Autophagy and lipid metabolism -
E64d and Pepstatin A were from Peptide Institute Osaka, Japan. Twelve hours after plating, the medium was replaced with fresh medium containing adenovirus vector. The oxygen consumption rate OCR was measured simultaneously three times to establish a baseline rate.
For each measurement, with a total of 16 measurements, there was a 3-min mix followed by a 3-min wait time to restore normal oxygen tension and pH in the microenvironment surrounding the cells. Drug injection was performed throughout the assay. To delete Atg5 in the liver, Cre expression was induced in the liver by intraperitoneal injection of pIpC Sigma Chemical, St.
Louis, MO, USA. Mice were housed in specific pathogen-free facilities, and the Ethics Review Committee for Animal Experimentation of Niigata University, and of the University of Tokyo approved the experimental protocol.
We have complied with all relevant ethical regulations. Blood glucose and β-hydroxybutyrate were measured using a glucose meter Terumo, Tokyo, Japan and blood ketone body meter Abbott Laboratories, Chicago, IL, USA , respectively.
Free fatty acids in plasma were analyzed by SRL Tokyo, Japan. Livers were homogenized in 0. Nuclear and cytoplasmic fractions from livers and cultured cells were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Thermo Fisher Scientific.
Full size images are presented in Supplementary Figure 11 — The pulled-down protein complexes were collected by centrifugation and extensively washed with TNE buffer containing 1. The resultant precipitants were analyzed by immunoblotting.
Using the Transcriptor First-Strand cDNA Synthesis Kit Roche Applied Science, Indianapolis, IN, USA , cDNA was synthesized from 1 μg of total RNA. Quantitative PCR was performed using the LightCycler ® Probes Master mix Roche Applied Science on a LightCycler ® Roche Applied Science.
Signals from human and mouse samples were normalized against GAPDH glyceraldehydephosphate dehydrogenase and Gusb β-glucuronidase , respectively. The sequences of the primers used for analysis of mouse livers or human cell lines are provided in Supplementary Table 1. Alexa Fluor conjugated donkey anti-rabbit IgG Invitrogen, San Diego, CA, USA was used as secondary antibody.
After image acquisition, contrast and brightness were adjusted using Photoshop CS4 Adobe Systems, San Jose, CA, USA. To examine lipid droplets, the sections were also stained with Oil Red O, and then observed with a microscope BX51, Olympus.
Z -projection stack images were acquired with z steps of 0. Image contrast and brightness were adjusted using Photoshop CS4 Adobe System. Chromatin immunoprecipitation ChIP assay was performed with an anti-H3K27ac antibody MABI, MAB Institute, Inc.
Solubilized chromatin was incubated overnight with Dynabeads anti-mouse IgG Invitrogen prebound with control IgG or anti-H3K27ac antibody.
The precipitated DNA was PCR-amplified using primers listed in Supplementary Table 2. Enhancer regions with H3K27ac deposition for the PCR amplification were selected from the CPT1A and CPT2 loci based on HepG2 cell data in ENCODE database. Lipidome analysis was performed as described previously with slight modification The organic lower phase was transferred to a clean vial and dried under a stream of nitrogen.
The lipids were then resolubilized in methanol containing 0. The flow rate was 0. ESI capillary voltage was set at 1. For experiments tracing the metabolic fate of palmitate, mice received 13 C 16 -palmitate 2.
A portion of the liver lobule was sampled for snap-freezing using liquid nitrogen. Metabolite extraction from tissues for metabolome analyses was performed as described previously After centrifugation, the aqueous phase was ultrafiltered using an Ultrafree MC-PLHCC ultrafiltration tube Human Metabolome Technologies.
The filtrate was concentrated on a vacuum concentrator SpeedVac, Thermo. The IC was equipped with an anion electrolytic suppressor Thermo Scientific Dionex AERS , which converted the potassium hydroxide gradient into pure water before the sample entered the mass spectrometer.
Separation was performed using a Thermo Scientific Dionex IonPac ASHC, 4-μm particle size column. The IC flow rate was 0. The Q-Exactive focus mass spectrometer was operated under ESI-negative mode for all detections. Statistical analysis was performed using the unpaired t -test Welch test two-sided.
