Specific gene expression in pancreatic beta cells
Prof. Michael Walker
Sarah Weiss Reut Bartoov-Shifman Gabriela Ridner Tali Avnit-Sagi Keren Bahar
Introduction
Diabetes is a metabolic disease whose incidence has reached epidemic proportions: it is estimated that some 150 million people worldwide are afflicted with diabetes. The number is projected to double within 20 years, driven by a parallel epidemic of obesity. There are two major types of diabetes: type 1 (juvenile) diabetes is an autoimmune disease, caused by selective destruction of the insulin-producing beta cells of the pancreas. Type 2 (adult onset) diabetes results from a combination of impaired insulin production by beta cells and impaired insulin action. The hallmark of diabetes is a persistently elevated level of blood glucose. There is currently no cure for diabetes, and although treatments are available, these have many limitations. The goal of our research is to understand how pancreatic beta cells perform their unique functions. We focus on 1) the transcriptional mechanisms that permit selective expression of beta cell specific genes and 2) the signaling mechanisms that permit beta cells to couple the release of insulin to physiological needs. We believe that such approaches will lead to better treatments for diabetes and ultimately to a true cure.
Transcriptional regulationThe insulin gene is expressed exclusively in pancreatic beta cells. Control is exerted in large part at the transcriptional level through well defined elements located within the promoter region. Several transcription factors interact with these elements; we have shown that cotransfection of the factors PDX1, BETA2 and E2A synergistically activate transcription of a co-transfected insulin gene promoter by ~150 fold. We further showed that the transcription factor HNF4 (mutation of which leads to the rare form of diabetes MODY 1) can further activate the insulin gene promoter 2 fold. On the other hand, measurement by quantitative RT-PCR of insulin mRNA levels in beta cells compared to non-beta cells indicates differential expression of at least 100,000 fold; thus, additional control mechanisms must be operating to confer the observed strict specificity of expression. We hypothesize that the chromatin environment of the insulin gene plays a critical role in this.
PDX1 is a homeodomain transcription factor essential for pancreatic development and mature beta cell function. Under certain circumstances it has been demonstrated that PDX1 can convert non-beta cells into insulin-producing cells capable of rescuing mice from type 1 diabetes. Thus far, this “trans-differentiation” phenomenon has been best examined in liver cells. We are studying the mechanism underlying this potentially important mechanism. Using the mouse liver cell line AML, we have shown that ectopic expression of the insulin gene in AML cells is accompanied by direct binding of PDX1 to the insulin gene promoter as well as changes in chromatin structure as determined by increased histone acetylation (Fig 1). We are further investigating the chromatin environment of the insulin gene and other beta cell specific genes (including GPR40 - see below) with a view to better understanding the molecular basis of trans-differentiation.
GPR40 - a fatty acid receptor selectively expressed in beta cells
To better understand beta cell function, we have used a differential cloning procedure to identify genes expressed differentially in pancreatic beta cells. One of the genes identified was GPR40, a member of the family of G-protein coupled receptors. Recently it has been shown that fatty acids can activate GPR40. This finding was of interest in light of the fact that fatty acids have important effects on beta cells including a short term stimulation of glucose-dependent insulin secretion and a long term chronic impairment of insulin secretion. We set out to determine whether GPR40 may play a role in mediating either of these effects on beta cells. Using RNAi procedures, we reduced GPR40 mRNA levels in INS1-E beta cell lines. We observed significantly reduced glucose-dependent intracellular Ca2+ levels and insulin secretion. Using pharmacological inhibitors, we showed that GPR40
>signaling depends on the Gαq-PLC pathway, resulting in release of Ca2+ from the endoplasmic reticulum, and leading to up-regulation of Ca2+ influx via L-type calcium channels. To examine the effect of reducing GPR40 in a more physiological context, we generated knockout mice in collaboration with the laboratory of Dr H. Edlund (Umea University, Sweden). Islets from GPR40 knockout mice responded poorly to fatty acid, confirming the importance of GPR40 in mediating acute effects of fatty acids. Although GPR40 knockout mice showed no striking phenotype under normal nutritional conditions, following 8 weeks on high fat diet, an interesting phenotype was observed: whereas wild type mice displayed many of the features of metabolic syndrome (glucose intolerance, insulin resistance, high blood lipid levels, fatty liver), GPR40 knockout mice showed striking protection from these effects. These results indicate that GPR40 may also be an important mediator of some of the long term effects of high fat diet in promoting diabetes. Further evidence in favor of this view came from transgenic mice that we generated in which GPR40 was over-expressed specifically in beta cells. Such mice showed early onset diabetes accompanied by severe beta cell dysfunction. These results strongly suggest that GPR40 is an important
>mediator of both the acute, stimulatory effects and the chronic, deleterious effects of fatty acids on beta cells. Furthermore the data indicate that GPR40 dependent hypersecretion of insulin may be a cause rather than a consequence of the insulin resistance observed in obesity (Fig. 2).
