Long noncoding RNA MALAT1 regulates generation of reactive oxygen species and the insulin responses in male mice

Abstract

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and its overexpression is associated with the development of many types of malignancy. MALAT1 null mice show no overt phenotype. However, in transcriptome analysis of MALAT1 null mice we found significant upregulation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulated antioxidant genes including Nqo1 and Cat with significant reduction in reactive oxygen species (ROS) and greatly reduced ROS-generated protein carbonylation in hepatocyte and islets. We performed lncRNA pulldown assay using biotinylated antisense oligonucleotides against MALAT1 and found MALAT1 interacted with Nrf2, suggesting Nrf2 is transcriptionally regulated by MALAT1. Exposure to excessive ROS has been shown to cause insulin resistance through activation of c-Jun N-terminal kinase (JNK) which leads to inhibition of insulin receptor substrate 1 (IRS-1) and insulin-induced phosphorylation of serine/threonine kinase Akt. We found MALAT1 ablation suppressed JNK activity with concomitant insulin-induced activation of IRS-1 and phosphorylation of Akt suggesting MALAT1 regulated insulin responses. MALAT1 null mice exhibited sensitized insulin-signaling response to fast-refeeding and glucose/insulin challenges and significantly increased insulin secretion in response to glucose challenge in isolated MALAT1 null islets, suggesting an increased insulin sensitivity. In summary, we demonstrate that MALAT1 plays an important role in regulating insulin sensitivity and has the potential as a therapeutic target for the treatment of diabetes as well as other diseases caused by excessive exposure to ROS.

Keywords: Insulin resistance; Long noncoding RNA; Oxidative stress; Reactive oxygen species; T2DM.

Fig. 1.

MALAT1 ablation inhibits ROS generation. (A) The ROS generation in mouse hepatocytes determined by immunofluorescence microscopy using DCFH-DA as a fluorescent dye. (B) ROS levels in MALAT1 null and wild type hepatocytes challenged with high glucose (HG), LPS and TNF-α for 12 h were determined by fluorescence quantification with the plate reader (BioTek). (C) and (D) Flow cytometric analysis of ROS generation in MALAT1 null and wild type hepatocytes. (E) ROS levels in isolated pancreatic islets from wild type and MALAT1 null mice challenged with high glucose, LPS and TNF-α determined by fluorescence quantification with the plate reader (BioTek). (F) Isolated mouse islets. The photos show the islets isolated from MALAT1 null mice and wild type mice, and then kept in cell culture medium. Scale bars represent 500 μm. Error bars show standard deviations. *P < 0.05. Data are representative of at least three independent experiments.

Fig. 2.

Measurement of total protein carbonylation in hepatocytes and pancreas of MALALT1 null and wild type mice. (A) and (B) Protein carbonylation determined by Western blotting. Equal amount of protein from primary hepatocytes (A) and pancreas (B) from the MALAT1 null and wild type control treated with high glucose (HG, 2 g/kg, 3 times in 24 h) or LPS (30 mg/kg, 2 h). (C) and (D) Quantification of protein carbonylation of hepatocytes and pancreas determined by ELISA (Absorbance was read at 375 nm OD). Error bars show standard deviations. *P < 0.05; **P < 0.01. Data are representative of at least three independent experiments.

Fig. 3.

MALAT1 ablation inhibits JNK activation and enhances the insulin signaling capacity. (A) Western blotting analysis of the JNK phosphorylation as well as proteins levels of IRS1, p-Tyr of IRS1, Akt and p-Akt in primary hepatocytes from MALAT1 null and wild type mice. Hepatocytes were treated with high glucose (HG) and LPS. Quantifications (NIH ImageJ) of the Western blotting, right panel. (B) Mice fed on HFD (12 weeks) were injected with insulin (1 units/kg) through the portal vein and hepatocytes were harvested 5 min after insulin injection. Protein was extracted for Western blot analysis. Left: Protein extracted from liver was immunoprecipitated for IRS-1 and immunoblotted (IB) for phosphorylated proteins. Quantification are shown on the right panel. Data were presented as the ratios of the phosphorylated protein/un-phosphorylated protein. Error bars show standard deviations. *P < 0.05. Data are representative of at least three independent experiments.

Fig. 4.

MALAT1 ablation improves insulin signaling responses in mice (A) Blood glucose levels under fast-refeed challenge in MALAT1 null mice (N = 30) and wild type mice (N = 29). (B) Blood glucose levels of MALAT1 in glucose tolerance test (Data collected from 6 wild type and 6 MALAT1 null mice). (C) Blood glucose levels in insulin tolerance test (n = 6). (D) Blood insulin levels in glucose tolerant test (n = 6). *P < 0.01. Error bars show standard deviations. *P < 0.05; **P < 0.01.

Fig. 5.

Pancreatic islets morphology and cellularity of MALAT1 null mice (A) Glucose-stimulated insulin secretion (3.3mM and 16.7 mM) was measured by ELISA (Rat/Mouse Insulin ELISA kit, Millipore) in isolated islets from MALAT1 null and wild type control mice. (B) Representative sections of pancreas from 6 week-old MALAT1 null and wild type mice using HE staining. (C) Sections of pancreas from 6 week-old MALAT1 null and wild type mice using Heidenhain's AZAN trichrome staining. (D) Quantification of total endocrine cell number per total pancreatic area. (E) and (F) Percentage of α and β cells in total endocrine cells per pancreatic area. (G) Representative sections of pancreas from 6-week-old MALAT1 null and wild type mice visualized by double immunofluorescence staining with anti-insulin (red) and anti-glucagon (green) antibodies. Error bars show standard deviations. *P < 0.05; **P < 0.01. Data are representative of at least three independent experiments.

Fig. 6.

MALAT1 interacts with Nrf2 and inhibits the Nrf2/ARE-driven pathways. (A) Nrf2 expression in primary hepatocytes from MALAT1 ablated and wild type mice treated with high glucose (HG) and LPS. (B) Antioxidant genes expression in primary hepatocytes treated with HG and LPS. (C) Interaction between MALAT1 and Nrf2 in hepatocytes treated with LPS determined by the lncRNA pulldown assay. Western blot of Nrf2 expression pulled down by biotinylated MALAT1 oligos and LacZ oligos (negative control) from nuclear extracts from wild type mice treated with LPS. (D) Hypothesized mechanism for the interaction between MALAT1 and Nrf2 where MALAT1 acts as a negative modulator (riborepressor) inhibiting Nrf2-regulated transcription of antioxidant-related genes. Error bars show standard deviations. *P < 0.05. Data are representative of at least three independent experiments.

Fig. 7.

Proposed model illustrating how MALAT1 regulates insulin sensitivity and glucose homeostasis Proposed mechanisms for the improved insulin signaling capacity in the MALAT1 ablated mice: 1) MALAT1 ablation improves insulin responses by upregulating the antioxidant gene expression thereby quenching ROS, and improving the insulin signaling capacity through inhibiting JNK activation and enhancing the functions of IRS1 and Akt; 2) MALAT1 ablation creates a low ROS internal environment which enhances pancreatic endocrine functions by increasing insulin signaling capacity.