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. 2016 Dec 13;24(6):863-874.
doi: 10.1016/j.cmet.2016.10.012. Epub 2016 Nov 17.

Physiological Suppression of Lipotoxic Liver Damage by Complementary Actions of HDAC3 and SCAP/SREBP

Romeo Papazyan  1 , Zheng Sun  2 , Yong Hoon Kim  1 , Paul M Titchenell  1 , David A Hill  1 , Wenyun Lu  3 , Manashree Damle  1 , Min Wan  1 , Yuxiang Zhang  1 , Erika R Briggs  1 , Joshua D Rabinowitz  3 , Mitchell A Lazar  4
Affiliations

Physiological Suppression of Lipotoxic Liver Damage by Complementary Actions of HDAC3 and SCAP/SREBP

Romeo Papazyan et al. Cell Metab. .

Abstract

Liver fat accumulation precedes non-alcoholic steatohepatitis, an increasing cause of end-stage liver disease. Histone deacetylase 3 (HDAC3) is required for hepatic triglyceride homeostasis, and sterol regulatory element binding protein (SREBP) regulates the lipogenic response to feeding, but the crosstalk between these pathways is unknown. Here we show that inactivation of SREBP by hepatic deletion of SREBP cleavage activating protein (SCAP) abrogates the increase in lipogenesis caused by loss of HDAC3, but fatty acid oxidation remains defective. This combination leads to accumulation of lipid intermediates and to an energy drain that collectively cause oxidative stress, inflammation, liver damage, and, ultimately, synthetic lethality. Remarkably, this phenotype is prevented by ectopic expression of nuclear SREBP1c, revealing a surprising benefit of de novo lipogenesis and triglyceride synthesis in preventing lipotoxicity. These results demonstrate that HDAC3 and SCAP control symbiotic pathways of liver lipid metabolism that are critical for suppression of lipotoxicity.

