Diabetes Mellitus Type I (IDDMI):

1. Diabetes Mellitus Type I (IDDMI): Potential Gene Therapy

2. *The discovery of insulin by Banting and Best in 1922 represented a milestone in clinical medicine, and also contributed substantially to progress in the fields of molecular and comparative endocrinology. *My focus will be on IDDMI, and potential gene therapy.

3. *Diabetes Mellitus I covers a wide range of etiologies. An auto-immune disorder is only part of the story, and it is manifested among a subset of the population struck with Diabetes Mellitus Type I.

4. *The presence of HLA-DQ alleles (also known as MHC Class II) seems to be a requirement for the onset autoimmune diabetes. This is an allele that encodes for a protein that is displayed on the surface of B-islet cells of pancreas.

5. **It has been determined that a mutation in this particular protein complex (MHC-II or HLA-DQ) has resulted in the substitution of a certain conserved aspartic amino acid by a neutral amino acid.

6. *Apparently this defect leads to induction of some T-cell proliferation. Hence the patients become insulin deficient and hyperglycemic, which if it goes unchecked may lead to comma and eventually death.

7. *According to a team of researchers led by Dr. A. T. Cheung et al., from the Medical Center at the University of Alberta in Canada, the possibility of genetically engineering non-B cells to release insulin upon feeding could be a therapeutic modality for patients with diabetes Type I.

8. *The resultant transgenic mice gave fantastic results in terms of producing insulin, not only on cells other than the pancreas (in tissues such as in the small intestine and in the stomach). * But more interestingly the insulin produced was specifically induced by the ingestion of glucose by these transgenic mice.

9. *In this particular study the authors wanted to find out whether it would be possible to induce gut mouse tumor-derived K cells to express a human insulin transgene, and which would then give it the capacity to secrete insulin in response to glucose ingestion. First, they needed to choose a vector that would make the transfection (infection of a cell by a virus that then causes a foreign piece of DNA to be incorporated into the infected host cell).

10. They used a plasmid to carry out this function. Then, they also needed a promoter that would be induced to express the insulin gene in response to glucose ingestion. The glucose-dependent insulinotropic (GIP) promoter found in gut K cells would be an ideal promoter. These K cells express the glucokinase enzyme (NZ) just like pancreatic B cells do, and this is thought to give such cells the capacity to secrete GIP in the presence of glucose.

11. *This NZ is also recognized as the pancreatic “glucose sensor.” Consequently, such similarities in these two types of cells led the researchers to target these gut cells for insulin gene therapy. The in-vitro expression of human insulin in mouse tumor-derived gut K cells was successful. This is what one observes in Fig. # 1.

12. Fig. # 1) Expression of human insulin in tumor-derived K cells. (A) Immunofluorescence staining for glucokinase (GK, red) and GIP (green) in mouse duodenal sections. (B) northern blot analysis of GIP mRNA in STC-1(parental heterogeneous cell population), and GTC-1(PCR-RT expanded—isolated single clone). K-cell enrichment was determined by comparing the amount of GIP mRNA in the parental cell line (STC-1) with that of the newly subcloned k-cell lines. (C) Schematic diagram of the plasmid (GIP/ins) used for targeting human insulin expression to K cells. The rat GIP promoter (~2.5 kb) was fused to the genomic human preproinsulin gene, which comprises 1.6 kb of the genomic sequence extending from nucleotides 2127 to 3732.

14. In FIGURE # 2: A)   Again here, you observe the PCR-RT expansion of human insulin cDNA (of its respective mRNA). Then, they compare that to the amount of the same cDNA produced in certain tissues of the transgenic mice. Notice the amounts of this cDNA produced in the stomach and the duodenum correlate well with the amount produced in the human pancreatic cells.

15. B)   The letter M at the beginning of this micrograph simply indicates kinds of markers patterns employed. So, just ignore it. Then on the left side of the micrograph they are indicating the amount of insulin gene cDNA reproduced, again using PCR-RT.

16. (+ = presence of RT; - = absence of RT). M+ indicates the amount of cDNA reproduced in the mouse when a human based Specific Primer is used. The results show that the human insulin cDNA was not expressed in the mouse islet cells. However, it is noted that human insulin was produced in the duodenum of such Tr(+) mice.

17. When Mouse Specific Primers are employed it is observed that the transgenic transformation does not affect the expression of the mouse natural insulin in its B islet cells. C) Immunohistochemical staining for human insulin sections of stomach (left column) and duodenum (middle column).

18. Arrows indicate human insulin immunoreactive cells. Duodenal sections from the same animal were also examined by immunofluorescence microscopy (right column), for both the Ab-Insulin complex and the Ab-GIP. The top sections give a higher magnifications of the same item observed in the bottom at lower magnification.

20. This is what’s demonstrated in Figure # 3: A)   Oral glucose tolerance tests: As one can see, the curve on the top of the graph clearly indicates that upon an injection of a high dose of glucose administered orally the non-transgenic mice who were subjected to the B-Cell toxin, STZ showed considerably elevated levels of glucose or hyperglycemia. Surprisingly enough, the transgenic mice subjected to the same procedures showed that they handle the glucose challenge in a manner that greatly reflected that of the normal mice or mice with intact B-cells.

22. Moreover, the authors hope to design an insulin therapy, which would overcome the present insidious side effects of insulin injections.

23. CONCLUSION: Ideal blood glucose levels are rarely attainable in patients requiring insulin injections. Consequently, diabetic patients are still at risk for the development of serious long-term complications, such as cardiovascular disorders, kidney disease, and blindness.

24. *Total insulin in the pancreas in STZ-treated transgenic mice was only 0.5% that of the sham-treated controls. These STZ-treated transgenic mice disposed of oral glucose in the same way that normal mice do, despite having virtually no pancreatic B cells, which indicates that human insulin produced from the gut was sufficient to maintain normal glucose levels.

25. The authors claim that previous attempts to replace insulin by gene therapy prevented glucosuria and other lethal consequences of diabetes, such as ketoacidosis, but were unable to restore normal glucose tolerance. Their findings suggest that insulin production from gut K cells may correct diabetes to the extent of restoring normal glucose tolerance.