The authors declare that the data supporting the findings of this study are available within the article and its supplementary information.
A reporting summary for this article is available as a supplementary information file. Source data for Fig.
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The hypocotyls or cotyledons were observed under confocal microscopy. For transmission electron microscopy, leaf tissues were fixed with 2.
For chloroplast counting, leaf tissues were fixed and embedded. The number of chloroplasts was counted from at least 60 mesophyll cell cross sections for each time point of dark treatment. Colocalization analysis of OLE1-GFP and ATG8e-DsRed signals was done with the Coloc 2 plugin for ImageJ.
Background subtraction from image pairs was performed using rolling ball subtraction with a pixel ball size. Statistical significance of the PCC of the image pairs was analyzed using the Costes image randomization test as described previously Costes et al.
Regions of interest were selected for colocalization analysis with Costes randomizations using a point spread function of 3. Five-day-old seedlings grown on 0. The seedlings were then transferred to half-strength MS medium containing 0. The fixed hypocotyls were washed twice with 0. After dehydration, the tissues were embedded in LR White resin CA, Electron Microscopy Sciences, London Resin Company in gelatin capsules.
Resin polymerization was performed at 50 to 55°C. Ultrathin sections 70 to 90 nm of LR White—embedded hypocotyls were collected with formvar-coated mesh nickel grids. The grids were first washed with 1× PBS containing 0. After blocking, the grids were incubated with the primary antibody:rabbit polyclonal anti-ATG8a catalog no.
After rinsing with blocking solution five times, 1 min each, the grids were then incubated in the secondary antibody of goat anti-rabbit immunoglobulin G conjugated with nm gold particles catalog no.
G, lot no. SLBW, Sigma-Aldrich; dilution in blocking solution for 1 h at room temperature. Following washing with 1× PBS and 0. Supplemental Figure 1. Time course of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in wild-type developing embryos.
Supplemental Figure 2. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into TAG in developing embryos and seedlings. Supplemental Figure 3. Rate of the incorporation of radiolabel from 14 C-acetate or 3 H 2 O into total fatty acids in leaves.
Supplemental Figure 4. Rate of the incorporation of radiolabel from 14 C-acetate into total membrane lipids in leaves. Supplemental Figure 5. PDAT activity in microsomal membranes isolated from seedlings. Supplemental Figure 6. Disruption of autophagy reduces TAG content in mature leaves of 4-week-old PDAT1 -overexpressing transgenic plants.
Supplemental Figure 7. Increased accumulation of DsRed-ATG8e—labeled structures in leaves of tgd1 plants under dark treatment. Supplemental Figure 8. Accumulation of autophagosomes and autophagic vacuoles in mature leaves of 4-week-old sdp plants under dark treatment.
Supplemental Figure 9. The appearance of LDs in the central vacuole in wild-type seedlings after dark treatment in the presence of concA. Supplemental Figure Autophagic activity in 4-week-old sdp plants under dark-induced starvation.
TAG levels in mature leaves of 4-week-old wild type, atg and atg plants under dark-induced starvation. Membrane lipid levels in mature leaves of 4-week-old sdp , atg sdp , and atg sdp plants under dark-induced starvation.
Chloroplast number in mature leaves of sdp plants under dark-induced starvation. Supplemental Data Set. Results of statistical analyses. The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: SDP1 Gramene: At5g SDP1 Araport: At5g ATG10 Gramene: AT3G ATG10 Araport: AT3G ATG3 Gramene: AT5G ATG3 Araport: AT5G LAS Gramene: AT1G LAS Araport: AT1G ACT1 Gramene: AT2G ACT1 Araport: AT2G TGD1 Gramene: AT1G TGD1 Araport: AT1G ATG5 Gramene: AT5G ATG5 Araport: AT5G PDAT1 Gramene: at5g PDAT1 Araport: at5g ATG2 Gramene: AT3G ATG2 Araport: AT3G ATG8 Gramene: AT4G ATG8 Araport: AT4G This work was supported by the U.
