Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb;124(2):768-84.
doi: 10.1172/JCI69413. Epub 2014 Jan 16.

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

Lijun Liu, Minwoo Nam, Wei Fan, Thomas E Akie, David C Hoaglin, Guangping Gao, John F Keaney Jr, Marcus P Cooper

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

Lijun Liu et al. J Clin Invest. 2014 Feb.

Abstract

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Fasting coordinately induces mitochondrially encoded transcripts and OXPHOS in liver.
(A) Hepatic gene expression of mitochondrially encoded transcripts in 24-hour fasted or fed C57BL/6 mice (n = 3). (B) Expression of genes that regulate mitochondrial biogenesis, mitochondrial transcription, lipogenesis, and gluconeogenesis (n = 3). (C) Complex activity of mitochondria isolated from liver of 24-hour fasted or fed C57BL/6 mice (n = 3). CI–CV, complexes I–V; CS, citrate synthase. (D) Biochemical assessment of mitochondrial content using citrate synthase activity in whole liver homogenate (n = 3). (E) Genetic assessment of mitochondrial content using mtDNA content (n = 3). (F) Assessment of mitochondrial content by immunoblotting citrate synthase protein in whole liver homogenate (n = 3–4). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA, with (B and C) or without (A) Bonferroni post-test, or 2-tailed unpaired Student’s t test (D and E).
Figure 2
Figure 2. Glucagon induces mitochondrially encoded genes and proteins.
(A) Neither insulin/dexamethasone (Insulin+Dex) nor their withdrawal (Vehicle) had an effect on mitochondrially encoded gene expression; however, glucagon induced mitochondrially encoded gene expression. (B) Effect of glucagon on several nuclear encoded ETC subunits. (C) Effect of glucagon on fasting-responsive genes and regulators of mitochondrial content and function. (D) [35S]-methionine labeling of cytoplasmic proteins (left 2 lanes) and mitochondrially encoded translation products, which were evident after inhibition of cytoplasmic translation with cycloheximide (CHX) (middle 6 lanes). Consistent with mitochondrial translation products, chloramphenicol (CAP) blocked their translation (right 2 lanes). GCG, glucagon. (E) Coomassie brilliant blue staining of the [35S]-labeled gel, showing equal protein loading. (F) Quantification of mitochondrially encoded [35S]-labeled proteins by scintillation courting (n = 3). Data are mean ± SEM. **P < 0.01, ***P < 0.001, 2-way ANOVA, with (B and C) or without (A) Bonferroni post-test, or 2-tailed unpaired Student’s t test (F).
Figure 3
Figure 3. Glucagon-mediated induction of mitochondrially encoded genes requires SIRT3.
(A) Fasted mouse liver showed no change in gene expression for several nuclear encoded ETC genes (n = 3–4). (B) Representative immunoblot of several nuclear encoded ETC subunits. Freshly isolated mitochondria were alkaline extracted with carbonate buffer, permitting assessment of the membrane fraction (Pellet) or soluble fraction. (C) Quantification of the several nuclear encoded ETC subunits (n = 3–4). (D–H) In Sirt3 knockout primary hepatocytes, glucagon-mediated induction of fasting-responsive genes was unaltered (D); however, induction of mitochondrially encoded gene expression was completely abrogated (E; n = 3) and no longer accompanied by increased mitochondrially encoded translation products (F and H). (G) Coomassie brilliant blue staining of [35S]-labeled gel, showing equal protein loading. Similar results were obtained using nicotinamide (see Supplemental Figure 1, E and F). Data are mean ± SEM, except in C (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA with Bonferroni post-test (A, D, and E) or 2-tailed unpaired Student’s t test (C and H).
Figure 4
Figure 4. In liver, SIRT3 is necessary and sufficient for fasting-mediated induction of mitochondrial encoded transcripts and OXPHOS.
