Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.     

Hepatic ATGL mediates PPAR-α signaling and fatty acid channeling through an L-FABP independent mechanism

Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to β-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for β-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum β-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for β-oxidation or the nucleus for PPAR-α regulation.     

Indentification of β-oxidation enzymes among peroxisomal polypeptides: Increase in Coomassie blue-stainable protein after clofibrate treatment

Peroxisomes contain enzymes catalyzing the β-oxidation of fatty acids, which have been purified and partially characterized. Hypolipidemic drugs, including clofibrate, cause a marked proliferation of peroxisomes and a striking increase in the activity of their β-oxidation system. We have compared by sodium dodecyl sulfate—polyacrylamide gel electrophoresis the polypeptide patterns of normal and clofibrate-induced peroxisomes and the purified β-oxidation enzymes. The data allow a tentative identification of the β-oxidation enzymes among the peroxisomal polypeptides; these enzymes constitute only a small part of the protein of normal peroxisomes. A subset of peroxisomal polypeptides, including the β-oxidation enzymes, is preferentially increased by clofibrate.     

Hidden Disease Susceptibility and Sexual Dimorphism in the Heterozygous Knockout of Cyp51 from Cholesterol Synthesis

We examined the genotype-phenotype interactions of Cyp51^+/− mice carrying one functional allele of lanosterol 14α-demethylase from cholesterol biosynthesis. No distinct developmental or morphological abnormalities were observed by routine visual inspection of Cyp51^+/− and Cyp51^+/+ mice and fertility was similar. We further collected a large data-set from female and male Cyp51^+/− mice and controls fed for 16 weeks with three diets and applied linear regression modeling. We used 3 predictor variables (genotype, sex, diet), and 39 response variables corresponding to the organ characteristics (7), plasma parameters (7), and hepatic gene expression (25). We observed significant differences between Cyp51^+/− and wild-type mice in organ characteristics and blood lipid profile. Hepatomegaly was observed in Cyp51^+/− males, together with elevated total and low-density lipoprotein cholesterol. Cyp51^+/− females fed high-fat, high-cholesterol diet were leaner and had elevated plasma corticosterone compared to controls. We observed elevated hepatocyte apoptosis, mitosis and lipid infiltration in heterozygous knockouts of both sexes. The Cyp51^+/− females had a modified lipid storage homeostasis protecting them from weight-gain when fed high-fat high-cholesterol diet. Malfunction of one Cyp51 allele therefore initiates disease pathways towards cholesterol-linked liver pathologies and sex-dependent response to dietary challenge.     

Conjugated and free sterols from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors and their effect on lipid metabolism and cholesterol transport in rat primary hepatocytes

Phytosterols have been widely studied for their cholesterol-lowering effect. Conjugated phytosterol forms have been found more active than free moieties. There are no reports about the sterol profile of black bean seed coats neither its effects on cholesterol metabolism. The aim of this research was to identify and quantify phytosterols from black bean seed coats and to determine their effects on cholesterol micellar solubility and on mRNA and key protein levels involved in lipid/cholesterol metabolism and cholesterol transport in primary rat hepatocytes. Free phytosterols, acylated steryl glycosides, and steryl glycosides were extracted from black bean seed coats. They were identified through HPLC–MS–TOF and quantified through HPLC equipped with UV–visible and evaporative light-scattering detectors. Free and conjugated phytosterols from the coats significantly increased the inhibitory effect of cholesterol micelle formation compared with stigmasterol, which was used as control (P < 0.05). In addition, phytosterols of black bean seed coat decreased lipogenesis by the downregulation of lipogenic proteins such as sterol regulatory element-binding protein 1 and fatty acid synthesis (FAS) in primary rat hepatocytes. Regarding β-oxidation, phytosterols upregulated the expression of carnitine palmitoyltransferase I and promoted the β-oxidation of long-chain fatty acids. Phytosterols inhibited cholesterol micellar solubility and reduced the activation of the liver X receptor, decreasing hepatic FAS and promoting hepatic β-oxidation of long-chain fatty acids.     