Department of Energy , Office of Science, Office of Basic Energy Sciences DE-SC , specifically through the Physical Biosciences program of the Chemical Sciences, Geosciences and Biosciences Division. Use of the transmission electron microscope and the confocal microscope at the Center of Functional Nanomaterials was supported by the Office of Basic Energy Sciences, U.
Department of Energy DE-SC and J. designed the experiments. performed the research. and C. participate in data analysis. wrote the article with contributions from J. and L. Anding , A. Cleaning house: Selective autophagy of organelles.
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The lilid responsible liid distribution of materials integral to the findings presented Micronutrient-rich diet this article in accordance with the policy described in the Instructions for Radiology and MRI www. Autophagy and lipid metabolism is: Metqbolism Xu cxu bnl. Autophagy is a major catabolic pathway Autophagy and lipid metabolism Autophaagy constituents Snd lipid droplets LDsstorage compartments for neutral lipids, are delivered to the lysosome or vacuole for degradation. The autophagic degradation of cytosolic LDs, a process termed lipophagy, has been extensively studied in yeast and mammals, but little is known about the role for autophagy in lipid metabolism in plants. Organisms maintain a basal level of autophagy under favorable conditions and upregulate the autophagic activity under stress including starvation. Here, we demonstrate that Arabidopsis Arabidopsis thaliana basal autophagy contributes to triacylglycerol TAG synthesis, whereas inducible autophagy contributes to LD degradation.Autophagy and lipid metabolism -
Knockdown of p62 in ATG7 -deficient HepG2 cells suppressed expression of Nrf2-target genes, but had no effect on the repression of PPARα target genes Fig. In accordance with a previous study of liver-specific NCoR1 -knockout mice 35 , loss of NCoR1 in HepG2 cells led to prominent induction of PPARα target genes Fig.
In both wild-type and NCoR1 -knockout HepG2 cells, expression of PPARα target genes was repressed by overexpression of NCoR1 Fig. These results suggest that quantitative control of NCoR1 has an impact on expression of PPARα target genes.
NCoR1-dependent PPARα-inactivation in autophagy-incompetent cells. a , c Immunoblot analysis. Parental and ATG7 -knockout HepG2 cells 14 were treated with siRNA for p62 a and NCoR1 c. Thereafter, both nuclear and cytoplasmic fractions were prepared and subjected to immunoblotting with the indicated antibodies.
b , d Real-time PCR analysis. Total RNAs were prepared from cells described in b and d. Values were normalized against the amount of mRNA in parental HepG2 cells treated with control siRNA.
e Immunoblot analysis. GFP or NCoR1 was exogenously expressed in wild-type and NCoR1 -knockout HepG2 cells using the adenovirus system.
Forty-eight hours after infection, both nuclear and cytoplasmic fractions were prepared from cells and subjected to immunoblotting with the indicated antibodies.
Data shown are representative of three separate experiments. f Real-time PCR analysis. Total RNAs were prepared from cells described in e. Values were normalized against the amount of mRNA in GFP-expressing wild-type HepG2 cells.
Such sharp fluctuations were detected even in fasting conditions Fig. Concomitant loss of NCoR1 in Atg7 -knockout mouse livers abolished the suppression of PPARα target genes, albeit not completely Supplementary Figure 6a.
In stark contrast to the significant accumulation of NCoR1 in both the nuclear and cytoplasmic fractions of Atg7 p62 double-knockout livers Fig. Consequently, the reduction in the nuclear level of PPARα in Atg7 p62 double-knockout livers was rescued by simultaneous loss of NCoR1 Fig.
As expected, no nuclear signals were recognized in Atg7 p62 NCoR1 triple-knockout hepatocytes, which validated the signals observed in other genotypes Fig.
Although the expression of PPARα target genes was not influenced by single deletion of p62 , it was significantly induced in NCoR1 -knockout mouse livers Supplementary Figure 6b and c.
