Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta
Graphical abstract
Metformin inhibits phenylephrine (PE)-induced smooth muscle contraction through exaggerated AMPK phosphorylation (independent of CaMKKβ), which would occur via ↑AMP/ATP ratio upon coordinated inhibition of ATP synthesis and increase in ATP consumption.
Introduction
Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease [1]. With regard to hypertension, clinical studies reveal its blood pressure-lowering effects in some but not in all subjects with varying co-morbid conditions [cited in [2], [3]]. Studies with different animal models demonstrate that its antihypertensive effects are attributed to endothelium-dependent and endothelium-independent relaxation of the underlying vascular smooth muscle in the vessel wall [2], [4], [5], [6], [7]. Although the molecular mechanism of endothelium-dependent vasodilation by metformin has been extensively studied [8], [9], [10], it remains unclear as to how metformin regulates agonist-induced contractions in intact arterial smooth muscle.
In vascular endothelium, metformin activates AMP-activated protein kinase (AMPK), which phosphorylates endothelial nitric oxide synthase thereby increasing the production of nitric oxide, a potent vasodilator [10], [11]. In endothelium-denuded vessels, metabolic stress and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) have been shown to activate AMPK [12], [13], [14], [15]. Importantly, activation of AMPK results in diminished myosin light chain kinase activity thereby attenuating vascular smooth muscle contraction [16]. Although metformin has been shown to inhibit vascular smooth muscle contraction [17], the intermediary role of AMPK remains unclear especially during agonist-induced contractions. For instance, metformin does not activate AMPK in endothelium-denuded porcine carotid artery [12], but it induces AMPK phosphorylation in porcine and rat aortic smooth muscle cells in culture [17], [18]. Hence, it is critically important to further investigate metformin regulation of contractile function and AMPK phosphorylation in intact arterial smooth muscle ex vivo.
AMPK is a heterotrimeric protein consisting of a catalytic α subunit and β and γ regulatory subunits [19]. Its activity is regulated by increases in adenosine diphosphate (ADP) and/or adenosine monophosphate (AMP) levels. In addition, increase in AMPK activity occurs through phosphorylation of Thr172 residue, inhibition of dephosphorylation of phosphorylated AMPK, and allosteric activation. The major upstream kinases that phosphorylate AMPK include liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) [19], [20].
Metformin has been shown to inhibit mitochondrial respiratory chain complex 1 [21], [22], thereby diminishing ATP/ADP ratio [22] in hepatocytes. However, it does not affect AMP level or AMP/ATP ratio in skeletal muscle cells [23]. To date, metformin regulation of nucleotide levels has not been examined in vascular smooth muscle during agonist-induced contractions. Previously, we and several other investigators have shown that, in vascular smooth muscle cells, vasoconstrictors evoke a rise in cytosolic free Ca2+ [24], [25] that would facilitate the activation of CaMKKβ [20]. Accordingly, vasoactive peptides (e.g., GPCR agonists such as vasopressin, angiotensin II, and endothelin-1) promote an increase in AMPK phosphorylation through an intermediary activation of CaMKKβ in rat aortic smooth muscle cells [16]. The objectives of the present study are to determine: (i) the effects of metformin and/or phenylephrine on nucleotide levels and AMPK phosphorylation; and (ii) the intermediary role of CaMKKβ toward AMPK phosphorylation in intact arterial smooth muscle during isometric contractions.
Using endothelium-denuded rat aortic rings, we performed isometric tension measurements to determine how metformin regulates phenylephrine-induced smooth muscle contraction. Under the same conditions, we performed LC–MS/MS MRM analysis to determine the changes in nucleotide levels. In addition, we performed immunoblot analysis to determine metformin and/or phenylephrine regulation of AMPK phosphorylation before and after treatment with compound C (AMPK inhibitor) or STO-609 (CaMKKβ inhibitor). To determine whether metformin regulation of contraction and AMPK phosphorylation occurs in a reversible manner, select studies included post-treatment washout protocols. To determine whether metformin treatment results in its uptake by smooth muscle, aortic tissue lysates were subjected to LC–MS/MS MRM analysis. The present findings demonstrate that metformin exaggerates phenylephrine-induced AMPK phosphorylation (independent of CaMKKβ), which is associated with diminished smooth muscle contraction.
Section snippets
Materials
Phenylephrine hydrochloride, serotonin (5-hydroxytryptamine hydrochloride), and acetylcholine chloride were purchased from Sigma–Aldrich (St. Louis, MO). Metformin hydrochloride, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside or acadesine), compound C (or dorsomorphin dihydrochloride), and STO-609 acetate were purchased from Tocris Bioscience (Minneapolis, MN). Phenformin hydrochloride and l-NMMA (l-NG-monomethyl arginine acetate) were purchased from Cayman Chemical (Ann Arbor, MI).