(A) Hepatic gene expression of mitochondrially encoded transcripts in 24-hour fasted Sirt3 KO mice (129S background) (n = 3). (B) Expression of genes that regulate mitochondrial biogenesis, mitochondrial transcription, lipogenesis, and gluconeogenesis in Sirt3 KO liver (n = 3). (C) Complex activity from liver of 24-hour fasted or fed Sirt3 KO mice (n = 3). (D) Genetic assessment of mitochondrial content using mtDNA content (n = 3). (E) Biochemical assessment of mitochondrial content using citrate synthase activity in whole liver homogenate (n = 3). (F) Assessment of mitochondrial content by immunoblotting citrate synthase protein in whole liver homogenate (n = 3). (G and H) Expression of (G) mitochondrially encoded transcripts and (H) genes that regulate mitochondrial biogenesis and mitochondrial transcription in H2.35 hepatoma cells stably expressing LacZ or SIRT3 (n = 3). (I) Immunoblot showing expression of ectopically expressed myc-tagged SIRT3 protein. (J) Using a Clark-type oxygen electrode, cellular respiration — basal, proton leak, and maximal respiration (FCCP) — was assessed in H2.35 hepatoma cells stably expressing LacZ or SIRT3. (n = 4). See Supplemental Figure 6 for representative oxygen consumption curves. (K) Fluorescent assessment of mitochondrial content using MitoTracker Green FM (n = 3). (L) Genetic assessment of mitochondrial content using mtDNA content (n = 3). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA, with (A and G) or without (B and H) Bonferroni post-test, or 2-tailed unpaired Student’s t test (C–E and J–L).
Figure 5
Figure 5. SIRT3 induces mitochondrially encoded genes by stimulating mitochondrial transcription.
(A) Metabolic labeling with 4sU. This method quantifies de novo transcripts, permitting assessment of mitochondrial transcription in whole cells. It can also be used to monitor transcript degradation. (B) Transcription of Sirt3, driven by the viral CMV promoter, was induced in cells replete with SIRT3, whereas transcription of several housekeeping genes was unchanged (n = 3). (C) Cells stably expressing SIRT3 exhibited increased mitochondrial transcription (n = 3). (D) Uridine was used to chase the 4sU label and assess transcript half-life. Half-lives of mitochondrial transcripts in H2.35 hepatoma cells stably expressing SIRT3 were unchanged. Data are mean ± SEM. ****P < 0.0001, 2-way ANOVA, with (B) or without (C) Bonferroni post-test.
Figure 6
Figure 6. SIRT3-mediated induction of mitochondrial transcription requires LRP130.
(A–D) Gene expression of Lrp130 and Sirt3 (A), representative immunoblot of LRP130 and SIRT3 (B), gene expression of mitochondrially encoded transcripts (C), and expression of genes that regulate mitochondrial biogenesis and mitochondrial transcription (D) in H2.35 hepatoma cells replete with LacZ or SIRT3, superimposed with control (shCtrl) or Lrp130 knockdown (shLRP130) (n = 3). (E–G) H2.35 hepatoma cells replete with SIRT3 or LacZ, but deficient for LRP130, were used. (E) SIRT3 was no longer sufficient to influence maximal respiration, using a Clark-type oxygen electrode (n = 6). However, control shRNA SIRT3 cells retained increased respiratory capacity (see Supplemental Figure 6). Assessment of mitochondrial content was unchanged, as assessed by (F) MitoTracker Green FM or (G) mtDNA content (n = 3). (H–K) LRP130 liver-specific knockout (LRP130 LKO) mice were used. Hepatic gene (H) and protein (I) expression (n = 4). (J) Effect of fasting on mitochondrially encoded genes in liver. (K) Effect of fasting on fasting-responsive genes in liver. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA, with (A, D, H, and K) or without (C and J) Bonferroni post-test, or 2-tailed unpaired Student’s t test (E–G).
Figure 7
Figure 7. During the fasted response, SIRT3 deacetylates LRP130 in liver mitochondria.
