Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.     

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.     

β2-Adrenergic receptor (β2-AR) agonist formoterol suppresses differentiation of L6 myogenic cells by blocking PI3K–AKT pathway

β₂-Adrenergic receptor (β₂-AR) is implicated in muscle metabolic activities such as glycogen metabolism, glucose uptake, lipolysis and muscle growth. However, the functional role of β₂-AR in the differentiation of skeletal muscle is largely unknown. Here, we examined the functional role of β₂-AR in L6 myoblast differentiation using the long-term-acting β₂-AR-specific agonist formoterol. We observed that formoterol treatment strongly suppressed L6 myoblast differentiation and the expression of myosin heavy chain (MHC) in a dose- and time-dependent manner. Showing that both long-acting agonist (formoterol) and short-acting agonist (terbutaline) inhibited the induction of MHC protein, whereas β₂-AR antagonist (ICI-118,551) upregulated MHC expression, we clearly demonstrated that β₂-AR is involved in L6 myoblast differentiation. Furthermore, our pharmacological inhibition study revealed that the PI3K–AKT pathway is the main signaling pathway for myotube formation. Formoterol inhibited the activation of PI3K–AKT signaling, but not that of ERK signaling. Moreover, formoterol selectively inhibited AKT activation by IGF-I, but not by insulin. Collectively, our findings reveal a previously undocumented role of β₂-AR activation in modulating the differentiation of L6 myoblasts.     

Per1/Per2–Igf2 axis–mediated circadian regulation of myogenic differentiation

Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter–enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.     

Increased long noncoding RNA LASP1-AS is critical for hepatocellular carcinoma tumorigenesis via upregulating LASP1

Aberrant long noncoding RNAs (lncRNA) have been proved to be associated with the many types of malignant tumors (including hepatocellular carcinoma [HCC]). In this study, a lncRNAs and mRNAs microarray analysis was performed in three pairs of HCC patitents’ tumor. We found lncRNA LIM and SH3 protein 1 antisense (LASP1-AS) and its sense-cognate gene LIM and SH3 protein 1 (LASP1) were upregulated in HCC and both are correlated with poorer prognosis and lower survival of HCC patients. Meanwhile, the expression of LASP1-AS correlated positively with LASP1 expression in HCC tissues. LASP1-AS promoted the proliferation, migration, and invasion abilities of HCC in vitro and vivo by enhancing LASP1 expression. Our study explored lncRNA LASP1-AS as an oncogene in HCC and promoted proliferation and metastasis capabilities of HCC via increasing the expression of its sense-cognate gene LASP1. LncRNA LASP1-AS might be a potential valuable prognostic biomarker and potential therapeutic target of HCC.     

Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum

The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage.     

CEP2 Attenuates Myoblast Differentiation But Does Not Affect Proliferation

CEP2 (CDC42EP2) is a member of the CDC42 subfamily that belongs to the Rho family. The Rho family plays an important role in a variety of cellular processes including skeletal myogenesis. Here, we find the expression of CEP2 increased significantly during C2C12 myogenesis. Overexpression of CEP2 could attenuate myoblast differentiation, while knockdown of CEP2 by siRNA results in enhancing myogenesis. Furthermore, we demonstrate for the first time that CEP2 attenuates myoblast differentiation via suppression of muscle regulatory factors (MRFs) rather than influencing myoblast proliferation. These results indicate that CEP2 acts as a repressor during myogenesis, which provides new insights into the role of CEP2 in muscle development.     

Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides

The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo.     

A lncRNA promotes myoblast proliferation by up-regulating GH1

Long noncoding RNAs (lncRNAs) are key regulatory factors for gene expression in a variety of biological processes; however, the role of lncRNAs in muscle formation and development is poorly understood, particularly in cattle. Here, we identified a highly expressed lncRNA in muscle, lncYYW, by high-throughput sequencing in bovine longissimus, scapular, intercostal, and gluteus muscles. The expression of lncYYW increased gradually during myoblast differentiation. Overexpression of lncYYW increased the number of cells in the DNA synthesis (S) stage of the cell cycle and upregulated the expression of two well-established myogenic markers, myogenin and myosin heavy chain. A microarray analysis showed that lncYYW positively regulates the expression of growth hormone 1 and its downstream genes, AKT1 and PIK3CD, in bovine myoblasts. This discovery provides a good foundation for further study of the mechanism of action of lncYYW during bovine myoblast development. Taken together, our results reveal a novel lncRNA associated with bovine myoblast proliferation and differentiation. This lncRNA will play a crucial and critical role in future studies of bovine muscle development.     

Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price of limited formation of acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA is a key metabolic precursor for a number of added-value products, in this work we deployed an in silico-guided rewiring program of central carbon metabolism for upgrading P. putida as a host for acetyl-CoA–dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets in addition to manually-curated information of enzyme mechanisms, identified targets that would lead to increased acetyl-CoA levels. Based on these predictions, a set of plasmids based on clustered regularly interspaced short palindromic repeats (CRISPR) and dead CRISPR-associated protein 9 (dCas9) was constructed to silence genes by CRISPR interference (CRISPRi). Dynamic reduction of gene expression of two key targets (gltA, encoding citrate synthase, and the essential accA gene, encoding subunit A of the acetyl-CoA carboxylase complex) mediated an 8-fold increase in the acetyl-CoA content of rewired P. putida. Poly(3-hydroxybutyrate) (PHB) was adopted as a proxy of acetyl-CoA availability, and two synthetic pathways were engineered for biopolymer accumulation. By including cell morphology as an extra target for the CRISPRi approach, fully rewired P. putida strains programmed for PHB accumulation had a 5-fold increase in PHB titers in bioreactor cultures using glucose. Thus, the strategy described herein allowed for rationally redirecting metabolic fluxes in P. putida from central metabolism towards product biosynthesis—especially relevant when deletion of essential pathways is not an option.     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

Effects of the somatomedins and insulin on myoblast differentiation in vitro

The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffe's L6 line. Incubation for 48 hr with either insulin or Temin's multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10^?7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.     

Feeding a High Concentrate Diet Down-Regulates Expression of ACACA,LPL and SCD and Modifies Milk Composition in Lactating Goats

High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase α (ACACA, ACCα) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.