Pleckstrin homology-like domain family A, member 3 (PHLDA3) deficiency improves islets engraftment through the suppression of hypoxic damage

Abstract

Islet transplantation is a useful cell replacement therapy that can restore the glycometabolic function of severe diabetic patients. It is known that many transplanted islets failed to engraft, and thus, new approaches for overcoming graft loss that may improve the outcome of future clinical islet transplantations are necessary. Pleckstrin homology-like domain family A, member 3 (PHLDA3) is a known suppressor of neuroendocrine tumorigenicity, yet deficiency of this gene increases islet proliferation, prevents islet apoptosis, and improves their insulin-releasing function without causing tumors. In this study, we examined the potential use of PHLDA3-deficient islets in transplantation. We observed that: 1) transplanting PHLDA3-deficient islets into diabetic mice significantly improved their glycometabolic condition, 2) the improved engraftment of PHLDA3-deficient islets resulted from increased cell survival during early transplantation, and 3) Akt activity was elevated in PHLDA3-deficient islets, especially under hypoxic conditions. Thus, we determined that PHLDA3-deficient islets are more resistant against stresses induced by islet isolation and transplantation. We conclude that use of islets with suppressed PHLDA3 expression could be a novel and promising treatment for improving engraftment and consequent glycemic control in islet transplantation.

Fig 1. Condition of PHLDA3-deficient islets at 24 hs after isolation.

A. Islets from PHLDA3 KO and WT groups were stained by SYTO Green 11 (for viable cells) and ethidium bromide (for dead cells, indicated by arrow). More viable cells and fewer dead cells were seen in KO group than the WT group. The size of the scale bar is 100 μm. B. Quantification of islet viability showed significantly higher viability for PHLDA3-deficient (= KO: n = 38 islets) than wild-type islets (n = 31 islets). C. Volumes of released insulin following stimulation in low (3.3mM) and high (16.5mM) glucose solutions. Insulin release dependent on glucose concentration was seen in both the KO and WT groups, and the levels between the two groups were similar (n = 15 islets in both groups). For statistical analysis, Mann–Whitney U test was used for viability and two-way repeated measures ANOVA for insulin—releasing function.

Fig 2. Metabolic condition of diabetic mice after intrahepatic islet transplantation with PHLDA3-deficient or wild-type islets.

Blood glucose (A) and C-peptide (B) levels were measured after transplantation of PHLDA3-deficient or wild-type islets, and the difference between the KO (n = 17) and WT (n = 14) groups was statistically assessed by two-way repeated measures ANOVA. Levels of both were significantly improved in KO group in comparison with WT group. Blood glucose levels gradually decreased in both groups, but were significantly lower in the KO group compared to the WT group by postoperative day (POD) 28 (357.3 ± 35.7 mg/dL in KO group vs. 470.4 ± 35.4 mg/dL in WT group at POD 28, p = 0.04; 286.8 ± 33.3 mg/dL vs. 428.6 ± 42.4 mg/dL at POD 56, p = 0.02; A). Change in blood glucose levels after glucose stimulation (C) and AUC of the blood glucose levels after glucose stimulation (D) at POD 56 also revealed that the metabolic condition of the mice transplanted with PHLDA3-deficient islets was improved. The times of experiments were eight. For statistical analyses, the two-way repeated measures ANOVA for change of blood glucose level and Mann–Whitney U test for AUC were used.

Fig 3. Size and number of engrafted PHLDA3-deficient islets.

A. Engrafted islets at POD 56 and 180. The size was almost the same between the KO and WT groups at POD 56 (upper and middle lanes). Hyperplasia of PHLDA3-deficient islets was not detected at POD 180, but the size tended to be wider than that at POD 56 (bottom vs top panels). The size of the scale bar was 100 μm. B and C. Quantification of islet areas. There was no difference between the KO (n = 29 islets) and WT (n = 8 islets) groups at POD 56 (B). There was also no difference in sizes between POD 56 (n = 29 islets) and 180 (n = 7 islets) in the KO group, but the islet area at 180 tended to be wider than that at POD 56 (C). D. Numbers of engrafted islets at POD 56. More engrafted islets were seen in the KO group (n = 17) than the WT group (n = 14). Mann–Whitney U test was used for statistical assessment.

Fig 4. Condition of islets at 12 hs after transplantation.

A. Histological analyses of KO and WT islets at 12 hs after transplantation. Apoptosis (TUNEL-positive), necrosis (eosin-positive without nucleus area) and proliferation (Ki67-positive) were seen in both the KO (n = 5) and WT (n = 6) groups, but they were less prominent in the KO group. The images (insulin, TUNEL, HE, Ki67) were of the same location. The dashed lines indicate the border of the transplanted islets. The size of the scale bar was 100 μm. B. Quantification of the digitalized images showing apoptosis, necrosis and proliferation. The data confirms that apoptosis of transplanted islets at 12 hs was lower in the KO group. The statistical analysis was done using the Mann–Whitney U test.

Fig 5. Expression of HIF-1α in transplanted islets at 12 hs after transplantation and Western blotting of islets cultured under hypoxic conditions.

A. Histological analysis of transplanted PHLDA3-deficient (KO) islets. The expression of HIF-1α was not strong, but relatively higher than in the surrounding liver tissue. The dashed line indicates the border of the transplanted islet. The size of the scale bar was 100 μm. B and C. Density of HIF-1α—positive islet cells (B) and ratio of density between islet and HIF-1α-negative area (C). The numbers of mice were 5 in the KO group and 6 in the WT group. These data revealed that the expression of HIF-1α was significantly stronger in KO group. The statistical analysis was done using the Mann–Whitney U test. D-F. Expression of HIF-1α (D), total and phosphorylation (p) of Akt (E), total and phosphorylation (p) of Akt substrates (F, MDM2, GSK3-β, S6K and 4E-BP1) in PHLDA3-deficient (K) and wild-type (W) islets in normoxic (N) and hypoxic (H) conditions were assessed by Western blotting. Expression of β-actin is also assessed. Same β-actin blot appears for pAkt(S473) and pS6K, pAkt(T308), pMDM2 and pGSK3-β, and total GSK3-β, total S6K and p4E-BP1, since they derive from the same blot. Akt was strongly phosphorylated in PHLDA3-deficient islets under hypoxic conditions. Phosphorylation of Akt-substrate MDM2 and GSK3-β were also induced in PHLDA3-deficient islets. HIF-1α was stabilized in PHLDA3-deficient islets, especially under hypoxic conditions.

Fig 6. Proposed mechanism underlying the effects of PHLDA3 deficiency on islet transplantation.

Modulation of the PI3K / Akt signaling pathway is proposed to be responsible for improved cell growth and reduced apoptosis seen with transplanted PHLDA3-deficient islets under hypoxic conditions.