Control Of Lipid Synthesis Research Articles

Autocrine VEGF-B signaling maintains lipid synthesis and mitochondrial fitness to support T cell immune responses.

T cells rewire their metabolic activities to meet the demand of immune responses, but how to coordinate the immune response by metabolic regulators in activated T cells is unknown. Here, we identified autocrine VEGF-B as a metabolic regulator to control lipid synthesis and maintain the integrity of the mitochondrial inner membrane for the survival of activated T cells. Disruption of autocrine VEGF-B signaling in T cells reduced cardiolipin mass, resulting in mitochondrial damage, with increased apoptosis and reduced memory development. The addition of cardiolipin or modulating VEGF-B signaling improved T cell mitochondrial fitness and survival. Autocrine VEGF-B signaling through GA-binding protein α (GABPα) induced sentrin/SUMO-specific protease 2 (SENP2) expression, which further de-SUMOylated PPARγ and enhanced phospholipid synthesis, leading to a cardiolipin increase in activated T cells. These data suggest that autocrine VEGF-B mediates a signal to coordinate lipid synthesis and mitochondrial fitness with T cell activation for survival and immune response. Moreover, autocrine VEGF-B signaling in T cells provides a therapeutic target against infection and tumors as well as an avenue for the treatment of autoimmune diseases.

MiR-19b-3p regulated by estrogen controls lipid synthesis through targeting MSMO1 and ELOVL5 in LMH cells

miR-19b-3p is reported to undertake various biological role, while its function and action mechanism in chicken hepatic lipid metabolism is unclear. Conservation analysis and tissue expression pattern of miR-19b-3p and its target gene were evaluated, respectively. Dual luciferase reporter system and Western blot technologies were adopted to validate miR-19b-3p target gene. Overexpression and knockdown assays were done to explore the biological functions of miR-19b-3p and target gene in Leghorn Male Hepatoma cell line (LMH). Regulatory approaches of estrogen on miR-19b-3p and target gene expressions are analyzed through site-directed mutation combined with estrogen receptors antagonist treatment assays. The results showed that chicken miR-19b-3p mature sequences are highly conserved among Capra hircus, Columba livia, Rattus norvegicus, Mus musculus, Cricetulus griseus, Danio rerio, Danio novaehollandiae, Orycodylus porosus, Crocodylus porosus, Gadus morhua, and widely expressed in lung, ovary, spleen, duodenum, kidney, heart, liver, leg muscle, and pectoral muscle tissues. miR-19b-3p could significantly increase intracellular triglyceride (TG) content and decrease intracellular cholesterol (TC) content via targeting methylsterol monooxygenase 1 (MSMO1) and elongase of very long chain fatty acids 5 (ELOVL5), which are highly conserved among species, in both mRNA and protein levels. Estrogen could inhibit miR-19b-3p expression, but directly promoted MSMO1 transcription via estrogen receptor α (ERα) and indirectly regulated ELOVL5 expression at the transcription level. Meanwhile, estrogen could also upregulate MSMO1 and ELOVL5 expression through inhibiting miR-19b-3p expression at the post-transcription level. Taken together, these results highlight the role and regulatory mechanism of miR-19b-3p in hepatic lipid metabolism in chicken, and might produce useful comparative information for human obesity studies and biomedical research.

Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway

Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.

Ammonia stimulates SCAP/Insig dissociation and SREBP-1 activation to promote lipogenesis and tumour growth.

Tumorigenesis is associated with elevated glucose and glutamine consumption, but how cancer cells can sense their levels to activate lipid synthesis is unknown. Here, we reveal that ammonia, released from glutamine, promotes lipogenesis via activation of sterol regulatory element-binding proteins (SREBPs), endoplasmic reticulum-bound transcription factors that play a central role in lipid metabolism. Ammonia activates the dissociation of glucose-regulated, N-glycosylated SREBP-cleavage-activating protein (SCAP) from insulin-inducible gene protein (Insig), an endoplasmic reticulum-retention protein, leading to SREBP translocation and lipogenic gene expression. Notably, 25-hydroxycholesterol blocks ammonia to access its binding site on SCAP. Mutating aspartate D428 to alanine prevents ammonia binding to SCAP, abolishes SREBP-1 activation and suppresses tumour growth. Our study characterizes the unknown role, opposite to sterols, of ammonia as a key activator that stimulates SCAP-Insig dissociation and SREBP-1 activation to promote tumour growth and demonstrates that SCAP is a critical sensor of glutamine, glucose and sterol levels to precisely control lipid synthesis.

