Expression and secretion of chicken apolipoprotein AI in transfected COS cells

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.     

Sequence requirements for apolipoprotein B RNA editing in transfected rat hepatoma cells

Apolipoprotein (apo) B mRNA undergoes a novel tissue-specific editing reaction, which replaces a genomically templated cytidine with uridine. This substitution converts codon 2153 from glutamine (CAA) in apo B100 mRNA to a stop codon (UAA) in apoB48 mRNA (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840). To examine sequences in the human apoB mRNA required for the editing reaction, a series of deletion mutants around the cytidine conversion site was prepared and transfected into a rat hepatoma cell line (McArdle 7777). This cell makes both apoB100 and apoB48. Editing was detected by a primer extension assay on cDNA that had been amplified by the polymerase chain reaction. RNAs of between 2385 and 26 nucleotides spanning the conversion site underwent similar levels of conversion. Editing was confirmed by cloning and sequencing of cDNA corresponding to the transfected RNAs. Conversion did not occur in transfected human hepatoblastoma (HepG2) or epithelial carcinoma (HeLa) cell lines, which do not make apoB48. These results verify that apoB48 is generated by a genuine tissue-specific RNA editing reaction and show that 26 nucleotides of apoB mRNA are sufficient for editing.     

Stearoyl-CoA desaturase 1 deficiency increases insulin signaling and glycogen accumulation in brown adipose tissue

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of oleate (C18:1) and palmitoleate (C16:1), which are the main monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters, and cholesterol esters. Previously, we showed that SCD1 deficiency elevates insulin-signaling components and downregulates protein-tyrosine phosphatase-1B (PTP-1B) in muscle, a major insulin-sensitive tissue. Here we found that, in brown adipose tissue (BAT), another insulin-sensitive tissue, the basal tyrosine phosphorylations of insulin receptor (IR) and IR substrates (IRS-1 and IRS-2) were upregulated in SCD1(-/-) mice compared with wild-type mice. The association of IRS-1 and IRS-2 with the alpha-p85 subunit of phosphatidylinositol 3-kinase as well as Akt-Ser(473) and Akt-Thr(308) phosphorylation is also elevated in the SCD1(-/-) mice. The mRNA expression, protein levels, and activity of PTP-1B implicated in the attenuation of the insulin signal are reduced in the SCD1(-/-) mice. The content of GLUT4 in the plasma membrane increased 2.5-fold, and this was accompanied by a 6-fold increase in glucose uptake in BAT of SCD1(-/-) mice. The increased glucose uptake was associated with higher glycogen synthase activity and glycogen accumulation. In the presence of insulin, [U-(14)C]glucose incorporation into glycogen was increased in BAT of SCD1(-/-) mice. Taken together, these studies illustrate increased insulin signaling and increased glycogen metabolism in BAT of SCD1(-/-) mice.     

Heme biosynthesis in a chicken hepatoma cell line (LMH): comparison with primary chick embryo liver cells (CELC)

5-Aminolevulinic acid synthase (ALA synthase), the rate-controlling enzyme of hepatic heme biosynthesis, is feed-back repressed by heme. In the liver, chemicals such as barbiturates markedly induce ALA synthase, especially in the presence of partial defects of heme biosynthesis. The inducibility and regulation of ALA synthase have been investigated using a variety of models, including intact animals and liver cell culture systems. A widely used model that closely approximates what occurs in vivo and in humans is that of primary cultures of chick embryo liver cells (CELCs). However, CELCs have some limitations: the cells obtained are somewhat heterogeneous; isolation and culture must be repeated every week resulting in weekly variations; and cells are short-lived limiting the feasibility of time-course and transfection studies. The aim of this study was to determine if LMH cells, a chick hepatoma cell line, are a good model comparable to that of CELCs. In both cells similar patterns of response of, ALA synthase activities and mRNA levels, and of porphyrin accumulation were obtained following treatments known to affect heme biosynthesis. Similarly, heme repressed ALA synthase mRNA levels in both cell types and ALA synthase activities in LMH cells. We conclude that LMH cells are a useful model for the study of hepatic heme biosynthesis and regulation of ALA synthase.     

Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. influence of insulin,glucocorticoid, fatty acids and cordycepin

• 1.1. Stearoyl-CoA desaturase (Δ9-desaturase) activity was measured in chicken primary hepatocytes, as a function of time in culture.
• 2.2. When using fasted donor animals, the desaturase activity was low at the beginning of culture and then increased steadily to a maximum value between 30 and 70 hr of culture. When hepatocyte cultures were prepared from fed animals, enzyme activity was high at the beginning of culture and maintained thereafter at similar values to those obtained in cultured hepatocytes from fasted animals after 30 hr of culture.
• 3.3. Insulin significantly enhanced enzyme activity when added to the culture medium at a 10⁻⁹M concentration, and a small stimulating effect was also observed with 10⁻⁶M dexamethasone.
• 4.4. Linoleic acid (0.5 mM) added to the culture medium as albuminic complex partly inhibited Δ9-desaturase activity.
• 5.5. Cordycepin (3' deoxyadenosine) decreased enzyme activity when present at a 3 μg/ml concentration in the culture medium.
• 6.6. Taken together, the induction of enzyme activity in culture, its impairment by cordycepin and response to insulin and linoleic acid strongly suggest that synthesis and translation of the Δ9-desaturase mRNA occur in chicken hepatocytes in primary culture, and that this cellular model may be a useful tool for further studies on Δ9-desaturase regulatory mechanisms.

