BBA - Molecular and Cell Biology of Lipids (v.1821, #6)

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith–Lemli–Opitz syndrome by Libin Xu; Lowell G. Sheflin; Ned A. Porter; Steven J. Fliesler (877-883).
Smith–Lemli–Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ7-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d 7-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25 μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.► 7-DHC-derived oxysterols were identified in the retinas of AY9944-treated rats by HPLC-MS. ► Oxysterol levels were quantified by HPLC-MS-MS using d 7-DHCEO as an internal standard. ► Intravitreal injection of 7-ketocholesterol into normal eyes induced panretinal degeneration. ► The involvement of 7-DHC-derived oxysterols in retinal degeneration in this model is suggested.
Keywords: 7-dehydrocholesterol; Oxysterol; Oxidation; Smith–Lemli–Opitz Syndrome; Retinal degeneration; 7-ketocholesterol;

Glycosphingolipid synthesis is essential for MDCK cell differentiation by Lucila G. Pescio; Nicolás O. Favale; María G. Márquez; Norma B. Sterin-Speziale (884-894).
Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin–Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.► Hypertonicity induces the differentiation of the apical membrane in MDCK cells. ► Glycosphingolipid metabolism is regulated by hypertonicity. ► MDCK cell differentiation depends on glycosphingolipid synthesis.
Keywords: Glycosphingolipids; Hypertonicity; Cell differentiation; Apical membrane;

Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice by Werner J. Kovacs; Khanichi N. Charles; Katharina M. Walter; Janis E. Shackelford; Thomas M. Wikander; Michael J. Richards; Steven J. Fliesler; Skaidrite K. Krisans; Phyllis L. Faust (895-907).
Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. We previously demonstrated that peroxisomes are critical for maintaining cholesterol homeostasis, using peroxisome-deficient Pex2 −/− mice on a hybrid Swiss Webster × 129S6/SvEv (SW/129) genetic background. Peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, leading to dysregulation of the endogenous sterol response mechanism. Herein, we demonstrate a more profound dysregulation of cholesterol homeostasis in newborn Pex2 −/− mice congenic on a 129S6/SvEv (129) genetic background, and substantial differences between newborn versus postnatal Pex2 −/− mice in factors that activate ER stress. These differences extend to relationships between activation of genes regulated by SREBP-2 versus PPARα. The SREBP-2 pathway is induced in neonatal Pex2 −/− livers from 129 and SW/129 strains, despite normal hepatic cholesterol levels. ER stress markers are increased in newborn 129 Pex2 −/− livers, which occurs in the absence of hepatic steatosis or accumulation of peroxins in the ER. Moreover, the induction of SREBP-2 and ER stress pathways is independent of PPARα activation in livers of newborn 129 and SW/129 Pex2 −/− mice. Two-week-old wild-type mice treated with the peroxisome proliferator WY-14,643 show strong induction of PPARα-regulated genes and decreased expression of SREBP-2 and its target genes, further demonstrating that SREBP-2 pathway induction is not dependent on PPARα activation. Lastly, there is no activation of either SREBP-2 or ER stress pathways in kidney and lung of newborn Pex2 −/− mice, suggesting a parallel induction of these pathways in peroxisome-deficient mice. These findings establish novel associations between SREBP-2, ER stress and PPARα pathway inductions.► Hepatic ER stress pathways are induced in peroxisome-deficient Pex2 knock-out mice. ► ER stress deregulates the endogenous sterol response mechanism. ► ER stress is induced in the absence of hepatic steatosis or accumulation of peroxins. ► The induction of SREBP-2 and ER stress is independent of PPARα activation.
Keywords: Cholesterol homeostasis; Peroxisome; Pex2; SREBP-2; ER stress; PPARα;

Regulation of plasma cholesterol esterification by sphingomyelin: Effect of physiological variations of plasma sphingomyelin on lecithin-cholesterol acyltransferase activity by Papasani Venkata Subbaiah; Xian-Cheng Jiang; Natalia A. Belikova; Buzulagu Aizezi; Zhi Hua Huang; Catherine A. Reardon (908-913).
Although sphingomyelin (SM) is the most abundant phospholipid in the plasma, next to phosphatidylcholine (PC), its physiological function in plasma is unclear. Here we employed plasma from various genetic models of mice which naturally differ in their plasma SM/PC ratios, to study the role of SM as a modulator of LCAT, the enzyme responsible for HDL maturation and the synthesis of cholesteryl esters (CE) in normal plasma. Serine palmitoyltransferase deficient mice, and SM synthase deficient mice, both of which have below normal SM/PC ratios, showed significantly elevated LCAT activities when assayed with the endogenous substrates. On the other hand, LDL receptor knockout mice, and apo E knockout mice, both of which have high SM/PC ratios, had markedly reduced (− 80%) LCAT activities. The LCAT levels in plasma, as assayed with an exogenous substrate, were similar in all groups, except for a 45% decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 CE all of which are formed by LCAT, and a higher percentage of the atherogenic 18:1 CE which is mainly derived from the action of liver ACAT, showing that in vivo, the contribution of LCAT to plasma CE is reduced while that of liver ACAT is increased. These results show that SM is a physiological modulator of LCAT activity as well as plasma CE composition, and this may contribute to the previously reported pro-atherogenic effect of high plasma SM levels.► LCAT activity is significantly higher in SM-deficient mice (Sms2 −/− , and Sptlc2 +/− ). ► LCAT activity is markedly inhibited in SM-rich plasma (ApoE −/− , and Ldlr −/− ). ► CE species composition shows a more atherogenic profile in SM-rich plasma.
Keywords: Sphingomyelin synthase 2 deficiency; Serine palmitoyl transferase deficiency; Apo E deficient mouse; LDL receptor deficient mouse; LCAT activity; Cholesteryl ester composition;

Leucine-rich glioma inactivated 3 regulates adipogenesis through ADAM23 by Hyun A Kim; Woo-Jae Park; Hyo-Soon Jeong; Hyun-e Lee; Seung Hoon Lee; Nyoun Soo Kwon; Kwang Jin Baek; Dong-Seok Kim; Hye-Young Yun (914-922).
Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.► LGI3 and ADAM23 are expressed in adipose tissues and 3T3-L1 cells. ► The 60-kDa LGI3 protein is increased in the ob/ob adipose tissues. ► LGI3 binds ADAM23 in adipose tissues and 3T3-L1 cells. ► LGI3 suppresses adipogenesis through ADAM23. ► We propose LGI3 as a candidate adipokine.
Keywords: LGI3; ADAM23; Adipocyte; Adipogenesis; Adipokine;

In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of H. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C55 and C60 dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C60 dolichol phosphate than with C55 dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.► H. volcanii ΔHVO_1799 cells don't fully reduce phospho/glycolipid isoprene chains. ► ΔHVO_1799 cells do not saturate the dolichol phosphate ω-position isoprene. ► Dolichol phosphate with reduced α- and ω-isoprenes is preferentially glycosylated.
Keywords: Archaea; Dolichol phosphate; Geranylgeranyl reductase; Haloferax volcanii; Isoprene; Reductase;

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.► Cholesterol is enriched in the plasma membrane of C. elegans spermatids. ► Cholesterol and glycosphingolipids are essential for sperm activation in C. elegans. ► (GPI)-anchored proteins on the sperm surface are necessary for sperm activation. ► Before efflux, the cholesterol might be required for upstream signal sorting.
Keywords: Caenorhabditis elegans; Major sperm protein (MSP); Sperm activation; Sperm motility; Lipid microdomains;