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Genetically Transformed Liver Cells Produce Active Insulin
SAN FRANCISCO--Scientists at the Medical College of Virginia have genetically manipulated liver cells to function as pancreatic islets and produce insulin. The researchers are now working to alter the liver cells further so that they will release insulin in response to changes in blood sugar levels. The goal is to create factories of blood sugar-regulating cells in the liver that one day may be used in the treatment of patients with severe diabetes.
Hepatocytes, unlike islet cells that normally produce insulin in the pancreas, are not vulnerable to the autoimmune disease that destroys the islets in individuals with diabetes. Liver cells also are the only cells in the body that can continually regenerate. So if an insulin-producing factory can be created in hepatocytes, "that factory may stay open indefinitely," according to Robert A. Fisher, MD, FACS, director of the Liver Transplantation Program at Medical College of Virginia, Richmond.
In a research study reported upon at the 1999 Clinical Congress of the American College of Surgeons, Dr. Fisher and his colleagues demonstrated that they could incorporate the gene for insulin into hepatocytes and that the genetically altered hepatocytes would produce mature, biologically active insulin. "We were able to put human insulin DNA using a viral promoter in a well-known vector virus, which is like a little carrier, into the hepatocyte. We then showed that the insulin DNA can be read and sequenced by an enzyme in the liver into a form of insulin that does what it is supposed to doand that is to take a form of inactive insulin and turn it into active insulin," he explained.
Dr. Fisher and his associates are following this research path because of the need for alternative treatments in managing patients with difficult-to-control diabetes. He explained that insulin-dependent diabetic patients can gain good control of their disease by regularly monitoring blood sugar levels and administering insulin subcutaneously as needed. Blood sugar levels typically fluctuate widely, however, and it is difficult for diabetic patients to maintain blood sugar levels consistently within normal ranges. Therefore, aggressive insulin replacement therapy is often given to patients with severe forms of the disease in order to control long-term complications of the disease, such as vascular insufficiency, blindness, and renal failure. However, aggressive insulin-replacement therapy runs the risk of inducing complications because of excessively low levels of blood sugar (hypoglycemia).
Although the therapeutic role for genetically engineered hepatocytes is not clear at this point, Dr. Fisher can see the hepatocytes being tested in diabetic patients who also have liver disease and are awaiting a donor liver "as a bridge to solid organ transplantation," he said. The hepatocytes also may be given in lieu of solid organ transplantation, he suggested. "Hepatocyte transplantation requires much less immunosuppression than a solid organ transplantation, and it provides the potential for introducing a cell that produces insulin on response to control blood sugar precisely without making the patient hypoglycemic or getting him into trouble with other secondary disease disorders," Dr. Fisher said.
It is too early to tell whether genetically engineered hepatocytes may be effective in individuals with non-insulin-dependent diabetes. "I don't think anyone should speculate at this point, because we have to do all the work in the in vivo animal model before we venture into the clinical area," he said.
Dr. Fisher's research takes advantage of some unusual attributes of liver cells. Hepatocytes have an enzyme that converts inactive substances into their active form. Dr. Fisher has "tricked" this enzyme into cleaving inactive (proinsulin) into active insulin through a process known as site-directed mutagenesis. By using this process, Dr. Fisher has been able to place signals in the DNA of a natural form of human insulin. The signals essentially instruct the insulin to cleave or break down more easily. The DNA then is inserted into the hepatocyte, and the enzyme "reads" the signals. "The idea was to fix this native enzyme to make it a little better at doing what we wanted it to do and convert proinsulin [inactive insulin] into the real thing," he said. This objective was accomplished in the study reported at the Clinical Congress.
The next phase of the research is to act on another native liver enzyme so that it will recognize changes in blood sugar. "The enzyme will be tagged to the gene for human insulin so insulin will be released and processed by the hepatocytes when blood sugar is high and turned off when blood sugar is low," he said.
The liver transplantation team at Medical College of Virginia has transplanted non- genetically-manipulated hepatocytes into 17 patients with liver failure over the last few years to sustain them while they waited for a solid organ to become available for donation. The team also has transplanted hepatocytes into the spleen to act as a short-term auxiliary liver. "The present study is an example of taking the some of the work we've done with the liver cell and using it to defeat another disease process," Dr. Fisher said.
Joining Dr. Fisher in the study were Dawen Bu, MD; James M. Coghill, BA; John Tawes; BS, Melissa Thompson, BS; and Marc Posner, MD, FACS.