Limits...
eIF6 coordinates insulin sensitivity and lipid metabolism by coupling translation to transcription.

Brina D, Miluzio A, Ricciardi S, Clarke K, Davidsen PK, Viero G, Tebaldi T, Offenhäuser N, Rozman J, Rathkolb B, Neschen S, Klingenspor M, Wolf E, Gailus-Durner V, Fuchs H, Hrabe de Angelis M, Quattrone A, Falciani F, Biffo S - Nat Commun (2015)

Bottom Line: Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin.The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes.Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA.

View Article: PubMed Central - PubMed

Affiliation: INGM, 'Romeo ed Enrica Invernizzi', 20122 Milano, Italy.

ABSTRACT
Insulin regulates glycaemia, lipogenesis and increases mRNA translation. Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin. The role of insulin-regulated translation is unknown. Here we show that reduction of insulin-regulated translation in mice heterozygous for eIF6 results in normal glycaemia, but less blood cholesterol and triglycerides. eIF6 controls fatty acid synthesis and glycolysis in a cell autonomous fashion. eIF6 acts by exerting translational control of adipogenic transcription factors like C/EBPβ, C/EBPδ and ATF4 that have G/C rich or uORF sequences in their 5' UTR. The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes. Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA. Since obesity, type 2 diabetes, and cancer require a Fasn-driven lipogenic state, we propose that eIF6 could be a therapeutic target for these diseases.

No MeSH data available.


Related in: MedlinePlus

eIF6 translational activity controls the expression of lipogenic transcription factors.(a) Global initiation is reduced on eIF6 inhibition and insulin administration, as shown by polysome/80S ratio. Data are represented as mean±s.d. Statistical P values were calculated by two-tailed t-test (n=4. ***P value ≤0.001). (b) Outline of the analysis and quantitative results: a subset of mRNA is differentially depleted (green) from eIF6 shRNA polysomes. The normalized ratio was obtained by the quantile method. Correction taking in account the reduction of polysomal area shows that more genes are affected by eIF6 deficiency. (c) Gene Ontology analysis shows that metabolism predominates in eIF6-affected genes, from the normalized ratio pool (P value ≤10−12). (d) Structural 5′-UTR analysis of genes affected by eIF6 deficiency. Polysomes from eIF6-depleted cells show reduction of G/C rich 5′ mRNAs (green box) (e) Transcription factors involved in lipogenesis are depleted from polysomes upon eIF6 shRNA. Structural features of their 5′ UTR are included. (f) C/EBPδ protein expression is modulated by eIF6. eIF6 increase, by lentiviral administration, induces C/EBPδ. eIF6 depletion, by lentiviral-mediated shRNA, reduces C/EBPδ. Representative experiments done in triplicates on AML12 hepatocytes and reconstituted mesenchymal stem cells (g) Lipogenic transcription factor C/EBPβ (LIP isoform) is reduced in eIF6-depleted cells. Left, mRNA structure of C/EBPβ generating three isoforms by differential translation (arrows: translation start sites). Blue small box with blue arrows is the C/EBP uORF. Right panel show a blot with LAP, LIP and C/EBPβ isoforms: LIP isoform is specifically downregulated. (Representative experiment done in triplicates). (h) ATF4 reporter translation depends from eIF6. Reporter assay of reinitiation with luciferase (blue box) cloned downstream of natural ATF4 uORF. eIF6 upregulation increases ATF4 translational reporter activity, eIF6 downregulation decreases it. Data are normalized on translation of a cap-dependent firefly control. The large black arrow represents the stop codon of the second uORF. Small arrows are start codons. Blue box is the reporter. Statistical P values were calculated by two-tailed t-test, as above (*P value ≤0.05; **P≤0.01; ***P≤0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4595657&req=5

