SREBP-1a–stimulated lipid synthesis is required for macrophage phagocytosis downstream of TLR4-directed mTORC1

Lee, Jae-Ho,Phelan, Peter,Shin, Minsang,Oh, Byung-Chul,Han, Xianlin,Im, Seung-Soon,Osborne, Timothy F. National Academy of Sciences 2018 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.115 No.52

<▼1><P><B>Significance</B></P><P>There is a growing appreciation for a fundamental connection between lipid metabolism and the innate immune response. Phagocytosis is a key macrophage innate immune response to pathogen exposure, and cytoplasmic membrane expansion is required to surround and capture the target pathogen prior to internalization. Sterol regulatory element binding proteins (SREBPs) are gene regulatory factors that sense the intracellular lipid environment and modulate key genes that drive fatty acid and cholesterol synthesis to maintain lipid homeostasis. In this study, we show that, in mutant cells that lack a key SREBP isoform, phagocytosis is impaired, and we track the defect to altered lipid composition of membrane phospholipids that results in decreased interaction between membrane lipid rafts and the actin cytoskeletal network.</P></▼1><▼2><P>There is a growing appreciation for a fundamental connection between lipid metabolism and the immune response. Macrophage phagocytosis is a signature innate immune response to pathogen exposure, and cytoplasmic membrane expansion is required to engulf the phagocytic target. The sterol regulatory element binding proteins (SREBPs) are key transcriptional regulatory proteins that sense the intracellular lipid environment and modulate expression of key genes of fatty acid and cholesterol metabolism to maintain lipid homeostasis. In this study, we show that TLR4-dependent stimulation of macrophage phagocytosis requires mTORC1-directed SREBP-1a−dependent lipid synthesis. We also show that the phagocytic defect in macrophages from SREBP-1a−deficient mice results from decreased interaction between membrane lipid rafts and the actin cytoskeleton, presumably due to reduced accumulation of newly synthesized fatty acyl chains within major membrane phospholipids. We show that mTORC1-deficient macrophages also have a phagocytic block downstream from TLR4 signaling, and, interestingly, the reduced level of phagocytosis in both SREBP-1a− and mTORC1-deficient macrophages can be restored by ectopic SREBP-1a expression. Taken together, these observations indicate SREBP-1a is a major downstream effector of TLR4−mTORC1 directed interactions between membrane lipid rafts and the actin cytoskeleton that are required for pathogen-stimulated phagocytosis in macrophages.</P></▼2>

VDR mediated HSD3B1 to regulate lipid metabolism and promoted testosterone synthesis in mouse Leydig cells

Xue Zhen,Zhuang Jianan,Bai Hao,Wang Ling,Lu Hongzhao,Wang Shanshan,Zeng Wenxian,Zhang Tao 한국유전학회 2022 Genes & Genomics Vol.44 No.5

Background: The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis wherein the VDR transcriptionally regulates expression of the genes involved in this complex process. 3β-Hydroxysteroid dehydrogenase-1 (HSD3B1) is an absolutely necessary enzyme for androgen synthesis. Objective: The purpose of the present study was to explore the molecular mechanism of VDR mediated HSD3B1 regulation of lipid metabolism and testosterone synthesis. Methods: The levels of VDR, HSD3B1 and lipid metabolism associated protein were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA). Targeted relationship between VDR and Hsd3b1 was evaluated by dual-luciferase reporter assay. Results: Based on the data analysis of mouse testicular proteome, we found that the expression of HSD3B1 was significantly reduced after VDR deletion. Here, we identified that Hsd3b1 was widely expressed in different tissues of mice by RT-qPCR, and was highly expressed in testis, and mainly located in testicular Leydig cells. Dual-luciferase assay confirmed that VDR could bind candidate vitamin D responsive elements (VDREs) in upstream region of Hsd3b1, and enhance gene expression. Furthermore, over-expression VDR and HSD3B1 significantly increased testosterone synthesis in mice Leydig cells. Meanwhile, Lpl expression was significantly down-regulated and Angptl4 expression was significantly up-regulated in the present of HSD3B1 overexpression. Both LPL and ANGPTL4 play important roles in regulating lipid metabolism. Conclusions: The present study unveiled VDR mediated HSD3B1 to regulate lipid metabolism and promoted testosterone synthesis in mouse Leydig cells. These findings will greatly help us to understand the roles of VDR and HSD3B1 in testosterone synthesis and lipid metabolism.

