Metabolic Syndrome Abolishes Glucagon-Like Peptide 1 Receptor Agonist Stimulation of SERCA in Coronary Smooth Muscle

Metabolic syndrome (MetS) is defined as the presence of three or more of the following five risk factors: obesity, hypertension, glucose intolerance, insulin resistance, and dyslipidemia (1). Obesity, MetS, and type 2 diabetes are all independent risk factors for cardiovascular disease. The presence of MetS doubles the risk of experiencing an adverse cardiovascular event (2). Thus, it is important to understand the effect of diabetes and MetS treatment modalities on cardiovascular health.

Insulin-sensitizing drugs and those that enhance insulin secretion from pancreatic β-cells have been used in the treatment of type 2 diabetes and MetS. Others, such as dipeptidyl peptidase-4 inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists (exenatide, liraglutide, AC3174, etc.), increase both insulin sensitivity and insulin secretion (3,4). Recently, insulin-sensitizing thiazolidinediones have been implicated in increased risk of fluid retention, LDL and triglyceride accumulation, heart failure, and myocardial infarction (5–7). Hence, there is great attention on cardiovascular outcomes of antidiabetic agents, and the U.S. Food and Drug Administration requires cardiovascular safety profiles for all new agents (8).

GLP-1 receptor agonists are attractive treatment options for MetS because GLP-1 is an endogenous hormone that functions in normal physiology to increase insulin sensitivity, biosynthesis, and secretion (3). Additionally, GLP-1 receptor agonists reduce myocardial infarct size in ischemia/reperfusion injury (9), improve cardiac function in chronic heart failure (10), and attenuate neointimal formation after vascular injury (11). At the cellular level, GLP-1 receptor agonists improve endothelial calcium homeostasis after simulated ischemia/reperfusion (12). Further, GLP-1 receptor agonists improve sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) activity in multiple cell types (13,14). These studies provide rationale for a protective role of GLP-1 in the coronary vasculature.

A recent study revealed impaired GLP-1 stimulation of myocardial glucose uptake in human patients with type 2 diabetes and in MetS swine (15). This finding raises the question of potential resistance to GLP-1 in MetS and type 2 diabetes and highlights the need for rigorous examination of GLP-1 action on coronary artery disease (CAD), specifically in MetS. Given the stimulation of SERCA in macrophages and ventricular myocytes (13,14) and altered SERCA in coronary smooth muscle cells (CSM) in MetS and diabetes (16,17), potentially vasoprotective actions of GLP-1 could be mediated through cellular Ca²⁺ signaling. This study investigated the effect of GLP-1 receptor agonists on SERCA stimulation in CSM from lean swine and on CAD and CSM SERCA stimulation in MetS.

All protocols involving animals were approved by the Institutional Animal Care and Use Committee at Indiana University School of Medicine and fully complied with animal use standards (18,19). Ossabaw miniature swine were separated into two treatment groups: placebo (n = 10) and GLP-1 receptor agonist AC3174 treated (n = 10; 0.25 µg/kg body weight subcutaneously, twice daily; Amylin Pharmaceuticals, San Diego, CA), which displays nearly identical pharmacokinetic and pharmacodynamics profiles as the marketed GLP-1 receptor agonist exenatide (4). CAD and MetS were induced in both treatment groups as described in the Supplementary Material. One animal in the placebo group died prior to the collection of end point data.

Epicardial coronary arteries were cleaned of adherent tissue and CSM were isolated with a collagenase solution as previously described (16,17).

CSM were loaded with fura-2/AM, and whole-cell intracellular Ca²⁺ levels were measured as described in the Supplementary Material.

CSM from lean, healthy Ossabaw swine or a separate group of swine with MetS and CAD were treated with 100 nmol/L exenatide during assessment of intracellular Ca²⁺. The selective SERCA inhibitor cyclopiazonic acid (CPA; 10 μmol/L) was used as a negative control for SERCA function.

Intravenous glucose tolerance testing (IVGTT) was performed as previously described (16,20) and in the Supplementary Material.

Blood samples were obtained at time of IVGTT prior to intravenous injection of glucose. Lipid electrolyte and enzyme content were measured offsite (Antech Diagnostics, West Lafayette, IN).

After 12 months on the diet, intravascular ultrasound (IVUS) pullbacks were performed as described in the Supplementary Material.

Coronary arterial rings were placed in phosphate-buffered formalin at the time of euthanasia. Hematoxylin and eosin, Verhoeff-van Gieson, and trichrome staining were performed on sections of these rings. Plaque burden and collagen content were determined using commercially available software (ImageJ 1.48v; National Institutes of Health) as previously described (16).