From the Editor’s Desk...

From the Editor’s Desk...

E2F1 promotes proliferation and metastasis of clear cell renal cell carcinoma via activation of SREBP1-dependent fatty acid biosynthesis.

Enhanced synthesis or uptake of lipids contributes to rapid cancer cell proliferation and tumor progression. In recent years, cell cycle regulators have been shown to be involved in the control of lipid synthesis, in addition to their classical function of controlling the cell cycle. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and is characterized by lipid-rich cytoplasmic deposition. However, the relationship between altered lipid metabolism and tumor progression in ccRCC is poorly understood. Here, we demonstrated that E2F transcription factor 1 (E2F1), in addition to its key role in regulating the cell cycle, induces extensive lipid accumulation and elevated levels of lipogenic enzymes in ccRCC cells by upregulating sterol regulatory element-binding protein 1 (SREBP1). E2F1 knockdown or SREBP1 suppression attenuated fatty acid (FA) de novo synthesis, cell proliferation and epithelial-mesenchymal transition (EMT) in ccRCC cells. Furthermore, overexpression of E2F1 promoted lipid storage, tumor growth and metastasis in a mouse xenograft model, whereas E2F1 downregulation or SREBP1 inhibition reversed these effects. In ccRCC patients, high levels of E2F1 and SREBP1 were associated with increased lipid accumulation and correlated with poor prognosis. Our results demonstrate that E2F1 can increase proliferation and metastasis through SREBP1-induced aberrant lipid metabolism, which is a novel critical signaling mechanism driving human ccRCC progression.

Saccharomyces cerevisiae Δ9‐desaturase Ole1 Interacts with Lipid Biosynthetic Enzymes that Produce Storage Lipid, Phospholipid, and Sterol‐esters

Lipids are essential both for integrity of membranes and energy storage in a variety of organisms, from yeast to humans. The chemical properties of membrane phospholipids and triacylglycerol are determined by the composition and organization of their acyl chain components. Acyl-CoA molecules are delivered to the enzymes involved in lipid biosynthesis in a specific order, as is evidenced by the high prevalence of unsaturated acyl chains in the second position of phosphatidic acid and triacylglycerol in Saccharomyces cerevisiae. The mechanism controlling the specific nature of these lipids is not yet known, but we have previously demonstrated that acyltransferases that produce both storage and membrane lipid interact with the Δ9-desaturase, Ole1, in a complex we have called the desaturasome. We propose that these interactions determine the incorporation of unsaturated acyl chains into phospholipid and triacylglycerol and thus control the composition of membrane and storage lipids. Yeast two-hybrid and coimmunoprecipitation studies have revealed novel protein-protein interactions between Ole1 and storage lipid, phospholipid, and sterol-ester biosynthesis enzymes. Notably, Ole1 has been found to interact with the acyl-CoA:sterol acyltransferases Are1 and Are2, phosphatidate cytidylyltransferase Cds1, phosphatidylserine synthase Cho1, phosphatidylinositol synthase Pis1, phosphatidylethanolamine methyltransferase Cho2, and the rate limiting step in sterol synthesis, HMG-CoA reductase Hmg1. This research has determined the composition of an interactome that may exist to regulate the order of acyl chain incorporation into phospholipid, triacylglycerol, and sterol-esters. Future research aims to determine exactly how these interactions control lipid synthesis and composition via metabolic flux analysis and gene deletion studies. Investigating this lipid biosynthesis complex will have implications for a wide variety of applied research, from treating lipid dysregulation in humans to biofuel production by microbes.

The Drosophila E78 nuclear receptor regulates dietary triglyceride uptake and systemic lipid levels.