(A) Representative immunoblot and protein quantification showing reduced acetylation of LRP130 (Ac-LRP130) in mitochondria isolated from fasted mouse liver (n = 3 experiments). (B) Immunoblot and protein quantification showing hyperacetylation of LRP130 in mitochondria isolated from liver deficient for SIRT3 (n = 4). (C) Immunoblot showing deacetylation of LRP130 in H2.35 cells stably expressing SIRT3 upon treatment with 500 μM NAD+. (D) SIRT3 and LRP130 coimmunoprecipitated, using ectopically expressed SIRT3-FLAG protein and endogenous LRP130. (E) Using purified proteins, SIRT3 robustly deacetylated the C terminus of LRP130. Deacetylation of LRP130 was inhibited by 12.5 mM nicotinamide (NAM). Shown is 1 representative of 4 independent experiments. Similar, but less robust, deacetylation was obtained for the N terminus of LRP130 (not shown). (F) Acetylated LRP130 fragments were incubated with control buffer or purified SIRT3 protein, then subjected to mass spectrometry. Lysines showing greater than 50% deacetylation by SIRT3 are graphed (gray and black bars). Percent deacetylation was calculated as 1 – (SIRT3 signal/control signal). 7 lysines were mutated to arginines (gray bars), generating LRP130-7KR, which mimics deacetylated protein. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired Student’s t test (A and B).
Figure 8
Figure 8. LRP130-7KR stimulates mitochondrial transcription and promotes OXPHOS.
(A) LRP130-7KR, which mimics deacetylated protein, showed increased affinity for aas 1–710 of POLRMT, a fragment that contains the catalytic domain. See Supplemental Figure 10 for more detailed mapping. (B) In contrast, compared with LRP130-WT, LRP130-7KR showed no differential affinity for TFB2M. (C, D, and F) Using 293T cells, endogenous human LRP130 was knocked down >95%, then reconstituted with murine LRP130-WT or LRP130-7KR (see Supplemental Figure 11). The shRNA targeting human LRP130 does not target mouse Lrp130 (not shown). Endogenous NAD+-dependent sirtuin activity was inhibited with 10 mM nicotinamide for 16 hours. LRP130-7KR had greater (C) mitochondrially encoded gene expression (n = 3), (D) mitochondrial transcription (n = 3), and (F) maximal respiration (n = 5) versus LRP130-WT. (E) Immunoblot showing similar levels of ectopically expressed LRP130-WT and LRP130-7KR. See Supplemental Figure 11 for total LRP130 protein. Data are mean ± SEM. *P < 0.05, ***P < 0.001, 2-way ANOVA (C and D) or 2-tailed unpaired Student’s t test (F).
Figure 9
Figure 9. In fasted liver, the transcription machinery of mitochondria sense nutrient deprivation via SIRT3, culminating in enhanced energy metabolism.
In liver, SIRT3 is induced by nutrient deprivation. Specifically, glucagon, which activates cAMP signaling, increases SIRT3 activity, perhaps by indirectly increasing mitochondrial NAD+ levels. LRP130 is then deacetylated and activated by SIRT3, which strengthens the LRP130-POLRMT interaction. This culminates in increased mitochondrial transcription and attendant OXPHOS. Presumably, processes dependent on OXPHOS — fatty acid oxidation, gluconeogenesis, ketogenesis, and ureagenesis — are augmented by increased respiratory capacity. To simplify the model, deacetylation of enzymes involved in fatty acid oxidation and ketogenesis, as well as SIRT3 action on individual OXPHOS subunits, are not illustrated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Finck BN, et al. A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes. Cell Metab. 2005;1(2):133–144. doi: 10.1016/j.cmet.2005.01.006. - DOI - PubMed
    1. Koves TR, et al. Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metab. 2008;7(1):45–56. doi: 10.1016/j.cmet.2007.10.013. - DOI - PubMed
    1. Satapati S, et al. Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver. J Lipid Res. 2012;53(6):1080–1092. doi: 10.1194/jlr.M023382. - DOI - PMC - PubMed
    1. Houtkooper RH, Pirinen E, Auwerx J. Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol. 2012;13(4):225–238. - PMC - PubMed
    1. Hirschey MD, et al. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Nature. 2010;464(7285):121–125. doi: 10.1038/nature08778. - DOI - PMC - PubMed

Publication types

MeSH terms