Elevated hepatic expression of H19 long noncoding RNA contributes to diabetic hyperglycemia

Abstract

Excessive hepatic glucose production (HGP) contributes significantly to the hyperglycemia of type 2 diabetes; however, the molecular mechanism underlying this dysregulation remains poorly understood. Here, we show that fasting temporally increases the expression of H19 long noncoding RNA (lncRNA) in nondiabetic mouse liver, whereas its level is chronically elevated in diet-induced diabetic mice, consistent with the previously reported chronic hepatic H19 increase in diabetic patients. Importantly, liver-specific H19 overexpression promotes HGP, hyperglycemia, and insulin resistance, while H19 depletion enhances insulin-dependent suppression of HGP. Using genome-wide methylation and transcriptome analyses, we demonstrate that H19 knockdown in hepatic cells alters promoter methylation and expression of Hnf4a, a master gluconeogenic transcription factor, and that this regulation is recapitulated in vivo. Our findings offer a mechanistic explanation of lncRNA H19's role in the pathogenesis of diabetic hyperglycemia and suggest that targeting hepatic H19 may hold the potential of new treatment for this disease.

Keywords: Diabetes; Endocrinology; Glucose metabolism; Metabolism; Noncoding RNAs.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Effects of hepatic H19 upregulation on HGP and glucose homeostasis.

(A) Body weight of mice fed with normal chow (NC) or high-fat diet (HFD). (B) Fasting plasma glucose levels. (C) Results of glucose-tolerance tests (GTTs). (D) Scatter plot of H19 levels in the liver. The horizontal line depicts group median, and the whiskers mark the interquartile range. (E) Scatter plot of H19 levels in livers of mice injected with AAV-Vec or AAV-H19. (F) Fasting blood glucose levels from mice injected with AAV-Vec or AAV-H19. (G) Fasting plasma insulin levels of mice injected with AAV-Vec or AAV-H19. (H) PTT results from mice injected with AAV-Vec or AAV-H19. (I) GTT results from mice injected with AAV-Vec or AAV-H19. (J) ITT results from mice injected AAV-Vec or AAV-H19. n = 5–6 mice per group. Data are the mean ± SEM. *P < 0.05, **P < 0.01. Student t test is used to compare differences between quantitative variables.

Figure 2. Hyperinsulinemic/euglycemic clamp studies of WT and KO mice.

Compared with the WT mice, the KO mice showed an increased glucose infusion rate (A), unchanged whole-body glucose uptake (B), unchanged basal EGP (C), and increased insulin-stimulated EGP suppression (D). Western blots and corresponding densitometry show decreased gluconeogenic gene expression in KO versus WT mouse livers (E). n = 5–7 mice per genotype. Quantification is based on 3 independent experiments. Numbers are the mean ± SEM. *P < 0.05 based on Student t test.

Figure 3. Effects of H19 expression on gluconeogenic gene expression and glucose output in vitro.

(A) HepG2 cells were transfected with siCon or siH19, followed by RNA extraction and qPCR analysis 48 hours later. Relative RNA levels are presented. (B) HepG2 cells were transfected with siCon or siH19, and glucose output assays were performed 48 hours later. Relative glucose outputs are presented. (C) qPCR results of HepG2 cells transfected with siCon or siH19 at 48-hour time point. (D) HepG2 cells were transfected with siCon or siH19, followed by Western blot analysis 48 hours later. Representative gel images from 3 transfection experiments are shown, with fold decreases in siH19 compared with siCon transfected cells marked on the right. (E–G) Mouse primary hepatocytes were infected with AAV-Vec or AAV-H19 viruses, followed by qPCR and Western blot analyses at 48 hours and glucose output assays at 72 hours. Quantification is based on 3 independent transfection/infection experiments. Numbers are the mean ± SEM. *P < 0.05, **P < 0.01 based on Student t test.

Figure 4. Regulation of HNF4A promoter methylation via the H19/SAHH pathway.

(A) Sequences of DMRs in the conserved promoter region of human and mouse Hnf4a. The 3 differentially methylated cytosine residues are indicated. The numbers on top of the sequences mark the positions of the indicated nucleotides in the chromosomes. The 3 differentially methylated cytosine residues are indicated in pink with arrowheads. (B) HepG2 cells were transfected with siCon, siH19, or siH19 + DEA. Genomic DNAs were extracted 15 hours later and analyzed by QMSP. (C) HepG2 cells were treated as described in B. RNAs were extracted 24 hours later and analyzed by qPCR. (D) RIP with anti-SAHH or preimmune IgGs from extracts of mouse livers. Top panel: RNA levels in immunoprecipitates were determined by qPCR. Levels of H19 and Gapdh mRNA are presented as fold enrichment in anti-SAHH relative to IgG immunoprecipitates. Bottom panel: relative RNA levels of H19 and Gapdh in mouse livers. Quantification is based on 3 independent experiments. Data are the mean ± SEM. *P < 0.05, **P < 0.01 based on Student t test.

Figure 5. Effects of fasting on H19 and gluconeogenic gene expression.

(A) WT mice were fasted overnight. qPCR analysis was performed on liver samples collected from fasted and nonfasted (Ctr) animals. Scatter plot of RNA levels is shown. (B) WT mice were overnight fasted, followed by protein analysis of the indicated genes by Western blotting. (C) HepG2 cells were treated with the indicated reagents for 2 hours. Relative H19 levels are presented. (D) Scatter plot of Hnf4a methylation as assessed by QMSP in fasted and control livers. n = 8–9 animals. Quantification is based on 3 independent experiments. Data are the mean ± SEM. *P < 0.05, **P < 0.01 based on Student t test.

Figure 6. Effects of H19 expression on hepatic Hnf4a promoter methylation, gluconeogenic gene expression, and insulin signaling in vivo.

Genomic DNAs were extracted from livers of NC and HFD mice (A), AAV-Vec and AAV-H19 mice (D), and WT and KO mice (G) fed ad libitum. Hnf4a promoter methylation was assessed by QMSP. (B, E, H) Relative Hnf4a mRNA levels as assessed by qPCR. (C, F, I) Relative protein levels as assessed by Western blotting. (J) Effects of H19 overexpression on hepatic insulin signaling. Proteins were extracted from livers of AAV-Vec– or AAV-H19–infected mice fed ad libitum and subjected to Western blot analysis. Quantifications (top panels) of INSR protein levels and AKT phosphorylation at Ser473 of the Western blots (bottom panel) are shown. n = 4–5 animals per group. Numbers are the mean ± SEM. *P < 0.05, **P < 0.01 based on Student t test. (K) A proposed model.