Antihyperglycemic mechanism of metformin occurs via the AMPK/LXRα/POMC pathway

Abstract

Metformin is a first-line drug for treating type 2 diabetes. Although metformin is known to phosphorylate AMP-activated protein kinase (AMPK), it is unclear how the glucose-lowering effect of metformin is related to AMPK activation. The aim of this study was to identify the urinary endogenous metabolites affected by metformin and to identify the novel underlying molecular mechanisms related to its anti-diabetic effect. Fourteen healthy male subjects were orally administered metformin (1000 mg) once. First morning urine samples were taken before and after administration to obtain metabolomic data. We then further investigated the anti-diabetic mechanism of metformin in vitro and in vivo. The fluctuation of the metabolite cortisol indicated that the neuroendocrine system was involved in the anti-diabetic effect of metformin. Actually we found that metformin induced AMPK/liver X receptor α (LXRα) phosphorylation, followed by pro-opiomelanocortin (POMC) suppression in rat pituitary cells. We confirmed this result by administering metformin in an animal study. Given that cortisol stimulates gluconeogenesis, we propose the anti-hyperglycemic effect of metformin is attributed to reduced POMC/adrenocorticotropic hormone (ACTH)/cortisol levels following AMPK/LXRα phosphorylation in the pituitaries.

Figure 1. Untargeted metabolomic profiling using HPLC/Q-TOF MS generated PCA score plots discriminating the metformin treated healthy subjects group (red) from the control group (blue).

Cs are for monitoring the overall quality of the analysis procedure (green). Data are shown for (a) the positive and (b) the negative ESI datasets. A representative data from 3 independent experiments.

Figure 2. Metformin reduces urinary cortisol, hydroxyl cortisol, ACTH, and glucose levels in the subjects.

For quantification, (a) cortisol and its metabolite (b) hydroxycortisol, (c) ACTH, and (d) glucose levels were normalized to those of creatinine. Levels of ACTH were measured by ELISA. Data are expressed as the mean ± SE. (e) Correlation between the ratio of cortisol and ACTH levels showed that metformin reduced ACTH secretion and cortisol levels. A representative data from 3 independent experiments.

Figure 3. Metformin suppresses POMC protein levels through phosphorylation of AMPK and subsequently LXRα in vitro.

The rat pituitary adenoma GH3 cells were treated with metformin by using the indicated concentrations and treatment times, and total cell lysates were used for western blotting. (a and b) Metformin upregulated AMPK phosphorylation and downregulated POMC expression. (c) Total cell lysates were used for immunoprecipitation with anti-phopho-Thr antibody and western blotting with anti-LXRα antibody. (d) Reduced POMC after metformin treatment was restored when two siRNAs (#1 and #2) targeting AMPK were transfected in GH3 cells. (e) After metformin treatment, knockdown of LXRα by using two siRNAs (#1 and #2) targeting LXRα (upper) restored POMC expression, although p-AMPK was still enhanced (lower). A representative data from 3 independent experiments. Full-length blots are presented in Supplementary Figure S8.

Figure 4. Metformin activates AMPK and reduces POMC, ACTH, cortisol, and glucose levels in vivo.

(a) Immunohistochemical staining (dark brown) of paraffin-embedded pituitary sections showed metformin (20 mg/kg) induced AMPK phosphorylation, and inhibited POMC and ACTH expression (left). The images are of a representative section (original magnification, ×400. Bar, 50 μm). The number of cells immunoreactive for p-AMPK, POMC, or ACTH was normalized to the total number of cells (right). Data represent the mean ± SE (n = 6). Relative quantification of the creatinine normalized urinary (b) cortisol and (c) glucose in rats before and after metformin treatment (once-daily for 3 consecutive days). Data are expressed as the mean ± SE (n = 6). *P < 0.05, the Wilcoxon signed ranks test used.

Figure 5. Proposed mechanism of the antihyperglycemic action of metformin action via the AMPK/LXRα/POMC pathway.