Mouse Insulin Cells Expressing an Inducible RIPCre Transgene Are Functionally Impaired

Gladys Teitelman,Mamdouh Kedees

doi:10.1074/jbc.m114.615484

Abstract

We used cre-lox technology to test whether the inducible expression of Cre minimize the deleterious effect of the enzyme on beta cell function. We studied mice in which Cre is linked to a modified estrogen receptor (ER), and its expression is controlled by the rat insulin promoter (RIP). Following the injection of tamoxifen (TM), CreER- migrates to the nucleus and promotes the appearance of a reporter protein, enhanced yellow fluorescent protein (EYFP), in cells. Immunocytochemical analysis indicated that 46.6 ± 2.1% insulin cells of adult RIPCreER- EYFP expressed EYFP. RIPCreER-EYFP (+TM) mice were normoglycemic throughout the study, and their glucose tolerance test results were similar to control CD-1 mice. However, an extended exposure to reagents that stimulate insulin synthesis was detrimental to the survival of IN+EYFP+cells. The administration of an inhibitor of the enzyme dipeptidyl-peptidase (DPP4i), which prevents the cleavage of glucagon-like peptide (GLP-1), to adult RIPCreER-EYFP mice lead to a decrease in the percentage of IN+EYFP+ to 17.5 ± 1.73 and a significant increase in apoptotic cells in islets. Similarly, a 2-week administration of the GLP-1 analog exendin 4 (ex-4) induced an almost complete ablation of IN+ expressing a different reporter protein and a significant decrease in the beta cell mass and rate of beta cell proliferation. Since normal beta cells do not die when induced to increase insulin synthesis, our observations indicate that insulin cells expressing an inducible RIPCre transgene are functionally deficient. Studies employing these mice should carefully consider the pitfalls of the Cre-Lox technique.

Highlights

We tested whether insulin cells expressing an inducible rat insulin promoter (RIP)-Cre transgene display a normal phenotype
RIPCreER-enhanced yellow fluorescent protein (EYFP) mice were normoglycemic throughout the study, and the Intraperitoneal Glucose Tolerance Test (IPGTT) of 5-month-old bigenic mice revealed that their response to glucose was normal, indicating that they were glucose tolerant (Fig. 1A)
In contrast to mice fed with control diet, the percentage of INϩEYFPϩ cells decreased from 29% at three months to 17.5 Ϯ 1.73 (4 mice and a total of 9474 INϩ cells scored) in 6-month-old RIPCreER-EYFP mice that received DPP4i therapy during months 4 – 6 (Fig. 2E)

Summary

Background

We tested whether insulin cells expressing an inducible RIP-Cre transgene display a normal phenotype. Since normal beta cells do not die when induced to increase insulin synthesis, our observations indicate that insulin cells expressing an inducible RIPCre transgene are functionally deficient Studies employing these mice should carefully consider the pitfalls of the Cre-Lox technique. There is evidence that mice constitutively expressing Cre-recombinase in beta cells are glucose intolerant [1] and that the Cre transgene driven by the rat insulin promoter (RIP) is expressed in the hypothalamus [2], raising concern regarding the evaluation of studies exploring the effect of genes on metabolic function. We used a line, termed RIPCreER-EYFP, generated by crossing mice (RIP-CreER) harboring a transgene comprised of the RIP linked to Cre recombinase-estrogen receptor with a strain containing a floxed reporter gene encoding for Enhanced Yellow Fluorescent Protein (EYFP). Since the measurement of glucose responsiveness evaluates the response of all the beta cell

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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION