Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Catherine B. Chan,Feihan F. Dai,Vasilij Koshkin,Christine A. Robson-Doucette,Michael B. Wheeler

doi:10.1074/jbc.m705652200

Catherine B. Chan, Feihan F. Dai + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m705652200

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Abstract

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.

Highlights

The physiological and pathological significance of mitochondrial permeability transition (MPT) in pancreatic ␤-cells is currently under investigation by several groups
Moderate Fatty Acid Treatment Does Not Induce Apoptosis in Clonal ␤-Cell Lines—Cells were exposed to various concentrations of palmitate, a saturated fatty acids (FA), and assayed for the onset of apoptosis after 48 h using the annexin V assay
A Solute Selective Form of MPT in ␤-Cells—Low conductance MPT was initially defined in terms of pore size allowing passage of only ions, such as Hϩ, Kϩ, Ca2ϩ, but not larger molecules, including sucrose and nucleotides [6, 7]

Summary

EXPERIMENTAL PROCEDURES

For kinetic Ca2ϩ measurements, loaded cells were diluted in KRB/BSA buffer up to 0.6 – 0.7 mg of protein/ml and Fluo-4 fluorescence was monitored with a FluoroCount plate reader (Packard Instrument Co.) at excitation/emission wavelengths of 485/530 nm. Insulin Secretion Assay and Measurement of Akt Phosphorylation—MIN6 cells cultured in 24-well plates were washed and preincubated for two sequential 30-min periods in glucose-free KRB buffer (125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl2, 5.0 mM NaHCO3, 25 mM HEPES, and 0.1% (w/v) bovine serum albumin), followed by incubation for 1 h in the same buffer containing 1 or 20 mM glucose in the absence or presence of 5 ␮M cyclosporin A or 0.15 ␮M FK-506.

RESULTS

Insulin secretion from untreated cells in the presence of CsA and FK-506

DISCUSSION