Impact of dietary phytol on lipid metabolism in SCP2/SCPX/L-FABP null mice

In vitro studies suggest that liver fatty acid binding protein (L-FABP) and sterol carrier protein-2/sterol carrier protein-x (SCP2/SCPx) gene products facilitate uptake and metabolism and detoxification of dietary-derived phytol in mammals. However, concomitant upregulation of L-FABP in SCP2/SCPx null mice complicates interpretation of their physiological phenotype. Therefore, the impact of ablating both the L-FABP gene and SCP2/SCPx gene (L-FABP/SCP2/SCPx null or TKO) was examined in phytol-fed female wild-type (WT) and TKO mice. TKO increased hepatic total lipid accumulation, primarily phospholipid, by mechanisms involving increased hepatic levels of proteins in the phospholipid synthetic pathway. Concomitantly, TKO reduced expression of proteins in targeting fatty acids towards the triacylglycerol synthetic pathway. Increased hepatic lipid accumulation was not associated with any concomitant upregulation of membrane fatty acid transport/translocase proteins involved in fatty acid uptake (FATP2, FATP4, FATP5 or GOT) or cytosolic proteins involved in fatty acid intracellular targeting (ACBP). In addition, TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass. Since individually ablating SCPx or SCP2/SCPx elicited concomitant upregulation of L-FABP, these findings with TKO mice help to resolve the contributions of SCP2/SCPx gene ablation on dietary phytol-induced whole body and hepatic lipid phenotype independent of concomitant upregulation of L-FABP.     

Aberrant oxidation of the cholesterol side chain in bile acid synthesis of sterol carrier protein-2/sterol carrier protein-x knockout mice

Peroxisomal beta-oxidation plays an important role in the metabolism of a wide range of substrates, including various fatty acids and the steroid side chain in bile acid synthesis. Two distinct thiolases have been implicated to function in peroxisomal beta-oxidation: the long known 41-kDa beta-ketothiolase identified by Hashimoto and co-workers (Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8151-8158) and the recently discovered 60-kDa SCPx thiolase, that consists of an N-terminal domain with beta-ketothiolase activity and a C-terminal moiety of sterol carrier protein-2 (SCP2, a lipid carrier or transfer protein). Recently, gene targeting of the SCP2/SCPx gene has shown in mice that the SCPx beta-ketothiolase is involved in peroxisomal beta-oxidation of 2-methyl-branched chain fatty acids like pristanic acid. In our present work we have investigated bile acid synthesis in the SCP2/SCPx knockout mice. Specific inhibition of beta-oxidation at the thiolytic cleavage step in bile acid synthesis is supported by our finding of pronounced accumulation in bile and serum from the knockout mice of 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestane-24-one (which is a known bile alcohol derivative of the cholic acid synthetic intermediate 3alpha,7alpha,12alpha-trihydroxy-24-keto-cholestano yl-coenzyme A). Moreover, these mice have elevated concentrations of bile acids with shortened side chains (i.e. 23-norcholic acid and 23-norchenodeoxycholic acid), which may be produced via alpha- rather than beta-oxidation. Our results demonstrate that the SCPx thiolase is critical for beta-oxidation of the steroid side chain in conversion of cholesterol into bile acids.     

Sterol carrier protein-2

The compartmentalization of cholesterol metabolism implies target-specific cholesterol trafficking between the endoplasmic reticulum, plasma membrane, lysosomes, mitochondria and peroxisomes. One hypothesis has been that sterol carrier protein-2 (SCP2, also known as the non-specific lipid transfer protein) acts in cholesterol transport through the cytoplasm. Recent studies employing gene targeting in mice showed, however, that mice lacking SCP2 and the related putative sterol carrier known as SCPx, develop a defect in peroxisomal beta-oxidation. In addition, diminished peroxisomal alpha-oxidation of phytanic acid (3,7,11, 15-tetramethylhexadecanoic acid) in these null mice was attributed to the absence of SCP2 which has a number of properties supporting a function as carrier for fatty acyl-CoAs rather than for sterols.     

New aspects of sterol carrier protein 2 (Nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes

Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipidtransfer protein, and stimulates various steps of cholesterol metabolism in vitro. Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically. This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2. SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities. The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively. P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx. This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction. PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation. PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down. This article attempts to gain insight into the role of SPC2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes.     

