While Tinkering With the Beta-Cell...
A common feature of the two major forms of human diabetes is the partial or complete loss of insulin secretion from




-cells in the pancreatic islets of Langerhans. In this article, we review the development of a set of tools for studying



-cell biology and their application to understanding of fuel-mediated insulin secretion and enhancement of



-cell survival. Insights into these basic issues are likely to be useful for the design of new drug and cell-based diabetes therapies.


The pancreatic islets of Langerhans play a critical role in regulation of fuel homeostasis by secreting insulin and glucagon in a regulated fashion. Secretion of these hormones becomes dysregulated in both major forms of diabetes, via destruction of insulin-producing




-cells in type 1 diabetes and loss of normal regulation of insulin secretion in type 2 diabetes. Development of the next generation of therapeutic strategies for diabetes, including cell-based insulin replacement in type 1 diabetes or drug therapies for enhancing insulin secretion in type 2 diabetes, will depend on a detailed understanding of the molecular and biochemical mechanisms involved in islet



-cell function and survival.


In light of the inadequacies of insulin injection therapy, great effort has been expended over the past 30 years in the development of cell-based insulin-replacement strategies. The fundamental concept is that transplantation of pancreatic islets might allow better regulation of insulin delivery to diabetic patients. Very recently, a breakthrough occurred in this field in the form of a series of human islet transplants done by Shapiro et al. at the University of Edmonton. In this study, patients received ~800,000 human islets, culled from two to three pancreases per recipient, via injection into the portal vein. Patients also received a cocktail of mild, nonsteroidal immunosuppressive agents. This resulted in the impressive finding of insulin independence in seven consecutive patients over an average time of 12 months. This compares to the prior 10 years of experience with human islet transplantation, in which success, defined as insulin independence 1 year after transplant, was reported in only 8% of patients.

While there is no question that the Edmonton Trial represents a major advance, several difficult issues must be overcome before cell-based insulin replacement can be broadly applied. First, the number of human pancreases that become available for islet harvesting in the U.S. is on the order of several thousand per year. When one considers that there are ~1 million patients with type 1 diabetes in the U.S., the disparity between supply and demand becomes clear. Another important issue is the difficulty inherent in controlling fundamental variables such as insulin content, insulin secretion, and cell viability in human islet preparations. Finally, immunoprotection of transplanted cells remains an issue, despite the dramatic success of the Edmonton trial. This is because the long-term effects of generalized, systemic immunosuppression, even with a mixture of relatively mild agents, won't be known for several years. It is also unclear whether such generalized immunosuppression will be an appropriate therapy for children with type 1 diabetes. Therefore, continued development of more specific and targeted methods for immunoprotection of transplanted cells is clearly needed.
The fundamental approach taken by our laboratory for solving these very difficult problems is to try to develop a replenishable source of cells that can deliver insulin in a regulated fashion and to find ways of protecting such cells in the transplant setting. Possible cell sources include stem cells or, alternatively, immortalized versions of the pancreatic islet



-cell. Over the course of the past 15 years, our group and its collaborators have developed a conceptual plan for cell-based insulin replacement. As summarized in Fig. 1, this plan has three fundamental elements. The first is to use the tools of genetic engineering to create an expandable population of secretory cells that will deliver large amounts of insulin in response to appropriate physiological cues. The second element is a macro-encapsulation membrane or device that allows safe transplantation of the engineered cells into diabetic subjects. This device should be selective, in that it should allow rapid diffusion of nutrients, oxygen, and waste products and, of course, the rapid exit of insulin. The device should also be rigorously cell exclusionary, such that it prevents contact of the transplanted cells with host tissues, while at the same time preventing contact of the cellular components of the host immune system, such as macrophages and lymphocytes, with the transplanted cells. However, a device of such design is unlikely to be sufficient to fully protect transplanted cells, because small soluble mediators of immune destruction such as inflammatory cytokines and reactive oxygen species will gain entry through the device membrane. Therefore, the last component of the concept is to develop methods for protecting the transplanted cells against the effects of these small soluble toxins.


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Concept schematic: cell-based insulin replacement in diabetes using an encapsulated, expandable population of insulin secreting cells. The concept has the following three essential components: 1) an expandable population of cells engineered for appropriate regulation of insulin secretion by normal physiologic cues; 2) a retrievable macroencapsulation device that is rigorously cell impermeant but that allows entry and exit of nutrients, insulin, and other small molecules; 3) development of cells that are resistant to the cytotoxic effects of small, soluble mediators of the immune response, such as inflammatory cytokines and reactive oxygen species.





Our work on cell-based insulin replacement has involved three groups: our academic laboratory at the University of Texas Southwestern Medical Center and its collaborators; a biotechnology company that we formed in Dallas, BetaGene, Inc.; and a materials engineering group, Gore Hybrid Technologies, founded by Dr. Mark Butler. With regard to the encapsulation device technology, Dr. Butler and his team have developed a retrievable and selective device fulfilling the criteria just summarized. However, the focus of this article will be on new advances in the understanding of islet biology.

In seeking to achieve the goal of cell-based insulin therapy, our group has worked with a variety of human and animal cell lines. We believe that an expandable source of human rather than animal cells will be required for success in cell-based therapy of human diabetes. One approach to the development of human cells at BetaGene was to use human neuroendocrine cell lines for insulin delivery. In these studies, expression of the human proinsulin gene in a human lung neuroendocrine cell line resulted in secretion of correctly processed insulin in response to different agents like carbachol or the phorbol ester, PMA, but not in response to the key physiologic regulator, glucose (H. Hohmeier, T. Becker, A.Thigpen, et al., unpublished observations). In short, while it has been possible to create rodent cell lines that exhibit robust glucose-stimulated insulin secretion (see below), development of human cells that stably maintain this property has been much more elusive.

The difficulty associated with procurement of stable human cell lines for insulin replacement has prompted us in recent years to refocus our efforts toward gaining a deeper understanding of the genetic and biochemical mechanisms of




-cell function. Our work on



-cell biology has evolved such that we now utilize several technologies in an integrated fashion to gain insights into mechanisms of fuel-regulated insulin secretion and methods for enhancing



-cell survival. This includes novel cell models, techniques of gene discovery and genetic engineering, and, very recently, the application of nuclear magnetic resonance (NMR) for metabolic analysis of insulin-producing cells. Note that insights gained in these areas are also likely to be relevant to the understanding of



-cell dysfunction in type 2 diabetes, possibly leading to development of new therapeutic strategies for enhancing



-cell mass or performance in this disease.