지질의 첨가를 통한 포도당 기반 무세포 단백질 합성 시스템의 단백질 발현 효율 향상

이소정,김호철,김동명 한국화학공학회 2019 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.57 No.7

Cell-free protein synthesis utilizes the translational machinery in a cell extract. Unlike the conventional cell-based expression methods, not being affected by the conditions for cell growth, cell-free protein synthesis enables flexible manipulation of individual factors affecting the efficiency protein biosynthesis. However, the high cost and low stability of the energy sources to regenerate ATP have limited the use of cell-free synthesis for large-scale production of recombinant proteins. One of the approaches to address this problem is to use glucose as an alternative energy source to regenerate ATP through the glucose-metabolizing pathways in a cell extract. In this study, in an attempt to improve the efficiency of ATP regeneration by reinforcing oxidative phosphorylation process, we supplemented with cellular lipids to a glucose-fueled reaction mixture for cell-free protein synthesis. As a result of the lipid supplementation, the productivity of chloramphenicol acetyltransferase in a cell-free synthesis system using glucose increased more than 6 fold compared to when the lipid was not supplemented. 무세포 단백질 합성 시스템은 세포를 파쇄한 후 파쇄액 내의 단백질 합성기구들을 이용하여 단백질을 발현하는 시스템으로 기존의 세포 기반 재조합 단백질 발현 기법들과 달리 세포의 생장조건에 영향을 받지 않으면서 발현 조절에관한 다양한 인자들을 인위적으로 조절 할 수 있는 장점이 있다. 그러나, 단백질 합성 과정 중 소모되는 ATP의 연속적 재생을 위해 사용되는 에너지원의 높은 비용과 낮은 안정성은 재조합 단백질 대량생산에의 적용을 제약하는 요인으로 작용하여 왔다. 이러한 문제를 해결하기 위한 대안들 중의 하나로 포도당을 에너지원으로 사용하여 세포 파쇄액내 대사과정을 통해 ATP를 재생하는 방법이 있다. 본 연구에서는 포도당을 에너지원으로 이용한 무세포 합성 시스템에서의 단백질 합성 효율 향상을 위하여 대장균 파쇄액으로부터 회수된 지질을 추가적으로 첨가함으로써 산화적 인산화 과정에서의 ATP재생을 증진시키고자 하였다. 그 결과, 지질이 추가된 무세포 단백질 합성 시스템은 지질이 추가되지 않은 대조군에 비하여 6배 이상 향상된 단백질 생산성을 나타내었다.

지질의 첨가를 통한 포도당 기반 무세포 단백질 합성 시스템의 단백질 발현 효율 향상

이소정 ( So Jeong Lee ),김호철 ( Ho-cheol Kim ),김동명 ( Dong-myung Kim ) 한국화학공학회 2019 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.57 No.1

Cell-free protein synthesis utilizes the translational machinery in a cell extract. Unlike the conventional cell-based expression methods, not being affected by the conditions for cell growth, cell-free protein synthesis enables flexible manipulation of individual factors affecting the efficiency protein biosynthesis. However, the high cost and low stability of the energy sources to regenerate ATP have limited the use of cell-free synthesis for large-scale production of recombinant proteins. One of the approaches to address this problem is to use glucose as an alternative energy source to regenerate ATP through the glucose-metabolizing pathways in a cell extract. In this study, in an attempt to improve the efficiency of ATP regeneration by reinforcing oxidative phosphorylation process, we supplemented with cellular lipids to a glucose-fueled reaction mixture for cell-free protein synthesis. As a result of the lipid supplementation, the productivity of chloramphenicol acetyltransferase in a cell-free synthesis system using glucose increased more than 6 fold compared to when the lipid was not supplemented.

