Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is implicated in liver cell proliferation. However, its role in hepatic steatosis and insulin resistance remain poorly understood. The aim of this study was to investigate the effects of MALAT1 on hepatic lipid accumulation and its potential targets. As expected, MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Knockdown of MALAT1 expression dramatically suppressed palmitate-induced lipid accumulation and the increase of nuclear SREBP-1c protein in HepG2 cells. In addition, RNA immunoprecipitation and RNA pull-down assay confirmed that MALAT1 interacted with SREBP-1c to stabilize nuclear SREBP-1c protein. Finally, injection of si-MALAT1 prevented hepatic lipid accumulation and insulin resistance in ob/ob mice. In conclusion, our observations suggest that MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability.

Scientific RepoRts | 6:22640 | DOI: 10.1038/srep22640 steatosis has not been investigated. The present study was designed to evaluate the role of MALAT1 in hepatic lipid metabolism and insulin resistance both in vitro and in vivo.

Results
MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Previous study showed that MALAT1 expression is significantly upregulated in endothelial cell of diabetic mice 18 . Here we investigated the MALAT1 expression pattern in HepG2 cells and primary mouse hepatocytes exposed with different doses of palmitate for 24 h. MALAT1 expression was dose-dependently increased in HepG2 cells and primary mouse hepatocytes (Fig. 1A). In addition to elevated MALAT1, palmitate treatment led to increased levels of the mRNA and nuclear SREBP-1c (Fig. 1B). We next evaluated these results in vivo. The MALAT1 levels were obviously elevated in livers from ob/ob mice (Fig. 1C). SREBP-1c expression was also increased in ob/ob mice (Fig. 1D), which was consistent with previous report 9 .
Knockdown of MALAT1 reversed palmitate-induced lipid accumulation in HepG2 cells. To establish the role of MALAT1 in lipid accumulation, we treated HepG2 cells and primary mouse hepatocytes with palmitate to mimic fatty acid overload conditions. Consistent with other's report, palmitate induced high intracellular levels of triglycerides and cholesterol 12 . To identify whether MALAT1 was involved in the effect of palmitate on lipid accumulation, HepG2 cells were transfected with si-MALAT1. As shown in Fig. 2A, MALAT1 siRNA efficiently decreased the expression of MALAT1 in HepG2 cells. Notably, knockdown of MALAT1 significantly decreased the palmitate-induced lipid accumulation in HepG2 cells. Palmitate caused a 3-fold increase in intracellular levels of triglycerides, which were significantly reduced by silencing of MALAT1 (Fig. 2B). Similar results of intracellular levels of cholesterol were also observed (Fig. 2C). In addition, deletion of MALAT1 in primary mouse hepatocytes significantly decreased the palmitate-induced lipid accumulation ( Fig. 2D-F).
Knockdown of MALAT1 reversed palmitate-induced the increase of nuclear SREBP-1 protein in hepatocytes. As shown in Fig. 3A, the reduction of MALAT1 expression abolished palmitate-enhanced nuclear SREBP1c protein level but had no effect on SREBP1c precursor and SREBP1c mRNA in HepG2 cells (Fig. 3A). Similar results were observed in primary mouse hepatocytes (Fig. 3B). Consistent with this, MALAT1 knockdown resulted in a significant decrease in the expression of SREBP-1c target genes, including ACC1, ACLY, SCD1 and FAS in HepG2 cells (Fig. 3C) and primary mouse hepatocytes (Fig. 3D) exposed to palmitate. These results indicated that knockdown of MALAT1 reversed palmitate-induced the increase of nuclear SREBP-1c expression and transcriptional activity in hepatocytes. Besides, MALAT1 knockdown decreased the expression of HMGCoA reductase (HMGCR) and gluconeogenic genes such as PEPCK and G-6-pase (Fig. 3C,D).

