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

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

Recently, islet transplantation in the treatment of type 1 diabetes has been revisited with improved results. This approach has the potential to restore the regulatory unit of endocrine pancreas, but it cannot be a definite solution because of its limitation for the use of toxic immune-suppressive agents and limited number of donors. One possible way to restore the insulin secretion safely is with cell therapy using tissue engineering of a patient's own somatic cells by transduction of the corresponding gene of interest, in which extrapancreatic cells are engineered to secrete insulin. We constructed a somatic cell therapy system using a furin-cleavable insulin gene and somatic cells of mesenchymal origin. We also tested a semipermeable chamber that can contain allo- or xenogeneic insulin-secreting cells and found that the device could be helpful for cell therapy. This novel approach should provide a feasible method for treatment of diabetes mellitus without the use of any toxic agent or of embryonic stem cells that involve ethical implications.
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PMID:Gene and cell-based therapy for diabetes mellitus: endocrine gene therapeutics. 1285 4

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

PACE (Physician-based Assessment and Counseling for Exercise) is an individualized theory-based minimal intervention strategy aimed at the enhancement of regular physical activity. The aim of this study was to evaluate the effectiveness of a PACE intervention applied by general practitioners (GPs) on potential determinants of physical activity. A randomized controlled trial was conducted in 29 general practices with the following inclusion criteria for patients: aged between 18 and 70 years, diagnosed with hypertension, hypercholesterolemia and/or non-insulin-dependent diabetes mellitus, and not in maintenance stage for regular physical activity. The intervention consisted of two visits with the GP and two telephone booster calls by a physical activity counselor. Determinants of physical activity were assessed with questionnaires at baseline, and at 8-week (short), 6-month (medium) and 1-year (long) follow-up. A significant positive effect was observed on self-efficacy, and on the use of cognitive and behavioral processes of change, at both short- and medium-term follow-up. The intervention respondents also perceived fewer barriers for regular physical activity at short-term and used behavioral processes of change more at long-term follow-up. No intervention effect was observed for perceived benefits of physical activity. In conclusion, this GP-based PACE intervention resulted in positive changes in potential determinants of physical activity.
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PMID:The positive effect on determinants of physical activity of a tailored, general practice-based physical activity intervention. 1547 5

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

We assessed therapeutic potential of intramuscular insulin gene delivery in a diabetic murine model. The human proinsulin gene cDNA engineered with concensus furin cleavage sequences was inserted into an advanced lentiviral vector that contained CMV early promoter. After injection of concentrated lentiviral vector (3.5 microg p24 Gag antigen) carrying the insulin gene into the thigh muscle, treated rats demonstrated an increase in body weight, increased survivability, attenuated the hyperglycemic response as well as prevented the formation of ketoacidosis. For these reasons, the intraparenchymal injection of lentiviral vectors into the skeletal muscle to ectopically produce insulin may be an easy and therapeutic treatment modality for type 1 diabetes mellitus.
Diabetes Res Clin Pract 2006 Mar
PMID:Gene therapy for diabetes mellitus in rats by intramuscular injection of lentivirus containing insulin gene. 1617 85

Insulin production afforded by hepatic gene therapy (HGT) retains promise as a potential treatment for type 1 diabetes, but successful approaches have been limited. We employed a novel and previously untested promoter for this purpose, glucose transporter-2 (GLUT2) to drive insulin production via delivery by recombinant adeno-associated virus (rAAV). In vitro, the GLUT2 promoter was capable of robust glucose-responsive expression in transduced HepG2 human hepatoma cells. Therefore, rAAV constructs were designed to express the furin-cleavable human preproinsulin B10 gene, under the control of the murine GLUT2 promoter and packaged for delivery with rAAV expressing the type 5 capsid. Streptozotocin-induced diabetic mice were subjected to hepatic portal vein injection immediately followed by implantation of a sustained-release insulin pellet to allow time for transgenic expression. All mice injected with the rAAV5-GLUT2-fHPIB10 virus remained euglycemic for up to 35 days post-injection, with 50% euglycemic after 77 days post-injection. In contrast, mock-injected mice became hyperglycemic within 15 days post-injection following dissolution of the insulin pellet. Serum levels of both human insulin and C-peptide further confirmed successful transgenic delivery by the rAAV5-GLUT2-fHPIB10 virus. These findings indicate that the GLUT2 promoter may be a potential candidate for regulating transgenic insulin production for hepatic insulin gene therapy in the treatment of type I diabetes.
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PMID:Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice. 1622 91

Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.
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PMID:Heat-regulated production and secretion of insulin from a human artificial chromosome vector. 1640 45

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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