Diabetes Metab J.  2022 Mar;46(2):337-348. 10.4093/dmj.2021.0056.

DA-1241, a Novel GPR119 Agonist, Improves Hyperglycaemia by Inhibiting Hepatic Gluconeogenesis and Enhancing Insulin Secretion in Diabetic Mice

• Kim Y
• Lee SW
• Wang H
• Kim RH
• Park HK
• Lee H
• Kang ES

• KMID: 2527726
• DOI: http://doi.org/10.4093/dmj.2021.0056

Abstract

Background
We investigated the antidiabetic effects of DA-1241, a novel G protein-coupled receptor (GPR) 119 agonist, in vitro and in vivo.
Methods
DA-1241 was administrated to high-fat diet (HFD)-fed C57BL/6J mice for 12 weeks after hyperglycaemia developed. Oral/intraperitoneal glucose tolerance test and insulin tolerance test were performed. Serum insulin and glucagon-like peptide-1 (GLP-1) levels were measured during oral glucose tolerance test. Insulinoma cell line (INS-1E) cells and mouse islets were used to find whether DA-1241 directly stimulate insulin secretion in beta cell. HepG2 cells were used to evaluate the gluconeogenesis and autophagic process. Autophagic flux was evaluated by transfecting microtubule-associated protein 1 light chain 3-fused to green fluorescent protein and monomeric red fluorescent (mRFP-GFP-LC3) expression vector to HepG2 cells.
Results
Although DA-1241 treatment did not affect body weight gain and amount of food intake, fasting blood glucose level decreased along with increase in GLP-1 level. DA-1241 improved only oral glucose tolerance test and showed no effect in intraperitoneal glucose tolerance test. No significant effect was observed in insulin tolerance test. DA-1241 did not increase insulin secretion in INS-1E cell and mouse islets. DA-1241 reduced triglyceride content in the liver thereby improved fatty liver. Additionally, DA-1241 reduced gluconeogenic enzyme expression in HepG2 cells and mouse liver. DA-1241 reduced autophagic flow in HepG2 cells.
Conclusion
These findings suggested that DA-1241 augmented glucose-dependent insulin release via stimulation of GLP-1 secretion, and reduced hepatic gluconeogenesis, which might be associated with autophagic blockage, leading to improved glycaemic control.

Keyword

Autophagy; Glucagon-like peptide 1; Gluconeogenesis; G protein-coupled receptor; Insulin secretion

Figure

• 

  Fig. 1 Basal metabolic rate characterization. During the study period, body weight, random blood glucose levels, and food intake were monitored weekly. (A) Body weight. (B) Body weight after 12 weeks of study period. There was no significant difference in body weight among groups. (C) Food intake. (D) Average random blood glucose level. (E) Fasting blood glucose level after 12 weeks of study period. Random blood glucose level was not different among groups; however, fasting blood glucose level was considerably reduced in the DA-1241 group. Data are presented as the mean±standard error of the mean. aP<0.01, compared with high-fat (HF) group (chow, n=4; HF, n=10; DA-1241, n=24), bP<0.001.

• 

  Fig. 2 Effects of DA-1241 on glucose tolerance and insulin tolerance tests (ITTs) in high-fat (HF) diet-fed mice. (A, B) Blood glucose excursion and area under the curve (AUC) during oral glucose tolerance test (OGTT) at week 8. DA-1241 improved glucose tolerance with a substantial decrease in AUC during OGTT. (C, D) Blood glucose excursion and AUC during intraperitoneal glucose tolerance test (IPGTT) at week 11. (E, F) Glucose levels and AUC during ITT at week 10. No significant differences in AUC of intraperitoneal GTT and ITT were observed between the HF diet and DA-1241 groups. Data are presented as the mean±standard error of the mean. aP<0.05, bP<0.01, compared with HF group (chow, n=4; HF, n=10; DA-1241, n=24), cP<0.001.

• 

  Fig. 3 Measurement of insulin secretion and serum total glucagon-like peptide-1 (GLP-1) levels. (A) Serum insulin levels were determined at 0 and 30 minutes during oral glucose tolerance test (OGTT) at week 8. (B) Change in serum insulin levels during OGTT. The DA-1241 group exhibited enhanced insulin levels. (C) Insulin levels in insulinoma cell line (INS-1E) cells after incubation with DA-1241 for 1 hour under low- and high-glucose conditions. Measurements were carried out in triplicate. DA-1241 did not enhance insulin secretion from INS-1E cells in response to high glucose concentrations. (D) Islets were preincubated in low- (2.8 mM) glucose for 30 minutes and then, incubated in high (16.7 mM) glucose. Insulin levels in mouse islets after incubation with DA-1241 (1,000 nM) under high glucose conditions. Insulin group of five islets. (E) Serum total GLP-1 levels increased after DA-1241 treatment. Serum total GLP-1 levels were determined at 0 and 15 minutes during OGTT at week 8. Data are presented as the mean±standard error of the mean. aP<0.05, bP<0.01, compared with HF group (chow, n=4; high-fat [HF], n=10; DA-1241, n=24), cP<0.001.

• 

  Fig. 4 Effects of DA-1241 on fatty liver and serum lipid profile. Blood samples and liver tissues were collected from overnight-fasted mice after 12 weeks of study period. (A) Liver sections were analysed by H&E staining. Images were obtained using an electron microscope at 20× magnification. Hepatic lipid accumulation was markedly diminished in the DA-1241 group. (B) Liver triglyceride (TG), (C) serum cholesterol, and (D) serum TG contents were measured after 12 weeks of study period. The DA-1241 group showed a considerable decrease in liver TG and serum total cholesterol, but not in serum TG levels. Data are presented as the mean±standard error of the mean. aP<0.05, bP<0.01, compared with HF group (chow, n=4; high-fat [HF], n=10; DA-1241, n=24).

• 

  Fig. 5 Effects of DA-1241 on gluconeogenic and autophagic enzyme expression. (A) Protein expression levels of glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in HepG2 cells treated with DA-1241 (1,000 nM) for 24 hours under starvation conditions. DA-1241 reduced G6Pase and PEPCK expression in HepG2 cells. (B) Protein expression levels of G6Pase and PEPCK in liver tissue of chow, high-fat diet (HFD), and HFD-DA 1241 group. DA-1241 treatment reduced G6pase and PEPCK compared to HFD group. (C) Protein levels of LC3 and p62 in HepG2 cells treated with DA-1241 (1,000 nM) for 24 hours in the presence or absence of bafilomycin A1 (20 nM) for 2 hours. DA-1241 reduced LC3 levels and enhanced p62 levels in HepG2 cells.

• 

  Fig. 6 Monitoring of autophagic flux in HepG2 cells. HepG2 cells were transfected with microtubule-associated protein 1 light chain 3-fused to green fluorescent protein and monomeric red fluorescent (mRFP-GFP-LC3) expression vector. Then, HepG2 cells were treated with DA-1241 (1,000 nM) for 24 hours, and bafilomycin A1 (20 nM) was added for 2 hours. Cells under normal conditions were used as a control. (A) Images were obtained using a confocal microscope at 40× magnification. (B) Quantification of the number of LC3 and (C) mRFP/GFP-LC3 ratio. DA-1241 increased LC3 puncta (yellow) and decreased mRFP puncta (red) in HepG2 cells. Data are presented as the mean±standard error of the mean. aP<0.01, bP<0.001, compared with control (CTL) (white bars) (n=6).

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