cytes.
Previous studies have confirmed that lncRNAs could directly interact with target proteins and increase their stability 20 . To investigate whether MALAT1 regulates SREBP-1c expression in such a manner, we overexpressed MALAT1 in HepG2 cells (Fig. 4A). Then, RIP and RNA pull-down assays were performed to identify whether SREBP-1c associated with MALAT1. As shown in Fig. 4B, MALAT1 was selectively interacting with nuclear SREBP-1c. SREBP-1c was measured by Western blot analysis in RNA pull-down assays. We also observed MALAT1 enrichment in RIP using SREBP-1c antibody in nuclear extracts from HepG2 cells (Fig. 4C).
We further explored whether MALAT1 increased SREBP-1c expression at the post-transcriptional level. We transfected pcDNA-MALAT1 in HepG2 cells for 24 h, then treated with the protein synthesis inhibitor cycloheximide (CHX) for 0, 3 or 6 h. Overexpression of MALAT1 inhibited degradation of nuclear SREBP-1c protein in HepG2 cells treated with CHX ( Fig. 4D). To further analyze the effect of MALAT1 on nuclear SREBP-1c protein levels, the Myc-tagged nuclear form of SREBP-1c was overexpressed in HEK293 cells by transient transfection. As shown in Fig. 4E, overexpression of MALAT1 caused accumulation of the nuclear form of SREBP-1c, while depletion of MALAT1 decreased accumulation of the nuclear form of SREBP-1c. In addition, we found MALAT1 overexpression inhibited the ubiquitination of SREBP-1c (Fig. 4F). These results indicated that MALAT1 stabilized SREBP-1c by preventing ubiquitin-mediated degradation.

SREBP-1c is required for MALAT1-induced intracellular lipid accumulation. SREBP-1c is a key
transcription factor that regulates the development of fatty liver and dyslipidemia 21 . We therefore studied whether SREBP-1c activation is required for MALAT1-induced lipid accumulation. Overexpression of MALAT1 in HepG2 cells increased nuclear SREBP1c protein level, which was reversed by transfection of SREBP-1c siRNA (Fig. 5A). In the cells transfected with si-control, overexpression of MALAT1 increased intracellular levels of triglycerides and cholesterol. However, lipid accumulation was weakened in HepG2 cells transfected with si-SREBP-1c (Fig. 5B,C). These results suggested that SREBP-1c mediates MALAT1-induced intracellular lipid accumulation.
Knockdown of MALAT1 expression reversed aggregation lipid in ob/ob mouse liver. To explore the role of LncRNA MALAT1-induced lipid accumulation in the liver, we treated ob/ob mice with si-MALAT1 daily for 10 days. MALAT1 expression in the si-MALAT1 injection group was significantly reduced in liver (Fig. 6A). The ratio of liver to body weight was significantly lower in si-MALAT1-injected mice (4.8 ± 0.32%) than that of si-control-injected mice (6.7 ± 0.43%). Meanwhile, a significant decrease in lipid accumulation in the liver in si-MALAT1-injected mice compared to si-control-injected mice (Fig. 6B).
We next determined whether si-MALAT1 effectively reduced nuclear SREBP-1c protein level in vivo in the ob/ob mouse liver. As shown in Fig. 6C, the amount of nuclear SREBP-1c was reduced in the livers of si-MALAT1-injected ob/ob mice compared with si-control-injected ob/ob mice. Meanwhile, the levels of ACC1, ACLY, SCD1, FAS, HMGCR, PEPCK and G-6-pase were strongly reduced in the livers of si-MALAT1-treated ob/ob mice (Fig. 6D).
Scientific RepoRts | 6:22640 | DOI: 10.1038/srep22640 MALAT1 knockdown improves insulin sensitivity in ob/ob mice. Because hepatic steatosis has often been associated with hepatic insulin resistance 22 , we measured the effect of MALAT1 knockdown on insulin sensitivity. Figure 7A showed the effect of si-MALAT1 on IPGTT in ob/ob mice. Blood glucose levels of si-MALAT1-injected ob/ob mice were lower after intravenous glucose loading compared with si-control-injected mice. After administration of insulin (2 U/kg), si-MALAT1-injected ob/ob mice also exhibited lower levels of blood glucose than si-control-injected mice (Fig. 7B). Downregulation of MALAT1 expression in the liver had no effect on body weight and food intake of ob/ob mice (Fig. 7C,D). However, MALAT1 knockdown significantly decreased fasting blood glucose after 10 days injection (Fig. 7E). MALAT1 knockdown did not affect fasting blood then, SREBP-1c mRNA levels (white bars, HepG2 cells; black bars, mouse hepatocytes) and protein level (left, HepG2 cells; right, mouse hepatocytes) were determined. The MALAT1 levels (C) and SREBP-1c protein level (D) were measured in livers from ob/ob. **P < 0.01, compared to palmitate = 0 mmol/L or ob/ + mice.
insulin levels (Fig. 7F). These results indicated that improvement of glucose intolerance in si-MALAT1-injected ob/ob mice was due to enhancing insulin sensitivity but not stimulating insulin secretion.

