The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the ␣ subunit of AMPK in a dose-and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviralmediated expression of a constitutively active AMPK␣ or a kinase-inactive AMPK␣, respectively. An insulinresistant state was induced by exposing cells to 30 mM glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3␣/␤. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPK␣. The kinase-inactive AMPK␣ increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPK␣ negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance. AMP-activated protein kinase (AMPK)1 is a phylogenetically conserved intracellular energy sensor that has been implicated in the regulation of glucose and lipid homeostasis (1-4). AMPK is activated by physiological stimuli, such as exercise, muscle contraction, and hormones including adiponectin and leptin, as well as by pathological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock (2, 5). AMPK serine/threonine protein kinase is a heterotrimeric complex consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥) (5). Regulation of AMPK activity is complex; it involves allosteric activation by AMP, which increases during states of stress where ATP is depleted, and phosphorylation via the presumptive upstream activator AMPK kinase (6 -9), which may also be allosterically activated by AMP (5). Moreover, phosphorylation of Thr-172 within the activation loop of the catalytic domain of the ␣ subunit is necessary for AMPK activity because sitedirected mutagenesis of Thr-172 to Ala completely abolishes AMPK activity (10, 11). Once activated, AMPK phosphorylates its downstream substrates to reduce ATP-consuming anabolic pathways, including ...

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversedphase LC-MS or LC-tandem mass spectrometry using an Atlantis dC18 capillary column, and/or nanospray MS, to fully characterize the individual lipids. The methodology is applied here for the analysis of human apolipoprotein Bassociated lipids. This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. The methods described should be easily adaptable to most modern MS instrumentation. A large variety of methods have been published for the separation of lipids, either by TLC or by LC; the methods have usually been described for the analysis of specific classes of compounds (www.cyberlipid.org). MS methods for the characterization of lipid mixtures have also been published in recent years, most of them centered on the use of matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) MS and electrospray ionization (ESI) MS (1) (in addition to the references cited in the text, important Web-based sources of information were www. cyberlipid.org, www.lipidlibrary.co.uk, and www.lipidmaps. org). Sophisticated methods like the characterization of complex glycolipids directly from TLC plates by vibrationally cooled MALDI Fourier transform-ion cyclotron resonance MS (2) require instrumentation that is not yet widely available. A variety of elegant nanospray MS methods have been described (3-5) that are generally a good choice for the characterization of lipids, but they may not be fully capable of both qualitative and quantitative analysis of highly complex mixtures. LC-MS offers possibilities for a better determination of minor compounds whose signals might otherwise be suppressed. It also allows for an additional level of characterization of components based on their chromatographic behavior as well as the MS results.Existing HPLC methods for the separation of lipids are limited, however, in that they either target only selected classes or are not compatible with subsequent MS. Pulfer and Murphy (6) suggested that, for a complete separation of lipids, normal-and reversed-phase chromatography should be combined. Because of its high sensitivity and the additional information it provides, MS is widely recognized as a superior detection method compared with the classic methods of ultraviolet or light scattering. We demonstrate here a simple, robust, and reproducible methodology for lipid analysis, which has been achieved by adapting to LC-MS sev...

The N-termnal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pue-chase experiments showed that the depletion of intracellular apoB-17follows an apparent first-order kinetics with tl2 = 51 min.Under conditions of continuous labeling, >60% of the total synthesized apoB-17 was secreted in a soluble form, =98% lipid-poor and -2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5-and 2.5-fold increases in the amount of labeled apoB-17 in the p < 1.063 g/ml and 1.063 < p < 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p > 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/ apoB-17, 7:1 (wt/wt), are 239 ± 43 A in diameter and 61 ± 4 A thick and contain -2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% a-helix, 36% 13-sheet, 9% (3-turn, and 16% random coil.To be consistent with this model >70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.

