Altered desaturation and elongation of fatty acids in hormone-sensitive lipase null mice

Background

Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored lipids, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. The aim of this study was to define lipid profiles in plasma, white adipose tissue (WAT) and liver of HSL null mice, in order to better understand the role of this multifunctional enzyme.

Methodology/Principal Findings

This study used global and targeted lipidomics and expression profiling to reveal changed lipid profiles in WAT, liver and plasma as well as altered expression of desaturases and elongases in WAT and liver of HSL null mice on high fat diet. Decreased mRNA levels of stearoyl-CoA desaturase 1 and 2 in WAT were consistent with a lowered ratio of 16∶1n7/16∶0 and 18∶1n9/18∶0 in WAT and plasma. In WAT, increased ratio of 18∶0/16∶0 could be linked to elevated mRNA levels of the Elovl1 elongase.

Conclusions

This study illustrates the importance of HSL for normal lipid metabolism in response to a high fat diet. HSL deficiency greatly influences the expression of elongases and desaturases, resulting in altered lipid profiles in WAT, liver and plasma. Finally, altered proportions of palmitoleate, a recently-suggested lipokine, in tissue and plasma of HSL null mice, could be an important factor mediating and contributing to the changed lipid profile, and possibly also to the decreased insulin sensitivity seen in HSL null mice.     

Differentiation of human testicular embryonal carcinoma and teratocarcinoma grown in nude mice and soft-agar cultures

The differentiation pattern of two human germ cell tumors, grown in nude mice and in vitro is described. Tumor A was an embryonal carcinoma (EC) of borderline histology with characteristics of yolk sac tumor and of seminoma; tumor B was a teratocarcinoma with yolk sac elements and syncytiotrophoblastic giant cells. The morphology of an EC as well as cytogenetic characteristics were maintained during 20 passages in nude mice from tumor A and over 11 passages from tumor B. Tumor A did not grow in vitro. Cell suspensions prepared from xenografted tumor B grew into cystic embryoid bodies in semi-solid tissue culture medium. These embryoid bodies showed cuboidal and flattened cells with microvilli, junctional complexes, peripheral microfilaments, and annulated lamellae, reminiscent of the 'inner cell mass' of a blastula and of endoderm, respectively. When such colonies were transplanted into nude mice, however, only tumors with the morphology found in the transplants appeared.     

Microsomal desaturation of stearic acid in relation to lymphocyte activation

The conversion of stearic acid to oleic acid (delta 9-desaturase) was followed in mouse thymocytes stimulated by either concanavalin A or concanavalin A + interleukin-2 resulting in different rates of cell proliferation. To estimate the plasma membrane turnover of oleic acid as compared to that of a saturated fatty acid, double-label experiments ([14C]oleic acid, [3H]palmitic acid) were performed. Following an inhibition delta 9-desaturase was found to be activated from the fourth hour of stimulation. In the early period of cell activation this process proved to be independent of protein synthesis, whereas in the stage of proliferation it was dependent on it. Increased membrane fluidity in the first 30 min of activation is not likely due to enrichment of oleic acid. Cell proliferation and microsomal desaturation seem to be coupled and an increasing amount of oleic acid is at least one of the factors resulting in increased fluidity of the surface membrane of proliferating cells.     

Hydrogen isotopic fractionations during desaturation and elongation associated with polyunsaturated fatty acid biosynthesis in marine macroalgae

Compound-specific hydrogen isotopic compositions (deltaD) of saturated, monounsaturated and polyunsaturated fatty acids have been determined for natural marine macroalgae including two brown algae (Heterokontophyta) and two red algae (Rhodophyta). deltaD values of individual fatty acids from four macroalgae exhibit a wide variation ranging from -189% to +48%. Generally, stearic (18:0), arachidic (20:0) and behenic acids (22:0) are much more enriched in D by up to approximately 180% relative to myristic (14:0), palmitic (16:0), octatetraenoic [18:4(n-3)] and eicosapentaenoic acids [20:5(n-3)]. Other fatty acids such as oleic [18:1(n-9)], lenoleic [18:2(n-6)] and linolenic acids [18:3(n - 3)] fall isotopically between these fatty acids. This wide deltaD variation of fatty acids is probably explained by the hydrogen isotopic fractionation during desaturation being much larger than that during elongation in the network of polyunsaturated fatty acid biosynthesis. A large hydrogen isotopic fractionation during desaturation may cause D-enrichment in the remaining hydrogen of the residual fatty acids, which could be controlled by the relative flux into their desaturates.     

Tumor lipids and liver lipid metabolism in the model human lung carcinoma/nude mice

Tumor lipids were studied in the experimental model Human Lung Carcinoma/nude mice as well as the effect of this human neoplasm on the host liver lipid metabolism. Fatty acid profiles from tumoral lipids revealed the loss of specificity for fatty acid composition in triglycerides. Host liver fatty acid composition and cholesterol metabolism were affected by the implanted human lung tissue. A noticeable increase ratio between saturated/unsaturated fatty acids was observed in host liver fatty acid phospholipids (1.17 +/- 0.17) in comparison to control liver (0.84 +/- 0.04). Cholesterol synthesis was assessed "in vivo" by means of [14C]acetate incorporation. The specific radioactivity of [14C] cholesterol was increased by a factor of about 6 in host liver as compared with control liver. This observation along with the marked decrease in the cholesterol content of host liver and the hypocholesterolemia detected in the host mice led us to suggest an increase in the liver cholesterol catabolism promoted by the presence of the tumor.     

Mechanism of fatty acid desaturation: a bioorganic perspective

The desaturation of long chain fatty acids is a ubiquitous transformation which plays a critical role in the biosynthesis of lipids. Of particular interest to the bioorganic chemist is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in a chemo-, regio- and stereoselective manner. The mechanism of membrane-bound desaturases has been examined using regiospecifically labelled analogues bearing deuterium, sulfur or fluorine-substituted methylene isosteres. These probes have been applied in the study of several biomedically important desaturase systems including a prototypical yeast stearoyl CoA delta(9) desaturase. In all cases, it has been found that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain. These results point to a common active site architecture which is highly conserved among a wide range of membranous desaturases.     

Action of Ebselen on rat hepatic microsomal enzyme-catalyzed fatty acid chain elongation, desaturation, and drug biotransformation

In the previous study, the organoselenium-containing anti-inflammatory agent, Ebselen, was found to disrupt both hepatic microsomal NADH- and NADPH-dependent electron transport chains. In the current investigation, we focus on the action of Ebselen on three separate metabolic reactions, namely, fatty acid chain elongation, desaturation, and drug biotransformation, which utilize reducing equivalents via these microsomal electron transport pathways. Both NADH-dependent and NADPH-dependent chain elongation reactions showed (i) that the condensation step was inhibited by Ebselen; all three substrates, palmitoyl CoA (16:0), palmitoleoyl CoA (16:1), and gamma-linolenyl CoA (18:3), were differentially affected by Ebselen; for example, the apparent Ki's of Ebselen for the condensation of 16:0, 16:1, and 18:3 in the absence of bovine serum albumin (BSA) preincubation were 7, 14, and 34 microM, and those in the presence of BSA preincubation were 35, 62, and 150 microM, respectively, supporting earlier data for multiple condensing enzymes; (ii) that the beta-ketoacyl CoA reductase-catalyzed reaction step which appears to receive electrons, at least in part, from the cytochrome b5 system, was also markedly inhibited by varying Ebselen concentrations; and (iii) that similar results were obtained with the dehydrase and the enoyl CoA reductase. Hence, each of the four component steps was significantly inhibited by Ebselen. Another important fatty acid biotransformation reaction, delta 9 desaturation of stearoyl CoA to oleoyl CoA, was significantly inhibited (90%) by 30 microM Ebselen. This effect appeared to be directly related to the NADH-dependent electron transport chain rather than to a direct action on the desaturase enzyme. Last, Ebselen also inhibited both aminopyrine and benzphetamine N-demethylations, two cytochrome P450-catalyzed reactions, in untreated rats, in rats on a high carbohydrate diet, and in phenobarbital-treated rats.     

Cell proliferation kinetics in two human tumors grown in athymic nude mice.