Lipid levels are maintained by balancing lipid uptake, synthesis, and mobilization. Although many studies have focused on the control of lipid synthesis and mobilization, less is known about the regulation of lipid digestion and uptake. Here we show that the Drosophila E78A nuclear receptor plays a central role in intestinal lipid homeostasis through regulation of the CG17192 digestive lipase. E78A mutant adults fail to maintain proper systemic lipid levels following eclosion, with this effect largely restricted to the intestine. Transcriptional profiling by RNA-seq revealed a candidate gene for mediating this effect, encoding the predicted adult intestinal lipase CG17192. Intestine-specific disruption of CG17192 results in reduced lipid levels similar to that seen in E78A mutants. In addition, dietary supplementation with free fatty acids, or intestine-specific expression of either E78A or CG17192, is sufficient to restore lipid levels in E78A mutant adults. These studies support the model that E78A is a central regulator of adult lipid homeostasis through its effects on CG17192 expression and lipid digestion. This work also provides new insights into the control of intestinal lipid uptake and demonstrate that nuclear receptors can play an important role in these pathways.

The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

SREBPs in the control of intestinal homeostasis and tumorigenesis

Lipids are key regulators of the growth and differentiation of intestinal epithelial cells, including intestinal stem cells (ISC). High‐fat diets and obesity are serious risk factors for gastrointestinal cancers in humans. Sterol regulatory element‐binding proteins (SREBPs) are transcription factors that control lipid synthesis and uptake in the intestine. Our recent studies have shown that SREBPs and their major products, sterols (from SREBP‐2) and unsaturated fatty acids (from SREBP‐1) have a critical role in sustaining growth of the intestinal epithelia: (1) The loss of Scap, which controls intracellular cleavage events required for SREBP activation, reduces lipid synthesis and causes a lethal intestinal injury in mice characterized by a loss of intestinal crypts, which house ISCs. (2) When SREBP‐2 is inactivated in intestinal epithelium, hyperplasia of intestinal crypts and hypertrophy of the intestine results. In SREBP‐2 knockouts, sterol synthesis falls while unsaturated fatty acid synthesis does not, owing to an increase in SREBP‐1. (3) Despite this overgrowth phenotype, ablation of SREBP‐2 in intestinal epithelia of mice blocks the formation of adenomas caused by mutant APC. These findings indicate that SREBPs govern intestinal epithelial homeostasis in complex ways. We propose that SREBPs maintain homeostasis of intestinal epithelia by producing specific, yet‐to‐be identified lipid metabolites that sustain the proliferation of ISCs. These pathways control intestinal organ size and malignant potential, which bears on human diseases of under‐ or over‐proliferation of the intestine, such as short gut syndrome and colorectal cancer, the 3rd most common cause of cancer death in the U.S.Support or Funding InformationNIH‐R01DK121842, NIH‐K08DK102652

Evidence for a novel interaction between key enzymes in the lipid biosynthesis pathway in S. cerevisiae

Lipids are essential for energy storage and integrity of membranes in organisms ranging from humans to yeast. The biological properties of triglycerides and other lipids are determined by their composition and the order of assembly. Previous research has demonstrated that acyl‐CoA molecules are delivered to the enzymes involved in lipid biosynthesis in a specific order. It has been proposed that this ordered assembly is achieved by means of a membrane‐bound lipid synthesis complex in which the key enzymes interact through specific protein‐protein contacts. Preliminary data from yeast two‐hybrid and coimmunoprecipitation studies have revealed specific protein‐protein interactions between lipid synthesizing enzymes. Interactions were found between Ole1, the main desaturase in yeast, and Dga1, the terminal enzyme in the triglyceride synthesis pathway, as well as between Ole1 and Slc1, a protein that acylates lysophosphatidic acid. Future research aims to determine the other proteins in complex with Ole1 with a biotin ligase proximity assay and direct interactors via in vitro studies. This research is revealing the architecture of the enzyme complex and how it controls lipid synthesis and composition. Investigating the lipid biosynthesis complex will have implications for a wide variety of applied research, from treating lipid dysregulation in humans to biofuel production by microbes.Support or Funding InformationNSERC, BioIndustrial Innovates Canada, Alberta Innovates Biofutures, University of Alberta Faculty of Medicine and Dentistry

