miR-548ag promotes DPP4 expression in hepatocytes through activation of TLR(7/8)/NF-κB pathway

Abstract

Objective: In our previous study, we identified a notable increase in miR-548ag content after obesity, which contributes to the progression of Type 2 diabetes Mellitus(T2DM) through the up-regulation of Dipeptidyl Peptidase-4(DPP4) expression within the liver. However, the precise molecular mechanisms underlying the upregulation of DPP4 by miR-548ag remain elusive. Mature miRNAs rich in GU sequences can activate the TLR(7/8)/NF-κB signalling pathway, which transcriptionally activates DPP4 expression. Notably, the proportion of GU sequences in hsa-miR-548ag was found to be 47.6%. The study proposes a hypothesis suggesting that miR-548ag could potentially increase DPP4 expression in hepatocytes by activating the TLR(7/8)/NF-κB signalling pathway.

Methods: Male C57BL/6J mice were fed normal chow diet (NCD, n = 16) or high-fat diet (HFD, n = 16) for 12 weeks. For a duration of 6 weeks, NCD mice received intraperitoneal injections of a miR-548ag mimic, while HFD mice and db/db mice (n = 16) were administered intraperitoneal injections of a miR-548ag inhibitor. qRT-PCR and Western Blot were used to detect the expression level of miR-548ag, DPP4 and the activation of TLR(7/8)/NF-κB signalling pathway. HepG2 and L02 cells were transfected with miR-548ag mimic, miR-548ag inhibitor, TLR7/8 interfering fragment, and overexpression of miR-548ag while inhibiting TLR7/8, respectively.

Results: (1) We observed elevated levels of miR-548ag in the serum, adipose tissue, and liver of obese mice, accompanied by an upregulation of TLR7/8, pivotal protein in the NF-κB pathway, and DPP4 expression in the liver. (2) miR-548ag promotes DPP4 expression in hepatocytes via the TLR(7/8)/NF-κB signalling pathway, resulting in a reduction in the glucose consumption capacity of hepatocytes. (3) The administration of a miR-548ag inhibitor enhanced glucose tolerance and insulin sensitivity in db/db mice.

Conclusions: MiR-548ag promotes the expression of DPP4 in hepatocytes by activating the TLR(7/8)/NF-κB signalling pathway. MiR-548ag may be a potential target for the treatment of T2DM.

Fig. 1. The expression levels of miR-548ag, TLR7/8 and key proteins of NF-κB pathway and DPP4 in liver tissues of obese mice were detected.

A General morphology of mice; B Body weight of mice; C Lee’s index of mice; D Weight of mice liver and adipose tissue; E Morphology of mice liver and adipose tissue; F Fasting blood glucose; G Serum lipids content; H–J Content of miR-548ag in epididymal white adipose tissue, serum and liver tissue of mice; K Protein expression level of mice in liver tissue. (t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 2. miR-548ag upregulates the expression of TLR7/8, NF-κB pathway key proteins and DPP4 in liver tissues and inhibits glucose tolerance and insulin sensitivity in mice.

A General view of mice; B Body weight of mice; C Weight of mice liver and adipose tissue; D General morphology of mice liver and adipose tissue; E, F Expression level of miR-548ag in mice serum and liver tissues; G Protein expression level in mice liver tissue; H Fasting blood glucose of mice; I Serum lipids content of mice; J Glucose tolerance of mice; K Insulin sensitivity of mice. (Rank sum test and t-test, *P < 0.05, **P < 0.01, ***P < 0.001, difference was statistically significant).

Fig. 3. miR-548ag inhibitor down-regulates TLR7/8, NF-κB pathway key proteins and DPP4 expression in liver tissues of obese mice.

A General view of mice; B Body weight of mice; C Weight of mice liver and adipose tissue; D General morphology of mice liver and adipose tissue; E Protein expression level in mice liver tissue. F Fasting blood glucose of mice; G Serum lipids content of mice; H Glucose tolerance of mice; I Insulin sensitivity of mice. (t-test, **P < 0.01, ***P < 0.001).

Fig. 4. miR-548ag inhibitor down-regulates TLR7/8, NF-κB pathway key proteins and DPP4 expression in liver tissues of db/db mice.

