Downregulation of hepatic lncRNA Gm19619 improves gluconeogenesis and lipogenesis following vertical sleeve gastrectomy in mice

Figures

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  Fig. 1. Gm19619 is a hepatic nuclear lncRNA which can be upregulated in both obese-sham-operated and overnight-fasted mice livers. a Heatmap showing the several protein-coding metabolic genes involved in lipogenesis (Fasn, Scd1, Cd36, and Srebf1) and gluconeogenesis (G6pc and Pck1), and the top 40 differentially transcribed lncRNAs with a fold change over 1.67 (p < 0.05) of the counts in FPKM in the RNAseq data of sham or vertical sleeve gastrectomy (VSG) operated mice livers (n = 3). b The relative level of different lncRNAs in lean, sham, or VSG-operated mice liver by real-time PCR (n = 3). c, d The relative RNA level of Gm19619, G6pc, and Pck1 in overnight-fasted (16 h) (n = 5) and re-fed (4 h) (n = 7) mice liver (c), and in murine immortal hepatocytes AML12 cells after treatment of 10 μM forskolin for 7, 12, and 18 h (n = 3) (d). e The full length of Gm19619 RNA was determined by RT-PCR using mouse liver cDNA after its 5′ and 3′ sequence was confirmed by RACE-PCR. f Real-time PCR determined the distribution of Gm19619 RNA in nucleus and cytosol (n = 3). Rnu2 and Gapdh RNA served as the marker of the nucleus and cytosol, respectively. Two-tailed unpaired t-test. Error bars represent…

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  Fig. 2. Adenovirus-associated virus (AAV) mediated hepatic Gm19619 forced transcription promoted hepatic gluconeogenesis in chow-fed mice. a Schema of the AAV vector for hepatic Gm19619 transcription under the liver-specific TBG promoter and the experimental timeline. GFP was used as the control. b The relative RNA level of Gm19619, G6pc, and Pck1 in AAV-Gm19619 (n = 9) or AAV-GFP (n = 7) infected mice liver after 16 h fasting and 4 h refeeding by real-time PCR. c Western blot analysis of protein levels of G6PC and PCK1 in AAV-Gm19619 and AAV-GFP mice liver (n = 3). d The blood glucose level of AAV-Gm19619 (n = 18) and AAV-GFP (n = 11) mice after overnight fasting. e–g GTT (e) (n = 11), ITT (f) (n = 11), and PTT (g) (n = 8) assay of the AAV-Gm19619 and AAV-GFP mice. Two-tailed unpaired t-test. Error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Fig. 3. AAV-CasRx mediated Gm19619 knockdown in high-fat diet (HFD) fed mice significantly improved the mice glucose metabolism. a Schema of the AAV-EFS-CasRx vector for Gm19619 knockdown and the experimental timeline. b The relative RNA levels of hepatic Gm19619, G6pc, and Pck1 in Gm19619 knockdown (CasRx-19×3) (n = 10) and control (CasRx-ctrl) (n = 7) mice. c The fasting blood glucose levels in CasRx-19×3 mice (n = 10) and CasRx-ctrl (n = 7) mice. d–f The GTT (d), ITT (e), and PTT (f) assay of CasRx-19×3 (n = 10, PTT n = 8) and CasRx-ctrl (n = 7) mice. g The western blot results of G6PC and PCK1 in CasRx-19×3 and control group mice livers (n = 3). Two-tailed unpaired t-test. Error bars represent the SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

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  Fig. 4. AAV-CasRx mediated Gm19619 knockdown in HFD-fed mice significantly improved the mice lipid metabolism. a The body weight of Gm19619 knockdown (CasRx-19×3) (n = 11) and control (n = 8) mice. b Representative images of liver sections by H&E and oil red o staining, respectively. Scale bar, 100 μm. c The serum level of triacylglycerol (TG) in CasRx-19×3 (n = 11) and CasRx-ctrl (n = 6) mice. d The serum level of NEFA in CasRx-19×3 (n = 11) and CasRx-ctrl (n = 8) mice. e The relative mRNA levels of some lipogenesis genes and Cd36 in CasRx-19×3 (n = 11) and CasRx-ctrl (n = 8) mice. f The relative mRNA levels of several lipolysis and beta-oxidation genes in CasRx-19×3 and CasRx-ctrl mice (n = 8). Two-tailed unpaired t-test. Error bars represent the SEM. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

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  Fig. 5. ChIRP data analysis indicated Gm19619 binds to the chromatic DNA of leptin receptor and Foxo1 and modulates their expression. a The second structure of Gm19619 RNA with minimal free energy as predicted on the RNAfold website. The locations of the 14 ChIRP probes were shown. b The genomic distribution of ChIRP pulldown DNA fragments with a pileup over 5 and longer than 200 bp. c Pathway enrichment analysis of genes with surrounding regions bound by Gm19619 identified from ChIRP-seq. The gene list from overlapping peaks was used to run KEGG pathway analysis with IPA (Ingenuity Pathway Analysis). d The Circos plot showing the ChIRP enriched genes within the leptin receptor pathway. e, f, k ChIRP-seq signals indicate Gm19619 binding along its own exon locus (e), the leptin receptor Lepr (f), and the Foxo1 gene locus (k). H3K27ac, H3K4me1, and H3K4me3 signals from ENCODE mouse liver ChIP-seq data were shown as control, mm10 as the reference genome. g The relative mRNA levels of Lepr, Plcg1, and Foxo1 in Gm19619 forced expressed mice (n = 5–10). h Western blot analysis of protein levels of LEPR, FOXO1, and CREB1 in AAV-Gm19619 and AAV-GFP mice liver (n = 3). i The relative mRNA levels…

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  Fig. 6. Schema showing the potential function of Gm19619 in the liver. While VSG represses the transcription of hepatic Gm19619, both HFD and fasting induce Gm19619 RNA. Gm19619 may promote gluconeogenesis by activating the Foxo1-G6pc/Pck1 pathway and facilitate lipid accumulation by inhibiting the leptin receptor signaling pathway.