Metformin Therapy Effects on the Expression of Sodium-Glucose Cotransporter 2, Leptin, and SIRT6 Levels in Pericoronary Fat Excised from Pre-Diabetic Patients with Acute Myocardial Infarction

Abstract

Background and purpose: pericoronary fat over-inflammation might lead to the development and destabilization of coronary plaque in patients with pre-diabetes (PDM). Notably, pericoronary fat could over-express the sodium-glucose cotransporter 2 (SGLT2) and leptin, along with decreased sirtuin 6 (SIRT6) expression in PDM vs. normoglycemic (NG) patients undergoing coronary artery bypass grafting (CABG) for acute myocardial infarction (AMI). However, in the current study, we evaluated inflammatory markers, SGLT2, SIRT6, and leptin levels in pericoronary fat and, subsequently, 12-month prognosis comparing PDM to NG subjected to CABG for AMI. In addition, we evaluated in PDM patients the effects of metformin therapy on SIRT6 expression, leptin, and SGLT2 levels, and assessed its beneficial effect on nitrotyrosine and inflammatory cytokine levels.

Methods: we studied AMI patients referred for CABG, divided into PDM and NG-patients. PDM patients were divided into never-metformin users and metformin users. Finally, we evaluated major adverse cardiac events (MACE) at a 12-month follow-up.

Results: the MACE was 9.1% in all PDM and 3% in NG patients (p < 0.05). Metformin users presented a significantly lower MACE rate in PDM than never-metformin users (p < 0.05). PDM showed higher inflammatory cytokines, 3-nitrotyrosine levels, SGLT2, and leptin content, and decreased SIRT6 protein levels in pericoronary fat compared to NG-patients (p < 0.05). PDM never-metformin-users showed higher SGLT2 and leptin levels in pericoronary fat than current-metformin-users (p < 0.05).

Conclusions: metformin therapy might ameliorate cardiovascular outcomes by reducing inflammatory parameters, SGLT2, and leptin levels, and finally improving SIRT6 levels in AMI-PDM patients treated with CABG.

Keywords: acute myocardial infarction; leptin; metformin; pericoronary fat; pre-diabetes; sodium-glucose cotransporter 2 protein.

Figure 1

Sodium-glucose transporter 2 (SGLT2) levels in pericoronary fat patients. (A,B) SGLT2 protein level in pericoronary fat specimens from 150 normal glucose, 150 pre-diabetic patients, and 55 pre-diabetic current-metformin-users detected by western blotting analysis. Lane 1 = molecular markers; lane 2 = normal glucose patients; lane 3 = pre-diabetic patients; lane 4 = pre-diabetic patients with metformin. The expression level was normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal control using Image J software and values expressed as arbitrary units (AU). (C) SGLT2 level in pericoronary fat specimens from 150 normal glucose, 150 pre-diabetic patients, and 55 pre-diabetic current-metformin-users assessed by enzyme-linked immunosorbent assay (ELISA) assay on homogenates. Boxplots represent the median, 25th, and 75th percentile values, while the black symbols outside represent the 10th and 90th percentiles. For comparison between the cohorts, we used ANOVA test. * p < 0.05 vs. normal glucose patients; ‡ p < 0.05 vs. pre-diabetic patients.

Figure 2

Sirtuin 6 (SIRT6) levels in pericoronary fat patients. (A,B) Sirt6 protein level in pericoronary fat specimens from 150 normal glucose, 150 pre-diabetic patients, and 55 pre-diabetic current-metformin-users detected by western blotting analysis. Lane 1 = molecular markers; Lane 2 = normal glucose patients; lane 3 = pre-diabetic patients; lane 4 = pre-diabetic patients with metformin. The expression level was normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal control using Image J software and values expressed as arbitrary units (AU). (C) SIRT6 level in pericoronary fat specimens from 150 normal glucose, 150 pre-diabetic patients and 55 pre-diabetic current-metformin-users assessed by ELISA assay on homogenates. Boxplots represent the median, 25th and 75th percentiles values while the black symbols outside the 10th and 90th percentiles. For comparison between the cohorts we used the ANOVA test. * p < 0.05 vs. normal glucose patients; ‡ p < 0.05 vs. pre-diabetic patients.

Figure 3

Leptin levels and inflammatory status in pericoronary fat patients. (A) Leptin and (B) 3-nitrotyrosine level in pericoronary fat specimens from 150 normal glucose, 150 prediabetic patients, and 55 prediabetic current-metformin-users assessed by enzyme-linked immunosorbent assay (ELISA) assay on homogenates. Boxplots represent the median, 25th, and 75th percentiles values, while the black symbols outside represent the 10th and 90th percentiles. (C) Assessment of cytokine levels in pericoronary fat specimens from 150 normal glucose, 150 prediabetic patients, and 55 prediabetic current-metformin-users assessed by the ELISA assay on homogenates. TNFα: tumor necrosis alpha; IL-6: interleukin 6; IL-1β: interleukin 1 beta; MCP-1: monocyte chemoattractant protein 1. For comparison between the cohorts, we used the ANOVA test. * p < 0.05 vs. normal glucose patients; ‡ p < 0.05 vs. prediabetic patients without metformin.

Figure 4

Regression analysis evidences a relationship between pericoronary fat leptin contents and sodium-glucose transporter 2 (SGLT2) in the overall study population.

Figure 5

(A) In the left (upper part), the Kaplan–Meier survival curves free from major adverse cardiac events (MACE) in normal glucose patients (blue color) vs. pre-diabetic patients (green color) at 1 year of follow-up. In the right (upper) part, the Kaplan–Meier survival curves free from MACE in pre-diabetic patients with metformin (blue color) vs. pre-diabetic patients without metformin therapy (green color) at 1 year of follow-up. (B) In the left (inferior) part, the Kaplan–Meier survival curves free from MACE, according to SGLT2 tertiles at 1 year of follow-up. In the right (inferior) part, the Kaplan–Meier survival curves free from MACE, according to leptin tertiles at 1 year of follow-up.