Intrinsic sterol- and phosphatidylcholine transfer activities of 17β-hydroxysteroid dehydrogenase type IV

Previous studies have shown that the 80 kDa 17β-hydroxysteroid dehydrogenase (17β-HSD) type IV comprises distinct domains, including an N-terminal region related to the short chain alcohol dehydrogenase multigene family and a C-terminal part related to the lipid transfer protein sterol carrier protein 2 (SCP2). In this study, we have investigated whether the SCP2-related part of the 80 kDa protein leads to an intrinsic sterol and phospholipid transfer activity, as shown earlier for the 60 kDa SCP2-related peroxisomal 3-ketoacyl CoA thiolase with intrinsic sterol and phospholipid transfer activity called sterol carrier protein x (SCPx). Our results indicate that a fraction rich in the 80 kDa form of 17β-HSD type IV exhibits high transfer activities for 7-dehydrocholesterol and phosphatidylcholine. In addition, a purified recombinant peptide derived from the SCP2-related domain of the 17β-HSD type IV has about 30% of the transfer activities for 7-dehydrocholesterol and phosphatidylcholine seen with purified recombinant human SCP2. We conclude that the 80 kDa type IV 17β-HSD represents a potentially multifunctional protein with intrinsic in vitro sterol and phospholipid transfer activity in addition to its enzymatic activity.     

Type-II 3-oxoacyl-CoA thiolase of the nematode Caenorhabditis elegans is located in peroxisomes, highly expressed during larval stages and induced by clofibrate.

We examined the expression and localization of type-II 3-oxoacyl-CoA thiolase in the nematode Caenorhabditis elegans. Type-II thiolase acts on 3-oxoacyl-CoA esters with a methyl group at the alpha carbon, whereas conventional thiolases do not. Mammalian type-II thiolase, which is also termed sterol carrier protein x (SCPx) or SCP2/3-oxoacyl-CoA thiolase, is located in the peroxisomes and involved in phytanic acid degradation and most probably in bile acid synthesis. The nematode enzyme lacks the SCP2 domain, which carries the peroxisomal-targeting signal, but produces bile acids in a cell-free system. Northern and Western blot analyses demonstrated that C. elegans expressed type-II thiolase throughout its life cycle, especially during the larval stages, and that the expression was significantly enhanced by the addition of clofibrate at 5 mM or more to the culture medium. Whole-mount in situ hybridization and immunostaining of L4 larvae revealed that the enzyme was mainly expressed in intestinal cells, which are multifunctional like many of the cell types in C. elegans. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected the enzyme in the matrix of peroxisomes. These results indicate the fundamental homology between mammalian SCPx and the nematode enzyme regardless of whether the SCP2 part is fused, suggesting their common physiological roles.     

Lauric acid dependent enhancement in hepatic SCPx protein requires an insulin deficient environment

Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treating with the fatty acid, lauric acid, induced SCPx mRNA levels in rat liver and in rat hepatoma H4IIE cells but enhanced protein levels of SCPx and the thiolase produced as a post-translational modification of SCPx were only seen in H4IIE cells. Further investigation revealed that the presence of insulin can mask lauric acid effects on the SCPx gene especially at the protein level. These data are in agreement with the findings that diabetes, a medical condition characterized by high levels of fatty acids in an insulin deficient environment, enhances the hepatic expression of SCPx.     

Biochemical composition of the Atlantic bonito Sarda sarda from the Aegean Sea (eastern Mediterranean Sea) in different stages of sexual maturity

The content (% wet mass) in water, ash, lipid, crude protein, DNA and RNA of different tissues was determined during sexual maturation of bonitos Sarda sarda from the Aegean Sea. A total of 220 specimens were collected in the following stages of sexual maturity: immature, resting, developing, mature, spawning and spent. Highest lipid levels in the white muscle, red muscle and liver were measured in immature specimens, while lowest levels were found in spawning bonitos. The gradual percentage of lipid reduction from immature to spawning bonitos was relatively higher in the liver (females 71·2% and males 64·4%) than in the white (females 59·2% and males 53·5%) and red (females 62·1% and males 51·7%) muscle. Lipid levels in the gonads increased gradually from the immature to spawning stage. The decrease of lipid in the somatic tissues was more intense in females than in males, and gonadal lipid content was higher in females than in males. There was a strong reverse correlation between water and lipid percentage in all tissues. Protein content decreased significantly only in spawning bonitos. The percentage of protein reduction from immature to spawning stage was relatively higher in males than in females in both white (females 3·4% and males 4·6%) and red (females 4·6% and males 5·1%) muscles. Protein content in the liver was significantly lower than in the other tissues, being highest in mature females. Gonadal protein content in females increased with maturation and decreased after spawning. The content in ash exhibited considerable stability. The RNA:DNA ratio exhibited a similar pattern of variation in both muscles. The RNA:DNA ratio increased during gonadal development gradually from the developing to spent stage. It was concluded that in S. sarda during gonadal development, there was an increase in gonadal lipid accompanied by a decrease in somatic tissue lipid reserves. Thus, reproductive inactive bonitos have more lipid in their edible part and a higher nutritional value than active ones.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Metabolic significance and expression of Caenorhabditis elegans type II 3-oxoacyl-CoA thiolase