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Figures

Figure 1
Figure 1. HDAC3 and SREBP1c independently bind near lipogenic genes to regulate their transcription
(A) Heat map of ChIP-seq peaks in mouse liver. C57Bl/6 mice (2–3 months old) were tail-vein injected with either AAV8:GFP or AAV8:HA-nSREBP1c and sacrificed 10 days later at ZT10. All ChIPs were performed in parallel. High-confidence peaks were clustered into 3 groups: common peaks where HA-nSREBP1c and HDAC3 peaks overlapped (purple), nSREBP1c-specfic peaks (red), and HDAC3-specific peaks (blue). (B) Average binding profiles in 2 kb windows. Black and brown lines represent HDAC3 peaks in AAV8:GFP and AAV8:HA-nSREBP1c injected mice, respectively. Green lines represent the HA-nSREBP1c peaks. (C) De novo motif analysis using 100 bp search windows. The top 6 motifs are shown in each group and ordered according to their percent enrichment (%) in peaks for each assigned group. See also Figure S1.
Figure 2
Figure 2. HDAC3 partially suppresses fatty liver formation in SCAP depleted livers
(A) Quantitative RT-PCR (RT-qPCR) analysis of livers from floxed mice fed a normal chow diet and harvested 10 days post AAV8 injection (Control, AAV8:GFP; LKO, AAV8:Cre). n = 4 to 5 mice per group. (B) Western blot of HDAC3, SCAP and full-length SREBP1 precursor in total liver lysates. Control mice are random littermate floxed mice injected with AAV8:GFP. (C) Liver triglyceride (TAG) measurements from normal chow fed mice described in (A). (D) RT-qPCR analysis of lipogenic genes in livers from normal chow fed mice described in (A). (E) Hepatic TAG measurements from mice fed a HFD for 8 weeks total (AAV8 injection at 6 weeks). These mice were 8 weeks old at the start of the HFD. n = 4 to 5 mice per group. (F and G) Representative H&E (F) and ORO (G) staining of livers from HFD fed mice described in (E). Scale bar, 100 μm. All error bars, s.e.m. Significance was determined by two-tailed Student’s t test (*P < 0.05). See also Figure S2.
Figure 3
Figure 3. Decreased fatty acid oxidation and accumulation of lipid intermediates in livers lacking HDAC3 and SCAP
(A) Fatty acid oxidation measured in primary hepatocytes. Control cells are from random littermate floxed mice injected with AAV8:GFP. SCAP LKO and DLKO cells are from SCAPf/f and HDAC3f/f/SCAPf/f mice, respectively, injected with AAV8:Cre. Cells were incubated with 3H-palmitate for 120 min with vehicle (water) or 100 μM etomoxir (an inhibitor of carnitine palmitoyltransferase-1 and fatty acid oxidation). 3H-labelled water (3H2O) was measured by scintillation counting. dpm, disintegrations per minute; 3H-palmitate > 3H2O, 3H-palmitate conversion to 3H2O. n = 6 wells (combined from 3 mice) per group. Significance was determined by one-way ANOVA with Tukey’s correction (*P < 0.05 for comparison between experimental and control cells). (B and C) Hepatic acylcarnitine (B) and free fatty acid (FFA) (C) content measured by liquid chromatography mass spectrometry. Average ion counts of each metabolite across all conditions are shown. n = 4 mice per group. Significance was determined by two-tailed Student’s t test followed by FDR correction (*P < 0.05). Ion counts are available in Table S1. (D and E) RT-qPCR analysis of lipid catabolic genes (D) and fatty acid esterification genes (E) in livers. n = 4 to 5 mice per group. Significance was determined by two-tailed Student’s t test (*P < 0.05). (F) Fatty acid metabolism pathway in DLKO mice. #Acyl-CoA levels were not measured. All error bars, s.e.m. Control, AAV8:GFP injected; LKO, AAV8:Cre injected. See also Figure S3.
Figure 4
Figure 4. Lack of HDAC3 and SCAP causes hepatic lipotoxicity, oxidative stress, and inflammation
(A) Relative ATP/ADP and ATP/AMP ratios in DLKO livers as measured by liquid chromatography mass spectrometry. n = 4 mice per group. (B) Liver thiobarbituric acid reactive substances (TBARS) assay showing levels of malondialdehyde (MDA) formation in 24 h fasted mice. n = 7 to 9 mice per group. (C and D) RT-qPCR analysis of antioxidant genes (C) and inflammation genes (D) identified by RNA-seq. n = 4 to 5 mice per group. (E) Western blot of total liver lysates. # denotes the subset of DLKO mice in which the pro-apoptotic factor JNK was activated by phosphorylation. (F) Number of immune cells from control (n = 4) or DLKO (n = 3) mice, as determined by flow cytometry and total cell number. Control and DLKO mice were sacrificed 15 days post AAV8 injections. All error bars, s.e.m. Control, AAV8:GFP injected; LKO, AAV8:Cre injected. Significance was determined by two-tailed Student’s t test (*P < 0.05). See also Figure S4.
Figure 5
Figure 5. Loss of hepatic HDAC3 and SCAP is synthetic lethal
(A) Kaplan-Meier survival curve of indicated LKO mice. n = 4 mice per group. Day 10 cohort (d10), mice were harvested 10 days post AAV8 injection. n = 4 to 5 mice per group. Moribund cohort (Pre-†), DLKO mice were monitored daily and were sacrificed when they presented with weight loss, inactivity, and non-responsiveness. Livers from control floxed mice in this cohort were harvested ~15 days post AAV8:GFP injection. n = 3 to 6 mice per group. (B) Body mass of mice from d10 and Pre-† mice. (C) Mass of epididymal white adipose tissue of Pre-† mice. (D) Levels of serum non-esterified fatty acids (NEFA) in d10 and Pre-† mice. (E) Levels of serum alanine transaminase (ALT) in d10 and Pre-† mice. (F) H&E staining of representative livers from d10 and Pre-† mice. Green arrowheads indicate immune cell infiltrates. Yellow arrowheads indicate damaged tissue. Scale bar, 100 μm. All error bars, s.e.m. Control, AAV8:GFP injected; LKO, AAV8:Cre injected. Significance was determined by two-tailed Student’s t test (*P < 0.05). See also Figure S5.
Figure 6
Figure 6. Restoration of DNL and TAG synthesis prevents lipotoxicity-induced liver damage
(A) RT-qPCR analysis of liver gene expression from Control (HDAC3f/f/SCAPf/f injected with AAV8:GFP + AAV8:Null), DLKO (HDAC3f/f/SCAPf/f injected with AAV8:Cre + AAV8:Null) and DLKO+nSREBP1c (HDAC3f/f/SCAPf/f injected with AAV8:Cre + AAV8:HA-nSREBP1c) mice sacrificed 10 days post AAV8 injection. n = 5 mice per group. (B) Hepatic TAG measurements in mice described in (A). (C) H&E staining of representative livers from Control, DLKO, and DLKO+nSREBP1c mice from (A). Green arrowhead points to a density of immune cell infiltrates that are lacking in Control and DLKO+nSREBP1c livers. Scale bar, 100 μm. (D) RT-qPCR analysis of liver antioxidant and inflammatory gene expression from the mice described in (A). (E) Kaplan-Meier survival curve of DLKO and DLKO+nSREBP1c mice. n = 4 mice per group. (F) Hepatic DNL rate in vivo measured by tracing hepatic 2H-palmitate synthesis from deuterated water during a 6-hour interval. n = 4 mice per group. (G and H) Hepatic levels of acylcarnitines (G) and other lipids (H) as measured by liquid chromatography mass spectrometry. Average ion counts of each metabolite across all conditions are shown in (G). Biological replicates are depicted in each row and each column represents a specific lipid metabolite in (H). The heat map highlights relative changes for each lipid metabolite with respect to its average ion counts in all three conditions. FFA, free fatty acids. MAG, monoacylglycerols. DAG, diacylglycerol. TAG, triacylglycerol. n = 4 to 5 mice per group. These mice were sacrificed 12 days after AAV8 injections. Ion counts and lipid identifications are available in Table S4. (I) Summary of lipid metabolism in HDAC3 LKO, SCAP LKO, and DLKO livers. All error bars, s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak correction (A,B) or Tukey’s correction (B,F) (*P < 0.05 for comparison between control and experimental mice). (G and H) Significance of lipidomics data was determined by one-way ANOVA followed by FDR correction (*P < 0.05). See also Figure S6.
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Comment in

  • A new pathway to eSCAPe lipotoxicity.
    Benhamed F, Postic C. Benhamed F, et al. Clin Res Hepatol Gastroenterol. 2018 Feb;42(1):3-5. doi: 10.1016/j.clinre.2017.10.005. Epub 2017 Nov 22. Clin Res Hepatol Gastroenterol. 2018. PMID: 29174379

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