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Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, the functional roles of CR in regulation of revascularization in response to ischemia have not been examined. Here we investigated whether CR modulates vascular response by employing a murine hindlimb ischemia model. Wild-type (WT) mice were randomly divided into two groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb ischemic surgery. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared with WT mice fed AL (AL-WT). Revascularization of ischemic hindlimb relative to the contralateral limb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow and capillary density analyses. CR-WT mice had significantly higher plasma levels of the fat-derived hormone adiponectin compared with AL-WT mice. In contrast to WT mice, CR did not affect the revascularization of ischemic limbs of adiponectin-deficient (APN-KO) mice. CR stimulated the phosphorylation of endothelial nitric-oxide synthase (eNOS) in the ischemic limbs of WT mice. CR increased plasma adiponectin levels in eNOS-KO mice but did not stimulate limb perfusion in this strain. CR-WT mice showed enhanced phosphorylation of AMP-activated protein kinase (AMPK) in ischemic muscle, and administration of AMPK inhibitor compound C abolished CR-induced increase in limb perfusion and eNOS phosphorylation in WT mice. Our observations indicate that CR can promote revascularization in response to tissue ischemia via an AMPK-eNOS-dependent mechanism that is mediated by adiponectin. Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, the functional roles of CR in regulation of revascularization in response to ischemia have not been examined. Here we investigated whether CR modulates vascular response by employing a murine hindlimb ischemia model. Wild-type (WT) mice were randomly divided into two groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb ischemic surgery. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared with WT mice fed AL (AL-WT). Revascularization of ischemic hindlimb relative to the contralateral limb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow and capillary density analyses. CR-WT mice had significantly higher plasma levels of the fat-derived hormone adiponectin compared with AL-WT mice. In contrast to WT mice, CR did not affect the revascularization of ischemic limbs of adiponectin-deficient (APN-KO) mice. CR stimulated the phosphorylation of endothelial nitric-oxide synthase (eNOS) in the ischemic limbs of WT mice. CR increased plasma adiponectin levels in eNOS-KO mice but did not stimulate limb perfusion in this strain. CR-WT mice showed enhanced phosphorylation of AMP-activated protein kinase (AMPK) in ischemic muscle, and administration of AMPK inhibitor compound C abolished CR-induced increase in limb perfusion and eNOS phosphorylation in WT mice. Our observations indicate that CR can promote revascularization in response to tissue ischemia via an AMPK-eNOS-dependent mechanism that is mediated by adiponectin. Obesity is closely associated with the development of metabolic syndrome and type 2 diabetes (1Reilly M.P. Rader D.J. Circulation. 2003; 108: 1546-1551Crossref PubMed Scopus (418) Google Scholar), which contribute to microvascular rarefaction and impaired collateral vessel growth under ischemic conditions (2Yilmaz M.B. Biyikoglu S.F. Akin Y. Guray U. Kisacik H.L. Korkmaz S. Int. J. Obes. Relat. Metab. Disord. 2003; 27: 1541-1545Crossref PubMed Scopus (63) Google Scholar, 3Schiekofer S. 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Caloric restriction (CR) 4The abbreviations used are: CR, caloric restriction; AL, ad libitum; WT, wild-type; KO, knockout; eNOS, endothelial nitric-oxide synthase; HR, heart rate; LDBF, laser Doppler blood flow; AMO PK, AMP-activated protein kinase; pfu, plaque-forming unit; NOS, nitric-oxide synthase; Ad-APN, adenovirus expressing adiponectin. has been shown to extend the life span of multiple species by retarding the aging process (5Ingram D.K. Anson R.M. de Cabo R. Mamczarz J. Zhu M. Mattison J. Lane M.A. Roth G.S. Ann. N. Y. Acad. Sci. 2004; 1019: 412-423Crossref PubMed Scopus (171) Google Scholar). In obese subjects CR has been shown to reduce visceral fat accumulation and also decrease body weight (6Fontana L. Klein S. J. Am. Med. Assoc. 