     

Structure and expression of a chicken gene coding for U1 RNA

We have isolated and sequenced a genomic fragment containing sequences complementary to chicken U1 RNA. The sequence of this genomic U1 gene is completely homologous and colinear with that of chicken U1 RNA. This U1 gene is part of a multigene family (6--10 copies per haploid genome), and these loci do not appear to be closely clustered. Sequences complementary to other snRNAs are not present within the 2.5 kb genomic fragment containing the U1 gene. We have determined that U1 RNA is synthesized by polymerase II; however, a "Hogness box" is not present upstream from its cap site at the position usually observed for mRNA genes. The synthesis of U1 RNA in oviduct nuclei during different states of hormonal induction also appears to be constitutive.     

Stearoyl-CoA desaturase 2 is required for peroxisome proliferator-activated receptor gamma expression and adipogenesis in cultured 3T3-L1 cells

Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPARgamma protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPARgamma protein but decreased the metabolic labeling of PPARgamma protein using [(35)S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPARgamma protein levels and adipogenesis in 3T3-L1 cells.     

Stearoyl-CoA desaturase is involved in the control of proliferation, anchorage-independent growth, and survival in human transformed cells

Saturated and monounsaturated fatty acids are the most abundant fatty acid species in mammalian organisms, and their distribution is regulated by stearoyl-CoA desaturase, the enzyme that converts saturated into monounsaturated fatty acids. A positive correlation between high monounsaturated fatty acid levels and neoplastic transformation has been reported, but little is still known about the regulation of stearoyl-CoA desaturase in cell proliferation and apoptosis, as well as in cancer development. Here we report that simian virus 40-transformed human lung fibroblasts bearing a knockdown of human stearoyl-CoA desaturase by stable antisense cDNA transfection (hSCDas cells) showed a considerable reduction in monounsaturated fatty acids, cholesterol, and phospholipid synthesis, compared with empty vector transfected-simian virus 40 cell line (control cells). hSCDas cells also exhibited high cellular levels of saturated free fatty acids and triacylglycerol. Interestingly, stearoyl-CoA desaturase-depleted cells exhibited a dramatic decrease in proliferation rate and abolition of anchorage-independent growth. Prolonged exposure to exogenous oleic acid did not reverse either the slower proliferation or loss of anchorage-independent growth of hSCDas cells, suggesting that endogenous synthesis of monounsaturated fatty acids is essential for rapid cell replication and invasiveness, two hallmarks of neoplastic transformation. Moreover, apoptosis was increased in hSCDas cells in a ceramide-independent manner. Finally, stearoyl-CoA desaturase-deficient cells were more sensitive to palmitic acid-induced apoptosis compared with control cells. Our data suggest that, by globally regulating lipid metabolism, stearoyl-CoA desaturase activity modulates cell proliferation and survival and emphasize the important role of endogenously synthesized monounsaturated fatty acids in sustaining the neoplastic phenotype of transformed cells.     

Organization of coding and intervening sequences in the chicken ovalbumin split gene

The interruptions in the chicken ovalbumin gene which were reported previously (Breathnach, Mandel and Chambon, 1977) are shown to be due to the presence of intervening sequences which separate the messenger-coding sequences. We present evidence for an additional interruption of the gene, which, together with those reported earlier and by Garapin et al. (1978b), make a total of six intervening sequences. All of these intervening sequences are located in the DNA region that corresponds to the part of the ov mRNA which codes for amino acids. The seven coding fragments of the split ovalbumin gene are arranged in the same order and relative orientation as in the ovalbumin double-stranded cDNA. All the sequences coding for ov mRNA are contained in a chromosomal DNA region of 6000 bp, which is more than 3 times longer than ov mRNA. The general organization of the ovalbumin split gene is discussed.     

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.     

Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA

COS-1 cells bearing FcgammaRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-delta (PKCdelta) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcgammaRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcgammaRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.     

Stearoyl-CoA desaturase 1 deficiency increases CTP:choline cytidylyltransferase translocation into the membrane and enhances phosphatidylcholine synthesis in liver

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.     

Activities of enzymes of lipid metabolism in Morris hepatoma 7800 C1 cells

(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.