DDX39B facilitates the malignant progression of hepatocellular carcinoma via activation of SREBP1-mediated de novo lipid synthesis.

PURPOSE: The detailed molecular mechanisms of aberrant lipid metabolism in HCC remain unclear. Herein, we focused on the potential role of DDX39B in aberrant lipogenesis and malignant development in HCC. METHODS: DDX39B expression in HCC and para-cancer tissues was measured by immunohistochemistry. CCK-8, colony formation and Transwell assays were utilized to detect HCC cell proliferation, migration and invasion in vitro. Oil red O and Nile red staining and triglyceride and cholesterol detection were used to measure lipogenesis. Coimmunoprecipitation was used to detect interactions between DDX39B and SREBP1. Immunofluorescence assays were performed to investigate the impact of DDX39B on SREBP1 nuclear translocation. A luciferase assay was used to explore the transcriptional activity of SREBP1. The subcutaneous and orthotopic xenograft models in nude mice were generated to verify the contribution of the DDX39B/SREBP1 axis to tumor growth, lung metastasis and lipid synthesis in vivo. RESULTS: DDX39B is upregulated in HCC tissues and predicts a worse prognosis. Upregulated DDX39B contributes to the proliferation, metastasis and lipogenesis of HCC cells. Mechanistically, DDX39B directly interacts with SREBP1, and silencing DDX39B impairs the stabilization of the SREBP1 protein through FBXW7-mediated ubiquitination and degradation of SREBP1. Furthermore, DDX39B deficiency decreases the nuclear translocation and activation of SREBP1 and transcription of SREBP1 downstream genes, resulting in reduced lipid accumulation. CONCLUSIONS: Our study reveals a novel mechanism by which DDX39B facilitates the malignant progression of HCC via activation of SREBP1-mediated de novo lipogenesis, implicating DDX39B as both a potential predictor of recurrence and prognosis and a promising therapeutic target.

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

circCAPRIN1 interacts with STAT2 to promote tumor progression and lipid synthesis via upregulating ACC1 expression in colorectal cancer.

BACKGROUND: Circular RNAs (circRNAs) generated by back-splicing of precursor mRNAs (pre-mRNAs) are often aberrantly expressed in cancer cells. Accumulating evidence has revealed that circRNAs play a critical role in the progression of several cancers, including colorectal cancer (CRC). However, the current understandings of the emerging functions of circRNAs in CRC lipid metabolism and the underlying molecular mechanisms are still limited. Here, we aimed to explore the role of circCAPRIN1 in regulating CRC lipid metabolism and tumorigenesis. METHODS: circRNA microarray was performed with three pairs of tumor and non-tumor tissues from CRC patients. The expression of circRNAs were determined by quantitative PCR (qPCR) and in situ hybridization (ISH). The endogenous levels of circRNAs in CRC cells were manipulated by transfection with lentiviruses overexpressing or silencing circRNAs. The regulatory roles of circRNAs in the occurrence of CRC were investigated both in vitro and in vivo using gene expression array, RNA pull-down/mass spectrometry, RNA immunoprecipitation assay, luciferase reporter assay, chromatin immunoprecipitation analysis, and fluorescence in situ hybridization (FISH). RESULTS: Among circRNAs, circCAPRIN1 was most significantly upregulated in CRC tissue specimens. circCAPRIN1 expression was positively correlated with the clinical stage and unfavorable prognosis of CRC patients. Downregulation of circCAPRIN1 suppressed proliferation, migration, and epithelial-mesenchymal transition of CRC cells, while circCAPRIN1 overexpression had opposite effects. RNA sequencing and gene ontology analysis indicated that circCAPRIN1 upregulated the expressions of genes involved in CRC lipid metabolism. Moreover, circCAPRIN1 promoted lipid synthesis by enhancing Acetyl-CoA carboxylase 1 (ACC1) expression. Further mechanistic assays demonstrated that circCAPRIN1 directly bound signal transducer and activator of transcription 2 (STAT2) to activate ACC1 transcription, thus regulating lipid metabolism and facilitating CRC tumorigenesis. CONCLUSIONS: These findings revealed the oncogenic role and mechanism of circCAPRIN1 in CRC. circCAPRIN1 interacted with STAT2 to promote CRC tumor progression and lipid synthesis by enhancing the expression of ACC1. circCAPRIN1 may be considered as a novel potential diagnostic and therapeutic target for CRC patients.

Lysosomal lipid switch sensitises to nutrient deprivation and mTOR targeting in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. DESIGN: Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. RESULTS: PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. CONCLUSION: Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss.