f3: eIF6 translational activity controls the expression of lipogenic transcription factors.(a) Global initiation is reduced on eIF6 inhibition and insulin administration, as shown by polysome/80S ratio. Data are represented as mean±s.d. Statistical P values were calculated by two-tailed t-test (n=4. ***P value ≤0.001). (b) Outline of the analysis and quantitative results: a subset of mRNA is differentially depleted (green) from eIF6 shRNA polysomes. The normalized ratio was obtained by the quantile method. Correction taking in account the reduction of polysomal area shows that more genes are affected by eIF6 deficiency. (c) Gene Ontology analysis shows that metabolism predominates in eIF6-affected genes, from the normalized ratio pool (P value ≤10−12). (d) Structural 5′-UTR analysis of genes affected by eIF6 deficiency. Polysomes from eIF6-depleted cells show reduction of G/C rich 5′ mRNAs (green box) (e) Transcription factors involved in lipogenesis are depleted from polysomes upon eIF6 shRNA. Structural features of their 5′ UTR are included. (f) C/EBPδ protein expression is modulated by eIF6. eIF6 increase, by lentiviral administration, induces C/EBPδ. eIF6 depletion, by lentiviral-mediated shRNA, reduces C/EBPδ. Representative experiments done in triplicates on AML12 hepatocytes and reconstituted mesenchymal stem cells (g) Lipogenic transcription factor C/EBPβ (LIP isoform) is reduced in eIF6-depleted cells. Left, mRNA structure of C/EBPβ generating three isoforms by differential translation (arrows: translation start sites). Blue small box with blue arrows is the C/EBP uORF. Right panel show a blot with LAP, LIP and C/EBPβ isoforms: LIP isoform is specifically downregulated. (Representative experiment done in triplicates). (h) ATF4 reporter translation depends from eIF6. Reporter assay of reinitiation with luciferase (blue box) cloned downstream of natural ATF4 uORF. eIF6 upregulation increases ATF4 translational reporter activity, eIF6 downregulation decreases it. Data are normalized on translation of a cap-dependent firefly control. The large black arrow represents the stop codon of the second uORF. Small arrows are start codons. Blue box is the reporter. Statistical P values were calculated by two-tailed t-test, as above (*P value ≤0.05; **P≤0.01; ***P≤0.001).

Mentions: Data from Figs 1 and 2 suggest that eIF6 drives the translation of specific factors that regulate a lipogenic switch. We analysed how eIF6 modulates insulin-stimulated translation by using a fast doxycycline-inducible system, combined with polysomal microarray. Forty-eight hours after doxycycline treatment, AML12 cells with inducible eIF6 shRNA presented a reduction of eIF6 (Supplementary Fig. 3a) and resistance to insulin-stimulated methionine incorporation (Supplementary Fig. 3b). Polysomal analysis of insulin-stimulated cells showed that eIF6 downregulation reduced the polysome/80S ratio as expected by the loss of eIF6 anti-association activity (Fig. 3a). In unstimulated conditions eIF6 deficiency did not affect polysome peaks (Supplementary Fig. 3c).


eIF6 coordinates insulin sensitivity and lipid metabolism by coupling translation to transcription.

Brina D, Miluzio A, Ricciardi S, Clarke K, Davidsen PK, Viero G, Tebaldi T, Offenhäuser N, Rozman J, Rathkolb B, Neschen S, Klingenspor M, Wolf E, Gailus-Durner V, Fuchs H, Hrabe de Angelis M, Quattrone A, Falciani F, Biffo S - Nat Commun (2015)

eIF6 translational activity controls the expression of lipogenic transcription factors.(a) Global initiation is reduced on eIF6 inhibition and insulin administration, as shown by polysome/80S ratio. Data are represented as mean±s.d. Statistical P values were calculated by two-tailed t-test (n=4. ***P value ≤0.001). (b) Outline of the analysis and quantitative results: a subset of mRNA is differentially depleted (green) from eIF6 shRNA polysomes. The normalized ratio was obtained by the quantile method. Correction taking in account the reduction of polysomal area shows that more genes are affected by eIF6 deficiency. (c) Gene Ontology analysis shows that metabolism predominates in eIF6-affected genes, from the normalized ratio pool (P value ≤10−12). (d) Structural 5′-UTR analysis of genes affected by eIF6 deficiency. Polysomes from eIF6-depleted cells show reduction of G/C rich 5′ mRNAs (green box) (e) Transcription factors involved in lipogenesis are depleted from polysomes upon eIF6 shRNA. Structural features of their 5′ UTR are included. (f) C/EBPδ protein expression is modulated by eIF6. eIF6 increase, by lentiviral administration, induces C/EBPδ. eIF6 depletion, by lentiviral-mediated shRNA, reduces C/EBPδ. Representative experiments done in triplicates on AML12 hepatocytes and reconstituted mesenchymal stem cells (g) Lipogenic transcription factor C/EBPβ (LIP isoform) is reduced in eIF6-depleted cells. Left, mRNA structure of C/EBPβ generating three isoforms by differential translation (arrows: translation start sites). Blue small box with blue arrows is the C/EBP uORF. Right panel show a blot with LAP, LIP and C/EBPβ isoforms: LIP isoform is specifically downregulated. (Representative experiment done in triplicates). (h) ATF4 reporter translation depends from eIF6. Reporter assay of reinitiation with luciferase (blue box) cloned downstream of natural ATF4 uORF. eIF6 upregulation increases ATF4 translational reporter activity, eIF6 downregulation decreases it. Data are normalized on translation of a cap-dependent firefly control. The large black arrow represents the stop codon of the second uORF. Small arrows are start codons. Blue box is the reporter. Statistical P values were calculated by two-tailed t-test, as above (*P value ≤0.05; **P≤0.01; ***P≤0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595657&req=5

