ATP-Binding Cassette Transporter Family C Protein 10 Participates in the Synthesis and Efflux of Hexosylceramides in Liver Cells.

In addition to sphingomyelin and ceramide, sugar derivatives of ceramides, hexosylceramides (HexCer) are the major circulating sphingolipids. We have shown that silencing of ABCA1 transmembrane protein function for instance in cases of loss of function of ABCA1 gene results in low levels of HDL as well as a concomitant reduction in plasma HexCer levels. However, proteins involved in hepatic synthesis and egress of HexCer from cells is not well known although ABCA1 seems to be indirectly controlling the HexCer plasma levels by supporting HDL synthesis. In this study, we hypothesized that protein(s) other than ABCA1 are involved in the transport of HexCer to HDL. Using an unbiased knockdown approach, we found that ATP-binding cassette transporter protein C10 (ABCC10) participates in the synthesis of HexCer and thereby affects egress to HDL in human hepatoma Huh-7 cells. Furthermore, livers from ABCC10 deficient mice had significantly lower levels of HexCer compared to wild type livers. These studies suggest that ABCC10 partakes in modulating the synthesis and subsequent efflux of HexCer to HDL in liver cells.

ATP-binding cassette protein ABCA7 deficiency impairs sphingomyelin synthesis, cognitive discrimination, and synaptic plasticity in the entorhinal cortex.

Sphingomyelin (SM) is an abundant plasma membrane and plasma lipoprotein sphingolipid. We previously reported that ATP-binding cassette family A protein 1 (ABCA1) deficiency in humans and mice decreases plasma SM levels. However, overexpression, induction, downregulation, inhibition, and knockdown of ABCA1 in human hepatoma Huh7 cells did not decrease SM efflux. Using unbiased siRNA screening, here, we identified that ABCA7 plays a role in the biosynthesis and efflux of SM without affecting cellular uptake and metabolism. Since loss of function mutations in the ABCA7 gene exhibit strong associations with late-onset Alzheimer's disease across racial groups, we also studied the effects of ABCA7 deficiency in the mouse brain. Brains of ABCA7-deficient (KO) mice, compared with WT, had significantly lower levels of several SM species with long chain fatty acids. In addition, we observed that older KO mice exhibited behavioral deficits in cognitive discrimination in the active place avoidance task. Next, we performed synaptic transmission studies in brain slices obtained from older mice. We found anomalies in synaptic plasticity at the intracortical synapse in layer II/III of the lateral entorhinal cortex but not in the hippocampal CA3-CA1 synapses in KO mice. These synaptic abnormalities in KO brain slices were rescued with extracellular SM supplementation but not by supplementation with phosphatidylcholine. Taken together, these studies identify a role of ABCA7 in brain SM metabolism and the importance of SM in synaptic plasticity and cognition, as well as provide a possible explanation for the association between ABCA7 and late-onset Alzheimer's disease.

Model systems for studying the assembly, trafficking, and secretion of apoB lipoproteins using fluorescent fusion proteins.

apoB exists as apoB100 and apoB48, which are mainly found in hepatic VLDLs and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive the assembly, intracellular trafficking, secretion, and function of Blps remains challenging. Our understanding of the intracellular and intraorganism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. We designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins colocalized with calnexin and were only secreted if cells were cotransfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. The availability of fluorescently labeled apoB proteins will facilitate the exploration of the assembly, degradation, and transport of Blps and help to identify novel compounds that interfere with these processes via high-throughput screening.

ATP binding cassette family A protein 1 determines hexosylceramide and sphingomyelin levels in human and mouse plasma.