Potential Novel Role of UDP Glucuronosyltransferases 2B11 and 2B28 in Crosstalk Between Androgen and Lipid Signalling

Androgens play critical roles in both prostate and breast cancer progression. Recent work has revealed extensive crosstalk between androgen and lipid signalling, including the ability of the androgen receptor to control lipid synthesis and uptake to fuel prostate cancer growth [1, 2]. Members of the UDP‐glucuronosyltransferase (UGT) enzyme family can metabolically inactivate multiple lipophilic small molecules including steroids such as androgens, which modulates both prostate and breast cancer progression [3, 4]. Moreover, transcriptional induction of UGTs by steroid‐liganded nuclear receptors (e.g. AR and ER) can create a feedback loop to control steroid signalling [3].The UGT2B11 and UGT2B28 enzymes are dramatically induced by androgens in prostate and breast cancer cells (up to 200‐fold); however, unlike some other UGTs they have little or no activity towards androgens and hence their functions have been enigmatic. We now have evidence that UGT2B11 and UGT2B28 function via two novel mechanisms that together indirectly drive cancer cell growth downstream of androgen signals. First, UGT2B11 and UGT2B28 control the activity of the SREBP lipid‐sensing and signalling pathway leading to increased expression of target genes involved in lipid synthesis/metabolism. They also appear to influence feedback regulation of nuclear SREBP via proteolytic turnover. Second, UGT2B11 and UGT2B28 heterodimerize with and inhibit known androgen‐metabolizing UGT enzymes, thus preventing androgen inactivation and clearance. These findings suggest a new mechanism for crosstalk between androgen and lipid signalling pathways in breast and prostate cancer cells, and prompt further investigation of UGTs as therapeutic targets.Support or Funding InformationNational Health and Medical Research Council of Australia, Flinders Foundation

AMP-activated protein kinase controls lipid and lactose synthesis in bovine mammary epithelial cells

The synthesis of milk components in bovine mammary epithelial cells (BMEC) requires an adequate supply of energy. The AMP-activated protein kinase (AMPK) is a cellular energy gauge that controls anabolic and catabolic processes to maintain a balance between energy supply and demand. The objectives of this study were to assess the role of AMPK on de novo lipid and lactose synthesis, as well as its regulation by glucose and acetate availability in BMEC. We isolated primary BMEC from the mammary tissue of 3 lactating Holstein cows and differentiated them with lactogenic hormones for 4 d. We measured protein abundance, site-specific phosphorylation, and proteolytic processing by immunoblotting. We quantified the expression of genes involved in lipid and lactose synthesis using real-time quantitative PCR. We measured de novo lipid and lactose synthesis by incorporation of radioactive substrates. We analyzed data by ANOVA using a randomized complete block design with PROC MIXED in SAS. To assess the effect of AMPK activation on milk component synthesis, we treated BMEC with 100 μM A-769662 (A76; an allosteric activator of AMPK) or vehicle control for 16 h. Consistent with activation of AMPK, A76 increased phosphorylation of its downstream targets ACC Ser79 and TSC2 Ser1387 by 144% and 26%, respectively. Activation of AMPK decreased lipid synthesis by 19%. This effect was accompanied by increased expression of FABP3. Activation of AMPK reduced the proportion of mature SREBP-1c. In addition, AMPK activation reduced lactose synthesis by 24% and lowered the expression of SLC2A1, the gene encoding GLUT1. To assess the regulation of AMPK by energy substrate availability, we incubated BMEC in a control medium containing 4 mM D-glucose and 1 mM sodium acetate, or medium lacking glucose or acetate, for 4 h. Compared with the control medium, deprivation of glucose or acetate promoted AMPKα phosphorylation at Thr172 by 84% or 58%, respectively. Activation of AMPK was significantly increased in BMEC only when the medium was devoid of glucose for at least 4 h. We concluded that activation of AMPK inhibits de novo lipid and lactose synthesis in BMEC. Further studies are needed to assess the physiological relevance of AMPK activation for milk composition in vivo and to identify the mechanisms mediating its effects on milk component synthesis.