On the basis of those in vitro and in vivo results, we concluded that downregulation of lipid-oxidation in autophagy-deficient livers is mainly due to accumulation of NCoR1. NCoR1-dependent PPARα-inactivation in autophagy-deficient livers.
a , c Gene expression of enzymes related to lipid oxidation in Atg7 p62 double- a and Atg7 p62 NCoR1 triple-knockout livers c. b , d NCoR1 level in Atg7 p62 double- b and Atg7 p62 NCoR1 triple-knockout livers d. Total homogenate, as well as nuclear and cytoplasmic fractions, were prepared from livers of mice of the indicated genotypes and subjected to immunoblotting with the indicated antibodies.
f Blood β-OHB in mice described in a. How does autophagy affect the level of NCoR1? First, we examined the kinetics of NCoR1 in response to starvation under autophagy-intact or -defective conditions.
Amino-acid starvation caused a marked decrease in NCoR1 levels in both nuclear and cytoplasmic fractions of control siRNA-treated HepG2 cells Fig. This reduction was largely inhibited by knockdown of ATG7 Fig.
The treatment of wild-type HepG2 cells with the lysosomal protease inhibitors E64d and pepstatin A caused a marked increase in the NCoR1 level Fig. Starvation promoted the ATG7-mediated decrease in the NCoR1 level Fig. Oxygen consumption rate OCR in ATG7 -deficient HepG2 cells under β-oxidation conditions tended to be lower than in cells expressing wild-type ATG7.
Moreover, the addition of etomoxir, which binds irreversibly to the CPT1A transporter and inhibits fatty-acid oxidation, barely had an effect on the OCR Fig.
Degradation of NCoR1 in autophagy-lysosomal pathway. Both nuclear and cytoplasmic fractions were prepared from ATG7 -knockdown HepG2 cells under nutrient-rich and deprived conditions and subjected to immunoblotting with the indicated antibodies.
b Immunoblot analysis. HepG2 cells were cultured in the presence or absence of E64d and Pepstatin A E. Subsequently, both nuclear and cytoplasmic fractions were prepared from the HepG2 cells and subjected to immunoblotting with the indicated antibodies.
GFP, wild-type ATG7, or ATG7 CS was expressed in ATG7 -knockout HepG2 14 cells by adenovirus system. Forty-eight hours after infection, the cells were cultured under nutrient-rich or -deprived conditions. d Oxygen consumption rate OCR. Arrows indicate the time when etomoxir was added to the cells.
The graphs represent the average OCR at four time points. Immunofluorescence staining revealed that endogenous NCoR1 and an autophagosome-localizing protein, GABARAP gamma-aminobutyric acid receptor-associated protein , co-localized in cytoplasmic puncta structures of HepG2 cells Fig.
Most Consistent with the biochemical results Fig. We also carried out immunofluorescent analysis with anti-LAMP1 and anti-NCoR1 antibodies. As expected, both proteins were co-localized in wild-type but not Atg7- deficient HepG2 cells Fig.
In ATG7 -knockout HepG2 cells, NCoR1 formed small punctate structures in both nucleus and cytoplasm, in addition to the large cytoplasmic puncta Fig. Among these structures, GABARAP mainly co-localized to large cytosolic NCoR1-positive structures, regardless of nutrient conditions Fig. Unlike wild-type HepG2 cells, the abundance of these structures did not decrease upon starvation Fig.
Our previous genetic studies showed the formation of LC3-dots through the interaction of LC3 with p62 even in Atg7 -deficient mouse hepatocytes 4 , Likewise, excessive accumulation of NCoR1 might sequester GABARAP due to their physical interaction, irrespective of the lipidation of GABARAP.
Localization of NCoR1 on GABARAP-positive structures. a , b Immunofluorescence analysis. Wild-type or ATG7 -deficient HepG2 cells were cultured under nutrient-rich or -poor conditions, and then immunostained with anti-GABARAP and anti-NCoR1 a or anti-Lamp1 and anti-NCoR1 antibodies b.
The number of cytoplasmic NCoR1 and GABARAP or of cytoplasmic NCoR1 and Lamp1 double-positive dots per 20 cells was counted. Each inset is a magnified image. Bar: 2. HEKT cells were utilized for the pull-down assays due to their high transfection efficiency and protein production.
In agreement with previous reports 38 , these assays confirmed the specific binding of ULK1 to GABARAP family proteins, as well as binding of p62 to both LC3B and GABARAP family proteins Fig.