f3: eIF6 translational activity controls the expression of lipogenic transcription factors.(a) Global initiation is reduced on eIF6 inhibition and insulin administration, as shown by polysome/80S ratio. Data are represented as mean±s.d. Statistical P values were calculated by two-tailed t-test (n=4. ***P value ≤0.001). (b) Outline of the analysis and quantitative results: a subset of mRNA is differentially depleted (green) from eIF6 shRNA polysomes. The normalized ratio was obtained by the quantile method. Correction taking in account the reduction of polysomal area shows that more genes are affected by eIF6 deficiency. (c) Gene Ontology analysis shows that metabolism predominates in eIF6-affected genes, from the normalized ratio pool (P value ≤10−12). (d) Structural 5′-UTR analysis of genes affected by eIF6 deficiency. Polysomes from eIF6-depleted cells show reduction of G/C rich 5′ mRNAs (green box) (e) Transcription factors involved in lipogenesis are depleted from polysomes upon eIF6 shRNA. Structural features of their 5′ UTR are included. (f) C/EBPδ protein expression is modulated by eIF6. eIF6 increase, by lentiviral administration, induces C/EBPδ. eIF6 depletion, by lentiviral-mediated shRNA, reduces C/EBPδ. Representative experiments done in triplicates on AML12 hepatocytes and reconstituted mesenchymal stem cells (g) Lipogenic transcription factor C/EBPβ (LIP isoform) is reduced in eIF6-depleted cells. Left, mRNA structure of C/EBPβ generating three isoforms by differential translation (arrows: translation start sites). Blue small box with blue arrows is the C/EBP uORF. Right panel show a blot with LAP, LIP and C/EBPβ isoforms: LIP isoform is specifically downregulated. (Representative experiment done in triplicates). (h) ATF4 reporter translation depends from eIF6. Reporter assay of reinitiation with luciferase (blue box) cloned downstream of natural ATF4 uORF. eIF6 upregulation increases ATF4 translational reporter activity, eIF6 downregulation decreases it. Data are normalized on translation of a cap-dependent firefly control. The large black arrow represents the stop codon of the second uORF. Small arrows are start codons. Blue box is the reporter. Statistical P values were calculated by two-tailed t-test, as above (*P value ≤0.05; **P≤0.01; ***P≤0.001).
Mentions: Data from Figs 1 and 2 suggest that eIF6 drives the translation of specific factors that regulate a lipogenic switch. We analysed how eIF6 modulates insulin-stimulated translation by using a fast doxycycline-inducible system, combined with polysomal microarray. Forty-eight hours after doxycycline treatment, AML12 cells with inducible eIF6 shRNA presented a reduction of eIF6 (Supplementary Fig. 3a) and resistance to insulin-stimulated methionine incorporation (Supplementary Fig. 3b). Polysomal analysis of insulin-stimulated cells showed that eIF6 downregulation reduced the polysome/80S ratio as expected by the loss of eIF6 anti-association activity (Fig. 3a). In unstimulated conditions eIF6 deficiency did not affect polysome peaks (Supplementary Fig. 3c).

Bottom Line: Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin.The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes.Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA.

View Article: PubMed Central - PubMed

Affiliation: INGM, 'Romeo ed Enrica Invernizzi', 20122 Milano, Italy.

ABSTRACT
Insulin regulates glycaemia, lipogenesis and increases mRNA translation. Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin. The role of insulin-regulated translation is unknown. Here we show that reduction of insulin-regulated translation in mice heterozygous for eIF6 results in normal glycaemia, but less blood cholesterol and triglycerides. eIF6 controls fatty acid synthesis and glycolysis in a cell autonomous fashion. eIF6 acts by exerting translational control of adipogenic transcription factors like C/EBPβ, C/EBPδ and ATF4 that have G/C rich or uORF sequences in their 5' UTR. The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes. Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA. Since obesity, type 2 diabetes, and cancer require a Fasn-driven lipogenic state, we propose that eIF6 could be a therapeutic target for these diseases.

No MeSH data available.


Related in: MedlinePlus