P38alpha-selective mitogen-activated protein kinase inhibitor for improvement of cultured human islet recovery

Abstract

Objectives: We investigated whether the recovery of cultured human islets is improved through the addition of a p38alpha-selective mitogen-activated protein kinase inhibitor, SD-282, to clinically used serum-free culture medium.

Methods: Immediately after isolation, islets were cultured for 24 hours in medium alone (control) or medium containing dimethyl sulfoxide, 0.1 microM SD-282, or 0.3 microM SD-282. Cytokine expression, apoptotic beta-cell percentage, and islet function were assessed postculture.

Results: Expression of p38 and phosphorylated p38 in islets increased during culture. Interleukin 6 mRNA expression in cultured islets, as well as IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor released into the medium, was significantly reduced by adding SD-282. The apoptotic beta-cell percentage was significantly lower in islets cultured with 0.1 microM SD-282, but not 0.3 microM, as compared with the control. Stimulation indices measured in vitro were higher but without significance (P = 0.06); the function of transplanted islets in diabetic NOD-scid mice was also better in 0.1-microM SD-282 group as compared with control.

Conclusions: Better islet function was obtained by adding 0.1 microM SD-282 to the serum-free culture medium. This improvement was associated with suppression of cytokine production and prevention of beta-cell apoptosis. However, this beneficial effect was diminished at a higher concentration.

Fig. 1. p38 expression increased during islet culture, as confirmed by Western blot

Western blot analysis of p38MAPK activation in pre-culture, 2h-, 24h-culture islets with or without SD-282. A: total p38, phosphorylated p38, phosphorylated HSP-27 and β actin in pre- and post-cultured islets. The blot is representative of 3 tests using different islet batches; B: An average level of phophorylated p38 normalized by total p38 in each culture condition at each time points (n=3); C: An average level of p38 substrate, phosphorylated HSP-27, in each culture condition at each time points (n=3, *p<0.01 and **p<0.005).

Fig. 2. β cell apoptosis in cultured human islets is prevented by supplementing the medium with 0.1 μM SD-282 as determined by Laser Scanning Cytometry

Islet sections were labeled by TUNEL to identify apoptotic cells, immunostained for insulin to identify β cells, and quantified using laser scanning cytometry. A: Representative merged images of islets cultured for 24 hours (left – medium alone (control), right – medium supplemented with 0.1 μM SD-282). Green – TUNEL-positive, red – insulin-positive, yellow – TUNEL-insulin double-positive, and blue – DAPI DNA-staining; B: Scattergrams and histograms obtained by scanning the adjacent slides; C: Percentages of apoptotic cells apoptotic β cell percentages were calculated by dividing the TUNEL-insulin double-positive cell number by the total insulin-positive cell number in each section. TUNEL-positive/insulin-negative cells represent apoptotic non-β cells. Percentages of non-β cells were calculated by dividing the TUNEL-positive/insulin-negative cell number by the total number of non-β cells. After 24 hours culture, the mean apoptotic non-β cells (%) significantly increased in the DMSO (p<0.01) and 0.3 μM SD-282 (p<0.05) groups as compared to pre (0h). The mean apoptotic β cells (%) significantly increased in the DMSO (p<0.05) group as compared to pre (0h). To determine SD-282 effects, the relative ratio was obtained by dividing the post-culture apoptotic cell percentage by that of the corresponding medium alone control group; D: Relative ratios of apoptotic non-β cells – Culture medium supplemented with 0.1 μM SD-282 significantly prevented non-β cell apoptosis as compared to control (p<0.001); E: Relative ratios of apoptotic β cells – β cell apoptosis was significantly decreased with 0.1 μM SD-282 in the medium (p<0.01). This SD-282 effect was not observed at a concentration of 0.3 μM (n=5).

Fig. 3. Human islets cultured with 0.1 μM SD-282 respond to glucose stimulation in vitro with higher stimulation indices than controls

The effect of SD-282 on islet glucose responsiveness was assessed in vitro using an insulin release assay in a perifusion system. Insulin release profiles obtained from 150 IEQ cultured for 24 hours with medium alone or medium containing 0.1 μM SD-282. A: representative insulin release curve; B: Stimulation indices of control and SD-282-treated islets. SD-282-treated islets responded to high glucose with higher stimulation indices than untreated controls, but no statistical significance was obtained (p=0.06, n=6).

Fig. 4. Human islets cultured with 0.1 μM SD-282 function better than controls in vivo after transplantation into diabetic NODscid mice

Freshly isolated islets were cultured at 37°C either with or without SD-282. After 24 hours, a marginal number of islets (1200 IEQ) were transplanted under the renal capsule of a diabetic NODscid mouse. A: Blood glucose (mg/dl) of each animal in the control group after transplantation. B: Blood glucose (mg/dl) of each animal in the 0.1 μM SD-282 treated group. – All five mice receiving islets cultured with 0.1 μM SD-282 reversed diabetes by week 3 of transplantation, whereas only one of four mice receiving control islets reversed diabetes. Removal of the islet grafts (⇩) at twenty-seven days after transplantation caused recurrence of diabetes in all mice. C: Glucose tolerance curves obtained by intraperitoneal glucose tolerance tests on week 4 – The mice which received islets treated with 0.1μM SD-282 showed significantly better response to glucose challenge than the mice receiving control islets (n=4 for control group and n=5 for SD-282 group).