Differential modulation of ACAT1 and ACAT2 transcription and activity by long chain free fatty acids in cultured cells.

Fatty acyl CoA and cholesterol are the substrates for cholesteryl ester synthesis by acyl coenzyme A:cholesterol acyltransferase (ACAT). Two ACAT genes have been identified; ACAT1 is expressed ubiquitously while ACAT2 is primarily expressed in intestine and liver. We tested effects of different free fatty acids (FFAs) on ACAT1 and ACAT2 expression and activity in HepG2 human hepatocytes and THP1 human macrophages. Incubation of oleic acid, arachidonic acid, or eicosapentaenoic acid, but not 25-hydroxycholesterol, induced ACAT1 mRNA levels 1.5--2-fold in HepG2, with no affect on ACAT2 mRNA. FFA had no affect on ACAT1 mRNA in THP1 cells. To determine if FFAs affect ACAT1 or ACAT2 posttranscriptionally, cells were labeled with [(3)H]cholesterol in the presence of the different FFAs for 1--5 h. Both HepG2 and THP1 cells showed the greatest cholesteryl ester production with oleic acid. This was also confirmed by the observation that more [(3)H]oleic acid incorporated into CE compared to [(3)H]eicosapentaenoic acid, even though there was no difference in the total uptake of these FFAs. In ACAT-deficient SRD4, CHO cells stably transfected with human ACAT1 or ACAT2, ACAT1 expressing cells showed a strong preference for oleic acid while ACAT2 expressing cells utilized unsaturated FFAs. Acyl CoA substrate specificity was further tested in microsomes isolated from these cells as well as HepG2 and THP1. THP1 and ACAT1 cells utilized oleoyl CoA preferentially. In contrast, HepG2 and ACAT2 microsomes utilized linolenoyl CoA as well. We conclude that FFAs increase ACAT1 mRNA levels in a cell specific manner, and furthermore that the ACAT reactions exhibit differential FFA utilization.

A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast.

The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes.

The enzyme acyl coenzyme A:cholesterol acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial component of intracellular lipid homeostasis. Two enzymes catalyze this activity in Saccharomyces cerevisiae (yeast), and several lines of evidence suggest multigene families may also exist in mammals. Using the human ACAT1 sequence to screen data bases of expressed sequence tags, we identified two novel and distinct partial human cDNAs. Full-length cDNA clones for these ACAT related gene products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2) cDNA library. ARGP1 was expressed in numerous human adult tissues and tissue culture cell lines, whereas expression of ARGP2 was more restricted. In vitro microsomal assays in a yeast strain deleted for both esterification genes and completely deficient in sterol esterification indicated that ARGP2 esterified cholesterol while ARGP1 did not. In contrast to ACAT1 and similar to liver esterification, the activity of ARGP2 was relatively resistant to a histidine active site modifier. ARGP2 is therefore a tissue-specific sterol esterification enzyme which we thus designated ACAT2. We speculate that ARGP1 participates in the coenzyme A-dependent acylation of substrate(s) other than cholesterol. Consistent with this hypothesis, ARGP1, unlike any other member of this multigene family, possesses a predicted diacylglycerol binding motif suggesting that it may perform the last acylation in triglyceride biosynthesis.

Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2).

Primary bile acid malabsorption (PBAM) is an idiopathic intestinal disorder associated with congenital diarrhea, steatorrhea, interruption of the enterohepatic circulation of bile acids, and reduced plasma cholesterol levels. The molecular basis of PBAM is unknown, and several conflicting mechanisms have been postulated. In this study, we cloned the human ileal Na+/bile acid cotransporter gene (SLC10A2) and employed single-stranded conformation polymorphism analysis to screen for PBAM-associated mutations. Four polymorphisms were identified and sequenced in a family with congenital PBAM. One allele encoded an A171S missense mutation and a mutated donor splice site for exon 3. The other allele encoded two missense mutations at conserved amino acid positions, L243P and T262M. In transfected COS cells, the L243P, T262M, and double mutant (L243P/T262M) did not affect transporter protein expression or trafficking to the plasma membrane; however, transport of taurocholate and other bile acids was abolished. In contrast, the A171S mutation had no effect on taurocholate uptake. The dysfunctional mutations were not detected in 104 unaffected control subjects, whereas the A171S was present in 28% of that population. These findings establish that SLC10A2 mutations can cause PBAM and underscore the ileal Na+/bile acid cotransporter's role in intestinal reclamation of bile acids.