The technique of labelled mitoses was used to investigate the cell proliferation kinetics in two human neoplasms, one a malignant melanoma and one a fibrosarcoma, transplanted to and grown serially in the athymic nude mutant (ANM) mouse. The experimental data obtained codrealted well with a theoretical percentage labelled mitoses curve based on the assumption that the time spent by a cell in each of the phases M, G1, S and G2 is described by four independent log-normal distributions. However, no unique second wave was defined by the experimental results. This means that only the deductions made about the duration of the G2 and S phases are reliable. The median duration of tma and the fibrosarcoma, respectively. By comparing these results with results published on cell cycle studies of transplantable animal tumors and human tumors in situ, it is concluded that the cell cycle parameters of a human tumor grown in the ANM mouse are close to those of the same tumor in the donor patient.     

Modification by clofibric acid of acyl composition of glycerolipids in rat liver. Possible involvement of fatty acid chain elongation and desaturation

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) to rats induced a marked change in acyl composition of hepatic glycerolipids; a considerable increase in the proportion of octadecenoic acid (18:1) was accompanied by a marked decrease in the proportion of octadecadienoic acid (18:2). Among the glycerolipids, the changes in the proportions of 18:1 and 18:2 were the most marked in phosphatidylcholine. The change in the acyl composition of phosphatidylcholine paralleled the change in free fatty acid composition in microsomes. The treatment of rats with clofibric acid resulted in a 2.3-fold increase in activity of microsomal palmitoyl-CoA chain elongation and a 4.8-fold increase in activity of stearoyl-CoA desaturation. The activities of acyl-CoA synthetase, 1-acylglycerophosphate acyltransferase and 1-acylglycerophosphorylcholine acyltransferase in hepatic microsomes were increased approx. 3-, 1.7- and 3.6-times, respectively, by the treatment of rats with clofibric acid. These findings are discussed with respect to the role of fatty acid modification systems in the regulation of acyl composition of phosphatidylcholine.     

Establishment of human colon carcinoma lines in nude mice.

After subcutaneous inoculation into nude mice of 24 human colon adenocarcinomas, growth, defined as histopathologically confirmed tumor growth which has been passed, was observed in 13 cases (54%). Tumors from metastatic sites showed higher take rates (58%) than tumors from primary sites or recurrent tumors (50%). Nine continuous tumor lines were established (69% of growing tumors) with metastatic tumors establishing more readily (100% of growing tumors) than primary tumors (40%). The average period in primary transplant was shorter for metastasis (8.3 weeks), than for primary tumors (18.5 weeks); total material 10.6 weeks. Average periods between passages were shorter than primary transplant times; these periods were shorter for metastases (6.6 weeks) than for primary tumors (9.4 weeks); total material 7.4 weeks. Of four growing tumors not established as continuous lines, three were primary and one a recurrent tumor, and the loss of tumor growth occurred in very early passages, not later than passage 3. All nude mouse-grown colon tumors were moderately well differentiated.     

Secretion of ceruloplasmin by a human clear cell carcinoma maintained in nude mice

Ceruloplasmin is the best known but least understood copper protein. Studies preliminary to investigating the control of ceruloplasmin synthesis have utilized a human renal cell carcinoma maintained in nude mice for 73 passages over a 5-year period. In vitro cultures of these cells were accomplished and the mRNAs were extracted prior to microinjection into Xenopus oocytes. The media examined by SE-HPLC and immunological techniques demonstrated that (1) after in vitro culture, ceruloplasmin was secreted as an uncleaved polypeptide chain with a MW of 135,000; (2) the translational product of ceruloplasmin mRNA injected into Xenopus oocytes was cleaved into fragments with MWs of 110,000, 67,000, and 50,000. The results indicate that mRNA for human ceruloplasmin can be obtained to serve as a template for the synthesis of a cDNA probe to investigate the control of human ceruloplasmin's synthesis.     

Myeloperoxidases of human myeloid leukemia cells HL-60 grown in culture and in nude mice

Human myeloid leukemia HL-60 cells were grown either in suspension culture or in nude mice. The two types of myeloperoxidase, the small and the large type, in crude extracts of these cells were analyzed by sucrose density gradient centrifugation. The proportions of the small and the large myeloperoxidase varied markedly depending on the growth conditions of cells. In cells in culture, the small myeloperoxidase amounted to 80% of the total myeloperoxidase, whereas in the solid tumors it amounted to only about 30% of the total. Both in cultured cells and solid tumors, 40% of the total myeloperoxidase was found in the soluble fraction and the rest in the granule fraction. However, adult and fetal blood granulocytes contained only the large myeloperoxidase, which was mainly recovered in the granule fraction. Antiserum prepared against purified large myeloperoxidase of HL-60 solid tumors reacted with the small myeloperoxidase as well as the large enzyme of HL-60 cells in culture. The antiserum also precipitated myeloperoxidase of adult and fetal blood granulocytes. An Ouchterlony double immunodiffusion test also revealed their immunological identity.     