Very low-density lipoproteins (VLDL) are metabolic precursors of low-density lipoproteins (LDL) and a risk factor for atherosclerosis. Human VLDL are heterogeneous complexes containing triacylglyceride-rich apolar lipid core and polar surface comprised of phospholipids, a nonexchangeable apolipoprotein B, and exchangeable apolipoproteins E and Cs. We report the first stability study of VLDL. Circular dichroism and turbidity data reveal an irreversible heat-induced VLDL transition that involves formation of larger particles and repacking of apolar lipids but no global protein unfolding. Heating rate effect on the melting temperature indicates a kinetically controlled reaction with high activation energy, E a . Arrhenius analysis of the turbidity data reveals two kinetic phases with E a =53±7 kcal/mol that correspond to distinct morphological transitions observed by electron microscopy. One transition involves VLDL fusion, partial rupture and dissociation of small spherical particles (d=7-15 nm), and another involves complete lipoprotein disintegration and lipid coalescence into droplets accompanied by dissociation of apolipoprotein B. The small particles, which are unique to VLDL denaturation, are comparable in size and density to high-density lipoproteins (HDL); they have apolar lipid core and polar surface comprised of exchangeable apolipoproteins (E and possibly Cs) and phospholipids. We conclude that, similar to HDL and LDL, VLDL are stabilized by kinetic barriers that prevent particle fusion and rupture and decelerate spontaneous inter-conversion among lipoprotein classes and subclasses. In addition to fusion, VLDL disruption involves transient formation of HDL-like particles that may mimic protein exchange among VLDL and HDL pools in plasma.Plasma lipoproteins, including high-, low-, intermediate-, and very-low density lipoproteins (HDL, LDL, IDL and VLDL), are macromolecular complexes of lipids and proteins (termed apolipoproteins) that mediate lipid transport and metabolism and are central in the development of coronary artery disease. HDL are anti-atherogenic, LDL are pro-atherogenic, and VLDL are not only direct metabolic precursors of LDL but also an independent risk factor for atherosclerosis (1)(2)(3)(4)(5)(6)(7)(8). VLDL are the major carriers of triacylglycerides (TG) in plasma. Human VLDL form heterogeneous population of spherical particles that contain apolar core comprised mainly of TG and cholesterol esters (CE) and polar surface comprised of cholesterol-containing phospholipid monolayer and proteins. The proteins include one copy of non-exchangeable apolipoprotein B (apoB, 550 kD) and multiple copies of exchangeable apolipoproteins, mainly apoE (34 kD) and apoCs (6-9 kD), that comprise over 50% of the total VLDL protein content. Metabolic remodeling by lipolytic enzymes converts VLDL into LDL that contain apoB as their sole protein, while the dissociated apoE and apoCs enter the HDL pool (2,4). Structural Our recent thermal and chemical denaturation analyses have shown that HDL and LDL...

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a “hierarchical” manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1–3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both “naked” and phospholipid-coated oil–water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1–3 to LDs.

We have previously demonstrated that endoplasmic reticulum (ER)-resident molecular chaperones interact with apolipoprotein B-100 (apoB) during its maturation. The initial stages of apoB folding occur while it is bound to the ER membrane, where it becomes partially lipidated to form a primordial intermediate. We determined whether this intermediate is dependent on the assistance of molecular chaperones for its subsequent folding steps. To that end, microsomes were prepared from HepG2 cells and luminal contents were subjected to KBr density gradient centrifugation. Immunoprecipitation of apoB followed by Western blotting showed that the luminal pool floated at a density of 1.12 g/ml and, like the membrane-bound pool, was associated with GRP94, ERp72, BiP, calreticulin, and cyclophilin B. Except for calreticulin, chaperone/apoB ratio in the lumen was severalfold higher than that in the membrane, suggesting a role for these chaperones both in facilitating the release of the primordial intermediate into the ER lumen and in providing stability. Subcellular fractionation on sucrose gradients showed that apoB in the Golgi was associated with the same array of chaperones as the pool of apoB recovered from heavy microsomes containing the ER, except that chaperone/apoB ratio was lower. KBr density gradient fractionation showed that the major pool of luminal apoB in the Golgi was recovered from 1.02 < d < 1.08 g/ml, whereas apoB in ER was recovered primarily from 1.08 < d < 1.2 g/ml. Both fractions were associated with the same spectrum of chaperones. Together with the finding that GRP94 was found associated with sialylated apoB, we conclude that correct folding of apoB is dependent on the assistance of molecular chaperone, which play multiple roles in its maturation throughout the secretory pathway including distal compartments such as the trans-Golgi network.The correct folding of apolipoprotein B (apoB) 1 into its mature, secretion-competent form is a complex process that leads to the formation and secretion of triacylglycerol (TAG)-rich lipoproteins, chylomicrons, and the atherogenic lipoproteins, very low density lipoproteins (VLDL) (1, 2). A two-step model for the formation of VLDL was originally proposed on the basis of electron microscopic studies in rat liver (3). This model was further supported by latter studies in rat liver (4), in rat hepatoma cells (McA-RH7777; Refs. 5-8), and in transgenic mice lacking the gene for apoB in the intestine (9). According to this model, the first step involves partial lipidation of apoB to form a primordial intermediate with HDL/LDL-like density. In the second step this intermediate fuses with a large apoB-free lipid droplet composed primarily of TAG to form nascent VLDL or chylomicrons. In HepG2 cells, however, these distinct steps were not clearly demonstrated. Nonetheless, the initial steps of lipidation are thought to be similar to other systems in that they are mediated by microsomal triglyceride transfer protein (MTP) (reviewed in Refs. 10 -12) and occur co-translationally,...