A Simple Evaporation Method for Large-Scale Production of Liquid Crystalline Lipid Nanoparticles with Various Internal Structures

Kim, Do-Hoon,Lim, Sora,Shim, Jongwon,Song, Ji Eun,Chang, Jong Soo,Jin, Kyeong Sik,Cho, Eun Chul American Chemical Society 2015 ACS APPLIED MATERIALS & INTERFACES Vol.7 No.36

<P>We present a simple and industrially accessible method of producing liquid crystalline lipid nanoparticles with various internal structures based on phytantriol, Pluronic F127, and vitamin E acetate. Bilayer vesicles were produced when an ethanolic solution dissolving the lipid components was mixed with deionized water. After the evaporation of ethanol from the aqueous mixture, vesicles were transformed into lipid-filled liquid crystalline nanoparticles with well-defined internal structures such as hexagonal lattices (mostly inverted cubic <I>Pn</I>3<I>m</I>), lined or coiled pattern (inverted hexagonal H<SUB>2</SUB>), and disordered structure (inverse microemulsion, L<SUB>2</SUB>), depending on the compositions. Further studies suggested that their internal structures were also affected by temperature. The internal structures were characterized from cryo-TEM and small-angle X-ray scattering results. Microcalorimetry studies were performed to investigate the degree of molecular ordering/crystallinity of lipid components within the nanostructures. From the comparative studies, we demonstrated the present method could produce the lipid nanoparticles with similar characteristics to those made from a conventional method. More importantly, the production only requires simple tools for mixing and ethanol evaporation and it is possible to produce 10 kg or so per batch of aqueous lipid nanoparticles dispersions, enabling the large-scale production of the liquid crystalline nanoparticles for various biomedical applications.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/aamick/2015/aamick.2015.7.issue-36/acsami.5b06413/production/images/medium/am-2015-06413p_0008.gif'></P>

Monoacylglycerol O-acyltransferase 1 (MGAT1) localizes to the ER and lipid droplets promoting triacylglycerol synthesis

( Yoo Jeong Lee ),( Jae-woo Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.7

Monoacylglycerol acyltransferase 1 (MGAT) is a microsomal enzyme that catalyzes the synthesis of diacylglycerol (DAG) and triacylglycerol (TAG). However, the subcellular locali-zation and catalytic function domain of this enzyme is poorly understood. In this report, we identified that murine MGAT1 localizes to the endoplasmic reticulum (ER) under normal conditions, whereas MGAT1 co-localize to the lipid droplets (LD) under conditions of enriching fatty acids, contributing to TAG synthesis and LD expansion. For the enzyme activity, both the N-terminal transmembrane domain and catalytic HPHG motif are required. We also show that the transmembrane domain of MGAT1 consists of two hydrophobic regions in the N-terminus, and the consensus sequence FLXLXXXn, a putative neutral lipid-binding domain, exists in the first transmembrane domain. Finally, MGAT1 interacts with DGAT2, which serves to synergistically increase the TAG biosynthesis and LD expansion, leading to enhancement of lipid accumulation in the liver and fat. [BMB Reports 2017; 50(7): 367-372]

Doxorubicin Attenuates Free Fatty Acid-Induced Lipid Accumulation via Stimulation of p53 in HepG2 Cells

( Chawon Yun ),( Sou Hyun Kim ),( Doyoung Kwon ),( Mi Ran Byun ),( Ki Wung Chung ),( Jaewon Lee ),( Young-suk Jung ) 한국응용약물학회 2024 Biomolecules & Therapeutics(구 응용약물학회지) Vol.32 No.1

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 μg/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

Lipid-coated ZnO nanoparticles synthesis, characterization and cytotoxicity studies in cancer cell

