Hydrogen peroxide‐induced translocation of glycolipid‐anchored (c)AMP‐hydrolases to lipid droplets mediates inhibition of lipolysis in rat adipocytes

Abstract

Background: The insulin-independent inhibition of lipolysis by palmitate, the anti-diabetic sulphonylurea glimepiride and H 2 O 2 in rat adipocytes involves stimulation of the glycosylphosphatidylinositol (GPI)-specific phospholipase-C (GPI-PLC) and subsequent translocation of the GPI-anchored membrane ectoproteins (GPI-proteins), Gce1 and cluster of differentiation antigen (CD73), from specialized plasma membrane microdomains (DIGs) to cytosolic lipid droplets (LDs). This results in cAMP degradation at the LD surface and failure to activate hormone-sensitive lipase. Reactive oxygen species (ROS) may trigger this sequence of events in response to palmitate and glimepiride. Experimental approach: The effects of various inhibitors of ROS production on the release of H 2 O 2 , GPI anchor cleavage and translocation of the photoaffinity-labelled or metabolically labelled Gce1 and CD73 from DIGs to LD and inhibition of lipolysis by different fatty acids and sulphonylureas were studied with primary rat adipocytes. Key results: Glimepiride and palmitate induced the production of H 2 O 2 via the plasma membrane NADPH oxidase and mitochondrial complexes I and III, respectively. Inhibition of ROS production was accompanied by the loss of (i) GPI-PLC activation, (ii) Gce1 and CD73 translocation and (iii) lipolysis inhibition in response to palmitate and glimepiride. Nonmetabolizable fatty acids and the sulphonylurea drug tolbutamide were inactive. NADPH oxidase and GPI-PLC activities colocalized at DIGs were stimulated by glimepiride but not tolbutamide. Conclusions and implications:The data suggest that ROS mediate GPI-PLC activation at DIGs and subsequent GPI-protein translocation from DIGs to LD in adipocytes which leads to inhibition of lipolysis by palmitate and glimepiride. This insulinindependent anti-lipolytic mechanism may be engaged by future anti-diabetic drugs.

Adipocyte expression of PU.1 transcription factor causes insulin resistance through upregulation of inflammatory cytokine gene expression and ROS production

“…The mechanism underlying PU.1's regulation of lipolysis could also be through the changes in ROS and TNF␣. It was documented that H 2 O 2 treatment increases basal lipolysis but suppresses ␤-adrenergic-stimulated lipolysis in adipocytes (37,50) through the activation of cAMP hydrolysis at the surface of lipoid droplets by H 2 O 2 (51,52). Additionally, TNF␣ also causes an increase in basal lipolysis and a decrease in stimulated lipolysis through downregulation of perilipin and cell death-inducing DFFA-like effector C (3, 15, 48).…”

NAD(P)H oxidase participates in the palmitate‐induced superoxide production and insulin secretion by rat pancreatic islets

“…Palmitate increases production of ROS in aortic smooth muscle cells, endothelial cells (Inoguchi et al, 2000), skeletal muscle cells (Lambertucci et al, 2008), and adipocytes (Muller et al, 2008) through a process that is dependent on NAD(P)H oxidase. In pancreatic islets, palmitate was demonstrated herein and in a previous study (Morgan et al, 2007) to increase superoxide content in a short‐time exposure through activation of NAD(P)H oxidase in the presence of 5.6 mM glucose.…”

“…The intensified ROS production involved in the streptozotocin‐induced experimental diabetes leads to generation of lipid peroxides and hydroperoxides, by oxidative degradation of polyunsaturated fatty acids, that damage proteins and DNA (Palsamy and Subramanian, 2010). The stimulating effect of FFA on ROS production has been demonstrated in several cell types (Inoguchi et al, 2000; Lambertucci et al, 2008; Muller et al, 2008). Fatty acids and their derivatives may modulate the efficiency of the mitochondrial respiratory chain by the inhibition of complexes I and III, thereby promoting generation of ROS (Schonfeld and Wojtczak, 2008).…”

Upregulation of Lipid Synthesis in Small Rat Adipocytes by Microvesicle‐Associated CD73 From Large Adipocytes

“…These are prepared for ongoing net deposition of TAG as a consequence of simultaneous upregulation of esterification and downregulation of lipolysis. A novel signaling pathway (20,21,22,23,24,25,26,27,28,29) for the regulation of lipid metabolism in rat adipocytes has recently been elucidated for the physiological lipogenic stimuli, palmitate, and H 2 O 2 , the antidiabetic sulfonylurea drug, glimepiride, and the insulin‐mimetic phosphoinositolglycan (PIG) compounds. The underlying mechanisms encompass (i) the release of small vesicles, so‐called microvesicles, which harbor glycosylphosphatidylinositol‐anchored proteins (GPI‐proteins), among them the (c)AMP‐degrading phosphodiesterase Gce1 and 5′‐nuceotidase CD73, from donor adipocytes, (ii) the interaction of the released microvesicles with acceptor adipocytes, (iii) the translocation of Gce1 and CD73 from the microvesicles to intracellular LD of the acceptor adipocytes, and (iv) the degradation of (c)AMP at the LD surface zone by Gce1 and CD73.…”

“…The underlying mechanisms encompass (i) the release of small vesicles, so‐called microvesicles, which harbor glycosylphosphatidylinositol‐anchored proteins (GPI‐proteins), among them the (c)AMP‐degrading phosphodiesterase Gce1 and 5′‐nuceotidase CD73, from donor adipocytes, (ii) the interaction of the released microvesicles with acceptor adipocytes, (iii) the translocation of Gce1 and CD73 from the microvesicles to intracellular LD of the acceptor adipocytes, and (iv) the degradation of (c)AMP at the LD surface zone by Gce1 and CD73. This series of events leads to the coordinated upregulation of the esterification of fatty acids into TAG and downregulation of their release from TAG in the acceptor adipocytes (20,21,22,23,24,25,26,27,28,29) and thereby guarantees TAG storage in and size gain of small adipocytes. Gce1 and CD73, which apparently function as critical components of the microvesicles on basis of their transfer from donor to acceptor adipocytes, are modified by a highly conserved GPI glycolipid structure, that is added post‐translationally to the carboxy terminus of eukaryotic GPI‐proteins (30,31).…”