A General view of mice; B Body weight of mice; C Weight of mice liver and adipose tissue; D General morphology of mice liver and adipose tissue; E Weights of liver and adipose tissueSerum lipids content of mice. F Protein expression level in mice liver tissue; G Fasting blood glucose of mice; H Glucose tolerance of mice; I Insulin sensitivity of mice. (t-test, **P < 0.01, ***P < 0.001).

Fig. 5. miR-548ag promotes DPP4 expression in HepG2 through activation of TLR(7/8)/NF-κB pathway.

mRNA expression levels of (A) miR-548ag, (B) TLR7/8, DPP4 in HepG2 cells after transfection with miR-548ag; C Protein expression levels of TLR(7/8), key proteins of NF-κB pathway, DPP4 in HepG2 cells after transfection with miR-548ag (D) HepG2 glucose consumption levels. E mRNA expression levels of TLR7/8, DPP4 in HepG2 cells after transfection with miR-548ag inhibitor; F Protein expression levels of TLR(7/8), key proteins of NF-κB pathway, DPP4 in HepG2 cells after transfection with miR-548ag inhibitor; G HepG2 glucose consumption levels; H Protein expression levels of TLR(7/8), key proteins of NF-κB pathway, DPP4 in HepG2 cells after transfection with TLR7 interference fragment; I Cell glucose consumption of HepG2; J The protein expression levels of HepG2 cells were transfected with TLR7 interference fragments after miR-548ag was modulated; K Glucose consumption of HepG2; L Protein expression level of HepG2 cells after transfection with TLR8 interference fragment; M Cell glucose consumption of HepG2; N Protein expression levels of HepG2 cells after transfection of TLR8 interference fragments with up-regulated miR-548ag; O Glucose consumption of HepG2 (Rank sum test, *P < 0.05, **P < 0.01, ***P < 0.001, the difference was statistically significant).

Fig. 6. miR-548ag promotes DPP4 expression in L02 through activation of TLR(7/8)/NF-κB pathway.

mRNA expression levels of (A) miR-548ag, (B) TLR7/8, DPP4 in L02 cells after transfection with miR-548ag; C Protein expression levels of TLR(7/8), key proteins of NF-κB pathway, DPP4 in L02 cells after transfection with miR-548ag (D) L02 glucose consumption levels; E mRNA expression levels of TLR7/8, DPP4 in L02 cells after transfection with miR-548ag inhibitor; F Protein expression levels of TLR(7/8), key proteins of NF-κB pathway, DPP4 in L02 cells after transfection with miR-548ag inhibitor; G L02 glucose consumption levels; H Protein expression levels of TLR(7/8), key proteins of NF-κB pathway, DPP4 in L02 cells after transfection with TLR7 interference fragment; I Cell glucose consumption of L02; J The protein expression levels of L02 cells were transfected with TLR7 interference fragments after miR-548ag was modulated; K Glucose consumption of L02; L Protein expression level of L02 cells after transfection with TLR8 interference fragment; M Cell glucose consumption of L02; N Protein expression levels of L02 cells after transfection of TLR8 interference fragments with up-regulated miR-548ag; O Glucose consumption of L02 (Rank sum test, *P < 0.05, **P < 0.01, ***P < 0.001, the difference was statistically significant).

Fig. 7. The levels of miR-548ag and DPP4 were significantly positively correlated in human serum.

A BMI in normal weight individuals and individuals with obesity; B Serum miR-548ag levels were significantly higher in individuals with obesity (n = 20) than in normal-weight individuals; C Serum DPP4 levels were significantly higher in individuals with obesity (n = 20) than in normal-weight individuals; D There was a positive correlation between serum miR-548ag and DPP4 levels in individuals with obesity (n = 40). (t-test, *P < 0.05, **P < 0.01, ***P < 0.001; Pearson’s correlation analysis).

Fig. 8. Injection of miR-548ag inhibitor and gavage of DPP4 inhibitor were both effective in improving glucose tolerance and insulin sensitivity in mice.

A Body weight of mice; B ITT and area under the ITT curve for injection of miR-548ag inhibitor and gavage of DPP4 inhibitor; C GTT and area under the GTT curve for injection of miR-548ag inhibitor and gavage of DPP4 inhibitor. (t-test, *P < 0.05, **P < 0.01, ***P < 0.001).