Similar to ULK1, NCoR1 bound to GABARAP family proteins, but not LC3B Fig. To determine which domain of NCoR1 is required for its interaction with GABARAP family proteins, we constructed a series of NCoR1-deletion mutants Fig.
NCoR1 N aa 1— and NCoR1 ΔC aa 1— were clearly detected in the precipitant Fig. However, deletion mutants NCoR1 M aa — , NCoR1 C aa — , and NCoR1 ΔN — exhibited a marked decrease in binding to GABARAP Fig. To narrow down the interaction domain, we prepared a series of deletions starting from NCoR1 ΔC N1—N4 Fig.
These assays revealed that NCoR1 N1, covering amino acids 1—, is sufficient for the interaction between NCoR1 and GABARAP Fig. elegans Fig. Specific interaction of NCoR1 with GABARAP. a Pull-down assay. One-Strep-FLAG OSF -tagged LC3B or GABARAP family proteins were expressed in HEKT cells.
Precipitates generated with Strep-Tactin Sepharose were subjected to immunoblot analysis with indicated antibodies. ULK1 and p62 bind to GABARAP family and all Atg8 family proteins, respectively, and were therefore used as positive controls.
Data are representative of three independent experiments. b Diagrams of the deletion-mutation constructs of NCoR1 left panel and the corresponding pull-down assays middle and right panels. Middle panel: Each FLAG-tagged NCoR1-deletion mutant and OSF-GABARAP was co-expressed in HEKT cells.
Right panel: OSF-GABARAP immobilized on Strep-Tactin Sepharose was mixed with lysates prepared from cells expressing each NCoR1-deletion mutant. The resultant precipitates were subjected to immunoblotting with anti-FLAG antibody.
Black and gray boxes indicate identical amino-acid residues with complete and partial conservation, respectively. d Pull-down assay. The assay was carried out as described in a. Both wild-type and mutant were efficiently expressed in the cells Fig.
Similar to the dynamics of endogenous NCoR1 Fig. Furthermore, GIM-deleted NCoR1 has a higher suppressive effect on the expression of PPARα target genes than wild-type NCoR1 under amino-acid starvation conditions Fig. Autophagic degradation of NCoR1 dependent on the GABARAP-interaction.
Immunoblot analysis. Forty-eight hours after transfection, the cells were cultured in nutrient-rich medium, or nutrient-deprived medium.
Thereafter, cytoplasmic and nuclear fractions were prepared and subjected to immunoblotting with the indicated antibodies. b Immunofluorescence analysis. c Real-time PCR analysis. Total RNAs were prepared from cells described in a.
Values were normalized against the amount of mRNA in NCoR1 -deficient HepG2 cells expressing FLAG. d Model of PPARα transactivation through selective autophagic degradation of NCoR1. NCoR1 serves as scaffold that facilitates interaction of several docking proteins to fine-tune transactivation of transcription factors, in particular nuclear receptors that have important roles in metabolic control.
Interaction of NCoR1 with nuclear receptors and histone deacetylases is vital for nuclear receptor-mediated downregulation of gene expression In this study, we found that autophagy also participates in regulation of the activity of the nuclear receptor PPARα through degradation of NCoR1, and that suppression of liver autophagy is accompanied by defective β-oxidation and ketogenesis.
Under nutrient-rich conditions, mechanistic target of rapamycin complex 1 mTORC1 phosphorylates ribosomal protein S6 kinase 2 S6K2. NCoR1 forms a complex with the S6K2, and the resultant complex translocates into the nucleus to suppress genes encoding enzymes involved in β-oxidation mTORC1 also binds and phosphorylates TFEB, a master transcription factor for a battery of Atg and lysosomal genes, causing it to be retained in the cytoplasm 43 , 44 , In response to nutrient deprivation and subsequent mTORC1 inactivation, nuclear translocation of NCoR1 is inhibited, TFEB is concomitantly dephosphorylated, and ULK1 kinase, an upstream factor involved in autophagosome formation, is activated.