Epidermal mammalian target of rapamycin complex 2 controls lipid synthesis and filaggrin processing in epidermal barrier formation

Perturbation of epidermal barrier formation will profoundly compromise overall skin function, leading to a dry and scaly, ichthyosis-like skin phenotype that is the hallmark of a broad range of skin diseases, including ichthyosis, atopic dermatitis, and a multitude of clinical eczema variants. An overarching molecular mechanism that orchestrates the multitude of factors controlling epidermal barrier formation and homeostasis remains to be elucidated. Here we highlight a specific role of mammalian target of rapamycin complex 2 (mTORC2) signaling in epidermal barrier formation. Epidermal mTORC2 signaling was specifically disrupted by deleting rapamycin-insensitive companion of target of rapamycin (Rictor), encoding an essential subunit of mTORC2 in mouse epidermis (epidermis-specific homozygous Rictor deletion [RicEKO] mice). Epidermal structure and barrier function were investigated through a combination of gene expression, biochemical, morphological and functional analysis in RicEKO and control mice. RicEKO newborns displayed an ichthyosis-like phenotype characterized by dysregulated epidermal de novo lipid synthesis, altered lipid lamellae structure, and aberrant filaggrin (FLG) processing. Despite a compensatory transcriptional epidermal repair response, the protective epidermal function was impaired in RicEKO mice, as revealed by increased transepidermal water loss, enhanced corneocyte fragility, decreased dendritic epidermal T cells, and an exaggerated percutaneous immune response. Restoration of Akt-Ser473 phosphorylation in mTORC2-deficient keratinocytes through expression of constitutive Akt rescued FLG processing. Our findings reveal a critical metabolic signaling relay of barrier formation in which epidermal mTORC2 activity controls FLG processing and de novo epidermal lipid synthesis during cornification. Our findings provide novel mechanistic insights into epidermal barrier formation and could open up new therapeutic opportunities to restore defective epidermal barrier conditions.

Fra-1 and c-Fos N-Terminal Deletion Mutants Impair Breast Tumor Cell Proliferation by Blocking Lipid Synthesis Activation.

Tumor cells require high rates of lipid synthesis to support membrane biogenesis for their exacerbated growth. The only two proteins known that activate phospholipid synthesis are Fra-1 and c-Fos, two members of the AP-1 family of transcription factors. These proteins that are overexpressed in human breast malignant tumors increase the rate of phospholipid synthesis at the endoplasmic reticulum through a mechanism independent of their nuclear function. The aim of this study was to inhibit breast tumor cell proliferation by modulating c-Fos and Fra-1 and regulate membrane biogenesis by controlling lipid synthesis rates. The molecular mechanism by which Fra-1 and c-Fos activate phospholipid synthesis was examined. Both proteins physically associate with the rate limiting enzyme CDP-DAG synthase through their N-terminus domain and activate it through their basic domain; neither protein associates to or activates the enzyme phosphatidylinositol synthase as determined through in vitro enzymatic reactions and FRET experiments. The N-terminus domain of both proteins act as negative dominant peptides that physically associate with CDP-DAG synthase but do not activate it. Proliferation of MDA-MB231 and 4T1 cells was impaired in vitro after inducing them to proliferate in the presence of the negative dominant peptides derived from Fra-1 and c-Fos. When tumors generated in Balb/c mice with the breast tumor cell line 4T1 were treated with these negative dominant peptides, a significant reduction in tumor growth was observed. Consequently, these Fra-1 and c-Fos negative dominant peptides can be exploited as a new therapeutic strategy to impair breast tumor cell proliferation.

MTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells.

The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.

