Exercise Reduces Insulin Resistance in Type 2 Diabetes Mellitus via Mediating the lncRNA MALAT1/MicroRNA-382-3p/Resistin Axis

Abstract

Insulin resistance (IR) is the primary pathological mechanism underlying type 2 diabetes mellitus (T2DM). Here, the study aimed to ascertain whether and how exercise mediates IR in T2DM. An in vivo mouse model of high-fat diet-induced IR and an in vitro high-glucose-induced IR model were constructed. High long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression was detected in T2MD and was positively correlated with HOMA-IR and resistin levels. Then, short hairpin RNA targeting MALAT1 (sh-MALAT1) or pcDNA-MALAT1 was delivered into human umbilical vein endothelial cells (HUVECs) to knock down or upregulate its expression, respectively. Silencing of MALAT1 resulted in reduced levels of resistin, Ang II, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelin-1 (ET-1), and p-insulin receptor substrate-1 (p-IRS)/ISR-1, and decreased cell migration, as well as enhanced glucose uptake and levels of nitric oxide (NO) and p-Akt/Akt. In the IR mouse model, exercise was observed to downregulate MALAT1 to reduce resistin, whereby exercise reduced homeostatic model assessment-insulin resistance (HOMA-IR). Besides, exercise also elevated microRNA-382-3p (miR-382-3p) expression in the serum of IR mice. Dual-luciferase reporter and RNA binding protein immunoprecipitation (RIP) assays identified that MALAT1 could bind to miR-382-3p to upregulate resistin. Collectively, the key observations of the study provide evidence that inhibition of MALAT1 elevates miR-382-3p to repress resistin, which consequently underlies the mechanism of exercise protecting against IR, highlighting a direction for T2DM therapy development.

Keywords: exercise; insulin resistance; metastasis-associated lung adenocarcinoma transcript 1; microRNA-382-3p; resistin; type 2 diabetes mellitus; vascular endothelial cells.

Figure 1

MALAT1 Is Highly Expressed in Patients with T2DM and Positively Correlates to HOMA-IR and Resistin Levels (A) MALAT1 expression in serum from healthy individuals and T2DM patients determined using qRT-PCR. (B) Serum resistin level from healthy individuals and T2DM patients measured using ELISA. (C) FBG in serum from healthy individuals and T2DM patients detected by a biochemical analyzer. (D) HOMA-IR level in serum from healthy individuals and T2DM patients. (E) Pearson’s correlation analysis of MALAT1 expression with HOMA-IR level. (F) Pearson’s correlation analysis of MALAT1 expression and resistin level. (G) Pearson’s correlation analysis of resistin level and HOMA-IR level; *p < 0.05, versus healthy individuals; n = 30 in healthy individuals; n = 90 in T2DM patients; measurement data were depicted as mean ± SD; comparisons between two groups were analyzed using unpaired t test.

Figure 2

Exercise Ameliorates IR by Inhibiting MALAT1 and Resistin Levels (A) HOMA-IR level in serum of IR mice subjected to exercise or IR mice treated with sh-MALAT1. (B) Resistin level in serum of IR mice subjected to exercise or IR mice treated with sh-MALAT1 measured using ELISA. (C) MALAT1 expression in serum of IR mice subjected to exercise or IR mice treated with sh-MALAT1 determined using qRT-PCR; *p < 0.05, versus mice fed with a normal diet; #p < 0.05, versus IR mice; measurement data were depicted as mean ± SD; comparisons among multiple groups were analyzed using one-way ANOVA; n = 10.

