Mouse insulin cells expressing an inducible RIPCre transgene are functionally impaired

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.

Keywords: Apoptosis; Beta Cells; Gene Knockout; Insulin Synthesis; Pancreatic Islet; Transgenic Mice.

FIGURE 1.

DPP4i induce an increase in apoptotic cells. A, IPGTT-Blood glucose levels after administration of glucose (ip) to 5-month-old CD-1 mice(dashed lines) or RIPCreER-EYFP (solid line) mice. n = 5 animals/group. No statistical difference was found between the results from the two different lines. B, body weight of bigenic mice fed control diet (dashed lines) or pellets containing the DPP4i (solid lines) through an 12-week-period; n = 4 animals/group. C, beta cell mass is similar in bigenic mice receiving control diet or pellets containing MK0626. At least 25,000 points were scored per group (n = 4). D, percentage of apoptotic cells per islet area is significantly higher in bigenic mice receiving a diet containing a DPP4i than in those fed a control diet. At least 20 islets were scored/group (n = 4 mice/group). E, islet of a DPP4i treated RIPCreER-EYFP mice immunostained for visualization of activated caspase-3 (ac-3; green). Arrow indicates a cell expressing cytoplasmic activated caspase 3. This cell is shown magnified in F. Asterisk is located between two apoptotic bodies; these bodies are shown magnified in G. H, islet of a bigenic mouse injected with saline. Note the absence of apoptotic figures.

FIGURE 2.

IN+EYFP+ cells are ablated by the DPP4i. A and B, EYFP histofluorescence (green) in pancreatic islets of RIPCreER-EYFP mice (+TM) fed with control (A) or DPP4i (B) diet; C, D, pancreatic islets of RIPCreER-EYFP mice (+TM) fed with control (C) or DPP4i (D) diet immunostained for insulin (red) and EYFP (green). E, percentage of EYFP+ cells in RIPCreER-EYFP mice receiving control or DPP4i diet. n = 3/line. Note that the percentage of IN+EYFP+ cells increase with time in mice fed with control diet. In contrast, the percentage of IN+EYFP+ cells decrease in bigenic mice receiving DPP4i. *, p < 0.005

FIGURE 3.

The decrease in the percentage of EYFP+IN+ cells is not due to a reduction in their rate of proliferation. A–C, islet of RIPCreER-EYFP (+TM) mouse immunostained for visualization of insulin (blue), BrdU(red), and EYFP(green). A, insulin +BrdU; B, IN + EYFP, and C, IN + EYFP+ BrdU. Arrow indicates an IN+ EYFP+BrdU+ cell; arrowhead indicates an IN+EYFP-BrdU+ cell and asterisk in B indicates two IN+EYFP+BrdU- cells. D, percentage of cells expressing BrdU is similar in the EYFP+IN+ and EYFP-IN+ cell populations. 7338 IN+EYFP cells and 943 IN+EYFP+ cells were scored/mouse (n = 3). E, the percentage of IN+ BrdU+ cells is lower in mice receiving a diet containing MK0626 than in mice fed a control diet. *, p < 0.05. At least 5000 IN cells were scored/group (n = 3 mice/group).

FIGURE 4.

Exendin-4 ablates IN+ cells expressing a RIPCre transgene. A, percentage of IN+PLAP+ cells is significantly reduced in RIPCreER-PLAP mice receiving exendin-4. At least 20 islets were scored/mice/3 mice/group. ***, p < 0.001. B, immunohistochemical visualization of insulin(red) and PLAP(green) in islets of RIPCreER-PLAP mice injected with saline (B) or with exendin-4 (C and D). Note that islets of mice receiving the incretin analog contain few (C) or almost no (D) PLAP+ cells. Bars, B and C: 20 um; D: 40 μm. E, Exendin-4 induced a 50% reduction in the beta cell mass compared with controls. Over 15,000 points were scored in sections (5 mice/group); ***, p < 0.001. F, percentage of insulin-positive cells expressing Ki67 is dramatically lower in RIPCreER-PLAP mice receiving exendin-4 than in mice injected with saline (over 3000 cells were scored per mice/5 mice per group). No statistical error was calculated for results from RIPCreER-PLAP mice injected with exendin-4 because most islets did not contain Ki67+ cells insulin cells.