The stability of wild-type and furin-cleavable proinsulin mRNA in transfected C(2)C(12) cells was measured by RT-PCR. Comparison of the decay rates in the presence of actinomycin D showed no significant difference between the two species of mRNA. A furin-cleavable proinsulin cDNA was created to contain the same distribution of CpG islands as wild-type proinsulin. Comparison of insulin-like immunoreactivity in all three cell lines transfected with either this construct or a widely used furin-cleavable proinsulin containing additional CpG islands showed that the presence of the extra CpG islands had no effect. Studies to examine amino acid substitutions used to create furin consensus sequences showed that the addition of basic residues at the C-peptide/A-chain junction was responsible for the reduced production of furin-cleavable proinsulin. Using this information, we engineered a cDNA for furin-cleavable rat proinsulin I that was efficiently processed to mature insulin and expressed at the same level as wild-type proinsulin.
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PMID:Enhanced expression of a furin-cleavable proinsulin. 1466 19

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH(2)-termini of its precursor, proIAPP, likely by the beta-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH(2)-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3(-/-)). These islets contained not only fully processed IAPP as in PC1/3(+/+) islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH(2)- or COOH-terminal cleavage sites.
Diabetes 2004 Jan
PMID:Role of beta-cell prohormone convertase (PC)1/3 in processing of pro-islet amyloid polypeptide. 1469 8

Embryonic stem (ES) cells can differentiate into many cell types. Recent reports have shown that ES cells can differentiate into insulin-producing cells. However, the differentiation is not efficient enough to produce insulin-secreting cells for future therapeutic use. Pdx-1, a homeodomain-containing transcription factor, is a crucial regulator for pancreatic development. We established an ES cell line in which exogenous pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus. Using this cell line, we examined the effect of pdx-1 expression during in vitro differentiation via embryoid body formation. The results showed that pdx-1 expression clearly enhanced the expression of the insulin 2, somatostatin, Kir6.2, glucokinase, neurogenin3, p48, Pax6, PC2, and HNF6 genes in the resulting differentiated cells. Immunohistochemical examination also revealed that insulin was highly produced in most of the differentiated ES cells. Thus, exogenous expression of pdx-1 should provide a promising approach for efficiently producing insulin-secreting cells from human ES cells for future therapeutic use in diabetic patients.
Diabetes 2004 Apr
PMID:Regulated expression of pdx-1 promotes in vitro differentiation of insulin-producing cells from embryonic stem cells. 1504 18

Long-term secretion of insulin by host muscle following transduction with an insulin gene construct offers the potential of gene therapy for diabetes without immunosuppression. Clinical implementation will be dependent on proof of principle in human tissue and a system for safely regulating basal insulin levels. Liposomal co-transfection with a tetracycline-responsive wild type human preproinsulin (pTRE-hppI1) or mutant construct (pTRE-hppI4), in which PC2 and PC3 cleavage sites were altered to form tetrabasic consensus sites for furin, together with pTet-off (coding for a transactivating protein) was evaluated in the C2C12 mouse myoblast cell line and human myoblasts following establishment in primary culture. In the absence of tetracycline, (pro)insulin secretion in C2C12 and human myoblasts transfected with tetracycline-responsive hppI1 and hppI4 constructs was comparable to that following transfection with equivalent constructs under the control of a constitutively active cytomegaloviral promoter. Percentage processing to mature insulin was <5% in C2C12 and human myoblasts transfected with pTet-off/pTRE-hppI1 but >90% in C2C12 cells and 45-60% in human myoblasts on transfection with pTet-off/pTRE-hppI4. Incremental dose-responsive suppression of proinsulin secretion was demonstrated in C2C12 and human myoblasts expressing pTet-off/pTRE-hppI1 following incubation with tetracycline (0-100 microg/ml) for up to 72 h. Reversibility was confirmed following tetracycline withdrawal. Dose-responsive tetracycline-inducible repression of mature insulin secretion was confirmed in C2C12 cells following transfection with pTet-off/pTRE-hppI4. Regulation of human proinsulin biosynthesis and secretion has been attained in vivo following plasmid-mediated gene transfer to rat skeletal muscle and oral tetracycline administration. In conclusion, processing to mature insulin has been confirmed following plasmid-mediated gene transfer to human muscle in addition to in vitro- and in vivo-regulated human proinsulin secretion employing the safe and well-tolerated antibiotic, tetracycline.
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PMID:Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle. 1582 Nov 5

The amyloid present in the islets of Langerhans in type 2 diabetes is polymerized islet amyloid polypeptide (IAPP). The precursor protein proIAPP is posttranslationally modified, a process involving the removal of NH2- and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH2-terminal processing site, and PC1/3 processes proIAPP exclusively at the COOH-terminal site. Little is known regarding the exact circumstances leading to islet amyloid formation. In this study, we have examined the possible significance of aberrant processing of proIAPP on amyloid formation in several in vitro cellular systems. In our studies, human (h)-proIAPP was transfected into beta-TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally, h-proIAPP was transfected into three different pituitary-derived cell lines with different prohormone convertase profiles: AtT-20 cells (deficient in PC2), GH3 cells (deficient in PC1/3), and GH4C1 cells (deficient in both convertases). We followed the processing of h-proIAPP with antibodies specific for the respective cleavage sites and stained the cells with Congo red to verify the accumulation of amyloid. Incomplete processing of h-proIAPP that occurs in AtT-20 and GH4C1 cells resulted in the formation of intracellular amyloid. No amyloid developed in beta-TC-6 and GH3 cells lines with full processing of proIAPP. An intracellular increase in proIAPP and/or its metabolic products may thus promote intracellular amyloid formation, thereby causing cell death. When extracellularly exposed, this amyloid might act as template for continuing amyloid formation from processed IAPP released from the surrounding beta-cells.
Diabetes 2005 Jul
PMID:Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation. 1598 13

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.
Diabetes 2006 Feb
PMID:Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1). 1644 80


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