The present study was undertaken to identify and characterize molecular chaperones that assist in the folding of apolipoprotein (apo) B, a secretory protein that requires assembly with lipids (lipidation) for its secretion. Both HepG2 cells, normally secreting fulllength apoB (apoB-100), and C127 cells transfected to secrete truncated forms of apoB, apoB-41, apoB-29, and apoB-17, respectively, were employed. C127 cells were used to determine whether chaperone binding is dependent on apoB lipidation as they secrete both unlipidated and lipidated apoB forms despite their lack of microsomal triglyceride transfer protein (MTP), which mediates lipidation of apoB in HepG2 cells. The endoplasmic reticulum (ER)-resident molecular chaperones GRP94, calreticulin, and ERp72 were co-immunoprecipitated with apoB-100 from HepG2 cell lysates following cross-linking of proteins in living cells. The same chaperones including BiP/GRP78 were also associated with all truncated forms of apoB. Sequential immunoprecipitation with antibodies to MTP and apoB revealed the presence of ternary complexes containing apoB-100, MTP, and ERp72. However, MTP is not obligatory for the binding of ERp72 as it was associated with all truncated forms of apoB in C127 cells that lack MTP. The interactions between apoB-100 and ERp72 or GRP94 persisted for at least 2 h following a 30-min pulse. Thus, BiP/ GRP78, calreticulin, ERp72, and GRP94 may participate in critical steps in the folding of apoB before any substantial lipidation occurs. ERp72 and GRP94 may also mediate the folding of more advanced folding intermediates and/or target the misfolded underlipidated pool of apoB for degradation. Apolipoprotein B (apoB)1 is an atypical secretory protein. Its folding into a mature, secretion-competent form necessitates assembly with triacylglycerols (TAG) and phospholipids to produce TAG-rich lipoproteins such as very low density lipoproteins and chylomicrons (1). A major fraction of nascent apoB is degraded intracellularly (2) when lipid availability is limited or when lipidation of apoB is inhibited (reviewed in Refs. 3 and 4). It was proposed that distinct amphipathic elements in the secondary structure of apoB may initiate the binding of lipids to apoB co-translationally (5). Further, growing evidence suggests that the process of apoB maturation in the cell is mediated by molecular chaperones (3, 4), which by binding nascent polypeptides prevent their aggregation and direct their folding into their mature, native form (6, 7). Indeed, the recruitment of lipids to the nascent lipoprotein is significantly aided by microsomal triglyceride transfer protein (MTP) localized to the lumen of the endoplasmic reticulum (ER) (reviewed in Ref. 8). MTP is a heterodimer composed of a 97-kDa subunit (MTPL) and protein-disulfide isomerase (PDI). The latter belongs to the family of the abundant thioredoxin-like proteins in the ER lumen, which catalyze the formation and rearrangement of disulfide bonds. PDI also functions as a molecular chaperone, and this function is inde...

of LDL cholesterol and, particularly, apoB are the strongest predictors of atherosclerosis and its causative agents ( 3 ). In atherosclerosis, LDL lipids are deposited in the arterial intima; according to the response-to-retention paradigm ( 4, 5 ), LDL retention by the arterial matrix proteoglycans triggers a cascade of pro-atherogenic events culminating in formation of atherosclerotic plaque ( 6-8 ). These events include biochemical modifi cations of LDL, such as oxidation and/or hydrolysis by the resident proteases and lipases, e.g., phospholipase A 2 and sphingomyelinase, which can reduce LDL affi nity for the LDL receptor, increase LDL affi nity for the proteoglycans, and promote LDL fusion (7)(8)(9)(10)(11)(12)(13)(14). In addition, ionic interactions with proteoglycans reduce LDL stability and promote their fusion and rupture (i.e., release of core lipids) ( 15 ). Fusion of lipoproteins prevents their exit from the arterial intima and thereby augments their further modifi cations, enhances LDL uptake by the arterial macrophages, and initiates the formation of atherosclerotic lesions ( 9, 16 ). Therefore, the pro-atherogenic potential of LDL is thought to be linked to their ability to fuse ( 9,10,17 ). Dissecting the pathogenic pathway of LDL fusion and identifying key factors that promote or inhibit this pathway can help obtain new therapeutic targets for atherosclerosis.Structural analysis of intact and modifi ed LDL has been limited to low resolution ( у 16 Å) by the large size and hydrophobicity of apoB and by LDL heterogeneity ( 1,(18)(19)(20)(21). Human plasma LDL consist of subclasses differing Abstract Fusion of modifi ed LDL in the arterial wall promotes atherogenesis. Earlier we showed that thermal denaturation mimics LDL remodeling and fusion, and revealed kinetic origin of LDL stability. Here we report the fi rst quantitative analysis of LDL thermal stability. Turbidity data show sigmoidal kinetics of LDL heat denaturation, which is unique among lipoproteins, suggesting that fusion is preceded by other structural changes. High activation energy of denaturation, E a = 100 ± 8 kcal/mol, indicates disruption of extensive packing interactions in LDL. Size-exclusion chromatography, nondenaturing gel electrophoresis, and negativestain electron microscopy suggest that LDL dimerization is an early step in thermally induced fusion. Monoclonal antibody binding suggests possible involvement of apoB N-terminal domain in early stages of LDL fusion. LDL fusion accelerates at pH < 7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the fi rst kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase t...