The authors cloned the cDNA of the nematode Caenorhabditis elegans encoding a 44-kDa protein (P-44), which is similar to sterol carrier protein x (SCPx). Genomic DNA data and Northern blot analysis excluded the possibility of P-44 forming SCPx-like fusion protein. P-44 is required in the formation of bile acid in vitro from CoA esters of their enoyl-form intermediate in the presence of d-3-hydroxyacyl-CoA dehydratase/d-3-dehydrogenase bifunctional protein. Also, rat SCPx converts 24-hydroxy-form intermediate to bile acid under similar conditions. From this and other evidence, P-44 and SCPx were categorized as type II thiolase. The mRNA encoding P-44 was detected in every developmental stage of C. elegans: egg, larval stages, and adult. P-44, therefore, seems essential for the normal functioning of this organism.     

Biogenesis of nonspecific lipid transfer protein and sterol carrier protein x: studies using peroxisome assembly-defective pex cell mutants

Nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We investigated which of these two potential topogenic sequences functions in biogenesis of nsLTP and SCPx. Morphological and biochemical analyses, making use of Chinese hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective pex7, as well as green fluorescent protein chimeras, revealed that both pre-nsLTP and SCPx are imported into peroxisomes by the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains in the cytosol, as assessed by subcellular fractionation of the wild-type Chinese hamster ovary cells. In an in vitro binding assay, only mature nsLTP, but not pre-nsLTP, from the cell lysates interacted with the Pex5p. It is likely, therefore, that modulation of the C-terminal PTS1 by the presequence gives rise to cytoplasmic localization of pre-nsLTP.     

Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice

Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.     

Sexual maturation in the black seabream Mylio macrocephalus Teleostei,Sparidae: changes in pituitary gonadotropes,hepatocytes and related biochemical constituents in liver and serum

Summary The black seabream, Mylio macrocephalus, exhibits the phenomenon of sex segregation. Immature fish are developing hermaphrodites, and show a considerable overlap with mature males in their body weights. Mature females tend to be the heaviest group. Fish can be classified into immature, developing and mature groups, with a further division into definitive males or females in the two latter groups. The developing groups still have bisexual gonads, whereas mature males have testes with only a comparatively inconspicuous portion of ovarian tissue, and mature females possess ovaries with vitellogenic oocytes and a greatly regressed testicular component. In the present study, monthly samples were collected over a 3-year period, and changes in pituitary gonadotropes and liver tissue studied by light, and electron microscopy. Seasonal changes in serum constituents were also studied by biochemical techniques.Gonadotropes increased in number and became hypertrophied during sexual maturation, showing an enhanced cytoplasmic vacuolation and degranulation of alcian blue- and periodic acid-Schiff-positive material. The levels of various biochemical constituents in the liver and serum of developing fish tended to be intermediate between those recorded in the immature and mature groups. In mature fish, the serum levels of glucose, sodium and calcium were elevated, but hepatic glycogen content was less than the developing group, and hepatocytes contained activated mitochondria. The seasonal changes in pituitary cytology, hepatic ultrastructure and serum constituents, could be correlated with the metabolic adaptations to sexual maturation.     

Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene

Although a role for liver fatty acid protein (L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABP–/– mice had significantly more fatty lipid droplets than male L-FABP–/– mice, whereas in phytol-fed wild-type L-FABP+/+ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABP–/– mice were significantly higher than in male L-FABP–/– mice. Furthermore, livers of phytol-fed female L-FABP–/– mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol. gene targeting; phytanic acid     

Growth differences and dimorphic expression of growth hormone (GH) in female and male Cynoglossus semilaevis after male sexual maturation

Half-smooth tongue sole, Cynoglossus semilaevis, is an ideal model to investigate the regulatory mechanisms of sexual growth dimorphism in fish species. The aim of the study was to investigate the effect of differential age of sexual maturity for females and males on growth and GH mRNA expression in C. semilaevis. The body weight differences between the sexes were not significant in C. semilaevis at age 5 months when females and males were all immature. Significant differences in body weight between the sexes were found after early sexual maturation of males at the age of 9 months. The body weight of 21-month-old females (621.4 ± 86.4g), still not immature, was even 3.28 times higher than that of the males (189.7 ± 14.4g). The cDNAs encoding GH in C. semilaevis was cloned. The GH gene is 2924bp long and consists of six exons and five introns. The results of qRT-PCR showed that GH mRNA levels of the immature females were not significantly different from that of immature males at age 5 months. However, GH mRNA levels of the immature females were significantly higher compared with those of the mature males at age 9 months (P<0.05). At age 11 months, GH mRNA levels of females were even 6.4-fold higher than that of males. In conclusion, for the first time we show that early sexual maturity of males is the main cause of sexual growth dimorphism in C. semilaevis and exert significant effect on GH mRNA expression.     

Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.     

Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.