This study describes the generation of a new model of human β-cell growth and differentiation and its application to HTS for compounds that affect the activation state of the insulin promoter, a key feature of the differentiated β-cell. The principal finding from this small screen of known drugs was that a subset of phenothiazine neuroleptics was able to acutely activate the insulin promoter, and chronic treatment led to downregulation of the insulin promoter.
A key to the development of the T6PNE β-cell line model described here was the finding that E47 controls both proliferation, through direct binding to the Kip2 promoter, and β-cell differentiation. Thus, selection for growth, as inevitably occurs in cells cultured in vitro, resulted in downregulation of E47 activity and loss of β-cell differentiation, including insulin expression. Of note, the effect of E47 on insulin and other β-cell genes was replicated in another cell line that we have studied, blox5, which also loses differentiated gene expression over time.15
The low levelE47 activity observed of in T6PNE compared with T6PN produces a small effect on proliferation, but this is sufficient to result in selection against maintenance of E47MER
expression over time. Thus, it was important to start periodically with earlier passage or cloned T6PNE cells.
Although glucose-responsive insulin secretion is not seen with induction of E47 activity in T6PNE, the upregulation of many β-cell genes in this system provides a model to study some aspects of β-cell differentiation and modulation of the insulin promoter. The microarray analysis revealed that T6PNE cells express a large number of the genes that are preferentially expressed in islets. However, it is clear that the level of expression is much lower in T6PNE than in primary β-cells.
For most purposes, the low level of expression of β-cell genes, particularly insulin, in T6PNE would be regarded as a drawback. However, it was precisely this low-level expression that made it possible to use HTS for insulin promoter modulators. Detecting differences in expression from the insulin promoter–GFP transgene is straightforward in T6PNE because the level of GFP can be titrated by adjusting the tamoxifen concentration. However, in primary β-cells or even conventional insulinoma cell lines such as MIN6, where the insulin gene is expressed at an enormous level, detecting the rapid change in GFP expression that is required for a practical high-throughput screen in primary islets is problematic.
It was surprising that the only class of drugs within the 1040-compound library that influenced insulin gene expression was the phenothiazines, a class of drugs known to predispose to diabetes upon chronic treatment.14
It should be noted that the screen was of short duration, with only 2 days of exposure to compound. Prolonged treatment with ethopropazine in T6PNE led to repression of insulin promoter activity. This could result from desensitization of a pathway that plays an important role in β-cell function and could contribute to the deleterious effects on β-cells that are found in most published studies, in which phenothiazines have been shown to inhibit glucose-responsive insulin secretion.16
Thus, as with other diabetogenic molecules such as fatty acids, acute exposure can stimulate aspects of β-cell function, whereas chronic exposure is harmful.2
Unfortunately, it was not possible to perform such chronic exposure studies with primary islets due to toxicity of the compounds. Using lower doses that are not toxic would require much longer times of exposure, exceeding the time in which islets can be maintained in vitro. However, longer term in vivo studies of the effects of phenothiazines on islet function would be of value.
The targets of phenothiazine neuroleptics are diverse, having been shown to affect a variety of proteins, including dopamine receptors, protein kinase C, calmodulin, and others.17
We have not as yet identified the target in T6PNE that signals to the insulin promoter. However, it is interesting to note that only a subset of phenothiazines was able to increase insulin promoter activity and that a specific structural feature of the terminal amine determined the level of activity. This suggests not only that a specific molecule is being targeted but that it is also likely to be a molecule that is different from those relevant to the psychiatric actions of the phenothiazines. There is poor concordance between the phenothiazines that we have found to act on the insulin promoter with those that have been reported to inhibit insulin secretion.16
With additional understanding of the target that is involved in insulin promoter modulation, it may be possible to design compounds that are more specific for the psychiatrically relevant target, with fewer effects on glucose homeostasis.