Activation of ADRB2/PKA Signaling Pathway Facilitates Lipid Synthesis in Meibocytes, and Beta-Blocker Glaucoma Drug Impedes PKA-Induced Lipid Synthesis by Inhibiting ADRB2.

Meibomian gland dysfunction is one of the main causes of dry eye disease and has limited therapeutic options. In this study, we investigated the biological function of the beta 2-adrenergic receptor (ADRB2)/protein kinase A (PKA) pathway in lipid synthesis and its underlying mechanisms in human meibomian gland epithelial cells (HMGECs). HMGECs were cultured in differentiation media with or without forskolin (an activator of adenylate cyclase), salbutamol (an ADRB2 agonist), or timolol (an ADRB2 antagonist) for up to 4 days. The phosphorylation of the cAMP-response element-binding protein (CREB) and the expression of peroxisome proliferator activator receptor (PPAR)γ and sterol regulatory element-binding protein (SREBP)-1 were measured by immunoblotting and quantitative PCR. Lipid synthesis was examined by LipidTOX immunostaining, AdipoRed assay, and Oil Red O staining. PKA pathway activation enhanced PPARγ expression and lipid synthesis in differentiated HMGECs. When treated with agonists of ADBR2 (upstream of the PKA signaling system), PPARγ expression and lipid synthesis were enhanced in HMGECs. The ADRB2 antagonist timolol showed the opposite effect. The activation of the ADRB2/PKA signaling pathway enhances lipid synthesis in HMGECs. These results provide a potential mechanism and therapeutic target for meibomian gland dysfunction, particularly in cases induced by beta-blocker glaucoma drugs.

Metabolomic Evaluation of N-Acetyl-p-Benzoquinone Imine Protein Adduct Formation with Therapeutic Acetaminophen Administration: Sex-based Physiologic Differences.

BACKGROUND: Acetaminophen (APAP)-associated transaminase elevation, induced by N-acetyl-p-benzoquinone imine (NAPQI) protein adduction, remains an area of research interest. Distinct from known genetic, physiologic, and dosage associations dictating severity of hepatic injury, no known factors predict an absence of protein adduct formation at therapeutic APAP dosing. HYPOTHESIS: Sex-based physiology is predictive of APAP-induced protein adduct formation and differential metabolite expression at therapeutic doses. METHODS: This retrospective study interrogated serum samples collected for a prior study investigating fluctuations of alanine aminotransferase (ALT) over time with 4G daily APAP dosing for ≥ 16 days in subjects from Denver, Colorado. Subjects were grouped by adduct formation (n = 184) vs no adducts (n = 20). Samples were run on ultra-high-performance liquid chromatography mass spectrometry from study days 0, 7, 16, and 31. Significant metabolite expressions were identified using t-tests with false discovery rate correction (FDR), partial least squares discriminant, and ANOVA simultaneous comparison analyses. Demographic and clinical data were explored using t-tests with FDR (age, weight, BMI, ALT) and Chi-square (sex, ethnicity, race) analyses. RESULTS: In pre-treatment samples, relative quantitation caprylic acid was expressed ninefold higher and 6-carboxyhexanoate was expressed threefold lower in subjects who did not develop adducts. Lactate had greater expression in the no adducts group (p = 0.001). Using absolute quantitation, glutathione was expressed 2.6-fold greater among no adduct subjects. Odds of males developing NAPQI protein adducts at therapeutic APAP dosing were 5.91 times lower than females (95% CI = 2.3-14.9; p = 0.0001). CONCLUSION: Multiple metabolites were differentially expressed based on adduct group and sex. Metabolites were identified unique to adduct development independent of sex. At therapeutic APAP dosing, males were less likely to develop APAP protein adducts. Further research into lipid biosynthesis and metabolism may provide further insight into physiology associated with adduct production.

MicroRNA-206 inhibits HCV proliferation through depressing ACC1 lipid synthesis signalling pathway.

MicroRNAs are considered to play important roles in cell biological and pathological progress. microRNA-206 (miR-206) was reported to participate in lipogenesis, and lipid droplets were necessary for the life cycle of HCV proliferation. Whether miR-206 was associated with HCV proliferation and the potential mechanism are not clear. In this study, we firstly identified that miR-206 could inhibit HCV proliferation at the RNA and protein level. Bioinformatical prediction of target genes binding to miR-206 was performed to investigate whether inhibiting function was due to a lipogenesis pathway. Then, the acetyl-CoA carboxylase 1 (ACC1) gene was selected as target gene of miR-206. The dual-luciferase reporter assay results showed that luciferase significantly decreased in cells transfected with 3'-UTR of the ACC1 gene and miR-206. The RNA and protein levels of the ACC1 gene and its pathway genes were significantly lower in cells transfected with miR-206 than in controls. Furthermore, the lipid droplet numbers also significantly decreased in cells transfected with miR-206. In conclusion, miR-206 could inhibit HCV proliferation through depressing ACC1 lipogenesis pathway and decreasing the lipid droplet numbers. miR-206 might be used as anti-HCV biochemical drug in the future.

Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Protein C receptor maintains cancer stem cell properties via activating lipid synthesis in nasopharyngeal carcinoma.

Metastasis and recurrence account for 95% of deaths from nasopharyngeal carcinoma (NPC). Cancer stem cells (CSCs) are regarded as one of the main reasons for tumor cell resistance to clinical therapy, and cancer metastasis or recurrence, while little is known about CSCs in NPC. The present study uncovers a subpopulation of cells labeled as CD45-EPCAM+PROCR+ in NPC biopsy samples that exhibit stem cell-like characteristics. A relatively low number of these cells initiate xenograft tumors in mice. Functional analysis reveals that protein C receptor (PROCR) not only serves as a stem cell marker in NPC, but also maintains tumor cells' stemness potential through regulating lipid metabolism and mitochondrial fission. Epistatic studies reveal that cAMP-protein kinase A stimulates Ca2+ release to manipulate lipid metabolism related genes' expression. Finally, in a cohort of 207 NPC samples, PROCR expression is correlated with tumor metastasis or recurrence, and predicts poor prognosis. These novel findings link PROCR labeled CSCs with lipid metabolism and mitochondrial plasticity, and provides new clinical target against metastatic or recurrent NPC.

Citrate Promotes Excessive Lipid Biosynthesis and Senescence in Tumor Cells for Tumor Therapy.

Metabolic disorder is one of the hallmarks of cancers, and reprogramming of metabolism is becoming a novel strategy for cancer treatment. Citrate is a key metabolite and critical metabolic regulator linking glycolysis and lipid metabolism in cellular energy homeostasis. Here it is reported that citrate treatment (both sodium citrate and citric acid) significantly suppresses tumor cell proliferation and growth in various tumor types. Mechanistically, citrate promotes excessive lipid biosynthesis and induces disruption of lipid metabolism in tumor cells, resulting in tumor cell senescence and growth inhibition. Furthermore, ATM-associated DNA damage response cooperates with MAPK and mTOR signaling pathways to control citrate-induced tumor cell growth arrest and senescence. In vivo studies further demonstrate that citrate administration dramatically inhibits tumor growth and progression in a colon cancer xenograft model. Importantly, citrate administration combined with the conventional chemotherapy drugs exhibits synergistic antitumor effects in vivo in the colon cancer models. These results clearly indicate that citrate can reprogram lipid metabolism and cell fate in cancer cells, and targeting citrate can be a promising therapeutic strategy for tumor treatment.

Heterogeneous fitness landscape cues, pknG low expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth.

Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB-S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRplus 2.0-genotyped isolates (with nontyped, S441, H445 and S450 positions), only one S450L isolate (R9, rpoB-S450L ATCC 25618, RR 1 µg/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB-S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt-rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

MicroRNA-432 inhibits milk fat synthesis by targeting SCD and LPL in ovine mammary epithelial cells.

The microRNA (miR)-432 is differentially expressed in the mammary gland of two breeds of lactating sheep with different milk production traits, and between the non-lactating and peak-lactation periods, but there have been no reports describing the molecular mechanisms involved. In this study, the effect of miR-432 on the proliferation of ovine mammary epithelial cells (OMECs) and the target genes of miR-432 were investigated. The effects of miR-432 on the expression of the target genes and the content of triglycerides in the OMECs were also analyzed. Transfection with a miR-432 mimic was found using CCK8 and Edu assays, to inhibit the viability of OMECs and reduce the number of proliferated OMECs. In contrast, a miR-432 inhibitor had the opposite effect to the miR-432 mimic, and together these results suggest that miR-432 inhibits the proliferation of OMECs. A dual luciferase assay revealed that the genes for stearoyl-CoA desaturase (SCD) and lipoprotein lipase (LPL) are targeted by miR-432. The transfection of miR-432 mimic into OMECs resulted in decreases in the expression of SCD and LPL, and three other milk fat synthesis marker genes; FABP4, LPIN1 and ACACA. The mimic also decreased the content of triglycerides. The miR-432 inhibitor had the opposite effect to the mimic on the expression of these genes and the level of triglycerides. This is the first study to reveal the biological mechanisms by which miR-432 inhibits milk fat synthesis in sheep.

High-Throughput Single-Cell Mass Spectrometry Reveals Abnormal Lipid Metabolism in Pancreatic Ductal Adenocarcinoma.