Figure 3

Silencing of MALAT1 Alleviates IR in HUVECs by Suppressing Resistin Expression (A) MALAT1 expression in IR HUVECs determined using qRT-PCR. (B) Western blot analysis of resistin level in IR HUVECs. (C) Glucose uptake in IR HUVECs (200×). (D) Migration of IR HUVECs detected using Transwell assay (200×). (E) serum levels of NO and Ang II in supernatant of IR HUVECs measured using ELISA. (F) Serum levels of TNF-α, IL-6, sICAM-1, and sVCAM-1 in supernatant of IR HUVECs measured using ELISA. (G) Western blot analysis of ET-1, p-IRS (Ser), ISR-1, p-Akt, and Akt proteins in IR HUVECs; *p < 0.05, versus cells cultured with DMEM supplemented with 5.5 mmol/L glucose (control); #p < 0.05, versus cells cultured with DMEM supplemented with 33 mmol/L glucose and transfected with sh-NC (IR +sh-NC); measurement data were depicted as mean ± SD; comparisons among multiple groups were analyzed by one-way ANOVA; the experiment was repeated three times.

Figure 4

Silencing of MATAT1 Upregulates miR-382-3p to Inhibit Resistin Expression (A) Putative miRNAs regulating resistin predicted by miRDB, miRSearch and DIANA. (B) Comparison of sequence homology of miR-382-3p in mice and human beings using BLAST. (C) Potential binding sites between MALAT1 and miR-382-3p predicted by the DIANA and starBase databases. (D) Relationship among MALAT1, miR-382-3p, and resistin confirmed by dual-luciferase reporter assay. (E) Expression of MALAT1 and resistin in response to transfection with miR-382-3p mimic determined using qRT-PCR. (F) Resistin expression in response to transfection with miR-382-3p mimic or sh-MALAT1, and/or pcDNA-MALAT1 detected using western blot analysis and qRT-PCR. (G) Correlation of MALAT1 and miR-382-3p with Ago2 using RIP assay; *p < 0.05, versus cells transfected with miR-NC, sh-NC, IgG, or vector plasmids; #p < 0.05, versus cells co-transfected with miR-NC and pcDNA-MALAT1 plasmids; measurement data were depicted as mean ± SD; comparisons between two groups were analyzed using unpaired t test; comparisons among multiple groups were analyzed by one-way ANOVA; the experiment was repeated three times.

Figure 5

Exercise Relieves IR by Elevating miR-382-3p Expression (A) miR-382-3p expression in healthy individuals (n = 30) and patients with T2DM (n = 90) determined using qRT-PCR; *p < 0.05, versus healthy individuals. (B) miR-382-3p expression in serum of IR mice or IR mice subjected to exercise detected using qRT-PCR; *p < 0.05, versus mice fed a normal diet; #p < 0.05, versus IR mice; measurement data were depicted as mean ± SD; comparisons between two groups were analyzed using unpaired t test; comparisons among multiple groups were analyzed by one-way ANOVA.

Figure 6

MALAT1/miR-382-3p/Resistin Axis Mediates IR in HUVECs (A) Expression of MALAT1 determined using qRT-PCR. (B) Expression of miR-382-3p determined using qRT-PCR. (C) Western blot analysis of resistin protein. (D) Glucose uptake of HUVECs determined by glucose detection kit (200×). (E) HUVEC migration detected using Transwell assay (200×). (F) Serum level of NO in HUVECs measured using ELISA. (G) Serum level of Ang II in HUVECs measured using ELISA; *p < 0.05, versus cells incubated on DMEM supplemented with 5.5 mmol/L glucose; #p < 0.05, versus cells incubated on the DMEM supplemented with 33 mmol/L glucose and transfected with vector plasmid; &p < 0.05, versus cells incubated on the DMEM supplemented with 33 mmol/L glucose and co-transfected with pcDNA-MALAT1 and miR-NC plasmids; measurement data were depicted as mean ± SD; comparisons among multiple groups were analyzed by one-way ANOVA; the experiment was repeated three times.

Figure 7

A Molecular Mechanism Map Depicting the Involvement of MALAT1/miR-382-3p/Resistin Axis in the Inhibitory Effect of Exercise on IR Exercise could alleviate IR of HUVECs via reducing MALAT1 and resistin, and increasing miR-382-3p.