ApoA-I Protects Pancreatic -Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin

BACKGROUND:High cholesterol levels in pancreatic beta-cells cause oxidative stress and decrease insulin secretion. beta-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves beta-cell insulin secretion by reducing oxidative stress.Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-beta-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by beta-cells was monitored by flow cytometry. The effects of apoA-I internalization on beta-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the beta-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in beta-cells and isolated islets with MitoSOX and confocal microscopy.An F1-ATPase beta-subunit on the beta-cell surface was identified as the main apoA-I-binding partner. beta-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase beta-subunit-dependent. beta-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase beta-subunit levels than beta-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E beta-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function.These results establish that beta-cells are functionally heterogeneous, and apoA-I restores insulin secretion in beta-cells with elevated cholesterol levels by improving mitochondrial redox balance.BACKGROUND:High cholesterol levels in pancreatic beta-cells cause oxidative stress and decrease insulin secretion. beta-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves beta-cell insulin secretion by reducing oxidative stress.Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-beta-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by beta-cells was monitored by flow cytometry. The effects of apoA-I internalization on beta-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the beta-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in beta-cells and isolated islets with MitoSOX and confocal microscopy.An F1-ATPase beta-subunit on the beta-cell surface was identified as the main apoA-I-binding partner. beta-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase beta-subunit-dependent. beta-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase beta-subunit levels than beta-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E beta-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function.These results establish that beta-cells are functionally heterogeneous, and apoA-I restores insulin secretion in beta-cells with elevated cholesterol levels by improving mitochondrial redox balance.BACKGROUND:High cholesterol levels in pancreatic beta-cells cause oxidative stress and decrease insulin secretion. beta-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves beta-cell insulin secretion by reducing oxidative stress.Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-beta-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by beta-cells was monitored by flow cytometry. The effects of apoA-I internalization on beta-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the beta-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in beta-cells and isolated islets with MitoSOX and confocal microscopy.An F1-ATPase beta-subunit on the beta-cell surface was identified as the main apoA-I-binding partner. beta-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase beta-subunit-dependent. beta-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase beta-subunit levels than beta-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E beta-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function.These results establish that beta-cells are functionally heterogeneous, and apoA-I restores insulin secretion in beta-cells with elevated cholesterol levels by improving mitochondrial redox balance.BACKGROUND:High cholesterol levels in pancreatic beta-cells cause oxidative stress and decrease insulin secretion. beta-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves beta-cell insulin secretion by reducing oxidative stress.Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-beta-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by beta-cells was monitored by flow cytometry. The effects of apoA-I internalization on beta-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the beta-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in beta-cells and isolated islets with MitoSOX and confocal microscopy.An F1-ATPase beta-subunit on the beta-cell surface was identified as the main apoA-I-binding partner. beta-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase beta-subunit-dependent. beta-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase beta-subunit levels than beta-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E beta-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function.These results establish that beta-cells are functionally heterogeneous, and apoA-I restores insulin secretion in beta-cells with elevated cholesterol levels by improving mitochondrial redox balance.