Linolenic acid desaturation and chain elongation and rapid turnover of phospholipid n - 3 fatty acids in isolated rat liver cells

The desaturation and chain elongation of [1-14C]linolenic acid was studied in isolated liver cells from rats fed a diet deficient in essential fatty acids. 14C-labelled 18:4, 20:3, 20:4, 20:5, 22:5 and 22:6, all n - 3 fatty acids, were formed. In the presence of lactate relatively large amounts of 20:5, 22:5 and 22:6 were formed. 20:5 was mainly present in phospholipids, 22:5 and 22:6 were present in both phospholipids and triacylglycerols. (+)-Decanoylcarnitine and (-)-hydroxycitrate decreased the formation of 20:5, 22:5 and 22:6 and increased the recovery of 18:4. The unchanged 18:3 substrate was also initially rapidly incorporated both in the phospholipids and in the triacylglycerol fraction. During long incubation periods, continued after nearly all the [14C]linolenic acid substrate had been metabolized either by esterification or by oxidation, the phospholipid content of labelled 18:3 and 18:4 decreased while the content of 20:5, 22:5 and 22:6 increased markedly, suggesting a remodeling of the phospholipid n - 3 fatty acid content by a series of deacylations-reacylations. The n - 3 fatty acid pattern in the triacylglycerol fraction changed little. 22:5 and 22:6 appeared in the VLDL fraction secreted by the isolated liver cells.     

An ascites form of human mammary carcinoma transplantable in nude mice

Mammary carcinoma cells from pleural effusion of a patient were inoculated into the peritoneal cavity of nude mice, and a large amount of ascites was produced about 120 days later. From the ascites, serial passages in the same form were successful in nude mice by intraperitoneal injection of 10(7) or more cells. The ascites cells retained the morphology almost similar to that of the patient tumor cells, whereas specific estrogen-binding proteins in the cytoplasm disappeared after growing in male nude mice. The results were compared with those of other established human cancer cell lines in nude mice.     

Fatty acid desaturation and microsomal lipid fatty acid composition in experimental hypothyroidism.

We have studied the influence of experimental hypothyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty acid composition. Hypothyroid rats demonstrated an 80% decrease in delta 9 (stearate) desaturation and a 43% decrease in delta 6 (linoleate) desaturation. Liver microsomal fatty acid composition was altered in the hypothyroid animals with a significantly decreased proportion of arachidonate and increased proportions of linoleate, eicosa-8,11,14-trienoate, eicosapentaenoate and docosahexaenoate. The bulk of these changes occurred in both of the two major phospholipid components, phosphatidylcholine and phosphatidylethanolamine. All of the changes were corrected by treatment of the hypothyroid rat with 25 micrograms of tri-iodothyronine/100 g body wt. twice daily. The diminished delta 9 desaturation did not lead to any changes in fatty acid composition. The increased linoleate and decreased arachidonate levels may be due to the diminished delta 6 desaturase activity, the rate-controlling step in the conversion of linoleate into arachidonate. The increases in the proportions of the other polyunsaturated fatty acid components cannot be explained by changes in the synthesis of unsaturated fatty acids, but are probably due to diminished utilization of these fatty acids.     

Sex-related differences in desaturation and chain elongation of essential fatty acids studied in isolated rat hepatocytes

When [14C]linoleic acid (18:2(n-6)) or [14C]dihomogammalinolenic acid (20:3(n-6)) was incubated with isolated liver cells from rats fed an essential fatty acid deficient diet, delta 6- and delta 5-desaturation, chain elongation and synthesis of 14C-labelled C14-C18 fatty acids (from [14C]acetate) were enhanced in female cells compared with male ones. No sex difference in total secretion of very low density lipoproteins (VLDL) was observed. However, VLDL secreted from female cells contained significantly more C16-C18 fatty acids than male cells. It is suggested that the observed sex differences, at least in part, may be related to the different content of fatty acid binding proteins in female cells compared with males.