Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.Insulin rapidly stimulates glucose transport into fat and muscle cells by causing the insertion of additional glucose transporters of the GLUT4 isotype into the plasma membrane, in a process referred to as GLUT4 translocation. The overall process consists of generation of the specialized vesicles containing GLUT4 from the endosomal system, the movement of these vesicles from the perinuclear region to the plasma membrane, and the fusion of the vesicles with the plasma membrane (1). The steps in this process that insulin accelerates, and the complete signaling pathways from the insulin receptor that lead to their acceleration, have not yet been fully defined. One partial insulin signaling pathway that has been established to be required for GLUT4 translocation is the pathway that proceeds from the receptor through tyrosine phosphorylation of the insulin receptor substrates to activation of phosphatidylinositol 3-kinase and generation of phosphatidylinositol 3,4,5-trisphosphate. The latter leads to the activation of the protein kinase Akt and also protein kinase C /, and there is evidence that GLUT4 translocation requires the activation of one or both of these kinases (reviewed in Refs. 2 and 3). However, although a substrate linking either kinase to GLUT4 translocation has been sought for several years, hitherto none has been clearly identified.Recently, we reported the discovery of a new substrate for insulin-activated Akt in 3T3-L1 adipocytes, which was designated AS160 for Akt subtrate of 160 kDa (4). The most prominent feature of AS160 is the presence of a GTPase activating domain for a Rab. Since Rabs are small G proteins that play critical roles in vesicle formation, movement, and fusion (5), we investigated the role of AS160 in GLUT4 translocatio...

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.

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Background: Hormone-sensitive lipase (HSL) is a multifunctional enzyme that is critically involved in regulating energy homeostasis. Results: HSL catalyzes the hydrolysis of cholesteryl esters and plays an indispensable role in the regulation of Bt 2 cAMP-induced steroidogenic acute regulatory protein (StAR) expression. Conclusion:The mode of action of HSL in cAMP/PKA-mediated regulation of steroidogenesis involves multiple signaling, including the LXR-regulatory pathway. Significance: The present findings can be useful for better understanding a number of physiopathological processes.