Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.

Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here, we provide evidence that the biophysical and functional characteristics of the two sterols differ and that this is because the double bond at C24 significantly weakens the sterol ordering potential. In model membranes, desmosterol was significantly weaker than cholesterol in promoting the formation or stability of ordered domains, and in mammalian cell membranes, desmosterol associated less avidly than cholesterol with detergentresistant membranes. Atomic scale molecular dynamics simulations showed that the double bond gives rise to additional stress in the tail, creating a rigid structure between C24 and C27 and favoring tilting of desmosterol distinct from cholesterol. Functional effects of desmosterol in cell membranes were assessed upon acutely exchanging ϳ70% of cholesterol to desmosterol. This led to impaired raft-dependent signaling via the insulin receptor, whereas non-raft-dependent protein secretion was not affected. We suggest that the choice of cholesterol synthesis route may provide a physiological mechanism to modulate raft-dependent functions in cells.In model membranes, cholesterol associates preferentially with long, saturated acyl chains, such as those in sphingolipids, thus reducing the area per lipid molecule (1, 2). There is substantial evidence to suggest that ordered lipid domains (rafts) composed of sterol and saturated lipids also exist in eukaryotic cell membranes and play important roles in numerous biological processes (3, 4). Lipid rafts are considered to exist in a liquid-ordered (L o ) 2 state characterized by tight ordering but relatively high lateral mobility of lipids and operationally often defined as detergent-resistant membranes (DRMs) (5, 6). Instead, unsaturated phospholipids are loosely packed, forming a liquid-disordered (L d ) membrane that is solubilized upon the addition of mild detergents. At least in model membranes, cholesterol is able to promote the separation of L o and L d domains (7-9). In cells, cholesterol levels influence the domain partitioning and biological activity of proteins that co-isolate in detergent-resistant membranes (DRMs) (10, 11).Taking the postulated critical role for cholesterol in raft formation and the diversity of sterols in biological materials, the sterol structural requirements for promoting ordered domain formation are highly relevant. Until now, the effects of sterol/steroid structure have mostly been addressed in model membranes. Slight modifications of the cholesterol structure (e.g. a shift of the double bond in the sterol ring or alteration of the 3-OH group) change the domain-forming properties of the molecule (12-14). Among the structurally closest relatives of cholesterol are its immediate biosynthetic precursors. The only difference ...

Niemann-Pick type C (NPC) disease is a neuro-visceral cholesterol storage disorder caused by mutations in the NPC-1 or NPC-2 gene. In the present paper, we studied IR (insulin receptor) activation and the plasma-membrane lipid assembly in primary hepatocytes from control and NPC1-/- mice. We have previously reported that, in hepatocytes, IR activation is dependent on cholesterol-sphingolipid rafts [Vainio, Heino, Mansson, Fredman, Kuismanen, Vaarala and Ikonen (2002) EMBO Rep. 3, 95-100]. We found that, in NPC hepatocytes, IR levels were up-regulated and the receptor activation was compromised. Defective IR activation was reproduced in isolated NPC plasma-membrane preparations, which displayed an increased cholesterol content and saturation of major phospholipids. The NPC plasma membranes were less fluid than control membranes as indicated by increased DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence anisotropy values. Both in NPC hepatocytes and plasma-membrane fractions, the association of IR with low-density DRMs (detergent-resistant membranes) was increased. Moreover, the detergent resistance of both cholesterol and phosphatidylcholine were increased in NPC membranes. Finally, cholesterol removal inhibited IR activation in control membranes but restored IR activation in NPC membranes. Taken together, the results reveal a lipid imbalance in the NPC hepatocyte, which increases lipid ordering in the plasma membrane, alters the properties of lipid rafts and interferes with the function of a raft-associated plasma-membrane receptor. Such a mechanism may participate in the pathogenesis of NPC disease and contribute to insulin resistance in other disorders of lipid metabolism.

Mammalian cells have evolved complex feedback mechanisms to ensure sufficient supply of cholesterol and to prevent its excessive accumulation. During the process of atherosclerosis, these homeostatic mechanisms fail in macrophages. Uncontrolled cholesterol deposition is promoted by scavenger functions of the macrophages and the adaptive mechanisms elicited are not sufficient to process the lipid load. Consequently, a lipid-laden 'foam cell' is formed. In this review, we summarize key aspects of intracellular cholesterol processing in the special case of macrophages, including mechanisms of lipoprotein cholesterol uptake, fate of the internalized cholesterol and mechanisms implicated in cholesterol efflux. The importance of inflammatory cues, the cellular compartmentalization of cholesterol homeostatic responses and the increasing information on the transcriptional control of cholesterol balance are discussed.