TY - JOUR

T1 - Inhibition of lysosomal function in macrophages incubated with elevated glucose concentrations

T2 - a potential contributory factor in diabetes-associated atherosclerosis

AU - Moheimani, Fatemeh

AU - Kim, Christine H J

AU - Rahmanto, Aldwin Suryo

AU - van Reyk, David M

AU - Davies, Michael Jonathan

N1 - Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

PY - 2012/7

Y1 - 2012/7

N2 - OBJECTIVE: People with diabetes have an elevated risk of atherosclerosis. The accumulation of lipid within macrophage cells in the artery wall is believed to arise via the uptake and subsequent processing of modified low-density lipoproteins (LDL) via the endo-lysosomal system. In this study the effects of prolonged exposure to elevated glucose upon macrophage lysosomal function was examined to determine whether this contributes to modulated protein catabolism.METHODS: Human monocytes were isolated from white-cell concentrates and differentiated, in vitro, into monocyte-derived macrophages over 11 days in medium containing 5-30 mmol/L glucose. Murine macrophage-like J774A.1 cells were incubated similarly. Lysosomal cathepsin (B, D, L and S) and acid lipase activities were assessed using fluorogenic substrates; cathepsin protein levels were examined by Western blotting. Lysosomal numbers were examined using the lysomotropic fluorescent dye LysoTracker DND-99, measurement of aryl sulfatase activity, and quantification of lysosome-associated membrane glycoprotein-1 (LAMP-1) by Western blotting.RESULTS: Exposure to elevated glucose, but not mannitol, resulted in a concentration-dependent decrease in the activity, and to a lesser extent protein levels, of four lysosomal cathepsins. Acid lipase activity was also significantly reduced. Arysulfatase activity, LAMP-1 levels and lysosomal numbers were also decreased at the highest glucose concentrations, though to a lesser extent.CONCLUSION: Long term exposure of human and murine macrophage cells to elevated glucose levels result in a depression of lysosomal proteolytic and lipase activities. This may result in decreased clearance and cellular accumulation of (lipo)proteins and contribute to the accumulation of modified proteins and lipids in diabetes-associated atherosclerosis.

AB - OBJECTIVE: People with diabetes have an elevated risk of atherosclerosis. The accumulation of lipid within macrophage cells in the artery wall is believed to arise via the uptake and subsequent processing of modified low-density lipoproteins (LDL) via the endo-lysosomal system. In this study the effects of prolonged exposure to elevated glucose upon macrophage lysosomal function was examined to determine whether this contributes to modulated protein catabolism.METHODS: Human monocytes were isolated from white-cell concentrates and differentiated, in vitro, into monocyte-derived macrophages over 11 days in medium containing 5-30 mmol/L glucose. Murine macrophage-like J774A.1 cells were incubated similarly. Lysosomal cathepsin (B, D, L and S) and acid lipase activities were assessed using fluorogenic substrates; cathepsin protein levels were examined by Western blotting. Lysosomal numbers were examined using the lysomotropic fluorescent dye LysoTracker DND-99, measurement of aryl sulfatase activity, and quantification of lysosome-associated membrane glycoprotein-1 (LAMP-1) by Western blotting.RESULTS: Exposure to elevated glucose, but not mannitol, resulted in a concentration-dependent decrease in the activity, and to a lesser extent protein levels, of four lysosomal cathepsins. Acid lipase activity was also significantly reduced. Arysulfatase activity, LAMP-1 levels and lysosomal numbers were also decreased at the highest glucose concentrations, though to a lesser extent.CONCLUSION: Long term exposure of human and murine macrophage cells to elevated glucose levels result in a depression of lysosomal proteolytic and lipase activities. This may result in decreased clearance and cellular accumulation of (lipo)proteins and contribute to the accumulation of modified proteins and lipids in diabetes-associated atherosclerosis.

KW - Animals

KW - Arylsulfatases

KW - Atherosclerosis

KW - Blotting, Western

KW - Cathepsins

KW - Cell Line

KW - Diabetic Angiopathies

KW - Down-Regulation

KW - Glucose

KW - Humans

KW - Lysosome-Associated Membrane Glycoproteins

KW - Lysosomes

KW - Macrophages

KW - Mice

KW - Microscopy, Fluorescence

KW - Sterol Esterase

KW - Time Factors

U2 - 10.1016/j.atherosclerosis.2012.04.026

DO - 10.1016/j.atherosclerosis.2012.04.026

M3 - Journal article

C2 - 22658253

VL - 223

SP - 144

EP - 151

JO - Atherosclerosis

JF - Atherosclerosis

SN - 0021-9150

IS - 1

ER -