Global-run on sequencing identifies Gm11967 as an Akt-dependent long noncoding RNA involved in insulin sensitivity

Figures

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  Figure 1. Feeding and the Akt pathway profoundly regulate hepatic gene transcription (A) Scatterplot of GRO-seq results comparing nascent transcription in livers of overnight fasted mice with that of mice that were overnight fasted and then refed normal chow for 2 h. Genes with an RPKM over 1 that are significantly upregulated in refeeding with a false discovery rate less than 0.05 are highlighted in red, whereas downregulated genes with a false discovery rate less than 0.05 are highlighted in blue. (B) Gene ontology performed on the sets of up- and downregulated genes identified by the GRO-seq analysis. The top five biological processes for each group are shown along with the respective genes that correlates with each process. (C) Heatmap of all the feeding-regulated genes identified in (A) and further showing results of GRO-seq from 2-h refed L-AktDKO and L-AktFoxo1TKO mice. The two clusters correlate with genes that are downregulated (pink) or upregulated (orange) upon refeeding. The final cluster (cyan) are upregulated by refeeding as well as dependent upon Akt inhibition of FoxO1. Two known feeding/Akt-regulated genes, Gck and IGFBP1, are highlighted on the heatmap.

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  Figure 2. eRNA analysis of GRO-seq data demonstrates a complex system of gene regulation in response to refeeding and changes in Akt signaling (A) Scatterplot of GRO-seq results comparing eRNA expression in livers of overnight fasted mice with that of mice that were overnight fasted and then refed normal chow for 2 h eRNA with an RPKM greater than 0.5 that are significantly upregulated in refeeding (at least 2-fold increased) are highlighted in red, whereas downregulated genes (at least 2-fold decreased) are highlighted in blue. (B) GREAT gene ontology analysis performed on the sets of up- and downregulated genes identified by the GRO-seq analysis. The top five biological processes for each group are shown. Each process is represented by two values; the first represents the number of eRNAs identified within it and the second is the number of genes associated with those eRNAs within this pathway. Example genes are shown for each pathway. (C) Heatmap of all the feeding-regulated genes identified in (A) and further results of GRO-seq from 2-h refed L-AktDKO and L-AktFoxo1TKO mice. Six clusters were identified, three from the feeding-stimulated eRNAs and three from the feeding-repressed that…

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  Figure 3. Identification of feeding and Akt-dependent lncRNAs by GRO-seq (A) Scatterplot of all Gencode lncRNAs that were identified by GRO-seq in 16 h overnight fasted and 16 h overnight fasted then 2-h refed mice. lncRNAs that are upregulated in refeeding with a false discovery rate less than 0.05 are highlighted in red, whereas downregulated lncRNAs with a false discovery rate less than 0.05 are highlighted in blue. Gm11967 is boxed in green, whereas Gm16551 and Gm15441 are boxed in red. (B) Heatmap of the 37 feeding-regulated lncRNAs and further comparison of their expression in 2-h refed L-AktDKO and L-AktFoxo1TKO mice. These data were grouped into three clusters; pink represents genes downregulated by refeeding, cyan represents feeding-induced, Akt-dependent genes, and orange represents feeding-induced, Akt-independent genes. Gm16551 and Gm15441 are boxed in red, whereas Gm11967 is highlighted in green. (C) qPCR analysis of Gm11967 and Gck in overnight fasted and refed mice (fasted n = 10 and refed n = 12). (D) qPCR analysis of Gm11967 and Gck in 12-week high-fat-diet-fed mice (normal chow n = 5 and high-fat diet n = 5). (E) qPCR analysis of Gm11967 and Gck expression in 16-h…

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  Figure 4. Gm11967 overexpression does not affect lipid gene expression in L-AktDKO mice (A) Gm11967 expression levels in ad libitum fed control and L-AktDKO mice as well as L-AktDKO mice overexpressing Gm11967 via a hepatocyte-specific AAV (for all conditions n = 5). (B) Body weights of control, L-AktDKO, and Gm11967 overexpressing L-AktDKO mice immediately after sacrificing along with the ratio of the liver weight to total body weight, represented as percentage of total body weight. Liver weight was recorded immediately after harvesting (for all conditions n = 5). (C) mRNA expression levels of Pparα and several of its target genes involved in fatty acid oxidation in ad libitum fed control, L-AktDKO, and L-AktDKO + Gm11967 mice (for all conditions n = 5). (D) mRNA expression levels of genes involved in de novo lipogenesis in ad libitum fed control, L-AktDKO, and L-AktDKO + Gm11967 mice (for all conditions n = 5). Data are represented as mean ± SEM Statistical significance was determined with an ordinary one-way ANOVA with Tukey’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 5. Gm11967 partially rescues insulin sensitivity in L-AktDKO mice (A) Blood glucose measurements of control, L-AktDKO, and L-AktDKO + Gm11967 after a 16-h fast (control n = 9, L-AktDKO n = 10, L-AtkDKO + Gm11967 n = 8). (B) Intra-peritoneal glucose tolerance test performed on 16-h fasted control, L-AktDKO, and L-AktDKO + Gm11967 (control n = 8, L-AktDKO n = 10, L-AtkDKO + Gm11967 n = 8). (C) Serum insulin levels of control, L-AktDKO, and L-AktDKO + Gm11967 immediately prior to and 15 min into a glucose tolerance test (control n = 8–9, L-AktDKO n = 10, L-AtkDKO + Gm11967 n = 8). (D) Western blot analysis of phospho-Akt2 and phospho-PRAS40 in 16-h fasted control, L-AktDKO, and L-AktDKO + Gm11967 that were injected with insulin for 20 min prior sacrificing. Control mice not injected with insulin were used to compare insulin stimulation. Hsp90 was used as a loading control for all samples. (n = 3 for all conditions). Data are represented as mean ± SEM (A–C). Statistical significance was determined with an ordinary one-way ANOVA with Tukey’s multiple comparison test (A and C) or two-way ANOVA with Tukey’s multiple comparison test (B). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 6. Gm11967 regulates hepatic Gck translation (A) Gck mRNA expression levels in ad libitum fed control, L-AktDKO, and L-AktDKO + Gm11967 mice (for all conditions n = 5). Data are represented as mean ± SEM Statistical significance was determined with an ordinary one-way ANOVA with Tukey’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Western blot analysis of Akt2 and Gck in ad libitum fed control, L-AktDKO, and L-AktDKO + Gm11967 (n = 3 for all conditions.). Hsp90 was used as a loading control. (C) Polysome profiling on livers from control, L-AktDKO, and L-AktDKO + Gm11967 mice followed by RT-PCR on the fractions to determine Gck enrichment in each fraction. A representative profile (repeated n = 3) is depicted along with representative RT-PCR for each condition. Gck enrichment in each fraction is shown as both a percentage of total RNA in the sample as well as an absolute measure in each fraction.