Cardioprotection Resulting from Glucagon-Like Peptide-1 Administration Involves Shifting Metabolic Substrate Utilization to Increase Energy Efficiency in the Rat Heart

Abstract

Previous studies have shown that glucagon-like peptide-1 (GLP-1) provides cardiovascular benefits independent of its role on peripheral glycemic control. However, the precise mechanism(s) by which GLP-1 treatment renders cardioprotection during myocardial ischemia remain unresolved. Here we examined the role for GLP-1 treatment on glucose and fatty acid metabolism in normal and ischemic rat hearts following a 30 min ischemia and 24 h reperfusion injury, and in isolated cardiomyocytes (CM). Relative carbohydrate and fat oxidation levels were measured in both normal and ischemic hearts using a 1-13C glucose clamp coupled with NMR-based isotopomer analysis, as well as in adult rat CMs by monitoring pH and O2 consumption in the presence of glucose or palmitate. In normal heart, GLP-1 increased glucose uptake (↑64%, p<0.05) without affecting glycogen levels. In ischemic hearts, GLP-1 induced metabolic substrate switching by increasing the ratio of carbohydrate versus fat oxidation (↑14%, p<0.01) in the LV area not at risk, without affecting cAMP levels. Interestingly, no substrate switching occurred in the LV area at risk, despite an increase in cAMP (↑106%, p<0.05) and lactate (↑121%, p<0.01) levels. Furthermore, in isolated CMs GLP-1 treatment increased glucose utilization (↑14%, p<0.05) and decreased fatty acid oxidation (↓15%, p<0.05) consistent with in vivo finding. Our results show that this benefit may derive from distinct and complementary roles of GLP-1 treatment on metabolism in myocardial sub-regions in response to this injury. In particular, a switch to anaerobic glycolysis in the ischemic area provides a compensatory substrate switch to overcome the energetic deficit in this region in the face of reduced tissue oxygenation, whereas a switch to more energetically favorable carbohydrate oxidation in more highly oxygenated remote regions supports maintaining cardiac contractility in a complementary manner.

Fig 1. Infarct assessment following cardiac ischemia-reperfusion injury in rat.

Sprague-Dawley rats (n = 5-6) were subjected to a 30 min LAD coronary artery occlusion followed by 24 hr period of reperfusion. Hearts were harvested for assessment of area at risk and infarct size. Representative photographs of heart sections stained with TTC and Evans Blue dye are shown for both Vehicle (A) and GLP-1 (B) groups. The areas of myocardial infarct are white, areas at risk (AAR) are the combined white and red regions, and area not at risk (ANAR) are dark blue. Infarct size and area at risk are presented as percentage of AAR and left ventricle, respectively (C). Data are presented as mean±SEM. *p<0.05 vs Vehicle.

Fig 2. Tissue cAMP levels in the right ventricle, AAR, and ANAR of left ventricle after myocardial ischemia/reperfusion injury.

Tissues were harvested following a 30 min ischemia/24 h reperfusion period and extracted as described in the text for cAMP analysis. Data comparing Vehicle (n = 3-6) with GLP-1 (300 pmol/kg/min) (n = 3-6) treatment groups are shown, with mean ±SEM as indicated. ***p<0.001 vs ANAR *p<0.05 vs Vehicle.

Fig 3. In vivo [³H]-2-DG uptake in rat myocardium.

Kinetics of [³H]-2-DG clearance from plasma after a single bolus injection for Vehicle (, n = 8), GLP-1 (300 pmol/kg/min) (∆, n = 8), or insulin (3 U/kg) (▼, N = 5) (A). Myocardial glucose uptake measured at the end of study; i.e. 30 min following tracer administration (B). Data are presented as mean±SEM. *p<0.05, ***p<0.001 vs Vehicle.

Fig 4. In vivo intermediary glucose metabolism in AAR and ANAR of rat left ventricle following a euinsulinemic-hyperglycemic clamp over 2 hr with 1-[¹³C] glucose infusion.

Left ventricle intermediary metabolite ¹³C enrichments of alanine, lactate and glutamate are presented for AAR or ANAR myocardial tissue from Vehicle (n = 6) and GLP-1 (300 pmol/kg/min group, n = 6) treatment groups (A). Relative carbohydrate oxidation versus fat oxidation in Vehicle and GLP-1 treated AAR and ANAR myocardial tissue (B). The relative carbohydrate versus fat oxidation is calculated using isotopomer analysis of alanine and glutamate enrichments as described in the methods section. Data are presented as mean±SEM. *p<0.05 vs respective Vehicle.

Fig 5. Glucose utilization and reserve capacity in cultured CMs.

Glucose utilization was assessed by examining percent change in ECAR and the reserve capacity assessed as percent change in OCR following FCCP challenge in the presence of indicated concentrations of GLP-1 or insulin at 70 nM. A typical seahorse plot representing changes in ECAR over time following acute treatment with 100 nM GLP-1 (maximal effective dose) or insulin optimal media (A) or suboptimal media (B). Percentage change in ECAR, 10 min post injection of GLP-1 (1, 10, 100 nM) or insulin with optimal media (C) and suboptimal media (D). Typical seahorse plots representing changes in OCR over time following acute treatment with 100 nM GLP-1 or insulin with optimal (E) or suboptimal (F) media. Percentage change in OCR, 80 min post injection of GLP-1 (1, 10, 100 nM) or insulin in optimal (G) and suboptimal (H) media. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. Data are presented as mean±SEM of 3–5 replicates per treatment from 2–4 individual experiments. ***p<0.001, *p<0.05, vs Control.

Fig 6. Effect of fatty acid oxidation on GLP-1 induced oxygen consumption in cultured CMs.

OCR changes following stimulation with 100 nM GLP-1 (maximal effective dose) or 70 nM insulin are presented following palmitate challenge. GLP-1 or insulin was injected at 21 min and Vehicle (bovine serum albumin) or Palmitate was injected at 52 min following initiation of data collection. The responses are presented as percentage changes in OCR before and after Vehicle or Palmitate injection (A). Quantitation of percentage changes before (left bars; first arrow in panel A) and after (right bars; second arrow in Panel A) Palmitate or Vehicle challenge (B). Data are presented as mean ±SEM of 3–4 replicates per treatment. *p<0.05, **p<0.01, ***p<0.001.