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Urocortin 2 Autocrine/Paracrine and Pharmacologic Effects to Activate AMP-Activated Protein Kinase in the Heart

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Urocortin 2 Autocrine/Paracrine and Pharmacologic Effects to Activate AMP-Activated Protein Kinase in the Heart Urocortin 2 autocrine/paracrine and pharmacologic effects to activate AMP-activated protein kinase in the heart Ji Lia, Dake Qib, Haiying Chengc, Xiaoyue Hub, Edward J. Millerd, Xiaohong Wub, Kerry S. Russellb, Nicole Mikushb, Jiasheng Zhangb, Lei Xiaoe, Robert S. Sherwinc, and Lawrence H. Youngb,1 aDepartment of Pharmacology and Toxicology, University at Buffalo, The State University of New York, Buffalo, NY 14214; bSection of Cardiovascular Medicine and cSection of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; dDepartment of Medicine, Boston University, Boston, MA 02118; and eUniversity of Florida Shands Cancer Center, Department of Anatomy and Cell Biology, College of Medicine, Gainesville, FL 32610 Edited* by Gerald I. Shulman, Howard Hughes Medical Institute and Yale University, New Haven, CT, and approved August 9, 2013 (received for review July 11, 2013) Urocortin 2 (Ucn2), a peptide of the corticotropin-releasing factor Ucn2 (5). Signaling pathways have substantial cross-talk, and (CRF) family, binds with high affinity to type 2 CRF receptors pharmacologic inhibitor studies suggest a possible association (CRFR2) on cardiomyocytes and confers protection against ische- between activation of PKCe and the energy-stress kinase AMP- mia/reperfusion. The mechanisms by which the Ucn2-CRFR2 axis activated protein kinase (AMPK) (10). AMPK is activated by mitigates against ischemia/reperfusion injury remain incompletely changes in cellular energetics, but its activity is also modulated by delineated. Activation of AMP-activated protein kinase (AMPK) other factors, including adiponectin, leptin, and macrophage also limits cardiac damage during ischemia/reperfusion. AMPK is migration inhibitory factor (11). Activated AMPK has important classically activated by alterations in cellular energetics; however, physiologic effects during ischemia, so that inactivation of AMPK hormones, cytokines, and additional autocrine/paracrine factors in mouse hearts interferes with energy generation and increases also modulate its activity. We examined the effects of both the their susceptibility to injury during ischemia/reperfusion (12). endogenous cardiac Ucn2 autocrine/paracrine pathway and Ucn2 CRF family peptides, including Ucn, also play a role in modu- treatment on AMPK regulation. Ucn2 treatment increased AMPK lating energy homeostasis (13). Ucn2 binds with high affinity to the activation and downstream acetyl-CoA carboxylase phosphorylation CRFR2 in cardiomyocytes (14). Thus, we hypothesized that an and glucose uptake in isolated heart muscles. These actions were endogenous cardiac Ucn2 autocrine/paracrine pathway might blocked by the CRFR2 antagonist anti–sauvagine-30 and by a PKCe modulate AMPK activation during ischemia. We also postulated translocation-inhibitor peptide (eV1-2). Hypoxia-induced AMPK acti- that pharmacologic treatment with Ucn2 might augment the vation was also blunted in heart muscles by preincubation with ei- intrinsic response of the AMPK pathway during ischemia, con- – e ther anti sauvagine-30, a neutralizing anti-Ucn2 antibody, or V1-2. tributing to its cardioprotective effect. Treatment with Ucn2 in vivo augmented ischemic AMPK activation and reduced myocardial injury and cardiac contractile dysfunction Results after regional ischemia/reperfusion in mice. Ucn2 also directly acti- Ucn2 Treatment Stimulates AMPK Activation and Glucose Uptake. To vatedAMPKinexvivo-perfused mouse hearts and diminished determine whether Ucn2 treatment stimulates cardiac AMPK injury and contractile dysfunction during ischemia/reperfusion. signaling, we initially incubated rat heart muscles with rat Ucn2 Thus, both Ucn2 treatment and the endogenous cardiac Ucn2 auto- (100 nmol/L) for 10 min. Immunoblots of muscle homogenates crine/paracrine pathway activate AMPK signaling pathway, via demonstrated that Ucn2 stimulated the phosphorylation of the a PKCe-dependent mechanism, defining a Ucn2-CRFR2-PKCe-AMPK activating Thr172 site on the AMPK α subunit (Fig. 1A)aswellits pathway that mitigates against ischemia/reperfusion injury. direct downstream target acetyl-CoA carboxylase (ACC) on Ser79 (Fig. 1A). Ucn2 also stimulated heart muscle glucose uptake (Fig. metabolism | cardiac function 1B), another well-established metabolic consequence of AMPK activation (15). Heart muscles expressed CRFR2 (Fig. 1A). To rocortin (Ucn) is a peptide that is closely related to the determine the mechanism by which Ucn2 treatment stimulates Uneuroendocrine hypothalamic hormone, corticotrophin- heart AMPK, we used the CRFR2 antagonist anti–sauvagine-30 (a- releasing factor (CRF). Ucn was originally identified in the rat SVG-30), an N-terminally truncated version of Ucn2 (14). Pre- midbrain (1) and has an appetite-suppressing effect (2). The incubation with a-SVG-30 abolished Ucn2-stimulated