Progress 10/01/06 to 09/30/10
Outputs
OUTPUTS: An absolute or functional deficit in beta-cell mass is a key factor in the pathogenesis of diabetes. We model obesity-driven beta-cell mass expansion by studying the diabetes-resistant C57BL/6-Leptinob/ob mouse.Wepreviously reported that cholecystokinin (Cck)wasthe most up-regulatedgenein obese pancreatic islets. We now show that islet cholecystokinin (CCK) is up-regulated 500-fold by obesity and expressed in both beta- and beta-cells. We bred a null Cck allele into the C57BL/6-Leptinob/ob background and investigated beta-cell mass and metabolic parameters of Cck-deficient obese mice. Loss of CCK resulted in decreased islet size and reduced beta-cell mass through increased beta-cell death. CCK deficiency and decreased beta-cell mass exacerbated fasting hyperglycemia and reduced hyperinsulinemia. We further investigated whether CCK can directly affect beta-cell death in cell culture and isolated islets. CCK was able to directly reduce cytokine- and endoplasmic reticulum stress-induced cell death. In summary, CCK is up-regulated by islet cells during obesity and functions as a paracrine or autocrine factor to increase beta-cell survival and expand beta-cell mass to compensate for obesityinduced insulin resistance. PARTICIPANTS: Jeremy A. Lavine, Philipp W. Raess, Donald S. Stapleton, Mary E. Rabaglia, Joshua I. Suhonen, Kathryn L. Schueler, James E. Koltes, John A. Dawson, Brian S. Yandell, Linda C. Samuelson, Margery C. Beinfeld, Dawn Belt Davis, Marc K. Hellerstein, Mark P. Keller, and Alan D. Attie TARGET AUDIENCES: People interested in islet biology, diabetes, and endocrinology. PROJECT MODIFICATIONS: This project has ended.
Impacts
The student working on this project, Jeremy Lavine, has graduated. We do not plan to extend this project further.
Publications
- Lavine, J.A., Raess, P.W., Stapleton, D.S., Rabaglia, M.E., Suhonen, J.I., Schueler, K.L., Koltes, J.E., Dawson, J.A., Yandell, B.S., Samuelson, L.C., Beinfeld, M.C., Davis, D.B., Hellerstein, M.K., Keller, M.P., And Attie A.D. (2010) Cholecystokinin is Up-regulated in obese islets and expands Beta-cell mass by increasing Beta-cell survival. Endocrinology 151,3577-3588.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have studied factors that stimulate beta cell proliferation. We have identified a hormone, cholecystokinin, which can induce the proliferation of rat, but not mouse or human beta cells. The species differences in response to this hormone may enable us to better understand why it is more difficult to stimulate beta cell proliferation in human than in rodent islets. In another study, we found that FoxM1, when overexpressed in either rodent or human islets, stimulates beta cell proliferation. This finding might have practical application in the context of islet transplantation. PARTICIPANTS: Jeremy Lavine, Dawn Belt Davis, Joshua Suhonen, Mary Rabaglia, Mark Keller, Christopher Newgard, Marjorie Beinfeld, Alan Kopin, Kathryn Schueler. The work is part of Jeremy Lavine's PhD thesis. It also formed the basis for a successful K08 award for Dawn Belt Davis, which also helped her to win an appointment as a tenure-track Assistant Professor of Medicine, beginning July 1, 2009. TARGET AUDIENCES: This work of of interest to the entire diabetes community of researchers and those involved in producing therapeutics for diabetes. PROJECT MODIFICATIONS: We are pursuing studies to understand the species differences in response to CCK in order to gain further insight on the relative resistance of human beta cells to agents that stimulate proliferation in rodent islets.
Impacts
Our findings might have application in both type 1 and type 2 diabetes. In type 1 diabetes, there is an unmet need to supply more islets and to better enhance their survival after transplantation. Treatments of the islets ex vivo prior to implantation might contribute the enhanced survival and function of the islets after implantation. We are currently negotiating a research agreement with the Genomics Institute of Novartis to explore these therapeutic applications of our work. In relation to type 2 diabetes, there is an unmet need to find agents that can expand beta cell mass. We hope that our work on the pathways that lead to beta cell proliferation might provide a sound rationale for drug screens aimed at discovering small molecules that can specifically and safely trigger beta cell proliferation. We also have to report an unfortunate setback in our research on this project. After publication of the article listed below, we discovered that the adenovirus we used to express the cholecystokinin gene also contained wild type virus, which expresses a potent mitogen, the E1A gene. Further work showed that the proliferative effect on human and mouse islets was due to E1A, not CCK. However, we did find that rat islets are indeed responsive to CCK. In February, we will publish an article that corrects the record and provides cautionary advice on the handling of adenoviruses.