2007; 297: 986-994Crossref PubMed Scopus (403) Google Scholar). CR have also been reported to lead to a reduction of hyperglycemia and hyperlipidemia that are major risk factors for ischemic heart diseases (7Zimmerman J. Kaufmann N.A. 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Ageing Dev. 2002; 123: 1121-1131Crossref PubMed Scopus (104) Google Scholar), pathological cardiac hypertrophy (13Seymour E.M. Parikh R.V. Singer A.A. Bolling S.F. J. Mol. Cell. Cardiol. 2006; 41: 661-668Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and ischemia-induced myocardial damage (14Shinmura K. Tamaki K. Saito K. Nakano Y. Tobe T. Bolli R. Circulation. 2007; 116: 2809-2817Crossref PubMed Scopus (153) Google Scholar). These findings suggest that CR counteracts the unfavorable features of obese complications. However, the consequences of CR on vascular responses to tissue ischemia have not been examined. Adipose tissue secretes a variety of bioactive molecules, referred to as adipokines, that directly affect obesity-linked disorders in remote organs (15Lago F. Dieguez C. Gomez-Reino J. Gualillo O. Nat. Clin. Pract. Rheumatol. 2007; 3: 716-724Crossref PubMed Scopus (449) Google Scholar). 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Lu L.X. Bernier M. DeCabo R. Lane M.A. Roth G.S. Ingram D.K. Exp. Gerontol. 2004; 39: 1049-1059Crossref PubMed Scopus (147) Google Scholar). Thus, it is plausible that adiponectin mediates the salutary actions of CR in the setting of obesity-linked vascular complications. Here we investigated whether CR modulates the process of ischemia-induced revascularization in vivo. We first examined the effect of CR on revascularization using a mouse model of vascular insufficiency. We also examined the potential contribution of adiponectin to CR-mediated revascularization process in ischemic muscles. Our observations indicate that CR promotes revascularization in response to tissue ischemia via modulation of adiponectin production. Materials—Endothelial nitric-oxide synthase (eNOS) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-eNOS (Ser-1177), phospho-AMPK (Thr-172), and pan-α-AMPK antibody were purchased from Cell Signaling Technology (Beverly, MA). Tubulin antibody was from Oncogene (Cambridge, MA). Adenovirus vectors containing the gene for β-galactosidase (Ad-βgal) and full-length mouse adiponectin (Ad-APN) were described previously (20Matsuda M. Shimomura I. Sata M. Arita Y. Nishida M. Maeda N. Kumada M. Okamoto Y. Nagaretani H. Nishizawa H. Kishida K. Komuro R. Ouchi N. Kihara S. Nagai R. Funahashi T. Matsuzawa Y. J. Biol. Chem. 2002; 277: 37487-37491Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar). Compound C was purchased from Calbiochem. CR Protocols—Studies using wild-type (WT), eNOS-deficient (eNOS-KO), and adiponectin-deficient (APN-KO) mice in a C57/BL6 background were approved by the Institutional Animal Care and Use Committee at Nagoya University. Male mice at the ages of 6 weeks were housed in individual cages and fed ad libitum (AL) on a normal chow for 2 weeks. Food was provided at the same time (3:00 p.m.), and food intake of individual mice was measured daily. The average value of caloric intake was calculated from daily food intake for 2 weeks. After that, mice were randomly divided into two groups. The AL group was fed ad libitum for an additional 4 weeks. CR mice were fed with 65% of the average caloric intake of control AL diet for the next 4 weeks. At 4 weeks after the CR or control AL diet, mice were subjected to unilateral hindlimb surgery. All mice were weighed at weekly intervals. Heart rate (HR) and systolic blood pressure were determined using a tail-cuff pressure analysis system in the conscious state. Mouse Model of Revascularization—Mice were subjected to unilateral hindlimb surgery under anesthesia with sodium pentobarbital (50 mg/kg intraperitoneally). In this model, the entire left femoral artery and vein were removed surgically as described previously (21Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Investig. 