Consequently, both autophagy and β-oxidation followed by production of ketone body occur at the same time At this time, the autophagic degradation of NCoR1 contributes to PPARα-activation to effectively promote β-oxidation in response to physiological fasting Fig.
Previously, autophagy was thought to contribute to lipid oxidation by increasing the supply of free fatty acids lipophagy. However, here we propose that autophagy is integrated into a highly sophisticated regulatory mechanism for a nuclear factor, PPARα, and that for this reason, impairment of autophagy in the liver causes defects in β-oxidation and ketogenesis.
We observe a marked reduction in PPARα in autophagy-deficient mouse livers. However, the phenotypes of liver-specific Atg7 -knockout mice are likely to be different from those of liver-specific PPARα -knockout mice, which have higher levels of liver triglyceride and cholesterol ester during fasting and exhibit severe steatosis In fact, we recognized a slight increase in the levels of some molecular species of triglyceride and cholesterol esters in Atg7 -deficient mouse livers Supplementary Figure 1.
Repression of the transactivation of nuclear receptors by NCoR1 in livers is not restricted to PPARα. For example, NCoR1 modulates transactivation of nuclear receptors such as LXRα and estrogen-related receptor α ERRα other than PPARα Thus, suppression of liver autophagy should be accompanied by repression of multiple nuclear receptors.
Indeed, LXRα target genes that encode enzymes involved in lipogenesis are downregulated in mouse livers lacking Rb1cc1 also called Fip , a component of the ULK1 kinase complex We verified the NCoR1-dependent suppression of LXRα-targets Supplementary Figure 8a—d and of LXRα protein Supplementary Figure 8e—h in autophagy-deficient livers.
Given the concurrent suppression of genes related to both lipogenesis and lipid oxidation upon loss of Atg7, lipid metabolism in the mutant livers is apparently stable under normal conditions, but should be stagnant under conditions in which fatty acids are mobilized.
Indeed, liver steatosis under physiological fasting and high-fat diet conditions was abolished by loss of Rb1cc1 , Atg7 , or Atg5 19 , 20 , 21 , In sharp contrast, loss of NCoR1 in mouse livers causes induction of gene sets regulated by LXRα, PPARα, and ERRα, leading to concurrent induction of lipogenesis and lipid oxidation In Atg7 p62 NCoR1 triple-knockout livers, the expression of PPARα target genes recovered up to control levels Fig.
In contrast to NCoR1 -knockout livers, however, we did not observe higher induction of the targets Supplementary Figure 4 , implying the presence of a still-hidden suppressive mechanism.
Recently, Sinha et al. We examined the phosphorylation level of RPS6KB1 in Atg7 -deficient hepatocytes and found that the level was comparable to that in control cells Supplementary Figure 10 , implying that the nuclear accumulation of NCoR1 in Atg7 -knockout livers depends on a distinct mechanism from the RPS6KB1-mediated translocation of NCoR1.
In addition, Sinha et al. showed that the nuclear accumulation of NCoR1 downregulates the expression of the SCD1 gene encoding stearoyl-CoA desaturase, which converts saturated fatty acids SFAs to mono-unsaturated fatty acids MUFAs.
In general, selective substrates for autophagy are tagged with a molecular marker that includes ubiquitin, leading to assembly of receptor proteins that bind to both marker molecules and the ATG8 family proteins including LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2 near the cargos 50 , Thus, cargo labeling and the transfer of receptor proteins to cargos mainly regulate selective autophagy.
In the case of selective autophagy for NCoR1, ubiquitination may serve as a signaling tag. NCoR1 is ubiquitinated by the ubiquitin ligase TBLR1 and subsequently degraded by the proteasome 54 , Because NCoR1 translocates from the nucleus to cytoplasm in response to nutrient starvation, ubiquitination of NCoR1 may be a signal not only for proteasomal degradation but also for selective autophagy, both of which favor the exchange of co-repressors for co-activators.
Because fasting decreased the levels of both nuclear and cytoplasmic NCoR1 even in Atg7 -knockout livers Fig. Therefore, NCoR1 is a hybrid protein with characteristics of both autophagy substrates and receptors; accordingly, receptor protein s might be dispensable for this type of selective autophagy.
