Study of insulin vascular sensitivity in aortic rings and endothelial cells from aged rats subjected to caloric restriction: Role of perivascular adipose tissue

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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

Study of insulin vascular sensitivity in aortic rings and endothelial cells from aged rats subjected to caloric restriction: Role of perivascular adipose tissue S. Amora, B. Martín-Carroa, C. Rubiob, J.M. Carrascosab, W. Huc, Y. Huangc, A.L. García-Villalóna, M. Granadoa,d,⁎ a

Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain c School of Biomedical Sciences, Institute of Vascular Medicine, Faculty of Medicine, Chinese University of Hong Kong, China d CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain b

A R T I C L E I N F O

A B S T R A C T

Keywords: Aging Insulin Akt Perivascular adipose tissue Aorta eNOS

The prevalence of metabolic syndrome is dramatically increasing among elderly population. Metabolic syndrome in aged individuals is associated with hyperinsulinemia and insulin resistance both in metabolic tissues and in the cardiovascular system, with this fact being associated with the cardiometabolic alterations associated to this condition. Caloric restriction (CR) improves insulin sensitivity and is one of the dietetic strategies most commonly used to enlarge life and to prevent aging induced cardiovascular alterations. The aim of this study was to analyze the possible beneficial effects of CR in aging-induced vascular insulin resistance both in aortic rings and in primary culture of endothelial cells. In addition, the inflammatory profile of perivascular adipose tissue (PVAT) and its possible role in the impairment of vascular insulin sensitivity associated with aging was also assessed. Three experimental groups of male Wistar rats were used: 3 (3 m), 24 (24 m) fed ad libitum and 24 months old rats subjected to 20% CR during their three last months of life (24 m–CR). Aorta rings surrounded or not by PVAT were mounted in an organ bath and precontracted with phenylephrine (10− 7.5 M). Changes in isometric tension were recorded in response to cumulative insulin concentrations (10− 8–10− 5.5 M) in the presence or absence of L-NAME (10− 4 M). Aortic rings and primary aortic endothelial cells were incubated in presence/absence of insulin (10− 7 M) and the activation of the PI3K/Akt and MAPK pathways as well as nitrite and nitrates concentrations and the mRNA levels of eNOS, insulin receptor, and GLUT-4 were assessed. CR prevented the aging-induced decrease in the vasodilator response to insulin and the aging-induced increase in the vasoconstrictor response to high insulin concentrations. Changes between 24 m and 24 m–CR aorta rings were abolished in the presence of L-NAME. CR induced-improvement in insulin vascular sensitivity was related with activation of the PI3K/Akt both in aortic rings and in aortic endothelial cells in response to insulin. CR attenuated the overexpression of iNOS, TNF-α and IL-1β in the PVAT of aged rats although aortic rings surrounded by PVAT from 24 m rats showed and increased vasorelaxation in response to insulin compared to aortic rings from 3 m and 24 m–CR rats. In conclusion, a moderate protocol of CR improves insulin vascular sensitivity and prevents the aging induced overexpression of pro-inflammatory cytokines in PVAT.

1. Introduction Aging is the major risk factor for cardiovascular diseases, as nearly 90% of incident CV events occur in adults over 55 years of age (Gu and Xu, 2013). Aging is associated with several cardiovascular alterations such as endothelial dysfunction and arterial stiffness that lead to an impairment on cardiovascular function (Tuomilehto, 2004). Some of these alterations are linked to metabolic syndrome whose prevalence is increased among elderly men and women. Indeed, the ratio for

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metabolic syndrome among men and women of 65 years old of age and older is approximately fivefold higher than among those aged 20–34 years old (Park et al., 2003; Schulman et al., 2007). A large body of evidence demonstrates that aging and metabolic syndrome share several metabolic alterations that include an altered distribution, expansion, and endocrine function of adipose tissue (Bonomini et al., 2015; Haffner, 2000), as well as hyperinsulinaemia and insulin resistance (Armani et al., 2017). Indeed, the onset of insulin resistance and type 2 diabetes is a hallmark of aging (Lopez-Lluch and

Corresponding author at: Department of Physiology, Faculty of Medicine, Universidad Autónoma de Madrid, C/Arzobispo Morcillo no. 2, 28029 Madrid, Spain. E-mail address: [email protected] (M. Granado).

http://dx.doi.org/10.1016/j.exger.2017.10.017 Received 4 August 2017; Received in revised form 15 October 2017; Accepted 16 October 2017 0531-5565/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Amor, S., Experimental Gerontology (2017), http://dx.doi.org/10.1016/j.exger.2017.10.017