1998; 101: 2567-2578Crossref PubMed Scopus (1085) Google Scholar). In some experiments, 2 × 108 plaque-forming units (pfu) of Ad-APN or Ad-βgal were systemically injected into the jugular vein of mice 3 days before the ischemic hindlimb surgery (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In some experiments, we intraperitoneally injected NOS inhibitor l-NAME (20 in or into WT mice to surgery and daily In some experiments, AMPK inhibitor compound C (20 in or was intraperitoneally injected into the of WT mice before the T. Y. K. M. N. K. T. PubMed Scopus Google Scholar, Z. Li H. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of blood flow was determined using a laser Doppler blood flow were on and before surgery and on days and flow was shown as in the laser using analysis of blood flow was as the of left to to of and (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). of density in was by were in compound and in in were and with by with antibody to The were and by endothelial were by the number of and the number of in randomly from in tissue (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). on were in containing sodium and inhibitor was determined by the The same of protein (50 were with The were with the at a by the antibody with at a were using of density and levels were measured with levels were measured with Adiponectin levels were determined using was by heart from mice on 4 weeks of are as analysis was by analysis of by A value of was as of CR on Body in WT reduction in body weight the first 2 weeks of CR, and the reduction in body weight was 2 and 6 weeks The WT mice fed AL showed a increase in body weight of this time At the of the time the in body weight AL-WT mice and WT mice fed CR diet was 26% and systolic blood pressure did not the two groups with L.B. Denny C.A. Seyfried T.N. Lipids Health Dis. 2006; 5: 13Crossref PubMed Scopus (73) Google Scholar, O. Y. G. M. Stein Y. 2003; PubMed Scopus Google Scholar), was a reduction in and plasma levels in the CR-WT mice compared with AL-WT of AL-WT and CR-WT mice were from WT mice fed AL or CR diet for 4 weeks value is heart rate systolic blood pressure density plasma AL-WT AL-WT AL-WT AL-WT mice in a CR Revascularization in to in WT and CR-WT mice of unilateral hindlimb ischemia 4 weeks after the of All mice and the of hindlimb blood flow before surgery and at time after surgery in the and CR-WT mice. In AL-WT mice, hindlimb perfusion after increased to of the limb by and to of the limb by CR-WT mice showed a increase in limb flow at and days after hindlimb surgery compared with AL-WT mice the of revascularization at the capillary density was measured in from the ischemic of ischemic are shown in analysis that the capillary density was significantly increased in CR-WT mice compared with AL-WT mice at days after surgery of Adiponectin in CR-mediated has been shown to increase levels of adiponectin (14Shinmura K. Tamaki K. Saito K. Nakano Y. Tobe T. Bolli R. Circulation. 2007; 116: 2809-2817Crossref PubMed Scopus (153) Google Scholar, 19Zhu M. Miura J. Lu L.X. Bernier M. DeCabo R. Lane M.A. Roth G.S. Ingram D.K. Exp. Gerontol. 2004; 39: 1049-1059Crossref PubMed Scopus (147) Google Scholar), and we have shown that adiponectin promotes blood flow in response to ischemia (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). These findings to that the increase in circulating adiponectin levels to the vascular response in the We plasma adiponectin levels in WT with AL or At the time of CR increased plasma adiponectin levels to a higher compared with AL-WT mice in AL-WT mice and in CR-WT mice, the of adiponectin in ischemia-induced revascularization by CR, we investigated the effect of CR on blood flow of ischemic of mice on and mice had a blood flow of ischemic limbs in with (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). CR did not affect ischemic limb perfusion of mice after surgery as compared with In the of mice with an adenovirus expressing adiponectin (Ad-APN) to a increase in plasma adiponectin In this Ad-APN is systemically 3 days to surgery. to of the and adiponectin can be in R. Sato K. Kumada M. Y. M. Kihara S. Ouchi N. Walsh K. 2007; PubMed Scopus Google Scholar). circulating levels of adiponectin are not in mice and mice, Ad-APN increased circulating levels to in mice at after surgery that is to AL-WT mice. of circulating adiponectin to an of limb perfusion after hindlimb surgery suggest that the effect of CR on ischemia-induced revascularization is mediated by the of adiponectin. eNOS for CR-induced eNOS an in revascularization hindlimb ischemia (21Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Investig. 