Because both NCoR1 and GABARAP family are ubiquitously expressed, it is plausible that selective autophagy of NCoR1 occurs in most tissues. However, the corresponding nuclear receptors for NCoR1 differ according to tissue type, implying that accumulation of NCoR1 due to loss of autophagy has distinct effects among tissues.
Suppression of autophagy in metabolic tissues is associated with degeneration and defective differentiation Although the former is thought to be due to impairment of cellular homeostasis and to pmediated Nrf2 activation in the case of liver , the reason for the latter remains unclear.
Recent work with tissue-specific NCoR1 -knockout mice revealed that loss of NCoR1 results in the activation of distinct transcription factors in specific tissues. For example, expression of PPARγ target genes is specifically induced in adipocyte-specific NCoR1 -knockout mice, which exhibit increased insulin sensitivity in liver, fat, and muscle, and develop obesity and expansion of fat tissue on a high-fat diet due to an increase in the number of small adipocytes and reduced inflammation in adipose tissue By contrast, adipogenesis is impaired in adipocyte-specific Atg7 or Atg5 -knockout mice, leading to a lean phenotype 58 , Loss of Atg5 or Atg7 in mouse skeletal muscle causes muscle atrophy and weakness, as well as accumulation of degenerated mitochondria 60 , These phenotypes could be explained by the accumulation of NCoR1 and subsequent deregulation of transcription networks, in addition to defective cellular homeostasis.
HepG2 cells were co-transfected with vectors pX and pEGFP-C1 , Clontech Laboratories, Mountain View, CA, USA , and cultured for 2 days. Thereafter, GFP-positive cells were sorted and expanded.
Loss of ATG7 or NCoR1 was confirmed by heteroduplex mobility assay followed by immunoblot analysis with anti-ATG7 or anti-NCoR1 antibody.
NCoR1 was expressed using a helper-dependent adenovirus vector system 62 containing loxP at position To exogenously express GFP, ATG7, or ATG7 CS , we used the Adenovirus Expression Vector Kit Takara Bio, Kusatsu, Japan.
The medium was replaced with fresh medium containing adenovirus with a multiplicity of infection MOI of E64d and Pepstatin A were from Peptide Institute Osaka, Japan.
Twelve hours after plating, the medium was replaced with fresh medium containing adenovirus vector. The oxygen consumption rate OCR was measured simultaneously three times to establish a baseline rate. For each measurement, with a total of 16 measurements, there was a 3-min mix followed by a 3-min wait time to restore normal oxygen tension and pH in the microenvironment surrounding the cells.
Drug injection was performed throughout the assay. To delete Atg5 in the liver, Cre expression was induced in the liver by intraperitoneal injection of pIpC Sigma Chemical, St. Louis, MO, USA. Mice were housed in specific pathogen-free facilities, and the Ethics Review Committee for Animal Experimentation of Niigata University, and of the University of Tokyo approved the experimental protocol.
We have complied with all relevant ethical regulations. Blood glucose and β-hydroxybutyrate were measured using a glucose meter Terumo, Tokyo, Japan and blood ketone body meter Abbott Laboratories, Chicago, IL, USA , respectively.
Free fatty acids in plasma were analyzed by SRL Tokyo, Japan. Livers were homogenized in 0. Nuclear and cytoplasmic fractions from livers and cultured cells were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Thermo Fisher Scientific.
Full size images are presented in Supplementary Figure 11 — The pulled-down protein complexes were collected by centrifugation and extensively washed with TNE buffer containing 1. The resultant precipitants were analyzed by immunoblotting.
Using the Transcriptor First-Strand cDNA Synthesis Kit Roche Applied Science, Indianapolis, IN, USA , cDNA was synthesized from 1 μg of total RNA. Quantitative PCR was performed using the LightCycler ® Probes Master mix Roche Applied Science on a LightCycler ® Roche Applied Science.
Signals from human and mouse samples were normalized against GAPDH glyceraldehydephosphate dehydrogenase and Gusb β-glucuronidase , respectively. The sequences of the primers used for analysis of mouse livers or human cell lines are provided in Supplementary Table 1.
