Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 mM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing b-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.-

The fatty acid transport proteins (FATP) and longchain acyl coenzyme A synthetase (ACSL) proteins have been shown to play a role in facilitating long-chain fatty acid (LCFA) transport in mammalian cells under physiologic conditions. The involvement of both FATP and ACSL proteins is consistent with the model of vectorial acylation, in which fatty acid transport is coupled to esterification. This study was undertaken to determine whether the functions of these proteins are coordinated through a protein-protein interaction that might serve as a point of regulation for cellular fatty acid transport. We demonstrate for the first time that FATP1 and ACSL1 coimmunoprecipitate in 3T3-L1 adipocytes, indicating that these proteins form an oligomeric complex. The efficiency of FATP1 and ACSL1 coimmunoprecipitation is unaltered by acute insulin treatment, which stimulates fatty acid uptake, or by treatment with isoproterenol, which decreases fatty acid uptake and stimulates lipolysis. Moreover, inhibition of ACSL1 activity in adipocytes impairs fatty acid uptake, suggesting that esterification is essential for fatty acid transport. Together, our findings suggest that a constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation. A central aspect of adipocyte biology is the ability of these specialized cells to efficiently take up and store longchain fatty acids (LCFAs) in response to nutritional and hormonal cues. In the "fed" state, higher serum levels of insulin stimulate an increase in fatty acid uptake and triglyceride storage in adipose tissue. On the other hand, during "starvation," b-adrenergic receptor agonists, such as epinephrine, stimulate pathways that activate hormonesensitive lipase in adipocytes, resulting in the hydrolysis of triglycerides and the release of free fatty acids and glycerol. In obesity and the metabolic syndrome, dysregulation of these processes may contribute to increases in serum free fatty acids and the genesis of type 2 diabetes (1, 2).The movement of LCFAs across the plasma membrane is tightly coupled to thioesterification, a process referred to as vectorial acylation (3). Early evidence for this mechanism came from genetic studies of Escherichia coli, in which fatty acid transport requires FadL, an outer membrane LCFA transporter, and FadD, an inner membraneassociated acyl CoA synthetase (4, 5). Vectorial acylation provides cells with an efficient means of rapidly metabolizing incoming fatty acids and contributes to decreasing the intracellular concentration of free fatty acids to favor import. Thioesterification of LCFAs is also an essential initial metabolic step for many downstream metabolic pathways of LCFA use, such as b-oxidation or triglyceride synthesis. The coupled transport and metabolism of LCFAs is analogous to glucose transport in mammalian cells, a process in which GLUT4-mediated translocation of glucose across the plasma membrane is coordinated with rapid phosphorylation by hexokin...