Sphingolipids, including ceramide, SM, and hexosylceramide (HxCer), are carried in the plasma by lipoproteins. They are possible markers of metabolic diseases, but little is known about their control. We previously showed that microsomal triglyceride transfer protein (MTP) is critical to determine plasma ceramide and SM, but not HxCer, levels. In human plasma and mouse models, we examined possible HxCer-modulating pathways, including the role of ABCA1 in determining sphingolipid plasma concentrations. Compared with control samples, plasma from patients with Tangier disease (deficient in ABCA1) had significantly lower HxCer (-69%) and SM (-40%) levels. Similarly, mice deficient in hepatic and intestinal ABCA1 had significantly reduced HxCer (-79%) and SM (-85%) levels. Tissue-specific ablation studies revealed that hepatic ABCA1 determines plasma HxCer and SM levels; that ablation of MTP and ABCA1 in the liver and intestine reduces plasma HxCer, SM, and ceramide levels; and that hepatic and intestinal MTP contribute to plasma ceramide levels, whereas only hepatic MTP modulates plasma SM levels. These results identify the contribution of ABCA1 to plasma SM and HxCer levels and suggest that MTP and ABCA1 are critical determinants of plasma sphingolipid levels.

Sphingolipids and Lipoproteins in Health and Metabolic Disorders.

Sphingolipids are structurally and functionally diverse molecules with significant physiologic functions and are found associated with cellular membranes and plasma lipoproteins. The cellular and plasma concentrations of sphingolipids are altered in several metabolic disorders and may serve as prognostic and diagnostic markers. Here we discuss various sphingolipid transport mechanisms and highlight how changes in cellular and plasma sphingolipid levels contribute to cardiovascular disease, obesity, diabetes, insulin resistance, and nonalcoholic fatty liver disease (NAFLD). Understanding of the mechanisms involved in intracellular transport, secretion, and extracellular transport may provide novel information that might be amenable to therapeutic targeting for the treatment of various metabolic disorders.

Hepatic Tm6sf2 overexpression affects cellular ApoB-trafficking, plasma lipid levels, hepatic steatosis and atherosclerosis.

The human transmembrane 6 superfamily member 2 (TM6SF2) gene has been implicated in plasma lipoprotein metabolism, alcoholic and non-alcoholic fatty liver disease and myocardial infarction in multiple genome-wide association studies. To investigate the role of Tm6sf2 in metabolic homeostasis, we generated mice with elevated expression using adeno-associated virus (AAV)-mediated gene delivery. Hepatic overexpression of mouse Tm6sf2 resulted in phenotypes previously observed in Tm6sf2-deficient mice including reduced plasma lipid levels, diminished hepatic triglycerides secretion and increased hepatosteatosis. Furthermore, increased hepatic Tm6sf2 expression protected against the development of atherosclerosis in LDL-receptor/ApoB48-deficient mice. In cultured human hepatocytes, Tm6sf2 overexpression reduced apolipoprotein B secretion and resulted in its accumulation within the endoplasmic reticulum (ER) suggesting impaired ER-to-Golgi trafficking of pre-very low-density lipoprotein (VLDL) particles. Analysis of two metabolic trait-associated coding polymorphisms in the human TM6SF2 gene (rs58542926 and rs187429064) revealed that both variants impact TM6SF2 expression by affecting the rate of protein turnover. These data demonstrate that rs58542926 (E167K) and rs187429064 (L156P) are functional variants and suggest that they influence metabolic traits through altered TM6SF2 protein stability. Taken together, our results indicate that cellular Tm6sf2 level is an important determinant of VLDL metabolism and further implicate TM6SF2 as a causative gene underlying metabolic disease and trait associations at the 19p13.11 locus.

Targeting microsomal triglyceride transfer protein and lipoprotein assembly to treat homozygous familial hypercholesterolemia.