Publications
- Lavine, J.A., Raess, P.W., Davis, D.B., Rabaglia, M.E., Presley, B.K., Keller, M.P., Beinfeld, M.C., Kopin, A.S., Newgard, C.B., and Attie, A.D. (2008) Overexpression of pre-pro-cholecystokinin stimulates Beta-cell proliferation in mouse and human islets with retention of islet function. Mol. Endocrinol. 22,2716-2728.
- Rieck, S. White, P., Schug, J., Fox, A.J., Smirnova, O., Gao, N., Gupta, R.K., Wang, Z.V., Scherer, P.E., Keller, M.P., Attie, A.D., and Kaestner, K.H. (2009) The transcriptional response of the islet to pregnancy in mice. Mol. Endocrinol. 23,1702-1712.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Type 1 and Type 2 diabetes result from a deficit in insulin production and Beta-cell mass. Methods to expand Beta-cell mass are under intensive investigation for the treatment of Type 1 and Type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote Beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured 3H-thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine if CCK overexpression stimulates Beta-cell proliferation. We studied islet function by glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating Beta-cells by quantitative RT-PCR and western blot analysis. Overexpression of CCK stimulated 3H-thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold into mouse and human Beta-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal 3H-thymidine incorporation. CCK is a Beta-cell proliferative factor that is effective in both mouse and human islets. CCK triggers Beta-cell proliferation without disrupting islet function, upregulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors. Shortly after this work was published, we discovered that the virus we used to express CCK was contaminated with wild type adenovirus. Thus, it is highly likely that the proliferative effects we attributed to CCK are in fact due to the adenovirual E1A gene. We are presently purifying both viruses to resolve this issue and will publish a correction as soon as we obtain the results. PARTICIPANTS: Training participants: Jeremy Lavine (grad student); Dawn Belt Davis (post-doc); Collaborator: Christopher Newgard (Duke) TARGET AUDIENCES: Biochemical, biophysical and chemical scientific research communities with the intent of increasing knowledge. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The project is at a point where we believe there might be some important therapeutic applications. We are negotiating with a pharmaceutical company for a potential collaboration aimed at developing therapy to promote beta cell proliferation.
Publications
- Lavine, J.A., Raess, P.W., Davis, D.B., Rabaglia, M.E., Presley, B.K., Keller, M.P., Beinfeld, M.C., Kopin, A.S., Newgard, C.B., and Attie, A.D. (2008) Overexpression of pre-pro-cholecystokinin stimulates cell proliferation in mouse and human islets with retention of islet function. Mol. Endocrinol. 22,2716-2728.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Obese mice and obese humans, compensate for insulin resistance by expanding Beta-cell mass. The failure to expand Beta-cell mass leads to the development of diabetes. Recent data from the use of GLP-1 analogs suggests that it is possible to stimulate the replication of Beta-cells in adult individuals. A major challenge will be to identify pathways and agents that stimulate Beta-cell replication without causing cancer. Our studies have shown that the CCK gene is dramatically induced in Beta-cells of obese animals. We have found that overexpression of pre-pro-CCK in isolated islets induces Beta-cell proliferation. Thus, our hypothesis is that CCK can be employed as an endogenous Beta-cell mitogen and presumably may constitute a safe therapeutic way to induce Beta-cell proliferation. For type 1 diabetes, a safe way to promote Beta-cell proliferation would potentially expand the yield and survivability of islets used for transplantation. Our studies have already shown that a
CCK-derived peptide, when expressed in human islets, provokes a strong proliferative response. However, we have not found a CCK-derived peptide that can stimulate Beta-cell proliferation when the protein is incubated with islets rather than produced by the islets. A key goal of the studies described in this proposal is to gain a better understanding of this paradoxical result. We find that adenovirus-mediated overexpression of pre-pro-CCK in mouse islets that lack both the CCK1R or CCK2R still causes Beta-cell proliferation. This suggests that the relevant CCK peptide is acting through a novel receptor and/or pathway. The characteriization of this pathway may provide important therapeutic targets. It is possible that this novel pathway does not have CCK as its primary agonist. Thus, our studies to better understand this pathway may lead to a novel agonist. There are sufficient differences between mouse and human islet biology to make extrapolation from the mouse to the human a risky
proposition. However, as described below, our in vitro experiments with human islets show a robust proliferative response to CCK, substantially better than we have seen in any of our mouse islet studies. Consequently, if we are successful in developing a virus-independent therapeutic based on this pathway, there is an immediate application to human islet transplantation and perhaps eventually, to type 2 diabetes treatment.
PARTICIPANTS: Training participants: Jeremy Lavine (grad student), Dawn Belt Davis (post-doc) Collaborator: Christopher Newgard (Duke)
TARGET AUDIENCES: Biochemical, biophysical and chemical scientific research communities with the intent of increasing knowledge.
Impacts
The project is at a point where we believe there might be some important therapeutic applications. We are negotiating with a pharmaceutical company for a potential collaboration aimed at developing therapy to promote beta cell proliferation.
Publications
- No publications reported this period
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