We investigated the acute effect of the GLP-1 receptor agonist, exenatide, on intracellular Ca²⁺ handling in CSM isolated from lean, healthy Ossabaw swine. Sarcoplasmic reticulum (SR) Ca²⁺ stores were released with caffeine in the absence of extracellular Ca²⁺ in the presence and absence of 100 nmol/L exenatide (Fig. 1A), and the subsequent undershoot of cytosolic Ca²⁺ during recovery was assessed. Exenatide treatment did not alter resting cytosolic Ca²⁺ (Fig. 1B) or Ca²⁺ influx through voltage-gated Ca²⁺ channels (Fig. 1C). Exenatide elicited improved recovery of cytosolic Ca²⁺ below baseline levels, and this effect was completely ablated in the presence of the SERCA inhibitor CPA (Fig. 1D). The GLP-1 receptor agonist liraglutide also demonstrated improved recovery of cytosolic Ca²⁺, which was prevented by the GLP-1 receptor antagonist, exendin (9-39) (Supplementary Fig. 1). In the presence of caffeine, SERCA buffering of cytosolic Ca²⁺ is functionally inhibited because of the much more rapid release of Ca²⁺ through ryanodine receptors on the SR membrane. Therefore, we assessed the rate of recovery to 50% recovery to baseline (t₅₀) in the presence of caffeine as a measure of Ca²⁺ extrusion and/or Ca²⁺ uptake into caffeine-insensitive intracellular stores, and to 100% recovery to baseline (t₁₀₀) in the absence of caffeine as another measure of SERCA activation. Exenatide significantly decreased time to 100%, but not 50%, recovery to baseline (Fig. 1E).

We then investigated the chronic effect of GLP-1 receptor agonism in vivo. To examine the effects of diet and GLP-1 receptor treatment on glucose metabolism, kidney function, and plasma electrolytes, blood profiles of these parameters were obtained (Supplementary Table 1) and IVGTTs were performed. Overall, feeding of an excess-calorie, atherogenic diet increased body weight, which was significantly attenuated by AC3174 treatment (Fig. 2A). Blood pressure was not altered by diet or AC3174 treatment. Whereas fasting plasma glucose was not altered by diet or AC3174 treatment (Supplementary Table 1), IVGTT assessment of glucoregulation revealed improved glucose handling in AC3174-treated swine, compared with placebo (Fig. 2B–D). This was corroborated by augmented plasma insulin levels after intravenous glucose challenge in AC3174-treated animals, compared with placebo (Fig. 2E–G).

IVUS was used to examine severity of CAD in vivo and was confirmed by histology. Both wall coverage and percent plaque burden were assessed as previously described (16) in placebo and AC3174-treated pigs. AC3174 treatment did not alter CAD severity, as indicated by either wall coverage or percent plaque burden (Fig. 3C and D). In vitro, histological examination of coronary arterial rings revealed no effect of AC3174 treatment on plaque burden (Fig. 3E, F, I, J, and H) or collagen deposition (Fig. 3G, K, and L).

We have previously demonstrated alterations in CSM Ca²⁺ handling in MetS-induced CAD (16,17,21). We therefore investigated whether chronic, in vivo AC3174 treatment resulted in improved intracellular Ca²⁺ handling in CSM. Figure 4A shows the Ca²⁺ signaling protocol to assess baseline Ca²⁺ levels, SR Ca²⁺ store release assessed by the caffeine-induced Ca²⁺ peak in the absence of extracellular Ca²⁺, and SERCA activity assessed by subsequent recovery of cytosolic Ca²⁺ levels and undershoot of cytosolic [Ca²⁺] below baseline levels. Chronic AC3174 treatment did not alter intracellular Ca²⁺ handling in CSM from swine with MetS-induced CAD (Fig. 4B–D).

Further, we assessed the acute, direct effect of GLP-1 receptor agonists on Ca²⁺ handling in CSM isolated from swine with MetS and CAD. Cells were exposed to the GLP-1 receptor agonist exenatide (100 nmol/L), which has an identical pharmacological profile as AC3174 (4) (Fig. 4A), for 3 min prior to treatment with high K⁺. We again assessed caffeine-induced SR Ca²⁺ store release by peak Ca²⁺ response to caffeine and SERCA activity by the subsequent undershoot of cytosolic Ca²⁺ during recovery. Exenatide did not alter either SR Ca²⁺ store release or the undershoot of cytosolic Ca²⁺ below baseline (Fig. 4E and F).