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2. Material and methods

Navas, 2016). Aging-induced alterations in cardiovascular function are related to impairment in insulin sensitivity both in metabolic tissues (Escriva et al., 2007) and in the cardiovascular system (Boudina, 2013), where insulin exerts direct effects in physiological conditions, both in the heart and in the vasculature (Muniyappa et al., 2007). In the myocardium insulin increases heart contractility (positive inotropic effect) through activation of Ca channels both in vivo (von Lewinski et al., 2005) and in vitro (Maier et al., 1999). In the vasculature insulin exerts vasodilatory actions through the activation of the insulin receptor (IR) tyrosine kinase, leading to tyrosine phosphorylation of IR substrate (IRS)-1, which binds and activates phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates Akt at serine 473, which directly activates endothelial nitric oxide (NO) synthase (eNOS) via phosphorylation at serine 1177. In blood vessels, the activation of the PI3K/Akt pathway in response to insulin leads, in the first place, to an increase in blood flow via capillary recruitment and, in the second place, to the vasodilation of larger blood vessels (Dimmeler et al., 1999; Steinberg et al., 1994). The impairment of insulin signaling in the vasculature is related to a reduced activation of the PI3K/Akt pathway which induces a decrease in NO bioavailability and an increase in the adhesion of mononuclear cells to the endothelium which, in the end, accelerate the atherosclerotic process and promotes a deleterious effect in cardiovascular function (Rask-Madsen et al., 2010). Interestingly, it is reported that in situations of hyperinsulinaemia and vascular insulin resistance there is a selective inhibition of the PI3K/Akt pathway whereas the activation of the mitogen-activated protein kinases (MAPKs) pathway, which mediates the proliferative and vasoconstrictor effects of insulin through the production of endothelin-1 (ET-1), remains unaffected (Cusi et al., 2000; Jiang et al., 1999; Muniyappa et al., 2007). Thus, the antihypertensive effects of insulin mediated by NO production are reduced under conditions of insulin resistance. Vascular insulin resistance in a context of metabolic syndrome may be linked, at least in part, to the secretion of proinflammatoy mediators by perivascular adipose tissue, since it is reported that in the metabolic syndrome this tissue becomes highly inflamed and induces vascular dysfunction through an augmented secretion of pro-inflammatory adipokines and vasoconstrictive factors such as the components of reninangiotensinogen-aldosterone system and reactive oxygen species (Szasz et al., 2013; Szasz and Webb, 2012). Caloric restriction (CR) is a dietary intervention that delays aging and extends lifespan in diverse species (Anderson and Weindruch, 2010) due, at least in part, to a promotion in cardiovascular health (Lopez-Lluch and Navas, 2016). Moreover, CR with adequate nutrition improves several deleterious conditions present in elder individuals such as insulin resistance, increased fasting glucose and insulin concentrations and prevents obesity, type 2 diabetes, hypertension and chronic inflammation (Soare et al., 2014). Thus, CR is considered a promising strategy to treat/prevent the metabolic and cardiovascular alterations associated with both aging and metabolic diseases. However the severity of CR plays a major in role in its beneficial effects (Vitousek et al., 2004), being reported in rodents that a protocol of 20% reduction in food intake for 3 months is effective decreasing adiposity, improving insulin sensitivity, both centrally and peripherally (Escriva et al., 2007; García-San Frutos et al., 2007), and preventing some of the aging induced alterations in cardiovascular function (Amor et al., 2017; Granado et al., 2014). However it is unknown weather this specific protocol of moderate CR is able to modify the inflammatory profile of perivascular adipose tissue and to prevent the aging-induced alterations in vascular insulin sensitivity in rats. Therefore the aim of this work was to analyze insulin vascular sensitivity in aortic rings and vascular endothelial cells from aged rats subjected to caloric restriction, as well as the possible role of perivascular adipose tissue in this response.

2.1. Animals Three (n = 12) and 24-months-old (n = 24) male Wistar rats from our in-house colony (Centre of Molecular Biology, Madrid, Spain) were used throughout this study. Rats were housed in climate-controlled quarters with a 12 h light cycle and fed ad libitum a standard laboratory chow A04–10 Rodent Maintenance Diet (SAFE, Spain) and water. Handling of animals was performed according to European Union laws and the guidelines of the National Institutes of Health). Experimental procedures were approved by the Institutional Committee of Research Ethics. 2.2. Caloric restriction Half of the 21-months-old rats (n = 12) were assigned to undergo a caloric restriction protocol as previously described (Perez et al., 2004). Animals were placed in individual cages and fed daily an amount of chow equivalent to 80% of normal food intake without supplementation with additional minerals or vitamins. Caloric restriction was prolonged during three months. 2.3. Experiments of vascular reactivity Restricted (CR) and ad libitum fed rats were decapitated at the ages of 3 (3 m) and 24 (24 m) months old previously anesthetized with sodium pentobarbital (100 mg/kg i.p.). Immediately after death, aorta was carefully dissected, cut in 2 mm segments and kept in cold isotonic saline solution. Each segment was mounted in a 4 ml organ bath containing modified Krebs–Henseleit solution at 37 °C (mM): NaCl, 115; KCl, 4.6; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 11. The solution was equilibrated with 95% oxygen and 5% carbon dioxide to a pH of 7.3–7.4. Briefly, two fine steel wires (100 μm diameter) were passed through the lumen of the vascular segment. One wire was fixed to the organ bath wall and the other wire was connected to a strain gauge for isometric tension recording (Universal Transducing Cell UC3 and Statham Microscale Accessory UL5, Statham Instruments, Inc.). This arrangement permits passive tension to be applied in a plane perpendicular to the long axis of the vascular cylinder. The changes in isometric force were recorded using a PowerLab data acquisition system (ADInstruments, Colorado Springs, CO, USA). After applying an optimal passive tension of 1 g, vascular segments were allowed to equilibrate for 60–90 min. Afterwards, segments were stimulated with potassium chloride (KCl 100 mM) to determine the contractility of smooth muscle. Segments which failed to contract at least 0.5 g to KCl were discarded. After equilibration, the segments were precontracted with 10− 7,5M phenylephrine (Sigma-Aldrich, St. Louis, MO, USA) to subsequently perform a cumulative dose-response curve in response to insulin (10− 8–10− 5.5 M) (Sigma-Aldrich, St. Louis, MO, USA). The relaxation in response to insulin was determined based on the percentage of the active tone achieved by the NO donor sodium nitroprusside (10− 5 M) (Sigma-Aldrich, St. Louis, MO, USA). The highest concentrations of insulin in the dose-response curve induced contraction instead of vasodilation in some aorta segments. This contraction was measured as the increase in tension over the level reached at the end of the vasodilation, and expressed as percentage of the contraction to potassium chloride (100 mM). To study the mechanism of the vasodilation in response to insulin some segments were preincubated for 30 min with the inhibitor of the nitric oxide synthase L-NAME (10− 4 M) (Nω-Nitro-L-arginine methyl ester hydrochloride) (Sigma-Aldrich, St. Louis, MO, USA). Moreover, to study the mechanism of vasoconstriction in response to high doses of insulin other segments were preincubated for 30 min with the antagonist of the angiotensin II receptor type 1 Losartan (10− 5 M) (2-Butyl-4chloro-1-[2′-(1H–tetrazol-5-yl)(1,1′-biphenyl)-4-yl]methyl 1H2