AMPK and actions of Ucn and CRF are mediated by binding to two distinct downstream ACC phosphorylation (Fig. 1C). In addition, a-SVG- receptors, type 1 CRF receptor (CRFR1) and CRFR2. The CRF 30 also eliminated Ucn2-stimulated glucose transport (Fig. 1D). family also includes two related peptides, Ucn2 and Ucn3. Ucn2 and the CRFR2 receptor are expressed in the heart (3) and Endogenous Ucn2 Regulates AMPK Activation and Glucose Uptake CRFR2 binds Ucn2 with 40-fold higher avidity than CRF (3). During Hypoxia. Ucn2 is highly expressed in the heart (7) and Ucn2 has diverse cardiovascular effects that are the focus of we hypothesized that endogenous secreted Ucn2 might activate recent interest because it protects the myocardium against ische- mic injury (4). The molecular mechanisms by which Ucn2 protect MEDICAL SCIENCES the heart are incompletely understood, but may include activation Author contributions: J.L., D.Q., K.S.R., L.X., and L.H.Y. designed research; J.L., D.Q., X.H., e e X.W., K.S.R., N.M., and J.Z. performed research; H.C. and L.X. contributed new reagents/ of the protein kinase C (PKC ) (5), phosphatidylinositol 3-kinase analytic tools; J.L., D.Q., H.C., X.H., E.J.M., X.W., K.S.R., N.M., J.Z., R.S.S., and L.H.Y. ana- (PI3K)/Akt (6), and p42/p44 mitogen-activated protein kinase (7) lyzed data; and J.L., D.Q., E.J.M., R.S.S., and L.H.Y. wrote the paper. signaling pathways. Ucn2 also activates mitochondrial ATP-sen- The authors declare no conflict of interest. sitive potassium channels (8) and prevents the opening of the mi- *This Direct Submission article had a prearranged editor. tochondrial permeability transition pore (8). 1To whom correspondence should be addressed. E-mail: [email protected]. e Activation of PKC protects the heart against ischemic injury This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (9) and plays an important role in the cardioprotective effect of 1073/pnas.1312775110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1312775110 PNAS | October 1, 2013 | vol. 110 | no. 40 | 16133–16138 Downloaded by guest on October 2, 2021 ABC Ucn2 _ 172 50 + a-SVG-30 p-AMPK (Thr ) * 40 p-AMPK (Thr172) AMPK 30 AMPK p-ACC (Ser79) 20 p-ACC (Ser79) ACC (pmol/min/mg) 10 Glucose uptake ACC 0 CRFR2 Fig. 1. Effects of Ucn2 treatment and the endoge- nous cardiac Ucn pathway on AMPK signaling. (A) Rat heart left ventricular papillary muscles were in- E D cubated with or without Ucn2 (100 nmol/L) for 10 50 min and AMPK and downstream ACC phosphoryla- Control Hypoxia 30’ * tion were assessed. Representative immunoblots 40 172 _ _ _ _ † of phospho-AMPK Thr (p-AMPK), total AMPKα + + a-SVG-30 α 79 _ _ _ _ 30 (AMPK ), phospho-ACC Ser (p-ACC), total ACC, and + + Ucn Ab 20 CRFR2 in heart homogenates. (B) The heart muscles were incubated with or without Ucn2 (100 nmol/L) 172 10 3 p-AMPK (Thr ) (pmol/min/mg) for 10 min before measuring 2-deoxy-[1- H] glucose Glucose uptake Glucose 0 uptake. (C and D) Heart muscles were preincubated AMPK Control Ucn2 with CRFR2 antagonist (a-SVG-30, 100 nmol/L) for 15 min, before incubation with or without Ucn2 (100 p-ACC (Ser79) nmol/L) for 10 min. (E and F) Heart muscles were preincubated with a-SVG-30 (100 nmol/L) or Ucn2 Ab μ ACC G (0.2 g/mL) for 15 min, then incubated in normoxic Vehicle (control) or hypoxic (hypoxia) buffer for 30 min. (C Compound C 80 and E) Representative immunoblots show p-AMPK, * AMPKα, p-ACC, and total ACC in heart muscle homogenates. (D and F) Glucose uptake measured 3 F 60 by 2-deoxy-[1- H] glucose. Values are means ± SE for < † < i * three to four experiments. *P 0.01 vs. control, P 75 * * 0.01 vs. Ucn + vehicle, §P < 0.05 vs. hypoxia + vehicle. i † * * *i 40 * (G) Heart muscles were preincubated with or with- Vehicle i† out compound C (10 μmol/L) for 15 min and then 50 a-SVG-30 Ucn Ab incubated with or without Ucn2 (100 nmol/L) in 20 oxygenated buffer for 10 min, or incubated in 25 oxygenated (control) or hypoxic (hypoxia) buffer for (pmol/min/mg) 30 min, or incubated with or without insulin (10 U/mL) Glucose uptake 0 in oxygenated buffer for 30 min, before measuring 0 Glucose uptake (pmol/min/mg) 2-[1-3H]deoxyglucose uptake. Values are means ± SE Control Hypoxia for four experiments. *P < 0.01 vs. control, §P < 0.05 † vs. control, P < 0.01 vs. Ucn or hypoxia alone. AMPK in an autocrine/paracrine fashion. Thus, we pretreated (Fig. 2A), and Ucn2 concentrations increased in the venous heart muscles with a-SVG-30 or neutralizing Ucn2 antibody effluents of ex vivo-isolated perfused hearts during postischemic (Ucn2 Ab) before 30 min of hypoxia. Both a-SVG-30 and Ucn2 reperfusion (Fig. 2B). These studies indicate that endogenous Ab partially inhibited hypoxic AMPK activation as well as Ucn2 was released during ischemia and are consistent with downstream ACC phosphorylation (Fig. 1E) and glucose uptake previous results in isolated cardiomyocytes during hypoxia (18). (Fig. 1F). These results indicate that endogenous cardiac Ucn2 We also measured serum Ucn2 concentrations during in vivo plays a role in modulating hypoxia-stimulated AMPK pathway regional ischemia.
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