1998; 101: 2567-2578Crossref PubMed Scopus (1085) Google Scholar). the potential of eNOS in CR-induced the and phosphorylation of eNOS in ischemic were by The of eNOS protein in ischemic did not AL and However, phosphorylation of eNOS at in ischemic was significantly in CR-WT mice in AL-WT mice A and the contribution of adiponectin to regulation of eNOS by CR, we the phosphorylation of eNOS in ischemic tissue of mice with AL or In contrast to the of CR on eNOS phosphorylation in WT mice, CR did not eNOS phosphorylation in mice A and whether increased of adiponectin phosphorylation of eNOS in ischemic muscle, Ad-βgal or Ad-APN was to and WT mice. adiponectin levels were in in in and in on with Ad-APN significantly increased eNOS phosphorylation in ischemic of WT and mice at after surgery A and the of eNOS in revascularization by CR, we examined the of CR on blood flow of ischemic in eNOS-KO mice. mice had increased plasma adiponectin levels compared with mice in mice and in mice, analysis that in limb perfusion were and mice on and eNOS-KO mice ischemia-induced vascular which is by we limb and tissue using a system J. Zhuang Z. M. K. T. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). the of of tissue ischemia after hindlimb surgery did not and mice We also the effect of CR on blood flow of ischemic in WT mice NOS inhibitor l-NAME or CR-WT mice showed increased of blood flow compared with AL-WT mice, with l-NAME the increase in limb perfusion in CR-WT mice suggest that revascularization is to eNOS that is in adiponectin production. of AMPK in CR-induced the of AMPK in CR-induced the and phosphorylation of AMPK in ischemic were by The of AMPK protein in ischemic did not AL and CR mice. However, phosphorylation of AMPK in ischemic was significantly in CR-WT mice in AL-WT mice at after the A and the of AMPK in CR-induced we examined the effect of CR on blood flow of ischemic in WT mice AMPK inhibitor compound C or CR-WT mice compound C or had increased plasma adiponectin levels compared with AL-WT mice in AL-WT mice, in CR-WT mice, and in compound CR-WT mice, of CR-WT mice with compound C increased limb perfusion by CR compared with CR-WT mice with compound C significantly CR-induced increase in eNOS phosphorylation in ischemic tissue and These suggest that AMPK is in CR-induced eNOS and that CR promotes ischemia-induced revascularization in a hindlimb model. CR enhanced blood flow and capillary formation in ischemic of wild-type mice, which was by increased levels of eNOS phosphorylation and plasma adiponectin. The actions of CR on and eNOS phosphorylation of ischemic limbs were abolished in mice. CR did not flow in ischemic limbs of eNOS-KO mice. The of CR to increase adiponectin levels is to contribute to the of revascularization under experimental We have shown that adiponectin revascularization of ischemic limbs in wild-type mice (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Our observations that CR increased plasma adiponectin levels in wild-type mice, in with (14Shinmura K. Tamaki K. Saito K. Nakano Y. Tobe T. Bolli R. Circulation. 2007; 116: 2809-2817Crossref PubMed Scopus (153) Google Scholar, 19Zhu M. Miura J. Lu L.X. Bernier M. DeCabo R. Lane M.A. Roth G.S. Ingram D.K. Exp. Gerontol. 2004; 39: 1049-1059Crossref PubMed Scopus (147) Google Scholar). CR ischemia-induced revascularization in wild-type mice but not mice. adiponectin promotes vascular and under conditions of (22Shibata R. Ouchi N. Kihara S. Sato K. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 28670-28674Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, H. Ouchi N. Kihara S. Walsh K. Kumada M. Y. Funahashi T. Matsuzawa Y. 2004; PubMed Google Scholar), we that adiponectin as an of vascular it was reported that CR to myocardial injury by adiponectin levels (14Shinmura K. Tamaki K. Saito K. Nakano Y. Tobe T. Bolli R. Circulation. 