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Fig. 1. PDX1 expression in liver AML cells alters chromatin structure of the insulin gene promoter as measured by extent of histone acetylation using chromatin immunoprecipitation assay. |
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Fig. 2. Models to explain steps leading from obesity to type 2 diabetes. Model A represents the conventional view, whereas model B is suggested by our GPR40 knockout and transgenic mouse models, emphasizing the role of GPR40 leading to hypesecretion of insulin and lipotoxocity-dependent beta cell dysfunction. |
Significance
Improved treatments and an eventual cure for diabetes will require better understanding of the basic mechanisms controlling beta cell function. Thus a knowledge of how non-beta cells can be induced to undergo trans-differentiation will permit manipulation of the efficiency of the process and its exploitation for clinical purposes. Likewise, an understanding of the mechanisms whereby GPR40 mediates the actions of fatty acids, in particular its long term toxic actions, may permit development of inhibitors of the process. We therefore believe our research will lead to valuable new tools for prevention and treatment of diabetes.
Selected Publications
Arava, Y., Adamsky, K., Ezerzer, C., Ablamunits, V. and Walker, M.
D. (1999) Specific gene expression in pancreatic b-cells: cloning and characterization of differentially expressed genes. Diabetes 48, 552-556.
Arava, Y., Seger, R. and Walker, M. D. (1999) GRFb, a novel regulator of calcium signaling, is expressed in pancreatic b cells and brain. J. Biol. Chem. 274, 24449- 24452.
Glick, E., Leshkowitz, D. and Walker, M. D. (2000) Transcription factor b2 acts cooperatively with E2A and PDX1 to activate the insulin gene promoter. J. Biol. Chem. 275, 2199-2204.
Bartoov-Shifman, R., Hertz, R., Wang, H., Wollheim, C. B., Bar-Tana, J., and Walker, M. D. (2002) Activation of the insulin gene promoter through a direct effect of hepatocyte nuclear factor 4 alpha J Biol Chem 277, 25914-9.
Liberzon, A., Ridner, G., and Walker, M. D. (2004) Role of intrinsic DNA binding specificity in defining target genes of the mammalian transcription factor PDX1. Nucleic Acids Res. 32, 54-64.
Shapiro, H., Shachar, S., Sekler, I., Hershfinkel, M. and Walker,
M.D. (2005) Role of GPR40 in fatty acid action on the beta cell
line INS-1E. Biochem Biophys Res Commun, 335, 97-104. Steneberg, P., Rubins, N., Bartoov-Shifman, R., Walker,
M.D. and Edlund, H. (2005) The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metab, 1, 245-258.
Acknowledgements
MDW is the incumbent of the Marvin Meyer and Jenny Cyker Chair of Diabetes Research. Supported by grants from the Israel Science Foundation and D Cure (Diabetes Care in Israel); Supported by the Mitchell Caplan Fund for Diabetes Research