β-Ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Impairment of 8-iso-PGF2ALPHA isoprostane metabolism by dietary conjugated linoleic acid (CLA)

8-iso-PGF_2α isoprostane (IP) is one of the most-used markers of lipid peroxidation in experimental models and humans. After its formation, it is promptly metabolized to 2,3 dinor (DIN) in peroxisomes.Conjugated linoleic acid (CLA) is preferentially β-oxidized in peroxisomes which may compete with IP, and thereby may affect its metabolism.In order to verify whether CLA is able to influence IP formation and/or metabolism and to explain the mechanism, we challenged rats supplemented with CLA or with triolein (as a control fatty acid), with a single dose of carbon tetrachloride (CCl₄) or of bacterial lipopolysaccharide (LPS). The results showed that IP and its precursor arachidonic acid hydroperoxide, as well as malondialdheyde (MDA), increase significantly in the liver of rats challenged with CCl₄, irrespective of the diet, while in LPS-treated rats only nitrites in liver and isoprostane in plasma increase. On the other hand, the peroxisomal β-oxidation products of IP, the DIN, is significantly lower in the CLA group with respect to control and triolein groups.To further investigate whether this is due to competition between CLA and IP at the cellular level, we incubated human fibroblasts from healthy subjects or patients with adrenoleukodystrophy (ALD), with CLA and/or commercially available IP. The rationale of this approach is based on the deficient peroxisomal β-oxidation of fibroblasts from ALD patients, leading to a reduced formation of DIN. In both normal and ALD cells, the presence of CLA significantly inhibits the formation of DIN from IP.We may conclude that both in vitro and in vivo studies strongly suggest that CLA may impair IP catabolism in peroxisomes. Consequently an increase of IP, as a sole result of CLA intake, cannot be considered as a marker of lipid peroxidation.     

Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica

3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached 0.50. β-Oxidation fluxes are very dependent on the presence of oxygen and conditions of accumulation of 3-hydroxy-γ-decalactone with very high yields were identified. These results are an important step in the production of the two decenolides. Moreover, they show the high dependence of β-oxidation fluxes on environmental conditions and relate these conditions to the accumulation of intermediates, results that are of interest to all the processes using yeast on lipids or alkanes.     

Effects of Dietary Inclusion of β-Hydroxy-β-Methylbutyrate on Growth Performance,Fat Deposition,Bile Acid Metabolism,and Gut Microbiota Function in High-Fat and High-Cholesterol Diet-Challenged Layer Chickens

Excessive lipid deposition in layer chickens due to inappropriate feeding adversely affects egg production; however, nutritional manipulation methods to deal with this issue are still limited. β-hydroxy-β-methylbutyrate (HMB), a metabolite of L-leucine, was recently reported as a lipid-lowering nutrient in mice and pigs, although its role in layers had not been investigated. Here, we employed high-fat and high-cholesterol diet (HFHCD)−challenged growing layers as an obese model to explore HMB function in the regulation of lipid metabolism and the potential mechanisms involved. We found that dietary supplementation with (0.05% or 0.10%) HMB significantly reduced HFHCD−induced bodyweight growth in layers, mainly due to reduction in abdominal fat deposition. Mechanistically, HMB supplementation enhanced hepatic bile acid synthesis from cholesterol through elevating expression of Cyp7a1, a gene coding a key enzyme in bile acid synthesis. Furthermore, 16S rRNA gene sequencing revealed that HMB supplementation remodeled the diversity and composition of the layers’ cecal microbiota, and the abundance of Bacteroidetes at the phylum level were especially affected. Correlation analysis further indicated a strong negative association between Bacteroidetes abundance and lipid metabolism−related parameters. Taken together, these data suggest that dietary HMB supplementation could improve abdominal fat deposition in layers, probably through modulating hepatic bile acid synthesis and gut microbiota function.     

Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents

Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11β-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone = 0.735% and corticosterone = 0.045%), and for B assay (aldosterone = 0.14%, 11-dehydro-B = 0.006%, cortisol = 0.016% and DOC = 0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y = 1.092X − 0.054, R² = 0.988; B ELISA: Y = 1.047X − 0.226, R² = 0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.     