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100 μl of culture medium on a 96- well plate. Immediately after, the Griess reagent (1:1 mixture of 1% sulfanilamide (Merck Millipore, Darmstadt, Germany), and 0.1% naphthylethylenediamine dihydrochloride (Merck Millipore, Darmstadt, Germany)) was added to each well and incubated at 37 °C for 30 min. The absorbance was measured at 540 nm. Nitrite and nitrate concentration was calculated using a NaNO2 standard curve and was expressed in μM.

imidazole-5-methanol monopotassium salt) (Sigma-Aldrich, St. Louis, MO, USA), the antagonist of endothelin type A receptor BQ-123 (10− 6 M) (Cyclo-Asp-Pro-Val-Leu-Trp) (Sigma-Aldrich, St. Louis, MO, USA) and the antagonist of the thromboxane synthase Furegrelate (10− 6 M) (5-(3-pyridinylmethyl)-2-benzofurancarboxylic acid, sodium salt) (Cayman Chemical, Ann Arbor, MI, USA). For each dose–response curve, the logarithm of the concentration producing 50% of the maximal response (ED50) was calculated by geometric interpolation. Finally, the effect of perivascular adipose tissue (PVAT) in the vascular reactivity in response to insulin (10− 8–10− 5.5 M) was assessed in 2 mm segments of thoracic aorta coated with PVAT. In these segments the vasodilator response to insulin was studied in the absence or presence of L-NAME (10− 4 M). Segments were precontracted with phenylephrine (10− 7.5 M) and the relaxation in response to insulin was determined based on the percentage of the active tone achieved by the NO donor sodium nitroprusside (10− 5 M).

2.7. Immunoblotting Arterial tissue and endothelial cells were homogenized using RIPA buffer, and total protein content was analyzed by the Bradford method (Bradford, 1976). For each assay, 100 μg of protein were loaded in each well and resolving gels with SDS acrylamide (10%) were used. After electrophoresis, proteins were transferred to polyvinylidine difluoride (PVDF) membranes (BioeRad, Hércules, CA, USA) and transfer efficiency was determined by Ponceau red dyeing. Membranes were then blocked with Tris-buffered saline (TBS) containing 5% (w/v) non-fat dried milk or bovine serum albumin (for phosphorylated proteins) and incubated with the appropriate primary antibody: endothelial oxide nitric synthase (eNOS) (1:1000; BD Bioscience, San José, CA, USA); phospho-endothelial oxide nitric synthase (p-eNOS) (1:500; Merck Millipore, Darmstadt, Germany); Akt (1:1000; Merck Millipore, Darmstadt, Germany); phospho-Akt (1:500; Cell signaling Technology, Danvers, MA, USA); MAPK (1:1000; Merck Millipore, Darmstadt, Germany); phospho-MAPK (1:500; Merck Millipore, Darmstadt, Germany). Membranes were subsequently washed and incubated with the corresponding secondary antibody conjugated with peroxidase (1:2000; Pierce, Rockford, IL, USA). Peroxidase activity was visualized by chemiluminescence and quantified by densitometry using BioRad Molecular Imager ChemiDoc XRS System. All data are normalized to the housekeeping protein GAPDH and referred to % of control values on each gel.