The principle finding of this study is that MetS confers vascular resistance to the effects of GLP-1 receptor agonists. AC3174 did improve several cardiovascular risk factors, such as the metabolic factors, glucose tolerance, and insulin secretion, indicating that longer-term treatment may have indirect benefits on cardiovascular health. Treatment of MetS and type 2 diabetes is confounded by potential secondary and detrimental cardiovascular effects. Previous studies in lean, healthy animals indicate that GLP-1 receptor agonists provide a potential cardioprotective treatment for type 2 diabetes and MetS (9–11). In the current study, we examined possible Ca²⁺ regulatory mechanisms for GLP-1 receptor agonist action in CSM from lean, healthy Ossabaw swine. Our finding that GLP-1 receptor agonists exert a positive effect on SERCA activity in CSM from lean Ossabaw swine is in agreement with findings that GLP-1 receptor agonists enhance SERCA activity in other cell types, including endothelial cells (12), macrophages (13), and cardiomyocytes (14), and is the first study examining the effect of GLP-1 receptor agonists in CSM. The kinetics of the Ca²⁺ transient shown in Fig. 1E demonstrate a lack of exenatide effect on Ca²⁺ extrusion. We have previously shown these kinetic measurements to be an appropriate assay of Ca²⁺ extrusion (17). Reduced SERCA activity has been implicated in CSM proliferation and neointimal formation in the progression of CAD (22,23). One possible mechanism explaining this phenomenon is induction of endoplasmic reticulum (ER) stress. As the single means by which Ca²⁺ may enter the ER, SERCA is a crucial regulator of ER Ca²⁺ homeostasis, and SERCA inhibition elicits an ER stress response through depletion of ER Ca²⁺ (24). ER stress is associated with the development of atherosclerosis (13). Inhibition of ER stress through heightened activation of SERCA could provide a novel means by which to treat CAD.

We also examined the effect of GLP-1 receptor agonists on CAD progression in MetS. This study was needed because of recent evidence of MetS-induced resistance to the cardioprotective effects of GLP-1 (15). The improvement of systemic glucoregulation with AC3174 treatment provides essential positive evidence for GLP-1 receptor agonist action in the Ossabaw swine model of MetS and CAD. Further, we demonstrated that GLP-1 receptor agonist AC3174 has no effect on MetS-induced CAD. This is in contrast with the recent finding that GLP-1 prevents myocardial ischemia–reperfusion injury (9) and injury-induced neointimal hyperplasia (11), providing evidence that the MetS phenotype itself confers cardiovascular resistance to the beneficial effects of GLP-1. It is important to note that although AC3174 treatment did not attenuate CAD progression, the absence of adverse effects of AC3174 provide a cardiovascular safety profile for GLP-1 receptor agonists.

Additionally, we examined the acute, direct effect of GLP-1 receptor agonists on intracellular Ca²⁺ regulation in CSM from Ossabaw swine with MetS and CAD who had not received any treatment with GLP-1 receptor agonists. Here, we found that exenatide had no effect on Ca²⁺ regulation in CSM from swine with MetS and CAD. This finding is in contrast with other studies in which GLP-1 receptor agonism resulted in increased SERCA activity (12–14). A study in humans with type 2 diabetes revealed that exenatide treatment enhanced endothelial-dependent vasodilation (25), although it is important to note that blood cholesterol levels were controlled in these patients. The current study, revealing a lack of effect of GLP-1 receptor agonism on SERCA activity in the setting of MetS, corroborates the earlier finding that MetS ablated the effect of GLP-1 receptor agonists in myocardium and highlights the need for additional studies to investigate specific aspects of MetS (obesity, hyperinsulinemia, glucose intolerance, hypertension, and dyslipidemia) that may underlie resistance of the coronary vasculature to GLP-1 receptor agonists.

Acknowledgments. The authors thank James P. Byrd, Brandy Sparks, and Josh Sturek (Indiana University School of Medicine) for technical assistance. The authors also thank Dr. Rebecca S. Bruning (Indiana University School of Medicine) for editorial assistance during preparation of the manuscript.

Funding. This study was supported by National Institutes of Health grant HL-062552.

Duality of Interest. This study was supported in part by Amylin Pharmaceuticals. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. S.L.D. researched and evaluated data and wrote the manuscript. M.L.M. researched data and reviewed and edited the manuscript. L.N.B. and A.M.F. researched and organized data and reviewed the manuscript. K.A.S. researched data and reviewed the manuscript. M.A., N.C., and M.S. conceptualized the chronic, in vivo study, researched data, and reviewed and edited the manuscript. M.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented at Experimental Biology 2013, Boston, MA, 20–24 April 2013, and at Experimental Biology 2014, San Diego, CA, 26–30 April 2014.