Expression Analysis of cPLA2 Alpha Interacting TIP60 in Diabetic KKAy and Non-Diabetic C57BL Wild-Type Mice: No Impact of Transient and Stable TIP60 Overexpression on Glucose-Stimulated Insulin Secretion in Pancreatic Beta-Cells

Abstract

In the present study we investigate the expression levels of cytosolic phospholipase A2 alpha (cPLA2alpha) interacting histone acetyl transferase proteins TIP60alpha and TIP60beta in non-diabetic C57BL wild-type mice and obese type 2 diabetic KKAy model mice. The aim was to test our hypothesis that TIP60 plays a regulatory role in glucose-stimulated insulin secretion from pancreatic beta-cells.

Material and methods: Ten obese diabetic KKAy mice and ten non-diabetic C57BL mice were fed a standard chow diet. After nine weeks, islet RNA was purified and used to measure TIP60 expression. We investigated the effect of TIP60alpha and TIP60beta on glucose-stimulated insulin secretion by transient and stable overexpression in the pancreatic mouse beta-cell line MIN6 and the rat beta-cell line INS-1E.

Results: We found that non-diabetic C57BL mice and diabetic KKAy mice have the same level of both the alpha and beta splice forms of TIP60. Furthermore, we demonstrated that transient and stable expression of TIP60 in INS-1E cells affects neither glucose-stimulated insulin secretion, insulin output nor cell insulin content. Also susceptibility to developing gluco-toxicity was unaffected.

Conclusion: TIP60 over-expression does not affect glucose stimulated insulin secretion, insulin content or abnormal beta-cell function during glucotoxicity.

Figure 1

Fasting plasma glucose (A) and insulin (B) in KKAy and C57BL mice before and after 9 weeks of feeding. The C57BL and KKAy control groups were fed standard chow diet. The animals were 5 weeks old when treatment began. Data are shown as mean ± SEM (n = 10 in each group). ^***p < 0.001 (unpaired), ^#p < 0.05 (paired), ^###p < 0.001 (paired).

Figure 2

RNA from pancreatic islets of C57BL and KKAy mice fed for 9 weeks with chow was isolated and the transcript abundance of TIP60α and TIP60β mRNA was measured using real-time RT-PCR. A: relative TIP60α expression. B: relative TIP60β expression. C: ratio of TIP60β mRNA and TIP60α mRNA. To evaluate TIP60 expression levels across tissues, RNA was prepared from liver and muscles from the C57BL group and the comparative transcript abundance of TIP60α (D) and TIP60β (E) mRNA in liver, muscles and MIN6 cells was measured. Measurements were carried out in triplicate for each sample and gene expressions were normalized to 18S rRNA expression (n = 4).

Figure 3

RNA from pancreatic islets of C57BL and KKAy mice fed for 9 weeks with chow was isolated and genes related to insulin secretion and regulation were studied for changes in their transcript abundance (TA) by real-time RT-PCR using TaqMan assays. Measurements were carried out in triplicate for each sample and gene expressions were normalized to 18S rRNA expression. Changes in TA were calculated for the KKAy group compared to the C57BL control group. ^*p < 0.05 (n = 4).

Figure 4

INS-1E cells were transiently transfected with EGFP-TIP60α and EGFP-TIP60β and empty EGFP vector. The transcript abundance of TIP60α (A) and TIP60β (B) mRNA was measured 48 hours after transfection using real time RT-PCR (n = 4). C: Western blot of 30 µg total protein lysate from EGFP (lane 1-2), EGFP-TIP60α (lane 3-4) and EGFP-TIP60β (lane 5-6) transfected INS-1E cells 48 h post transfection, probed with anti-TIP60 antibody. D: Western blot as figure C probed with actin antibody. E-H: Glucose-stimulated insulin secretion at 2 mM glucose (low) and 15 mM glucose (high) (E), stimulated GSIS going from low to high glucose (F), insulin output from culture medium (G) and cell insulin content (H) for passage 70 (upper panel) and passage 74 (lower panel) (n = 12). I-J: Fluorescence microscopy of fixated EGFP-TIP60α (I) and EGFP-TIP60β (J) transfected INS-1E cells 48 h after transfection.

Figure 5

INS-1E cells were transducted with the following retroviral expression plasmids, pSUPER-retro-puro, pSUPER-TIP60α and pSUPER-TIP60β followed by puromycin selection. The transcript abundance of TIP60α (A) and TIP60β (B) mRNA was measured using real time RT-PCR. (n = 4). C: Western blot of 30 µg total protein lysate from pSUPER (lane 1-3), pSUPER-TIP60α (lane 4-6) and pSUPER-TIP60β (lane 7-9) stably expressing INS-1E cell lines, probed with anti-TIP60 antibody. D: Western blot as in C, probed with actin antibody. Panels E, F and G show glucose-stimulated insulin secretion (E) at 2 mM glucose (low) and 15 mM glucose (high) stimulated GSIS going from low to high glucose (F) and insulin content (G) (n = 12).

Figure 6

Glucotoxicity of INS-1E cells stable overexpressing TIP60α. INS-1E stable transduced with empty pBabe (A) or pBabe-TIP60α (B) were seeded in 24-well plates 2 × 10⁵ cells/well. After 24 h, the culture medium was removed and replaced with culture medium containing 11.1 mM, 16.7 mM, 20.0 mM or 25.0 mM glucose and cultured for 48 h. Subsequently, we washed the cells for 15 min. in UB buffer and measured GSIS at 2 and 15 mM glucose Each bar represents the mean ± SEM from 12 incubation wells. ^* p < 0.05.