2.4. Incubation of aorta segments in presence/absence of insulin (10− 7 M) Some 2 mm thoracic aorta segments were incubated in 6well culture plates (3segments/well) with 1.5 ml of Dulbecco's Modified Eagle's Medium and Ham's F12 medium (DMEM/F-12) with glutamine from Gibco (1:1 mix; Invitrogen, Carlsbad, CA, USA), supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) or insulin (10− 7 M) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 95% O2 and 5% CO2 incubator. After 30 min of incubation, both the segments and the culture media were collected and stored at − 80 °C for further analysis. 2.5. Primary culture of aortic endothelial cells After sacrifice, aortas from animals from the three experimental groups were carefully dissected and washed in sterile PBS (Invitrogen, Carlsbad, CA, USA). The isolation of rat aortic endothelial cells was performed as previously described (Wong et al., 2011). Briefly, the rat aorta was incubated with type I collagenase (0.01 g/5 ml sterile PBS) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 15 min in an orbital shaker (Controltecnica, Boadilla del Monte, Madrid, Spain). The reaction was stopped with 5 ml of DMEM/F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Hampton, NH, USA). The aorta was discarded and the solution was then centrifuged at 1000 rpm for 5 min. After centrifugation, cells were resuspended in 5 ml of DMEM/F-12 supplemented with penicillin/streptomycin and 10% FBS and cultured at 37 °C in a 95% O2 and 5% CO2 incubator (Esco, Hatboro, PA, USA) in a 25 cm3 flask until 80–90% confluency. Afterwards, endothelial cell were washed with PBS, detached with trypsin (Thermo Fisher Scientific, Hampton, NH, USA), centrifugated, resuspended and cultured with DMEM/F-12 supplemented with penicillin/streptomycin and 10% FBS until 80–90% confluency in a 75 cm3 flask. At this moment, cells were trypsinized, centrifuged and seeded in 6 well/plates (106 cells/well) until confluency. Later on cells were fasted for 18 h (DMEM/F12 not supplemented with 10% FBS), and incubated at 37 °C in a 95% O2 and 5% CO2 in presence/ absence of insulin (10− 7 M) for 30 min. Finally, endothelial cells were trypsinized and both supernatants cells were collected and stored at − 80 °C for further analysis. The survival rate, measured by Trypan Blue dying, was superior to 90%.

2.8. RNA extraction and quantitative RT-PCR Total RNA was extracted from aorta segments, endothelial cells and PVAT according to the TRI Reagent protocol (Chomczynski, 1993). cDNA was then synthesized from 1 μg of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Afterwards, glucose transporter 4 (GLUT-4), insulin receptor (IR), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Cyclooxygenase 2 (COX-2), Tumor necrosis factor alpha (TNF-α), Interleukin 6 (IL-6) and Interleukin 1 beta (IL-1β) mRNA levels were assessed in aorta, endothelial cells or PVAT samples by quantitative real-time PCR. Quantitative real-time PCR was performed by using assay-on-demand kits (Applied Biosystems, Foster City, CA, USA) for each gene: GLUT-4 (Rn 00562597), IR (Rn 00690703), eNOS (Rn 02132634), iNOS (Rn 00561646), COX-2 (Rn 01483828), TNF-α (Rn 01525859), IL-6 (Rn 01489669) and IL-1β (Rn 00580432). TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used for amplification according to the manufacturer's protocol in a Step One machine (Applied Biosystems, Foster City, CA, USA). Values were normalized to the housekeeping gene 18S (Rn01428915). According to manufacturer's guidelines, the ΔΔCT method was used to determine relative expression levels. Statistics were performed using ΔΔCT values (Livak and Schmittgen, 2001). 2.9. Statistical analysis

2.6. Nitrites and Nitrates determination in the culture medium Values are expressed as the mean ± SEM. For the statistical analysis, GraphPad 5.0 Software was used. The effect of age was determined by comparing values of 3-months-old rats and 24-months-old rats fed ad libitum by unpaired Student's t-test. The effect of caloric restriction was determined by comparing values of 24-months-old rats fed ad libitum and 24-months-old rats with caloric restriction by

Nitrites and nitrates concentrations in the culture medium from both aorta segments incubations and aortic endothelial cell primary culture were measured by a modified method of the Griess assay, described by Miranda et al., 2001 (Miranda et al., 2001). Briefly, 100 μl of vanadium chloride (Sigma-Aldrich, St. Louis, MO, USA) was added to 3

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from 24 m–CR rats in the presence of Furegrelate 10− 6 M (P < 0.001). 3.2. Insulin-induced vasodilation in aortic rings in presence/absence of LNAME Relaxation of aortic rings from 3 m, 24 m and 24 m–CR in response to accumulative doses of insulin is represented in Fig. 2A. Insulin administration induced vasodilation in aortic segments from all experimental groups in a dose-dependent manner, with this vasodilation being significantly lower in aortic segments from 24 m compared to aortic segments from 3 m (P < 0.001) and 24 m–CR rats (P < 0.05). Accordingly the log of ED50 was significantly decreased in aged rats fed ad libitum compared to young rats (P < 0.05) and this effect was prevented by CR (P < 0.01). Finally, the pre-incubation of aortic rings with L-NAME (10− 4 M) significantly decreased insulin induced vasodilation in all experimental groups and abolished the differences between the relaxation of aortic segments from 24 m and 24 m–CR rats (Fig. 2B). 3.3. Nitrites and Nitrates concentrations in aorta segments in the presence/ absence of insulin (10− 7 M) Nitrites and Nitrates concentrations in aorta segments in the presence/absence of insulin are shown in Table 1. In aorta segments, nitrites and nitrates concentrations were decreased in 24 m rats compared to 3 m (P < 0.001). Incubation with insulin 10− 7 M increased NO2 + NO3 levels in aorta segments form 3 m and 24 m–CR rats (P < 0.05 for both) and decreased them in aorta segments from 24 m rats (P < 0.05). 3.4. Insulin activation of PI3K/Akt and MAPK pathways in aortic rings Fig. 1. (A) Contraction insulin dose-response curve and log of ED50 of aortic rings from 3month-old Wistar rats fed ad libitum (3 m; n = 12), 24-month-old Wistar rats fed ad libitum (24 m; n = 12) and 24-month-old Wistar rats subjected to caloric restriction (24 m–CR; n = 12). (B) Contractile response of aortic rings from 3-month-old Wistar rats fed ad libitum (3 m; n = 12), 24-month-old Wistar rats fed ad libitum (24 m; n = 12) and 24-month-old Wistar rats subjected to caloric restriction (24 m–CR; n = 12) in response to insulin (10–5,5M) after pre-incubation with vehicle, Losartan (10− 5 M), BQ-123 (10− 6 M) or Furegrelate (10− 6 M). Data are represented as mean ± S.E.M (n = 12 rats/ group). ⁎P < 0.05 vs 3 m; #P < 0.05 vs 24 m; $$P < 0.01 vs its respective control.