Identification of a mutation in the ileal sodium-dependent bile acid transporter gene that abolishes transport activity.

The ileal Na+/bile acid cotransporter plays a critical role in the reabsorption of bile acids from the small intestine. In the course of cloning and characterizing the human ileal Na+/bile acid cotransporter cDNA, a dysfunctional isoform was identified in a patient diagnosed with Crohn's disease. Expression studies using hamster-human ileal Na+/bile acid cotransporter cDNA chimeras narrowed the location of the defect to the carboxyl-terminal 94 amino acids. Comparison of the sequence of the dysfunctional isoform to that of a wild-type human ileal Na+/bile acid cotransporter genomic clone revealed a single C to T transition resulting in a proline to serine substitution at amino acid position 290. The inheritance of this mutation in the proband's family was confirmed by single-stranded conformation polymorphism analysis and DNA sequencing. In transfected COS-1 cells, the single amino acid change abolished taurocholate transport activity but did not alter the transporter's synthesis or subcellular distribution. This dysfunctional mutation represents the first known molecular defect in a human sodium-dependent bile acid transporter.

Cloning and chromosomal localization of the human ileal lipid-binding protein.

A human ileal lipid-binding protein (ILBP3) cDNA was isolated and shown to encode a 128 amino acid protein with homology to the fatty acid-binding protein gene family. The human ILBP amino acid sequence exhibited 78% identity to the rat ILBP sequence. Northern blot analysis revealed a single transcript of approx. 0.6 kb in small intestine, but no hybridization to liver, spleen, thymus, prostate, ovary, caecum, or colon. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human ILBP gene resides on chromosome 5 and is not linked to any other known fatty acid-binding protein family member.

Bile acid transporters.

The sodium-dependent bile acid transporters and sodium-independent organic anion transporters are integral membrane glycoproteins that function in the enterohepatic circulation of bile acids. The recent cloning and expression of different classes of bile acid transporters have provided insights into their structure and molecular mechanism.

Pituitary function tests: comparison of ACTH and 11-deoxy-cortisol responses in the metyrapone test and with the insulin hypoglycemia test.

PURPOSE: To compare the sensitivity of ACTH and 11-deoxy-cortisol (comp. S) responses in the short metyrapone test and the latter with the insulin hypoglycemia test. METHODS: Retrospective evaluation of 115 short metyrapone tests and comparison of 18 pairs of metyrapone and insulin tests. 20 healthy controls and 95 patients with confirmed pituitary disease were studied. All hormones were measured by sensitive radioimmunoassays. RESULTS: In patients with pituitary disease not requiring hydrocortisone substitution (n = 70), the ACTH response in the metyrapone test was subnormal in 47 cases (< 33 pmol/L), the comp. S response (< 200 nmol/L) in 21 cases only. Comparison of the relationship between ACTH and comp. S with an ACTH-cortisol dose-response curve obtained in normal subjects shows that subnormal ACTH responses after metyrapone in the range between 13 and 33 pmol/L still generate normal comp. S responses. The results of the metyrapone test correlated significantly with those of the insulin test. CONCLUSIONS: Measuring plasma ACTH in the scope of the metyrapone test makes the test more sensitive to detect secondary adrenal insufficiency than with steroid measurements alone. Results of the metyrapone test correlate significantly with the cortisol response to insulin hypoglycemia.

Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter.

Active uptake of bile acids from the lumen of the small intestine is mediated by an ileal Na(+)-dependent bile acid transport system. To identify components of this transport system, an expression cloning strategy was employed to isolate a hamster ileal cDNA that exhibits bile acid transport activity. By Northern blot analysis, mRNA for the cloned transporter was readily detected in ileum and kidney but was absent from liver and proximal small intestine. The transporter cDNA encoded a 348-amino acid protein with seven potential transmembrane domains and three possible N-linked glycosylation sites. The amino acid sequence was 35% identical and 63% similar to the rat liver Na+/bile acid cotransporter. After transfection into COS cells, the hamster cDNA transported taurocholate in a strict Na(+)-dependent fashion with an apparent Km of 33 microM. This taurocholate transport was inhibited by various bile acids but not by taurine or other organic anions. The Na+ dependence, saturability, and bile acid specificity of transport as well as the tissue specificity of mRNA expression strongly argue that the transporter cDNA characterized in this study is the Na+/bile acid cotransporter described previously in ileum.