Cao Dingding,Shu Xugang,Zhu Dandan,Liang Shengli,Hasan Murtaza,Gong Sheng 나노기술연구협의회 2020 Nano Convergence Vol.7 No.14

ZnO nanoparticles are widely used in biological, chemical, and medical fields, but their toxicity impedes their wide application. In this study, pristine ZnO NPs (~ 7 nm; ~ 18 nm; ~ 49 nm) and lipid-coated ZnO NPs (~ 13 nm; ~ 22 nm; ~ 52 nm) with different morphologies were prepared by chemical method and characterized by TEM, XRD, HRTEM, FTIR, and DLS. Our results showed that the lipid-coated ZnO NPs (~ 13 nm; ~ 22 nm; ~ 52 nm) groups improved the colloidal stability, prevented the aggregation and dissolution of nanocrystal particles in the solution, inhibited the dissolution of ZnO NPs into ­Zn 2+ cations, and reduced cytotoxicity more efficiently than the pristine ZnO NPs (~ 7 nm; ~ 18 nm; ~ 49 nm). Compared to the lipid-coated ZnO NPs, pristine ZnO NPs (~ 7 nm; ~ 18 nm; ~ 49 nm) could dose-dependently destroy the cells at low concentrations. At the same concentration, ZnO NPs (~ 7 nm) exhibited the highest cytotoxicity. These results could provide a basis for the toxicological study of the nanoparticles and direct future investigations for preventing strong aggregation, reducing the toxic effects of lipid-bilayer and promoting the uptake of nanoparticles by HeLa cells efficiently.

Synthesis of lipid-coated PLGA nanoparticles via emulsion solvent evaporation method for drug delivery platform

송인현,신승원,신우정,엄숭호 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0

Here, we demonstrate a new, core-shell nanoparticulate composed of three different components: an outer lipid membrane, a polymeric interfacial layer, and a drug-loading aqueous core. This novel nanoparticulate is synthesized by a new, double emulsion synthetic protocol that overcomes the weaknesses of the conventional emulsion method. The physicochemical properties of nanoparticulates can be varied; for instance, we replaced the outer membrane compartments with several lipid molecules. In addition, the interfacial polymeric layer can be modified by adding either hydrophobic PLGAs or hydrophilic nucleic acids with higher biocompatibility. A variety of surface functionalizations can also be achieved including several unique characteristics such as surface multifunctionality and sustained drug (e.g., ovalbumin) release. We believe that such versatile nanoparticulates are promising in vivo carriers for medications against several infectious diseases.

Functional in silico analysis of a non-synonymous SNP located in the coding region of the FASN gene in Korean native cattle

공홍식,이윤석 한국동물유전육종학회 2023 한국동물유전육종학회지 Vol.7 No.3

Bovine fatty acid synthase, an enzyme encoded by the FASN gene in cattle, is a multi-enzyme protein that catalyses fatty acid synthesis. This cytosolic enzyme catalyzes the synthesis of palmitate from acetylcoenzyme A and malonyl-coenzyme A in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). However, there is no previous verification study that each allele of SNPs related to lipid synthesis were impact on protein function in Korean native cattle and nsSNPs for the FASN gene have not yet been verified by computer analysis. Given the role of the FASN gene in beef quality traits in cattle, the study aimed to use computational analysis to narrow down the candidate nsSNPs for FASN that may affect protein structure and/or function, which may play an important role in lipid synthesis. These results predicted that the g.16039 T>C nsSNP at position R1957Y of FASN was functionally 'Deleterious' and 'PROBABLY DAMAGING' in non-synonymous SNP functional analysis, and the g.16039 T>C and g.17924 A>G nsSNPs at positions R1957Y and T2266A decrease the stability of a FASN protein and have two PTM sites for proteolytic cleavage and amidation. In addition, the R1957Y and T2266A variants of FASN were shown to have a direct effect on altering the protein structure. Therefore, we suggested that our results could be used as fundamental data for further studies related to functional verification of nsSNPs based on bovine cells.