2007; 116: 2809-2817Crossref PubMed Scopus (153) Google Scholar). CR also the and myocardial damage in obese mice with which is by increased adiponectin levels in plasma and T. S. T. H. S. T. K. M. Int. J. Cardiol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the of adiponectin by CR could a mechanism in the of cardiovascular to is that eNOS is beneficial for of vascular and metabolic diseases (21Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Investig. 1998; 101: 2567-2578Crossref PubMed Scopus (1085) Google Scholar, Kearney Int. J. Obes. Relat. Metab. Disord. 2002; PubMed Scopus Google Scholar, H. T. T. U. 2002; PubMed Scopus Google Scholar). In this CR increased the phosphorylation of eNOS in ischemic and the of CR to enhance revascularization was in eNOS-KO mice or WT mice NOS Thus, CR to promote revascularization in ischemic to is reported that CR in of mice, and are in eNOS-KO mice C. A. V. R. L. S. A. O. S. 2005; PubMed Scopus Google Scholar). observations suggest that eNOS as a of the protective actions of has been shown that adiponectin exerts vascular modulation of eNOS We have shown that adiponectin phosphorylation of eNOS, which is associated with enhanced endothelial and into H. Ouchi N. Kihara S. Walsh K. Kumada M. Y. Funahashi T. Matsuzawa Y. 2004; PubMed Google Scholar, N. H. Kihara S. Kumada M. Sato K. T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, R. C. Ouchi N. Galasso G. K. T. M. Kihara S. T. Walsh K. PubMed Scopus Google Scholar). Adiponectin by endothelial via the phosphorylation of eNOS N. H. Kihara S. Kumada M. Sato K. T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Huang Y. D. D. C. A. 2007; PubMed Scopus Google Scholar, H. M. Funahashi T. Shimomura I. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). we have shown that mice increased injury compared with WT mice, which is by eNOS in ischemic tissue M. Y. A. R. J. C. M.A. Ouchi N. Circulation. PubMed Scopus Google Scholar). administration of adiponectin and eNOS phosphorylation in ischemic the actions of adiponectin were in eNOS-KO mice, that eNOS is for salutary vascular responses to adiponectin in ischemic with this that mice have phosphorylation of eNOS in ischemic muscles. The CR-induced increase in eNOS phosphorylation ischemia was abolished in mice. the increased plasma adiponectin in eNOS-KO mice CR, CR had on perfusion of ischemic limbs in eNOS-KO mice. that the actions of CR are at in the Our that AMPK is for revascularization in ischemic CR stimulated the phosphorylation of AMPK in the ischemic and administration of an AMPK inhibitor CR-induced increase in ischemia-induced revascularization and eNOS AMPK is reported to directly eNOS at B.J. D. I. de PubMed Scopus Google Scholar). has also been shown that adiponectin phosphorylation of eNOS in endothelial to AMPK N. H. Kihara S. Kumada M. Sato K. T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. Funahashi T. Shimomura I. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). AMPK has also been shown to stimulate vascular endothelial growth in ischemic N. R. Walsh K. 2005; PubMed Scopus Google Scholar). However, vascular endothelial growth levels in ischemic did not AL and CR-WT mice not that the beneficial effect of CR on revascularization is not mediated by the of this be in CR-induced we the that CR-mediated increase in adiponectin has a salutary effect on the under conditions of ischemic by and promotes the revascularization diseases, including type 2 increased and of and peripheral artery diseases of microvascular rarefaction and impaired collateral vessel growth under ischemic conditions (2Yilmaz M.B. Biyikoglu S.F. Akin Y. Guray U. Kisacik H.L. Korkmaz S. Int. J. Obes. Relat. Metab. Disord. 2003; 27: 1541-1545Crossref PubMed Scopus (63) Google Scholar, A. A. S. S. H. A. Circulation. PubMed Scopus Google Scholar, R. H. G. J. PubMed Scopus Google Scholar). The findings reported suggest that CR could revascularization in response to tissue Thus, approaches at caloric intake could be for of ischemic heart and limb diseases.
Kondo et al. (Fri,) studied this question.