Homozygous familial hypercholesterolemia (HoFH) is a polygenic disease arising from defects in the clearance of plasma low-density lipoprotein (LDL), which results in extremely elevated plasma LDL cholesterol (LDL-C) and increased risk of atherosclerosis, coronary heart disease, and premature death. Conventional lipid-lowering therapies, such as statins and ezetimibe, are ineffective at lowering plasma cholesterol to safe levels in these patients. Other therapeutic options, such as LDL apheresis and liver transplantation, are inconvenient, costly, and not readily available. Recently, lomitapide was approved by the Federal Drug Administration as an adjunct therapy for the treatment of HoFH. Lomitapide inhibits microsomal triglyceride transfer protein (MTP), reduces lipoprotein assembly and secretion, and lowers plasma cholesterol levels by over 50%. Here, we explain the steps involved in lipoprotein assembly, summarize the role of MTP in lipoprotein assembly, explore the clinical and molecular basis of HoFH, and review pre-clinical studies and clinical trials with lomitapide and other MTP inhibitors for the treatment of HoFH. In addition, ongoing research and new approaches underway for better treatment modalities are discussed.

Structure-function analyses of microsomal triglyceride transfer protein missense mutations in abetalipoproteinemia and hypobetalipoproteinemia subjects.

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their functional effects. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. H297Q, D384A, and G661A mutants also supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP's three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, novel mutations that cause severe hypolipidemia probably exist in other genes in these patients, and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide.

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp(-/-)) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.

Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of the N-Terminal ß-Barrel in Microsomal Triglyceride Transfer Protein Function.

BACKGROUND: The use of microsomal triglyceride transfer protein (MTP) inhibitors is limited to severe hyperlipidemias because of associated hepatosteatosis and gastrointestinal adverse effects. Comprehensive knowledge about the structure-function of MTP might help design new molecules that avoid steatosis. Characterization of mutations in MTP causing abetalipoproteinemia has revealed that the central α-helical and C-terminal ß-sheet domains are important for protein disulfide isomerase binding and lipid transfer activity. Our aim was to identify and characterize mutations in the N-terminal domain to understand its function. METHODS AND RESULTS: We identified a novel missense mutation (D169V) in a 4-month-old Turkish male child with severe signs of abetalipoproteinemia. To study the effect of this mutation on MTP function, we created mutants via site-directed mutagenesis. Although D169V was expressed in the endoplasmic reticulum and interacted with apolipoprotein B (apoB) 17, it was unable to bind protein disulfide isomerase, transfer lipids, and support apoB secretion. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished protein disulfide isomerase heterodimerization, lipid transfer, and apoB secretion, without affecting apoB17 binding. Furthermore, mutants with preserved charges (D169E, K187R, and K189R) rescued these activities. CONCLUSIONS: D169V is detrimental because it disrupts an internal salt bridge leading to loss of protein disulfide isomerase binding and lipid transfer activities; however, it does not affect apoB binding. Thus, the N-terminal domain of MTP is also important for its lipid transfer activity.

Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia.

Mutations in microsomal triglyceride transfer protein (MTP) cause abetalipoproteinemia (ABL), characterized by the absence of plasma apoB-containing lipoproteins. In this study, we characterized the effects of various MTP missense mutations found in ABL patients with respect to their expression, subcellular location, and interaction with protein disulfide isomerase (PDI). In addition, we characterized functional properties by analyzing phospholipid and triglyceride transfer activities and studied their ability to support apoB secretion. All the mutants colocalized with calnexin and interacted with PDI. We found that R540H and N780Y, known to be deficient in triglyceride transfer activity, also lacked phospholipid transfer activity. Novel mutants S590I and G746E did not transfer triglycerides and phospholipids and did not assist in apoB secretion. In contrast, D384A displayed both triglyceride and phospholipid transfer activities and supported apoB secretion. These studies point out that ABL is associated with the absence of both triglyceride and phospholipid transfer activities in MTP.

Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

AIMS: Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. METHODS AND RESULTS: Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. CONCLUSION: Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

The effect of C1 inhibitor on intestinal ischemia and reperfusion injury.

