Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis of insulin granules

Abstract

Objective: The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from β-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking β-cell ABCA1.

Research design and methods: Calcium imaging, patch clamp, and membrane capacitance were used to assess the effect of ABCA1 deficiency on calcium flux, ion channel function, and exocytosis in islet cells. Electron microscopy was used to analyze β-cell ultrastructure. The quantity and distribution of proteins involved in insulin-granule exocytosis were also investigated.

Results: We show that a lack of β-cell ABCA1 results in impaired depolarization-induced exocytotic fusion of insulin granules. We observed disturbances in membrane microdomain organization and Golgi and insulin granule morphology in β-cells as well as elevated fasting plasma proinsulin levels in mice in the absence of β-cell ABCA1. Acute cholesterol depletion rescued the exocytotic defect in β-cells lacking ABCA1, indicating that elevated islet cholesterol accumulation directly impairs granule fusion and insulin secretion.

Conclusions: Our data highlight a crucial role of ABCA1 and cellular cholesterol in β-cells that is necessary for regulated insulin granule fusion events. These data suggest that abnormalities of cholesterol metabolism may contribute to the impaired β-cell function in diabetes.

FIG. 1.

Mice lacking β-cell ABCA1 show impaired glucose tolerance, impaired insulin secretion, impaired islet cholesterol efflux, and increased islet cholesterol levels. A: Plasma glucose levels during glucose tolerance test (n = 5). B: Glucose-stimulated insulin secretion from isolated islets. Values represent pooled data from three separate experiments; each consisted of pooled islets from three mice per genotype, and values are expressed as percent of islet content relative to basal secretion, which is arbitrarily set to 1. C: Cholesterol efflux toward apoA1. Values represent pooled data from three separate experiments. D: Islet cholesterol levels (n = 4). ***P < 0.001 compared with controls.

FIG. 2.

Ca²⁺ influx and ion channel activity unaltered in β-cells lacking ABCA1. A: Influence of 10 mmol/L glucose and 100 μmol/L tolbutamide (Tol) on [Ca²⁺]_i. [Ca²⁺]_i was monitored as the 340:380 nm fluorescence ratio. Representative traces of individual β-cells are shown. B: Ca²⁺ current density from β-cells: i) representative current density recording and ii) voltage clamp protocol used to elicit Ca²⁺ currents. C: Group data for control (n = 14) and ABCA1^−P/−P (n = 11) mice for peak current density, mean current density measured during the last 10 ms of the depolarization step (End), and total Ca²⁺ current density for entire depolarization step measured as area under the curve (AUC). D: K⁺ current density from β-cells: i) representative current density recording and ii) voltage clamp protocol used to elicit K⁺ currents. E: Group data for control (n = 8) and ABCA^−P/−P mice (n = 7) during different voltages.

FIG. 3.

Depolarization-induced exocytosis is impaired in β-cells lacking ABCA1. A: β-Cell membrane capacitance (C_m) and voltage-dependent Ca²⁺ currents (I_Ca) in response to a single 500-ms depolarization. B: The average exocytotic response, normalized to initial cell size (n = 40–58). C: FM1-43 destaining in response to 30 mmol/L KCl in ABCA1^+/+ (n = 23, ■) and ABCA1^−P/−P (n = 24, □) β-cells. AU, arbitrary unit. D: Capacitance measurements after a series of membrane depolarizations of progressively increasing duration. E: The average responses of ABCA1^+/+ (n = 40, ■) and ABCA1^−P/−P (n = 58, □) β-cells. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls.

FIG. 4.

β-Cells lacking ABCA1 show similar number of docked granules. A: Representative electron micrographs. Insert shows docked granules at the plasma membrane in more detail. B: The percentage of secretory granules localized to the plasma membrane (<100 nm) within ABCA1^+/+ and ABCA1^−P/−P β-cells (n = 13–19). (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Major alterations to Golgi organization in ABCA1^−P/−P β-cells. The Golgi region in β-cells from control mice (A and B) reflected the hallmark architecture of Golgi membranes organized as a series of “compact regions” of stacked cisternae connected laterally to form a ribbon. Golgi region in β-cells from ABCA1^−P/−P mice (C and D) demonstrated a tendency toward circular organization with more ordered and tightly stacked appearance, both at the level of increased cisternal stacking and increased lateral continuity along the length of the ribbon itself. GA, Golgi apparatus; M, mitochondrion; N, nucleus. Bars, 1 μm (except for inset, 500 nm).

FIG. 6.

Cholesterol accumulation alters membrane microdomain organization and impairs SNARE protein localization. A: SNAP-25, VAMP-2, syntaxin-1, and syntaxin-4 protein levels in isolated islets. Graphs represent pooled densitometric measurement of protein signal intensity from three separate experiments. Actin was used as the loading control. B: Representative Western blot of transferrin receptor (TfR), a marker for soluble fractions; flotillin, a marker for nonsoluble fractions; and SNAP-25 in lipid raft fractions of MIN6 cells treated with or without 2 mmol/L cholesterol (n = 2) for 30 min, followed by 10 mmol/L MβCD (n = 1) for an additional 30 min. Fractions 5–8 were designated as nonsoluble and band intensities quantified and expressed on the right panel. C: Representative Western blot of EGF-induced phosphorylation of AKT in isolated islets. Graphs represent pooled densitometric measurement of protein signal intensity from four separate experiments. **P < 0.01 compared with controls. (A high-quality color representation of this figure is available in the online issue.)

FIG. 7.

The exocytotic defect in ABCA1^−P/−P β-cells is rescued by acute intracellular cholesterol depletion. A: Whole-cell membrane capacitance from β-cells after intracellular dialysis with a low (10 μmol/L) concentration of MβCD to deplete cholesterol via the cell interior. B: Membrane capacitance during co-infusion of 200 nmol/L free–Ca²⁺ together with 10 μmol/L MβCD or an equal concentration of DMSO. C: The total capacitance increase, normalized to initial cell size, at 200 s after Ca²⁺ infusion (n = 13–16). D: The exocytotic response to a series of ten 500-ms depolarizations rescued in ABCA1^−P/−P β-cells by intracellular cholesterol depletion. E: The cumulative capacitance response of ABCA1^+/+ (n = 21, ○) and ABCA1^−P/−P β-cells dialyzed with DMSO (n = 23, □) or 10 μmol/L MβCD (n = 20, ■). For clarity, the ABCA1^+/+ + MβCD group is not shown, although this was not different from controls. *P < 0.05 and **P < 0.01 compared with controls.