Discussion
The present study firstly demonstrated that excess palmitate increased LncRNA MALAT1 expression, activated SREBP-1c, and induced intracellular lipid accumulation in hepatocytes. We found that MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. We also found that inhibition of the MALAT1 expression decreased nuclear SREBP1c level and lipid accumulation both in vitro and in vivo. In addition, the reduction of MALAT1 in the liver improved insulin sensitivity in ob/ob mice.
LncRNA MALAT1 has been discovered to be involved in liver cell proliferation 19 . However, the function of MALAT1 in the improvement of hepatic steatosis has not been studied. Our data demonstrated that the expression of MALAT1 was obviously increased in hepatocytes under fatty acid overload conditions. In addition, MALAT1 expression is markedly elevated in the livers of ob/ob mice. Gene silencing of MALAT1 attenuated lipid accumulation in palmitate-treated HepG2 cells. In ob/ob mice, knockdown of MALAT1 significantly reduced liver lipids and promoted insulin sensitivity. More importantly, overexpression of MALAT1 leading to lipid accumulation. These findings establish a novel role of MALAT1 in hepatic steatosis and insulin resistance. The data of the present study additionally demonstrated that increased MALAT1 is associated with nuclear SREBP-1c protein level. Deletion of MALAT1 abolished the increase of nuclear SREBP-1c protein level induced by palmitate but had no effect on SREBP-1c precursor and SREBP-1c mRNA. Therefore, MALAT1 regulated SREBP-1c expression at post-transcriptional level. Accumulating evidence has demonstrated that lncRNAs may interact with proteins and change the activity of these proteins 20,23 . MALAT1 is localized in the nuclear, implicating that its function serves as protein-coding RNAs 24 . Our data showed that MALAT1 directly bound nuclear SREBP-1c protein and increased stabilization of SREBP-1c protein. Our results also demonstrated that knockdown of MALAT1 abolished the increase of expression of ACC1, ACLY, SCD1 and FAS, which were the  SREBP-1c is the critical transcription factor that regulates cholesterol and fatty acid synthesis 7 . In obese patients, increased SREBP-1c expression was observed in liver 26 . Inhibition of SREBP-1c could suppress hepatic lipogenesis and lipid accumulation in liver 27 . Our present study found that downregulation of SREBP-1c expression effectively abolished the increase of intracellular levels of triglycerides and cholesterol induced by MALAT1. These finding indicated that the effect of MALAT1 on intracellular lipid accumulation is dependent on SREBP-1c. Moreover, we also found that MALAT1 had the effect on SREBP-2 mRNA stability (data not shown). Future studies are necessary to clarify how MALAT1 regulated SREBP-2 expression in vitro and in vivo.
In summary, MALAT1 induced hepatic lipid accumulation and insulin resistance by increasing SREBP-1c and target genes expression. This study suggested inhibition of MALAT1 has potential for the treatment of obesity and type 2 diabetes.