In basal conditions aging did not significantly downregulate the ratio p-Akt/Akt (Fig. 3A), but it significantly decreased the ratio peNOS/eNOS (P < 0.05; Fig. 3C) and increased the ratio p-MAPK/ MAPK (P < 0.01; Fig. 3B) in arterial tissue. CR did not prevent neither the aging induced decrease in the ratio p-eNOS/eNOS nor the aging induced increased in the ratio p-MAPK/MAPK, but it significantly increased the ratio p-Akt/Akt (P < 0.05). Incubation of aortic rings with insulin (10− 7 M) for 30 min did not modify the activation of Akt, MAPK or eNOS in 24 m rats, but it significantly upregulated the ratios p-Akt/ Akt in 3 m (P < 0.01) and 24 m–CR rats (P < 0.05), the ratio pMAPK/MAPK in 3 m rats (P < 0.01) and the ratio p-eNOS/eNOS in both 3 m and 24 m–CR rats (P < 0.05 for both).

unpaired Student's. Gene and protein expression in the presence/absence of insulin was compared by unpaired Student's t-test. P value of < 0.05 was considered significant. 3. Results

3.5. Insulin activation of insulin receptor (IR), endothelial nitric oxide synthase (eNOS) and glucose transporter four (GLUT-4) mRNA levels in arterial tissue

3.1. Insulin-induced vasoconstriction in aortic rings in presence/absence of Losartan, BQ-123 or Furegrelate At high concentrations insulin induced vasoconstriction in aortic rings from all experimental groups (Fig. 1A), with this vasoconstriction being significantly higher in the aortic rings from 24 m rats compared to those from 3 m rats at the doses of 10− 5.5 and 10− 6 M (P < 0.05 for both), and to those from 24 m–CR rats at the dose of 10− 5.5 M (P < 0.05). Likewise, the log of ED50 was significantly decreased in aged rats fed ad libitum compared to that of young rats (P < 0.01), whereas there were no significant changes in the log of ED50 between 24 m and 24 m–CR rats. To study the mechanism implied in insulin induced vasoconstriction at the dose of 10− 5.5 M some aortic rings from each experimental group were pre-incubated with the antagonist of type 1 angiotensin II receptors Losartan (10− 5 M), the antagonist of type A endothelin-1 receptors BQ-123 (10− 6 M) or the inhibitor of thromboxane synthase Furegrelate (10− 6 M) (Fig. 1B). None of the treatments prevented insulin induced vasoconstriction at the dose of 10− 5.5 M. On the contrary, insulin induced vasoconstriction significantly increased in aortic rings

In basal conditions aging was associated with a significant downregulation of IR (P < 0.05; Fig. 4A) and GLUT-4 (P < 0.05; Fig. 4C) mRNA levels in arterial tissue whereas the mRNA levels of eNOS were unchanged (Fig. 4B). CR did not modify the gene expression of eNOS and GLUT-4 but it significantly increased the mRNA levels of IR (P < 0.05). Incubation of aortic rings with insulin (10− 7 M) for 30 min significantly upregulated the mRNA levels of IR, eNOS and GLUT-4 in arterial tissue of 3 m rats (P < 0.05 for all) and the gene expression of GLUT-4 in aorta segments from 24 m–CR rats. 3.6. Nitrites and Nitrates concentrations in aortic endothelial cells in the presence/absence of insulin (10− 7 M) Nitrites and Nitrates concentrations in aortic endothelial cells in the presence/absence of insulin are shown in Table 1. In aortic endothelial cells, nitrites and nitrates concentrations were increased in 24 m rats compared to 3 m (P < 0.05). Incubation with insulin 10− 7 M 4

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Fig. 2. Relaxation insulin dose-response curve of aortic rings from 3-month-old Wistar rats fed ad libitum (3 m; n = 12), 24-month-old Wistar rats fed ad libitum (24 m; n = 12) and 24month-old Wistar rats subjected to caloric restriction (24 m–CR; n = 12) in the absence (A) or presence of the nitric oxide synthase inhibitor L-NAME (10− 4 M). Data are represented as mean ± S.E.M (n = 12 rats/group). ⁎P < 0.05 vs 3 m; ⁎⁎P < 0.01 vs. 3 m; ⁎⁎⁎P < 0.001 vs 3 m. $$P < 0.01 vs 24 m; #P < 0.05 vs its respective control.

increased the gene expression of this enzyme in endothelial cells from 24 m–CR rats. IR mRNA levels (Fig. 6B) were not modified in the different experimental groups neither in basal conditions nor in response to insulin administration (10−7 M). On the contrary, in basal conditions aging was associated with a significant decrease in the mRNA levels of GLUT-4 (P < 0.01) that was not attenuated by CR (Fig. 6C). Incubation of aorta rings with insulin significantly increased the gene expression of GLUT-4 in the arterial tissue of 3 m rats but not in arterial tissue from 24 m and 24 m–CR rats.