Mitochondrial 2,4-dienoyl-CoA Reductase Deficiency in Mice Results in Severe Hypoglycemia with Stress Intolerance and Unimpaired Ketogenesis

The mitochondrial β-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial β-oxidation of unsaturated fatty acids, we created a DECR–deficient mouse line. In Decr^−/− mice, the mitochondrial β-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr^−/− mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C_18:2), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr^−/− mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal β-oxidation and microsomal ω-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1α and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.     

Factors influencing α-tocopherol synthesis in pepper fruits

Determination of vitamin E in the fruit pericarp of green, yellow and red varieties of Capsicum annuum L. from the local market points to a parallel accumulation in pepper fruits of α-tocopherol with secondary carotenoids and triacylglycerols enriched in unsaturated fatty acids. Highest α-tocopherol concentrations of about 400 nmol per g of dry weight have been found in red fruits. Ripe yellow and red pepper fruits grown under greenhouse conditions were smaller and contained lower α-tocopherol contents than corresponding ones from the local market. An approximation to the α-tocopherol levels in market fruits has been observed, however, if the green plants had been treated with the bleaching herbicide norflurazone before fruit ripening, affecting the carotenoid pathway. Optimum herbicide efficiency has been obtained via watering of the green plants. In ripe fruits of the yellow and red varieties α-tocopherol contents were paralleled by increasing γ-tocopherol methyltransferase activities. In chromoplast preparations from pepper, methylation capacities have been found for γ- and δ-tocopherol as well as for the structurally related tocotrienols, the diterpene side chain of which consists of a geranylgeranyl- instead of the reduced phytyl residue found in tocopherols. β-Tocopherol was not methylated, which supports the position-specific methylation of prenylquinones at the 5 position of the tocopherol aromatic headgroup.     

Identification of critical amino acids involved in α1-β interaction in voltage-dependent Ca channels

In voltage-dependent Ca²⁺ channels, the α₁ and β subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AID_A GST fusion protein for the four interacting β_1b BID mutants was not significantly altered compared with the wild-type β_1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive β mutants with the full-length α_1A subunit only slightly modified the stimulation efficiency when compared with the wild-type β_1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the β subunit-induced current stimulation.     

Characterization of β-endorphin- and α-MSH-related peptides in rat heart

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that β-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to β-endorphin- and α-MSH-related peptides. Ion exchange HPLC analysis revealed that β-endorphin(1–31) was further processed to α-N-acetyl-β-endorphin(1–31), which comprised 35.9 ± 0.1% of total immunoreactivity, and smaller amounts of β-endorphin(1–27), β-endorphin(1–26), and their α-N-acetylated derivatives. The predominant α-MSH immunoreactive peptides coeluted with α-MSH and N,O-diacetyl-α-MSH by reverse-phase HPLC, although small amounts of ACTH(1–13)-NH₂ were also present. Thus, multiple forms of β-endorphin and α-MSH are localized in rat heart. β-Endorphin(1–31) is a minor constituent, however, indicating that nonopioid β-endorphin peptides predominate.     

6β,19-Bridged androstenedione analogs as aromatase inhibitors

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New 6β,19-bridged steroid analogs of androstenedione, 6β,19-epithio- and 6β,19-methano compounds 11 and 17, were synthesized starting from 19-hydroxyandrostenedione (6) and 19-formylandrost-5-ene-3β,17β-yl diacetate (12), respectively, as aromatase inhibitors. All of the compounds including known steroids 6β,19-epoxyandrostenedione (4) and 6β,19-cycloandrostenedione (5) tested were weak to poor competitive inhibitors of aromatase and, among them, 6β,19-epoxy steroid 4 provided only moderate inhibition (K_i: 2.2 μM). These results show that the 6β,19-bridged groups of the inhibitors interfere with binding in active site of aromatase.     

Structure of a novel highly branched α-glucan enzymatically produced from maltodextrin

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility α-glucan containing a large amylase-resistant portion. This α-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility α-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The α-glucan was found to be a novel highly branched α-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.     

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate

Using a monoclonal antibody against the entire C-terminal end of human APP₆₉₅ (643–695 sequence) and a monoclonal antibody directed against human β[1–40] amyloid peptide (βA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of βA. According to the nature of N- and C-terminal amino acids we identified endogenous β-, γ-, ε-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the β-amyloid sequence at same sites as those observed in human βA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study βA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human βA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.