Materials and Methods
Reagents. DMEM Cell culture. HepG2 cells were purchased from ATCC and maintained in DMEM supplemented with 10% FBS. Cells were cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO 2 . To generate the in vitro model of high fat-induced insulin resistance, HepG2 cells were treated with palmitate for 24 h as previously described 28 . Primary isolation and culture of hepatocytes. Hepatocytes were isolated and cultured from the livers of male C57BL/BKS mice as described previously 29 . Cell viability of primary hepatocytes was evaluated by the trypan blue exclusion test and was always higher than 70%. (A) ob/ob mice were injected with si-control or si-MALAT1 daily for 10 days, then, MALAT1 expression in liver was determined (n = 5 per group). (B) ob/ob mice were injected with si-control or si-MALAT1 daily for 10 days, then, Liver sections were stained with H-E (top row) or Oil red O (bottom row). Original magnification, ×20. Scale bar = 50 μm (n = 3 per group). (C) The nuclear SREBP-1c (nSREBP-1c) expression was measured in liver from ob/ob mice injected with si-control or si-MALAT1 daily for 10 days. (D) ACC1, ACLY, SCD1, FAS, HMGCR, LDLR, PEPCK and G-6-pase mRNA expression was measured in liver from ob/ob mice injected with si-control or si-MALAT1 daily for 10 days (n = 3 per group). Hepatic triglycerides (E) and cholesterol content (F) were measured in liver from ob/ob mice injected with si-control or si-MALAT1 daily for 10 days (n = 6 per group). (E) **P < 0.01, compared to si-control.
Scientific RepoRts | 6:22640 | DOI: 10.1038/srep22640 Animal studies. Mice were housed under 12/12 hr light/dark cycles and free access to water and a standard chow diet containing 60% carbohydrate, 13% fat, and 27% protein on a caloric basis. All animals studied were 8-to 12-week-old male ob/+ or ob/ob mice obtained from Model Animal Research Center of Nanjing University, China. Mice were anesthetized with pentobarbital sodium salt (35 mg/kg) and injected through the tail vein with 2.5 mg/kg body weight of lipid nanoparticles-formulated si-control or si-MALAT1 as described previously 30 . Body weight, fasting blood glucose levels and fasting blood insulin levels were measured after fasting the animals for 4 h (starting from 9:00 AM) on Day 0 (before injection), Day 5 and Day 10. Blood glucose concentrations were measured with the Hexokinase Method (Thermo Fisher Scientific, Lafayette, CO, USA). Blood insulin was measured using a rat/mouse insulin ELISA kit (Millipore, Billerica, MA). All animal studies were performed according Body weight (C), food intake (D), fasting blood glucose levels (E) and fasting blood insulin levels (F) were measured after fasting the animals for 4 h on Day 0, Day 5 and Day 10 (n = 8 per group). *P < 0.05, **P < 0.01, compared to si-control.
to guidelines established by the Research Animal Care Committee of Yangzhou University, China. Animals were treated humanely, using approved procedures in accordance with the guidelines of the Institutional Animal Care and Use Committee at Yangzhou University, China.

Measurement of intracellular cholesterol and triglyceride. Lipids in cultured HepG2 cells and pri-
mary mouse hepatocytes were extracted using a Folch extraction of lipids and resuspension in 0.1% Triton X-100 as described previously 28 . The intracellular cholesterol and triglyceride in extracted lipids were measured using the reagents from Cayman Chemical (Ann Arbor, MI) according to the manufacturer's instruction. The hepatic triglyceride and cholesterol content were normalized with tissue weight.
Real-time PCR assay. The mRNA was quantified by real-time PCR using a LightCycler480 II Sequence Detection System (Roche, Basel, Switzerland). All data were analyzed using β -Actin gene expression and GAPDH as internal standard. The sequences of primers used are available in supplemental table 1. Ubiquitination Assay. HepG2 cells were transfected with plasmids encoding Myc-SREBP1c, Flag-ubiquitin, and pcDNA-MALAT1. After transfection for 48 h, the cells were incubated with MG132 (10 μM) for 6 hours and lysed with cold RIPA buffer. Equal amounts of total cell lysates were incubated with the Myc antibodies for 2 hours at 4 °C. Immunocomplexes were collected using protein-A sepharose beads for 1 hour at 4 °C. Further, the immunoprecipitates were washed with RIPA buffer and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting analyses with anti-Flag antibodies.

Western blot analysis.
Expression plasmids construction. The MALAT1 expression plasmid (pcDNA-MALAT1, nucleotides 3207-8411 bp) was constructed as previous report 31 . The sequences of primers were as follow: M A L AT 1 , 5 ′ -T G AG T C G AG C T C T G C C A AG T C C T G G AG A A ATAG TAG -3 ′ ( for w ard ) an d 5′-AGTCATGGGCCCTGAAGACAGATTAGTAAAAGCA-3′ (reverse). he plasmid of pcDNA-MALAT1 was sequenced and confirmed to be correct.

Histological analysis.
For H-E, liver tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4 μm sections. For Oil red O staining, liver was frozen in liquid nitrogen and cut into 8 μm sections. Section were stained and analyzed at ×20 magnification using a Leica DMRB microscope.

Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT). IPGTT was per-
formed by an intraperitoneal injection of D-glucose (2 g/kg body weight, i.p.) after 6 h fast. Blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes post injection. For ITT, mice were fasted for 6 h and then injected with human insulin (Novo-Nordisk, Bagsvaerd, Denmark) at 0.75 U/kg body weight. Blood glucose levels were measured at 0, 15, 30, and 60 minutes. Statistical analysis. Statistical analyses were performed using statistical analysis software SPSS 13.0. Data were expressed as the mean ± SD. Analysis of variance (ANOVA) was used to determine the statistical differences among the groups. A P value of less than 0.05 and are provided in the figures. A P value < 0.05 was considered statistically significant.