Inhibition of p53 preserves Parkin-mediated mitophagy and pancreatic β-cell function in diabetes

Abstract

Mitochondrial compromise is a fundamental contributor to pancreatic β-cell failure in diabetes. Previous studies have demonstrated a broader role for tumor suppressor p53 that extends to the modulation of mitochondrial homeostasis. However, the role of islet p53 in glucose homeostasis has not yet been evaluated. Here we show that p53 deficiency protects against the development of diabetes in streptozotocin (STZ)-induced type 1 and db/db mouse models of type 2 diabetes. Glucolipotoxicity stimulates NADPH oxidase via receptor for advanced-glycation end products and Toll-like receptor 4. This oxidative stress induces the accumulation of p53 in the cytosolic compartment of pancreatic β-cells in concert with endoplasmic reticulum stress. Cytosolic p53 disturbs the process of mitophagy through an inhibitory interaction with Parkin and induces mitochondrial dysfunction. The occurrence of mitophagy is maintained in STZ-treated p53(-/-) mice that exhibit preserved glucose oxidation capacity and subsequent insulin secretion signaling, leading to better glucose tolerance. These protective effects are not observed when Parkin is deleted. Furthermore, pifithrin-α, a specific inhibitor of p53, ameliorates mitochondrial dysfunction and glucose intolerance in both STZ-treated and db/db mice. Thus, an intervention with cytosolic p53 for a mitophagy deficiency may be a therapeutic strategy for the prevention and treatment of diabetes.

Fig. 1.

p53^−/− mice were resistant to hyperglycemia in STZ-induced type 1 diabetes. (A) Blood glucose and (B) serum insulin concentrations from mice fed ad libitum were measured at the indicated time points after STZ treatment (n = 16–18). (C) Glucose and (D) insulin tolerance tests were performed 4 wk after STZ treatment (n = 12). (E) Serum insulin values were assessed before and at 15 and 30 min into the glucose tolerance test (n = 12). Data are shown as the means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Glucose oxidation capacity was preserved in STZ-treated p53^−/− mice. (A) Histological analysis of insulin immunostaining of islets 4 wk after STZ treatment. (Scale bars, 500 μm.) The β-cell area relative to the pancreatic area was quantified (n = 4). (B) Representative results of the OCR of islets assessed using the Seahorse XF24 extracellular flux analyzer. The amplification rate of OCR at high (22.2 mM) glucose was calculated relative to basal (2.8 mM) glucose stimulation from five replicates per sample (n = 4). Data are shown as the means ± SD. *P < 0.05; ***P < 0.001.

Fig. 3.

Cytosolic p53 inhibits Parkin-mediated mitophagy in β-cells. (A) The endogenous Parkin–p53 complex in β-cells. Cytosolic lysate of MIN6 β-cells exposed to 30 mM high glucose (HG) and 0.3 mM palmitate (PA) for 1 wk was immunoprecipitated with anti-p53, Parkin, and control IgG antibodies. (B) Representative images of GFP-LC3–overxpressing MIN6 β-cells exposed to HG and PA. Cells were transfected with siRNA targeting p53 and/or Parkin and were treated to underscore mitophagy with 100 nM Baf-A1, a specific inhibitor of H⁺-ATPases of the vacuolar type, for 6 h before the immunostaining of mitochondria with anti-TOM20 (red) (original magnification, ×1,000). (Scale bars, 10 μm.) (Right) Colocalization between LC3 and TOM20. A minimum of 50 GFP-positive cells were scored in three independent experiments. (C and D) MIN6 β-cells were stimulated with 2.8 or 22.2 mM glucose for 30 min. Insulin secretion (C) and ATP contents (D) were measured by ELISA and a luminometer, respectively, in duplicate (n = 4). (E) Glucose-stimulated insulin secretion was measured by ELISA in the context of mitophagy deficiency induced by the transfected with siRNA targeting Atg5 or PINK1. The experiments were performed in duplicate (n = 4). Data are shown as the means ± SD. *P < 0.05; **P < 0.01.

Fig. 4.