Even populations of clonal cells are heterogeneous, which requires high-throughput analysis methods with single-cell sensitivity. Here, we propose a rapid, label-free single-cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK-293T, PANC-1, CFPAC-1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

Glucose-sensitive acetylation of Seryl tRNA synthetase regulates lipid synthesis in breast cancer.

Abnormally enhanced de novo lipid biosynthesis has been increasingly realized to play crucial roles in the initiation and progression of varieties of cancers including breast cancer. However, the mechanisms underlying the dysregulation of lipid biosynthesis in breast cancer remain largely unknown. Here, we reported that seryl tRNA synthetase (SerRS), a key enzyme for protein biosynthesis, could translocate into the nucleus in a glucose-dependent manner to suppress key genes involved in the de novo lipid biosynthesis. In normal mammary gland epithelial cells glucose can promote the nuclear translocation of SerRS by increasing the acetylation of SerRS at lysine 323. In SerRS knock-in mice bearing acetylation-defective lysine to arginine mutation, we observed increased body weight and adipose tissue mass. In breast cancer cells the acetylation and nuclear translocation of SerRS are greatly inhibited. Overexpression of SerRS, in particularly the acetylation-mimetic lysine to glutamine mutant, dramatically inhibits the de novo lipid synthesis and hence greatly suppresses the proliferation of breast cancer cells and the growth of breast cancer xenografts in mice. We further identified that HDAC4 and HDAC5 regulated the acetylation and nuclear translocation of SerRS. Thus, we identified a SerRS-meditated inhibitory pathway in glucose-induced lipid biosynthesis, which is dysregulated in breast cancer.

LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance.

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

Effect of antioxidant treatment with n-acetylcysteine and swimming on lipid expression of sebaceous glands in diabetic mice.

The sebaceous gland (SG) is involved in different inflammatory, infectious and neoplastic processes of the skin and can be related to specific diseases, e.g., diabetes mellitus. Sometimes, the histological diagnosis requires complementary tests due to the ability of diseases to mimic other tumors. We evaluated the sebaceous gland density in Non-obese diabetic mice to analyze the N-acetylcystein effects and swimming exercise treatment in sebaceous glands healing, using specific staining in histochemistry and immunohistochemistry reactions in the identification of the lipid expression in the sebaceous gland. We investigated the intracytoplasmic lipid expression and analysis of gland density from SG in dorsal skin samples from the Non-obese diabetic (NOD mice) and diabetic animals submitted to antioxidant treatment and physical exercise. For histological analysis of the sebaceous glands, specific staining in histochemistry with sudan black and immunohistochemistry reaction with adipophilin were used in the evaluation. Statistical analysis showed significant proximity between the values of the control group and the diabetic group submitted to the swimming exercise (DS group) and similar values between the untreated diabetic group (UD group) and diabetic group treated with the antioxidant N-acetylcysteine (DNa group), which did not prevent possible differences where p < 0.01. Adipophilin (ADPH) immunohistochemistry permitted more intense lipid staining in SGs, the preservation of the SG in the control group, and a morphological deformed appearance in the UD and DNa groups. However, weak morphological recovery of the SG was observed in the DS-Na group, being more expressive in the DS group. In conclusion, the groups submitted to physical exercises showed better results in the recovery of the analyzed tissue, even being in the physiological conditions caused by spontaneous diabetes.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1-10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.

EEF1D facilitates milk lipid synthesis by regulation of PI3K-Akt signaling in mammals.

Mammal's milk is an abundantly foremost source of proteins, lipids, and micronutrients for human nutrition and health. Understanding the molecular mechanisms underlying synthesis of milk components provides practical benefits to improve the milk quality via systematic breeding program in mammals. Through RNAi with EEF1D in primary bovine mammary epithelial cells, we phenotypically observed aberrant formation of cytoplasmic lipid droplets and significantly decreased milk triglyceride level by 37.7%, and exploited the mechanisms by which EEF1D regulated milk lipid synthesis via insulin (PI3K-Akt), AMPK, and PPAR pathways. In the EEF1D CRISPR/Cas9 knockout mice, incompletely developed mammary glands at 9th day postpartum with small or unformed lumens, and significantly decreased triglyceride concentration in milk by 23.4% were observed, as well as the same gene expression alterations in the three pathways. For dairy cattle, we identified a critical regulatory mutation modifying EEF1D transcription activity, which interpreted 7% of the genetic variances of milk lipid yield and percentage. Our findings highlight the significance of EEF1D in mammary gland development and milk lipid synthesis in mammals.

BdFAR4, a root-specific fatty acyl-coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon.

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.