Activation of liver X receptor promotes fatty acid synthesis in goat mammary epithelial cells via modulation of SREBP1 expression

In bovine mammary tissue and cells, liver X receptor (LXR) regulates lipid synthesis mainly via transactivation of the transcription factor sterol regulatory element binding protein 1 (SREBP1). In the present work, we investigated the role of LXR in controlling lipid synthesis via transactivation of SREBP1 in goat primary mammary cells (GMEC). The GMEC were treated with a synthetic agonist of LXR, T0901317, and transactivation and transcription of SREBP1, expression of lipogenic genes, and fatty acid profiling and triacylglycerol (TAG) content of the cells were measured. A mild increase in the mRNA expression level of LXRα (NR1H3) was observed following treatment with different concentrations of T0901317, and a dose-dependent increase in mRNA and transactivation of SREBP1 was detected. Activation of LXR resulted in a significant increase in the mRNA expression of most of the measured genes related to de novo synthesis, desaturation, and transport of fatty acids; TAG synthesis; and transcription regulators. Compared with the control, total content of cellular TAG increased by more than 20% with T0901317 treatment. Furthermore, addition of T0901317 increased the proportion of unsaturated fatty acids (e.g., C16:1, C18:1, C20:1, and C22:1), and decreased the proportion of saturated fatty acids (e.g., C16:0, C18:0, C20:0, and C22:0). These results provide evidence that LXR regulates the expression and activity of SREBP1. Our results indicated that LXR participate in regulating the transcription of genes involved in milk fat synthesis in GMEC in an SREBP1-dependent fashion.

Transcriptional profiling of swine mammary gland during the transition from colostrogenesis to lactogenesis using RNA sequencing

BackgroundColostrum and milk are essential sources of antibodies and nutrients for the neonate, playing a key role in their survival and growth. Slight abnormalities in the timing of colostrogenesis/lactogenesis potentially threaten piglet survival. To further delineate the genes and transcription regulators implicated in the control of the transition from colostrogenesis to lactogenesis, we applied RNA-seq analysis of swine mammary gland tissue from late-gestation to farrowing. Three 2nd parity sows were used for mammary tissue biopsies on days 14, 10, 6 and 2 before (−) parturition and on day 1 after (+) parturition. A total of 15 mRNA libraries were sequenced on a HiSeq2500 (Illumina Inc.). The Dynamic Impact Approach and the Ingenuity Pathway Analysis were used for pathway analysis and gene network analysis, respectively.ResultsA large number of differentially expressed genes were detected very close to parturition (−2d) and at farrowing (+ 1d). The results reflect the extraordinary metabolic changes in the swine mammary gland once it enters into the crucial phases of lactogenesis and underscore a strong transcriptional component in the control of colostrogenesis. There was marked upregulation of genes involved in synthesis of colostrum and main milk components (i.e. proteins, fat, lactose and antimicrobial factors) with a pivotal role of CSN1S2, LALBA, WAP, SAA2, and BTN1A1. The sustained activation of transcription regulators such as SREBP1 and XBP1 suggested they help coordinate these adaptations.ConclusionsOverall, the precise timing for the transition from colostrogenesis to lactogenesis in swine mammary gland remains uncharacterized. However, our transcriptomic data support the hypothesis that the transition occurs before parturition. This is likely attributable to upregulation of a wide array of genes including those involved in ‘Protein and Carbohydrate Metabolism’, ‘Immune System’, ‘Lipid Metabolism’, ‘PPAR signaling pathway’ and ‘Prolactin signaling pathway’ along with the activation of transcription regulators controlling lipid synthesis and endoplasmic reticulum biogenesis and stress response.

Srebp-controlled glucose metabolism is essential for NK cell functional responses.

Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.

NPM-ALK Mediated Tyrosine Phosphorylation of ATP Citrate Lyase Regulates Lipid Metabolism and Promotes Oncogenesis of Anaplastic Large Cell Lymphoma

