p85α deficiency protects β-cells from endoplasmic reticulum stress-induced apoptosis

Abstract

In insulin resistant states such as type 2 diabetes, there is a high demand on the β-cell to synthesize and secrete insulin, which challenges the ability of the endoplasmic reticulum (ER) to synthesize and fold nascent proteins. This creates a state of ER stress that triggers a coordinated program referred to as the unfolded protein response (UPR) that attempts to restore ER homeostasis. We identified a role for the p85α regulatory subunit of PI3K to modulate the UPR by promoting the nuclear localization of X-box binding protein 1, a transcription factor central to the UPR. In the present study we demonstrate that reducing p85α expression in β-cells can markedly delay the onset and severity of the diabetic phenotype observed in Akita(+/-) mice, which express a mutant insulin molecule. This is due to a decrease in activation of ER stress-dependent apoptotic pathways and a preservation of β-cell mass and function. These data demonstrate that modulation of p85α can protect pancreatic β-cells from ER stress, pointing to a potentially therapeutic target in diabetic states.

Fig. 1.

Assessment of glucose homeostasis at 6 wk of age. mRNA expression of (A) p85α or (B) p85β were measured from islets isolated from study group mice at 20 wk of age. (C) Fasting glucose was measured after a 16- to 18-h fast (n = 6–12). (D) Fasting insulin was measured from serum after a 16- to 18-h fast (n = 5–8). (E) Random fed glucose was measured between 8:00 and 10:00 AM (n = 5–7). (F) Random fed serum insulin levels. n = 5–6. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Morphometric analysis of islets at 9 wk of age. (A) Hematoxylin and eosin staining and IHC using anti-insulin and anti-glucagon antibodies was performed on pancreas sections. Representative images are shown. (B) Islet area was determined using ImageJ, and average islet size is plotted. n = 4. (C) Total β-cell area as a percentage of total pancreas area. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Transmission electron microscopy (TEM) reveals a reduction in ER stress-associated abnormalities and restoration of insulin content in Akita/β-KO islets. TEM was performed on islets isolated from study group animals at 20 wk of age. Representative images are shown.

Fig. 4.

Measurement of proliferation and apoptosis at 9 wk of age. (A) Rates of apoptosis were determined by performing the TUNEL assay. n = 3 per group. (B) Isolated islets from control and β-KO animals were treated with vehicle or tunicamycin for 24 h before performing the TUNEL assay. (C) Proliferation of β-cells was determined by performing IHC using anti-insulin and anti-Ki67 antibodies. n = 3. *P < 0.05, **P < 0.01. (D) Quantification of proliferation rates was performed and is expressed as the percentage of insulin⁺/Ki67⁺ cells.

Fig. 5.

Islet gene expression reveals a profile indicative of reduced ER and oxidative stress and activation of the NLRP3 inflammasome pathway in Akita/β-KO islets. Quantitative RT-PCR was performed with gene-specific primers to determine the relative mRNA expression of (A) ER stress, (B) oxidative stress, or (C) NLRP3 inflammasome-associated genes in isolated islets from mice at 20 wk of age. n = 3–5. Gene expression was normalized to TBP mRNA expression. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

p85α stabilizes XBP-1 and regulates XBP-1–dependent apoptosis. (A) The MIN6 β-cell line was transduced with the indicated adenoviral constructs. Forty-eight hours later, whole-cell protein lysates were prepared, resolved by SDS/PAGE, and immunoblotted with the indicated antibodies. (B) The MIN6 β-cell line was transduced with the indicated adenoviral constructs. Twenty-four hours later, transduced cells were treated with vehicle, tunicamycin (5 μg/dL), or thapsigargin (200 nM) for an additional 24 h. Subsequently, whole-cell protein lysates were prepared, resolved by SDS/PAGE, and immunoblotted with the indicated antibodies. (C) The MIN6 β-cell line was transduced with the indicated adenoviral constructs. Forty-eight hours later, protein lysates were prepared, resolved by SDS/PAGE, and immunoblotted with the indicated antibodies.