An intracellular role for ABCG1-mediated cholesterol transport in the regulated secretory pathway of mouse pancreatic β cells
Jeffrey M. Sturek, … , Raghavendra G. Mirmira, Catherine C. Hedrick
Jeffrey M. Sturek, … , Raghavendra G. Mirmira, Catherine C. Hedrick
Published June 7, 2010
Citation Information: J Clin Invest. 2010;120(7):2575-2589. https://doi.org/10.1172/JCI41280.
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Research Article Metabolism

An intracellular role for ABCG1-mediated cholesterol transport in the regulated secretory pathway of mouse pancreatic β cells

Abstract

Cholesterol is a critical component of cell membranes, and cellular cholesterol levels and distribution are tightly regulated in mammals. Recent evidence has revealed a critical role for pancreatic β cell–specific cholesterol homeostasis in insulin secretion as well as in β cell dysfunction in diabetes and the metabolic response to thiazolidinediones (TZDs), which are antidiabetic drugs. The ATP-binding cassette transporter G1 (ABCG1) has been shown to play a role in cholesterol efflux, but its role in β cells is currently unknown. In other cell types, ABCG1 expression is downregulated in diabetes and upregulated by TZDs. Here we have demonstrated an intracellular role for ABCG1 in β cells. Loss of ABCG1 expression impaired insulin secretion both in vivo and in vitro, but it had no effect on cellular cholesterol content or efflux. Subcellular localization studies showed the bulk of ABCG1 protein to be present in insulin granules. Loss of ABCG1 led to altered granule morphology and reduced granule cholesterol levels. Administration of exogenous cholesterol restored granule morphology and cholesterol content and rescued insulin secretion in ABCG1-deficient islets. These findings suggest that ABCG1 acts primarily to regulate subcellular cholesterol distribution in mouse β cells. Furthermore, islet ABCG1 expression was reduced in diabetic mice and restored by TZDs, implicating a role for regulation of islet ABCG1 expression in diabetes pathogenesis and treatment.

Authors

Jeffrey M. Sturek, J. David Castle, Anthony P. Trace, Laura C. Page, Anna M. Castle, Carmella Evans-Molina, John S. Parks, Raghavendra G. Mirmira, Catherine C. Hedrick

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Figure 2

ABCG1-deficient islets have impaired insulin secretion in vitro.

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ABCG1-deficient islets have impaired insulin secretion in vitro. (A and ...
(A and B) Pancreata from WT and Abcg1–/– mice were fixed, sectioned, and immunostained for insulin, and the total β cell area as determined by insulin-positive staining was quantified (n = 8). Scale bars: 500 μm. (C) Islets were isolated from WT and Abcg1–/– mice, and glucose- and potassium-induced insulin secretions were measured in vitro (n = 9–22, groups of 50 islets). (D and E) Isolated islets were loaded with the calcium dye fura-2, and [Ca2+]i changes in response to glucose perifusion were measured over time. (D) Representative curves of [Ca2+]i oscillations from WT and Abcg1–/– islets. (E) Average net increases in [Ca2+]i from basal to glucose-stimulated (n = 33–43 islets). (F) Insulin content from isolated islets (n = 10). (G) mRNA was harvested from isolated islets, and qRT-PCR was performed for transcript levels of the unprocessed insulin pre-mRNA, the mature processed insulin mRNA (transcripts for both insulin genes I and II were captured), and the regulatory enzyme glucokinase (n = 5–10). (H and I) First- and second-phase insulin secretion from WT and Abcg1–/– islets was measured by successive static incubations (first phase, 5 minutes in 30 mM KCl; second phase, 10 minutes in 28 mM glucose) (n = 10–11, groups of 50 islets). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.