increased NO2 + NO3 levels in aorta segments form 3 m and 24 m–CR rats (P < 0.05 for both) but not in endothelial cells from 24 m rats. 3.7. Insulin activation of PI3K/Akt and MAPK pathways in primary cultures of aortic endothelial cells Fig. 5 shows the ratios between p-Akt/Akt (A) and p-MAPK/MAPK (B) in aortic endothelial cells from the different experimental groups. In basal conditions there were no differences in the ratios of p-Akt/Akt and p-MAPK/MAPK in the endothelial cells from the different experimental groups. However, in response to insulin administration (10−7 M), there was an up-regulation in the ratio p-Akt/Akt in endothelial cells from 3 m (P < 0.01), 24 M (P < 0.05) and 24 m–CR (P < 0.05) rats and an up-regulation in the ratio p-MAPK/MAPK in endothelial cells from 24 m rats (P < 0.05).

3.9. Gene expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin 1β (IL-1β) in perivascular adipose tissue (PVAT) Fig. 7 shows the gene expression of eNOS (A), iNOS (B), COX-2 (C), TNF-α (D), IL-6 (E) and IL-1β (F) in the periaortic adipose tissue from 3 m, 24 m and 24 m–CR rats. Aging did not modify the mRNA levels of COX-2 and IL-6 in the aorta but it significantly up-regulated the gene expression of eNOS (P < 0.001), iNOS (P < 0.01), TNF-α (P < 0.01) and IL-1β (P < 0.05). CR attenuated aging induced increase in eNOS (P < 0.05), iNOS (P < 0.05), TNF-α (P < 0.05) and IL-1β

3.8. Insulin activation of insulin receptor (IR), endothelial nitric oxide synthase (eNOS) and glucose transporter four (GLUT-4) mRNA levels in in primary cultures of aortic endothelial cells The gene expression of eNOS did not differ among experimental groups in basal conditions (Fig. 6A). However, insulin significantly

Table 1 Nitrites and nitrates (NO2 + NO3) concentrations released by aortic rings and endothelial cells from 3-month-old Wistar rats fed ad libitum (3 m; n = 24) and 24-month-old Wistar rats fed ad libitum (24 m; n = 20), or subjected to caloric restriction (24 m–CR; n = 25) in absence (culture medium) or presence of insulin 10− 7 M. Data are represented as mean ± S.E.M. Treatment

3m 24 m 24 m–CR

Aortic rings

Endothelial cells

Culture medium

Insulin 10− 7 M

Culture medium

Insulin 10− 7 M

15,11 ± 0,16 13,64 ± 0,14### 13,31 ± 0,37

16,31 ± 0,35⁎ 13,12 ± 0,12⁎ 14,39 ± 0,21⁎

2,48 ± 0,21 4,55 ± 0,92# 4,30 ± 1,04

7,27 ± 0,21⁎⁎ 5,02 ± 1,07 9,97 ± 3,99⁎

⁎

P < 0.05 vs. their respective control. P < 0.01 vs. their respective control. # P < 0.05 vs. 3 m. ### P < 0.001 vs. 3 m. ⁎⁎

5

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Fig. 3. (A) Relation of protein levels of phosphorylated Akt (p-Akt) to total Akt in aorta rings from 3- or 24- months-old rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction in absence (control) or presence of insulin (10− 7 M) for 30 min. (B) Relation of protein levels of phosphorylated MAPK (p-MAPK) to total MAPK in aorta rings from 3- or 24- monthsold rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction, in absence (control) or presence of insulin (10− 7 M) for 30 min. (C) Relation of protein of phosphorylated eNOS (p-eNOS) to total eNOs in aorta rings from 3- or 24- months-old rats fed ad libitum, or 24months-old rats after 3 months of caloric restriction, in absence (control) or presence of insulin (10− 7 M) for 30 min. Data are represented as mean ± S.E.M (n = 8 samples/group). # P < 0.05 vs 3 m; ##P < 0.01 vs 3 m; $ P < 0.05 vs 24 m; ⁎P < 0.05 vs its respective control; ⁎⁎P < 0.01 vs its respective control.

found in the literature as it has been reported that physiological doses of insulin induce vasodilation in different vascular beds through the activation of PI3K/Akt intracellular pathway and the subsequent eNOS phosphorylation and NO production, whereas supraphysiological insulin concentrations induce vasoconstriction (Schulman and Zhou, 2009). However this phenomenon depends on the type of arteries, being reported that low concentrations of insulin induce vasoconstriction and higher dosages of insulin induced vasodilation in certain vascular beds such as cerebral arteries (Katakam et al., 2009). The mechanisms by which insulin induces vasoconstriction are controversial and include both endothelium-dependent (Rebolledo et al., 2001) and independent mechanisms such as an activation of the sympathetic nervous system (Berne et al., 1992; Scherrer and Sartori, 1997). Regarding vasoconstriction, our results show that aging is associated with contraction of aorta segments in response to high concentrations of insulin and that this effect is prevented by CR. However we have not been able to elucidate the mechanism responsible for this phenomenon since the vasoconstrictor effect of insulin is not abolished in the presence of blockers of endothelin 1 type A receptors, angiotensin II type 1 receptors or thromboxane synthetase inhibitors. As expected physiological insulin concentrations induced vasodilation of aorta segments from all experimental groups, with this

(P < 0.05) mRNA levels. 3.10. Insulin-induced vasodilation in aortic rings surrounded by perivascular adipose tissue in presence/absence of L-NAME Fig. 8 shows the vascular response of aortic rings surrounded by perivascular adipose tissue in response to cumulative insulin concentrations in absence (A) or presence (B) of L-NAME (10−4 M). Insulin induced vasodilation in a dose dependent manner in aortic rings from all experimental groups with this vasodilatory effect being significantly higher in the aortic rings from 24 m rats compared to 3 m and 24 m–CR rats (P < 0.01). Blockade of NO by L-NAME did not modify the relaxation in response to insulin in aortic rings from none of the experimental groups. 4. Discussion In this study we report for the first time the beneficial effects of a moderate protocol of CR preventing the aging induced alterations in vascular insulin sensitivity. These alterations include a decreased vasoconstrictor response to high doses of insulin and an increased vasodilator response to low doses of insulin. Our results agree with others 6

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Fig. 4. Gene expression of insulin receptor (IR;A), endotelial nitric oxide synthase (eNOS;B) and glucose transporter type 4 (GLUT-4;C), in the aorta from 3- or 24- months-old rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction, in absence (control) or presence of insulin (10− 7 M) for 30 min. Data are represented as mean ± S.E.M (n = 8 samples/group). #P < 0.05 vs 3 m; $P < 0.05 vs 24 m; ⁎P < 0.05 vs its respective control.

Fig. 5. (A) Relation of protein levels of phosphorylated Akt (p-Akt) to total Akt in primary culture of endothelial cells from 3- or 24- monthsold rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction in absence (control) or presence of insulin (10− 7 M) for 30 min. (B) Relation of protein levels of phosphorylated MAPK (p-MAPK) to total MAPK in primary culture of endothelial cells from 3- or 24months-old rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction, in absence (control) or presence of insulin (10− 7 M) for 30 min. (C) Relation of protein of phosphorylated eNOS (p-eNOS) to total eNOs in primary culture of endothelial cells from 3- or 24months-old rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction, in absence (control) or presence of insulin (10− 7 M) for 30 min. Data are represented as mean ± S.E.M (n = 8 samples/group). ⁎ P < 0.05 vs its respective control; ⁎⁎P < 0.01 vs its respective control.

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Fig. 6. Gene expression of insulin receptor (IR;A), endotelial nitric oxide synthase (eNOS;B) and glucose transporter type 4 (GLUT-4;C), in primary culture of endothelial cells from 3- or 24- months-old rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction, in absence (control) or presence of insulin (10− 7 M) for 30 min. Data are represented as mean ± S.E.M (n = 8 samples/group). ⁎P < 0.05 vs its respective control; ##P < 0.01 vs. 3 m.

differences between our results and those published by Schulman et al. may be due to the measurement of mRNA vs protein levels and/or to the different strains of rats used (Wistar vs Sprague-Dawley). Insulin significantly increased de ratio p-Akt/Akt and the ratio peNOS/eNOS in arterial tissue from 24 m–CR which clearly shows increased insulin sensitivity compared to 24 m rats. Although this is the first study reporting the beneficial effects of CR on vascular insulin sensitivity in aged rats, we and other authors have reported that CR prevents aging induced insulin resistance in other organs and tissues such as the heart (unpublished data from our group), the hypothalamus (Garcia-San Frutos et al., 2012) the adipose tissue (Sierra Rojas et al., 2016) or the skeletal muscle (Wang et al., 2016). In addition, the beneficial effect of CR increasing insulin-induce vasodilation in aged rats seems to be mediated by the release of NO since preincubation of aorta segments with L-NAME abolish the differences in the vasodilation between aorta segments from 24 and 24 m rats. Similarly NO has been reported to mediate the beneficial effects of CR on aging-induced endothelial dysfunction in rodents (Chou et al., 2010; Rippe et al., 2010; Yang et al., 2004; Zanetti et al., 2010) as it prevents the aging-induced decrease in vasodilation in response to

vasodilation being significantly greater in aorta segments of 3 m and 24 m–CR rats compared to those from 24 m rats. Moreover, this effect was associated with a higher release of NO in response to insulin both in aorta segments and in primary aorta endothelial cells. Likewise other authors have reported that vascular insulin sensitivity decreases with age due to an impaired activation of the PI3K/Akt intracellular pathway and eNOS phosphorylation which is related to a decreased NO production and to an increased secretion of endothelin-1 (Li et al., 2009; Schulman et al., 2007; Wang et al., 2011). Our results agree with those reported by other authors as they show that aging is associated with decreased levels of p-Akt and p-eNOS in arterial tissue, and with a decreased release of nitrites and nitrates by aorta segments in response to insulin (Yang et al., 2004). The decrease in eNOS phosphorylation in response to insulin in the aorta of aged rats may be related, at least in part, with the decreased mRNA levels of IR in the arterial tissue. In addition, the gene expression of IR increases in response to insulin in the arterial tissue of 3 m rats but not in the arterial tissue of 24 m rats, which indicates a state of vascular insulin resistance. However other authors have reported that IR protein levels are not modified in the aorta of aged rats in basal conditions (Schulman et al., 2007). The 8