Alexa Fluor conjugated donkey anti-rabbit IgG Invitrogen, San Diego, CA, USA was used as secondary antibody. After image acquisition, contrast and brightness were adjusted using Photoshop CS4 Adobe Systems, San Jose, CA, USA.
To examine lipid droplets, the sections were also stained with Oil Red O, and then observed with a microscope BX51, Olympus. Z -projection stack images were acquired with z steps of 0. Image contrast and brightness were adjusted using Photoshop CS4 Adobe System.
Chromatin immunoprecipitation ChIP assay was performed with an anti-H3K27ac antibody MABI, MAB Institute, Inc. Solubilized chromatin was incubated overnight with Dynabeads anti-mouse IgG Invitrogen prebound with control IgG or anti-H3K27ac antibody.
The precipitated DNA was PCR-amplified using primers listed in Supplementary Table 2. Enhancer regions with H3K27ac deposition for the PCR amplification were selected from the CPT1A and CPT2 loci based on HepG2 cell data in ENCODE database.
Lipidome analysis was performed as described previously with slight modification The organic lower phase was transferred to a clean vial and dried under a stream of nitrogen.
The lipids were then resolubilized in methanol containing 0. The flow rate was 0. ESI capillary voltage was set at 1. For experiments tracing the metabolic fate of palmitate, mice received 13 C 16 -palmitate 2.
A portion of the liver lobule was sampled for snap-freezing using liquid nitrogen. Metabolite extraction from tissues for metabolome analyses was performed as described previously After centrifugation, the aqueous phase was ultrafiltered using an Ultrafree MC-PLHCC ultrafiltration tube Human Metabolome Technologies.
The filtrate was concentrated on a vacuum concentrator SpeedVac, Thermo. The IC was equipped with an anion electrolytic suppressor Thermo Scientific Dionex AERS , which converted the potassium hydroxide gradient into pure water before the sample entered the mass spectrometer.
Separation was performed using a Thermo Scientific Dionex IonPac ASHC, 4-μm particle size column. The IC flow rate was 0. The Q-Exactive focus mass spectrometer was operated under ESI-negative mode for all detections.
Statistical analysis was performed using the unpaired t -test Welch test two-sided. The authors declare that the data supporting the findings of this study are available within the article and its supplementary information. A reporting summary for this article is available as a supplementary information file.
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Autophagy is a lipld catabolic process Autophsgy delivers intracellular proteins Autophagy and lipid metabolism organelles to visceral fat reduction methods lysosome for degradation Autophagy and lipid metabolism recycling. Evidences over Autophgy past decades have proved that autophagy participates metaabolism cell fate Autophagt and also plays a key role Micronutrient-rich diet Autiphagy cellular energy and nutrient stores. Lipid droplets LDs are the main lipid storage form in living organisms. The process of autophagic degradation of LDs is referred to lipophagy or macrolipophagy. Lipophagy is not only indispensable for the cellular lipid metabolism but also closely associated with several metabolic disorders such as obesity, hepatic steatosis, atherosclerosis, and so on. Here, we summarize recent progress in understanding the molecular mechanisms of lipophagy regulation and the emerging roles of lipophagy in various biological processes and metabolic disorders.
The anv is the Autophagyy organ of lipid metabolism and plays a vital role in cellular metabolisms, such as lipid digestion, absorption, liid and decomposition [ Autophagy and lipid metabolism ]. Chronically disturbed hepatic metabolixm may Micronutrient-rich diet oipid obesity and metabolic syndrome, causing Micronutrient-rich diet fatty Plyometric training for athletes disease NAFLD [ 2 ]. Approximately 1 in 30 patients diagnosed with NAFLD develops cirrhosis or a liver-associated complication [ 3 ]. Autophagy plays a crucial role during hepatic lipid metabolism. Diet, environment, and drugs strongly affect hepatic lipid metabolism through autophagy [ 4 ]. Therefore, a further understanding of the specific molecular mechanisms of autophagy and various cell types involved has been deeply explored. The current article reviews the main pathways and regulatory mechanisms of hepatic autophagy and the related effects on lipid metabolism. 
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