| Research Abstract |
For engineering insulin-producing surrogate cells for diabetes gene therapy, two types of cells are available : neuroendocrine and non-neuro-endocrine cells. Neuroendocrine cells are equipped with a processing mechanism that converts a propeptide into a mature bioactive peptide, as well as a regulatory secretion system by which a mature peptide is secreted in response to extracellular stimuli. Thus, when neuroendocrine cells such as anterior pituitary corticotroph-derived endocrine cells AtT20 are used for diabetes gene therapy, the primary focus has been oriented to engineer an insulin secretion mechanism that responds to a physiological range of glucose stimuli. On the other hand, when non-neuroendocrine cells are used as insulin-producing cells, the cells need to be engineered with a processing mechanism and a regulatory secretion system.
In pancreatic beta cells, proinsulin is processed to mature insulin by prohormone convertases PC2 and PC3 (also named PC1) and carboxypeptidase H (
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CPH). These two proteolytic reactions appeared specific to secretory granule-containing neuroendocrine cells. However, when we replaced the processing sites of proinsulin with those cleavable by a yeast Kex2 family endoprotease furin, the non-neuroendocrine cell lines including COS-7, HepG2, CHO,and NIH3T3 produced insulin with the same size as synthetic human insulin. Next, the furin-cleaved proinsulin required the removal of basic residues by carboxypeptidases for its maturation. Although non-neuroendocrine cells expressed different quantities of carboxypeptidase HmRNA,these cells contained considerable levels of carboxypeptidase activity. The insulins resulting from these cell lines were eluted as a single peak at the mature insulin position on a cation-exchange chromatography column. Thus, non-neuroendocrine cells are able to produce correctly processed insulin if proinsulin is first mutated to possess furin-cleavable processing sites.
For developing an insulin expression system that can be regulated by extracellular stimili, most desirable system is to use glucose as a stimulator. Alternative regulator may be insulin itself for down-regulating the expression. Since non-neuroendocrine cells do not carry secretory granules and secrete proteins and peptides through a constitutive pathway without their retention in the cytoplasm, a regulatory step for the production of insulin is limited to a gene transcription level. Many genes are known to be regulated their expression by glucose. For the regulatory expression of these genes target cells are required to equip with both glucose transporter type 2 and glucokinase for responding to a physiological range of glucose consentrations. For this criterion the primarily cultured hepatocytes are the cell of choice for assessing the expression of these genes. However, hepatocytes are relatively inefficient for DNA transfer by conventional gene transfer methods. Furthermore, hepatocyte culture cell lines with glucose transporter type 2 and glucokinase are not avalable at the present time. Thus, we attempted to generate a regulatory expression system by insulin itself using a hepatoma cell line and a phosphoenolpyruvate carboxykinase (PEPCK) promoter. The PEPCK is expressed in hepatocytes and catalyzes oxaloacetate to phosphoenolpyruvate by taking phosphate from GTP.This enzyme acts as a key step for hepatic glucose production. In diabetic subjects this enzyme is highly activated by the lack of insulin. The PEPCK promoter is known to be up-regulated by cAMP,glucocorticoids, retinoic acid (RA), and down-regulated by insulin and phorbol ester. We examined the insulin production using a PEPCK promoter plus insulin DNA construct in H4Ell cells.
The H4llE cells secreted immunoreactive insulin (IRI) constantly at a level of 2-3 fmol/10^6 cells/h. IRI was increased about 2-fold upon stimulation with 0.5 mM cAMP,further 3-fold with cAMP-dependent phosphodiesterase inhibitor IBMX.IRI was elevated only 2-fold by 5 to 700 nM dexamethasone, but 10-12-fold by the further addition of cAMP and IBMX.Retinoic acid induced IRI by 4-fold. Addition of exogenous insulin to the culture medium decreased insulin mRNA expresion strikingly on Northern blot. However, together with cAMP,IBMX,and dexamethasone, the inhibitory effect of exogenous insulin became weakened. In the insulin-producing H4llE cells the production was augmented by the addition of wortmannin, a phosphatidylinositol (PI) -3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. When the cells were perifused, insulin secretion was elevated 2h after the stimulation. Thus, the secretory pattern from the H4llE cells may correspond to the action mode of intermediate-acting insulin.
We then introduced the insulin expression unit into an adenoviral vector Adex. Using a recombinant adenoviral vector, we confirmed the production of mature insulin from primarily cultured rat hepatocytes. The insulin secretion from hepatocytes was also well regulated by dbcAMP,IBMX,glucagon, and dexamethasone. We moved onto an in vivo experiment using streptozotocin (STZ) -induced diabetic rats. After injecting STZ at 100 or 150mg/Kg body weight, rats exhibited hyperglycemia of 400-600mg/dl blood glucose. We administered the recombinant adenovirus from a rat tail vein, through where most of viruses are thought to accumulate in the liver. Insulin level went up to 2-3ng/ml around 10 days after the injection, and blood glucose went down to 300-400mg/dl. Although we could not attain normoglycemia in the recombinant virus-injected rats, we think that this insulin expression system improves diabetes mildly. We are currently determining a best virul titer and an appropriate method of virus administration. Less
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