Complement activation and neutrophil stimulation are two major components in events leading to ischemia and reperfusion (IR) injury. C1 inhibitor (C1INH) inhibits activation of each of the three pathways of complement activation and of the contact system. It is also endowed with anti-inflammatory properties that are independent of protease inhibition. The goal of these studies was to investigate the role and mechanism of C1INH in alleviating IR-induced intestinal injury. C57BL/6, C1INH-deficient (C1INH(-/-)), bradykinin type 2 receptor-deficient (Bk2R(-/-)), and C3-deficient mice (C3(-/-)) were randomized into three groups: sham operated control, IR, and IR + C1INH-treated groups. Ischemia was generated by occlusion of the superior mesenteric artery followed by reperfusion. C1INH or reactive center-cleaved inactive C1INH (iC1INH) was injected intravenously before reperfusion. IR resulted in intestinal injury in C57BL/6, C1INH(-/-), Bk2R(-/-), and C3(-/-) mice with significantly increased neutrophil infiltration into intestinal tissue. In each mouse strain, C1INH treatment reduced intestinal tissue injury and attenuated leukocyte infiltration compared with the untreated IR group. C1INH inhibited leukocyte rolling in the mesenteric veins of both C57BL/6 and C3-deficient mice subjected to IR. C1INH treatment also improved the survival rate of C57BL/6 and C1INH(-/-) mice following IR. Similar findings were observed in the IR animals treated with iC1INH. These studies emphasize the therapeutic benefit of C1INH in preventing intestinal injury caused by IR. In addition to the protective activities mediated via inhibition of the complement system, these studies indicate that C1INH also plays a direct role in suppression of leukocyte transmigration into reperfused tissue.

ADAMTS13: a new link between thrombosis and inflammation.

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats-13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13(-/-) mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13(-/-) compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13(-/-) but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.

The combined roles of ADAMTS13 and VWF in murine models of TTP, endotoxemia, and thrombosis.

Ultralarge von Willebrand factor (UL-VWF) multimers are thought to play a central role in pathogenesis of the disease thrombotic thrombocytopenic purpura (TTP); however, experimental evidence in support of this hypothesis has been difficult to establish. Therefore, to examine directly the requirement for VWF in TTP pathogenesis, we generated ADAMTS13-deficient mice on a TTP-susceptible genetic background that were also either haploinsufficient (Vwf+/-) or completely deficient (Vwf-/-) in VWF. Absence of VWF resulted in complete protection from shigatoxin (Stx)-induced thrombocytopenia, demonstrating an absolute requirement for VWF in this model (Stx has been shown previously to trigger TTP in ADAMTS13-deficient mice). We next investigated the requirements for ADAMTS13 and VWF in a murine model of endotoxemia. Unlike Stx-induced TTP findings, LPS-induced thrombocytopenia and mortality were not affected by either VWF or ADAMTS13 deficiency, suggesting divergent mechanisms of thrombocytopenia between these 2 disorders. Finally, we show that VWF deficiency abrogates the ADAMTS13-deficient prothrombotic state, suggesting VWF as the only relevant ADAMTS13 substrate under these conditions. Together, these findings shed new light on the potential roles played by ADAMTS13 and VWF in TTP, endotoxemia, and normal hemostasis.

Prothrombotic effects of fibronectin isoforms containing the EDA domain.

OBJECTIVE: Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury. FN containing Extra Domain A (EDA+ FN) is absent from normal plasma, but elevated plasma levels of EDA+ FN are found in several pathological conditions. We hypothesized that EDA+ FN plays a special role in thrombosis. METHODS AND RESULTS: We used mouse strains constitutively including (EDA+/+) or excluding (EDA-/-) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA+ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA+/+ mice thrombi (>30 microm) grew faster when compared with EDA(WT/WT) (6.6+/-0.2 minutes versus 8.3+/-0.6 minutes, P<0.05) and the mean vessel occlusion time was shorter (9.9+/-0.4 minutes versus 14.6+/-1.7 minutes, P<0.05). However, the presence of EDA+ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA+/+ mice after collagen/epinephrine infusion was twice that of EDA(WT/WT) or EDA-/- mice. CONCLUSIONS: Our findings reveal that EDA+ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated.