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SIRT1-metabolite binding histone macroH2A1.1 protects hepatocytes against lipid accumulation.

Pazienza V, Borghesan M, Mazza T, Sheedfar F, Panebianco C, Williams R, Mazzoccoli G, Andriulli A, Nakanishi T, Vinciguerra M - Aging (Albany NY) (2014)

Bottom Line: The functional significance of this binding is unknown.Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation.MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Gastroenterology Unit, IRCCS "Casa Sollievo della Sofferenza" Hospital, San Giovanni Rotondo, Italy.

ABSTRACT
Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development.

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Overexpression of macroH2A1 isoforms (macroH2A1.1 or macroH2A1.2) and lipid accumulation in Hepa1-6 cells and HepG2 cells. Upper panels: cells were transiently transfected with Lipofectamine with either an empty vector (control, CTL) or with Cherry-tagged macroH2A1.1 and macroH2A1.1 constructs. 24 hours post transfection cells were exposed to a 100 mM 1:1 mixture of oleic acid/linoleic acid (FFA) conjugated with albumin, for an additional 24 hours. Cells were then fixed, nuclei stained with DAPI (blue) and lipids with ORO. Overlay of Cherry tagged macroH2A1-transfected nuclei and DAPI staining is observed in pink. Lower panel: quantifications of ORO stained areas are means ± SEM of 1000 cells per condition. *p<0.05.
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Figure 2: Overexpression of macroH2A1 isoforms (macroH2A1.1 or macroH2A1.2) and lipid accumulation in Hepa1-6 cells and HepG2 cells. Upper panels: cells were transiently transfected with Lipofectamine with either an empty vector (control, CTL) or with Cherry-tagged macroH2A1.1 and macroH2A1.1 constructs. 24 hours post transfection cells were exposed to a 100 mM 1:1 mixture of oleic acid/linoleic acid (FFA) conjugated with albumin, for an additional 24 hours. Cells were then fixed, nuclei stained with DAPI (blue) and lipids with ORO. Overlay of Cherry tagged macroH2A1-transfected nuclei and DAPI staining is observed in pink. Lower panel: quantifications of ORO stained areas are means ± SEM of 1000 cells per condition. *p<0.05.

Mentions: MacroH2A1.2, but not of macroH2A1.1, is upregulated in the liver of NAFLD in vivo models [21]; however the function of these isoforms in NAFLD is unknown. We examined the effect of macroH2A1 isoforms on lipid accumulation in two well established hepatic cell lines, Hepa1-6 and HepG2 cells [26, 27]. HepG2 cells expressed more abundant endogenous levels of macroH2A1.1 and macroH2A1.2 than Hepa1-6 cells. (Figure 1B) However, within each cell line similar endogenous levels of macroH2A1.1 and macroH2A1.2 were expressed (Figure 1B): this not being confounding factor, we ectopically over-expressed one or the other isoform. Transient transfection with cherry-tagged macroH2A1.1 or macroH2A1.2 constructs (Figure 1C, left) yielded a 30-40% efficiency (Figure 1C, right) and did not have any effect on the cell cycle, as measured by the percentage of cells gated in the G0/G1, S and G2/M phases by flow cytometry in Hepa1-6 cells (Table I, n=3). Similar results were obtained in HepG2 cells (data not shown). 24 hours post-trasfection Hepa1-6 and HepG2 cells were treated with a 100 μM 1:1 mixture of FFA (oleic acid and linoleic acid) for an additional 24 hours, when cells were fixed and lipids were stained using ORO. Upon counterstaining with DAPI (blue), nuclei of Cherry-tagged macroH2A1.1 and macroH2A1.2 transfected cells were observed in pink/orange (Figures 2A and B). Morphometric evaluation of cytoplasmic ORO staining showed that macroH2A1.1-overexpressing Hepa 1-6 (Figure S1, left upper panels) or HepG2 (Figure 2, right upper panels) cells were protected from lipid accumulation as compared with control cells transfected with an empty vector, while macroH2A1.2-overexpressing cells only slightly enhanced lipid content. In Hepa1-6 this trend was statistically significant in FFA-treated macroH2A1.1- or macroH2A1.2-overexpressing cells (Figure 2, lower left panel), while in HepG2 significativity was obtained for FFA-treated macroH2A1.1-overexpressing cells (Figure 2, lower right panel). Intrahepatic lipid droplets as observed in NAFLD are constituted mainly of triglycerides (TG), synthesized upon FFA intake/synthesis and cholesteryl-esters, which are instead synthesized upon augmented levels of free cholesterol. MacroH2A1.1-overexpressing Hepa1-6 (Figure 1D, left panel) or HepG2 (Figure 1D, right panel) cells consistently displayed a decreased content of TG, compared to macroH2A1.2-overexpressing and control cells. As for cholesterol content, macroH2A1.1-overexpressing Hepa1-6 and HepG2 cells showed a lower content when compared to control cells upon FFA exposure (Figure 1E). MacroH2A1.1 overexpressing HepG2 cells showed lower levels of cholesterol also in absence of FFA (Figure 1E). These data demonstrate for the first time that metabolite-binding histone variant macroH2A1.1, but not macroH2A1.2, protects against hepatic lipid accumulation in vitro.


SIRT1-metabolite binding histone macroH2A1.1 protects hepatocytes against lipid accumulation.

Pazienza V, Borghesan M, Mazza T, Sheedfar F, Panebianco C, Williams R, Mazzoccoli G, Andriulli A, Nakanishi T, Vinciguerra M - Aging (Albany NY) (2014)

Overexpression of macroH2A1 isoforms (macroH2A1.1 or macroH2A1.2) and lipid accumulation in Hepa1-6 cells and HepG2 cells. Upper panels: cells were transiently transfected with Lipofectamine with either an empty vector (control, CTL) or with Cherry-tagged macroH2A1.1 and macroH2A1.1 constructs. 24 hours post transfection cells were exposed to a 100 mM 1:1 mixture of oleic acid/linoleic acid (FFA) conjugated with albumin, for an additional 24 hours. Cells were then fixed, nuclei stained with DAPI (blue) and lipids with ORO. Overlay of Cherry tagged macroH2A1-transfected nuclei and DAPI staining is observed in pink. Lower panel: quantifications of ORO stained areas are means ± SEM of 1000 cells per condition. *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927808&req=5

Figure 2: Overexpression of macroH2A1 isoforms (macroH2A1.1 or macroH2A1.2) and lipid accumulation in Hepa1-6 cells and HepG2 cells. Upper panels: cells were transiently transfected with Lipofectamine with either an empty vector (control, CTL) or with Cherry-tagged macroH2A1.1 and macroH2A1.1 constructs. 24 hours post transfection cells were exposed to a 100 mM 1:1 mixture of oleic acid/linoleic acid (FFA) conjugated with albumin, for an additional 24 hours. Cells were then fixed, nuclei stained with DAPI (blue) and lipids with ORO. Overlay of Cherry tagged macroH2A1-transfected nuclei and DAPI staining is observed in pink. Lower panel: quantifications of ORO stained areas are means ± SEM of 1000 cells per condition. *p<0.05.
Mentions: MacroH2A1.2, but not of macroH2A1.1, is upregulated in the liver of NAFLD in vivo models [21]; however the function of these isoforms in NAFLD is unknown. We examined the effect of macroH2A1 isoforms on lipid accumulation in two well established hepatic cell lines, Hepa1-6 and HepG2 cells [26, 27]. HepG2 cells expressed more abundant endogenous levels of macroH2A1.1 and macroH2A1.2 than Hepa1-6 cells. (Figure 1B) However, within each cell line similar endogenous levels of macroH2A1.1 and macroH2A1.2 were expressed (Figure 1B): this not being confounding factor, we ectopically over-expressed one or the other isoform. Transient transfection with cherry-tagged macroH2A1.1 or macroH2A1.2 constructs (Figure 1C, left) yielded a 30-40% efficiency (Figure 1C, right) and did not have any effect on the cell cycle, as measured by the percentage of cells gated in the G0/G1, S and G2/M phases by flow cytometry in Hepa1-6 cells (Table I, n=3). Similar results were obtained in HepG2 cells (data not shown). 24 hours post-trasfection Hepa1-6 and HepG2 cells were treated with a 100 μM 1:1 mixture of FFA (oleic acid and linoleic acid) for an additional 24 hours, when cells were fixed and lipids were stained using ORO. Upon counterstaining with DAPI (blue), nuclei of Cherry-tagged macroH2A1.1 and macroH2A1.2 transfected cells were observed in pink/orange (Figures 2A and B). Morphometric evaluation of cytoplasmic ORO staining showed that macroH2A1.1-overexpressing Hepa 1-6 (Figure S1, left upper panels) or HepG2 (Figure 2, right upper panels) cells were protected from lipid accumulation as compared with control cells transfected with an empty vector, while macroH2A1.2-overexpressing cells only slightly enhanced lipid content. In Hepa1-6 this trend was statistically significant in FFA-treated macroH2A1.1- or macroH2A1.2-overexpressing cells (Figure 2, lower left panel), while in HepG2 significativity was obtained for FFA-treated macroH2A1.1-overexpressing cells (Figure 2, lower right panel). Intrahepatic lipid droplets as observed in NAFLD are constituted mainly of triglycerides (TG), synthesized upon FFA intake/synthesis and cholesteryl-esters, which are instead synthesized upon augmented levels of free cholesterol. MacroH2A1.1-overexpressing Hepa1-6 (Figure 1D, left panel) or HepG2 (Figure 1D, right panel) cells consistently displayed a decreased content of TG, compared to macroH2A1.2-overexpressing and control cells. As for cholesterol content, macroH2A1.1-overexpressing Hepa1-6 and HepG2 cells showed a lower content when compared to control cells upon FFA exposure (Figure 1E). MacroH2A1.1 overexpressing HepG2 cells showed lower levels of cholesterol also in absence of FFA (Figure 1E). These data demonstrate for the first time that metabolite-binding histone variant macroH2A1.1, but not macroH2A1.2, protects against hepatic lipid accumulation in vitro.

Bottom Line: The functional significance of this binding is unknown.Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation.MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Gastroenterology Unit, IRCCS "Casa Sollievo della Sofferenza" Hospital, San Giovanni Rotondo, Italy.

ABSTRACT
Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development.

Show MeSH
Related in: MedlinePlus