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Fig. 7. Gene expression of endotelial nitric oxide synthase (eNOS; A), inducible nitric oxide synthase (iNOS; B), ciclooxygenase 2 (COX-2; C), tumor necrosis factor alpha (TNF-α; D), interleukin 6 (IL-6; E) and interleukin 1 beta (IL-1β; F) in perivascular adipose tissue (PVAT) from 3- or 24- months-old rats fed ad libitum, or 24-months-old rats after 3 months of caloric restriction. Data are represented as mean ± S.E.M (n = 8 samples/group). ⁎P < 0.05 vs. 3 m; ⁎⁎P < 0.01 vs. 3 m; ⁎⁎⁎P < 0.001 vs. 3 m #P < 0.05 vs. 24 m.

et al., 2003). Our results demonstrate that the periaortic adipose tissue of 24 m rats show a proinflammatory profile as it overexpresses iNOS, TNF-α and IL-1β compared to the PVAT from young rats. These results agree with those reported by Fleenor et al. (2014) who found a greater production of superoxide and pro-inflammatory proteins in PVAT from old mice. On the contrary the PVAT from aged rats subjected to CR showed decreased mRNA levels of iNOS, TNF-α and IL-1β compared to the PVAT from aged rats fed ad libitum. To our knowledge this is the first study reporting the beneficial effect of CR decreasing the proinflamatory profile of PVAT in aged rats. However it has already been reported that CR decreases macrophage infiltration and the mRNA levels of different proinflammatory cytokines in other adipose tissue depots such as the visceral, the subcutaneous and the brown adipose tissue, with this fact being related with the improvement in insulin sensitivity (Sierra Rojas et al., 2016). Interestingly, and against what we were expecting, the increased gene expression of proinflammatory cytokines in PVAT from 24 m was not associated with a decreased vasodilation in response to insulin in aortic rings. In fact, aortic rings from 24 m rats surrounded by PVAT

acetylcholine in different vascular beds such as the aorta (Zanetti et al., 2010), cerebral (Walker et al., 2014) or carotid arteries (Donato et al., 2013; Rippe et al., 2010). Likewise, the increased release of nitrites and nitrates in response to insulin in primary cultures of endothelial cells from 24 m–CR rats clearly shows both an improvement in aging induced endothelial dysfunction and an improvement in insulin sensitivity directly in the vascular endothelium. Similarly, it is reported that CR directly exerts antioxidative, pro-angiogenic, and anti-inflammatory effects in rat cerebromicrovascular endothelial cells (Csiszar et al., 2014). Finally, since PVAT is reported to secrete a number of vasoactive substances that affect the vascular tone, and as it has been suggested that inflammation of PVAT may be implicated in vascular dysfunction by causing the disappearance of its anticontractile effect, we aimed to investigate the inflammatory profile of PVAT and its possible relation with the aging induced impairment of insulin induced vasodilation in aorta rings. This study is relevant since the secretion of proinflamatory markers by PVAT, and specifically the secretion of TNF-α, is reported to affect vascular insulin sensitivity in rats (Yudkin et al., 2005; Zhang 9

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Fig. 8. Relaxation insulin dose-response curve of aortic rings surrounded by perivascular adipose tissue (PVAT) from 3-month-old Wistar rats fed ad libitum (3 m; n = 12), 24-month-old Wistar rats fed ad libitum (24 m; n = 12) and 24-month-old Wistar rats subjected to caloric restriction (24 m–CR; n = 12) in the absence (A) or presence of the nitric oxide synthase inhibitor L-NAME (10− 4 M). Data are represented as mean ± S.E.M. (n = 12 rats/group). ⁎P < 0.05 vs 3 m; ⁎⁎P < 0.01 vs. 3 m; ⁎⁎⁎P < 0.001 vs 3 m. $P < 0.05 vs 24 m; $$P < 0.01 vs 24 m; #P < 0.05 vs its respective control.

Funding

exerted a greater insulin-induced vasodilation than aortic rings from both 3 m and 24 m–CR rats. This effect could be related with the upregulation in the gene expression of both eNOS and iNOS in the PVAT of aged rats fed ad libitum. However, preincubation of aorta segments surrounded by PVAT with the nitric oxide synthase inhibitor L-NAME only attenuated insulin-induced vasodilation of aortic rings from 3 m rats but not insulin-induced vasodilation of aortic rings from 24 m and 24 m–CR rats, which indicates that the relaxation in response to insulin in these segments is independent of NO. Accordingly, it has been reported that PVAT induced vasorelaxation is mediated by potassium channels (Lynch et al., 2013; Tsvetkov et al., 2016), although the identity of the K + channel subtype(s) involved is still a matter of debate (Tano et al., 2014). Although the specific mechanism implied in the increased insulin-induced vasorelaxation of aorta segments surrounded by PVAT in 24 m rats and its prevention by CR requires further investigation, it is possible that the increased amount of PVAT surrounding the aorta of 24 m rats may secrete higher concentrations of the releasing adipocyte-derived relaxing (ADRF), whose identity is still unknown. In addition, in this study we have just analyzed the role of the PVAT surrounding the thoracic aorta which seems to be more similar to brown adipose tissue rather than to visceral adipose tissue (Victorio et al., 2016), and is reported to be less affected by aging, both functionally and morphologically, than the PVAT surrounding the abdominal aorta (Padilla et al., 2013). In conclusion, a moderate protocol of CR consisting in a 20% reduction of food intake for 3 months attenuates aging-induced alterations in insulin vascular sensitivity and prevents aging-induced overexpression of proinflammatory cytokines in thoracic periaortic adipose tissue in rats. If these results could be extrapolated to humans, CR may be a promising dietetic strategy to prevent/treat aging induced cardiovascular alterations related to increased vascular insulin resistance.

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