Introduction: Anaplastic large cell lymphoma (ALCL) is characterized by recurrent translocations involving anaplastic lymphoma kinase (ALK) and resulting in oncogenic chimeric fusion tyrosine kinases such as NPM-ALK. We integrated mass-spectrometry-driven phosphoproteomics and metabolomics of ALK+ ALCL and discovered that ATP citrate lyase (ACLY) is a candidate substrate of NPM-ALK at residue Y682. ACLY is a critical enzyme responsible for synthesis of acetyl-CoA and connects vital biosynthetic pathways such as carbohydrate and lipid metabolism. Functional regulation of ACLY by tyrosine phosphorylation is unknown and the significance of tyrosine phosphorylation of ACLY on cancer cell growth and metabolism have never been described.Methods: To identify novel substrates of NPM-ALK, we performed phospho-proteomic analyses of 3 ALK+ALCL derived cell lines. The MS-phosphoproteomic data were subjected to custom bioinformatic algorithms to identify highly statistically correlated phosphotyrosine proteins and phosphosites regulated by ALK kinase activity. We generated a phosphospecific antibody against a highly-ranked candidate (pACLY Y682) and utilized it in western blot analyses on ALK+ and ALK- ALCL lymphoma cell lines. Immunohistochemistry (IHC) on well-characterized primary human ALK+ (n=20) and ALK-ALCL biopsies (n=28) were performed. Co-immunoprecipitations (Co-IP) with HA-tagged ACLY-WT and ACLY-Y682F and ALK antibodies were performed and analyzed by western blot using the pACLY Y682 antibody. In vitro kinase assays were performed to investigate the direct role of NPM-ALK in phosphorylation of ACLY at Y682 using GST-tagged ACLY-WT or ACLY-Y682F peptides in the presence or absence of immunocomplexes containing active NPM-ALK or the kinase-defective K210R mutant. To study the impact of Y682F mutant on lipid metabolism, we generated ALCL-derived cell lines stably transduced to express GFP-tagged ACLY-WT or ACLY-Y682F and subjected them to metabolomic analysis for flux, β-oxidation or lipid synthesis using liquid chromatography/tandem mass spectrometry or gas chromatography platforms. The Seahorse XF24 Flux analyzer was used to measure oxygen consumption rates (OCR) in DEL stably expressing ACLY-WT and ACLY-Y682F. Cells stably expressing ACLY-WT and ACLY-Y682F were evaluated for cell proliferation, colony formation and tumor formation in SCID-BEIGE mouse xenograft models.Results: Phosphoproteomic analyses yielded a dataset containing a total of 626 tyrosine phosphoproteins. Bioinformatic analysis identified pACLY Y682 as a novel candidate substrate of NPM-ALK. Western blot analysis corroborated phosphoproteomic studies and demonstrated that phosphorylation of ALCY at Y682 is regulated by NPM-ALK activity. Expression of active NPM-ALK increased the phosphorylation of ACLY at Y682 in HEK293T cells compared to the K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates ACLY at Y682. Immunoprecipitation (IP) of ALK and co-IP (HA) demonstrated interaction of NPM-ALK and ACLY in co-transfected HEK293T cell lysates. Stable expression of ACLY-Y682F or pharmacologic antagonism of ALK with crizotinib resulted in decreased ACLY activity, decreased lipid synthesis and increased fatty acid β-oxidation when compared to ACLY-WT. β-oxidation of 13 C-oleic acid-labeled fatty acid demonstrated increased labeling of +2-citrate (p<0.01) and +18-Oleyol carnitine (p<0.001) in ACLY-Y682F cells when compared to ACLY-WT. Similarly, the OCR was significantly increased in cells expressing ACLY-Y682F (p<0.001). IHC studies of ALCL biopsies (ALK+ and ALK-) demonstrated significant correlation between NPM-ALK and ACLY pY682- expression (p<0.01). Expression of ACLY-Y682F in the DEL cell line resulted in dramatically decreased cell proliferation, impaired clonogenic potential as observed by small colony size in colony formation assay and abrogated tumor growth in in vivo xenograft model when compared to ACLY-WT.Conclusion: We identify for the first time a direct tyrosine kinase-mediated mechanism for ACLY regulation. In this regard, our studies demonstrate a novel oncogenic mechanism whereby NPM-ALK controls lipid synthesis and b-oxidation by phosphorylation of ACLY at Y682 and this mechanism contributes to ALCL pathogenesis. Our findings have significant implications for novel therapies targeting tumor metabolism in ALCLs. DisclosuresNo relevant conflicts of interest to declare.