Parkin-mediated mitophagy maintains insulin secretion under diabetic conditions. (A) Construction of the stable MIN6 β-cell lines overexpressing Parkin. Representative immunoblots are shown from three independent experiments. (B) Excitation ratio (590/430 nm) of pMT-mKeima-Red–overexpressing stable cell lines incubated with HG and PA. Baf-A1–treated cells were used as a negative control for the induction of mitophagy. The experiments were performed in eight replicates (n = 3). (C) The sequential state III OCR of isolated mitochondria from stable cell lines was assessed using the Seahorse XF24 analyzer. Representative results are shown, and the OCR of each complex was calculated from five replicates per sample (n = 3). (D) Glucokinase (GK) and hexokinase (HK) activities in stable cell lines. Results are shown from four independent experiments performed in duplicate. (E) ATP contents and (F and G) insulin secretion were measured by a luminometer and ELISA, respectively. The experiments were performed in duplicate (n = 4). Data are shown as the means ± SD. ^†P < 0.05 versus stable cell lines carrying control vectors under the same condition.

Fig. 5.

Parkin-mediated mitophagy links p53 deficiency and protection against diabetes. (A) Representative electron micrographs of STZ-treated islets. Arrows indicate abnormal mitochondria defined by marked swelling associated with an increased number of disarrayed or disappeared cristae and by the reduced electron density of the matrix (original magnification ×5,000). (Scale bars, 2 μm.) Magnified photograph represents an autophagic vacuole containing mitochondria (red box) (original magnification ×12,000). (Scale bar, 500 nm.) (Right) Abnormal mitochondria and mitochondria incorporated into the autophagic vacuole were quantified from at least 10 β-cells (n = 3). (B and C) Batches of 10 pancreatic islets isolated from STZ-treated WT, p53^−/−, Parkin^−/−, and p53^−/−Parkin^−/− mice were stimulated with 2.8 or 22.2 mM glucose for 30 min. ATP contents (B) and insulin secretion (C) were measured by a luminometer and ELISA, respectively. The amplification rates of ATP contents and insulin secretion at high glucose were calculated relative to basal glucose stimulation. Results are shown from duplicate experiments per sample (n = 6). (D) Blood glucose levels and (E) serum insulin levels from mice fed ad libitum (n = 11–20). (F) Glucose tolerance tests were performed after 4 wk of PFT-α or the carrier DMSO treatment (n = 12). (G) Serum insulin values were assessed before and at 15 and 30 min into the glucose tolerance test (n = 12). (H) The OCR of islets was assessed using the Seahorse XF24 analyzer. The amplification rate of OCR at high glucose was calculated relative to basal glucose stimulation from five replicates per sample (n = 4). Data are shown as the means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

PFT-α preserves β-cell integrity in db/db mice. (A) Six-week-old mice were injected with PFT-α or the carrier DMSO three times per week for 4 wk. Blood glucose levels were measured at the indicated time points (n = 14). (B) Serum insulin levels from mice fed ad libitum were measured after 4 wk of PFT-α or the carrier DMSO treatment (n = 14). (C) Glucose and (D) insulin tolerance tests were performed after 4 wk of treatment (n = 8). (E) Serum insulin values were assessed before and at 15 and 30 min into the glucose tolerance test (n = 8). (F) Representative electron micrographs of islets in db/db mice treated with PFT-α or the carrier DMSO. Arrows indicate abnormal mitochondria (original magnification ×5,000). (Scale bars, 2 μm.) Magnified photograph represents an autophagic vacuole containing mitochondria (red box) (original magnification ×12,000). (Scale bar, 500 nm.) (Lower) Abnormal mitochondria and mitochondria incorporated into the autophagic vacuole were quantified from at least 10 β-cells (n = 3). (G) Batches of five pancreatic db/db mice islets were stimulated with 2.8 or 22.2 mM glucose. The OCR of islets was assessed using the Seahorse XF24 analyzer. The amplification rate of OCR at high glucose was calculated relative to basal glucose stimulation from five replicates per sample (n = 4). (H) ATP contents and (I) insulin secretion were measured by a luminometer and ELISA, respectively. The amplification rates of ATP contents and insulin secretion at high glucose were calculated relative to basal glucose stimulation. Results are shown from duplicate experiments per sample (n = 4). Data are shown as the means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.