Long-term metformin treatment, at 100 μM or 1 mM concentration, inhibits PE-induced smooth muscle contractility in rat aorta ex vivo
Previous studies have shown that oral administration of metformin for up to 11 weeks results in diminished vascular reactivity in rats [32]. In particular, the maximal contractile response to norepinephrine is diminished in the mesenteric arteries (with or without endothelium) isolated from metformin-treated rats [32]. In the present study, we examined whether ex vivo incubation of endothelium-denuded aorta with metformin for an extended period of time (18 h) alters vascular smooth muscle
Discussion
The principal findings of the present study using ex vivo endothelium-denuded rat aorta include: (i) metformin accumulation in intact arterial smooth muscle; (ii) a modest but significant increase in AMPK phosphorylation by phenylephrine but not by metformin per se; (iii) an exaggerated increase in AMPK phosphorylation with an associated elevation of AMP/ATP ratio by phenylephrine after metformin treatment; (iv) compound C (AMPK inhibitor) inhibition of AMPK phosphorylation induced by
Acknowledgements
This work was supported by the National Heart, Lung, and Blood Institute/National Institutes of Health Grant (R01-HL-097090), University of Georgia Research Foundation, and University of Georgia RC Wilson Pharmacy Fund to L. Segar.
References (60)
- et al.
Effect of metformin treatment on multiple cardiovascular disease risk factors in patients with type 2 diabetes mellitus
Metabolism
(2004) - et al.
Vascular effects of metformin. Possible mechanisms for its antihypertensive action in the spontaneously hypertensive rat
Am J Hypertens
(1996) - et al.
Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocin-induced diabetic rats
Life Sci
(2006) - et al.
Improved endothelial function with metformin in type 2 diabetes mellitus
J Am Coll Cardiol
(2001) - et al.
Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species
J Biol Chem
(2004) - et al.
AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase
J Biol Chem
(2008) - et al.
Metformin-induced AMP-activated protein kinase activation regulates phenylephrine-mediated contraction of rat aorta
Biochem Biophys Res Commun
(2012) - et al.
Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I
J Biol Chem
(2000) - et al.
The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways
J Biol Chem
(2002) - et al.
Rapid determination of metformin in human plasma by liquid chromatography–tandem mass spectrometry method
J Chromatogr B: Analyt Technol Biomed Life Sci
(2004)
Quantification of metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats treated with lipopolysaccharide
Pharmacol Rep
Decreased vascular reactivity in metformin-treated fructose-hypertensive rats
Metabolism
Effects of guanidine derivatives on mitochondrial function. 3. The mechanism of phenethylbiguanide accumulation and its relationship to in vitro respiratory inhibition
J Biol Chem
Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation
Cell Metab
Involvement of organic cation transporter-3 and plasma membrane monoamine transporter in serotonin uptake in human brain vascular smooth muscle cells
Front Pharmacol
Characterization of the CaMKKbeta-AMPK signaling complex
Cell Signal
Exercise-induced AMPK activity in skeletal muscle: role in glucose uptake and insulin sensitivity
Mol Cell Endocrinol
Opposing adrenergic actions of intravenous metformin on arterial pressure in female spontaneously hypertensive rats
Cardiovasc Res
The effect of metformin on blood pressure, plasma cholesterol and triglycerides in type 2 diabetes mellitus: a systematic review
J Intern Med
Metformin decreases plasma insulin levels and systolic blood pressure in spontaneously hypertensive rats
Am J Physiol
Metformin improves vascular function in insulin-resistant rats
Hypertension
Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase
Diabetes
AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633
Circ Res
Metabolic activation of AMP kinase in vascular smooth muscle
J Appl Physiol
Activation of AMP kinase alpha1 subunit induces aortic vasorelaxation in mice
J Physiol
Endothelium-dependent vasorelaxation to the AMPK activator AICAR is enhanced in aorta from hypertensive rats and is NO and EDCF dependent
Am J Physiol Heart Circ Physiol
AMP-activated protein kinase activator AICAR acutely lowers blood pressure and relaxes isolated resistance arteries of hypertensive rats
J Hypertens
AMP-activated protein kinase inhibits IGF-I signaling and protein synthesis in vascular smooth muscle cells via stimulation of insulin receptor substrate 1 S794 and tuberous sclerosis 2 S1345 phosphorylation
Mol Endocrinol
AMPK: a nutrient and energy sensor that maintains energy homeostasis
Nat Rev Mol Cell Biol
Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP
Biochem J
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